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Maternal-fetal disposition, fetal pharmacodynamics and comparative pharmacokinetics of diphenhydramine… Yoo, Sun Dong 1989

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"MATERNAL-FETAL DISPOSITION, FETAL PHARMACODYNAMICS AND COMPARATIVE PHARMACOKINETICS OF DIPHENHYDRAMINE IN PREGNANT AND NONPREGNANT SHEEP by SUN DONG YOO B . S c . (Pharm.), Sungkyunkwan U n i v e r s i t y , 1980 M.Sc. (Pharm.), The U n i v e r s i t y of Manitoba, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Facul ty of Pharmaceutical Sciences D i v i s i o n of Pharmaceutics and Biopharmaceutics) We accept t h i s t h e s i s as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA MAY, 1989 Copyright by Sun Dong Yoo, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmaceutical Sciences The University of British Columbia Vancouver, Canada Date June 21, 1989  DE-6 (2/88) ABSTRACT A c a p i l l a r y GC/NPD assay method which provides improved s e n s i t i v i t y and s e l e c t i v i t y fo r diphenhydramine i s repor ted . The assay method involves s i n g l e drug ex t rac t ion and s p l i t l e s s sample i n j e c t i o n . Standard curves are l i n e a r in the range of 2-320 ng/mL of diphenhydramine, which represents an amount of the drug from -40 pg to 6.4 ng at the de tec tor . Th is assay method was used in the subsequent s tudies of the materna l - fe ta l d i s p o s i t i o n and comparative pharmacokinetics of the drug in pregnant and nonpregnant sheep. Pharmacokinetics of the drug were charac ter i zed in the nonpregnant sheep a f te r i . v . bolus i n j e c t i o n of 25, 50, 100 and 200 mg doses on a crossover b a s i s . The to ta l body c learance and dose-normalized AUC remained unchanged over the dose range s t u d i e d . There were, however, s i g n i f i c a n t increases in the e l im ina t ion h a l f - l i f e and volume of d i s t r i b u t i o n a f t e r a 200 mg dose, as compared to those a f t e r 25 mg dose. The plasma f ree f r a c t i o n of the drug (0.229) was independent of drug concentrat ions over the range of 10-2,000 ng/mL. The d i s p o s i t i o n of diphenhydramine in the maternal and fe ta l plasma, f e t a l t racheal and amniotic f l u i d s was studied in the c h r o n i c a l l y ca the te r i zed materna l - fe ta l sheep fo l lowing maternal i . v . bolus i n j e c t i o n or maternal and f e t a l i . v . i n f u s i o n s . Fol lowing maternal i . v . bolus i n j e c t i o n , the drug was r a p i d l y t rans fe r red from th ewe to the f e t u s , r e s u l t i n g in s i g n i f i c a n t f e t a l drug exposure ( fe ta l /materna l AUC r a t i o , - 0 . 9 ) . There was no s i g n i f i c a n t d i f f e r e n c e i i i in the pharmacokinetic parameters between the pregnant and nonpregnant ewes. Plasma f ree f r a c t i o n of the drug in the pregnant sheep was not s i g n i f i c a n t l y d i f f e r e n t from that in the nonpregnant sheep, but was smal ler than that in the fetus (0.141 vs. 0.277). The t o t a l body c learance in the fe tus (472.7 mL/min) was smal ler than in the mother (3426.1 mL/min). The t ransp lacenta l c learance from fetus to mother (264.4 mL/min) was -3 times higher than that from mother to fetus (82.4 mL/min). Maternal nonplacental c learance (3343.8 mL/min) accounted for -98% of the maternal t o t a l body c learance , whereas f e t a l nonplacental c learance (208.4 mL/min) accounted fo r -45% of the f e t a l to ta l c lea rance . Therefore , in the ewe, the drug appeared to be e l iminated mainly by nonplacental pathways. However, in the f e t u s , both p lacenta l and nonplacental pathways are important f o r drug e l i m i n a t i o n . Maternal drug in fus ions r e s u l t e d in f e t a l CNS depressant e f f e c t s , whereas f e t a l i n f u s i o n s r e s u l t e d in s t i m u l a t i o n ; t h i s d i f f e r e n c e could be a t t r ibu ted to the d i f f e r e n t f e t a l plasma drug concentrat ions achieved. Diphenhydramine accumulated both in the amniotic and f e t a l t racheal f l u i d s . Fol lowing i n j e c t i o n of diphenhydramine into the amniotic c a v i t y , an average of 63% was e l iminated by the mother, of which, -47% was rece ived from the fetus and the res t (16%) from the amniotic f l u i d via d i r e c t d i f f u s i o n across the uterus . A l s o , -37% of the to ta l a v a i l a b l e drug was el iminated by the f e t u s , which i s greater than that obtained a f t e r maternal i . v . bolus i n j e c t i o n (-5%) or maternal in fus ion (-2%) but i s comparable to that a f te r f e t a l i n f u s i o n (-45%). Diphenhydramine in jec ted into the amniotic sac , the re fo re , appeared to be p r e f e r e n t i a l l y taken up by the f e t u s , r e s u l t i n g in much greater degree of f e t a l drug exposure than maternal exposure. The f e t a l i v pulmonary ex t rac t ion r a t i o of the drug at s teady-s ta te averaged -0 .08 and the d e l i v e r y of the drug to the lungs via the pulmonary c i r c u l a t i o n seemed to be s u f f i c i e n t to account f o r drug accumulation in the f e t a l t racheal f l u i d . V TABLE OF CONTENTS Chapter ABSTRACT LIST OF TABLES LIST OF FIGURES LIST OF SCHEMES LIST OF ABBREVIATIONS ACKNOWLEDGEMENTS 1. INTRODUCTION 1 1.1. General Background 1 1 .1 .1 . Pharmacology and Therapeutics 1 1 .1 .2 . T o x i c i t y 3 1.2. Bas ic Pharmacokinetics 4 1 .2 .1 . Absorpt ion 4 1 .2 .2 . D i s t r i b u t i o n 4 1 .2 .3 . Plasma Prote in Binding 5 1.2.4. Metabolism 5 1 .2 .5 . Excret ion 7 1.2.6. In teract ions with Other Drugs 7 1.3. A n a l y t i c a l Methods 9 1.4. Ant ih is tamines in Pregnancy 10 1 .4 .1 . Ant ih istamine Use in Pregnancy 10 1.4.2. Placental t r a n s f e r of Ant ih istamines 11 1 .4 .3 . Safety of Ant ih istamines in Pregnancy 12 1.4.4. E f f e c t s of Drugs on the Fetal Central Nervous System 13 1.5. Rat ionale and S p e c i f i c Aims of the Research 16 1 .5 .1 . Rat ionale 16 1.5.2. S p e c i f i c Aims 18 2. EXPERIMENTAL 19 Page i i x xi i i xvi xvi i xx i vi 2 .1 . Development of a C a p i l l a r y GC/NPD Assay Method f o r Diphenhydramine 19 2 . 1 . 1 . Mater ia ls 19 2 .1 .2 . Preparat ion of Stock So lu t ions 19 2 . 1 . 3 . Instrumentation and Opt imizat ion of Gas Chromatographic Condi t ions 20 2 . 1 . 3 . 1 . Equipment 20 2 . 1 . 3 . 2 . Opt imizat ion of Gas Chromatographic Condi t ions 20 2 .1 .4 . Drug Ext rac t ion Studies 22 2 .1 .5 . Routine Procedures fo r Diphenhydramine A n a l y s i s 22 2 . 1 . 5 . 1 . Operating GC Condi t ions 22 2 . 1 . 5 . 2 . Drug Ext rac t ion from B i o l o g i c a l Samples 23 2 . 1 . 5 . 3 . Preparat ion of the C a l i b r a t i o n Curve 23 2 . 1 . 5 . 4 . Gas Chromatography-Mass Spectrometry 24 2 . 1 . 5 . 5 . A p p l i c a t i o n of the Developed Assay Method to a Placental Transfer Study 25 2 .2 . Pharmacokinetics of Diphenhydramine a f te r Dose Ranging in Nonpregnant Sheep 25 2 . 2 . 1 . Animals and Surg ica l Preparat ion 25 2 .2 .2 . Drug Admin is t ra t ion and Experimental Protocol 26 2 . 2 . 3 . Pharmacokinetic Ana lys is 27 2 .2 .4 . Plasma Prote in Binding 28 2 .2 .5 S t a t i s t i c a l Ana lys is 30 2 .3 . Pharmacokinetics of Diphenhydramine in the Pregnant Ewe and Fetal Lamb: i . v . Bolus In ject ion Studies 30 2 . 3 . 1 . Animals and Surg ica l Preparat ion 30 2 . 3 . 2 . Drug Admin is t ra t ion and Experimental Protocol 31 2 . 3 . 3 . Pharmacokinetic A n a l y s i s 32 2 .4 . Transplacenta l C learance, Nonplacental Clearance and Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep: Maternal and Fetal Infusions to Steady-State 34 2 . 4 . 1 . Animals and Surg ica l Preparat ion 34 2 .4 .2 . Drug Admin is t ra t ion and Experimental Protocol 35 2 . 4 . 3 . Pharmacokinetic Ana lys is 36 2 .4 .4 . Plasma Protein Binding Studies 38 2 . 4 . 5 . Recording and A n a l y t i c a l Techniques 38 2 . 4 . 6 . S t a t i s t i c a l A n a l y s i s 40 2 .5 . R e c i r c u l a t i o n of Diphenhydramine from Amniotic F l u i d to the Ewe and Fetal Lamb: In t ra-amniot ic Bolus In ject ion Studies 40 2 . 5 . 1 . Animals and Surg ica l Preparat ion 40 2 .5 .2 . Drug Admin is t ra t ion and Experimental Protocol 41 2 . 5 . 3 . Data Ana lys is 42 2.6 . Pulmonary D i s p o s i t i o n of Diphenhydramine in the Fetal Lamb: Maternal or Fetal Infusions to Steady-State 42 2 . 6 . 1 . Animals and Surg ica l Preparat ion 42 2 .6 .2 . Drug Admin is t ra t ion and Experimental Protocol 43 2 . 6 . 3 . Pharmacokinetic Ana lys is 44 RESULTS 45 3 .1 . A n a l y t i c a l Development 45 3 . 1 . 1 . Opt imizat ion of C a p i l l a r y GC Condi t ions 45 3 .1 .2 . Opt imizat ion of Drug Ext rac t ion Procedures 48 3 . 1 . 3 . A p p l i c a t i o n of the Developed Assay Method to Placental Transfer Studies in Pregnant Sheep 53 3 .2 . Pharmacokinetics of Diphenhydramine a f te r Dose Ranging in Nonpregnant Sheep 59 3 .3 . Pharmacokinetics of Diphenhydramine in the Pregnant Ewe and Fetal Lamb: i . v . Bolus In ject ion Studies 68 3 .4 . Transplacenta l C learance, Nonplacental Clearance and Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep: Maternal and Fetal Drug Infusions to Steady-State 80 3 . 4 . 1 . Transplacenta l and Nonplacental Clearances in Materna l -Feta l Unit 80 3 . 4 . 1 . 1 . Achievement of Steady-State Diphenhydramine Concentrat ions in Maternal and Fetal Plasma 80 3 . 4 . 1 . 2 . Plasma Protein Binding in Maternal and Fetal Sheep 87 3 . 4 . 1 . 3 . Drug D i s p o s i t i o n in Fetal Tracheal and Amniotic F l u i d s 87 3 . 4 . 1 . 4 . Transplacenta l and Nonplacental Clearances 94 3 .4 .2 . Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep 101 3 . 4 . 2 . 1 . Maternal Diphenhydramine Infusion 101 3 . 4 . 2 . 2 . Fetal Diphenhydramine Infusion 105 3 . 4 . 2 . 3 . Control Sa l ine Infusion 113 3 .5 . R e c i r c u l a t i o n of Diphenhydramine from Amniotic F l u i d to the Ewe and Fetus: In t ra-amniot ic Bolus In ject ion Studies 115 3 .6 . Pulmonary Ext rac t ion of Diphenhydramine in Fetal Lamb Fol lowing Maternal or Fetal Infusion to Steady-State 123 DISCUSSION 129 4 . 1 . Development of a C a p i l l a r y GLC/NPD Assay Method 129 VI 11 4 .2 . Pharmacokinetics of Diphenhydramine a f t e r Dose Ranging in Nonpregnant Sheep 132 4 . 2 . 1 . D o s e - L i n e a r i t y of Pharmacokinetics of Diphenhydramine in Nonpregnant Sheep 132 4 . 2 . 2 . Plasma Prote in Binding in Nonpregnant Sheep 134 4 .3 . Pharmacokinetics of Diphenhydramine in Pregnant Sheep Fol lowing i . v . Bolus Admin is t ra t ion 136 4 . 3 . 1 . P lacenta l T rans fe r of Diphenhydramine and the Extent of Fetal Drug Exposure 136 4 . 3 . 2 . D i s p o s i t i o n of Diphenhydramine in Maternal and Fetal Sheep 136 4 . 3 . 3 . Comparisons of Diphenhydramine Pharmacokinetics Between Pregnant and Nonpregnant Sheep 137 4 . 3 . 4 . Extent of Drug E l im ina t ion from the Fetal Lamb Fol lowing Maternal i . v . Bolus Dosing 137 4 . 3 . 5 . Drug Accumulation in Fetal Tracheal and Amniotic F l u i d s 138 4 .4 . Transplacenta l C learance, Nonplacental Clearance and Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep: i . v . Infusion to Steady-State 143 4 . 4 . 1 . Transplacenta l and Nonplacental Clearances 4 . 4 . 2 . Drug Accumulation in Fetal Tracheal and Amniotic F l u i d s 4 . 4 . 3 . Drug D i s p o s i t i o n in Maternal and Fetal Sheep Fol lowing Maternal and Fetal Infusions 4 . 4 . 4 . Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep 4 . 5 . Drug R e c i r c u l a t i o n from Amniotic F l u i d to the Ewe and Fetal Lamb Fol lowing In t ra -amniot ic Bolus Admin is t ra t ion 4 . 5 . 1 . Amniotic F l u i d Dynamics 4 . 5 . 2 . Extent of Drug Excret ion from Amniotic F l u i d by Fetal Swallowing 4 . 5 . 3 . D i s p o s i t i o n of Diphenhydramine in the Fetal Tracheal F l u i d s , Materna l -Feta l Drug In jec t ion into Amniotic Cavi ty 4 . 5 . 4 . Extent of Drug E l im ina t ion from the Ewe and Fetus Fol lowing Drug In ject ion into Amniotic Cav i ty Amniotic and Plasma a f t e r 4 . 6 . Pulmonary D i s p o s i t i o n of Diphenhydramine in the Fetal Lamb Fol lowing Maternal or Fetal Infusions to Steady-State 143 151 152 155 162 162 164 165 168 171 SUMMARY AND CONCLUSIONS 175 REFERENCES 1 8 6 X LIST OF TABLES Table 1. C a l i b r a t i o n curve data fo r sheep plasma. 56 Table 2. Mean values of a r t e r i a l P o 2 , 0 2 content and hematocrit obtained a f te r i . v . bolus i n j e c t i o n of diphenhydramine to 6 nonpregnant sheep. 60 Table 3. Estimates of NONLIN pharmacokinetic constants of diphenhydramine obtained a f te r i . v . bolus i n j e c t i o n of 25, 50, 100 and 200 mg doses of the drug to 6 nonpregnant sheep. 63 Table 4. Pharmacokinetic parameters of diphenhydramine obtained a f t e r i . v . bolus i n j e c t i o n s to 6 nonpregnant sheep. 64 Table 5. Free f r a c t i o n of diphenhydramine unbound to plasma prote in determined in samples obtained a f t e r i . v . bolus i n j e c t i o n of the 200 mg doses to nonpregnant sheep. 70 Table 6. Physica l c h a r a c t e r i s t i c s of ind iv idua l pregnant sheep used in the maternal i . v . bolus i n j e c t i o n study. 71 Table 7. The e f f e c t of maternal diphenhydramine admin is t ra t ion on f e t a l a r t e r i a l pH, P c o 2 , P o 2 , 0 2 content and hematocrit fo l lowing 100 mg i . v . bo lus . 73 Table 8. Estimates of NONLIN pharmacokinetic constants obtained from maternal plasma diphenhydramine data fo l lowing a 100 mg i . v . dose of the drug to ind iv idua l ewes. 76 Table 9. Pharmacokinetic parameters obtained a f te r a 100 mg i . v . dose of diphenhydramine to ind iv idua l ewes. 77 Table 10. Percentage of the int ravenously administered maternal dose (100 mg bolus) of diphenhydramine e l iminated by the fetus via nonplacental pathways. 79 Table 11. Physical c h a r a c t e r i s t i c s of ind iv idua l pregnant sheep used in the s teady-s ta te in fus ion study. 82 Table 12. S teady-s ta te diphenhydramine concentrat ions in the ewe and fetus fo l lowing maternal and f e t a l drug admin is t ra t ions . 85 Table 13. Maternal to f e ta l or f e ta l to maternal diphenhydramine concentrat ion r a t i o s at s teady-s ta te fo l lowing maternal and f e t a l drug i n f u s i o n . 86 Table 14. Plasma f ree f r a c t i o n of diphenhydramine in the pregnant ewe and fe tus at s teady-s ta te fo l lowing maternal or f e t a l drug a d m i n i s t r a t i o n . 88 xi Table 15. Values f o r the observed maximum drug concentrat ions ( C m a x ) of diphenhydramine and time to reach C m a x ( T m a x ) in t racheal (TF) and amniotic (AF) f l u i d s fo l lowing maternal and f e t a l in fus ions to s t e a d y - s t a t e . 92 Table 16. Average r a t i o s of the f e t a l t racheal (TF) to a r t e r i a l (FA) diphenhydramine concentrat ions and the e l i m i n a t i o n h a l f - l i f e ( t j j of the drug obtained in maternal (MA) and f e t a l (FA) plasma, t racheal (TF) and amniotic (AF) f l u i d s obtained a f t e r maternal and f e t a l in fus ions to steady-state .93 Table 17. Transplacenta l and nonplacental c learances of diphenhydramine based on to ta l drug concentrat ions in the ewe and fetus fo l lowing maternal and f e t a l in fus ions to s t e a d y - s t a t e . 95 Table 18. Total body c learances of diphenhydramine and percent c o n t r i b u t i o n of nonplacental c learances to to ta l drug e l im ina t ion in the ewe and fetus based on to ta l drug concent ra t ion . 96 Table 19. Transplacenta l and nonplacental c learances of diphenhydramine based on f ree drug concentrat ions in the ewe and fe tus fo l lowing maternal and f e t a l i n f u s i o n s to s t e a d y - s t a t e . 97 Table 20. Total body c learances of diphenhydramine and percent c o n t r i b u t i o n of the nonplacental c learances to to ta l drug e l im ina t ion in the ewe and fetus based on f ree drug concent ra t ions . 98 Table 21. Area under the plasma diphenhydramine concentrat ion vs. time curves in the ewe and fetus and amount of the drug that was e l iminated from the mother and fetus via the nonplacental pathway a f te r maternal and f e t a l i n f u s i o n s . 100 Table 22. Maternal a r t e r i a l pH, Pco?, P 0 2 , O2 content and hematocrit be fore , dur ing and a f te r diphenhydramine in fus ion to the ewe and fetus (n = 6). 102 Table 23. Fetal a r t e r i a l pH, P C O 2 , P 0 2 , Oo content and hematocrit be fore , dur ing and a f te r diphenhydramine i n f u s i o n to the ewe and fetus (n = 8). 103 Table 24. E f f e c t s of diphenhydramine on the fe ta l heart rate and a r t e r i a l pressure c a l c u l a t e d over 30 min i n t e r v a l s before , during and a f t e r drug in fus ions to the ewe and fe tus (n = 8). 104 Table 25. Overa l l inc idence of low, high and intermediate vol tage ECoG episodes in the fetus p r i o r t o , dur ing and a f t e r i n f u s i o n of diphenhydramine to the ewe and f e t u s . 106 xi i Table 26. Percentage of f e t a l breathing in low, high and intermediate vol tage ECoG p r i o r t o , during and a f t e r i n f u s i o n of diphenhydramine to the ewe and fetus (n = 8 ) . 109 Table 27. Percentage of f e t a l e l e c t r o - o c u l a r a c t i v i t y (EoG) in low, high and intermediate voltage ECoG p r i o r t o , dur ing and a f t e r i n f u s i o n of diphenhydramine to the ewe and f e t u s . 110 Table 28. Durat ion of low, high and intermediate vol tage ECoG episodes in the fetus p r i o r t o , dur ing and a f t e r in fus ion of diphenhydramine to the ewe and fetus (n = 8 ) . I l l Table 29. Physica l c h a r a c t e r i s t i c s of ind iv idua l pregnant sheep used in diphenhydramine in t ra -amnio t ic bolus i n j e c t i o n study. 116 Table 30. Values fo r the observed maximum drug concentrat ions ( C m a x ) and time to reach C m a x ( T r a a x ) in maternal (MA) and f e t a l (FA) a r t e r i a l plasma and fe ta l t racheal (TF) and amniotic (AF) f l u i d s fo l lowing i n j e c t i o n of 50 mg diphenhydramine hydrochlor ide into the amniotic c a v i t y 118 Table 31. Average r a t i o s of the maternal a r t e r i a l (MA), umbi l ica l venous (UV) and f e t a l t racheal (TF) to f e t a l a r t e r i a l (FA) drug concentrat ions and the e l i m i n a t i o n h a l f - l i f e ( t i . ) of the drug obtained a f te r i n j e c t i o n of 50 mg diphenhydramine hydrochlor ide in to the amniotic c a v i t y . 120 Table 32. Total area under the drug concentrat ion v s . time curves f o r the maternal and f e t a l a r t e r i a l and umbi l ica l venous plasma, f e t a l t racheal and amniotic f l u i d s a f t e r i n j e c t i o n of 50 mg diphenhydramine hydrochlor ide in to the amniotic c a v i t y . 122 Table 33. The s teady-s ta te diphenhydramine concentrat ions and l e v e l s in the hour fo l lowing cessat ion of the maternal or f e t a l in fus ion in fe ta l pulmonary a r t e r i a l (PA), pulmonary venous (PV), c a r o t i d a r t e r i a l (CA) b lood, f e t a l t racheal (TF) and amniotic (AF) f l u i d s and maternal a r t e r i a l blood (MA). 126 Table 34. Fetal pulmonary ex t rac t ion and the to ta l body c learance of diphenhydramine c a l c u l a t e d at s teady-s ta te a f t e r maternal or f e t a l drug admin is t ra t ion . 128 xi i i LIST OF FIGURES F i g . 1. Hydrogen flow rate-NPD response curves: c a r r i e r gas (helium) flow rate constant , 1 mL/min; and a i r flow rate constant , 60 mL/min. 46 F i g . 2. E f f e c t of a i r f low rate on NPD response and peak width: hydrogen flow rate constant , 3 mL/min; helium flow rate constant , 1 mL/min; and peak width = peak area count/peak he ight . 47 F i g . 3. E f f e c t of column pressure on NPD response and peak width: hydrogen flow rate constant , 3 mL/min; a i r flow rate constant , 50 mL/min; and peak width = peak area count/peak he ight . 49 F i g . 4. Re la t ionsh ip between height equivalent to t h e o r e t i c a l p la tes ( H . E . T . P . ) vs. l i n e a r v e l o c i t y of the helium c a r r i e r gas determined f o r diphenhydramine. Column head pressures were 7, 9, 11, 13, 15 and 17 p s i . 50 F i g . 5. E f f e c t of purge a c t i v a t i o n time on NPD response: hydrogen flow rate constant , 3 mL/min; a i r f low ra te constant , 50 mL/min; and helium flow rate constant , 1 mL/min. 51 F i g . 6. E f f e c t of the add i t ion of t r ie thy lamine (TEA) on ex t rac t ion of diphenhydramine (hatched bar) and orphenadrine (open bar) with dichloromethane (IAA, isoamyl a l c o h o l ) . 52 F i g . 7. Ex t rac t ion time vs. NPD response curves: dichloromethane was used f o r drug e x t r a c t i o n . 54 F i g . 8. Representat ive c a p i l l a r y gas chromatograms obtained from blank sheep plasma (A) and a plasma sample from a pregnant sheep (B) a f t e r admin is t ra t ion of 100 mg of diphenhydramine hydrochlor ide in t ravenous ly . 55 F i g . 9. A semilogar i thmic p lo t of the diphenhydramine concentrat ion vs. time p r o f i l e s obtained in maternal and f e t a l plasma fo l lowing a 100 mg i . v . bolus dose to a pregnant ewe. 58 F i g . 10. Average plasma drug concentrat ion vs. time curves obtained a f t e r i . v . admin is t ra t ion of 25, 50, 100 and 200 mg doses of diphenhydramine hydrochlor ide to 6 nonpregnant sheep. 61 F i g . 11. Changes in the terminal e l im ina t ion rate constant (lambda n) of diphenhydramine as a funct ion of dose obtained in 6 nonpregnant sheep. Each symbol represents each animal and the hor izonta l l i n e s represent mean va lues . 65 F i g . 12. S teady-s ta te volume of d i s t r i b u t i o n (Vd^*) of diphenhydramine as a funct ion of dose obtained in 6 nonpregnant sheep. 66 xiv F i g . 13. Re la t ionsh ip between the area under the plasma drug concentrat ion vs. time curve and the i . v . bolus doses of diphenhydramine given on a crossover bas is to 6 nonpregnant sheep. 67 F i g . 14. Determination of equ i l ib r ium time used in the study of plasma prote in binding of diphenhydramine by e q u i l i b r i u m d i a l y s i s (n = 3 ) . 69 F i g . 15. Representat ive c a p i l l a r y gas chromatograms of the maternal (MA) and f e t a l (FA) a r t e r i a l plasma, f e t a l t racheal (TF) and amniotic (AF) f l u i d s obtained from c o n t r o l - and drug-t reated pregnant sheep. 74 F i g . 16. Representat ive semilogar i thmic p lo ts of the maternal and f e t a l a r t e r i a l plasma, f e ta l t racheal and amniotic f l u i d diphenhydramine concentrat ion vs. time p r o f i l e s fo l lowing a 100 mg bo lus . 75 F i g . 17. Average maternal and f e t a l plasma diphenhydramine concentrat ion vs. time curves obtained fo l lowing i . v . i n f u s i o n of the drug to the ewe (n = 8 ) . 83 F i g . 18. Average maternal and f e t a l plasma diphenhydramine concentrat ion vs. time curves obtained fo l lowing i . v . i n f u s i o n of the drug to the fetus (n = 8 ) . 84 F i g . 19. Representat ive semilogar i thmic p lo ts of the maternal and f e t a l a r t e r i a l plasma, f e t a l t racheal and amniotic f l u i d diphenhydramine concentrat ion vs. time curves in a pregnant ewe and fetus fo l lowing drug in fus ion to the ewe. 90 F i g . 20. Representat ive semilogar i thmic p lo ts of the maternal and f e t a l a r t e r i a l plasma, f e ta l t racheal and amniotic f l u i d diphenhydramine concentrat ion vs. time curves in a pregnant ewe and fetus fo l lowing drug in fus ion to the f e t u s . 91 F i g . 21. High speed chart recordings of h igh , low and intermediate vol tage ECoG patterns in a f e t a l lamb. 107 F i g . 22. Representat ive examples of the recording of breath ing , ECoG and e l e c t r o - o c u l o g r a p h i c (EoG) a c t i v i t y in the f e t a l lamb during the contro l per iod (A) and during f e t a l i n f u s i o n of diphenhydramine (B). 108 F i g . 23. Examples of the large amplitude breathing a c t i v i t y present in the i n i t i a l phases of diphenhydramine in fus ions to the f e t u s . 114 F i g . 24. Average diphenhydramine concentrat ion vs. time curves fo r maternal and fe ta l a r t e r i a l plasma, f e t a l t racheal and amniotic f l u i d s fo l lowing admin is t ra t ion of 50 mg diphenhydramine hydrochlor ide in to the amniotic c a v i t y in to 5 pregnant sheep. 117 XV F i g . 25. Representat ive diphenhydramine concentrat ion vs. time curves fo r maternal and f e t a l a r t e r i a l and umbi l ica l venous plasma, f e t a l t racheal and amniotic f l u i d s fo l lowing i n t r a -amniotic bolus i n j e c t i o n of 50 mg diphenhydramine hydrochlor ide to a pregnant ewe (ewe no. 287). Inset f i g u r e shows the f e t a l a r t e r i a l and umbi l ica l venous plasma and f e t a l t racheal drug concentrat ion vs. time curves over the i n i t i a l 6 hr per iod a f te r drug admin is t ra t ion . 121 F i g . 26. P lo ts of the fe ta l pulmonary a r t e r i a l (PA), pulmonary venous (PV) and c a r o t i d a r t e r i a l (GA) and maternal a r t e r i a l (MA) diphenhydramine concentrat ion vs. time p r o f i l e s fo l lowing maternal drug i n f u s i o n . 124 F i g . 27. Representat ive p lo ts of the fe ta l pulmonary a r t e r i a l (PA), pulmonary venous (PV) and c a r o t i d a r t e r i a l (CA) and maternal a r t e r i a l (MA) diphenhydramine concentrat ion vs. time p r o f i l e s fo l lowing fe ta l drug i n f u s i o n . 125 xvi LIST OF SCHEMES Scheme 1. Chemical s t ruc ture of diphenhydramine. 1 Scheme 2. A two-compartment open model used in the c a l c u l a t i o n of t ransp lacenta l and nonplacental c learances in the materna l - fe ta l uni t (Szeto et al., 1982). 37 xvii LIST OF ABBREVIATIONS AAG ct j -acid g lycopro te in AF amniot ic f l u i d ANOVA a n a l y s i s of var iance AUC area under the plasma drug concentra t ion vs. time curve AUMC area under the f i r s t moment curve AUTOAN a decis ion-making pharmacokinetic computer program °C "cent igrade Cf s teady -s ta te f e t a l plasma t o t a l (bound and unbound) drug concentra t ion a f t e r maternal i . v . i n f u s i o n Cf/ s teady -s ta te f e t a l plasma to ta l drug concentra t ion a f t e r f e t a l i . v . i n f u s i o n ^ f , f r e e s teady -s ta te f e t a l plasma unbound drug concentra t ion a f te r maternal i . v . i n fus ion c f ' , f r e e s teady -s ta te f e t a l plasma unbound drug concentra t ion a f t e r f e t a l i . v . i n f u s i o n CI-MS chemical ion iza t ion-mass spectrometry CL f f t o t a l drug c learance from the f e t a l compartment C L f m t r ansp lacenta l c learance of drug from the f e t a l to maternal compartment C L f 0 nonplacental c learance of drug from the f e t a l compartment C L m f t ransp lacenta l c learance of drug from the maternal to f e ta l compartment C L m m t o t a l drug c learance from the maternal compartment C L m o nonplacental c learance o f drug from the maternal compartment C/.jg t o t a l body c learance of to ta l drug (bound and unbound) xvi i i cm centimeter C m s teady-s ta te maternal plasma to ta l (bound and unbound) drug concentrat ion a f te r maternal i . v . i n f u s i o n C m / s teady-s ta te maternal plasma to ta l drug concentrat ion a f te r f e t a l i . v . i n fus ion C m a x maximum drug concentrat ion Cm,free s teady-s ta te maternal plasma unbound drug concentrat ion a f t e r maternal i . v . i n fus ion Cm' , f ree s teady-s ta te maternal plasma unbound drug concentrat ion a f t e r f e ta l i . v . i n fus ion CNS cent ra l nervous system C.V . c o e f f i c i e n t of v a r i a t i o n ECoG e l e c t r o c o r t i c o g r a p h i c EEG e l e c t r o e n c e p h a l o g r a p h ^ EOG e l e c t r o - o c u l o g r a p h i c EI e lec t ron impact EI-MS e lec t ron impact-mass spectrometry eq. equation ER ex t rac t ion r a t i o FA f e t a l a r t e r i a l F i g . f i g u r e g gram g a c c e l e r a t i o n of g r a v i t y GC gas chromatography GC-MS gas chromatography-mass spectrometry GC/NPD gas chromatography with nitrogen-phosphorus detec t ion H 2 hydrogen gas xix HC1 hydroch lor ic ac id He helium gas H. E . T . P . height equivalent to a t h e o r e t i c a l p la te Hg mercury HPLC high performance l i q u i d chromatography hr hour I. D. in terna l diameter i .m. intramuscular I .U. in te rna t iona l uni t i . v . intravenous kg ki logram k 0 drug in fus ion rate to the mother k Q / drug in fus ion rate to the fetus K H 2 P O 4 monopotassium phosphate L l i t e r M molar ( m o l e s / l i t e r ) m meter MA maternal a r t e r i a l mg mi l l ig ram Ug microgram mL m i l l i l i t e r mm mi l l ime te r min minute m.w. molecular weight N 2 0 n i t rogen oxide NaOH sodium hydroxide ng nanogram NONLIN computer program fo r non - l inea r l e a s t squares regress ion a n a l y s i s of pharmacokinetic systems NPD nitrogen-phosphorus detector 0 2 oxygen gas O.D. outer diameter Pco 2 p a r t i a l pressure of carbon d iox ide in the blood pH negative logari thm of hydrogen ion concentrat ion P o 2 p a r t i a l pressure of oxygen in the blood PTFE po ly te t ra f luoroe thy lene Qum umbi l ica l blood flow r c o r r e l a t i o n c o e f f i c i e n t r^ c o e f f i c i e n t of determination REM rap id eye movement s . c . subcutaneous S .D . standard dev ia t ion sec second tk h a l f - l i f e t m a x time taken to reach maximum drug concentrat ion TP t racheal pressure t^ re tent ion time of the peak vs. versus * s i g n i f i c a n t l y d i f f e r e n t (p<0.05) ACKNOWLEDGEMENTS I would l i k e to s i n c e r e l y thank Dr. James E. Axelson f o r h is s u p e r v i s i o n , encouragement and f i n a n c i a l support throughout my Ph.D. program. I am deeply indebted to Dr. Dan W. Rurak f o r h is guidance, c r i t i c a l commentary and a l l the support with the animal experiments. I would a lso l i k e to thank my superv isory committee members, Dr. Frank S. Abbott , Dr. Jim M. Orr and Dr. John G. S i n c l a i r f o r t h e i r encouragement and c o n s t r u c t i v e c r i t i c i s m throughout the program. I thank Ms. Sandy M. T a y l o r , Dr. K. Wayne Riggs, Mr. Eddie Kwan and Ms. Marlene Van der Weyde fo r t h e i r help with the animal preparat ion and experiments and Barbara A. McErlane and David Main f o r t h e i r ass is tance with drug a n a l y s i s . I a lso thank Ms. Grace Chan and Mr. Matthew R. Wright f o r t h e i r cons t ruc t i ve c r i t i c i s m . Thanks a lso to my col leagues in the l a b o r a t o r y , George R. Tonn, J ing Wang, Krishnaswamy Yeleswaram fo r t h e i r encouragement and f r i e n d s h i p . Graduate Teaching A s s i s t a n t s h i p , Graduate Research A s s i s t a n t s h i p , U n i v e r s i t y Graduate Fe l lowsh ip , U n i v e r s i t y Travel Fund and CIDA Travel Grant are g r a t e f u l l y acknowledged. Th is pro jec t was supported by the Medical Research Counci l of Canada. xxi i This t h e s i s i s dedicated to my mother, Yong Ok Ahn and my s i s t e r , Dong Ju Yoo fo r t h e i r constant l o v e , encouragement and support from the other s ide of the world and a lso to the memory of my f a t h e r , Tae Seung Yoo. 1 1. INTRODUCTION 1.1. General Background Diphenhydramine, 2- (diphenylmethoxy)-N,N-dimethylethylamine, i s a potent histamine H j - receptor antagonist that possesses a n t i c h o l i n e r g i c , ant iemet ic and sedat ive proper t ies (Douglas, 1985). Diphenhydramine was synthesized in the 1940s ( R i e v e s c h l , 1947) and has been in c l i n i c a l use for over 40 y e a r s . Th is compound i s a v a i l a b l e , without p r e s c r i p t i o n , as an a n t i h i s t a m i n i c , ant iemet ic and sedat ive agent. Diphenhydramine i s a weakly bas ic amine with molecular weight (m.w.) of 255.4 and a pKa of 9.0 (Marsha l l , 1955; de Roose et al., 1970). Its s t r u c t u r a l formula i s shown in Scheme 1. Scheme 1. Chemical s t ruc ture of diphenhydramine. 1 .1 .1 . Pharmacology and Therapeutic E f f e c t s A number of review a r t i c l e s descr ibe the pharmacological act ions of diphenhydramine (Loew, 1947; M e l v i l l e , 1973; Drouin , 1985). Diphenhydramine appears to block histamine H j - receptors compet i t i ve ly and 2 r e v e r s i b l y (Rocha e S i l v a , 1978). Diphenhydramine i s capable of antagoniz ing the a b i l i t y of histamine to contract smooth muscle in the g a s t r o i n t e s t i n a l t r a c t and lungs of guinea p i g s , and to induce v a s o d i l a t o r e f f e c t s in cats and dogs ( M e l v i l l e , 1973; Douglas, 1985). Diphenhydramine a lso i n h i b i t s the act ion qf histamine-mediated a l l e r g i c responses such as wheal format ion, edema and increased c a p i l l a r y permeab i l i t y . It suppresses s a l i v a r y and lacr imal secre t ion caused by histamine but f a i l s to antagonize the ac t ion of histamine to mediate g a s t r i c secre t ion ( M e l v i l l e , 1973). The act ion of histamine in the s t imula t ion of g a s t r i c ac id secre t ion i s thought to be mediated via h^-receptors (Black et al., 1972). Diphenhydramine has a b iphas ic act ion on mast c e l l s : at low doses, i t i n h i b i t s the re lease of histamine and at higher doses, i t induces histamine re lease (Mota and Dias Da S i l v a , 1960; Lau and Pearce, 1985). Diphenhydramine is known to be e f f e c t i v e in r e l i e v i n g a l l e r g i c symptoms such as hay fever , a l l e r g i c r h i n i t i s , u r t i c a r i a , dermatoses and p r u r i t i s (Douglas, 1985). It i s a lso known to reduce post -opera t ive nausea and vomi t ing , and to reduce vomiting due to malignancy or a n t i n e o p l a s t i c drug use. It i s recommended that diphenhydramine be administered as a 25-50 mg oral dose and as a 10-50 mg i . v . or i .m. dose, with a maximum dose of 400 mg per day (Krogh, 1987). Diphenhydramine is cont ra ind ica ted in newborn i n f a n t s , and in pat ients exper iencing an asthmatic attack or h y p e r s e n s i t i v i t y to the drug. Safety has not been proven fo r use of diphenhydramine in pregnancy and l a c t a t i o n and, as a r e s u l t , the use of the drug dur ing pregnancy i s not recommended (Krogh, 1987). A good c o r r e l a t i o n has been reported between plasma 3 diphenhydramine concentrat ions and the suppression of histamine- induced sk in wheal reac t ion or the sedat ive e f f e c t s of the drug in humans (Carruthers et al., 1978). Fol lowing i . v . or oral admin is t ra t ion of 50 mg diphenhydramine hydroch lor ide , a n t i h i s t a m i n e e f f e c t s can l a s t fo r ~7 hr , whereas s leep iness or sedat ive e f f e c t s are observed during the f i r s t ~3 hr (Carruthers et al., 1978). Sleep and/or drowsiness seem to occur when plasma drug l e v e l s are above -70-100 ng/mL (Albert et al., 1975; Carruthers et al., 1978). It appears that a n t i h i s t a m i n e e f f e c t s can be e l i c i t e d at a therapeut ic range of -25-50 ng/mL, without s i g n i f i c a n t sedat ion . 1 .1.2. T o x i c i t y Studies of acute diphenhydramine t o x i c i t y in animals show that the i . v . L D 5 0 of the drug i s 42-46 mg/kg in the r a t , 98 mg/kg in mice, 10-11 mg/kg in rabb i ts and 24-30 mg/kg in the dog. The oral L D 5 0 of diphenhydramine ranges from 500-545 mg/kg in ra ts and from 164-167 mg/kg in mice (Rieveschl and Gruhz i t , 1945; Gruhzi t and F isken , 1947). In these animal s p e c i e s , t o x i c doses may cause excitement, tremors, convuls ions and r e s p i r a t o r y and card iac f a i l u r e r e s u l t i n g in death (Rieveschl and Gruhz i t , 1945; Gruhzi t and F isken , 1947). In humans, diphenhydramine poisoning has been f requent ly reported, accounting f o r -5% of the to ta l number of drug i n t o x i c a t i o n s (Koppel and Ibe, 1987). Acute symptoms of diphenhydramine poisoning in humans include impaired consc iousness , h a l l u c i n a t i o n , mydr ias is , t a c h y c a r d i a , se izure and convuls ions (wyngaarden and Seevers, 1951; Hausmann et al., 1983). In the reported cases of human diphenhydramine i n t o x i c a t i o n , approximately 6-40 4 times the therapeut ic dose (50 mg) appears to have been ingested . Topical a p p l i c a t i o n of diphenhydramine can a lso e l i c i t t o x i c symptoms, such as d e l i r i u m and h a l l u c i n a t i o n s in humans ( F i l l o u x , 1986). Acute t o x i c i t y of diphenhydramine can be t reated by g a s t r i c lavage, terminat ion of convuls ions with b a r b i t u r a t e s , diphenylhydantoin or paraldehyde, and v e n t i l a t i o n fo r c a r d i o r e s p i r a t o r y depression (Re iche lder fe r et al, 1955; Hestand and Teske, 1977; Hausmann et a / . , 1983). 1.2. Bas ic Pharmacokinetics 1 .2 .1 . Absorpt ion Fol lowing admin is t ra t ion of a s i n g l e oral dose (50 mg) of diphenhydramine hydrochlor ide in humans, peak plasma drug concentrat ions occurred at 2-4 h r s , with maximum drug concentrat ions of -40-80 ng/mL (Carruthers et al., 1978; Ber l inger et al., 1982). M u l t i p l e oral admin is t ra t ion of 50 mg q.i.d. to human volunteers resu l ted in average s teady -s ta te plasma drug concentrat ions of -100 ng/mL (Glazko et al., 1974). A large f i r s t - p a s s e f f e c t has been noted a f t e r ora l diphenhydramine admin is t ra t ion in humans, with approximately 30-60% of the dose being metabolized by the l i v e r before i t reaches the systemic c i r c u l a t i o n (Albert et al., 1975; Carruthers et al., 1978; Ber l inger et al., 1982; Meredith et al., 1984; Blyden et al., 1986). 1 .2 .2 . D i s t r i b u t i o n Diphenhydramine t i s s u e d i s t r i b u t i o n s t u d i e s , performed fo l lowing 5 subcutaneous ( s . c . ) adminis t ra t ion in the ra t and guinea p i g , showed that the highest concentrat ion was found in lung t i s s u e , fol lowed by sp leen , k idney, l i v e r and muscle (Glazko and D i l l , 1949a). Hausmann et al. (1983) have reported diphenhydramine concentrat ions determined in var ious t i s s u e s of a human pat ient fo l lowing f a t a l overdosage of the drug. The highest concentrat ion was a lso found in the lungs , fol lowed by kidney, l i v e r , sp leen , muscle and plasma. The drug concentrat ion in the lungs was -10 times higher than that found in plasma. 1 .2 .3 . Plasma Prote in Binding A pre l iminary study of diphenhydramine plasma prote in binding determined in two human subjects showed that the drug prote in binding was extensive (-98%, A lber t et al., 1975). Spector et al. (1980) have reported the plasma prote in binding of ^-d iphenhydramine to be -76% and -85% in O r i e n t a l s and Caucasians. Diphenhydramine binds to human serum albumin but the degree of binding i s low, suggesting that diphenhydramine may bind to plasma prote in (s ) other than albumin. However, the plasma prote in binding c h a r a c t e r i s t i c s of the drug have not been ex tens ive ly studied in e i t h e r human or animal s p e c i e s . 1 .2.4. Metabolism The l i v e r appears to be the p r i n c i p a l s i t e of diphenhydramine degradation in r a t s , although the lung and kidney are a lso ac t ive (Glazko and D i l l , 1949b). Diphenhydramine i s r e a d i l y demethylated by rat l i v e r microsome preparat ions in vitro (Roozemond et al., 1965). A study 6 employing paper p a r t i t i o n chromatography of ur ine obtained a f t e r admin is t ra t ion of ^C-diphenhydramine to ra ts showed that at l e a s t s i x d i f f e r e n t r a d i o a c t i v e compounds were present (Glazko et al., 1949). Drach et al. (1970) l a t e r reported the presence of unchanged diphenhydramine and i t s N-demethylated metabol i tes , N,N-didemethyl- and N-demethyl-diphenhydramine, in ra t u r i n e . In the rhesus monkey, N-demethylation fol lowed by ox ida t ive deamination to diphenylmethoxyacetic ac id i s the main metabol ic pathway (Drach and Howell , 1968). The mono- and di-demethylated d e r i v a t i v e s of diphenhydramine, the N-oxide d e r i v a t i v e and the deaminated c a r b o x y l i c ac id d e r i v a t i v e have been i d e n t i f i e d as diphenhydramine metabol i tes in monkey ur ine (Drach and Howell , 1968; Drach et al., 1970). Diphenylmethoxyacetic ac id i s fu r ther conjugated with glutamine in the Rhesus monkey (Drach and Howell , 1968) or with g l y c i n e in the dog (Drach et al., 1970), but ne i ther the f ree nor conjugated form was found in ra t ur ine (Drach et al., 1970). The deaminated c a r b o x y l i c ac id d e r i v a t i v e , diphenylmethoxyacetic a c i d , has been found to be a major metabol i te in monkey plasma and urine (Drach and Howell , 1968; Drach et al., 1970). Benzhydrol had been prev ious ly suggested to be a metabol i te of diphenhydramine in vitro (Glazko and D i l l , 1949b). However, only a t race amount of benzhydrol was detected in monkey urine (Drach and Howell , 1968). The metabol ic pathway(s) in man has not been f u l l y es tab l i shed but the i d e n t i f i e d ur inary excret ion products fo r diphenhydramine, measured by gas chromatography-mass spectrometry (GC-MS), inc lude small amounts of unchanged drug and a primary amine metabol i te , N ,N-didemethyl-diphenhydramine, large amounts of a secondary amine, N-demethyl-diphenhydramine, and diphenylmethoxyacetic ac id as the major metabol i te (Chang et al., 1974). Diphenylmethoxyacetic ac id i s excreted as a f ree or 7 as a conjugated form in humans but the exact nature of the conjugate has not been repor ted . There appears to be no information on diphenhydramine metabolism in sheep. 1 .2 .5 . Excret ion Fol lowing admin is t ra t ion of diphenhydramine (10 mg/kg s . c . ) to r a t s , 4-6% of the dose was excreted in ur ine as unchanged drug (Glazko and D i l l , 1949a). In r a b b i t s , -21% of the administered dose (25-100 mg s . c . ) was excreted in ur ine as unchanged drug (Hald, 1947). In man, ur inary diphenhydramine excre t ion accounts fo r a small por t ion (2-4%) of the administered dose (Hald, 1947; A lber t et al., 1975), and the sum of the ur inary excre t ion of diphenhydramine and i t s metabol i tes accounts f o r -50-60% a f t e r ora l drug adminis t ra t ion (Glazko et al., 1974). In c o n t r a s t , -33% of the to ta l r a d i o a c t i v i t y was recovered in ur ine fo l lowing s . c . admin is t ra t ion of ^C-diphenhydramine to ra ts with the remainder being recovered in the f e c e s , suggesting the p o s s i b i l i t y of b i l i a r y excre t ion (Glazko et al., 1949). 1 .2 .6 . In teract ions with Other Drugs Diphenhydramine i s known to potent ia te e f f e c t s of other c e n t r a l l y a c t i v e drugs such aS jethanol , temazepam and butorphanol (Burns and Moskowitz, 1983; Brademas, 1984; Kargas et al., 1985; Cohen et al., 1987). Since diphenhydramine can cause sedation and mild euphoria at high doses in man, t h i s compound i s subject to abuse (Feldman and Behar, 1986). Diphenhydramine has been reported to reduce the oral absorpt ion of several 8 drugs such as sodium p-aminosa l icy la te in ra ts (Lavigne and Marchand, 1973), acetaminophen and su l f i somid ine in rabb i ts ( Ichibagase et al., 1981; Imamura et al., 1981). This reduct ion of oral b i o a v a i l a b i l i t y was speculated to be due to delay in the g a s t r i c emptying in these s p e c i e s . When administered together with methaqualone, diphenhydramine can a l t e r methaqualone pharmacokinet ics: the e l im ina t ion h a l f - l i f e was increased by ~2 f o l d and the to ta l body c learance decreased by one -ha l f in the ra t (Gupta et al., 1982). The a l t e r a t i o n s in the pharmacokinetics of methaqualone were a t t r ibu ted to an i n h i b i t o r y e f f e c t of diphenhydramine on the hepat ic microsomal b iotransformat ion of the former drug. It i s not c l e a r whether diphenhydramine induces drug-metabol iz ing enzymes. Burns et al. (1963) t reated three dogs with d a i l y oral dose of 50 mg/kg of diphenhydramine. Forty days a f te r the diphenhydramine treatment, they administered 25 mg/kg oral dose of diphenhydramine and measured the drug plasma concentrat ions over 7 hr and found that these were cons iderab ly lower than those measured during the contro l p e r i o d . These i n v e s t i g a t o r s speculated that chronic diphenhydramine admin is t ra t ion resu l ted in enzyme induct ion of i t s own metabolism. The same group of i n v e s t i g a t o r s (Conney and Burns, 1960) a lso reported that pretreatment in ra ts with diphenhydramine (50 mg/kg/day f o r 4 days) shortened the durat ion of zoxazolamine p a r a l y s i s and suggested induct ion of l i v e r microsomal enzymes, although they d id not d i r e c t l y measure the a c t i v i t y of the zoxazolamine-metabol iz ing enzyme system f o r diphenhydramine. There appears to be no d i r e c t evidence fo r the induct ion of l i v e r microsomal enzyme a c t i v i t y by diphenhydramine. 9 1.3. A n a l y t i c a l Methods A number of a n a l y t i c a l methods have been reported fo r the determinat ion of diphenhydramine in b i o l o g i c a l f l u i d s , inc lud ing UV spectrophotometry (Wallace et a 7 . , 1966), f luorescence dye techniques (Glazko et a 7 . , 1962; Glazko et al., 1974), high performance l i q u i d chromatography (HPLC) (Skof i tsch and Lembeck, 1983), GC-MS (Chang et al., 1974; Carruthers et al., 1978) and gas chromatography ( B i l z e r and Gundert-Remy, 1973; A l b e r t et al., 1974; Baugh and C a l v e r t , 1976; Abernethy and Greenbla t t , 1983; Barni Comparini et al., 1983; C h i a r o t t i et al., 1983; Lutz et al., 1983; Meatheral l and Guay, 1984). UV spectrometry and f luorescence dye methods lack s u f f i c i e n t s p e c i f i c i t y and s e n s i t i v i t y fo r t race l eve l drug measurement. The HPLC method i s convenient s ince no e x t r a c t i o n procedure or d e r i v a t i z a t i o n step i s r e q u i r e d , but s e n s i t i v i t y l i m i t a t i o n s requ i re the use of r e l a t i v e l y large volumes of plasma and i n j e c t i o n of the e n t i r e sample volume in to the chromatograph. GC methods employing conventional packed columns have been used fo r monitoring plasma concentrat ions in s i n g l e dose pharmacokinetic s tudies ( B i l z e r and Gundert-Remy, 1973; A l b e r t et al., 1974; Baugh and C a l v e r t , 1976; Abernethy and Greenbla t t , 1983). These methods requi re the use of plasma volumes la rger than that normally c o l l e c t e d during the study of p lacenta l t r a n s f e r of drugs. GC methods employing g lass c a p i l l a r y columns and f l a m e - i o n i z a t i o n de tec t ion have been reported fo r the determination of a number of drugs, i n c l u d i n g diphenhydramine (Barni Comparini et al., 1983; C h i a r o t t i et al., 1983). The combination of n i t r o g e n / p h o s p h o r u s - s p e c i f i c detect ion (NPD) 10 with c a p i l l a r y GC has improved the detect ion l i m i t s f o r diphenhydramine to f a c i l i t a t e s tudies of the pharmacokinetics of the drug in man (Lutz et al., 1983; Meatheral l and Guay, 1984). While these ni t rogen s e l e c t i v e methods provide good s e n s i t i v i t y and s e l e c t i v i t y , there appeared to be two s i g n i f i c a n t l i m i t a t i o n s in these methods: they required large plasma volumes (1 .0-3 .0 mL) fo r ex t rac t ion and a very small f i n a l r e c o n s t i t u t i o n volume (10 nl) f o r i n j e c t i o n (Lutz et al., 1983; Meatheral l and Guay, 1984). Due to these l i m i t a t i o n s in the e x i s t i n g assay methods, there was s t i l l the requirement fo r an assay method with improved s e n s i t i v i t y , which could be r o u t i n e l y appl ied to p lacenta l drug t r a n s f e r s tudies where the sample volume i s l i m i t e d . 1.4. Ant ih is tamines in Pregnancy 1 .4 .1 . Ant ih is tamine Use in Pregnancy Surveys of drug use in pregnancy publ ished between 1963 and 1987 i n d i c a t e that on average -20% of pregnant women take ant ih is tamine-conta in ing preparat ions at some point during pregnancy (Peckham and King , 1963; For fa r and Nelson, 1973; H i l l , 1973; Doering et al., 1978; Brocklebank et al., 1978; Rayburn et al., 1982; Piper et al., 1987). Among a n t i h i s t a m i n i c agents, diphenhydramine appears to be taken by -23% of pregnant women (Piper et al., 1987). The predominant therapeut ic uses of ant ih is tamines during pregnancy are in the treatment of a l l e r g i e s , nausea and vomi t ing , and as cough and co ld remedies. Ant ih is tamine therapy (diphenhydramine or chlorpheniramine) i s e f f e c t i v e in a l l e v i a t i n g symptoms of pregnancy-re la ted u r t i c a r i a l condi t ions (Read, 1977; Ahmed and Kaplan, 11 1981). Nausea and vomiting seem to be r e l a t i v e l y common problems in ear ly pregnancy. The s l i g h t l y higher consumption of ant ih is tamines in the f i r s t t r i m e s t e r , as opposed to l a t e r , i s probably due to the use of the drugs in antinausea and ant iemetic therapy (For far and Nelson, 1973). The common co ld appears to be the most frequent r e s p i r a t o r y complaint in pregnancy, with an o v e r a l l incidence of 17% (Peckham and K ing , 1963). In a d d i t i o n , nasal congest ion r e s u l t i n g from c a p i l l a r y engorgement of the r e s p i r a t o r y t r a c t i s common (Pra t t , 1981) and would exacerbate the symptoms r e s u l t i n g from a l l e r g i c r h i n i t i s and c o l d s . Thus, ant ih istamines appear to be taken at a l l stages of pregnancy. There are l i m i t e d data on the durat ion of ant ih is tamine therapy in pregnancy, although For far and Nelson (1973) report a mean durat ion of 15 days. 1 .4 .2 . P lacenta l Transfer of Ant ih istamines Drugs cross the placenta l a r g e l y by simple d i f f u s i o n , and fo r l i p i d so lub le molecules at l e a s t , there i s no s i g n i f i c a n t b a r r i e r to the t r a n s f e r of compounds with a molecular weight l e s s than -600 (Reynolds, 1979). Most ant ih istamines have a cons iderab ly small molecular weight ( e . g . , diphenhydramine m.w.= 255) and, the re fo re , should r e a d i l y cross the p lacen ta . However, there is very l i t t l e information on the rate and/or extent of p lacenta l t r a n s f e r of the an t ih is tamines . B r i e f mention has been made of materna l - fe ta l t r a n s f e r of promethazine in humans, but there appears to be no publ ished quant i ta t i ve data (Burt , 1971). Placental t r a n s f e r of 3 5 S-promethaz ine seems to occur in the mouse (Hansson and Schmiterlow, 1961). The p lacenta l t r a n s f e r of c imet id ine (Mihaly et al., 1983; Ching et al., 1985) and r a n i t i d i n e (Mihaly et al., 1982) has been 12 reported in sheep. Rapid p lacenta l t r a n s f e r occurs but the f e t a l drug leve l i s cons iderab ly lower than that in maternal plasma fo r both drugs fo l lowing maternal drug admin is t ra t ion . As d iscussed in the fo l lowing s e c t i o n , other i n d i r e c t evidence of p lacenta l t r a n s f e r e x i s t s in the form of apparent f e t a l e f f e c t s of some ant ih istamines fo l lowing adminis t ra t ion to the mother. 1 .4 .3 . Safety of Ant ih istamines in Pregnancy There i s l i t t l e information on harmful e f f e c t s of ant ih istamines in the embryo and f e t u s , e i t h e r in terms of the potent ia l of the drugs to i n t e r f e r e with embryogenesis in the f i r s t t r imester and thus y i e l d te ra togen ic e f f e c t s , or the potent ia l to a f f e c t f e t a l funct ions l a t e r in pregnancy. lams and Rayburn (1982) have reported that there are no known harmful e f f e c t s of ant ih istamines at any stage of pregnancy. In the case of diphenhydramine, there is some disagreement as to whether the drug e l i c i t s harmful e f f e c t s in the f e t u s . No tera togenic e f f e c t s of diphenhydramine have been reported in ra ts and rabb i ts (King et al., 1965; McColl et al., 1967; Schardein et al., 1971). However, other s tudies reported harmful e f f e c t s of the drug, such as reduced l i t t e r s i z e , f e ta l body weight, re ta rda t ion in the development of body organs and a l t e r a t i o n s in pa la te development in mice (Naranzo and Naranzo, 1968; J o n e s - P r i c e et al., 1983). The main source of human data comes from the C o l l a b o r a t i v e Per ina ta l Pro ject (Heinonen et al., 1977), which examined tera togenic drug e f f e c t s in 50,282 mother -ch i ld p a i r s . Of the ant ih istamines s p e c i f i c a l l y examined (chlorpheniramine, pheniramine, diphenhydramine, methapyri lene, t r ipelennamine and bromopheniramine), only bromopheniramine was associated 13 with a higher than expected incidence of f e t a l anomalies. However, in a r e t r o s p e c t i v e F inn ish study (Saxen, 1974) where 559 c h i l d r e n with oral c l e f t s were matched with a contro l group, diphenhydramine intake during the f i r s t t r imester was greater in mothers of the c l e f t group. Parkin (1974) g ives c l i n i c a l d e t a i l s of an in fant whose mother had taken 150 mg of diphenhydramine hydrochlor ide d a i l y during pregnancy f o r treatment of a p r u r i t i c r a s h . On the f i f t h postnatal day, the in fant developed genera l i zed tremulousness and d i a r r h e a , which p e r s i s t e d f o r several days. Subsequently, the drug was shown to be present in the blood of the infant on the f i f t h day. These features are cons is ten t with diphenhydramine withdrawal symptoms. 1.4.4. E f f e c t s of Drugs on the Fetal Central Nervous System Although there i s very l i m i t e d information on the cent ra l nervous system (CNS) e f f e c t s of ant ih istamines in the f e t u s , there are numerous reports on the e f f e c t s of other c e n t r a l l y ac t ive agents (Hutchings, 1978; Dai ley et al., 1982). The developing CNS seems p a r t i c u l a r l y vulnerable to t o x i c damage, p a r t l y because of i t s extended per iod of development. Depending on the durat ion of exposure, dose and t o x i c po ten t ia l of the compound in ques t ion , the r e s u l t i n g CNS damage can range from gross morphologic anomalies to subt le a l t e r a t i o n s in CNS funct ion (Hutchings, 1978). These l a t t e r anomalies are termed behavioural tera togenic e f f e c t s and invo lve d e f i c i t s in CNS a c t i v i t i e s such as motor f u n c t i o n , learn ing and memory. Major tera togenic CNS l e s i o n s occur mainly dur ing the per iod of organogenesis, but funct iona l impairment can occur l a t e r in pregnancy, p a r t i c u l a r l y dur ing the per iod of the brain growth spurt (Dobbing, 1976). 14 Th is per iod of CNS maturation spans the per ina ta l p e r i o d , occur r ing at d i f f e r e n t stages of development in d i f f e r e n t s p e c i e s . In humans, i t begins at about midgestat ion and continues perhaps in to the t h i r d year of l i f e (Dobbing, 1976). The major i ty of work in behavioural te ra to logy has been c a r r i e d out with rodent models (Coyle et al., 1980; Kimmel, 1981). A wide var ie ty of behavioural parameters, such as a c t i v i t y , motor c o o r d i n a t i o n , sensory funct ion and learn ing and memory a b i l i t i e s have been examined in the neonata l , adolescent and adult stages of development in r e l a t i o n to prenatal drug exposure. Two areas of behavioural te ra to logy that have rece ived l e s s a t tent ion than the postnatal eva luat ion of neuro log ic func t ion are f e t a l CNS funct ion and behaviour dur ing the actual per iod of drug exposure, and the r e l a t i o n s h i p between f e t a l l e v e l s of the drug and the observed e f f e c t s (Kimmel, 1981). Such studies employing small animal models are t e c h n i c a l l y d i f f i c u l t , i f not imposs ib le . However, with the development of la rge animal models, p a r t i c u l a r l y those using pregnant sheep, a la rge v a r i e t y of p h y s i o l o g i c a l , endocrine and behavioural parameters can be monitored from the undisturbed fetus in utero over the l a s t t h i r d of g e s t a t i o n . It i s a lso p o s s i b l e to car ry out d e t a i l e d pharmacokinetic ana lys is of p lacenta l drug t r a n s f e r and materna l - fe ta l drug d i s p o s i t i o n (Szeto et al., 1982a). A number of f e t a l neuro log ica l and behavioural parameters have been recorded in these prepara t ions , inc lud ing e l e c t r o c o r t i c a l a c t i v i t y , eye movements, electromyographic a c t i v i t y in l imb, neck and trunk muscles, limb and body movements and breathing a c t i v i t y (Dawes et al., 1972; Natale et al., 1981; Rigatto et a / . , 1982). These var ious s tudies have ind icated that f e t a l behavioural s tates become 15 recognizable by about 0.8 of ges ta t ion (term, 145 days ) , at l e a s t as r e f l e c t e d by the appearance of a l t e r n a t i n g per iods of high and low voltage e l e c t r o c o r t i c a l a c t i v i t y . The high vol tage pat tern i s s i m i l a r to quiet s leep a f t e r b i r t h , whi le the bulk of the low vol tage a c t i v i t y appears analogous to rap id eye movement (REM) s l e e p . A l s o , the fetus may be awake fo r a b r i e f per iod of time (Rigatto et al., 1982), although t h i s i s d isputed (Harding, 1984). Linked to the c y c l e of e l e c t r o c o r t i c a l a c t i v i t y i s the ep isod ic occurrence of b r e a t h i n g - l i k e a c t i v i t y , which normally is present only dur ing the low voltage ECoG pattern (Jansen and Chernick , 1983). Limb movements a lso exh ib i t an element of p e r i o d i c i t y as they occur in high vol tage e l e c t r o c o r t i c o g r a p h i c (ECoG) states or in the t r a n s i t i o n s between high and low vol tage states (Natale et al., 1981). A number of s tudies have demonstrated profound e f f e c t s of c e n t r a l l y a c t i v e drugs on ECoG and breathing a c t i v i t y in f e t a l lambs (P iercy et al., 1977; Jansen et al., 1983; Dawes, 1984; Umans and Szeto, 1985). CNS depressants ( e . g . , pento tha l , meperidine, ethanol) administered to the ewe in doses that have minimal maternal e f f e c t s suppress fe ta l breathing a c t i v i t y and low vol tage ECoG a c t i v i t y (Boddy et al., 1976; Ruckebusch et al., 1976; P iercy et al., 1977; Pa t r i ck et al., 1985). In c o n t r a s t , var ious CNS st imulants r e s u l t in c o r t i c a l arousal (low voltage ECoG a c t i v i t y ) and increased f e t a l breathing a c t i v i t y (Jansen et al., 1983; Szeto , 1983). Indomethacin and 5-hydroxytryptophan d i s r u p t the normal l i n k between f e t a l breathing and REM sleep (Kitterman et al., 1979; Q u i l l i g a n et al., 1981). However, only in a few of these s t u d i e s , namely those i n v o l v i n g methadone and e thano l , were the measurements of f e t a l behaviour accompanied by a pharmacokinetic ana lys is of materna l - fe ta l drug 16 d i s p o s i t i o n (Szeto et al., 1982a; McLeod et al., 1983; Szeto , 1983; Pa t r ick et al., 1985). Recent s tudies with human f e t u s e s , which have employed noninvasive monitor ing techniques, p a r t i c u l a r l y rea l - t ime ul trasound viewing, have ind ica ted that behavioural s tates s i m i l a r to those in the fe ta l lamb a lso occur in the human p e r i n a t a l l y (Ni jhuis et al., 1982). C e n t r a l l y ac t ive drugs can have profound acute e f f e c t s on f e t a l CNS funct ion and behaviour in humans (Dai ley et al., 1982; Jansen et al., 1983; Szeto, 1983). However, the postnatal e f f e c t s of drug- induced prenatal behavioural a l t e r a t i o n s are not yet known, although Szeto (1983) has suggested that the l a t e postnatal e f f e c t s of prenatal methadone exposure may be in par t , long-term consequences of the d i r e c t ac t ion of methadone on f e t a l ECoG a c t i v i t y and cerebra l metabolism. It i s a lso poss ib le that d i s r u p t i o n s of the normal pattern of f e t a l r e s p i r a t o r y a c t i v i t y could have long-term e f f e c t s on the r e s p i r a t o r y system, s ince to ta l or p a r t i a l reduct ion in breathing a c t i v i t y by phrenic nerve sect ion or experimental growth re ta rda t ion r e s u l t s in reduced lung weight at b i r t h (Fewell et al., 1981; Malony et al., 1982). 1.5. Rat ionale and S p e c i f i c Aims of the Research 1 .5 .1 . Rat ionale In the preceeding s e c t i o n s , a case has been made fo r the fo l lowing p o i n t s : 1) there is a r e l a t i v e l y common usage of ant ih is tamines during pregnancy, 2) c l a s s i c a l histamine H j - receptor antagonists probably cross 17 the p lacen ta , and may exert depressant or sedat ive e f f e c t s in the f e t u s , 3) there i s a shortage of data on the extent of p lacenta l t r a n s f e r and fe ta l e f f e c t s of a l l an t ih is tamines . In regard to the l a s t p o i n t , there are ser ious techn ica l and e th ica l d i f f i c u l t i e s in obta in ing such data from pregnant humans and, f o r technica l reasons, i t i s impract ica l to obtain d e t a i l e d pharmacokinetic and p h y s i o l o g i c a l information from small animal models f o r pregnancy. Large animal models would seem to be more appropr ia te , and of these, the pregnant sheep i s the most commonly employed. There are c l e a r d i f f e r e n c e s in p lacenta l s t ruc ture and permeabi l i ty between sheep and humans, but f o r l i p i d so lub le molecules, the d i f f e r e n c e s are of l e s s importance than fo r h y d r o p h i l i c substances. The f e t a l lamb i s more mature at b i r t h than i s the human f e t u s , but in both species there appear to be s i m i l a r behavioural s ta tes in the fetus towards the end of g e s t a t i o n . Hence, the pregnant sheep would seem to be an acceptable model to use in the proposed study, and would provide data that should be useful in assessing the safety of the a n t i h i s t a m i n e drug use in pregnancy Diphenhydramine i s t y p i c a l of most histamine H j - receptor antagonists in that i t possesses a n t i c h o l i n e r g i c , ant iemet ic , a n t i t u s s i v e and sedat ive proper t ies (Douglas, 1985). This compound has been f requent ly used dur ing pregnancy fo r the treatment of var ious a l l e r g i c d isorders ( e . g . , a l l e r g i c r h i n i t i s ) and problems s p e c i f i c to pregnancy, such as nausea and vomiting in the f i r s t t r imester (For far and Nelson, 1973) and a pregnancy-re la ted u r t i c a r i a l cond i t ion in the t h i r d t r imester (Ahmed and Kaplan, 1981). Diphenhydramine i s a weakly basic amine compound (pKa = 9.0) and possesses high 1 i p o p h i l i c i t y , low molecular weight (255.4) and CNS 18 a c t i v i t y . There fore , i t seems probable that t h i s compound would cross the p lacenta and cause CNS e f f e c t s in the f e t u s . 1 .5 .2 . S p e c i f i c Aims The primary ob jec t ives of the present i n v e s t i g a t i o n are: 1) to develop a c a p i l l a r y g a s - l i q u i d chromatographic method (GC/NPD), with improved s e n s i t i v i t y and s e l e c t i v i t y , s u i t a b l e fo r studies where b i o l o g i c a l sample volumes are l i m i t e d . 2) to examine dose dependency of diphenhydramine pharmacokinetics in nonpregnant sheep. 3) to study the p lacenta l t r a n s f e r and d i s p o s i t i o n of diphenhydramine in the materna l - fe ta l sheep fo l lowing i . v . bolus i n j e c t i o n of the drug to the pregnant ewe. 4) to study t ransp lacenta l and nonplacental c learances of diphenhydramine fo l lowing separate maternal and fe ta l drug in fus ions to s teady-s ta te plasma drug concent ra t ion . 5) to study the e f f e c t of diphenhydramine admin is t ra t ion on fe ta l behavioural s t a t e s , i.e., e l e c t r o c o r t i c a l and e l e c t r o - o c u l a r a c t i v i t y and breathing movements, and c a r d i o r e s p i r a t o r y v a r i a b l e s , i.e., heart r a t e , blood pressure and blood gas tensions in r e l a t i o n to f e t a l drug exposure in the l a t t e r t h i r d of gesta t ion in sheep. 6) to examine the p o s s i b i l i t y of the r e c i r c u l a t i o n of diphenhydramine wi th in the materna l - fe ta l uni t a f t e r bolus i n j e c t i o n of the drug in to the amniotic c a v i t y in the pregnant sheep. 7) to study the pulmonary ex t rac t ion of diphenhydramine in the f e t a l lamb fo l lowing maternal or f e t a l drug i n f u s i o n to s t e a d y - s t a t e . 19 2. EXPERIMENTAL 2 .1 . Development of a C a p i l l a r y GC/NPD Assay Method fo r Diphenhydramine 2 . 1 . 1 . Mater ia ls Diphenhydramine hydroch lor ide , 2-(diphenylmethoxy)-N,N-dimethylethylamine (Parke Dav is , Lot No. C429419, Montreal , Quebec) and orphenadrine hydroch lor ide , N,N-dimethyl -2- [ (2-methylphenyl) phenyl -methoxy] ethanamine ( P f a l t z and Bauer, Lot No. D352, S tan ford , CT) were used as reference standards. Diphenhydramine hydrochlor ide fo r i n j e c t i o n (Benadryl^ In jec tab le , Parke Dav is , Lot No. AD 613-1, B r o c k v i l l e , Ontar io) was obtained from the Hospita l Pharmacy, Health Sciences Center H o s p i t a l , Vancouver, B r i t i s h Columbia. Tr ie thylamine (TEA) was purchased from Pierce Chemical C o . , Rockford, IL. Toluene, dichloromethane, benzene, heptane and hexane, d i s t i l l e d in g l a s s , were purchased from Caledon Laborator ies (Georgetown, Ontario) and isoamyl a lcohol from A l l i e d Chemical Co. (Morristown, NJ) . A s o l u t i o n of 1 M sodium hydroxide was prepared from ACS reagent grade chemical (American S c i e n t i f i c and Chemical , S e a t t l e , WA). De ionized, d i s t i l l e d water was used in the preparat ion of stock so lu t ion throughout the a n a l y s i s . 2 . 1 . 2 . Preparat ion of Stock So lu t ions Diphenhydramine hydrochlor ide (160 ng/mL, equiva lent to base) and the in te rna l standard, orphenadrine hydrochlor ide (1.0 fig/ml, equiva lent to base) were prepared by d i s s o l v i n g these compounds in 20 d i s t i l l e d water. The so lu t ions were stored at 4°C fo r up to two months. 2 . 1 . 3 . Instrumentation and Opt imizat ion of Gas Chromatographic Condi t ions 2 . 1 . 3 . 1 . Equipment A Model 5830A Hewlett-Packard (HP, Avondale, PA) gas chromatograph equipped with a n i t rogen /phosphorus -se lec t i ve de tec to r , a Model 18835B HP c a p i l l a r y i n l e t system, a Model 18850 HP in tegra tor system fo r peak area in tegra t ion and quant i ta t ion and a 25 m x 0.31 mm I.D. c r o s s - l i n k e d f u s e d - s i l i c a c a p i l l a r y column (5% phenylmethyl s i l i c o n e , f i l m th ickness 0.52 |zm, Hewlett Packard) was used fo r a l l ana lyses . Thermogreen^ LB-2 s i l i c o n e rubber septa (Supelco, B e l l a f o n t e , PA) were used to provide low bleed at high i n l e t temperature. A Model 7671A HP automatic sampler was used fo r sample i n j e c t i o n into the gas chromatograph in the s p l i t l e s s i n j e c t i o n mode. 2 . 1 . 3 . 2 . Opt imizat ion of Gas Chromatographic Condi t ions Samples fo r the pre l iminary s tudies of opt imiz ing GC condi t ions were prepared as f o l l o w s . To 2.0 mL of diphenhydramine stock so lu t ion (160 ng/mL) were added 0.2 mL of the in terna l standard orphenadrine s o l u t i o n (1 Mg/mL) and 0.5 mL of 1 M NaOH. To t h i s mixture, 6 mL of toluene was added and the aqueous phase was extracted by shaking the mixture f o r 20 min on a rotary shaker (Labquake Tube Shaker, Model 415-110, Lab Indus t r i es , Berkeley, CA). The sample was cent r i fuged and then the aqueous layer was a s p i r a t e d . The remaining organic l ayer was 21 t r a n s f e r r e d to a cu l tu re tube and dr ied under a gent le stream of n i t rogen in a 35°C water bath. The residue was recons t i tu ted to a volume of 100 nl with to luene . A l iquo ts of 2 /zL were in jec ted in to the gas chromatograph. Samples were in jec ted in s p l i t l e s s i n j e c t i o n mode under GC c o n d i t i o n s : i n j e c t i o n port temperature, 250°C; oven temperature, 200°C; and detect ion port temperature, 300°C . Under these GC c o n d i t i o n s , the fo l lowing instrument parameters were var ied and tested f o r the nitrogen-phosphorus detector response. 1) hydrogen gas (make-up gas) f low: 2 .0 , 2 .5 , 2.75, 3 .0 , 3 .25, 3 .5 , 3 .75, 4.0 and 4.25 mL/min at constant rates of the helium c a r r i e r gas (1.0 mL/min) and a i r (60 mL/min). 2) a i r (make-up gas) f low: 30, 40, 45, 50, 60 and 80 mL/min at constant ra tes of the helium c a r r i e r gas (1.0 mL/min) and hydrogen (3.0 mL/min). 3) column pressure : 8, 10, 13, 15, 18, 20 and 25 psi at constant flow rates of the hydrogen gas (3.0 mL/min) and a i r (50 mL/min). 4) purge a c t i v a t i o n t ime: 0 .1 , 0 .2 , 0 .6 , 1.0, 1.4 and 1.6 min at constant flow rates of hydrogen gas (3.0 mL/min), a i r (50 mL/min) and helium gas (1.0 mL/min). The e f f i c i e n c y of a GC column i s expressed by the number of t h e o r e t i c a l p la tes (n): n = 1 6 ( t R / W b ) 2 (eq. 1) where t R i s the re ten t ion time of the peak and Wh i s the peak width at base (Rooney, 1981). The t h e o r e t i c a l p la te i s a hypothet ica l uni t of the column in which one equ i l ib r ium of the solute occurs between the c a r r i e r gas and l i q u i d phase of the column. The hypothet ica l uni t expressed in terms of column length i s c a l l e d the height equivalent to t h e o r e t i c a l p la te ( H . E . T . P . ) which can be c a l c u l a t e d by d i v i d i n g the column length by the p la te number. In the present study, the number of t h e o r e t i c a l p la tes was c a l c u l a t e d fo r diphenhydramine at column pressures , 7, 9, 11, 13, 15 and 17 p s i , and then the van Deemter curve showing the r e l a t i o n s h i p between H . E . T . P . and l i n e a r v e l o c i t y of the helium c a r r i e r gas was p lo t ted fo r t h i s compound. 2 .1 .4 . Drug Ext rac t ion Studies The fo l lowing ex t rac t ion parameters were examined. 1) drug ex t rac t ion e f f i c i e n c y of s o l v e n t s : heptane, hexane, to luene, benzene and dichloromethane. 2) e f f e c t of the addi t ion of t r ie thy lamine to dichloromethane on i t s drug ex t rac t ion e f f i c i e n c y : 0, 0 .01, 0.005, 0.1 and 0.5 M t r i e t h y l a m i n e . 3) ex t rac t ion t ime: 5, 10, 15, 20, 30, 40, 60 and 80 min. 2 . 1 . 5 . Routine Procedures fo r Diphenhydramine A n a l y s i s 2 . 1 . 5 . 1 . Operating GC Condi t ions The operat ing GC condi t ions fo r rout ine sample a n a l y s i s were: i n j e c t i o n port temperature, 250°C; i n i t i a l oven temperature, 190°C; programming r a t e , 2°C/min at 1 min and 5°C/min at 8.5 min; f i n a l oven temperature, 240°C; n i t rogen /phosphorus -se lec t ive detector temperature, 3 0 0 ° C ; c a r r i e r gas (helium) flow r a t e , 1.0 mL/min; septum purge flow 23 r a t e , 0.6 mL/min; make-up gas flow r a t e , 2 mL/min; hydrogen/a i r f low rate r a t i o , 3:30. 2 . 1 . 5 . 2 . Drug Ex t rac t ion from B i o l o g i c a l Samples To 0 .3 -0 .5 mL of plasma were added 0.2 mL of in terna l standard (1.0 jug/mL) and 0.5 mL of 1 M sodium hydroxide in a 15 mL d isposable cu l tu re tube with a screw cap l i n e d with po ly te t ra f luoroe thy lene (PTFE) (Corning Glass Works, Corning, NY). The aqueous phase was adjusted to a to ta l volume of 2.5 mL (pH -13) with d i s t i l l e d water and mixed on a vortex mixer. To t h i s mixture, 6.0 mL of 0.05 M t r ie thy lamine in dichloromethane were added and the aqueous phase was extracted by shaking the mixture fo r 20 min on a rotary shaker (Labquake Tube Shaker, Model 415-110, Lab Indust r ies , Berkeley, CA). The samples were placed in a f reezer ( - 2 0 ° C ) fo r 5 min and then cent r i fuged at 2,300 g fo r 10 min. A f t e r c e n t r i f u g a t i o n , the aqueous layer was asp i ra ted and d i s c a r d e d . The remaining organic l ayer was t r a n s f e r r e d to a c lean 15 mL c u l t u r e tube and d r ied under a gent le stream of n i t rogen in a 30°C water bath. The residue was recons t i tu ted to a volume of 100 /iL with to luene. The sample was mixed on a vortex mixer fo r 10 sec . A l i q u o t s of 2 /zL were used fo r GC/NPD a n a l y s i s . 2 . 1 . 5 . 3 . Preparat ion of the C a l i b r a t i o n Curve A 0.5 mL sample of blank sheep plasma was spiked with varying amounts of diphenhydramine (2, 4, 8, 16, 32, 64, 128, 192, 256 and 320 ng) using the prepared stock so lu t ion (160 ng/mL) and d i l u t e d stock 24 s o l u t i o n (20 ng/mL), and then 0.2 mL of the orphenadrine s o l u t i o n (1.0 /jg/mL) and 0.5 mL of 1 M sodium hydroxide were added. The aqueous phase was adjusted to a to ta l volume of 2.5 mL (pH -13) with d i s t i l l e d water and the samples were extracted as descr ibed above. Quant i ta t ive est imat ion of diphenhydramine in plasma was accomplished by p l o t t i n g the area r a t i o s of diphenhydramine and orphenadrine against the range of ind ica ted diphenhydramine concent ra t ions . For recovery study, diphenhydramine was d i s s o l v e d in methanol to provide concentrat ions ranging from 2 to 320 ng/mL. The amount of diphenhydramine extracted from plasma by dichloromethane was c a l c u l a t e d using c a l i b r a t i o n curves obtained from d i r e c t i n j e c t i o n of the prepared methanolic s o l u t i o n s . 2 . 1 . 5 . 4 . Gas Chromatography-Mass Spectrometry C a p i l l a r y GC e lect ron- impact mass spectrometry (EI-MS) and chemical i o n i z a t i o n mass spectrometry (CI-MS) were c a r r i e d out using a Model 5987A HP gas chromatograph-mass spectrometer equipped with a 25 m x 0.32 mm I.D. (0.25 nm f i l m th ickness) 0V-1701 f u s e d - s i l i c a column (Quadrex S c i e n t i f i c , New Haven, CT) . Methane was used as the i o n i z i n g gas and the s p l i t l e s s i n j e c t i o n mode was employed in the study. The fo l low ing cond i t ions were used fo r GC-MS of diphenhydramine and orphenadrine: i n i t i a l oven temperature, 50°C; time 1, 0 min; r a t e , 3 0 ° C / m i n ; f i n a l oven temperature, 260°C; time 2, 10 min; i n j e c t i o n port temperature, 240°C; c a r r i e r gas (helium) flow r a t e , 1.0 mL/min; e lec t ron m u l t i p l i e r vo l tage , 2200 V; emission cur ren t , 300 /xA. 25 2 . 1 . 5 . 5 . A p p l i c a t i o n of the Developed Assay Method to a Placental T rans fe r Study A pre l iminary experiment on the p lacenta l t r a n s f e r of diphenhydramine was c a r r i e d out in a pregnant ewe c h r o n i c a l l y implanted with maternal and fe ta l vascular catheters (Rurak and Gruber, 1983). A 100 mg i . v . dose of diphenhydramine hydrochlor ide was administered over a 30 sec per iod via a maternal femoral venous catheter and the catheter f lushed with 5.0 mL of hepar in ized s a l i n e . Blood samples fo r diphenhydramine determination were simultaneously withdrawn from the maternal and f e t a l femoral a r t e r i a l catheters at - 5 , 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min. The blood samples were t r a n s f e r r e d to hepar in ized Vacutainer^ c o l l e c t i o n tubes (Becton-D i c k i n s o n , M iss issauga , Ontar io) and immediately c e n t r i f u g e d . Fol lowing c e n t r i f u g a t i o n , the plasma was t rans fe r red to a 15 mL PTFE- l ined screw-capped d isposab le c u l t u r e tube, and stored at -20°C u n t i l ext racted and analyzed. 2 .2 . Pharmacokinetics of Diphenhydramine a f t e r Dose Ranging in Nonpregnant Sheep 2 . 2 . 1 . Animals and Surg ica l Preparat ion Six nonpregnant Dorset or Su f fo lk ewes of 2-3 years of age, with a mean body weight of 90 + 16 (S.D.) kg, were used in the study. The animals were from the same f l o c k (Department of Animal Sc ience , U n i v e r s i t y of B r i t i s h Columbia) and were ra ised and fed under the same c o n d i t i o n s . Surgery was performed a s e p t i c a l l y using 1-2% halothane 26 (Ayerst Labora to r ies , Montreal , Quebec) and 60% n i t rous oxide anesthes ia , fo l lowing induct ion of anaesthesia with 1 g of i . v . sodium pentothal (Abbott Labora tor ies , Chicago, IL ) . S i l i c o n e rubber c a t h e t e r s , with an O.D. of 2.2 mm (Dow Corning, Midland, MI), were implanted into the femoral ar tery and v e i n . A m p i c i l l i n (500 mg, Ayerst L a b o r a t o r i e s , Montreal , Quebec) was given to the ewe i .m. d a i l y fo r 3 days a f t e r surgery . The catheters were f lushed d a i l y with 3 mL of hepar in ized i s o t o n i c s a l i n e (12 IU/mL). A f t e r surgery , the animals were allowed to recover fo r at l e a s t 3 days p r i o r to exper imentat ion. 2 . 2 .2 . Drug Admin is t ra t ion and Experimental Protocol Each animal rece ived 25, 50, 100 and 200 mg doses of diphenhydramine hydrochlor ide (Benadryl 1* In jec tab le , 50 mg/mL, Parke Dav is , Lot No. 01565-1, Scarborough, Ontar io) on a randomized crossover b a s i s , with each experiment separated by a minimum of a 2 day per iod fo r drug washout. Drug was d i l u t e d in i s o t o n i c s a l i n e to 5 mL and in jec ted as an i . v . bolus dose via the venous catheter over 30 sec and the catheter subsequently f lushed with 5 mL of i s o t o n i c s a l i n e . A r t e r i a l blood samples (3.0 mL) were taken via the femoral a r t e r i a l catheter at - 5 , 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min fo l lowing drug admin is t ra t ion . Blood samples were t r a n s f e r r e d to V a c u t a i n e r R c o l l e c t i o n tubes and cent r i fuged immediately at 3,500 g fo r 10 min. The plasma was t rans fe r red to g lass cu l tu re tubes and frozen at -20°C and stored u n t i l a n a l y s i s . A r t e r i a l blood samples (0.6 mL) were c o l l e c t e d at - 5 , 5, 10 and 60 min fo r the measurement of P o 2 , P c o 2 , 0 2 content and hematocr i t . A r t e r i a l P o 2 , Pco 2 and pH were measured using 27 IL micro 13 blood gas analyzer ( Instrumentation Labora tor ies , Lexington, M A ) and correc ted to 3 9 ° C . Blood oxygen content was measured by Lex-C^-con-k oxygen content analyzer (Lexington Instrument, Watham, MA) and hematocrit by a m i c r o - c a p i l l a r y cent r i fuge ( Internat ional Equipment C o . , Needham, MA). Plasma diphenhydramine concentrat ions were measured by a c a p i l l a r y gas chromatographic method employing nitrogen-phosphorus detec t ion (see sec t ion 2 . 1 . 5 ) . 2 . 2 . 3 . Pharmacokinetic A n a l y s i s Plasma drug concentrat ion vs. time data were used f o r curve f i t t i n g by the computer program AUTOAN (Sedman and Wagner, 1976) and the obtained i n i t i a l estimates of the pharmacokinetic parameters were subsequently analyzed using the nonl inear l e a s t squares computer program NONLIN (Metzler et al., 1974). The i n i t i a l d i s t r i b u t i o n h a l f - l i f e (t%, lambda^) and the terminal e l im ina t ion h a l f - l i f e {tk, lambda n) were c a l c u l a t e d by the fo l lowing equat ions: tk, lambda! = 0.693/lambdaj (eq. 2) tk, lambda n = 0.693/1ambda n (eq. 3) where lambdaj and lambda n are the l a rges t and the smal lest d i s p o s i t i o n rate constants obtained from mult iexponent ia l plasma drug concentrat ion vs. time decay curves , r e s p e c t i v e l y (Gibald i and P e r r i e r , 1982). The area under the plasma drug concentrat ion vs. time curves , A U C Q , was c a l c u l a t e d using the equat ion: A U C Q = A U C Q + A U C * (eq. 4) where t represents the time when the l a s t sample was taken. The f i r s t term, A U C Q was c a l c u l a t e d using the t rapezoida l r u l e , and the second 28 term was c a l c u l a t e d using the equat ion: AUC^ = C t / l a m b d a n (eq. 5) where C .^ i s the concentrat ion of the l a s t sample. The AUC obtained from 00 0 to t was >95% of the to ta l AUC Q . There fore , the p o s s i b l e e r ror in 00 AUC t a r i s i n g from an est imat ion of lambda n i s minimal . The to ta l body c learance (CLjg) was obtained from: C/_TB= Dose/AUCg (eq. 6) The apparent volume of d i s t r i b u t i o n ( f t / a r e a ) was obtained from: ^ a r e a = Dose/(lambda n-AUCo) (eq. 7) The volume of d i s t r i b u t i o n at s teady-s ta te [Vdss) was c a l c u l a t e d (Giba ld i and P e r r i e r , 1982) e i t h e r by an equation which requi res curve f i t t i n g : Vdss = Dose ' l^C^lambdayVt i^Cz/lambda,-)] 2 (eq. 8) or by a model-independent method: Vdss = (Dose-AUMCo)/(AUCo) 2 (eq. 9) oo where AUMC0 i s the to ta l area under the f i r s t moment of the plasma concentra t ion vs. time curve from time 0 to i n f i n i t y , c a l c u l a t e d by the t rapezo ida l ru le with a c o r r e c t i o n made fo r ex t rapo la t ion to i n f i n i t y beyond the l a s t data po in t : A U M C Q = AUMCg + C t ' t / l a m b d a n + C t / l a m b d a n 2 (eq. 10) 2 .2 .4 . Plasma Prote in Binding Diphenhydramine hydrochlor ide (Sigma Chem. C o . , Lot No. D-3630, S t . L o u i s , MO) was d i s s o l v e d in Sorensen's i s o t o n i c phosphate buf fer (0.067 M, pH = 7.4) to prepare 10, 25, 50, 100, 250, 500, 1,000, 2,000, 4,000 and 10,000 ng/mL drug s o l u t i o n s . Optimum e q u i l i b r i u m time was 29 determined at the two drug concent ra t ions , 500 and 10,000 ng/mL, by d i a l y z i n g the drug-conta in ing buf fer so lu t ions against an equiva lent volume of blank sheep plasma and by analyz ing drug concentra t ion in the plasma compartment at 0 .5 , 1.0, 2 .0 , 3 .0 , 4.0 and 6.0 hr . An optimum e q u i l i b r i u m time of 3 hr was determined and appl ied f o r fu r ther binding s t u d i e s . D i a l y s i s was c a r r i e d out at 39°C in P lex ig lass ' * d i a l y s i s c e l l s in which buf fer and plasma were separated by cel lophane d i a l y s i s membrane (Sigma Chem. C o . , Lot No. 39C-6131, molecular weight c u t o f f = 12,000 Dal tons , S t . L o u i s , MO). The d i a l y s i s membrane was b o i l e d in d i s t i l l e d water fo r 1 hr and soaked in i s o t o n i c phosphate buf fe r f o r 1 hr before i t was mounted wi th in the d i a l y s i s c e l l . At the end of the d i a l y s i s , both buf fe r and plasma were t r a n s f e r r e d to g lass tubes and kept f o r drug a n a l y s i s . The p o s s i b i l i t y of leakage of prote in across the membrane was tested by adding a drop of the d i a l y s i s buf fe r to 0.1 mL of 3% t r i c h l o r o a c e t i c a c i d . The absence of t u r b i d i t y was an i n d i c a t o r of membrane i n t e g r i t y . The f ree f r a c t i o n of diphenhydramine was determined in vitro by d i a l y z i n g 1.0 mL of the prepared drug s o l u t i o n s (10 to 10,000 ng/mL), against equivalent volumes of blank plasma from s u r g i c a l l y unaltered nonpregnant sheep. The f ree f r a c t i o n of diphenhydramine was a lso measured in vivo in the 6 nonpregnant animals, by analyz ing drug concentrat ions in the 5 and 60 min plasma samples taken from experiments invo lv ing the admin is t ra t ion of the 200 mg dose. In two animals, the f ree f r a c t i o n of diphenhydramine was determined in vivo in a l l the samples c o l l e c t e d during the experiments fo l lowing admin is t ra t ion of the 200 mg dose. The f ree f r a c t i o n of the drug was c a l c u l a t e d by the equat ion: f ree f r a c t i o n = C u / C p (eq. 11) 30 where C u and Cp are buf fer and plasma concentrat ions of diphenhydramine a f te r d i a l y s i s , r e s p e c t i v e l y . 2 . 2 . 5 . S t a t i s t i c a l Ana lys is S t a t i s t i c a l ana lys is was performed by One-way A n a l y s i s of Variance and Tukey mul t ip le comparison t e s t . The s i g n i f i c a n c e leve l was p<0.05. Values are given as the mean + 1 S .D . 2 .3 . Pharmacokinetics of Diphenhydramine in the Pregnant Ewe and Fetal Lamb: i . v . Bolus In ject ion Studies 2 . 3 . 1 . Animals and Surg ica l Preparat ion Four pregnant Dorset ewes were s u r g i c a l l y prepared f o r experimentation at 122-129 days of g e s t a t i o n . A l l animals were fasted overnight p r i o r to surgery. P reopera t ive ly , 3 mg of atropine s u l f a t e (Glaxo L a b o r a t o r i e s , Montreal , Quebec) was administered to the ewe via the jugu la r vein 5-10 min before induct ion of anaesthesia with the i . v . i n j e c t i o n of 1 g of th iopenta l sodium (Abbott Labora to r ies , Chicago, IL ) . Fol lowing in tuba t ion , anaesthesia was maintained with halothane (1.5-2.0%) (Ayerst Labora tor ies , Montreal , Quebec) and n i t rous oxide (60%) in oxygen. Asep t i c techniques were employed throughout the s u r g i c a l procedure. The maternal abdomen was opened by a mid l ine i n c i s i o n , and access to the fetus was gained through a small i n c i s i o n in a region of 31 the uterus that was f ree from major blood v e s s e l s . S i l i c o n e rubber catheters (Dow Corning, Midland, MI) were implanted in to the f e t a l femoral a r t e r y , l a t e r a l t a rsa l v e i n , t rachea , and amniotic c a v i t y . The t racheal catheter (2.2 mm O . D . ) , which was inser ted through a small i n c i s i o n 1-2 cm below the larynx and advanced 4 to 5 cm, d id not obstruct lung f l u i d e f f l u x from the airway into the pharynx. Fol lowing c a t h e t e r i z a t i o n of the f e t u s , the uter ine i n c i s i o n s were c losed in two l a y e r s . Catheters were then implanted in a maternal femoral ar tery and v e i n . A l l ca theters were f i l l e d with hepar in ized s a l i n e (12 IU/mL) and tunneled subcutaneously and e x t e r i o r i z e d through a small i n c i s i o n in the f lank of the ewe, where they were stored in a c lean denim pouch. The maternal abdominal i n c i s i o n was c losed in l a y e r s . Amniotic f l u i d l o s t dur ing surgery was replaced with i r r i g a t i o n s a l i n e (Travenol Canada Inc . , M iss issauga , O n t a r i o ) . Immediately fo l lowing surgery , 500 mg A m p i c i l l i n (Ayerst Labora tor ies , Montreal , Quebec) was given to the fetus via the t a r s a l venous ca the ter , into the amniotic c a v i t y via the amniotic catheter and to the ewe in t ramuscu lar ly . A m p i c i l l i n was given to the ewe i .m. f o r the f i r s t 3 days a f t e r surgery and d a i l y in to the amniotic c a v i t y fo r the durat ion of the prepara t ion , u n t i l d e l i v e r y . Fol lowing surgery , the ewes were kept in pens in the company of other sheep and were given f ree access to food and water. Animals were allowed to recover f o r a minimum of three days before exper imentat ion. 2.3.2 Drug Admin is t ra t ion and Experimental Protocol For each experiment, the ewe was placed in a monitoring cage adjacent to the holding pen in f u l l view of companion ewes and with free 32 access to food and water. A 100 mg dose of diphenhydramine hydrochlor ide (Benadry l R In jec tab le , Parke Dav is , Lot No. AD 613-1, B r o c k v i l l e , Ontar io) was in jec ted in i s o t o n i c s a l i n e via the maternal femoral venous catheter over a per iod of 30 sec , and the catheter was f lushed with 5 mL of hepar in ized s a l i n e . Maternal (3.0 mL) and fe ta l (1.5 mL) a r t e r i a l blood samples were withdrawn simultaneously at - 5 , 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min a f t e r drug a d m i n i s t r a t i o n . At i n t e r v a l s dur ing the experiment, the f e t a l blood c o l l e c t e d was replaced with an equal volume of blood from the ewe. Amniotic and tracheal f l u i d samples (1.5 mL each) were a lso c o l l e c t e d simultaneously together with the maternal and f e t a l blood samples. The blood samples were c o l l e c t e d in hepar in ized c o l l e c t i o n tubes and immediately cent r i fuged at 3,500 g f o r 10 min. The plasma was t r a n s f e r r e d to a 15 mL d isposable g lass cu l tu re tube with a screw cap l i n e d with po ly te t ra f luoroe thy lene and frozen at - 20 ° u n t i l the time of ex t rac t ion and assay. Diphenhydramine concentrat ions in b i o l o g i c a l f l u i d s were determined using the nitrogen/phosphorus d e t e c t i o n - c a p i l l a r y gas chromatographic method (see sect ion 2 . 1 . 5 ) . 2 . 3 . 3 . Pharmacokinetic A n a l y s i s I n i t i a l estimates of the pharmacokinetic parameters a f te r i . v . admin is t ra t ion were obtained by computer ana lys is using the program AUT0AN (Sedman and Wagner, 1976). Using the i n i t i a l est imates provided by AUT0AN, the data was then f i t t e d using the program NONLIN (Metzler et al., 1974). A l l data points were weighted using the r e c i p r o c a l of drug concen t ra t ion . Maternal plasma concentrat ion vs. time curves were 33 f i t t e d using the biexponent ia l equation descr ibed as f o l l o w s : C p = A e " a t + B e - ^ (eq. 12) where Cp i s the plasma concentrat ion at time t , a and 0 are the apparent d i s t r i b u t i o n a l and terminal e l im ina t ion rate constants , and A and B are in te rcep ts of the a and /} phases, r e s p e c t i v e l y . The d i s t r i b u t i o n a l and terminal e l im ina t ion h a l f - l i v e s , t i n and ti , a, were c a l c u l a t e d : tj, a = 0.693/a (eq. 13) t % ^ = 0.693/0 (eq. 14) oo The area under the plasma drug concentrat ion vs. time curves , A U C 0 , was c a l c u l a t e d using the equations 4 and 5. The to ta l body c learance (C/.jg) was obtained by the equation 6. The apparent volume of d i s t r i b u t i o n was c a l c u l a t e d by the equations 7 and 9. The percentage of the maternal ly administered diphenhydramine that i s e l iminated by the fetus via the nonplacental pathway was c a l c u l a t e d by the fo l lowing equat ion: Percentage of administered dose that i s e l iminated by the fetus = A U C F o ' C L f o - 1 0 0 / ( A U C M o - C L m o + A U C F o ' C L f o ) (eq. 15) CD 00 where AUCp 0 and AUC^ 0 are the to ta l area under the plasma drug concentra t ion vs. time curves in the fetus and ewe, r e s p e c t i v e l y , and C L f 0 and C L m o are the nonplacental c learances of diphenhydramine from the f e t a l and maternal compartments, r e s p e c t i v e l y . Mean values (n = 8) of the C L m o (3343.8 mL/min) and C L f o (208.4 mL/min) f o r diphenhydramine were obtained from the s teady-s ta te drug in fus ion experiments (Sect ion 3 .4 .1 .4 ) and used in the c a l c u l a t i o n . Values in the text and tab les are given as the mean ± 1 S .D . 34 2.4 Transplacenta l C learance, Nonplacental Clearance and Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep: Maternal and Fetal Infusions to Steady-State 2 . 4 . 1 . Animals and Surg ica l Preparat ion Studies were performed on 12 t ime-dated pregnant sheep (Dorset and Su f fo lk breeds) . At 119-129 days gesta t ion (term 145 days ) , surgery was performed a s e p t i c a l l y in the animals under halothane (1-2%) and n i t rous oxide (60%) in 0 2 anaesthesia , fo l lowing induct ion of anaesthesia with i . v . sodium pentothal (1 g) and in tubat ion of the ewe. Access to the fetus was gained through a mid l ine abdominal i n c i s i o n in the ewe and an i n c i s i o n through the uter ine wall f ree from placentomes and major blood v e s s e l s . S i l i c o n e rubber catheters (Dow Corn ing , Mid land, MI) were implanted in a f e t a l femoral a r tery and l a t e r a l t a r s a l v e i n , in the f e t a l t rachea and in the amniotic c a v i t y . In one animal (ewe no. 130), a catheter was a lso implanted in the common umbi l ica l vein (Rurak and Gruber, 1983). E lect rodes of T e f l o n R - c o a t e d s t a i n l e s s - s t e e l wire (Cooper C o r p . , Chatsworth, CA) were implanted b i p a r i e t a l l y on the dura to record the f e t a l electroencephalogram and through the o r b i t a l r idge of the zygomatic bone of each eye fo r e l e c t r o -ocu la r r e c o r d i n g s . Catheters were a lso implanted in a maternal femoral a r tery and v e i n . A l l catheters and e lect rodes were tunneled subcutaneously in the maternal abdominal wall to ex i t through a small i n c i s i o n in the f l a n k . They were stored in a denim pouch in the ewe's f l ank when not in use. A l l vascular catheters were f lushed d a i l y with 2 mL of s t e r i l e 0.9% NaCl so lu t ion conta in ing 12 IU of heparin per mL. 35 A n t i b i o t i c prophylax is was as descr ibed in sec t ion 2 . 3 . 1 . A f t e r surgery , the animals were kept in holding pens with other sheep and given f ree access to food and water. The sheep were allowed to recover f o r 3-14 days before experimentat ion. To car ry out an experiment, the ewe was t rans fe r red to a monitoring car t adjacent to and in f u l l view of companion sheep. 2 . 4 .2 . Drug Admin is t ra t ion and Experimental Protocol A to ta l of 20 experiments were c a r r i e d out on the 12 animals at 126 to 142 days gesta t ion (Table 11). Sixteen of these were c a r r i e d out on e ight of the sheep and involved separate maternal and f e t a l i . v . i n f u s i o n s of diphenhydramine, given 48 hr apar t . The remaining four experiments involved contro l i . v . i n fus ions of p h y s i o l o g i c a l s a l i n e to the fetus (n = 3) or ewe (n = 1) . Infusions of drug and s a l i n e were c a r r i e d out using a Harvard pump (Model 944, Harvard Apparatus, M i l l i s , MA); the volume in fus ion rate was 0.17 mL/min fo r 90 min. For drug i n f u s i o n s , diphenhydramine hydrochlor ide (Benadry l R In jec tab le , Parke Davis , Lot No. 01565-1, Scarborough, Ontar io) was d i l u t e d to the appropriate concentrat ion with 0.9% s a l i n e immediately before admin is t ra t ion . For maternal i n f u s i o n s , the drug was given as a 20 mg loading dose, fol lowed by a continuous i n f u s i o n rate of 0.67 mg/min. The drug was administered to the fetus as a 5 mg loading dose, fol lowed by a continuous in fus ion rate of 0.17 mg/min. 36 During the drug i n f u s i o n experiments, pa i red maternal (2.5 mL) and f e t a l (1.5 mL) a r t e r i a l b lood , f e t a l t racheal and amniotic f l u i d s (1.5 mL each) samples were c o l l e c t e d at - 5 , 5, 15, 30, 45, 60, 75, 90, 95, 100, 105, 110, 120, 135, 150, 180, 210, 240, 270, 300, 330 and 390 min f o r the est imat ion of diphenhydramine concent ra t ion . In one animal (ewe no. 130), umbi l ica l venous blood in add i t ion to the maternal and f e t a l a r t e r i a l blood was sampled during maternal and f e t a l i n f u s i o n s . Samples f o r maternal and fe ta l blood gas and pH measurements were c o l l e c t e d at - 5 , 5, 30, 60, 90 and 150 min in both the drug and s a l i n e i n f u s i o n experiments. The to ta l volume of f e t a l blood removed in the drug i n f u s i o n experiments was 38 mL, whereas in the cont ro l s t u d i e s , the volume was 5 mL. In a l l experiments, the volume of f e t a l blood c o l l e c t e d was replaced by an equal volume of maternal blood in three d iv ided and equal doses, two during the experiment (a f te r 45 and 120 min samples) and one at the end. Plasma samples f o r diphenhydramine ana lys is were stored at -20°C un t i l a n a l y s i s . 2 . 4 . 3 . Pharmacokinetic A n a l y s i s A two-compartment open model was used to descr ibe the d i s p o s i t i o n of the drug in the materna l - fe ta l sheep, with drug e l i m i n a t i o n occur r ing from both the maternal and f e t a l compartments (Scheme 2) . 37 C L M F M F C L f M • C L C L . MO FO M = MATERNAL COMPARTMENT F = FETAL COMPARTMENT Scheme 2. A two-compartment open model used in the c a l c u l a t i o n of t ransp lacenta l and nonplacental c learances in the maternal f e t a l un i t (Szeto et al., 1982a). Transplacenta l and nonplacental c learances of diphenhydramine were c a l c u l a t e d as descr ibed by Szeto et al. (1982a): C Lmm - k 0 / [ C m s s " C f s s ( C m ' s s / C f ' „ ) ] (eq. 16) C L f f = k 0 ' / [ C f ' s s - C m ' s s ( C f s s / C m s s ) ] (eq. 17) C L m f = C L f f ( C f s s / C m s s ) (eq. 18) CLfm - C L m m ( C m ' s s / C f ' s s ) (eq. 19) C L mo - c l mm " C L m f <e<- 2 0 ) C L f 0 = C L f f - C L f m (eq. 21) where C l m m and CL f f are the t o t a l c learances from the mother and f e t u s , r e s p e c t i v e l y , C L m f i s the t ransp lacenta l c learance from the maternal to f e t a l compartment, C L f m i s the t ransp lacenta l c learance from the f e t a l to maternal compartment, and C L m o and C L f 0 are the nonplacental c learances from the mother and f e t u s , r e s p e c t i v e l y . By using the values f o r the i n f u s i o n rates to the mother (k 0 ) and fetus (kg') a n d t n e s teady -s ta te drug concentrat ions in the mother and fe tus fo l low ing maternal i n f u s i o n (Cm s s and C f s s ) and f e t a l i n f u s i o n ( C m ' s s and C f s s ) , 38 a l l the c learances were c a l c u l a t e d . Separate ly , the to ta l body c learance from the mother and f e t u s , C L m m and C L f f , were a lso c a l c u l a t e d by the fo l lowing equations (Gibald i and P e r r i e r , 1982): C L m m = k 0 / C m s s (eq. 22) C L f f = k 0 7 C f ' s s (eq. 23) 2 .4 .4 . Plasma Prote in Binding Studies Prote in binding of diphenhydramine in the maternal and fe ta l plasma was determined by equ i l ib r ium d i a l y s i s as descr ibed in the Sect ion 2 .2 .4 . B r i e f l y , maternal and fe ta l plasma samples c o l l e c t e d at 75 and 90 min from each animal were d ia lyzed against an equal volume (0.3 mL) of the phosphate buf fe r so lu t ion (Sorensen's i s o t o n i c b u f f e r , 0.067 M, pH = 7.4) f o r 3 hr . D i a l y s i s was c a r r i e d out at 39°C f o r maternal plasma and 3 9 . 5 ° C fo r f e t a l plasma in P lex ig lass ' * d i a l y s i s c e l l s in which buf fer and plasma were separated by cel lophane d i a l y s i s membrane (Sigma Chem. C o . , Lot No. 39C-6131, molecular weight c u t o f f = 12,000 Daltons S t . L o u i s , M0). The f ree f r a c t i o n was c a l c u l a t e d by equation 11. 2 . 4 . 5 . Recording and A n a l y t i c a l Techniques For at l e a s t 90 min p r i o r to and fo r at l e a s t 90 min a f t e r the i n f u s i o n p e r i o d , f e t a l a r t e r i a l , amniotic and t racheal p ressures , heart ra te and e l e c t r o c o r t i c a l and e l e c t r o - o c u l a r a c t i v i t y were recorded cont inuously on a Beckman R612 recorder (Sensormedics C o r p . , Anaheim, CA). Fetal heart rate was obtained from the a r t e r i a l pulse using a 39 cardie-tachometer (Model 9857, Sensormedics C o r p . , Anaheim, CA). Mean f e t a l a r t e r i a l pressure was corrected f o r i n t r a - u t e r i n e pressure , measured from the amniotic ca the ter , by s u b t r a c t i o n . Episodes of f e ta l breathing a c t i v i t y were detected from the t racheal pressure t r a c e . The minimum breath amplitude analyzed was 1 mm Hg and the minimum durat ion of breathing a c t i v i t y accepted as a breathing episode was 10 sec . Fetal e l e c t r o c o r t i c a l (ECoG) and e l e c t r o - o c u l a r (EoG) s i g n a l s were recorded using a type 9806A AC/DC coupler (Sensormedics C o r p . , Anaheim, CA) with the low and high frequency c u t o f f f requencies set to approximately 0.5 and 50 Hz, r e s p e c t i v e l y . Recordings of f e ta l ECoG a c t i v i t y were d iv ided in to per iods of low, intermediate and high vol tage ampli tude, with vol tage ranges of 25 to 50, 80 to 110, and 125 to 175 M V , r e s p e c t i v e l y . The normal recording speed was 0.1 mm/sec, but o c c a s i o n a l l y higher speeds (5-10 mm/sec) were used to obtain q u a l i t a t i v e information on the frequency c h a r a c t e r i s t i c s of the ECoG a c t i v i t y . Episodes of rap id eye movements (REM) were detected from the e l e c t r o - o c u l a r a c t i v i t y t r a c e . Sect ions of the records where ana lys is of the ECoG and EoG waveforms was impossible because of maternal or f e ta l movement were excluded from a n a l y s i s . The to ta l excluded was 11 min, or 0.2% of the to ta l minutes analyzed. For the en t i re c o n t r o l , in fus ion and p o s t i n f u s i o n per iods , the percentage of time spent in h igh , low and intermediate ECoG a c t i v i t y and the percentage time with EoG a c t i v i t y was c a l c u l a t e d . Maternal and fe ta l P 0 2 , PCO2 and pH were measured using an IL model 1306 automated blood gas analyzer ( Instrumentation Labora tor ies , Lexington, MA) and corrected to temperatures of 39.0 and 3 9 . 5 ° C fo r the ewe and f e t u s , r e s p e c t i v e l y . Blood oxygen content was measured by Lex-40 02-con-k oxygen content analyzer and hematocrit by a m i c r o c a p i l l a r y c e n t r i f u g e . Measurement of plasma diphenhydramine concentrat ions was accomplished with the use of a c a p i l l a r y g a s - l i q u i d chromatograph with ni trogen/phosphorus s e l e c t i v e detect ion (see sect ion 2 . 1 . 5 ) . 2 . 4 . 6 . S t a t i s t i c a l Ana lys is S t a t i s t i c a l ana lys is was performed using Student 's paired t-t e s t f o r pa i red data and the unpaired t - t e s t fo r unpaired da ta . Changes in the c a r d i o r e s p i r a t o r y s t a t e s , blood gas s t a t u s , pH, blood pressures and heart ra tes during the course of the experiments were tested fo r s t a t i s t i c a l s i g n i f i c a n c e using 2-way ana lys is of var iance and Duncan's m u l t i p l e range t e s t . The s i g n i f i c a n c e leve l was p<0.05. Values are given as the mean ± 1 S .D. 2 .5 . R e c i r c u l a t i o n of Diphenhydramine from Amniotic F l u i d to the Ewe and Fetal Lamb: In t ra-amniot ic Bolus In ject ion Studies 2 . 5 . 1 . Animals and Surg ica l Preparat ion Five pregnant Dorset or Su f fo lk ewes with a mean body weight of 76 ± 8 kg were employed in the study. Surgery was performed at a ges ta t iona l age of 120 ± 5 days as descr ibed prev ious ly (see sect ion 2 . 4 . 1 ) . S i l i c o n e rubber catheters were implanted in to the f e t a l femoral a r t e r y , l a t e r a l t a r s a l vein and t rachea . Two catheters were placed into the amniotic c a v i t y , one catheter near the hindlimbs and the other near the f o r e l i m b s . Catheters were a lso implanted in a maternal femoral 41 ar tery and v e i n . In one animal (ewe no. 287), a catheter was implanted in to the umbi l ica l vein as w e l l . A n t i b i o t i c prophylax is was as descr ibed p rev ious ly (sect ion 2 .3 .1 ) . Animals were allowed to recover f o r a minimum of 4 days (8 ± 4 days) before exper imentat ion. 2 . 5 .2 . Drug Admin is t ra t ion and Experimental Protocol For each experiment, a 50 mg dose of diphenhydramine hydrochlor ide (Benadryl 1* In jec tab le , Parke Dav is , Lot No. 01866, Scarborough, Ontar io) was d i l u t e d in i s o t o n i c s a l i n e to 5 mL, in jec ted as an i . v . bolus dose via the amniotic catheter placed near the fore l imbs over 30 sec and subsequently f lushed with 10 mL of i s o t o n i c s a l i n e . Maternal a r t e r i a l (3.0 mL), f e t a l a r t e r i a l (1.5 mL) and umbi l ica l venous (1.5 mL) blood and f e t a l t racheal (1.5 mL) and amniotic (1.5 mL) f l u i d samples were withdrawn simultaneously at - 5 , 5, 10, 15, 20, 30, 45 min, and 1.0, 1.5, 2 .0 , 2 .5 , 3 .0 , 3 .5 , 4 .0 , 5 .0, 6 .0 , 8 .0 , 10.0, 12.0, 20.0 and 28.0 hr fo l lowing drug a d m i n i s t r a t i o n . The blood samples were c o l l e c t e d in hepar in ized c o l l e c t i o n tubes and immediately cent r i fuged at 3,500 g fo r 10 min. The plasma was t r a n s f e r r e d to a 15 mL g lass c u l t u r e tube and frozen at -20°C un t i l a n a l y s i s . Samples fo r the measurement of f e t a l a r t e r i a l pH, Pco2, P02, O2 content and hematocrit were c o l l e c t e d at - 5 , 5, 10, 30 and 60 min fo l lowing drug a d m i n i s t r a t i o n . These var iab les were determined as descr ibed in the Sect ion 2 .4 .5 . At i n t e r v a l s during the experiment, the f e t a l blood c o l l e c t e d was replaced with an equal volume of blood from the ewe. 42 2 . 5 . 3 . Data A n a l y s i s The terminal e l im ina t ion h a l f - l i f e of diphenhydramine in the maternal and f e t a l a r t e r i a l and umbi l ica l venous plasma, and t racheal and amniotic f l u i d s was c a l c u l a t e d by l i n e a r regress ion ana lys is of the drug concentrat ion vs. time p r o f i l e during the terminal e l im ina t ion phase. The r a t i o of the fe ta l t r a c h e a l , maternal a r t e r i a l and umbi l ica l venous to f e t a l a r t e r i a l drug concentrat ions was c a l c u l a t e d by d i v i d i n g drug concentrat ions in each f l u i d by corresponding f e t a l a r t e r i a l drug concentrat ions at each sampling time and the average r a t i o was determined f o r each animal . Area under the drug concentrat ion vs. time curves in each b i o l o g i c a l f l u i d was c a l c u l a t e d by eq. 4 and 5. S t a t i s t i c a l ana lys is was performed using Student 's paired t - t e s t . The s i g n i f i c a n c e l eve l was p<0.05. Values are given as the mean ± 1 S .D . 2 .6 . Pulmonary D i s p o s i t i o n of Diphenhydramine in the Fetal Lamb: Maternal or Fetal Infusions to Steady-State 2 . 6 . 1 . Animals and Surg ica l Preparat ion Three pregnant Dorset or Su f fo lk ewes were s u r g i c a l l y prepared at ges ta t iona l age of 124 to 129 days. Surgery was performed a s e p t i c a l l y on the animals using halothane (1-2%) and n i t rous oxide (60%) anesthesia (See Sect ion 2 . 4 . 1 . ) . S i l i c o n e rubber catheters were implanted in a f e t a l jugu la r vein (JV) , c a r o t i d ar tery (CA), the main pulmonary trunk (PA), a branch of the l e f t pulmonary vein (PV) and the t rachea (TF) . Catheters were a lso implanted in the maternal femoral 43 ar tery (MA) and vein (MV) and in the amniotic c a v i t y (AF) . A m p i c i l l i n was given to the ewe and fetus as descr ibed in sec t ion 2 . 3 . 1 . Animals were allowed to recover f o r a minimum of 3 days before experimentat ion. For each experiment, the ewe was t rans fe r red to a monitor ing cage adjacent to and in f u l l view of the holding pen. 2 . 6 .2 . Drug Admin is t ra t ion and Experimental Protocol Experiments were c a r r i e d on the 3 animals at 128-133 days g e s t a t i o n . Two of the experiments involved fe ta l drug in fus ions and one experiment involved maternal drug i n f u s i o n . Drug in fus ions were c a r r i e d out in these animals as descr ibed prev ious ly in the Sect ion 2 .4 .2 . B r i e f l y , fo r f e t a l i n f u s i o n , diphenhydramine hydrochlor ide (Benadryl In jectable , Parke Dav is , Lot No. 01866, Scarborough, Ontar io) was in jec ted as a 5 mg loading dose via the f e t a l jugu la r vein ca the ter , fo l lowed by a continuous i n f u s i o n , with the drug i n f u s i o n rate of 0.17 mg/min and the i n f u s i o n volume of 0.17 mL/min f o r 90 min. For maternal i n f u s i o n , the drug was given as a 20 mg loading dose via the maternal femoral vein ca the te r , fol lowed by a continuous i n f u s i o n , with the drug i n f u s i o n rate of 0.67 mg/min and the i n f u s i o n volume of 0.17 mL/min fo r 90 min. During the drug in fus ion experiments, f e t a l pulmonary a r t e r i a l , pulmonary venous and c a r o t i d a r t e r i a l blood samples (1.5 mL each) were c o l l e c t e d simultaneously at - 5 , 60, 75, 90, 120 and 150 min. In a d d i t i o n , f e ta l t racheal and amniotic f l u i d s (1.5 mL each) and maternal a r t e r i a l blood (3.0 mL) were a lso taken at the same t ime. The to ta l 44 volume of f e t a l blood c o l l e c t e d was replaced by an equal volume of maternal blood in two d iv ided doses, one a f te r 75 min and one at the end of the experiment. A l l the samples were stored at -20°C f o r drug a n a l y s i s (see sec t ion 2 .1 .5 ) . 2 . 6 . 3 . Pharmacokinetic A n a l y s i s Total body clearances of diphenhydramine in the ewe (CL m m ) and fetus ( C L ^ ) were c a l c u l a t e d by the fo l lowing equat ions: C L m m = Maternal drug in fus ion r a t e / C m a ) S S ( e q . 24) CL f f = Fetal drug in fus ion r a t e / C c a s s (eq. 25) where C m a s s and C c a s s are the s teady-s ta te drug concentrat ions measured in the maternal femoral a r t e r i a l and f e t a l c a r o t i d a r t e r i a l plasma, r e s p e c t i v e l y . Fetal pulmonary ex t rac t ion r a t i o (ERp U-|m) of diphenhydramine was c a l c u l a t e d by the equat ion: E R p u l m = ( c p a , s s " c p v , s s ) / c p a , s s (eq.26) where C p a s s and C p V S S are the pulmonary a r t e r i a l and venous drug concentrat ions at s t e a d y - s t a t e , r e s p e c t i v e l y . Values are expressed as the mean ± 1 S .D. 45 3. RESULTS 3 .1 . A n a l y t i c a l Development 3 . 1 . 1 . Opt imizat ion of C a p i l l a r y GC Condi t ions The e f f e c t s of changes in the flow rates of the make-up gases, hydrogen and a i r , and the c a r r i e r gas , hel ium, on N/P detector response were examined. At constant rates of helium c a r r i e r gas flow (1.0 mL/min) and a i r f low (60.0 mL/min), hydrogen gas flow was increased from 2.0 to 4.25 mL/min ( F i g . 1). Although the maximum s e n s i t i v i t y of diphenhydramine and orphenadrine was obtained at hydrogen flow r a t e s , 3 .5-3 .75 mL/min ( F i g . 1) , a hydrogen flow rate of 3.0 mL/min was chosen and u t i l i z e d f o r fu r ther s tudies s ince at higher flow r a t e s , the chromatographic base l ine became unstable , with increased d r i f t and n o i s e . The e f f e c t of changes in a i r flow rate (range, 30.0 to 80.0 mL/min) on N/P detector response and peak width of diphenhydramine and orphenadrine were examined under constant rates of hydrogen gas flow (3.0 mL/min) and helium gas flow (1.0 mL/min) ( F i g . 2 ) . As the a i r flow ra te was i n c r e a s e d , the N/P detector response decreased. Peak width remained r e l a t i v e l y unchanged over the a i r flow rate range examined. An optimum a i r f low rate of 50.0 mL/min was appl ied f o r fu r ther study s ince the r e s o l u t i o n was bet ter at a i r f low rates >50.0 mL/min without a apprec iab le loss of s e n s i t i v i t y ( F i g . 2) . 46 F i g . 1. Hydrogen flow rate-NPD response curves: c a r r i e r gas (helium) flow rate constant , 1 mL/min; and a i r f low rate constant , 60 mL/min. E r ror bars represent mean ± 1 S .D. (n = 4, mean of two i n j e c t i o n s f o r each sample). 47 *h— 1 1 1 i i ' 30 40 50 60 70 80 Air Flow Rate (ml/min) F i g . 2. E f f e c t of a i r f low rate on NPD response and peak width: hydrogen flow rate constant , 3 mL/min; helium flow rate constant , 1 mL/min; and peak width = peak area count/peak he ight . E r ror bars represent mean ± 1 S .D . (n = 3-4, mean of two i n j e c t i o n s f o r each sample). 48 F i g . 3 shows the e f f e c t of column pressure on N/P detector response and peak width fo r diphenhydramine and orphenadrine at constant flow rates of hydrogen (3.0 mL/min) and a i r (50.0 mL/min). N/P detector response i s greatest at column pressure of 13 psi (equivalent to 1.0 mL/min). The van Deemter curves f o r diphenhydramine and orphenadrine are shown in F i g . 4. The lowest H . E . T . P . value was a lso obtained at -13 p s i . The column pressure of 13 p s i , the re fo re , was chosen to provide optimum helium c a r r i e r gas flow r a t e . The r e l a t i o n s h i p between purge a c t i v a t i o n time and NPD response f o r diphenhydramine and orphenadrine i s shown in F i g . 5. The purge a c t i v a t i o n time of 1.0 min allows enough time fo r the so lu tes in jec ted in to the i n j e c t i o n port in s p l i t l e s s mode to be t rans fe r red onto the column ( F i g . 5) . 3 . 1 .2 . Opt imizat ion of Drug Ext rac t ion Procedures Various s o l v e n t s , inc lud ing heptane, hexane, to luene, benzene and dichloromethane, in order of increas ing p o l a r i t y , were examined fo r t h e i r r e l a t i v e ex t rac t ion e f f i c i e n c y . Among the solvents examined, dichloromethane was found to be the most e f f i c i e n t solvent fo r s i n g l e -step ex t rac t ion of diphenhydramine and the in terna l standard, orphenadrine. Furthermore, the add i t ion of t r ie thy lamine in dichloromethane improved the ex t rac t ion e f f i c i e n c y of both diphenhydramine and orphenadrine by -5 f o l d over that of dichloromethane alone ( F i g . 6 ) . A hexane:isoamyl alcohol mixture (98:2) , which has been p r e v i o u s l y used fo r diphenhydramine ex t rac t ion (Abernethy and F i g . 3. E f f e c t of column pressure on NPD response and peak width: hydrogen flow rate constant , 3 mL/min; a i r f low rate constant , 50 mL/min; and peak width = peak area count/peak he ight . Er ror bars represent mean + 1 S .D. (n = 3-4, mean of two i n j e c t i o n s f o r each sample). 50 0.500-, 0.400 E 0.300 ui 0.200-x 0.100-0.000 10 20 30 40 Linear Velocity (Cm/sec) 50 F i g . 4. Re la t ionsh ip between height equiva lent to t h e o r e t i c a l p la tes (H .E .T .P ) vs. l i n e a r v e l o c i t y of the helium c a r r i e r gas determined fo r diphenhydramine. Column head pressures were 7, 9, 11, 13, 15 and 17 p s i . 51 45n — i 1 1 1 1 1 1 I i 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Purge Ac t i va t ion Time (min) F i g . 5. E f f e c t of purge a c t i v a t i o n time on NPD response: hydrogen flow ra te constant , 3 mL/min; a i r f low ra te constant , 50 mL/min; and helium flow rate constant , 1 mL/min. Er ror bars represent mean ± 1 S .D . (n = 3-4, mean of two i n j e c t i o n s f o r each sample). 0.0 0.001 0 .005 0.01 0.1 T E A Cone (M) in Dichloromethane 0.5 (98:2) Hexane: IAA E f f e c t of the add i t ion of t r i e thy lamine (TEA) on ex t rac t ion of diphenhydramine (hatched bar) and orphenadrine (open bar) with dichloromethane (IAA, isoamyl a l c o h o l ) . E r r o r bars represent mean ± 1 S .D . (n = 4, mean of two i n j e c t i o n s f o r each sample). 53 Greenbla t t , 1983), resu l ted in improved ex t rac t ion e f f i c i e n c y as compared to dichloromethane on ly , although the extent of increase in ex t rac t ion e f f i c i e n c y i s l e s s than that by dichloromethane with t r i e thy lamine ( F i g . 6 ) . F i g . 7 shows the r e l a t i o n s h i p between NPD response and ex t rac t ion t ime, ranging from 10 to 80 min. Both diphenhydramine and orphenadrine appear to be maximally extracted by -10 min and fu r ther sample ex t rac t ions were conducted with the ex t rac t ion time of 20 min. 3 . 1 . 3 . A p p l i c a t i o n of the Developed Assay Method to Placenta l Transfer Studies in Pregnant Sheep Representat ive GC chromatograms fo r maternal plasma obtained from a diphenhydramine-treated pregnant sheep, together with a corresponding contro l chromatogram i s shown in F i g . 8. Extraneous peaks from endogenous const i tuents d id not i n t e r f e r e with the ana lys is ( F i g . 8 ) . The peaks with re tent ion times (t^) of 7.85 and 9.31 min were i d e n t i f i e d by GC-MS as diphenhydramine and orphenadrine, r e s p e c t i v e l y . The data f o r a representa t ive c a l i b r a t i o n curve used in the quant i ta t ion of diphenhydramine in maternal and f e t a l sheep plasma are presented in Table 1. L i n e a r i t y was observed over the concentrat ion range studied (2-320 ng/mL) with the l i n e of best f i t being descr ibed by y = 0.0050x + 0.0004 with a c o r r e l a t i o n c o e f f i c i e n t (r) of 0.9996. An average diphenhydramine recovery of 103.2% ( c o e f f i c i e n t of v a r i a t i o n , 5.2%) was obtained over the concentrat ion range s t u d i e d . F i g . 7. Ex t rac t ion time vs. NPD response curves: dichloromethane was used f o r drug e x t r a c t i o n . E r ror bars represent mean ± 1 S.D. (n = 4, mean of two i n j e c t i o n s fo r each sample). 55 B *1—' in 00 W1 0 J L 15 15 F i g . 8. Representat ive c a p i l l a r y gas chromatograms obtained from blank sheep plasma (A) and a plasma sample from a pregnant sheep (B) a f t e r admin is t ra t ion of 100 mg of diphenhydramine hydrochlor ide in t ravenous ly . The plasma sample from the drug t reated sheep contained diphenhydramine (Rj. = 7.85 min) and spiked in terna l s tandard, orphenadrine (R t = 9.31 min) . At tenuat ion 2 5 , NPD-Voltage 14V. 56 Table 1. C a l i b r a t i o n curve data f o r sheep p lasma 3 . Amount of Diphenhydramine/Orphenadrine C o e f f i c i e n t Diphenhydramine area r a t i o , of Spiked (ng) (mean ± 1 S . D . ) ° V a r i a t i o n (%) 2 0.010 + 0.001 10.0 4 0.018 + 0.001 5.6 8 0.037 + 0.003 8.1 16 0.080 + 0.006 7.5 32 0.152 + 0.010 6.6 64 0.324 + 0.025 7.7 128 0.662 + 0.038 5.7 192 0.956 + 0.024 2.5 256 1.238 + 0.047 3.8 320 1.609 + 0.024 1.5 a. L inear regress ion l i n e fo r diphenhydramine: y = 0.0500x + 0.0004, r = 0.9996. b. Number of samples, n = 4 (mean of two i n j e c t i o n s ) . 57 Pure authent ic diphenhydramine samples, diphenhydramine-spiked contro l sheep plasma and plasma samples c o l l e c t e d from diphenhydramine-t reated sheep have been examined using GC-MS techniques. The t o t a l - i o n current mass chromatograms fo r diphenhydramine obtained from GC-MS s tud ies of these samples a l l showed a s i n g l e peak fo r diphenhydramine in the p o s i t i v e i o n i z a t i o n mode, prov id ing evidence f o r the p u r i t y of the compound. Confirmation of the i d e n t i t y of diphenhydramine was obtained using both EI and CI mass spectra from authent ic drug and drug-spiked plasma. When Cl-mass spectra were obtained and evaluated throughout the diphenhydramine peak e l u t i o n from dosed sheep plasma, the r e s u l t s a lso confirmed the i d e n t i t y of diphenhydramine and peak homogeneity. Prominent ion fragments fo r diphenhydramine were observed at m/e 167, 256, 209 and 254 in the CI mode and at m/e 58, 73, 167, 165, 45, 168, 44 and 42 in the EI mode. The fragmentation patterns of diphenhydramine were s i m i l a r to those reported e a r l i e r ( C o s t e l l o , 1981). Semilogar i thmic p lo ts of diphenhydramine concentrat ion in maternal and f e t a l sheep plasma vs. time data fo l lowing an i . v . bolus i n j e c t i o n of 100 mg diphenhydramine hydrochlor ide to the ewe are shown in F i g . 9. I n i t i a l estimates of the pharmacokinetic parameters for diphenhydramine a f t e r an i . v . dose were obtained by computer ana lys is using the program AUTOAN (Sedman and Wagner, 1976). The data were l a t e r f i t t e d using the program NONLIN (Metzler et al., 1974) and these i n i t i a l es t imates . The d e c l i n e in the maternal and fe ta l plasma diphenhydramine concentra t ion with time was observed to fo l low a biexponent ia l decay descr ibed by the fo l lowing equat ion: Cp = A e " a t + B e " ^ , where Cp is the plasma concentrat ion at any t ime, t . A and B are the in te rcepts of the 58 —i 1 1 1 1 1 60 120 180 240 300 360 T I M E ( m i n ) F i g . 9. A semi logar i thmic p lo t of the diphenhydramine concentra t ion vs. time p r o f i l e s obtained in maternal (•) and f e t a l (A) plasma fo l low ing a 100 mg i . v . bolus dose to a pregnant ewe. The terminal e l i m i n a t i o n h a l f - l i f e was c a l c u l a t e d to be 48 and 44 min in the ewe and f e t u s , r e s p e c t i v e l y . 59 a and fi phases, r e s p e c t i v e l y . The parameters a and fi represent the d i s t r i b u t i o n and terminal e l im ina t ion rate constants , r e s p e c t i v e l y . The maternal h a l f - l i f e of the a phase (t i , , a ) was c a l c u l a t e d to be 7 min, i n d i c a t i n g rap id d i s t r i b u t i o n of diphenhydramine fo l lowing a d m i n i s t r a t i o n . A terminal e l im ina t ion h a l f - l i f e ( t ^ ^) of 48 min was c a l c u l a t e d f o r diphenhydramine in maternal plasma. The p lacenta l t r a n s f e r of diphenhydramine to the fetus was r a p i d , with peak f e t a l plasma concentrat ion being at ta ined in l e s s than 5 min fo l lowing drug a d m i n i s t r a t i o n . The f e t a l diphenhydramine decay curve fol lowed a pattern s i m i l a r to that observed in maternal plasma. D i s t r i b u t i o n and terminal e l im ina t ion h a l f - l i v e s of 8 and 44 min were c a l c u l a t e d f o r diphenhydramine in the f e t u s . 3 .2 . Pharmacokinetics of Diphenhydramine a f t e r Dose Ranging in Nonpregnant Sheep Control values fo r the mean a r t e r i a l pH and Pco2 were 7.481 + 0.015 and 34.3 + 1.4 mm Hg, r e s p e c t i v e l y , and no s i g n i f i c a n t changes were observed fo r these va r iab les over the dose range s t u d i e d . Changes in mean a r t e r i a l P02, O2 content and hematocrit are shown in Table 2. Mean a r t e r i a l P02 decreased s i g n i f i c a n t l y at 5 min fo l lowing i n j e c t i o n of 100 and 200 mg doses and therea f te r returned to the contro l range. There were s i g n i f i c a n t increases in 0 2 content and hematocrit at 5 and 10 min a f t e r admin is t ra t ion of the 200 mg i . v . dose but these parameters returned to contro l values by 1 hr fo l lowing drug a d m i n i s t r a t i o n . Figure 10 shows the average plasma diphenhydramine concent ra t ion vs. time curves obtained a f te r i . v . bolus i n j e c t i o n of 25, 60 Table 2. Mean values of a r t e r i a l P02, 0 2 content and hematocrit obtained a f te r i . v . bolus i n j e c t i o n of diphenhydramine to 6 nonpregnant sheep. Diphenhydramine Hydrochlor ide Dose (mg) Time (min) 25 50 100 200 Po 2 -5 111.2(12.8)a 120.8(3.7) 118.8(6.7) 121.5(6.7) 5 114.7(9.0) 110.2(12.5) 106.4(6.1)* 110.5(3.8)* 10 109.4(12.9) 116.7(13.7) 109.7(6.1) 113.1(9.9) 60 113.8(11.4) 111.5(6.7) 115.7(10.5) 116.9(7.1) 0 2 content -5 12.4(0.9) 13.2(0.7) 12.9(0.8) 12.6(0.6) 5 12.5(1.0) 12.7(1.0) 13.3(0.9) 14 .9(1 .5 ) * 10 12.3(0.8) 12.7(0.7) 13.0(1.1) 14 .2(1 .9)* 60 12.4(1.2) 13.1(0.9) 12.6(1.0) 12.9(1.2) Hematocrit -5 25.7(2.4) 27.4(1.5) 27.1(2.6) 26.8(2.4) 5 25.9(2.8) 27.1(2.5) 28.2(2.1) 31 .3 (3 .8 ) * 10 26.0(2.1) 26.0(2.2) 28.6(2.8) 30 .6 (4 .6 ) * 60 26.4(3.4) 29.5(3.7) 26.5(2.9) 28.0(3.3) a. values given as mean + 1 S .D. * p<0.05. 61 F i g . 10. Average plasma drug concentra t ion vs. time curves obtained a f t e r i . v . admin is t ra t ion of 25 (O), 50 (•) , 100 (A) and 200 (•) mg doses of diphenhydramine hydrochlor ide to 6 nonpregnant sheep. 62 50, 100 and 200 mg dose of the drug to 6 nonpregnant sheep on a crossover b a s i s . Plasma drug concentrat ion vs. time curves were best descr ibed by biexponent ia l equations in a l l but two of the experiments, where t r i exponent ia l decay was observed in one animal a f t e r the 100 mg dose and in another animal a f te r the 200 mg dose. Values f o r the intercompartmental t r a n s f e r rate constants are shown in Table 3. Pharmacokinetic parameters c a l c u l a t e d are shown in Table 4. The average i n i t i a l d i s t r i b u t i o n a l and the terminal e l im ina t ion h a l f - l i v e s were increased from 5.4 to 8.9 min and from 34.0 to 67.5 min, r e s p e c t i v e l y , as dose increased . The average e l im ina t ion h a l f - l i f e a f t e r a 200 mg dose was s i g n i f i c a n t l y longer than a f te r e i t h e r the 25 or the 50 mg dose: converse ly , the terminal e l im ina t ion rate constant , lambda n , decreased s i g n i f i c a n t l y a f t e r the 200 mg dose ( F i g . 11). There was a lso an increase in the volume of d i s t r i b u t i o n (2.8 to 5.5 L/kg f o r Vdss), with increas ing dose. The Vdss obtained from the 200 mg dose was s i g n i f i c a n t l y greater than that from the 25 mg dose ( F i g . 12). However, wi th in the dose range of 25-100 mg, there was no s i g n i f i c a n t d i f f e r e n c e in e i t h e r the e l im ina t ion h a l f - l i f e or the volume of d i s t r i b u t i o n . Values f o r Vdss were c a l c u l a t e d by two d i f f e r e n t methods, namely, compartmental ana lys is using intercompartmental microconstants (eq. 8 ) , and moment a n a l y s i s using AUC and AUMC (eq. 9 ) . VdS5 values c a l c u l a t e d by these two methods were in c lose agreement, with 2-3% v a r i a t i o n (Table 4 ) . The average CLJQ (~5 L/hr /kg) remained r e l a t i v e l y unchanged regard less of dose (Table 4) . Although there were changes in the k i n e t i c parameters of the d i s t r i b u t i o n and e l im ina t ion h a l f - l i v e s and OO the volume of d i s t r i b u t i o n , there was a proport ional increase in AUC 0 as oo dose increased ( F i g . 13) and therefore dose-normal ized AUC 0 remained Table 3. Estimates of NONLIN pharmacokinetic constants of diphenhy-dramine obtained a f t e r i . v . bolus i n j e c t i o n of 25, 50, 100 and 200 mg doses to 6 nonpregnant sheep (mean ± 1 S . D . ) . Parameter Diphenhydramine Hydrochlor ide Dose (mg) 25 50 100 a 200 a Aj (ng/mL) 188.3 (93.9) 275.3 (69.9) 426.7 (133.8) 488.6 (215.6) A 2 (ng/mL) 53.4 (29.5) 90.3 (32.7) 142.3 (47.8) 258.8 (73.7) lambda^ (min - * ) 0.1545 (0.0638) 0.1326 (0.0633) 0.1074 (0.0549) 0.0861 (0.0399) lambda 2 (min - * ) 0.0240 (0.0091) 0.0203 (0.0047) 0.0159 (0.0036) 0.0117 (0.0022) k 1 0 (min" 1 ) 0 .0696 6 (0.0241) 0.0532 c (0.0102) 0.0407 (0.0049) 0.0270 (0.0097) k j 2 ( m i n - 1 ) 0.0537 (0.0260) 0.0493 (0.0370) 0.0392 (0.0312) 0.0323 (0.0200) k 2 j ( m i n - 1 ) 0.0552 6 (0.0297) 0.0504 (0.0231) 0.0433 (0.0254) 0.0386 (0.0190) r 2 0.992 d (0.017) 0.998 (0.003) 0.995 (0.004) 0.998 (0.003) a. obtained from 5 animals because one set plasma drug concent ra t ion-time data f i t to t r i exponent ia l decay. b. s t a t i s t i c a l l y d i f f e r e n t from the value obtained a f t e r 100 and 200 mg doses (p<0.05). c . s t a t i s t i c a l l y d i f f e r e n t from the value obtained from 200 mg dose (p<0.05). d. c o r r e l a t i o n c o e f f i c i e n t obtained from NONLIN nonl inear l e a s t squares regress ion a n a l y s i s . 64 Table 4. Pharmacokinetic parameters of diphenhydramine obtained a f te r i . v . bolus i n j e c t i o n s to 6 nonpregnant sheep. Diphenhydramine Hydrochlor ide Dose (mg) Parameter 25 50 100 200 tk, lambdaj (min) 5.4 ( 2 . 6 ) a 6.7 (4.1) 7.1 (3.6) 8.9 (4.6) tk, lambda n (min) 34.0 (16.5) 36.5 (12.5) 52.3 (19.2) 6 7 . 5 b (18.7) CZ. T B (L /hr /kg ) 5.0 (1.7) 4.8 (0.7) 5.0 (0.8) 4.6 (0.8) ""area ( L / k 9> 4.1 (2.8) 4.1 (1.0) 6.5 (3.3) 7.3 (1.5) Vdss ( L / k g ) c 2.8 (1.5) 3.1 (0.9) 4.5 (1.9) 5.5" (1.5) Vdss ( L / k 9 ) e 2.8 (1.7) 3.0 (0.8) 4.4 (1.7) 5 . 4 d (1.6) A U C Q (/xg-h/L) 60.4 (12.9) 119.9 (16.8) 229.4 (39.4) 504.4 (116.9) a. values given as mean + ( S . D . ) . b. value d i f f e r e n t from that obtained a f t e r 25 and 50 mg doses (p<0.05). c . c a l c u l a t e d by: Dose-AUMCo/(AUCo) 2 . d. value d i f f e r e n t from that obtained a f te r 25 mg dose (p<0.05). e. c a l c u l a t e d by: Dose-jL (C 7- / lambda 7-) /[ JL 7=1 7 7 7=1 (C 7-/lambda 7-)] 2 65 2.0 n 1.5 -• O c 1.0 0.5 -• A A O 0.0 25 50 100 200 Dose (mg) F i g . 11. Changes in the terminal e l i m i n a t i o n rate constant (lambda n) of diphenhydramine as a funct ion o f dose obtained in 6 nonpregnant sheep. Each symbol represents each animal and the hor izonta l l i n e s represent mean va lues . S i g n i f i c a n t l y d i f f e r e n t from 25 and 50 mg doses (p<0.05). 8 i 66 CP c o •J—' D cn Q M — o 0) 6-4 -2 -A • A A • • • o > 0 — i 1 1 1 2 5 5 0 1 0 0 2 0 0 Dose ( m g ) F i g . 12. S teady-s ta te volume of d i s t r i b u t i o n (^cf s s) of diphenhydramine as a funct ion of dose obtained in 6 nonpregnant sheep. * S i g n i f i c a n t l y d i f f e r e n t from a 25 mg dose (p<0.05). 67 F i g . 13. Re la t ionsh ip between the area under the plasma drug oo concentra t ion vs. time curve (AUC 0) and i . v . bolus doses of diphenhydramine given on a crossover bas is to 6 nonpregnant sheep. Parameters fo r the l i n e a r regress ion l i n e were: y = 2.54x - 9.52 (r = 0 .95) . 68 unchanged. Plasma prote in binding of diphenhydramine in nonpregnant sheep was determined by equ i l ib r ium d i a l y s i s . Equ i l ib r ium between the plasma and buf fer compartments was obtained by 3 hr ( F i g . 14). Drug adsorpt ion onto the sur face of the d i a l y s i s c e l l and membrane was minimal , with an average recovery of 99 + 5% over the drug concentrat ion range of 10-10,000 ng/mL. In vitro prote in binding experiments showed that there was no s i g n i f i c a n t change in the f ree f r a c t i o n over a drug concentrat ion range from 10-2,000 ng/mL, with a mean of 0.332 + 0.057. The f ree f r a c t i o n , however, was increased to 0.489 at 4,000 ng/mL and 0.570 at 10,000 ng/mL. The extent of plasma prote in binding was a lso determined in the 6 animals in vivo in 5 and 60 min samples obtained a f t e r admin is t ra t ion of a 200 mg dose. The average f ree f r a c t i o n was 0.229 + 0.080 in the nonpregnant ewes employed (Table 5) . In two animals, the f ree f r a c t i o n of diphenhydramine was determined in a l l samples c o l l e c t e d a f t e r a 200 mg dose. The unbound f r a c t i o n remained unchanged regard less of drug concent ra t ion , with a mean of 0.303 ± 0.033 over the concentra t ion range, 30-567 ng/mL in one animal (ewe no. 3) and 0.170 + 0.020 over the concentrat ion range, 31-783 ng/mL in the other animal (ewe no. 132). 3 .3 . Pharmacokinetics of Diphenhydramine in the Pregnant Ewe and Fetal Lamb: i . v . Bolus In ject ion Studies The physica l c h a r a c t e r i s t i c s of ind iv idua l ewes employed in the study are shown in Table 6. The gesta t iona l age at the time of the 14. Determination of e q u i l i b r i u m time used in the study of plasma pro te in binding of diphenhydramine by e q u i l i b r i u m d i a l y s i s (n = 3 ) . I n i t i a l buf fe r concent ra t ion : A500 ng/mL, 0 10,000 ng/mL. 70 Table 5. Plasma f ree f r a c t i o n of diphenhydramine determined in samples obtained a f t e r i . v . bolus i n j e c t i o n of the 200 mg dose to nonpregnant sheep. Free Frac t ion Animal No. 5 min 60 min Average 3 0.284 (567.0) 0.282 (169.1) 0.283 15 0.347 (477.6) 0.324 (111.2) 0.335 128 0.174 (702.7) 0.237 (96.6) 0.205 132 0.179 (782.7) 0.147 (131.6) 0.163 905 0.214 (434.0) 0.318 (109.4) 0.266 906 0.122 (1072.1) 0.124 (183.6) 0.123 mean + S .D . 0.220 ± 0.082 0.239 + 0.086 0.229 ± 0.080 Numbers in brackets are the to ta l drug concentrat ions (ng/mL) in the plasma sample employed. 71 Table 6. Physical c h a r a c t e r i s t i c s of ind iv idua l pregnant Dorset ewes used in the maternal diphenhydramine i . v . bolus i n j e c t i o n study. Maternal Gestat ional Age Fetal Body No. DPHM HC1 Ewe Body (days) Weight of i .v No. Weight in utero Fetuses Dose (kg) ( k g ) a (mg/kg) Surgery Experiment 104 89.1 122 129 3.0 2 1.122 62 60.0 126 139 2.5 1 1.667 98 61.8 129 140 2.9 1 1.618 110 67.3 122 130 2.1 2 1.486 69.6 125 135 2.6 1.473 ( 1 3 . 4 ) 6 (3) (6) (0.4) (0.246) a. c a l c u l a t e d by the equat ion: log ( fe ta l body weight in utero) = log (b i r th weight) - (0.0153 x number of days between experiment and b i r t h ) . b. Numbers in brackets are ± 1 S .D. 72 experiment ranges from 129-140 days (mean 135 days ) , and the maternal body weight ranged from 60-89 kg (mean 70 kg). Changes in the f e t a l a r t e r i a l blood gas s t a t u s , pH, 0 2 content and hematocrit are shown in Table 7. Fetal descending a o r t i c P o 2 , P c o 2 , pH, 0 2 content and hematocrit in the contro l per iod averaged 17.1 ± 4.3 mm Hg, 49.0 ± 9.0 mm Hg, 7.399 ± 0.108, 8.4 ± 2.5 vol % and 39.9 ± 3.6 vol %, r e s p e c t i v e l y . These values are in the normal range fo r f e t a l lambs. In a l l four f e t u s e s , Po 2 f e l l fo l lowing drug admin is t ra t ion to a low of 12.7 ± 2.9 mm Hg at 5 min, and there was a corresponding f a l l in blood oxygen content . By 30 min fo l lowing drug i n j e c t i o n , Po 2 had returned to contro l l e v e l s . No other changes in fe ta l blood gas status were observed. F i g . 15 shows representa t ive c a p i l l a r y gas chromatograms obtained from maternal and fe ta l plasma, f e ta l t racheal and amniotic f l u i d s of a pregnant sheep fo l lowing a 100 mg i . v . bolus i n j e c t i o n of diphenhydramine hydroch lor ide . Extraneous peaks from endogenous const i tuents were n e g l i g i b l e and d id not i n t e r f e r e with the a n a l y s i s . Semi logar i thmic p l o t s of concentrat ion vs. time data in maternal and f e t a l plasma, and t racheal and amniotic f l u i d s fo l lowing adminis t ra t ion of a 100 mg i . v . dose to a ewe, are shown in F i g . 16. Each set of maternal plasma concentrat ion vs. time data was best descr ibed by a b iexponent ia l equat ion . The intercompartmental t r a n s f e r ra te constants and the pharmacokinetic parameters obtained from the ind iv idua l ewes a f t e r maternal drug admin is t ra t ion are shown in Tables 8 and 9. The t r a n s f e r of the drug to the fetus was r a p i d , with the 73 Table 7. The e f f e c t of maternal diphenhydramine admin is t ra t ion on fe ta l a r t e r i a l pH, P c o 2 , P o 2 , 0 2 content and hematocrit fo l lowing 100 mg i . v . bo lus . Parameter Control 5 min 10 min 30 min PH 7 .399 a 7.391 7.394 7.393 (0.108) (0.101) (0.109) (0.096) PcOo 49.0 50.6 49.9 50.1 (mm Hg) (9.0) (5.5) (6.8) (7.1) POo 17.1 * 12.7 15.2 17.9 (mm Hg) (4.3) (2.9) (2.3) (5.3) 0 2 Content 8.4 5.2* 6.4 8.7 (% v /v ) (2.5) (1.5) (2.3) (2.3) Hematocrit 39.9 40.7 41.1 40.7 (% v/v) (3.6) (3.1) (2.9 (3.3) a. mean ± 1 S .D . (n = 4 ) . * P<0.05. 74 L O 0 0 MA ro cn LO oo ro art FA L O CO 15 0 TF ro AF ro CTl LO 00 LJJLJ ^ j L Lr »J—^  15 0 15 0 15 F i g . 15. Representat ive c a p i l l a r y gas chromatograms of the maternal (MA) and f e t a l (FA) a r t e r i a l plasma, f e t a l t racheal (TF) and amniot ic (AF) f l u i d s obtained from c o n t r o l - and drug- t rea ted pregnant sheep. The samples contained diphenhydramine (R t = -7 .85 min) and spiked in te rna l standard orphenadrine (R .^ = -9.31 min) . 75 100Ch c £ 1 0 0 : c (J C o o V c D X ) >, x: c x : C L 10 60 120 180 240 Time (min) 300 360 F i g . - 1 6 . Representat ive semi logar i thmic p l o t s of the maternal (O) and f e t a l (•) a r t e r i a l plasma, f e t a l t racheal (•) and amniotic (•) f l u i d diphenhydramine concentrat ion vs. time p r o f i l e s fo l lowing a 100 mg i . v . bo lus . 76 Table 8. Estimates of NONLIN pharmacokinetic constants obtained from maternal plasma diphenhydramine data fo l lowing a 100 mg i . v . dose of the drug to ind iv idua l ewes. Parameter Ewe No. mean ± S .D. 104 62 98 110 A (ng/mL) 764.3 277.6 521.7 1586.7 787.6 ± 568.6 b B (ng/mL) 442.6 142.7 265.5 542.3 348.3 ± 178.6 a ( m i n - 1 ) 0.0997 0.0822 0.0567 0.0932 0.0830 ± 0.0189 P ( m i n - 1 ) 0.0145 0.0125 0.0162 0.0112 0.0136 ± 0.0022 1.000 0.998 0.999 0.998 0.999 ± 0.001 k 1 0 ( m i n - 1 ) 0.0317 0.0285 0.0307 0.0302 0.0303 ± 0.0013 kj2 ( m i n - 1 ) 0.0368 0.0302 0.0123 0.0380 0.0293 ± 0.0119 k 2 i ( m i n - 1 ) 0.0457 0.0362 0.0298 0.0258 0.0344 ± 0.0088 a. c o e f f i c i e n t of regress ion obtained from NONLIN nonl inear l e a s t squares regress ion a n a l y s i s . b. Values are expressed as the mean ± 1 S .D. 77 Table 9. Pharmacokinetic parameters obtained a f t e r a 100 mg i . v . dose of diphenhydramine to ind iv idua l ewes. Parameter Ewe No. mean ± 1 S.D. 104 62 98 110 Maternal parameter H, a (min) 7.0 8.4 12.2 7.4 8.8 ± 2.4 H, p (min) 47.8 55.4 42.9 62.1 52.0 ± 8.5 ^area ( L / k 9 ) 2.2 9.3 4.1 2.3 4.5 ± 3.3 Vdss (L/kg) 1.6 6.7 2.7 1.7 3.2 ± 2.4 C/1TB (L /h r /kg ) 1.9 7.0 4.0 1.5 3.6 ± 2.5 AUCM™ (mg-hr/L) 599.9 240.0 406.0 973.6 554.9 ± 315.5 Fetal parameter thf a (min) 7.9 15.5 3.1 _a 8.8 ± 6.3 p (min) 43.8 60.3 37.7 43.3 46.3 ± 9.8 AUC Fo (mg-hr/L) 252.6 185.9 561.0 789.0 447.1 ± 280.4 A U C F > U C M ; 0.42 0.78 1.38 0.81 0.85 ± 0.40 a. Value indeterminate because concentrat ion vs. time course d id not f i t b iexponent ia l decay. 78 highest f e t a l plasma cencentrat ions observed at the e a r l i e s t sampling time (5 min) in three animals. Three of the four sets of f e t a l data were descr ibed by a biexponent ia l equat ion, with a d i s t i n c t but short d i s t r i b u t i o n phase fol lowed by a rap id e l i m i n a t i o n . In the remaining animal (ewe no. 110), however, peak fe ta l plasma drug concentrat ion occurred 20 min a f te r drug admin is t ra t ion , preventing the i d e n t i f i c a t i o n of a p o s s i b l e short d i s t r i b u t i o n a l phase preceeding the terminal e l i m i n a t i o n phase in t h i s f e t u s . The average f e t a l drug e l im ina t ion h a l f - l i f e , determined from the terminal phase (46.3 ± 9.8 min) , was not s i g n i f i c a n t l y d i f f e r e n t from that in the ewe (52.0 + 8.5 min). During the e n t i r e sampling p e r i o d , the fe ta l plasma concentrat ions c l o s e l y p a r a l l e l e d that in the mother, with an overa l l c o r r e l a t i o n c o e f f i c i e n t between maternal and f e t a l plasma drug concentrat ions of 0.857. The average f e t a l to maternal drug concentrat ion r a t i o was 0.90 ± 0.49, but in one experiment, diphenhydramine concentrat ion in the fetus was s l i g h t l y , but c o n s i s t e n t l y higher than in the ewe. The r a t i o of f e t a l to maternal plasma AUC values obtained from four sheep averaged 0.85 ± 0.40. The mean of the percentage of diphenhydramine administered to the ewe that i s e l iminated by the fetus via nonplacental e l im ina t ion pathways (eq. 15) was 5.0% (Table 10). A l t e r n a t i v e l y , most of the drug (95.0%) administered to the pregnant sheep was e l iminated by the ewe via maternal nonplacental pathways. In the t racheal f l u i d , the average time to reach the maximum drug concentra t ion was 28 min (range, 10-45 min) a f t e r drug a d m i n i s t r a t i o n , with an average peak concentrat ion of 551.2 ng/mL (range, 476.7-699.0 ng/mL). The f e t a l t racheal to a r t e r i a l plasma drug 79 Table 10. Percentage of the int ravenously administered maternal dose (100 mg bolus) of diphenhydramine e l iminated by the fetus via nonplacental pathways. A U C F o ' C L f o 6 A U C F ^ - C L f o - 1 0 0 / (mg) ( A U C M o ' C L m o ^ A U C F ; . C L f o ) A U C M o - C L m o a Ewe No. (mg) 104 120.4 3.2 2.6 62 48.2 2.3 4.6 98 81.5 7.0 7.9 110 195.3 9.9 4.8 111.4 ± 63.3 5.6 ± 3.5 5.0 ± 2.2 a. Mean value (n = 8) fo r the nonplacental c learance of diphenhydramine from the maternal compartment, C L m o (3343.8 mL/min), was obtained from s teady-s ta te in fus ion experiments (Sect ion 3 . 4 . 1 . 4 ) . b. Mean value ((n = 8) fo r the nonplacental c learance of diphenhydramine from the f e t a l compartment, C L f o (208.4 mL/min), was obtained from s teady-s ta te i n f u s i o n experiments (Sect ion 3 . 4 . 1 . 4 ) . 80 concentra t ion r a t i o was c a l c u l a t e d by d i v i d i n g t racheal drug concentrat ions by corresponding fe ta l plasma drug concentrat ions at each sampling t ime. The average r a t i o of the f e t a l t racheal to a r t e r i a l plasma diphenhydramine concentrat ions c a l c u l a t e d was 2.7 ± 2 .4 . S i m i l a r l y , diphenhydramine concentrat ions in amniotic f l u i d rose p r o g r e s s i v e l y , with i t s peak concentrat ion (mean, 113.1 ng/mL) occurr ing from 1.5 to 3.0 hr a f te r drug admin is t ra t ion . Approximately 3 .0 -3 .5 hr a f t e r drug dos ing , diphenhydramine l e v e l s in the amniotic f l u i d exceeded those in t racheal f l u i d and maternal and f e t a l plasma ( F i g . 16). The highest diphenhydramine concentrat ion u l t imate ly achieved in amniotic f l u i d was s u b s t a n t i a l l y l e s s than the highest concentrat ions in f e t a l plasma in each experiment (mean r a t i o , 0 .34) . The average apparent terminal e l i m i n a t i o n h a l f - l i f e in the t racheal f l u i d (38.7 ± 1.5 min) determined by l i n e a r regress ion ana lys is was s i g n i f i c a n t l y shor ter than those in maternal (52.0 ± 8.5 min) and f e t a l (46.3 ± 9.8 min) plasma. Diphenhydramine i s e l iminated from the amniotic f l u i d more slowly than from the maternal and f e t a l plasma and t racheal f l u i d , with an average e l i m i n a t i o n h a l f - l i f e of 168 min. 3 .4 . Transplacenta l C learance, Nonplacental Clearance and Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep: Maternal and Fetal Drug Infusions to Steady-State 3 . 4 . 1 . Transplacenta l and Nonplacental Clearances in the Maternal -Fetal Unit 3 . 4 . 1 . 1 . Achievement of Steady-State Diphenhydramine Concentrat ions in Maternal and Fetal Plasma 81 A to ta l of 16 diphenhydramine in fus ion experiments were c a r r i e d out in the 8 animals, invo lv ing separate maternal and f e t a l i . v . i n fus ions of the drug, given 48 hr apart . Information on the ind iv idua l pregnant ewes and fetuses employed in the study i s shown in Table 11. Maternal and f e t a l drug in fus ion experiments were c a r r i e d out over the ges ta t iona l age range of 127-136 (mean, 131 ± 3) and 126-136 (mean, 132 ± 4) days, r e s p e c t i v e l y . Diphenhydramine i s r a p i d l y t r a n s f e r r e d across the p lacenta from mother to fetus or from fetus to mother, with the highest drug concentrat ions observed at the e a r l i e s t sampling time (5 min) a f t e r e i t h e r the maternal or f e t a l drug a d m i n i s t r a t i o n . Average maternal and f e t a l plasma drug concentrat ion vs. time curves fo l lowing maternal and f e t a l in fus ions are shown in F i g . 17 and 18, r e s p e c t i v e l y . Plasma drug concentrat ions reached a plateau by 30-60 min a f t e r s t a r t i n g the i n f u s i o n and from 60-90 min, the drug l e v e l s remained r e l a t i v e l y constant . S teady-sta te diphenhydramine concentrat ions were c a l c u l a t e d as the mean of drug concentrat ions at 60, 75 and 90 min and are shown in Table 12. The average s teady-s ta te plasma diphenhydramine concentrat ion a f t e r maternal i n f u s i o n was 212.1 + 67.8 ng/mL in the mother and 36.3 ± 14.4 ng/mL in the f e t u s , r e s u l t i n g in a f e ta l to maternal concentrat ion r a t i o ( C f s s / C m s s ) of 0.19 ± 0.10 (Table 12 and 13). Fol lowing f e t a l i n f u s i o n s , maternal and f e t a l s teady-s ta te drug concentrat ions were 31.1 ± 11.6 and 447.6 ± 185.2 ng/mL, r e s p e c t i v e l y , with an average maternal to f e t a l concentrat ion r a t i o ( C m ' s s / C f ' s s ) of 0.08 ± 0.05 (Table 12 and 13). The mean c o e f f i c i e n t of v a r i a t i o n of the maternal and f e t a l s teady-s ta te drug concentrat ions between 60-90 min a f t e r the s t a r t of i n f u s i o n was 4.1 ± 3.3% and 4.8 ± 3.4%, r e s p e c t i v e l y , f o r maternal 82 Table 11. Physical c h a r a c t e r i s t i c s of ind iv idua l pregnant sheep used in the s teady-s ta te in fus ion study. Maternal Gestat ional Age Fetal Weight Number Ewe Breed Body (days) in utero (kg) of No. Weight Fetuses (kg) Surgery MI FI MI FI Average Drug Infusion Study 121 S u f f o l k 66.4 128 134 136 3.3 3.4 3.4 2 125 S u f f o l k 71.4 123 134 136 1.7 1.8 1.8 3 130 S u f f o l k 96.4 123 128 126 2.5 2.4 2.5 1 133 Su f fo lk 88.2 129 136 135 2.2 2.2 2.2 2 138 Dorset 61.4 122 132 134 2.5 2.7 2.6 1 202 S u f f o l k 89.1 119 132 130 1.5 1.4 1.5 4 204 Dorset 62.7 122 128 130 2.4 2.5 2.5 1 480 Dorset 85.0 120 127 129 1.3 1.3 1.3 2 77.6 (13.6) 123 (4) 131 (3) 132 (4) 2.2 (0.7) 2.2 (0.7) (2.2) (0.7) Sa l ine Infusion Study 135 Dorset 70.0 122 128 - 1.7 2 213 S u f f o l k 86.8 123 - 136 2.1 3 215 Dorset 86.4 129 - 142 2.4 1 482 Dorset 68.2 125 - 133 1.1 3 77.9 (10.1) 123 (2) 128 135 (6) 1.7 1.8 (0.6) Numbers in brackets are ± 1 S .D. MI, maternal i n f u s i o n ; F I , f e ta l i n f u s i o n . 83 F i g . 17. Average maternal (•) and f e t a l (O) plasma diphenhydramine concentra t ion vs. time curves obtained fo l lowing i . v . i n fus ion of the drug to the ewe (n = 8 ) . 84 F i g . 18. Average maternal (•) and f e t a l (o) plasma diphenhydramine concentra t ion vs. time curves obtained fo l lowing i . v . i n f u s i o n of the drug to the fetus (n = 8 ) . 85 Table 12. Steady-s ta te diphenhydramine concentrat ions in the ewe and fetus fo l lowing maternal and fe ta l drug admin is t ra t ions . Ewe Maternal Infusion (ng/mL) Fetal i n f u s i o n (ng/mL) No. C m s s c f s s Cm' ss C f ss 121 360.3 (17 .3 ) a 35.5 (5.9) 53.9 (2.6) 658.0 (108.6) 125 215.8 (23.7) 29.6 (5.1) 26.5 (2.9) 697.9 (120.7) 130 185.4 (13.3) 20.8 (6.3) 25.3 (1.8) 274.9 (82.8) 133 207.8 (18.1) 56.0 (12.4) 26.4 (2.3) 367.0 (81.5) 138 197.8 (33.2) 49.6 (16.2) 39.5 (6.6) 509.8 (166.2) 202 152.9 (24.0) 18.3 (6.7) 22.4 (3.5) 323.4 (117.7) 204 236.1 (45.6) 29.1 (11.7) 36.5 (7.1) 192.1 (77.2) 480 140.3 (41.1) 51.2 (13.5) 17.9 (5.3) 557.9 (146.7) x ± S .D . 212.1 ± 67.8 (27.0 ± 11.7) 36.3 ± 14.4 (9.7 ± 4.2) 31.1 (4.0 ± 11.6 ± 2.0) 447.6 (112.7 ± 185.2 ± 32.2) a. Values in brackets are the data based on f ree drug concent ra t ions . 86 Table 13. Maternal to f e t a l or fe ta l to maternal diphenhydramine concentrat ion r a t i o s at s teady-s ta te fo l lowing maternal and f e t a l drug i n f u s i o n . Ewe Maternal Infusion Fetal i n f u s i o n No. C f s s / C m s s C m s s / C f s s C m ' s s / C f ' s s C f ' s s / C m ' s s 121 0.10 (0.34) 10.15 (2.93) 0.08 (0.02) 12.21 (41.76) 125 0.14 (0.22) 7.29 (4.65) 0.04 (0.02) 26.34 (41.62) 130 0.11 (0.47) 8.91 (2.13) 0.09 (0.02) 10.87 (46.00) 133 0.27 (0.69) 3.71 (1.46) 0.07 (0.03) 13.90 (35.44) 138 0.25 (0.49) 3.99 (2.05) 0.08 (0.04) 12.91 (25.70) 202 0.12 (0.28) 8.36 (3.58) 0.07 (0.03) 14.44 (33.60) 204 0.12 (0.26) 8.11 (3.90) 0.19 (0.09) 5.26 (10.87) 480 0.37 (0.33) 2.74 (3.04) 0.03 (0.04) 31.17 (27.68) x ± S .D . 0.19 (0.39 ± 0.10 ± 0.16) 6.66 (2.97 ± 0.98 ± 1.06) 0.08 ± 0.05 (0.04 ± 0.02) 15.89 (32.83 ± 3.02 ± 11.32) Values in brackets are the data based on f ree drug concent ra t ions . 87 i n fus ions and 6.4 ± 4.2 and 4.5 ± 4.2%, r e s p e c t i v e l y fo r f e t a l i n f u s i o n s . 3 . 4 . 1 . 2 . Plasma Prote in Binding in Maternal and Fetal Sheep The f ree f r a c t i o n of diphenhydramine determined at s teady-s ta te was s i g n i f i c a n t l y higher in the fetus (0.277 ± 0.087) than in the mother (0.141 ± 0.079) (Table 14). The r a t i o of f e t a l to maternal f ree f r a c t i o n of the drug was greater than uni ty in a l l the animals except one, with a mean r a t i o of 2.37 ± 1.04. The s teady-s ta te concentrat ions of unbound diphenhydramine were c a l c u l a t e d accord ing ly and are shown in parentheses in Table 12. As with to ta l drug concent ra t ions , unbound diphenhydramine concentrat ions in the mother are s t i l l higher than in the fetus fo l lowing maternal i n f u s i o n , although the r a t i o of the fe ta l to maternal f ree drug concent ra t ions , C f S S } f r e e / C m s s > f r e e , increased as compared with the C f $ s / C m s s r a t i o (0.39 vs. 0.19) (Table 13). S i m i l a r l y , fo l lowing f e t a l drug i n f u s i o n s , s teady-s ta te unbound drug concentrat ions in the fetus were higher than in the mother and there was a fu r ther decrease in the maternal to f e t a l f ree drug concentrat ion r a t i o , C m S S 5 f r e e / C f ' s s ? f r e e > a s compared with the r a t i o based on the to ta l drug concentrat ion (0.04 vs. 0.08) (Table 13). 3 . 4 . 1 . 3 . Drug D i s p o s i t i o n in Fetal Tracheal and Amniotic F l u i d s Representat ive semilogari thmic p lo ts of the f e t a l t racheal and amniot ic f l u i d as well as maternal and f e t a l plasma diphenhydramine concentrat ion vs. time curves obtained a f t e r maternal and fe ta l 88 Table 14. Plasma f ree f r a c t i o n of diphenhydramine in the pregnant ewe and fetus at s teady-s ta te fo l lowing maternal or f e t a l drug a d m i n i s t r a t i o n 9 . Ewe Free Frac t ion Feta l /Maternal Ratio No. Ewe Fetus 121 0.048 0.165 3.44 125 0.110 0.173 1.57 130 0.072 0.301 4.18 133 0.087 0.222 2.55 138 0.168 0.326 1.94 202 0.157 0.364 2.32 204 0.193 0.402 2.08 480 0.293 0.263 0.90 x ± S.D 0.141 ± 0. 079 0.277 ± 0 .087 6 2.37 ± 1.04 a. Free f r a c t i o n of the drug was determined by e q u i l i b r i u m d i a l y s i s in 75 and 90 min maternal and f e t a l plasma samples obtained fo l lowing maternal and fe ta l drug i n f u s i o n s , r e s p e c t i v e l y . b. S i g n i f i c a n t l y d i f f e r e n t from the maternal value (p<0.05). 89 i n f u s i o n s are shown in F i g s . 19 and 20, r e s p e c t i v e l y . During maternal i n f u s i o n experiments, the average time required to reach the maximum diphenhydramine concentrat ions ( C m a x ) , T m a x , in the f e t a l t racheal f l u i d (95 ± 41 min) was not s i g n i f i c a n t l y d i f f e r e n t from that in the amniotic f l u i d (113 ± 55 min) (Table 15). The average C m a x in the t racheal f l u i d (124.7 ng/mL) was s i g n i f i c a n t l y higher than that in the amniotic f l u i d (16.6 ng/mL) (Table 15). The r a t i o of the f e t a l t racheal to plasma drug concentrat ions averaged 3.4 fo r the maternal in fus ion experiments (Table 16). However, the highest diphenhydramine concentra t ion u l t ima te ly achieved in amniotic f l u i d fo l lowing maternal i n f u s i o n was s u b s t a n t i a l l y l e s s than the highest drug concentrat ion in f e t a l plasma (mean r a t i o , 0.4 ± 0 .2 ) . Fol lowing fe ta l i n f u s i o n s , the time required to reach the C m a x in t racheal f l u i d (75 ± 31 min) was s i g n i f i c a n t l y l e s s than that in amniotic f l u i d (129 ± 41 min) (Table 15). However, these T m a x values were not s i g n i f i c a n t l y d i f f e r e n t from the corresponding values obtained from maternal i n f u s i o n s . As in the case of maternal i n f u s i o n s , the C m a x in the t racheal f l u i d (1790.4 ng/mL) was s i g n i f i c a n t l y greater than that in the amniotic f l u i d (139.9 ng/mL) (Table 15). The average fe ta l t racheal to plasma drug concentrat ion r a t i o was 5.4 ± 4.5 (Table 16). The C m a x in the amniotic f l u i d was lower than that in the f e t a l plasma in each experiment (mean r a t i o , 0.3 ± 0 .3 ) . The average e l im ina t ion h a l f - l i f e of diphenhydramine in the t racheal f l u i d obtained a f t e r maternal in fus ion (49 ± 17 min) was not d i f f e r e n t from that obtained a f te r f e ta l in fus ion (56 + 16 min) (Table F i g . 19. Representat ive semi logar i thmic p l o t s of the maternal (o) and f e t a l (•) a r t e r i a l plasma, f e t a l t racheal (A) and amniotic (A) f l u i d diphenhydramine concentrat ion vs. time curves in a pregnant ewe and fetus fo l lowing drug i n f u s i o n to the ewe. 91 5000 A ' A " A N ? 1000 rh A " A ' A c o c V o c o o Q) c *E D l _ T J >, x: c 0) a. Q ^•-•-•-•-•-•^A'A'A^ 100 10. / \T>4 boo-o-°-o' 0.r Infusion 60 120 180 240 300 360 420 480 Time (min) Fig. 20. Representative semi logarithmic plots of the maternal (o) and fetal (•) arterial plasma, fetal tracheal (A) and amniotic (A) fluid diphenhydramine concentration vs. time curves in a pregnant ewe and fetus following drug infusion to the fetus. 92 Table 15. Values fo r the observed maximum drug concentrat ions ( C m a x ) of diphenhydramine and time to reach C m a x ( T m a x ) in t racheal (TF) and amniotic (AF) f l u i d s fo l lowing maternal and fe ta l in fus ions to s t e a d y - s t a t e . Maternal Infusion Fetal Infusion Ewe No. ^max (ng/mL) Tmax (min) r °max (ng/mL) ^max (min) TF AF TF AF TF AF TF AF 121 71.9 6.4 95 105 3303.7 41.9 5 110 125 88.7 11.8 95 135 1066.1 98.6 95 90 130 31.6 . a 95 . a 551.9 179.9 90 135 133 66.3 9.9 90 150 887.8 35.7 75 100 138 100.4 25.2 135 180 2952.6 142.1 90 90 202 396.7 12.8 5 100 3132.4 128.9 95 150 204 123.3 20.8 135 120 732.7 120.7 90 150 480 118.5 29.4 110 5 1695.9 371.7 60 210 * 124.7 . ( 1 1 3 . 9 ) ° 16.6 (8.6) 95 (41) 113 (55) 1790.4* (1161.4) 139.9 (105.6) 75* (31) 129 (41) a. Amniotic f l u i d samples have not been taken. b. Numbers in brackets are ± 1 S .D. S i g n i f i c a n t l y d i f f e r e n t (P<0.05) from the corresponding value in the amniotic f l u i d . 93 Table 16. Average r a t i o s of the f e t a l t racheal (TF) to a r t e r i a l (FA) diphenhydramine concentrat ions and the e l im ina t ion h a l f - l i f e (t^) of the drug obtained in maternal (MA) and f e t a l (FA) plasma, t racheal (TF) and amniotic (AF) f l u i d s a f t e r maternal and f e t a l in fus ions to s t e a d y - s t a t e . Maternal Infusion Fetal Infusion Ewe No. TF /FA S (min) TF/FA h (min) MA TF FA A F a MA TF FA AF 121 2.1 (0.9) 51 27 31 1.5 (1.1) 32 37 69 161 125 3.0 (1.1) 39 47 32 1.7 (1.0) 36 50 62 99 130 1.4 (0.3) 51 67 49 1.3 (0.8) 11 64 122 118 133 1.1 (0.3) 63 36 83 2.6 (0.7) 25 39 42 154 138 1.9 (0.9) 66 35 39 - 11.0 (8.3) 36 44 71 -202 12.1 (6.0) 33 b 108 8.9 (4.9) 178 67 90 131 204 3.4 (2.6) 43 69 108 12.0 (15.3) 74 85 73 128 480 2.4 (1.0) 59 61 54 3.9 (2.2) 34 62 51 67 3.4 (3.6) 51 (12) 49 (17) 63 (32) 5.4 (4.5) 53 (53) 56 (16) 73 (25) 123 (32) a. The t^ in the amniotic f l u i d fo l lowing maternal in fus ion was not determined because amniotic DPHM concentrat ions were too low to c h a r a c t e r i z e the e l im ina t ion phase. b. Numbers in brackets are mean ± 1 S .D . 94 16). The e l im ina t ion h a l f - l i f e of diphenhydramine in the amniotic f l u i d a f t e r maternal in fus ions was not determined because drug concentrat ions in t h i s f l u i d were too low fo r the c h a r a c t e r i z a t i o n of the e l im ina t ion phase. However, the h a l f - l i f e of diphenhydramine in the amniotic f l u i d determined fo l lowing f e t a l in fus ions averaged 123 ± 32 min and was s i g n i f i c a n t l y greater than that in the t racheal f l u i d (56 ± 16 min) (Table 16). 3 . 4 . 1 . 4 . Transplacenta l and Nonplacental Clearances Transplacenta l and nonplacental c learances of diphenhydramine from both mother and fetus were ca lcu la ted based on the to ta l drug concentrat ions (Table 17 and 18) and f ree drug concentrat ions (Table 19 and 20). The c learance values normalized by the maternal or f e t a l body weight are a lso given in parentheses. Based on to ta l drug concent ra t ions , average to ta l body clearances from the mother (CL m m ) and fetus {CLff) were 3426.1 and 472.7 mL/min, r e s p e c t i v e l y (Table 18). Fetal to ta l body c learance of diphenhydramine i s r e l a t i v e l y small (13.8%) as compared with the maternal to ta l body c learance . However, when the CL f f was corrected for f e ta l body weight, the r e s u l t i n g c learance value was -5 f o l d greater than the maternal body weight cor rec ted C L m m (Table 18). When the to ta l body c learance was c a l c u l a t e d by the model-independent method (equation 22 and 23), the values were in very c lose agreement with those r e s u l t i n g from the compartmental a n a l y s i s , with an average c o e f f i c i e n t of v a r i a t i o n of 1.5%. The f e t a l to maternal t ransp lacenta l c learance , C L f m (264.4 ± 138.7 mL/min), was ~3 f o l d greater than the maternal to fe ta l t ransp lacenta l c learance , 95 Table 17. Transplacenta l and nonplacental c learances of diphenhydramine based on to ta l drug concentrat ions in the ewe and fetus fo l lowing maternal and fe ta l in fus ions to s t e a d y - s t a t e . Transplacenta l Clearance Nonplacental Clearance Ewe (mL/min) (mL/min) No. C L m f C L f m C L m o CLfo 121 25.2 ( 7 . 4 ) a 152.8 (44.9) 1840. .2 (27.7) 102. 6 (30.2) 125 32.9 (18.3) 117.9 (65.5) 3072 .6 (43.0) 122. 2 (67.9) 130 68.7 (27.5) 334.4 (133.8) 3564 .6 (37.0) 278. .2 (111.3) 133 124.8 (56.7) 235.3 (107.0) 3146 .8 (35.7) 227. ,8 (103.6) 138 125.4 (48.2) 266.3 (102.4) 3311 .8 (53.9) 233. .8 (89.9) 202 62.2 (41.5) 304.5 (203.0) 4334 .4 (48.7) 215, .2 (143.5) 204 109.5 (43.8) 549.4 (219.8) 2781 .9 (44.4) 339. .0 (135.6) 480 110.3 (84.9) 154.3 (118.7) 4697 .7 (55.3) 148 .0 (113.9) 82.4 ; t 40.5 b 264.4 ± 138.7 C 3343 .8 ± 890.7 208 .4 ± 8 0 . 4 C (41.0 : ± 24.1) (124.4 ± 6 0 . 9 ) c (43.2 ± 9.5) (99.5 ± 3 6 . 8 ) c a. Numbers in brackets are the c learances (mL/min/kg) normalized to maternal ( C L m o ) or f e ta l ( C L f m , C L m f and C L f o ) body weight. b. Values are given as mean + 1 S .D. c . S i g n i f i c a n t l y d i f f e r e n t from maternal value (p<0.05). 96 Table 18. Total body c learances of diphenhydramine and percent c o n t r i b u t i o n of nonplacental c learances to to ta l drug e l i m i n a t i o n in the ewe and fetus based on to ta l drug concent ra t ion . Ewe No. Total Body Clearance (mL/min) Nonplacental (' Cont r ibut ion %) CL O Lmm ^ f f Maternal Fetal 121 1865.4 (28 .1 ) a 255.4 (75.1) 98.7 40.2 125 3105.5 (43.5) 240.1 (133.9) 98.9 50.9 130 3633.3 (37.7) 612.6 (245.0) 98.1 45.4 133 3271.6 (37.1) 463.1 (210.5) 96.2 49.2 138 3437.2 (56.0) 500.1 (192.4) 97.6 46.8 202 4396.6 (49.4) 519.7 (346.5) 98.6 41.4 204 2891.4 (46.1) 888.4 (355.4) 96.2 38.2 480 4808.0 (56.6) 302.3 (232.5) 97.7 49.0 3426.1 ± 9 0 5 . 8 ° 472.7 + 215 .7 C 97.8 ± 1.1 45.1 ± 4 . 7 C (44.3 ± 9.8) (223.9 ± 9 5 . 8 ) c a. Numbers in brackets are the c learances (mL/min/kg) normalized to maternal (CL m m ) or fe ta l (CLff ) body weight. b. Values are given as mean + 1 S .D. c . S i g n i f i c a n t l y d i f f e r e n t from maternal value (p<0.05). 9 7 Table 19. Transplacenta l and nonplacental c learances of diphenhydramine based on f ree drug concentrat ions in the ewe and fetus fo l lowing maternal and f e t a l in fus ions to s t e a d y - s t a t e . Transplacenta l Clearance Nonplacental Clearance (mL/min) (mL/min) Ewe No. C L m f C L f m C L mo C L f o 121 527. ,7 (155.2) 930. 2 (273.6) 38325.2 (577.2) 617. 1 (181.5) 125 298. ,7 (165.9) 679. ,4 (377.4) 27976.9 (391.8) 708. 6 (393.7) 130 956. ,1 (382.4) 1092. ,7 (437.1) 49308.9 (511.5) 941. 0 (376.4) 133 1428. .6 (649.4) 1059. ,9 (481.8) 36130.0 (409.6) 1025. ,4 (466.1) 138 733. .2 (282.0) 796. .6 (306.4) 19735.8 (321.4) 706. ,0 (271.5) 202 398, .6 (265.7) 832, .9 (555.3) 27611.7 (309.9) 595. .0 (396.7) 204 567, .3 (226.9) 1377, .1 (550.8) 14405.9 (229.8) 834, .0 (333.6) 480 377 .7 (290.5) 593 .1 (456.2) 16037.7 (188.7) 556. .7 (428.2) 661 .0 ± 375.8 920 .2 ± 252.4 28691.5 ± 12067.0 750 .0 ± 170.0 (302.3 ± 158.0) (429.8 ± 104.4) (367.5 ± 132.8) (356.0 ± 91.8) a. Numbers in brackets are the c learances normalized to maternal or f e t a l body weight (mL/min/kg). b. Values are given as mean + 1 S .D . 98 Table 20. Total body c learances of diphenhydramine and percent con t r ibu t ion of the nonplacental c learances to to ta l drug e l im ina t ion in the ewe and fetus based on f ree drug concent ra t ions . Total Body Clearance Nonplacental Contr ibut ion (mL/min) (%) Ewe No. CL "''-mm CLf f Maternal Fetal 121 38852.9 ( 5 8 5 . l ) a 1547.3 (455.1) 98.6 39.9 125 28275.6 (396.0) 1388.0 (771.1) 98.9 51.1 130 50265.0 (521.4) 2033.7 (813.5) 98.1 46.3 133 37558.6 (425.8) 2085.3 (947.9) 96.2 49.2 138 20469.0 (333.4) 1502.6 (577.9) 96.4 47.0 202 28010.3 (314.4) 1427.9 (951.9) 98.6 41.7 204 14973.2 (238.8) 2211.1 (884.4) 96.2 37.7 480 16415.4 (193.1) 1149.8 (884.5) 97.7 48.4 29352.5 ± 1 2 2 5 2 . 9 ° 1668.2 ± 387.2 97.6 ± 1.2 45.2 ± 4.8 (376.0 ± 134.0) (785.8 ± 180.0) a. Numbers in brackets are the c learances normalized to maternal or f e t a l body weight (mL/min/kg). 6. Values are given as mean + 1 S .D. 99 C L m f (82.4 ± 40.5 mL/min) (Table 17). Average c o n t r i b u t i o n of the p lacenta l c learance of the drug accounted fo r 2.2% of the to ta l drug c learance in the mother ( C L m f / C L m m ) , whereas in the f e t u s , the average p lacenta l c learance accounted fo r 54.9% of the to ta l c learance from the fetus ( C L f m / C L f f ) (Table 18). In one animal preparat ion (ewe no. 130), the p lacenta l ex t rac t ion r a t i o (ER) of diphenhydramine was c a l c u l a t e d by the equat ion: Placental ER = (FA - Um v ) / (FA) (eq.27) where FA and Umv are the s teady-s ta te fe ta l femoral a r t e r i a l and umbi l ica l venous drug concentrat ions a f te r f e t a l i n f u s i o n , r e s p e c t i v e l y . The ER of diphenhydramine across the placenta on the f e t a l s ide was c a l c u l a t e d to be 0.64. Separate ly , the ex t rac t ion r a t i o of the drug by the fe tus was determined in the same animal a f t e r maternal drug i n f u s i o n : Fetal ER = (Umy - FA)/(Um v) (eq. 28) The fe ta l ex t rac t ion r a t i o of diphenhydramine c a l c u l a t e d by t h i s equation was 0.33. Absolute amounts of diphenhydramine e l iminated from the oo oo maternal compartment ( A U C ^ Q * C L M O ) and fe ta l compartment (AUCp 0 -CL f 0 ) were c a l c u l a t e d fo r each animal and are shown in Table 21. Fol lowing maternal i n f u s i o n , the amount of diphenhydramine e l iminated from the pregnant ewe and fetus averaged 78.6 ± 4.3 and 1.2 ± 0.6 mg, r e s p e c t i v e l y . Whereas, fo l lowing fe ta l i n f u s i o n , the amount of the drug e l iminated from the maternal and f e t a l compartments averaged 14.7 + 6.2 100 Table 21. Area under the plasma diphenhydramine concentrat ion vs. time GO 00 curves in the ewe (AUC^ 0) and fetus (AUCp 0) and amount of the drug that was e l iminated from the ewe and fetus via the nonplacental pathway a f t e r maternal and f e t a l i n f u s i o n s . Ewe No. A U C M o (Mg.h/L) A U C W C L Mo L L mo A U C F o (mg) (/ ig-h/L) A U C F ^ C L f 0 (mg) A U C F o . C L f o . 1 0 0 / ( A U C M o ' C L m o + A U C F ; - C L f 0 ) Maternal Infusion 121 698.9 77.2 81.4 0.5 0.6 125 416.3 76.7 63.6 0.5 0.6 130 359.2 76.8 57.6 1.0 1.2 133 398.6 75.3 145.8 2.0 2.6 138 445.4 88.5 121.4 1.7 1.9 202 310.2 80.7 58.8 0.8 0.9 204 455.3 76.0 85.0 1.7 2.2 480 276.5 77.9 125.4 1.1 1.4 420.1 78.6 92.4 1.2 1.4 (129.0) (4.3) (34.1) (0.6) (0.8) Fetal Infusion 121 128.2 14.2 1456.9 9.0 38.8 125 57.8 10.7 1562.4 11.5 51.8 130 47.6 10.2 1006.5 16.8 62.3 133 55.5 10.5 681.7 9.3 47.1 138 89.7 17.8 1063.3 14.9 45.6 202 109.7 28.5 926.0 12.0 29.5 204 86.8 14.5 403.9 8.2 36.2 480 38.6 10.9 1179.0 10.5 49.0 76.7 14.7 1035.0 11.5 45.0 (31.9) (6.2) (380.5) (3.0) (10.2) Numbers in brackets are ± 1 S .D. 101 and 11.5 ± 3.0 mg, r e s p e c t i v e l y . The mean percentage of diphenhydramine administered to the ewe that i s e l iminated by the fetus via the nonplacental pathway, A U C F o ' C L f o - 1 0 0 / ( A U C M Q - C L m o + A U C F Q - C L f o ) , averaged 1.4 ± 0.8%. However, in the case of f e t a l drug i n f u s i o n , the percentage of the dose administered to the fetus that i s e l iminated from the fetus via the nonplacental pathway averaged 45.0 ± 10.2%. 3.4.2 Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep 3 . 4 . 2 . 1 . Maternal Diphenhydramine Infusion Maternal i . v . in fus ions of diphenhydramine were given to 8 ewes at ges ta t iona l age ranging from 127-136 (mean, 131 ± 3) days (Table 11). Diphenhydramine concentrat ions in the ewe and fetus averaged 212.1 ± 67.8 (range, 140.3-360.3) and 36.3 + 14.4 (range, 18.3-56.0) ng/mL, r e s p e c t i v e l y (Table 12). In the contro l p e r i o d , maternal a r t e r i a l pH, Pco 2 and Po 2 averaged 7.499 ± 0.038, 34.1 ± 2.8 mm Hg and 114.2 ± 14.8 mm Hg, r e s p e c t i v e l y (Table 22). The fe ta l control values were 7.388 ± 0.037, 44.6 ± 3 . 3 mm Hg and 19.9 ± 3.9 mm Hg, r e s p e c t i v e l y (Table 23). There was a s i g n i f i c a n t r i s e in f e ta l Pco 2 during the in fus ion and decreases in 0 2 content and hematocrit a f te r the in fus ion (Table 23). Control f e t a l a r t e r i a l pressure and heart rate averaged 47.4 ± 3.0 mm Hg and 145.5 ± 18.5 beats /min , r e s p e c t i v e l y (Table 24). There were no changes in e i t h e r v a r i a b l e during or a f t e r the in fus ion p e r i o d . 102 Table 22. Maternal a r t e r i a l pH, P c o 2 , P o 2 , 0 2 content and hematocrit be fore , during and a f t e r diphenhydramine i n f u s i o n to the ewe and fetus (n = 6 ) . Time pH Pco 2 Po 2 O o Content Hematocrit (min) (mm Hg) (mm Hg) (%) (%) Maternal Infusion -5 7.499 + 0.038 34, .1 + 2.8 114.2 + 14.8 13, .7 + 0 . 8 a 29. 3 + 3. 4 5 7.483 + 0.024 35, .3 + 1.6 115.1 + 15.9 13, .6 ± 0.7 28. 7 + 3. 7 30 7.487 + 0.034 33, .7 + 2.7 117.9 ± 19.2 13 .2 + 1.1 28. 6 + 1. 7 60 7.503 + 0.034 32, .0 + 2.7 125.5 ± 31.5 13, .1 + 1.4 29. 9 + 2. 4 90 7.476 + 0.027 35 .5 + 4.1 118.1 ± 30.0 12 .3 + 0.9 28. 4 + 3. 3 p60 7.484 + 0.023 32 .5 + 2.2 123.4 ± 13.1 12 .8 + 0.5 28. 3 + 2. 8 Fetal Infusion -5 7.473 + 0.033 33 .7 ± 4.4 117.4 ± 5.4 13 .0 + 1 .7 6 27. .7 ± 3. .7 5 7.487 + 0.053 34 .0 + 4.0 113.7 ± 5.5 12 .5 + 1.6 26. .7 + 3. ,5 30 7.477 + 0.026 33 .8 + 4.0 123.6 ± 9.3 12 .2 + 1.4 28, .0 + 3. ,4 60 7.477 + 0.025 33 .3 + 3.5 119.2 ± 17.3 12 .6 + 1.6 27, .8 + 3. .2 90 7.488 + 0.043 33 .0 + 3.6 120.7 ± 8.7 12 .6 + 1.6 28, .2 ± 3. ,4 p60 7.491 + 0.066 33 .8 + 6.1 127.3 ± 12.2 12 .7 ± 1.5 28 .1 + 3. .6 a . n = 3 fo r 0 2 content measurements in the maternal i n f u s i o n study. b. n = 5 f o r 0 2 content measurements in the f e t a l i n f u s i o n study. 103 Table 23. Fetal a r t e r i a l pH, P c o 2 , P o 2 , 0 2 content and hematocrit be fore , dur ing and a f te r diphenhydramine i n f u s i o n to the ewe and fe tus (n = 8 ) . Time pH Pco 2 Po 2 0 2 Content Hematocrit (min) (mm Rg) (mm Hg) (%) (%) Maternal Infusion -5 7.388 ± 0.037 44.6 + 3.3 19.9 ± 3.9 7.2 + 1.6 a 36.4 + 3.1 5 7.367 ± 0.030 47.4 + * 3.8 17.8 + 5.0 6.4 + 1.7 36.8 + 3.2 30 7.371 ± 0.034 45.9 + 2.4 19.4 + 4.0 7.3 + 1.2 35.1 + 3.3 60 7.358 ± 0.069 45.7 + 3.1 18.6 + 3.9 6.8 + 1.3 35.9 + 3.1 90 7.381 ± 0.035 48.1 + 2.9 18.8 + 4.8 6.1 + 1.7 35.5 + 3.0 p60 7.391 ± 0.035 46.4 + 3.2 19.3 ± 4.7 5.7 + 1.0 34.5 + 2.8 Fetal Infusion -5 7.396 ± 0.074 45.8 + 5.5 19.7 + 5 . 6 ° 7.5 + 2.5 37.1 + 3.5 5 7.353 ± 0.045* 47.3 + 4.3 16.4 + 5.2* 5.2 + 2 .1 * 37.6 + 3.9 30 7.360 ± 0.047* 44.9 + 3.6 18.2 + 5.6 6.0 + 2.5* 36.6 + 3.2 60 7.358 ± 0.041* 44.1 + 5.0 18.8 + 5.5 5.8 + 2.0* 36.4 + 2.9* 90 7.380 ± 0.055 44.1 + 2.6 20.0 + 6.2 6.5 + 2.3 36.3 + 1.7* p60 7.375 ± 0.041 42.9 + 3.7* 21.1 ± 6.4 7.0 ± 2.2 34.8 + 2.4* a. n = 4 f o r 0 2 content measurements in the maternal i n f u s i o n study. 6. n = 6 f o r Po 2 measurements in the f e t a l in fus ion study. *p<0.05 104 Table 24. E f f e c t s of diphenhydramine on the fe ta l heart ra te and a r t e r i a l pressure c a l c u l a t e d over 30 min i n t e r v a l s before , dur ing and a f t e r drug in fus ions to the ewe and fe tus (n = 8 ) . Time In terva ls Maternal Infusion Fetal Infusion Fetal heart rate (beat/min) -60 ~ -31 min 144 + 17 156 ± 28 -30 - -1 min 147 + 20 152 ± 20 1 ~ 30 min 146 + 14 182 ± 34* 31 ~ 60 min 144 + 17 160 ± 20 61 - 90 min 145 ± 14 159 ± 23 91 - 120 min 144 + 17 155 ± 20 121 ~ 150 min 143 + 17 149 ± 14 Fetal a r t e r i a l pressure (mm Hq) -60 ~ -31 min 46.5 + 3.2 51.2 ± 6.5 -30 ~ -1 min 48.2 + 2.8 49.9 ± 7.4 1 ~ 30 min 46.9 + 4.5 51.2 ± 7.9 31 - 60 min 46.0 + 5.4 50.9 ± 9.1 61 ~ 90 min 46.8 + 4.8 51.6 ± 7.9 91 ~ 120 min 46.3 + 4.3 50.8 ± 7.6 121 ~ 150 min 45.9 + 5.1 48.6 ± 9.9 *P<0.05 105 In the contro l p e r i o d , a l l fetuses exh ib i ted regular a l t e r a t i o n s between low and high vol tage e l e c t r o c o r t i c a l a c t i v i t y (Table 25, F i g . 21-22). There were a lso b r i e f per iods of an intermediate vol tage p a t t e r n , usua l ly occurr ing in the t r a n s i t i o n s between low and high vol tage pa t te rns . The percentage of time spent in low, high and intermediate vol tage ECoG patterns averaged 55 ± 7, 35 ± 9 and 10 ± 6, r e s p e c t i v e l y (Table 25). Fetal breathing a c t i v i t y occurred fo r 42 ± 9% of the time and t h i s was l a r g e l y associated with low vol tage ECoG a c t i v i t y , as were REM (Table 26). During the in fus ion p e r i o d , there was a s i g n i f i c a n t d e c l i n e in the percentage of low vol tage ECoG pattern (55 ± 7 to 46 ± 12%) (Table 25), the percentage of low vol tage ECoG a c t i v i t y conta in ing REM (80 ± 11 to 55 ± 24%) (Table 27), in the o v e r a l l inc idence of f e t a l breathing (42 ± 9 to 21 ± 15%) (Table 26) and in the amount of breathing during low voltage ECoG a c t i v i t y (67 ± 13 to 36 ± 28%) (Table 26). There were no changes in the average durat ions of low and high vol tage ECoG episodes (Table 28). A f t e r cessa t ion of the i n f u s i o n , a l l the monitored behavioural and breathing v a r i a b l e s returned to t h e i r contro l ranges. A l l ewes were standing during and a f t e r the per iod of drug admin is t ra t ion . There were no d iscernab le a l t e r a t i o n s in maternal behaviour; in p a r t i c u l a r there was no evidence of drowsiness. 3 . 4 . 2 . 2 . Fetal Diphenhydramine Infusion Fetal i . v . in fus ions of diphenhydramine were c a r r i e d out in 8 sheep at gesta t iona l ages ranging from 126-136 (mean, 132 ± 4) days (Table 11). Maternal and f e t a l s teady-s ta te diphenhydramine 106 Table 25. Overa l l inc idence of low, high and intermediate vol tage ECoG episodes in the fetus p r i o r t o , dur ing and a f t e r i n f u s i o n of diphenhydramine to the ewe and fetus (n = 8 ) . % e l e c t r o c o r t i c a l a c t i v i t y Drug Infusion Low High Intermediate Maternal Infusion P r i o r to Infusion 55 + 7 35 + 9 10 + 6 During Infusion 46 + 12* 41 + 13 13 + 14 A f t e r Infusion 56 + 9 39 + 11 5 + 3 Fetal Infusion P r i o r to Infusion 51 ± 10 41 + 10 8 + 4 During Infusion 26 + 20 28 + 21 46 ± 31* A f t e r Infusion 50 + 12 34 + 6 17 + 15 *p<0.05 HIGH XX) LOW i ^ ^ ^ M ^ ^ INTBWIEDIATE High speed chart recordings of h igh , low and intermediate vol tage ECoG patterns in a f e t a l lamb. 108 TP 2 5 r (mmHej) o>-k. Control Period 4^ 5 min ECoG 100(A/C E O G iocw [ | » , i In ^ | I # ^ M * 1 B. Fetal Infusion Period TP 25r (mmHg) o L ECoG 10CW [ EOG 1CXW [ i .•j Uli. r « n . l « | . > te|4i Fig. 22. Representative examples of the recording of breathing, ECoG and electro-oculographic (EoG) a c t i v i t y in the fetal lamb during the control period (A) and during fetal infusion of diphenhydramine (B). TP, tracheal pressure. 109 Table 26. Percentage of f e t a l breathing in low, high and intermediate vol tage ECoG p r i o r t o , during and a f t e r i n f u s i o n of diphenhydramine to the ewe and fetus (n = 8 ) . % Fetal Breathing in ECoG Episodes Total Drug Infusion Breathing (%) Low High Intermediate Maternal Infusion P r i o r to Infusion 67 + 13 5 + 4 27 + 23 42 + 9 During Infusion 36 + * 28 5 + 8 32 + 46 21 + •k 15 A f t e r Infusion 59 + 25 3 + 4 21 + 34 37 + 15 Fetal Infusion P r i o r to Infusion 70 + 22 4 + 7 20 + 18 44 + 12 During Infusion 54 + 37 11 + 10 51 + 26* 47 + 22 A f t e r Infusion 53 + 24 8 + 8 16 + 15 34 + 13 *p<0.05 110 Table 27. Percentage of f e t a l e l e c t r o - o c u l a r a c t i v i t y (EoG) in low, high and intermediate vol tage ECoG p r i o r t o , dur ing and a f te r i n f u s i o n of diphenhydramine to the ewe and f e t u s . % Fetal EOG spent in ECoG Episodes Total EOG Drug Infusion (%) Low High Intermediate Maternal Infusion (n - 5) P r i o r to Infusion 80 ± 11 0 21 + 19 47 + 10 During Infusion 55 ± 24 0 34 + 33 35 + 13 A f t e r Infusion 86 ± 6 5 + 7 5 ± 7 48 + 12 Fetal Infusion (n = 3) P r i o r to Infusion 84 ± 9 2 + 3 8 + 8 47 + 1 During Infusion 85 ± 13 5 + 9 63 + 21* 53 + 26 A f t e r Infusion 87 ± 2 1 + 1 9 + 12 49 + 8 *p<0.05 I l l Table 28. Duration of low, high and intermediate vol tage ECoG episodes in the fetus p r i o r t o , during and a f t e r in fus ion of diphenhydramine to the ewe and fetus (n = 8 ) . Drug Infusion Mean Duration of ECoG Episodes (min) Low High Intermediate Maternal Infusion P r i o r to i n f u s i o n During Infusion A f t e r Infusion 16.4 ± 2.3 13.7 ± 5.0 20.9 ± 4.5 11.6 ± 3.0 10.8 ± 3.1 13.9 ± 3.7 5.8 ± 3.6 4.6 ± 2.1 6.4 ± 5.6 Fetal Infusion P r i o r to Infusion 15.0 + 4.0 13.2 + 3.8 5.9 + 1.5 During Infusion 8.8 ± 3.8 10.8 + 7.7 7.9 + 5.6 A f t e r Infusion 15.4 + 4.9 11.1 + 2.9 9.1 + 5.2 *p<0.05 112 concentrat ions averaged 31.1 ± 11.6 (range, 17.9-53.9) and 447.6 ± 185.2 (range, 192.1-697.9) ng/mL, r e s p e c t i v e l y (Table 12). Fetal a r t e r i a l Po 2 f e l l from 19.7 ± 5.6 to 16.4 ± 5.2 mm Hg in the f i r s t 5 min of the in fus ion and recovered t h e r e a f t e r . There was a d e c l i n e in both pH and 0 2 content , which was maintained fo r the f i r s t 60 min of drug admin is t ra t ion (Table 23). There was a lso a decrease in hematocrit fo l lowing drug admin is t ra t ion . Fetal Pco 2 was unchanged, except fo r a s l i g h t f a l l at the end of the experiment. Maternal blood gas v a r i a b l e s in the contro l per iod averaged 117.4 ± 5.4 mm Hg, 33.7 ± 4.4 mm Hg and 7.473 ± 0.033 fo r P o 2 , Pco 2 and pH, r e s p e c t i v e l y (Table 22). There were no changes during or a f t e r the i n f u s i o n p e r i o d . Fetal a r t e r i a l pressure and heart rate are given in Table 24. There was a s i g n i f i c a n t increase in heart rate during the f i r s t 30 min of the i n f u s i o n p e r i o d , from 154.0 ± 24.0 to 182.0 ± 34.0 beats /min , with a return to contro l l e v e l s t h e r e a f t e r . A r t e r i a l pressure was not s i g n i f i c a n t l y changed. Fetal ECoG, e l e c t r o - o c u l a r and breathing v a r i a b l e s are i l l u s t r a t e d in Table 25-27. In the contro l p e r i o d , the percentages of the time spent in low, high and intermediate voltage patterns were s i m i l a r to the values obtained before the maternal diphenhydramine i n f u s i o n s . During drug in fus ion to the f e t u s , there was a s i g n i f i c a n t f a l l in the amount of low voltage ECoG pattern (51 ± 10 to 26 ± 20%) and a s i m i l a r tendency f o r the amount of high voltage a c t i v i t y to d e c l i n e (41 ± 10 to 28 ± 21%), although t h i s change was not s t a t i s t i c a l l y 113 s i g n i f i c a n t (Table 25). Conversely , there was a marked r i s e in the incidence of the intermediate vol tage pa t te rn , from 8 ± 4 to 47 ± 31%, and a lso a la rge r i s e in the occurrence of REM during intermediate vol tage ECoG a c t i v i t y (8 ± 8 to 63 ± 22%) (Table 25 and 27). The increase in the amount of t h i s intermediate vol tage pat tern resu l ted in a d i s r u p t i o n in the normal c y c l i n g between high and low vol tage episodes ( F i g . 22). Th is was r e f l e c t e d in a marked f a l l in the average durat ion of low vol tage episodes during the in fus ion p e r i o d , from 15.0 ± 4.0 to 8.8 ± 3.8 min (Table 28). The overa l l incidence of f e t a l breathing movements in the contro l per iod averaged 44 ± 12% and was not a l te red during the i n f u s i o n . However, there was a s i g n i f i c a n t increase in the amount of f e t a l breathing during intermediate ECoG a c t i v i t y and t h i s was accompanied by a tendency fo r a f a l l in the incidence of breathing during the low vol tage ECoG pattern (Table 26). A l s o , in seven of the e ight experiments, there was an appearance of large amplitude f e t a l breathing movements wi th in 1 min of drug admin is t ra t ion , which l as ted f o r 5 to 20 min ( F i g . 23). In these seven f e t u s e s , a r t e r i a l Po 2 had f a l l e n by 3.9 ± 0.9 mm Hg by 5 min a f t e r the s t a r t of diphenhydramine i n f u s i o n . In c o n t r a s t , in the fetus in which large amplitude breathing a c t i v i t y d id not occur , Po 2 rose by 1 mm Hg at 5 min. 3 . 4 . 2 . 3 . Control Sa l ine Infusion The mean values fo r f e t a l blood gases, heart r a t e , a r t e r i a l p ressure , behavioural and breathing parameters in these four experiments were not d i f f e r e n t from those found before diphenhydramine i n f u s i o n . Furthermore, there were no changes in any of these v a r i a b l e s during 114 ARTEtlAl nu&sutE HEART BATE | TtACMEU PRESSURE (mmHg) FTO mm mm ECOG 100. J EOG WO, •ft-*. , I 1 I "fi'1" ft"1'*' F i g . 23. Examples of the large amplitude breathing a c t i v i t y present in the i n i t i a l phases of diphenhydramine i n f u s i o n s to the f e t u s . The arrow ind ica tes the time at which the i n f u s i o n was s t a r t e d . The drug was given by simultaneous i . v . bolus i n j e c t i o n (5 mg) and i n f u s i o n (0.17 mg/min f o r 90 min) to s t e a d y - s t a t e . 115 i n f u s i o n of 0.9% s a l i n e to the fetus or ewe. 3 .5 . R e c i r c u l a t i o n of Diphenhydramine from Amniotic F l u i d to the Ewe and Fetus: Intra-Amniot ic bolus In ject ion Studies A 50 mg dose of diphenhydramine hydrochlor ide was in jec ted as a bolus in to the amniotic c a v i t y in 5 t ime-dated pregnant sheep. C h a r a c t e r i s t i c s of ind iv idua l ewes employed in the study are summarized in Table 29. The gesta t iona l age at the time of the experiment ranged from 122-137 days (mean, 129 ± 7 days) and the maternal body weight ranged from 63-85 kg (mean, 76 ± 8 kg). Control values fo r the f e t a l a r t e r i a l blood gases, P 0 2 , PCO2 and 0 2 content averaged 22.3 ± 7.6 mm Hg, 47.3 ± 9.3 mm Hg and 6.8 ± 2 . 5 vol%. Average contro l f e t a l a r t e r i a l blood pH and hematocrit were 7.391 ± 0.056 and 31.2 ± 3.6 vol%. A l l these v a r i a b l e s are wi th in normal range in near-term f e t a l lamb. Drug admin is t ra t ion in to the amniotic c a v i t y resu l ted in no s i g n i f i c a n t change in these v a r i a b l e s when examined at 5, 10, 30 and 60 min a f te r drug i n j e c t i o n . F i g . 24 shows average drug concentrat ion vs. time curves fo r maternal and fe ta l a r t e r i a l plasma, f e ta l t racheal and amniotic f l u i d s obtained fo l lowing drug admin is t ra t ion into the amniotic c a v i t y in 5 pregnant sheep. The highest drug concentrat ion in the amniotic f l u i d was observed at the f i r s t sampling time (5 min) in a l l the animals, with a mean C m a x of 82,467.5 ± 80,918.0 ng/mL (Table 30). The observed mean C m a x in f e t a l plasma (228.0 ± 101.5 ng/mL) was s i g n i f i c a n t l y higher than that in maternal plasma (43.5 ± 15.6 ng/mL) (Table 30). A l s o , the drug 116 Table 29. Physical c h a r a c t e r i s t i c s of ind iv idua l pregnant sheep used in diphenhydramine in t ra -amnio t i c bolus i n j e c t i o n study. Ewe No. Breed Maternal body Weight (kg) Gestat ional (days) Age Fetal Body Weight in uteror (kg) Number of Fetuses Surgery Experiment 204 Dorset 62.7 122 137 3.2 1 208 Su f fo lk 80.9 117 126 1.2 3 279 Dorset 74.5 116 124 1.6 2 283 Dorset 79.1 128 136 3.1 1 287 Dorset 84.5 118 122 1.4 2 76.3 (8.4) 120 (5) 129 (7) 2.1 (1.0) a. Ca lcu la ted by the equat ion: log ( fe ta l body weight) = log (b i r th weight) - (0.0153 x number of days between experiment and d e l i v e r y ) . 6. Numbers in brackets are ± 1 S .D . 117 F i g . 24. Average diphenhydramine concentrat ion vs. time curves f o r maternal a r t e r i a l (O) and f e t a l a r t e r i a l (•) plasma, f e t a l t racheal (A) and amniotic (A) f l u i d s fo l lowing admin is t ra t ion of 50 mg diphenhydramine hydrochlor ide in to the amniotic c a v i t y in to 5 pregnant sheep. 118 Table 30. Values fo r the observed maximum drug concentrat ions ( C m a x ) and time to reach C m a x ( T m a x ) in maternal (MA) and f e t a l (FA) a r t e r i a l plasma and f e t a l t racheal (TF) and amniotic (AF) f l u i d s fo l lowing i n j e c t i o n of 50 mg diphenhydramine hydrochlor ide into the amniotic c a v i t y . Ewe No. °max (ng/mL) Tmax (min) MA FA TR AMN MA FA TR AMN 204 29.9 188.8 769.9 213700.9 90 90 300 5 208 38.6 129.8 3971.4 85991.2 90 120 10 5 279 66.3 150.3 1760.5 81449.8 45 90 45 5 283 52.1 355.5 5952.4 13137.3 45 90 10 5 287 30.4 315.7 1475.4 180585.4 90 90 45 5 43.5 (15.6) 228.0 (101.5) 2785.9 (2136.9) 82467.5 (80918.0) 72 (25) 96 (13) 82 (123) 5 (0) Values in brackets are ± 1 S .D. 119 concentra t ion in f e ta l a r t e r i a l plasma was c o n s i s t e n t l y and s i g n i f i c a n t l y higher than the corresponding value in maternal a r t e r i a l plasma throughout the sampling t ime, with the mean maternal to f e ta l plasma drug concentrat ion r a t i o of 0.23 ± 0.10 ( F i g . 24 and Table 31). The C m a x in t racheal f l u i d (2,785.9 + 2,136.9 ng/mL) was s i g n i f i c a n t l y greater than that e i t h e r in the maternal or in the f e t a l plasma. The time taken to reach the C m a x ( T m a x ) averaged 1.2 ± 0.4 hr in maternal plasma and 1.6 ± 0.2 hr in f e ta l plasma (Table 30). The average e l i m i n a t i o n h a l f - l i f e of the drug in maternal plasma (1.7 ± 0.5 hr) was s i g n i f i c a n t l y shor ter than that in f e ta l plasma (7.7 + 1.2 hr) (Table 31). The h a l f - l i f e in f e t a l t racheal and amniotic f l u i d s , 11.0 ± 4.5 hr and 10.8 ± 3.8 hr , r e s p e c t i v e l y , was s i g n i f i c a n t l y longer than that in e i t h e r the f e t a l or the maternal plasma. In one animal (ewe no. 287), drug concentrat ions were measured in umbi l ica l venous plasma as well as maternal and fe ta l a r t e r i a l plasma, f e t a l t racheal and amniotic f l u i d , and the drug concentrat ion- t ime curves obtained in t h i s animal are shown in F i g . 25. As seen in the other animals, drug concentrat ions in f e ta l plasma were s i g n i f i c a n t l y higher than in maternal plasma throughout the sampling t ime. Furthermore, the drug concentrat ion in umbi l ica l venous plasma was higher than in f e ta l a r t e r i a l plasma f o r most of the sampling per iod and the average d i f f e r e n c e in concentrat ion was s t a t i s t i c a l l y s i g n i f i c a n t (Fig 25). Total area under the drug concentra t ion- t ime curves f o r each f l u i d c a l c u l a t e d by the equations 4 and 5 and the amount of the drug e l iminated from the maternal and fe ta l compartments are shown in Table 32. The average AUCpQ i s -10 times greater than the CO average A U C M o . However, the amount of the drug e l iminated from the 120 Table 31. Average r a t i o s of the maternal a r t e r i a l (MA), umbi l ica l venous (UV) and f e t a l t racheal (TF) to f e t a l a r t e r i a l (FA) drug concentrat ions and the e l im ina t ion h a l f - l i f e (t^) of the drug obtained a f te r i n j e c t i o n of 50 mg diphenhydramine hydrochlor ide into the amniotic c a v i t y . Ewe No. Concentrat ion Ratio tk (hr) a MA/FA UV/FA TR/FA MA FA UV TF AF 204 0.16 (0 .09 ) D - 4.31 (4.39) 2.0 8.3 - 17.2 10.2 208 0.33 (0.19) - 45.85 (113.66) 1.6 7.8 - 8.2 9.5 279 0.33 (0.16) - 6.10 (3.15) 1.1 9.3 - 9.7 18.8 283 0.23 (0.24) - 21.47 (30.55) 1.3 6.1 - 10.6 9.3 287 0.11 (0.08) 2.01 (2.31) 4.18 (3.11) 2.3 7.1 7.5 8.1 7.2 0.23 (0.10) - 16.38 (17.99) 1.7 (0.5) 7.7 (1.2) - 10.8 (3.8) 11.0 (4.5) a. t\ i s determined by l i n e a r regress ion a n a l y s i s . b. Values are expressed as the mean (± 1 S . D . ) . 121 0 4 8 12 16 2 0 2 4 2 8 Time (hr) Fig. 25. Representative diphenhydramine concentration vs. time curves for maternal (o) and fetal arterial (•) and umbilical venous plasma (•), fetal tracheal (A) and amniotic (A) fluids following intra-amniotic bolus injection of 50 mg diphenhydramine hydrochloride to a pregnant ewe (ewe no. 287). Inset figure shows the fetal arterial and umbilical venous plasma and fetal tracheal drug concentration vs time curves over the init ial 6 hr period after drug administration. 122 Table 32. Total area under the drug concentrat ion vs. time curves fo r the maternal and f e t a l a r t e r i a l and umbi l ica l venous plasma, f e t a l t racheal and amniotic f l u i d s a f te r i n j e c t i o n of 50 mg diphenhydramine hydrochlor ide into the amniotic c a v i t y . Ewe No. Parameter mean (± S.D. ) 204 208 279 283 287 AUCMp (/zg-hr/L) 120.2 156. 0 191 .4 159 .0 156.9 156.7 (25.2) AUCpp ( /zg c hr/L) 1345.2 1233. 2 1064 .1 1636 .7 2108.6 1477.6 (409.8) (/zg-hr/L) - - - - 2987.6 -( /zg ! hr/L) 10064.8 10570. 5 6977 .3 52233 .7 10807.0 18130.7 (19126.3) (/zg-hr/L) 127180.0 7711. 5 49540 .1 35263 .4 126182.3 87175.5 (42849.1) A U C M o - C L m o (mg) 24.1 31. 3 38 .4 31 .9 31.0 31.4 (5.1) A U C y o - C L m f 0.6 0. 8 1 .0 0 .8 0.8 0.8 (0.1) AUC " - C L f 0 (mg) 16.8 15. 4 13 .3 20 .5 26.4 18.5 (5.1) A U C F o ' C L f m (mg) 21.3 19. 6 16 .9 26 .0 33.5 23.4 (6.5) Mean values (n = 8) fo r the maternal and f e t a l nonplacental c lea rances , C L m o 3343.8 mL/min and C L f 0 208.4 mL/min, and the t ransp lacenta l c learances between the ewe and f e t u s , C L m f 82.4 mL/min and C L f m 264.4 mL/min, were used in the c a l c u l a t i o n (see sec t ion 3 . 4 . 1 . 4 ) . 123 fetus via the nonplacental pathway (18.5 ± 5.1 mg) was s i g n i f i c a n t l y l e s s than that e l iminated from the maternal sheep (31.4 ± 5.1 mg). The sum of the amount of drug e l iminated from both the maternal and fe ta l compartments averaged 49.9 ± 6.4 mg. 3 .6 . Pulmonary Ext rac t ion of Diphenhydramine in Fetal Lamb Fol lowing Maternal or Fetal Infusion to Steady-State Surgery was performed in 16 t ime-dated pregnant sheep in the t h i r d t r imeste r of g e s t a t i o n , out of which, 4 were employed in a drug i n f u s i o n study and the res t (n = 12) were excluded s ince the pulmonary venous catheter mal funct ioned. Maternal or f e t a l diphenhydramine i n f u s i o n experiments were conducted in the 4 animal preparat ions over the ges ta t iona l age ranging from 128-133 days. A f t e r the drug i n f u s i o n experiments, the s u r g i c a l l y prepared fe ta l lambs were subjected to autopsy to conf i rm that the pulmonary a r t e r i a l and venous catheters were proper ly p laced . In one surg ica l p repara t ion , the pulmonary venous catheter t i p was found to be pos i t ioned in the l e f t atrium and the animal was subsequently excluded from the study. F i g . 26 and 27 show representa t ive p lo ts of the f e t a l pulmonary a r t e r i a l , pulmonary venous and c a r o t i d a r t e r i a l drug concentrat ion vs. time p r o f i l e s fo l lowing maternal and fe ta l i n f u s i o n s , r e s p e c t i v e l y . S teady-s ta te drug concentrat ions were c a l c u l a t e d as the mean of the drug concentrat ions in 60, 75 and 90 min samples (Table 33). During maternal i n f u s i o n , s teady-s ta te drug concentrat ion in c a r o t i d a r t e r i a l plasma was higher than in pulmonary a r t e r i a l plasma (60.6 vs. 51.2 ng/mL). Drug 124 F i g . 26. P lo ts of the f e t a l pulmonary a r t e r i a l (PA), pulmonary venous (PV) and c a r o t i d a r t e r i a l (CA) and maternal a r t e r i a l (MA) diphenhydramine concentrat ion vs. time p r o f i l e s fo l lowing maternal drug i n f u s i o n . 125 F i g . 27. Representat ive p l o t s of the f e t a l pulmonary a r t e r i a l (PA), pulmonary venous (PV) and c a r o t i d a r t e r i a l (CA) and maternal a r t e r i a l (MA) diphenhydramine concentra t ion vs. time p r o f i l e s fo l low ing f e t a l drug i n f u s i o n . Table 33. The s teady-s ta te diphenhydramine concentrat ions and l e v e l s in the hour fo l lowing cessat ion of the maternal or f e ta l i n f u s i o n in f e t a l pulmonary a r t e r i a l (PA), pulmonary venous (PV), c a r o t i d a r t e r i a l (CA) b lood, f e t a l t racheal (TF) and amniotic (AF) f l u i d s and maternal a r t e r i a l blood (MA). Ewe Route of Sampling Drug Cone. (ng/mL) Drug Cone. (ng/mL) No. Admin is t ra t ion S i t e at Post Infusion Steady-State 30 min 60 min 228 Infusion PA 726.7 323.3 145.0 Fetal PV 621.2 342.7 153.3 CA 521.0 278.2 127.7 TF 2278.6 1474.1 1414.7 AF - - -MA 31.8 16.6 8.0 148 Infusion PA 470.1 200.0 74.9 Fetal PV 451.0 246.6 77.3 CA 437.0 185.2 71.7 TF 2039.3 993.2 593.3 AF 78.9 74.1 86.9 MA - - -225 Infusion PA 51.2 27.7 13.1 Maternal PV 48.6 23.7 10.4 CA 60.0 28.0 12.9 TF 70.0 53.4 14.9 AF 6.2 10.7 9.3 MA 154.3 36.9 16.9 127 concentrat ion in the pulmonary a r t e r i a l plasma was c o n s i s t e n t l y higher than in the pulmonary venous plasma throughout the experiment ( F i g . 26 and Table 33). In c o n t r a s t , during f e t a l i n f u s i o n s , s teady-s ta te drug concentrat ion in pulmonary a r t e r i a l plasma (mean, 598.4 ng/mL) was higher than in c a r o t i d a r t e r i a l plasma (mean, 479.0 ng/mL) ( F i g . 27 and Table 33) . Diphenhydramine in pulmonary a r t e r i a l plasma was higher than in pulmonary venous plasma during the fe ta l in fus ion per iod (598.4 vs. 536.1 ng/mL). Fol lowing cessat ion of the f e t a l drug i n f u s i o n , however, there was a reversa l of the drug concentrat ion g rad ien t , r e s u l t i n g in higher l e v e l s in pulmonary venous plasma than in pulmonary a r t e r i a l plasma (205.0 vs. 186.1 ng/mL) (Table 33). The pulmonary ex t rac t ion r a t i o of diphenhydramine by the lung c a l c u l a t e d by equation 26 ranged from 0.04 to 0.14 (mean, 0.08 ± 0.06) (Table 34). Fol lowing e i t h e r maternal or f e t a l drug i n f u s i o n , f e t a l t racheal drug concentrat ion was higher than in fe ta l or maternal plasma, whereas amniotic drug concentrat ions were lower than the corresponding concentrat ions in maternal or f e t a l plasma (Table 33). Maternal and fe ta l to ta l body c learances were c a l c u l a t e d by equations 24 and 25 (Table 34). Maternal to ta l body c learance i s more than -10 times greater than f e t a l c learance (4402.1 vs. 357.7 mL/min). However, when normalized by maternal or f e t a l body weight, the CLf f (188.6 mL/min/kg) i s greater than C L m m (53.8 mL/min/kg) (Table 34). 128 Table 34. Fetal pulmonary ex t rac t ion and the to ta l body c learance of diphenhydramine c a l c u l a t e d at s teady-s ta te a f t e r maternal or f e t a l drug admin is t ra t ion . Ewe No Route of Fetal Pulmonary 3 Total Body Clearance Admin is t ra t ion Ext rac t ion (mL/min) Ratio CLmm CLf f 148 Fetal in fus ion 0.04 - 389.0 ( 1 8 5 . 2 ) ° 225 Maternal Infusion 0.05 4402.1 ( 5 3 . 8 ) c 228 Fetal Infusion 0.14 - 326.3 u (191.9)' 0.08 + 0.06 a . c a l c u l a t e d by [ (PA) - (PV) ]x l00 / (PA) . b. Normalized by the maternal body weight (mL/min/kg). c . Normalized by the fe ta l body weight (mL/min/kg). 129 4. DISCUSSION 4 . 1 . Development of a C a p i l l a r y GC/NPD Assay Method Open tubular c a p i l l a r y column GC has been ex tens ive ly used as an a n a l y t i c a l tool in the ana lys is of complex mat r ices , such as environmental samples, f o s s i l f u e l s , food and cosmetics and b i o l o g i c a l mater ia ls (Rooney, 1981). Several g lass c a p i l l a r y GC/NPD methods (Ch ia ro t t i et al., 1983; Barni Comparini et a / . , 1983) have been reported f o r the determination of diphenhydramine in s t ree t drug samples. Although g lass c a p i l l a r y columns are cons iderab ly more iner t than those of metal , they s t i l l possess s i g n i f i c a n t column surface a c t i v i t y lead ing to absorpt ive i n t e r a c t i o n s with the substances being chromatographed. The recent in t roduct ion of i n e r t f u s e d - s i l i c a c a p i l l a r y columns minimizes these problems (Lipsky et al. , 1980), prov id ing reproducib le and h igh ly s e n s i t i v e analyses of a v a r i e t y of drugs and t h e i r metabol i tes in t i s s u e s and b i o l o g i c a l f l u i d s . As a consequence, these methods are more f requent ly used f o r pharmacokinetic s t u d i e s , where small plasma volumes (0.5 mL or l ess ) are encountered. Lutz et al. (1983) reported a GC method f o r diphenhydramine measurement in human serum using a bonded-phase f u s e d - s i l i c a c a p i l l a r y column. Meatheral l and Guay (1984) l a t e r reported a GC/NPD method fo r the determinat ion of diphenhydramine employing d i r e c t sample i n j e c t i o n onto a f u s e d - s i l i c a c a p i l l a r y column. These methods provide good s e n s i t i v i t y and s e l e c t i v i t y , however, they s t i l l required the use of large plasma volumes (1 .0-3.0 mL) and a very small f i n a l sample 130 r e c o n s t i t u t i o n volume (10 /zL). In the present research , a GC/NPD method with improved s e n s i t i v i t y was developed. S p l i t l e s s i n j e c t i o n was employed to f a c i l i t a t e the automation of sample a n a l y s i s . This i n j e c t i o n mode has been found to be useful fo r t race ana lys is of d i l u t e samples without pre -concent ra t ion and fo r samples with components e l u t i n g near the solvent peak (Grob and Grob, 1969a, 1969b, 1974). A purge a c t i v a t i o n time of 1 min was chosen s ince longer purge a c t i v a t i o n times d id not provide increased peak area counts , suggesting no fu r ther t r a n s f e r of sample onto the column. As isothermal column operat ion caused peak broadening, m u l t i - s t e p temperature programming was used to improve peak shape and the r e s o l u t i o n of the drug and in terna l standard. Orphenadrine was chosen as an in terna l standard because of i t s s t r u c t u r a l s i m i l a r i t y to diphenhydramine. Resolut ion of diphenhydramine and orphenadrine i s e x c e l l e n t , as i l l u s t r a t e d in F i g . 9. The developed assay method has been found to show good l i n e a r i t y over the concentrat ion range studied (Table 1). Within-run p r e c i s i o n ( r e p e a t a b i l i t y ) of a representa t ive c a l i b r a t i o n curve showed good r e p r o d u c i b i l i t y with c o e f f i c i e n t s of v a r i a t i o n ranging from 1.5 to 10.0% (Table 1) . The s e n s i t i v i t y l i m i t of t h i s assay method (-40 pg at the detector ) was found to be s u i t a b l e fo r the subsequent pharmacokinetic assessment of plasma diphenhydramine concentrat ion vs. time p r o f i l e s in c h r o n i c a l l y ca the te r i zed maternal and f e t a l sheep. 131 Adsorpt ion of diphenhydramine from the organic s o l v e n t , by g lass s u r f a c e s , was observed to be a major source of drug l o s s during assay development. Solvents of var ious p o l a r i t i e s , inc lud ing heptane, hexane, to luene , benzene and dichloromethane, were examined fo r t h e i r ex t rac t ion e f f i c i e n c y . Dichloromethane was found to be the most e f f i c i e n t solvent fo r s i n g l e - s t e p plasma ex t rac t ion of diphenhydramine, prov id ing low- leve l drug quant i ta t ion without any in te r fe rence from plasma c o n s t i t u e n t s . The add i t ion of t r ie thy lamine in dichloromethane (0.01 M) minimized drug adsorpt ion losses during ex t rac t ion and evaporat ion processes . The formation of an emulsion between plasma and dichloromethane during ex t rac t ion occurred , but was e f f e c t i v e l y broken by p lac ing samples in a f reezer ( - 2 0 ° C ) fo r 5 min p r i o r to c e n t r i f u g a t i o n . Since c h l o r i n a t e d solvents cause a r e v e r s i b l e ni trogen/phosphorus detector s e n s i t i v i t y l o s s , toluene was used instead of dichloromethane fo r the r e c o n s t i t u t i o n of the plasma ext rac ts fo r sample i n j e c t i o n . A s i g n i f i c a n t ca r ry -over of diphenhydramine and orphenadrine from the automatic sampler syr inge needle was observed when toluene was used to wash the s y r i n g e . Among var ious solvents examined, acetone was found to e l iminate the ca r ry -over problem when the automatic sampler performed four wash c y c l e s . The use of automatic l i q u i d sample in t roduc t ion has made i t p o s s i b l e to perform rout ine ana lys is fo r the diphenhydramine pharmacokinetic s t u d i e s . 132 4 .2 . Pharmacokinetics of Diphenhydramine a f t e r Dose Ranging in Nonpregnant Sheep 4 . 2 . 1 . Dose -L inear i t y of Pharmacokinetics of Diphenhydramine in Nonpregnant Sheep D i s t r i b u t i o n of diphenhydramine a f t e r i . v . bolus i n j e c t i o n was rap id in sheep, with a mean d i s t r i b u t i o n h a l f - l i f e of 5-9 min. The average volume of d i s t r i b u t i o n (Vdss) obtained over the dose range studied (2 .8 -5 .5 L/kg) f a r exceeds the to ta l body water in sheep (-0.6 L/kg) (Macfar lane, 1975). Th is suggests that despi te moderately high plasma pro te in binding (77%), diphenhydramine d i s t r i b u t e s ex tens ive ly in to body t i s s u e s . In other animal s p e c i e s , such as ra ts and monkeys, diphenhydramine e x h i b i t s a very rap id d i s t r i b u t i o n into t i s s u e s , with the maximum t i s s u e uptake occurr ing at 1-3 min a f te r i . v . i n j e c t i o n (Drach et al., 1970; Wagner, 1973). In r a t s , t i s s u e drug uptake appears to be saturated over the dose range of 4-16 mg/kg, and the e l im ina t ion h a l f - l i f e tends to decrease with increas ing dose (Wagner, 1973). The decrease in the e l im ina t ion h a l f - l i f e in the ra t could be a r e f l e c t i o n of the t i s s u e sa tura t ion or a decrease in t i s s u e binding of the drug. In the present study, however, the drug dose employed was low (0 .2-2 .9 m9Ag) and there was a trend towards an increase in the mean Vdss (from 2.8 to 5.5 L/kg) and e l im ina t ion h a l f - l i f e (from 34 to 68 min) as the dose was increased . Therefore , i t i s not l i k e l y that t i s s u e satura t ion occurred in the sheep. The extent of plasma prote in binding of the drug d id not change over the drug concentrat ion range, 10-2,000 ng/mL in vitro. Since changes in apparent volume of d i s t r i b u t i o n (Vd) are a funct ion of both changes in plasma and t i s s u e b i n d i n g , i t may be 133 p o s s i b l e that t i s s u e drug uptake or binding was increased with no change in plasma pro te in binding (Gibald i et al., 1978). OO In the nonpregnant sheep, ne i ther dose-normal ized AUC 0 nor the t o t a l body c learance (C/. Tg) was a f fec ted by dose. However, the e l i m i n a t i o n h a l f - l i f e i s dependent upon both C/. T g and the volume of d i s t r i b u t i o n (0 .693 I V C / / C / . J B ) and s ince C/. T g remained unchanged while the volume of d i s t r i b u t i o n increased with increas ing dose, the net e f f e c t was an increase in the e l im ina t ion h a l f - l i f e . The average C/LTg of approximately 5 L /h r /kg i s 1.5 times the l i v e r blood flow (-3.3 L /hr /kg) (Katz and Bergman, 1969) and i s c lose to the to ta l ca rd iac output (-5 L /hr /kg) (Runciman et al., 1984) in sheep. Diphenhydramine i s , t h e r e f o r e , l i k e l y to be c leared by the l i v e r as well as by other t i s s u e s and organs. Diphenhydramine CZ.Tg in sheep i s at l e a s t -3 times greater than that found in humans (Spector et al., 1980; Ber l inger et al., 1982; Meredith et al., 1984). A s i m i l a r more rap id c learance in sheep, as compared with humans, has a lso been reported fo r a number of other drugs, i nc lud ing e t idoca ine (Pedersen et al., 1982), l i d o c a i n e (Bloedow et al., 1980), meperidine (Szeto et al., 1978) and metoclopramide (Riggs et al., 1988a). In the case of diphenhydramine, the high CZ.Tg in sheep i s cons is ten t with a much shorter e l im ina t ion h a l f - l i f e than in humans s ince the volumes of d i s t r i b u t i o n of diphenhydramine in these species are s i m i l a r . In the present study, there was a t rans ien t decrease in Po 2 at 5 min fo l lowing i n j e c t i o n of e i t h e r a 100 or a 200 mg dose. There was no change in 0 2 content and hematocrit over the dose range of 25-100 mg 134 but a f t e r 200 mg there were s i g n i f i c a n t decreases in these parameters. The increase in 0 2 content i s l i k e l y the r e s u l t of the increase in hematocrit which in turn may be mediated by a l t e r a t i o n s in c a p i l l a r y pe rmeab i l i t y . 4 . 2 . 2 . Plasma Prote in Binding in Nonpregnant Sheep The present study demonstrates that the plasma prote in binding of diphenhydramine i s independent of the to ta l drug concentrat ion in the range of 10-2,000 ng/mL in nonpregnant sheep plasma in vitro. Appropr iate volumes of the plasma samples were used fo r drug ana lys is so that drug concentrat ions f e l l wi thin the concentrat ion range of the standard curve . The in vivo determination of prote in binding in a l l the samples c o l l e c t e d from the two animals dosed with a 200 mg dose a lso confirmed that the extent of plasma prote in binding d id not change over the drug concentrat ion range encountered (30-780 ng/mL). Although the f ree f r a c t i o n of diphenhydramine appeared to increase at higher drug concentrat ions (4,000 and 10,000 ng/mL) in vitro, curve f i t t i n g of the binding p lo t was not attempted s ince the present binding study was not designed to comprehensively charac te r i ze the binding parameters. The extent of plasma prote in binding of diphenhydramine in the nonpregnant sheep (77%) seems comparable to that reported in humans (76-98%) (Albert et al., 1975; Spector et al., 1980; Meredith et al., 1984). Diphenhydramine binds to human serum albumin but the degree of binding i s low (Drach et al., 1970). Furthermore, there i s no c o r r e l a t i o n between the extent of prote in binding of the drug and serum albumin concentrat ions in man (Meredith et al., 1984). Diphenhydramine, l i k e 135 other bas ic amine drugs such as l i d o c a i n e and propranolol ( G i l l i s et al., 1985), may bind to a j - a c i d g l y c o p r o t e i n , but we have not been able to measure a j - a c i d g lycopro te in concentrat ions due to lack of commercial ly a v a i l a b l e an t ise ra s p e c i f i c to ovine a j - a c i d g l y c o p r o t e i n . Further i n v e s t i g a t i o n i s required to determine whether diphenhydramine binds to a j - a c i d g lycopro te in in human or sheep plasma. In summary, diphenhydramine showed nonl inear d i s t r i b u t i o n c h a r a c t e r i s t i c s over the dose range of 25-200 mg in the nonpregnant sheep. There were increases in the d i s t r i b u t i o n and e l i m i n a t i o n h a l f -l i v e s and the volume of d i s t r i b u t i o n , with increas ing dose. There was, oo however, a proport ional increase in AUC 0 , with no change in C/ . jg. Plasma pro te in binding of diphenhydramine d id not change over the concentra t ion range observed. The change in the volume of d i s t r i b u t i o n may be due to changes in t i s s u e drug uptake or b ind ing . 136 4.3 Pharmacokinetics of Diphenhydramine in Pregnant Sheep Fol lowing i . v . Bolus Admin is t ra t ion 4 . 3 . 1 . P lacenta l Transfer of Diphenhydramine and the Extent of Fetal Drug Exposure There appears to be no d e t a i l e d publ ished studies of the p lacenta l t r a n s f e r of histamine H j - receptor b lockers invo lv ing s e r i a l blood sampling from the mother and fetus in utero. The present i n v e s t i g a t i o n demonstrates rap id and extensive materna l - fe ta l t r a n s f e r of diphenhydramine in pregnant sheep. The extent of f e t a l exposure to a drug i s more accurate ly r e f l e c t e d by the f e t a l to maternal AUC r a t i o than by a comparison of f e t a l and maternal plasma drug concentrat ions at any t ime, t , a f t e r drug admin is t ra t ion (Levy and Hayton, 1973). The f e t a l to maternal AUC r a t i o of 0.85 obtained fo r diphenhydramine i n d i c a t e s s i g n i f i c a n t f e ta l exposure to the drug a f t e r maternal i . v . a d m i n i s t r a t i o n . Drug t r a n s f e r across the placenta appears to occur by simple d i f f u s i o n . Diphenhydramine has a r e l a t i v e l y low molecular weight (m.w. = 255) and high l i p i d s o l u b i l i t y (pKa = 9 .0 ) , both of which favor p lacenta l t r a n s f e r by t h i s process (Reynolds, 1979). 4 . 3 . 2 . D i s p o s i t i o n of Diphenhydramine in Maternal and Fetal Sheep Maternal plasma diphenhydramine concentrat ion vs. time curves show a biexponent ia l decay; a s i m i l a r pattern of decay has a lso been observed in humans (Albert et al., 1975; Meredith et al., 1984). However, the e l im ina t ion h a l f - l i f e i s shorter in pregnant sheep (0 .7-1.0 hr) than in humans (4 .1-9 .3 h r ) , (Abernethy and Greenbla t t , 1983; 137 Meredith et al., 1984). As d iscussed prev ious ly (sect ion 4 . 2 . 1 ) , a s i m i l a r , more rap id drug c learance in sheep, as compared with humans, e x i s t s f o r a number of drugs inc lud ing l i d o c a i n e (Bloedow et al., 1980), e t idoca ine (Pedersen et al., 1982), metoclopramide (Riggs et al., 1988a) and meperidine (Szeto et al., 1978; Kuhnert et al., 1980). In the l a t t e r case , there are data al lowing comparison between pregnant sheep and pregnant women. Hence, in s tudies of f e t a l drug e f f e c t s employing the pregnant sheep model, drug doses higher than normally used in humans may be r e q u i r e d . 4 . 3 . 3 . Comparisons of Diphenhydramine Pharmacokinetics between Pregnant and Nonpregnant Sheep The d i s t r i b u t i o n and e l im ina t ion h a l f - l i v e s in the pregnant ewes, 9 and 52 min, r e s p e c t i v e l y , are s i m i l a r to those obtained a f t e r 100 mg i . v . bolus i n j e c t i o n of the drug to the nonpregnant ewes (8 and 52 min) . In the nonpregnant sheep, there i s a tendency f o r higher values of Vdss (6.5 vs. 4.5 L/kg) and CZ. T B (5.0 vs. 3.6 L/h/kg) and fo r oo a lower value of AUC 0 (229.4 vs. 554.9 jtig.h/L) as compared with pregnant sheep (Table 4 and 9) . However, when tested fo r s t a t i s t i c a l s i g n i f i c a n c e (Mann-Whitney t e s t ) , there i s no s i g n i f i c a n t d i f f e r e n c e in any of the pharmacokinetic parameters between these two groups. 4 . 3 .4 . Extent of Drug E l iminat ion from the Fetal Lamb Fol lowing Maternal i . v . Bolus Dosing The percentage of diphenhydramine e l iminated i r r e v e r s i b l y by the fetus via the nonplacental pathways, e.g., hepat ic and/or renal 138 e l i m i n a t i o n , c a l c u l a t e d in the present study averaged 5.0 ± 2.2% of the t o t a l drug a v a i l a b l e in the materna l - fe ta l sheep (Table 10). The sum of the amount of diphenhydramine e l iminated by the maternal (111.4 mg) and f e t a l (5.6 mg) sheep c a l c u l a t e d by t h i s method i s greater than i s a n t i c i p a t e d , i.e., the amount of diphenhydramine administered i s 87.5 mg as a f ree base. However, t h i s d iscrepancy i s not s u r p r i s i n g cons ider ing that there i s a large i n t e r i n d i v i d u a l v a r i a b i l i t y in AUC in these animals (Table 9) and that the C L m o and C L f 0 used in the c a l c u l a t i o n are from a study employing a d i f f e r e n t group of pregnant sheep. It i s a lso p o s s i b l e that the drug might have been subject to r e c i r c u l a t i o n wi th in the materna l - fe ta l sheep (see sect ions 4 .3 .4 , 4 .4 .3 and 4.5 .4 f o r fu r ther d i s c u s s i o n ) . Never the less , i t seems that -5% of the to ta l drug a v a i l a b l e in the materna l - fe ta l sheep was e l iminated from the fe ta l compartment via the nonplacental pathway. When c a l c u l a t e d f o r metoclopramide (data from Riggs et al., 1987b and 1988a), the mean percentage of the amount of the drug that i s i r r e v e r s i b l y e l iminated by the fetus was -2% of the to ta l drug administered to the pregnant sheep, which i s l e s s than, but comparable t o , the diphenhydramine data (5%). There fore , i t appears that most of the drug administered to the maternal sheep via bolus dosing i s e l iminated by the mother. 4 .3 .5 Drug Accumulation in Fetal Tracheal and Amniotic F l u i d s Fol lowing diphenhydramine i . v . bolus i n j e c t i o n to the ewe, f e t a l t racheal drug concentrat ions were i n i t i a l l y lower than those in f e t a l plasma. However, by -10-45 min a f te r the drug i n j e c t i o n , t racheal diphenhydramine concentrat ions exceeded the corresponding f e t a l plasma 139 drug concent ra t ions . The average fe ta l t racheal to plasma drug concentra t ion r a t i o of 2.8 ind ica tes that a substant ia l amount of the drug i s accumulated in t racheal f l u i d . In the f e t a l lamb during the l a t t e r t h i r d t r imester of g e s t a t i o n , f e t a l t racheal f l u i d i s produced at a constant rate of approximately 4.5 mL/kg/hr from the lungs (Mescher et a 7 . , 1975). The f e t a l t racheal f l u i d i s l i k e l y to be formed as a r e s u l t of an ac t ive t ranspor t of c h l o r i d e ion from plasma and i n t e r s t i t i a l f l u i d across the pulmonary epi the l ium (Olver and Strang, 1974). In some r e s p e c t s , the f l u i d resembles an u l t r a f i l t r a t e of plasma, in regards to Na + and K + concent ra t ions . However, concentrat ions of several other e l e c t r o l y t e s , such as C l " and H C O 3 " , are s i g n i f i c a n t l y d i f f e r e n t from those in f e t a l plasma. P a r t i c u l a r l y , the to ta l prote in content in t racheal f l u i d (0.3 g/L) i s s u b s t a n t i a l l y lower than in f e t a l plasma (62.7 g/L) (Olver and St rang, 1974). The t racheal f l u i d i s known to leave the lungs , passes along the trachea into the pharynx, and the f l u i d i s then e i t h e r swallowed by the fetus or secreted into the amniotic c a v i t y (Harding et al., 1984). Poss ib le mechanisms respons ib le fo r accumulation of diphenhydramine in f e t a l t racheal f l u i d i n c l u d e ; 1) secre t ion of the drug across a l v e o l a r epi thel ium a f te r uptake by a l v e o l a r c e l l s , 2) secre t ion across the trachea and 3) d i f f u s i o n from s a l i v a / a m n i o t i c f l u i d down the airway. Based on the observat ions that pulmonary ex t rac t ion r a t i o of diphenhydramine [(PA-PV)/PA] examined at s teady-s ta te averages 0.08 in f e t a l lambs (sect ion 3.6) and that uptake of the drug by the 140 lungs i s s u f f i c i e n t to account fo r drug accumulation in f e t a l t racheal f l u i d (see sec t ion 4 .6 ) , secre t ion of the drug across the pulmonary ep i the l ium may be the major route of drug accumulation in the t racheal f l u i d . However, cons ider ing the low rate of t racheal f l u i d production in the fetus (-4.5 mL/kg /hr ) , t h i s process alone may not expla in the rap id appearance of diphenhydramine at the sampling s i t e , i.e., the t rachea , dur ing the i n i t i a l sampling p e r i o d . Secret ion of drug across the trachea may be an important mechanism fo r the i n i t i a l drug accumulat ion. In humans, diphenhydramine i s known to be secreted in s a l i v a , with a mean s a l i v a to plasma drug concentrat ion r a t i o of 0.33 (Sharp et al., 1983). Assuming that a s i m i l a r s i t u a t i o n e x i s t s in the f e t a l lamb, secre t ion via s a l i v a r y glands and d i f f u s i o n down the airway may play a minor r o l e in the tracheal drug accumulat ion. D i f f u s i o n from amniotic f l u i d along the airway may be an important route of drug accumulation in the case where the drug i s present in high concentrat ions in the amniotic f l u i d and the f l u i d i s ingested by the f e t u s , e.g., i n t ra -amnio t ic drug admin is t ra t ion (see sec t ion 4 . 5 . 3 ) . In the f e t u s , diphenhydramine that i s accumulated in the t racheal f l u i d could be e i t h e r swallowed by the fetus or t ran fe r red in to the amniotic sac fo r fu r ther r e c i r c u l a t i o n of the drug within the f e t u s . In a d u l t s , many amine-containing compounds are ex tens ive ly c leared by the lung through metabolism, excret ion or by i r r e v e r s i b l e binding ( C o l l i n s and Dedr ick, 1982; Hori et al., 1987). Using i s o l a t e d lung prepara t ions , s i g n i f i c a n t accumulation of several amine compounds, i nc lud ing p r o p r a n o l o l , l i d o c a i n e , imipramine, amphetamine and methadone, has been demonstrated (Anderson et al., 1974; Roth and Wiersma, 1977). 141 Fol lowing oral or s . c . admin is t ra t ion , accumulation of diphenhydramine in the lungs of adult guinea p i g , ra t and human has a lso been reported (Glazko and D i l l , 1949a; Hausmann et al., 1983). The c e l l type in lung respons ib le f o r accumulation of these drug substances has not been i d e n t i f i e d fo r a l l drugs although pulmonary endothe l ia l c e l l s appear to be involved in the uptake of a number of bas ic amine drugs (Roth, 1985). With reference to the pulmonary c learance of drugs, the fetus would appear to d i f f e r from the adult in at l e a s t two r e s p e c t s . F i r s t l y , the excre t ion of n o n v o l a t i l e , in tac t drug via lung f l u i d may be an add i t iona l route of e l im ina t ion a v a i l a b l e p r e n a t a l l y . Secondly, the marked accumulation of diphenhydramine occurs despi te a very low rate of pulmonary blood flow and r e c e i p t of a small f r a c t i o n of combined v e n t r i c u l a r output as opposed to the a d u l t , where the e n t i r e card iac output i s d e l i v e r e d to the lung . To date , metoclopramide as well as diphenhydramine are the only drugs that have been reported to be excreted into the fe ta l t racheal f l u i d , although the extent of t racheal drug excre t ion tends to be much higher fo r metoclopramide than the l a t t e r drug ( fe ta l t racheal to a r t e r i a l plasma drug concentrat ion r a t i o , 15.1 vs. 2.8) (Riggs et al., 1987a). At present , there are no data to expla in the greater accumulation of metoclopramide in to the t racheal f l u i d as compared to diphenhydramine. Tracheal f l u i d pH i s lower than that in plasma (6.23 vs. 7 .39) , but ion t rapping would not expla in the d i f f e r e n c e , given the fac t that both diphenhydramine and metoclopramide are weakly b a s i c , with pKa values of ~9. The higher plasma prote in binding of diphenhydramine (-70%) as compared with metoclopramide (-40%, Riggs et al., 1988a) may have been i n v o l v e d . The pulmonary d i s p o s i t i o n of diphenhydramine in the fe ta l lamb w i l l be fu r ther d iscussed in the 142 fo l lowing sec t ion (see sect ion 4 .6 ) . Fol lowing i . v . bolus i n j e c t i o n of diphenhydramine to the pregnant ewe, drug concentrat ions in the amniotic f l u i d rose p r o g r e s s i v e l y , reaching the peak concentrat ion (mean = 113.1 ng/mL) by 1.5-3.0 hr a f t e r drug admin is t ra t ion . However, the highest diphenhydramine concentrat ion in the amniotic f l u i d i s - 1 /3 of the highest drug concentrat ion in the f e t a l plasma, i n d i c a t i n g l e s s drug accumulation in t h i s f l u i d as compared with t racheal f l u i d . Diphenhydramine i s more slowly e l iminated from the amniotic f l u i d (t^, ^ = 168 min) than from e i t h e r t racheal f l u i d (39 min) or f e t a l plasma (46 min) . During the t h i r d t r imester of gesta t ion in both sheep and humans, the fetus i s known to swallow both amniotic and t racheal f l u i d , and probably s a l i v a r y secre t ions as well (Harding et a 7 . , 1984). For example, the fetus swallows amniotic f l u i d at an average rate of -300-1,000 mL/day in l a te ovine pregnancy (Harding et al., 1984; Tomoda et al., 1985) and -750 mL/day in near term human pregnancy (Lotger ing and Wallenburg, 1986). Therefore , i t would appear that diphenhydramine excreted in to amniotic f l u i d may be swallowed by the f e t u s , or otherwise t r a n s f e r r e d from the amniotic c a v i t y by d i f f u s i o n across the amnio-a l l a n t o i s in to the f e t a l or maternal c i r c u l a t i o n . The r e c i r c u l a t i o n of diphenhydramine from the amniotic f l u i d w i l l be d iscussed fu r ther in a subsequent sec t ion (4 .5 ) . 143 4.4 Transplacenta l C learance, Nonplacental Clearance and Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep: i . v . Infusion to Steady-State 4 . 4 . 1 . Transplacenta l and Nonplacental Clearances The pharmacokinetics of diphenhydramine were studied in the c h r o n i c a l l y instrumented materna l - fe ta l sheep model fo l lowing maternal and f e t a l drug in fus ions to s t e a d y - s t a t e . As seen in F i g s . 17 and 18, i t i s apparent that s teady-s ta te drug concentrat ions were obtained in both maternal and f e t a l plasma by 30-60 min a f t e r drug a d m i n i s t r a t i o n . Drug i n f u s i o n s , however, d id not r e s u l t in drug e q u i l i b r i u m between the mother and fetus at s teady -s ta te : the C f s s / C m s s r a t i o i s smal ler than uni ty on the bas is of e i t h e r the to ta l drug concentrat ion (0.19 ± 0.10) or the f ree drug concentrat ion (0.39 ± 0 .16) , i n d i c a t i n g that fe tus has an a b i l i t y to e l iminate diphenhydramine via nonplacental pathways. The present data i n d i c a t e that the nonplacental c learance accounts fo r an average of 45.1 ± 4.7% of the to ta l c learance of diphenhydramine from the f e t u s , which i s greater than that reported fo r metoclopramide (-24%) (Riggs et al., 1987b). The average fe ta l to maternal to ta l drug concentrat ion r a t i o at s teady-s ta te fo l lowing maternal in fus ion is s t r i k i n g l y smal ler than the average fe ta l to maternal concentrat ion r a t i o obtained a f t e r maternal i . v . bolus i n j e c t i o n (0.19 vs. 0 .90) . S i m i l a r s i t u a t i o n s , where the to ta l f e t a l to maternal drug concentrat ion r a t i o a f t e r i . v . i n fus ion i s smal ler than that a f t e r i . v . bolus i n j e c t i o n , e x i s t fo r a number of drugs, inc lud ing metoclopramide (0.55 vs. 0 .70) , methadone (0.14 vs. -0.30) and morphine (0.13 vs. 0.25) (Szeto et al., 1981; Szeto et al., 1982b; Golub et al., 1986; Riggs et 144 al., 1988a). There fore , comparisons of the extent of r e l a t i v e fe ta l drug exposure, determined as the C f s s / C m s s r a t i o or fe ta l /materna l oo concentra t ion or AUC 0 r a t i o (Mihaly and Morgan, 1984), should always be made under s i m i l a r experimental p r o t o c o l s . There are l i m i t e d data a v a i l a b l e on the p lacenta l t r a n s f e r of other an t ih is tamines . In the case of c imet id ine and r a n i t i d i n e , the f e t a l to maternal s teady-s ta te to ta l drug concentrat ion r a t i o a f t e r maternal i n f u s i o n i s -0 .03 in the pregnant sheep (Mihaly et al., 1982; Ching et al., 1985). Both c imet id ine and r a n i t i d i n e are h y d r o p h i l i c in nature and the extent of prote in binding of these drugs (-30-40%) i s lower than that of diphenhydramine (-72-86%) in sheep. However, the greater l i p o p h i l i c nature of diphenhydramine seems to favour more extensive f e t a l drug exposure as compared to the l a t t e r two drugs. Diphenhydramine i s known to bind to human serum albumin but the degree of b inding i s low (Drach et a / . , 1970). It i s not known whether the drug binds to a j - a c i d g lycopro te in in human or animal plasma. But the degree of binding of the drug does not seem to change over a wide range of drug concent ra t ions , i.e., -98% at -100 and -1100 ng/mL in man (Albert et al., 1975) and -70% over 30-2,000 ng/mL in sheep. The average f ree f r a c t i o n of diphenhydramine in the pregnant ewe (0.141 ± 0.079) i s s i g n i f i c a n t l y lower than that in the f e t a l lamb (0.277 ± 0.087) . A higher f ree f r a c t i o n in the f e t a l sheep as compared with pregnant ewe has been reported fo r a number of other bas ic l i p o p h i l i c drugs, such as p r o p r a n o l o l , methadone and l i d o c a i n e ( H i l l et al., 1986). Lower binding of these drugs in the fetus as compared with mother has 145 been speculated to be due to the lower a j - a c i d g lycopro te in concentrat ions in the fetus as compared with mother. We have not measured a j - a c i d g lycopro te in concentrat ions in sheep and, the re fo re , do not have data to support the c o r r e l a t i o n between the prote in concentra t ion and the extent of prote in binding of the drug. There was no s i g n i f i c a n t d i f f e r e n c e in the f ree f r a c t i o n of the drug between pregnant ewes (0.141 ± 0.079) and the nonpregnant ewes (0.229 ± 0.080). No c o r r e l a t i o n was observed between gesta t iona l age and the f e t a l nonplacental c learance of diphenhydramine or the f e t a l nonplacental con t r ibu t ion (%) to the to ta l f e t a l drug c lea rance . This l ack of c o r r e l a t i o n suggests that the development of the func t iona l metabol ic capac i ty of diphenhydramine d id not vary apprec iably over 126-136 days gesta t ion in the f e t u s . The f e t a l nonplacental c learance of the drug, C L f 0 (208.4 mL/min) i s -6% of the maternal nonplacental c l e a r a n c e , C L m o (3343.8 mL/min). The C L f 0 normalized by the f e t a l body weight (99.5 mL/min/kg) i s -20% of the f e t a l combined v e n t r i c u l a r output (-530 mL/min/kg) , whereas the C L m o normalized by the maternal body weight (43.2 mL/min/kg) i s -30% of the maternal card iac output (-150 mL/min/kg) in sheep (Rudolph and Heymann, 1970; Rosenfe ld , 1977). There fore , despi te the fac t that the weight normalized C L f 0 i s -2 times greater than the weight normalized C L m o , diphenhydramine appears to be c leared more e f f i c i e n t l y from the mother than from the f e t u s . P lacenta l c learance fo r several substances has been f requent ly determined by use of the F i c k ' s d i f f u s i o n p r i n c i p l e (Meschia et al., 1967). Meschia et al. (1967) have def ined the p lacenta l c learance as 146 the rate of drug d i f f u s i o n across the placenta d iv ided by the t ransp lacenta l concentrat ion grad ien t , and have formulated the concept of f l o w - l i m i t e d and d i f f u s i o n - l i m i t e d p lacenta l c lea rance . F low- l imi ted substances have a high p lacenta l permeabi l i ty and t h e i r p lacenta l t r a n s f e r i s p r i m a r i l y determined by the rate of drug d e l i v e r y to the p l a c e n t a . The permeabi l i ty of the p lacenta to d i f f u s i o n - l i m i t e d substances i s low and t r a n s f e r i s determined p r i m a r i l y by p lacenta l s t r u c t u r a l c h a r a c t e r i s t i c s . This method can be used f o r drugs that are m e t a b o l i c a l l y iner t but are c leared r a p i d l y by the p lacen ta , r e s u l t i n g in a measurable t ransp lacenta l concentrat ion g rad ien t . More r e c e n t l y , Anderson et al. (1980) reported a technique fo r the measurement of p lacenta l c learance of drugs without any assumption regarding p lacenta l metabolism or plasma prote in b ind ing , as long as drug t r a n s f e r across the p lacenta i s d i f f u s i o n - l i m i t e d . The p lacenta l c learance c a l c u l a t e d by t h i s method i s equivalent to C L m f . These two methods are based on one common assumption, that the rate of p lacenta l c learance i s the same in e i t h e r d i r e c t i o n across the p lacenta . The u n i d i r e c t i o n a l f e t a l -maternal p lacenta l c learance has been reported f o r a number of drugs, such as acetyl s a l i c y l i c ac id (Anderson et al., 1980), a glucose analogue, a-methyl -D-glucopyranoside (Stacey et al., 1978), a n t i p y r i n e , 3 H 2 0 , urea (Meschia et al., 1967) and ethanol (Bonds et al., 1980). Recent ly , Wang et al. (1986), using Sze to 's compartmental method, demonstrated that there i s no s i g n i f i c a n t d i f f e r e n c e between C L m f (60.8 mL/min) and C L f m (58.4 mL/min) fo r acetaminophen in sheep, suggesting a u n i d i r e c t i o n a l t ransp lacenta l c learance . However, s i t u a t i o n s where C L m f i s d i f f e r e n t from C L f m e x i s t fo r a number of other drugs such as methadone (Szeto et al, 1982a) and metoclopramide (Riggs et al., 1987b). 147 In the case of diphenhydramine, the C L m f (82.4 mL/min) i s s i g n i f i c a n t l y smal ler than C L ^ (264.4 mL/min). Diphenhydramine i s h igh ly l i p o p h i l i c and i t s p lacenta l t r a n s f e r may be a f fec ted l a r g e l y by the per fus ion c h a r a c t e r i s t i c s of the placenta and, to a l e s s e r extent , by the anatomic p roper t i es of the p lacenta or the p lacenta l permeab i l i t y , i.e., i t may be f l o w - l i m i t e d . A number of substances such as a n t i p y r i n e , 3 H 2 0 and ethanol have been prev ious ly c l a s s i f i e d by the F ick p r i n c i p l e [ Infusion R a t e / ( C f ' s s - C m ' s s ) ] as being f l o w - l i m i t e d , with the t ransp lacenta l c learance of these compounds ranging from -80-160 mL/min/kg (Meschia et al., 1967; Walker, 1977; Bonds et al., 1980; Owens et al., 1986). On the other hand, urea i s c l a s s i f i e d as a d i f f u s i o n - l i m i t e d substance, with a low t ransp lacenta l c learance (-20 mL/min/kg) (Meschia et al., 1967). When a modif ied equation of the F ick p r i n c i p l e [ ( C f s s x C L f m ) / ( C f s s - C m ' s s ) ] was a p p l i e d , the t ransp lacenta l value f o r diphenhydramine averaged -130 mL/min/kg, again suggesting that the p lacenta l c learance of diphenhydramine i s f l o w - l i m i t e d . In near-term sheep, maternal p lacenta l blood flow comprises -15% of maternal card iac output, whereas f e t a l p lacenta l blood flow i s -40% of f e t a l combined v e n t r i c u l a r output (Rudolph and Heymann, 1970; Rosenfe ld , 1977). There fore , with diphenhydramine, C L f m may be higher than C L m f because of the greater proport ion of card iac output d e l i v e r e d to the p lacenta in the fetus compared to the ewe. One potent ia l l i m i t a t i o n of the two-compartment model proposed by Szeto et al. (1982a) is that i t does not account f o r the p o s s i b i l i t y of p lacenta l drug metabolism in the t ransplacenta l c learance terms, C L m f and C L f m . According to t h i s model, both C L m f and C L f m are simply the 148 t r a n s f e r c learances and, i f there i s any drug metabolism occurr ing wi th in the p lacen ta , i t w i l l be r e f l e c t e d in the f e t a l or maternal nonplacental c lea rance , C L f 0 and C L m o . On the other hand, the p lacenta l c learance c a l c u l a t e d by the model-independent method using the F ick p r i n c i p l e w i l l represent the metabol ic c learance wi th in the placenta as well as the t r a n s f e r c learance across the placenta (Meschia et al., 1967; Wang et a7. 1986). Therefore , i f there i s any drug metabolism occur r ing wi th in the p lacenta , the p lacenta l c learance c a l c u l a t e d by the compartmental method may be an underestimate as compared to the p lacenta l c learance c a l c u l a t e d by the model-independent method. In case of acetaminophen, the t ransp lacenta l c learance (CL m f or C L f m ) c a l c u l a t e d by the two methods was s i m i l a r , and p lacenta l metabolism was, the re fo re , postu la ted to be n e g l i g i b l e (Wang et al., 1986). The ER of diphenhydramine across the placenta on the f e t a l s ide (0.64) c a l c u l a t e d in one experiment in the present study (eq. 23) i s ~5 times greater than that of acetaminophen (0.12) . Assuming a s i m i l a r rate of umbi l ica l blood flow in the two studies (-500 mL/min, Wang et al., 1986), the p lacenta l c learance of diphenhydramine from the f e t a l s i d e , C L p (ER x umbi l ica l blood f low) , must be -5 times greater than that of acetaminophen. In f a c t , the average C L f m c a l c u l a t e d f o r diphenhydramine (264.4 mL/min) i s - 5 f o l d greater than the C L f m reported fo r acetaminophen (58.4 mL/min). The f e t a l ER of diphenhydramine (0.33) c a l c u l a t e d by the eq. 24 represents the f r a c t i o n of the drug rece ived by the fetus that i s c leared by nonplacental mechanisms, such as fe ta l hepa t ic , r e n a l , pulmonary and other p o s s i b l e routes but excluding p lacenta l uptake/metabolism. In t h i s animal (no. 130), t h e r e f o r e , the d i f f e r e n c e (-11%) between the nonplacental con t r ibu t ion (45.4%) 149 c a l c u l a t e d by the compartmental method and the percentage f e t a l ex t rac t ion r a t i o (33%) determined a f t e r maternal in fus ion may represent a propor t ion of the percentage cont r ibu t ion of the p lacenta l metabolism to the t o t a l f e t a l c learance of diphenhydramine. However, fu r ther i n v e s t i g a t i o n i s required to determine the con t r ibu t ion of the p lacenta l metabolism to the to ta l f e ta l drug c learance . Placental and nonplacental c learances of a number of drugs, inc lud ing morphine, methadone (Szeto et al., 1982a and 1982b), acetaminophen (Wang et al., 1986) and metoclopramide (Riggs et al., 1987b), have been determined at s teady-s ta te in the c h r o n i c a l l y instrumented pregnant sheep prepara t ion . Methadone, metoclopramide and diphenhydramine are a l l weakly basic compounds with high 1 i p o p h i l i c i t y (pKa 9 . 0 - 9 . 3 ) . These drugs are known to bind to plasma pro te in more ex tens ive ly in the mother than in the f e t u s . The extent of p ro te in binding of diphenhydramine (86% in mother and 72% in fe tus) tends to be higher than that of methadone (75% in mother and 58% in fe tus) and metoclopramide (49% in mother and 39% in fe tus) (Szeto et al., 1982c; Riggs et al., 1988a). Respective values fo r the C L f f , C L f m , C L m f fo r diphenhydramine (472.7, 264.4 and 82.4 mL/min) are comparable to those fo r methadone (470.8, 312.8 and 93.5 mL/min) (Szeto et al., 1982a). Since the s teady-s ta te C f s s / C m s s r a t i o a f t e r maternal in fus ion i s a funct ion of both C L m f and CLf f ( C f s s / C m s s = C L m f / C L f f ) and C L m f and CLf f f o r diphenhydramine and methadone are s i m i l a r , the C f $ s / C m s s r a t i o fo r diphenhydramine (0.19) c l o s e l y approaches the C f s $ / C m s s r a t i o fo r methadone (0.18) (Szeto et al., 1982b). Riggs et al. (1987b) reported p lacenta l and nonplacental c learances of metoclopramide in sheep, as 150 body weight normalized va lues . The C L f m f o r diphenhydramine (124.4 mL/min/kg) i s s i m i l a r to the C L f m f o r metoclopramide (125.1 mL/min/kg) but the C L f 0 fo r diphenhydramine (99.5 mL/min/kg) i s ~ 2.5 f o l d greater than f o r metoclopramide (40.0 mL/min/kg) , r e s u l t i n g in the greater CLf f f o r diphenhydramine (223.9 mL/min/kg) than metoclopramide (165.1 mL/min/kg) . On the other hand, C L m f fo r metoclopramide i s ~2 f o l d greater than that f o r diphenhydramine (79.0 vs. 41.0 mL/min/kg) and the r e s u l t i n g C f s s / C m s s r a t i o f o r metoclopramide (0.55) i s much higher than that f o r diphenhydramine (0.19) . In c o n t r a s t , morphine and acetaminophen are much l e s s l i p i d so lub le than methadone, metoclopramide and diphenhydramine. The extent of prote in binding in mother and fetus i s about the same f o r morphine and acetaminophen, with the degree of b inding being <5% fo r the former and -95% f o r the l a t t e r . The t ransp lacenta l c l ea rances , C L f m and C L m f , and the to ta l f e t a l c learance , CL f f f o r these drugs are lower than f o r the more l i p o p h i l i c drugs d iscussed above. The CLf f f o r the more l i p o p h i l i c drugs, methadone, metoclopramide (assuming fe ta l body weight of 2.5 kg) and diphenhydramine (range, -410-470 mL/min) i s at l e a s t 2 f o l d greater than that f o r morphine (81.0 mL/min) and acetaminophen (184.0 mL/min). The higher CL f f f o r the more l i p o p h i l i c drugs seems to be a r e s u l t of greater C L f m of these drugs (range, 264.4—312.8 mL/min) as compared to the C L f m of acetaminophen (58.4 mL/min) and morphine (58.4 mL/min). There fore , i t i s l i k e l y that the 1 i p o p h i l i c i t y / h y d r o p h o b i c i t y of a drug i s a major determinant fo r the t ransplacenta l c l ea rances , C L f m and C L m f , whereas C L f 0 r e f l e c t s the metabolic capac i ty in the f e t u s . In regards to the l a t t e r view, i t i s i n t e r e s t i n g to note that acetaminophen, morphine and metoclopramide are metabolized mainly by g lucuron idat ion 151 and s u l f a t i o n and diphenhydramine and methadone are metabolized p r i m a r i l y by N-demethylat ion. The low C L f 0 fo r acetaminophen (22.6 mL/min), morphine (125.6 mL/min) and metoclopramide (-100 mL/min) as compared to diphenhydramine (208.4 mL/min) and methadone (158.0 mL/min) may r e f l e c t the funct iona l d i f f e r e n c e s in the metabol ic capac i ty in the f e t u s . Placental metabolism of acetaminophen i s n e g l i g i b l e in near-term sheep (Wang et al., 1986), whereas diphenhydramine could be metabolized by the p lacen ta , as demonstrated i n d i r e c t l y by the noncompartmental ex t rac t ion method (equation 27) in one animal experiment (ewe no. 130) in the present study. Diphenhydramine i s a lso known to be excreted into f e t a l lamb lung f l u i d (Riggs et al., 1987a). Pre l iminary s tudies of the f e t a l pulmonary d i s p o s i t i o n of diphenhydramine ind ica te that ex t rac t ion by the l e f t f e t a l lamb lung i s -8% at s teady-s ta te (see sec t ion 3 .6 ) . It i s a lso p o s s i b l e that t i s s u e s other than f e t a l lungs and p lacenta , such as f e t a l l i v e r , kidneys and/or i n t e s t i n e are respons ib le f o r the f e t a l diphenhydramine e l i m i n a t i o n . In c o n c l u s i o n , diphenhydramine is c leared from mother and fetus by p lacenta l and nonplacental pathways. Further work i s required to determine the primary pathways of the drug e l i m i n a t i o n , e . g . , f e ta l hepat ic metabolism, renal and pulmonary excret ion 4 . 4 . 2 . Drug Accumulation in Fetal Tracheal and Amniotic F l u i d s Fol lowing drug in fus ions to the ewe and f e t u s , diphenhydramine was found to be excreted into the fe ta l t racheal and amniotic f l u i d s ( F i g s . 19 and 20). In a l l the animals employed, the average f e t a l 152 t racheal to a r t e r i a l drug concentrat ion r a t i o was greater than 1.0 fo l lowing e i t h e r the maternal or f e ta l drug in fus ion (Table 16). The average r a t i o of the fe ta l t racheal to a r t e r i a l drug concentrat ions a f t e r maternal in fus ion (mean, 3.4 ± 3.6) was comparable to that obtained a f t e r f e t a l drug in fus ion (mean, 5.4 ± 4 .5 ) . A l s o , these r a t i o s are comparable to the r a t i o a f te r i . v . bolus i n j e c t i o n of the drug to pregnant sheep (mean, 2.7 ± 2 .4 ) . The C m a x in the tracheal f l u i d i s s i g n i f i c a n t l y greater than the C m a x in the amniotic f l u i d fo l lowing e i t h e r the maternal in fus ion (mean, 124.7 vs. 16.6 ng/mL) or the f e t a l in fus ion (mean, 1790.4 vs. 139.9 ng/mL) (Table 15). Diphenhydramine concentrat ions in the t racheal f l u i d are higher than the corresponding concentrat ions in the amniotic f l u i d f o r most of the sampling per iod (see F i g s . 19 and 20), hence i t i s p o s s i b l e that diphenhydramine excreted into fe ta l lung f l u i d in part accounts fo r the drug accumulation in the amniotic f l u i d s ince the t racheal f l u i d i s cont inuously produced at a constant rate from the lungs and flows out , and i s e i t h e r swallowed or secreted in to the amniotic sac (see sect ion 4 . 3 . 4 ) . 4 . 4 . 3 . Drug D i s p o s i t i o n in Maternal and Fetal Sheep Fol lowing Maternal and Fetal Infusions Fol lowing in fus ions of the drug to the ewe, an average of 78.6 ± 4.3 mg of diphenhydramine, as a f ree base, was c a l c u l a t e d to be oo el iminated from the maternal sheep ( A U C M o - C L m o ) and 1.2 ± 0.6 mg of diphenhydramine, as a f ree base, was e l iminated from the f e t a l lamb ( A U C F Q - C L f o ) (Table 21). Therefore , an average of 1.4% of the to ta l 153 drug a v a i l a b l e in the materna l - fe ta l sheep was e l iminated by the fetus [ A U C F o - C L f o ' 1 0 0 / ( A U C M o - C L m o + A U C F o ' C L f o ) ] and the res t (98.6%) was e l iminated by the ewe [AUC M Q-CL r n o - 100 / (AUC M Q-CL m o + A U C F Q - C L f o ] (Table 21). On the other hand, fo l lowing drug in fus ions to the f e t u s , an average of 14.7 ± 6.2 mg and 11.5 ± 3.0 mg of diphenhydramine, as a f ree base, was e l iminated from the ewe and f e t u s , r e s p e c t i v e l y (Table 21). There fore , in the case of f e ta l drug i n f u s i o n , an average of 45.0% of the t o t a l drug a v a i l a b l e in the materna l - fe ta l sheep was e l iminated by the f e t a l lamb and 55.0% of the to ta l drug by the maternal sheep (Table 21). P r e v i o u s l y , maternal and fe ta l t ransp lacenta l and nonplacental c learances of the drug were c a l c u l a t e d (eq. 16-21) by use of the values of the s teady-s ta te materna l - fe ta l drug concentrat ions fo l lowing maternal and f e t a l in fus ions and t h e i r in fus ion rates (sect ion 3 . 4 . 1 . 4 ) . The average percent con t r ibu t ion of maternal nonplacental c learance to maternal to ta l drug c learance , C L m o / ( C L m o + C L m f ) , was c a l c u l a t e d to be 97.8%, whereas the average percent con t r ibu t ion of the f e t a l nonplacental to f e ta l to ta l drug c lea rance , C L f 0 / ( C L f 0 + C L f m ) , i s 45.1% (Table 18). There i s a good agreement between the percent con t r ibu t ion of the maternal nonplacental to maternal to ta l drug c learance [ C L m o - 1 0 0 / ( C L m o + C L m f ) = 97.8%, Table 18] and the percentage of the t o t a l amount of the drug that was e l iminated from the mother via the nonplacental pathway fo l lowing maternal drug i n f u s i o n , c a l c u l a t e d by use of AUC and nonplacental c learance values [ A U C M Q ' C L m o ' 1 0 0 / ( A U C M Q - C L m o + 00 A U C F o ' C L f 0 ) = 98.6%, from Table 21]. S i m i l a r l y , there i s a lso a good agreement between the percent con t r ibu t ion of the f e t a l nonplacental to 154 f e t a l t o t a l drug c learance [ C L f o ' 1 0 0 / ( C L f o + C L f m ) = 45.1%, Table 18] and the percentage of the to ta l amount of the drug that was e l iminated from the fetus via the nonplacental pathway fo l lowing f e t a l drug i n f u s i o n [ A U C F o ' C L f o ' 1 0 0 / ( A U C M o ' C L m o + A U C F o ' C L f o ) = 45.0%, Table 21]. There fore , employing the two d i f f e r e n t methods, i t has been demonstrated that the percentage of the to ta l diphenhydramine a v a i l a b l e in the materna l - fe ta l sheep that was e l iminated by the f e t a l lamb averaged -1-2% fo l lowing maternal in fus ions and -45% fo l lowing f e t a l drug i n f u s i o n s . The amount of diphenhydramine e l iminated from the ewe and fetus via the nonplacental pathway averaged 78.6 mg and 1.2 mg, r e s p e c t i v e l y , fo r the maternal in fus ions (sum, 79.8 mg) and 14.7 mg and 11.5 mg, r e s p e c t i v e l y , f o r the fe ta l in fus ions (sum, 26.2 mg) (Table 21). It should be noted that the sum of the amount of the drug e l iminated from oo both the ewe and fetus via the nonplacental pathway, A U C ^ 0 ' C L m o + OO A U C p 0 ' C L f 0 , exceeded the administered dose. In the case of maternal i n f u s i o n , the c a l c u l a t e d amount of diphenhydramine that was e l iminated by the materna l - fe ta l uni t was 79.8 mg, whereas the administered dose was 70.0 mg (as a f ree base) . S i m i l a r l y , in the case of f e t a l drug i n f u s i o n , the c a l c u l a t e d amount of the drug that was e l iminated by the materna l - fe ta l un i t (26.2 mg) exceeded the administered dose (17.5 mg as a f ree base) . As stated in the proceeding sect ions (sect ions 4.3.2 and 4 . 4 . 2 ) , i t i s probable that the drug administered to the pregnant ewe or fe tus i s subject to r e c i r c u l a t i o n via f e ta l swallowing of the t racheal and amniotic f l u i d s and via drug t r a n s f e r from the c h o r i o - a l l a n t o i s back to the f e t a l or maternal c i r c u l a t i o n . Therefore , i t i s expected that 155 the c a l c u l a t e d amount of to ta l diphenhydramine a v a i l a b l e in the materna l - fe ta l sheep i s greater than the dose administered. A l s o , i t i s not s u r p r i s i n g that the c a l c u l a t e d amount of drug e l iminated by the ewe and fetus compared to the dose a c t u a l l y administered i s greater fo l lowing the f e t a l drug in fus ion (149.8%) as compared to the maternal i n f u s i o n (114.0%) s ince fo l lowing fe ta l i n f u s i o n , a greater amount of the drug could be accumulated in the f e t a l t racheal and amniotic f l u i d s f o r fu r ther drug r e c i r c u l a t i o n . 4 . 4 . 4 . Fetal E f f e c t s of Diphenhydramine in Pregnant Sheep In the l a s t quarter of g e s t a t i o n , the f e t a l lamb e x h i b i t s regu lar a l t e r a t i o n s between episodes of high vo l tage , low frequency and low vo l tage , high frequency ECoG a c t i v i t y (Dawes et al., 1972). The former a c t i v i t y bears many s i m i l a r i t i e s to quiet sleep a f t e r b i r t h , whereas the l a t t e r pa t te rn , the bulk of which i s associa ted with REM and rap id i r r e g u l a r breathing movements, appears equivalent to REM or paradoxical s l e e p . Various inves t iga to rs have reported b r i e f per iods (1-3 min) of an aroused s t a t e , with low voltage ECoG a c t i v i t y , lack of REM and vigorous breathing and body movements; t h i s may represent wakefulness (Ruckebusch et al., 1977; Harding, 1984). A t r a n s i t i o n a l s t a t e , between high and low voltage ep isodes , and vice versa, with an intermediate vol tage amplitude has a lso been reported (Natale et al., 1981). In the present i n v e s t i g a t i o n , we obtained ECoG r e s u l t s s i m i l a r to these f i n d i n g s , although we d id not attempt to i d e n t i f y per iods of f e t a l a r o u s a l . 156 The f e t a l e f f e c t s of diphenhydramine were examined at two d i f f e r e n t concentrat ion ranges, achieved by separate in fus ions of the drug to the ewe and f e t u s . The fe ta l s teady-s ta te concentrat ion of the drug during the maternal in fus ions averaged 36.3 ng/mL. This leve l i s below that (<50 ng/mL) associa ted with d iscernab le CNS e f f e c t s in adult humans (Carruthers et al., 1978). The f e t a l plasma diphenhydramine concentrat ion during fe ta l in fus ion averaged 447.6 ng/mL. This i s approximately 3 times the i n i t i a l plasma diphenhydramine concentrat ion r e s u l t i n g from i . v . i n j e c t i o n of 50 mg of the drug to human subjects (Carruthers et al., 1978; Blyden et al., 1986), but well below the leve l of ~5 Mg/mL found in a male subject who died from a diphenhydramine overdose (Hausmann et al., 1983). There do not appear to be any reports of the drug l e v e l s in c h i l d r e n who have had convuls ive episodes r e s u l t i n g from diphenhydramine po ison ing . The two modes of diphenhydramine adminis t ra t ion used in the present study resu l ted in two d i s t i n c t patterns of f e t a l behavioural e f f e c t s , which we ascr ibe to the markedly d i f f e r e n t f e t a l plasma drug concentrat ion achieved. During in fus ions of the drug to the ewe, there were reduct ions in the incidence of low voltage ECoG a c t i v i t y and the amount of REM associa ted with the low vol tage a c t i v i t y , i.e., a reduct ion in the amount of REM s l e e p . Coupled with t h i s was a f a l l in the amount of breathing a c t i v i t y . This l a t t e r e f f e c t is s i m i l a r to those reported fo r CNS depressants such as c h l o r a l o s e , p e n t o b a r b i t a l , diazepam and e thano l , which involve temporary suppression of f e t a l breathing movements (Dawes et al., 1972; Boddy et al., 1976; P iercy et al., 1977; Pa t r i ck et a7. ,1985) . In terms of the f e t a l ECoG e f f e c t s of 157 diphenhydramine during maternal i n f u s i o n s , these appear to be s i m i l a r t o , but l e s s s t r i k i n g than those e l i c i t e d by pentobarbi ta l a d m i n i s t r a t i o n , where there i s a marked reduct ion in the incidence of the low vol tage pattern and increase in the amount of high vol tage a c t i v i t y (Boddy et a 7 . , 1976). In c o n t r a s t , with ethanol admin is t ra t ion to pregnant sheep, there i s a replacement of both high and low voltage ECoG a c t i v i t i e s by an intermediate voltage pattern (Pa t r ick et a 7 . , 1985), a s i t u a t i o n s i m i l a r to the r e s u l t s obtained during in fus ion of diphenhydramine to the f e t u s . The fe ta l ECoG e f f e c t s of diazepam and c h l o r a l o s e do not appear to have been repor ted . In a d u l t s , the predominant centra l e f f e c t of diphenhydramine is seda t ion , r e f l e c t e d by i n a b i l i t y to concentrate , a l te red coord ina t ion and s leep iness (Carruthers et a 7 . , 1978; Roth et al., 1987). The assessment of the CNS e f f e c t s in a d u l t s , both humans and animals, has r e l i e d l a r g e l y on behavioural t es ts and sub jec t ive assessments of drowsiness by the experimental subjects (Carruthers et al., 1978; Winter, 1985). The r e s u l t s from studies of the EEG e f f e c t s of the drug in humans (Goldste in et a 7 . , 1968; Fink and Irwin, 1979) are cons is ten t with a sedat ive e f f e c t of the drug, although Roth et al. (1987) found that when diphenhydramine was given in the evening the percentages of the var ious sleep stages during the night were unal tered from the placebo r e s u l t s . However, these behavioural and EEG data from humans cannot be r e a d i l y compared with the information obtained from the f e t a l lamb. The main reason fo r t h i s i s the great d i f f e r e n c e in sleep state pattern in the fetus and adu l t . In the f e t u s , the awake state r a r e l y i f ever occurs (Harding, 1984), whereas in the a d u l t s , the assessment of 158 the CNS e f f e c t s of diphenhydramine has been accomplished by adminis ter ing the drug to awake sub jec ts . However, in view of the s i m i l a r i t i e s of the f e t a l e f f e c t s of diphenhydramine during the maternal i n f u s i o n s to those e l i c i t e d by other CNS depressant substances, i t seems reasonable to conclude tha t , at the plasma l e v e l s achieved during these experiments (36 ng/mL), the drug e l i c i t e d f e t a l sedat ive e f f e c t s . Whether higher f e t a l drug l e v e l s , in the range assoc ia ted with sedat ive ac t ions in a d u l t s , would r e s u l t in greater e f f e c t s appears worthy of i n v e s t i g a t i o n . When diphenhydramine was infused d i r e c t l y to the f e t u s , a d i s t i n c t l y d i f f e r e n t pattern of f e ta l behavioural e f f e c t was observed. These comprised a dramatic increase in the incidence of an intermediate vol tage ECoG pattern at the expense of both REM and quiet s l e e p . The net e f f e c t was a d i s r u p t i o n in the normal c y c l i n g of ECoG a c t i v i t y between REM and quiet sleep by a large increase in the amount of a t r a n s i t i o n a l ECoG s t a t e . The overa l l incidence of f e t a l breathing a c t i v i t y was una l te red , although there was an increase in the amount dur ing the intermediate ECoG pattern and the appearance of large amplitude breathing movements immediately a f t e r the i n i t i a l bolus i n j e c t i o n , when the plasma drug l e v e l s were at t h e i r h ighest . There was a lso modest and t rans ien t hypoxemia, acidemia and t a c h y c a r d i a . The average f a l l in Po 2 (3.3 mm Hg) and pH (0.043) i s s i m i l a r to that observed during vigorous f e t a l a c t i v i t y and breathing movements (Harding et a 7 . , 1983; Rurak and Gruber, 1983). These blood gas changes are probably a consequence of an increase in fe ta l oxygen consumption r e s u l t i n g from the large amplitude breathing a c t i v i t y , as s i m i l a r 159 changes and an increase in 0 2 consumption are observed during vigorous f e t a l b rea th ing , both when occurr ing spontaneously (Rurak and Gruber, 1983) and when st imulated by hypercapnia (Rurak et al., 1986). The r i s e in f e t a l heart rate i s s i m i l a r to the tachycard ia observed in adult dogs given an i . v . i n fus ion of diphenhydramine at a ra te (4 mg/kg/min) c lose to that (-5 mg/kg/min) used in the current s tudy, and has been a t t r ibu ted to the a n t i c h o l i n e r g i c act ions of the drug (Hirschowitz and Mol ina , 1983). A l t e r n a t i v e l y , po ten t i a t ion of the e f f e c t s of endogenous histamine by diphenhydramine could be involved in the t a c h y c a r d i a , inasmuch as in the i s o l a t e d gu inea-p ig hear t , diphenhydramine at 10"^ to 10"^ M potent ia tes the p o s i t i v e chronotropic e f f e c t s of low but not high doses of histamine (Levi and Kuye, 1974) and, in the current study, f e ta l s teady-s ta te concentrat ions of the drug averaged 1.8 x 10"° M during the fe ta l i n f u s i o n s . However, the card iac e f f e c t s of histamine and i t s antagonists are complex and vary between species (Levi et al., 1982), so that fur ther study i s needed to understand the card iac e f f e c t s of diphenhydramine in the f e t a l lamb. The ECoG changes during the in fus ion of diphenhydramine to the fetus are s i m i l a r to that observed a f te r acute ethanol admin is t ra t ion to pregnant sheep (Pat r ick et al., 1985). However, the e f f e c t s of these two agents on f e t a l breathing a c t i v i t y are d i f f e r e n t , because with e thano l , f e t a l breathing a c t i v i t y i s suppressed fo r 9 hr a f t e r drug admin is t ra t ion (Pat r ick et al., 1985). Other agents have st imulatory e f f e c t s in the f e t a l lamb, invo lv ing the occurrence of breathing a c t i v i t y in low and high voltage ECoG s t a t e s , as in the case of 160 indomethacin and other prostaglandin synthetase i n h i b i t o r s (Kitterman et al., 1979) and 5-hydroxytryptophan ( Q u i l l i g a n et al., 1981). Methadone, on the other hand, e l i c i t s a hyperact ive s ta te invo lv ing suppression of both quiet and REM sleep and continuous breathing and body movements (Szeto, 1983). The e f f e c t s of a l l these agents are un l ike those a f te r f e t a l i n f u s i o n of diphenhydramine and so the e f f e c t s of the l a t t e r would appear to be unique, at l e a s t in terms of the agents examined to date . During the f e t a l i n f u s i o n s , we obtained no evidence f o r the convu ls ive and se izure a c t i v i t y that occurs in c h i l d r e n subject to diphenhydramine overdose (Wyngaarden and Seevers, 1951; Reyes-Jacang and Wenzl, 1969) and adult cats given high doses of the drug (Faingold and Berry , 1972). E p i l e p t i f o r m se izure a c t i v i t y can be induced in the f e t a l lamb with l i d o c a i n e (Teramo et al., 1974); i t i s a lso observed in some fetuses before i n t r a - u t e r i n e death (Chapman et al., 1978). However, i t was not present at any point during the per iod of in fus ion of diphenhydramine to the f e t u s , in s p i t e of the r e l a t i v e l y high drug l e v e l s obta ined . Whether such a c t i v i t y can be e l i c i t e d with higher doses remains to be determined. A f t e r cessat ion of diphenhydramine a d m i n i s t r a t i o n , with both maternal and fe ta l i n f u s i o n s , f e t a l behavioural parameters qu ick ly returned to t h e i r contro l ranges, so that the values in the immediate p o s t - i n f u s i o n per iod were not d i f f e r e n t from those in the contro l p e r i o d . This i s probably a r e f l e c t i o n of the rapid rate of c learance of the drug from the maternal and f e t a l c i r c u l a t i o n s . The relevance of the present observat ions in the fe ta l lamb to the human fetus i s unclear at present , as there appear to be no reports 161 on the f e t a l behavioural e f f e c t s of diphenhydramine or other ant ih is tamines in humans. In t h i s regard , however, i t i s worth not ing that the e f f e c t s of ethanol on f e t a l breathing a c t i v i t y are very s i m i l a r in the sheep and human (McLeod et a 7 . , 1983; Pa t r i ck et al., 1985). As mentioned p r e v i o u s l y , diphenhydramine e l i c i t e d behavioural e f f e c t s in the f e t a l lamb at plasma drug l e v e l s lower than those associa ted with CNS e f f e c t s in adult humans. It would a lso appear that the f e t a l lamb i s more s e n s i t i v e to the CNS act ions of diphenhydramine than i s the pregnant ewe as no behavioural modi f ica t ions were noted in the l a t t e r , even during maternal admin is t ra t ion of the drug, when the maternal plasma l e v e l s of the drug were much higher than those needed to e l i c i t CNS e f f e c t s in adult humans. This s i t u a t i o n i s s i m i l a r to the e f f e c t s of pentobarbi ta l in pregnant sheep, where doses of the drug (4 mg/kg) which have no obvious e f f e c t s on the ewe cause suppression of REM sleep and breathing a c t i v i t y in the fetus (Boddy et al., 1976). S i m i l a r l y , ethanol at a dose of 0.25 mg/kg in pregnant women markedly suppresses f e t a l breathing a c t i v i t y with minimal e f f e c t s on the mother (McLeod et al., 1983). If diphenhydramine e l i c i t s behavioural e f f e c t s in the human f e t u s , t h i s could have two important consequences, p a r t i c u l a r l y i f the drug was e f f e c t i v e at lower concentrat ions in the fetus than in the a d u l t . The f i r s t r e s u l t s from the f a c t that the current d i a g n o s t i c t e s t s of human f e t a l w e l l - b e i n g , such as the nonstress tes t (Brown and P a t r i c k , 1981) and the fe ta l b iophys ica l score (Manning et al., 1980) monitor elements of f e t a l behaviour, such as body and breathing movements and movement-associated heart rate changes. If these monitored v a r i a b l e s are a f fec ted by diphenhydramine-induced a l t e r a t i o n s in f e t a l behaviour, there could be a misdiagnosis of f e t a l d i s t r e s s , 162 p a r t i c u l a r l y i f the phys ic ian were unaware that the pat ient was taking the drug. The second potent ia l consequence i s that prolonged exposure of the fetus to the drug could r e s u l t in long term a l t e r a t i o n s in CNS f u n c t i o n . Th is i s suggested by the observat ions of Parkin (1974) on apparent withdrawal symptoms in a newborn in fant whose mother had been t reated with 150 mg of diphenhydramine d a i l y f o r an unspec i f i ed per iod of time during pregnancy. In summary, the e f f e c t s of the ant ih is tamine , diphenhydramine, have been inves t iga ted in the f e t a l lamb at two d i f f e r e n t concentrat ion ranges, achieved by separate i . v . in fus ions of the drug to the ewe and f e t u s . With low f e t a l diphenhydramine concent ra t ions , sedat ive e f f e c t s were observed, comprising reduct ion in the amounts of REM sleep and f e t a l breathing a c t i v i t y . In c o n t r a s t , with higher drug l e v e l s , there was t r a n s i e n t vigorous breathing a c t i v i t y , hypoxemia, acidemia and tachycard ia and a marked increase in the amount of a t r a n s i t i o n a l ECoG pa t te rn . Moreover, f e t a l CNS e f f e c t s were observed at plasma concentrat ions of the drug below those associated with sedat ion in adult humans. Given the f a i r l y high frequency of use of ant ih istamines during pregnancy, fu r ther examination of t h e i r e f f e c t s on the f e t u s , in both animals and humans, i s warranted. 4 .5 . Drug R e c i r c u l a t i o n from Amniotic F l u i d to the Ewe and Fetal Lamb Fol lowing Intra-amniot ic Bolus Admin is t ra t ion 4 . 5 . 1 . Amniotic F l u i d Dynamics 163 In the l a s t t h i r d of gesta t ion in sheep, the fetus i s surrounded by amniotic f l u i d with an average volume of 300-1,000 mL (Wintour et al. 1978; Tomoda et a / . , 1985). Primary f a c t o r s c o n t r o l l i n g the amniotic f l u i d volume are thought to be fe ta l ur ine and lung f l u i d product ion , f e t a l swallowing and t ransp lacenta l f l u i d exchange (Mescher et al 1975; Harding et al., 1984; Tomoda et al., 1985; Ross et al., 1988; Wlodek et al., 1988). Near term, to ta l f e t a l ur inary output rate averages -700-1,300 mL/day ( H i l l and Lumbers, 1988; Wlodek et al., 1988) and approximately 40-60% of the f e t a l ur ine output, or -300-800 mL of f e t a l ur ine dra ins in to the amniotic c a v i t y via the urethra and the res t in to the a l l a n t o i c c a v i t y via the urachus (Ross et al., 1988; Wlodek et al., 1988). Fetal t racheal f l u i d i s produced at a r e l a t i v e l y constant ra te (4.5 mL/kg 'hr , Mescher et al., 1975) and t h e r e f o r e , in a 2 kg f e t u s , more than 200 mL of the f e t a l t racheal f l u i d enters the pharynx per day, and e i t h e r dra ins into the amniotic c a v i t y or i s swallowed by the f e t u s . A substant ia l quant i ty of f l u i d (mean, 300-1,100 mL/day) i s swallowed by the f e t a l lamb in utero in the l a s t t h i r d of gesta t ion (Bradley and M i s t r e t t a , 1973; Harding et al., 1984; Tomoda et al., 1985). Th is appears to be mainly a mixture of amniotic and fe ta l t racheal f l u i d s , although i t i s poss ib le that s a l i v a and secre t ions of the nasal mucosa are a lso swallowed (Harding et al., 1984). In the proceeding sect ions (sect ions 3.3 and 3 . 4 . 1 . 3 ) , i t has been demonstrated that diphenhydramine is excreted into the fe ta l t racheal and amniotic f l u i d s fo l lowing e i t h e r maternal or f e t a l drug i . v . admin is t ra t ion . One potent ia l consequence of these f i n d i n g s i s 164 r e c i r c u l a t i o n of the drug from the t racheal and amniotic f l u i d s back to the f e t u s , via several poss ib le rou tes , i.e., re-uptake from the lungs, swallowing of amniotic or lung f l u i d s and uptake from amniotic f l u i d by the f e t a l membranes. To examine t h i s p o s s i b i l i t y , we administered diphenhydramine in to the amniotic c a v i t y and monitored drug concentrat ions in the amniotic and f e t a l t racheal f l u i d s , f e t a l and maternal a r t e r i a l plasma in 5 c h r o n i c a l l y ca the te r i zed materna l - fe ta l sheep at 122-137 days g e s t a t i o n . 4 . 5 . 2 . Extent of Drug Excret ion from Amniotic F l u i d by Fetal Swallowing Tomoda et al (1985) measured the disappearance rate of r ad io iod ina ted (*^I) serum albumin from amniotic f l u i d fo l lowing i n j e c t i o n in to the amniotic c a v i t y . The disappearance rate of the l a b e l l e d albumin from the amniotic f l u i d averaged 4.9 ± 2.7 %/hr (n = 17), and the disappearance of the r a d i o a c t i v i t y was accounted fo r by f e t a l swallowing s ince the l a b e l l e d albumin disappears only through f e t a l swallowing: i t i s swallowed, d igested and absorbed into the fe ta l c i r c u l a t i o n as f ree iodine (Tomoda et al., 1985). The f ree iodine is r e a d i l y t r a n s f e r r e d from the fetus to the ewe across the p lacenta and eventua l ly a l l the r a d i o a c t i v i t y of the l a b e l l e d serum albumin can be recovered in maternal ur ine ( P r i t c h a r d , 1965), with n e g l i g i b l e amount of the a c t i v i t y found in fe ta l ur ine (Tomoda et al., 1985). There fore , the p o s s i b i l i t y of r e c i r c u l a t i o n of the l a b e l l e d albumin via f e t a l u r ina t ion in to the amniotic c a v i t y is excluded. When the disappearance rate was c a l c u l a t e d fo r diphenhydramine by use of the same p r i n c i p l e (Lingwood 165 and Wintour, 1984; Tomoda et al., 1985), i.e., disappearance rate expressed as percent per hour, disappearance rate (%/hr) = (1-10 s ) * 100, where s i s the slope of the l i n e a r regress ion l i n e of the terminal l i n e a r phase of the logar i thm of the amniotic drug concentrat ion vs. time data , the average disappearance rate was c a l c u l a t e d to be 6.7 ± 2.0 %/hr . The r a t i o of the l a b e l l e d albumin to diphenhydramine disappearance r a t e , 4 . 9 / 6 . 7 , g ives an est imat ion (-73%) of the propor t ion of the to ta l rate of loss of diphenhydramine that can be accounted f o r by fe ta l swallowing. However, i t should be noted that the disappearance rate from the amniotic f l u i d c a l c u l a t e d f o r diphenhydramine (6.7 %/hr) may not be accounted fo r by f e t a l swallowing alone and i f the drug i s subject to r e c i r c u l a t i o n wi th in the maternal-f e t a l sheep, the disappearance rate would be underestimated. A l s o , the c a l c u l a t e d percentage of the to ta l rate of l o s s from amniotic f l u i d accounted f o r by f e t a l swallowing (73%) may be an overest imat ion i f the drug i s t r a n s f e r r e d by d i r e c t d i f f u s i o n across the placenta from the amniot ic f l u i d to the maternal c i r c u l a t i o n or i f the drug i s taken up by the fetus via the f e t a l c i r c u l a t i o n in the c h o r i o - a l l a n t o i s and the p lacenta and via d i f f u s i o n across the fe ta l s k i n . 4 . 5 . 3 . D i s p o s i t i o n of Diphenhydramine in the Amniotic and Fetal Tracheal F l u i d s , Maternal -Feta l Plasma a f t e r Drug In ject ion into Amniotic Cavi ty In a l l the experiments, the drug concentrat ion in the fe ta l a r t e r i a l plasma was c o n s i s t e n t l y higher than the corresponding value in the maternal plasma throughout the sampling t ime, with an average fe ta l to maternal plasma drug concentrat ion r a t i o being - 4 . 5 . Szeto et al., 166 (1978) reported tha t , fo l lowing i n t r a - a m n i o t i c admin is t ra t ion of meperidine in pregnant sheep, the drug was t r a n s f e r r e d more ex tens ive ly to the mother than to the f e t u s . At t h i s t ime, i t i s not c l e a r what i s respons ib le fo r the d i f f e r e n c e in the t r a n s f e r pat tern of these two drugs. In our present study, diphenhydramine appeared to be taken up p r e f e r e n t i a l l y by the fetus and then fu r ther t r a n s f e r r e d from the fetus to the mother. S i m i l a r observat ions were made f o r metoclopramide: fo l lowing i n t r a - a m n i o t i c a d m i n i s t r a t i o n , the drug was found to be much more ex tens ive ly t r a n s f e r r e d to the f e t a l compartment than maternal compartment (Riggs et a 7 . , 1988b). Further d i s c u s s i o n on the extent of drug t r a n s f e r , i . e . , approximate estimates of the amount of drug t r a n s f e r r e d from the amniotic f l u i d to the fetus or to the mother across the p lacenta by d i r e c t d i f f u s i o n , i s made in the fo l lowing sec t ion (see sec t ion 4 . 5 . 4 ) . The apparent e l im ina t ion h a l f - l i f e of the drug in the amniotic f l u i d (11.0 ± 4.5 hr) i s comparable to that in the f e t a l t racheal f l u i d (10.8 ± 3.8 hr) but i s s i g n i f i c a n t l y longer than that observed in f e t a l (7.7 ± 1.2 hr) or maternal (1.7 ± 0.5 hr) plasma (Table 31). It i s worth not ing t h a t , in the present study, the drug was given e x t r a v a s c u l a r l y in to the amniotic c a v i t y , where drug absorpt ion took place from the amniotic f l u i d into the materna l - fe ta l c i r c u l a t i o n . The absorpt ion process i s r e f l e c t e d in the h a l f - l i f e of the drug in amniotic f l u i d (11.0 h r ) , which i s s i g n i f i c a n t l y longer than the e l i m i n a t i o n h a l f - l i f e in f e t a l (7.7 hr) or maternal (1.7 hr) plasma. A l l of the h a l f - l i v e s obtained f o r maternal and f e t a l plasma, t racheal and amniotic f l u i d in the i n t r a - a m n i o t i c bolus experiments are -2-10 times greater 167 than the corresponding h a l f - l i v e s obtained a f te r maternal or f e t a l i . v . admin is t ra t ion (Table 9, 16 and 31). It would appear that in the case of i n t r a - a m n i o t i c drug i n j e c t i o n experiments, the h a l f - l i f e of diphenhydramine in a l l four f l u i d s has been p r i m a r i l y in f luenced by the absorpt ion of drug across fe ta l and maternal membranes, 7*.e., absorpt ion i s the r a t e - l i m i t i n g s tep . A l l these h a l f - l i v e s are , t h e r e f o r e , l i k e l y an overest imat ion of the "true" h a l f - l i f e i f the drug was t r a n s f e r r e d from the amniotic c a v i t y to the maternal and f e t a l compartments over a prolonged per iod of t ime. The f e t a l t racheal to plasma drug concentrat ion r a t i o c a l c u l a t e d in the in t ra -amnio t ic i n j e c t i o n study averaged 16.4 ± 18.0 (range, 4 .2 -45 .9 ) : t h i s r a t i o i s greater than that obtained a f t e r e i t h e r the maternal i . v . bolus i n j e c t i o n (2.7 ± 2 .4 ) , maternal i . v . i n f u s i o n (3.4 ± 3.6) or f e t a l i . v . i n fus ion (5.4 ± 4 .5 ) . Fetal plasma C m a x obtained fo l lowing drug i n j e c t i o n in to the amniotic c a v i t y (mean, 228.0 ± 101.5 ng/mL) i s lower than the s teady-s ta te fe ta l plasma l e v e l s a f t e r f e t a l drug in fus ion (447.6 ± 112.7 ng/mL) and greater than that a f t e r maternal drug in fus ion (36.3 ± 14.4 ng/mL). Therefore , the greater extent of drug accumulation in the fe ta l t racheal f l u i d fo l lowing i n t r a - a m n i o t i c i n j e c t i o n , as compared to maternal or f e t a l i . v . a d m i n i s t r a t i o n , can not be explained by ex t rac t ion across the pulmonary epi the l ium a lone. It i s poss ib le that diphenhydramine in the amniotic f l u i d could have d i f f u s e d d i r e c t l y into the f e t a l t racheal f l u i d in the airway due to a large concentrat ion gradient between the amniotic and t racheal f l u i d s ( F i g s . 24 and 25), as the amniotic f l u i d enters the pharynx due to fe ta l swallowing. However, fu r ther study is 168 requ i red to i n v e s t i g a t e the pathways and mechanism f o r drug accumulation in to f e t a l lung f l u i d . 4 . 5 . 4 . Extent of Drug E l im ina t ion from the Ewe and Fetus Fol lowing Drug In jec t ion in to Amniotic Cavi ty co Using the maternal and f e t a l AUCQ values combined with the previous est imates of maternal and f e t a l t ransp lacenta l ( C L m f and C L f m ) and nonplacental c learances ( C L m o and C L f 0 ) , approximate est imates of var ious routes of drug e l i m i n a t i o n from amniotic f l u i d were obta ined . Fol lowing drug i n j e c t i o n in to the amniotic c a v i t y , an average of 18.5 ± 5.1 mg of diphenhydramine ( f ree base) was e l iminated from the fe tus oo ( A U C p 0 * C L f 0 ) and 31.4 ± 5.1 mg of the drug e l iminated from the ewe CO ( A U C ^ 0 ' C L m o ) (Table 32) . Therefore , the percentage o f the t o t a l drug a v a i l a b l e in the materna l - fe ta l sheep that was e l iminated by the f e t u s , A U C F o * C L f o - 1 0 0 / ( A U C M o - C L m o + A U C F o ' C L f o ) , averaged 37.1% . Th is (37.1%) i s greater than that a f t e r maternal i . v . bolus i n j e c t i o n (5.0%) or maternal i . v . i n f u s i o n (1.4%) and i s l e s s than, but comparable t o , that a f t e r d i r e c t i n f u s i o n o f the drug to the fetus (45.0%) (see sec t ions 4 .3 .3 and 4 . 4 . 3 ) . The amount of diphenhydramine e l iminated by the mother that was t r a n s f e r r e d d i r e c t l y from the amniotic f l u i d , A U C M Q * C L m o - AUCpQ*CLf m , averaged 8.0 mg (range from -2.1 to 21.5 mg). Hence, an average of 26% (range from -7 to 56%) of the t o t a l drug e l iminated by the mother was CO CO t r a n s f e r r e d d i r e c t l y from amniotic f l u i d [ ( A U C M o ' C L m o - A U C p 0 * C L f m ) • 1 0 0 / oo A U C ^ Q ' C L ^ ] . The r e s t of the t o t a l drug e l iminated by the mother (74 ± 169 20%) was rece ived from the fetus ( A U C F o - C L f m - 1 0 0 / A U C M " - C L m o ) . A l s o , i t appears that approximately 63 ± 8% of the to ta l drug a v a i l a b l e in the 03 materna l - fe ta l sheep was e l iminated by the mother [ A U C M o * C L m o * 1 0 0 / CO 00 ( A U C ^ Q ' C L C T Q + A U C p 0 ' C L f 0 ) ] , of which, an average of 47% was rece ived from the fe tus (eq. 29) and the res t (16± 18%) was rece ived from the amniot ic f l u i d via d i r e c t d i f f u s i o n across the p lacenta (eq. 30) . Percentage of t o t a l drug a v a i l a b l e in the materna l - fe ta l sheep e l iminated by the mother rece ived from the fetus = A U C p o ' C L f r n - 1 0 0 / ( A U C M o - C L r n o + A U C p o ' C L f o ) (eq. 29) Percentage of to ta l drug a v a i l a b l e in the materna l - fe ta l sheep e l iminated by the mother t r a n s f e r r e d d i r e c t l y from the amniot ic f l u i d = ( A U C M ^ C L m o - A U C F ^ C L f m ) - 1 0 0 / ( A U C M o - C L m o + A U C p o ' C L f o ) (eq. 30) O v e r a l l , d i r e c t d i f f u s i o n across the uterus appears to play a minor r o l e in the t r a n s f e r of diphenhydramine from the amniot ic f l u i d to the mother. Rather, i t seems that the t r a n s f e r of substances from the maternal c i r c u l a t i o n to the amniotic f l u i d i s mediated through the f e t u s . In the case of a m p i c i l l i n admin is t ra t ion to pregnant women, accumulation of drug in the amniotic f l u i d occurred , and by 2-3 hr amniot ic l e v e l s exceeded those of e i t h e r maternal or f e t a l plasma (Bray et ai., 1966). However, when the experiment was repeated with a dead f e t u s , only a small amount of a m p i c i l l i n ( less than -20% of that observed in the case of l i v i n g fetus) accumulated in the amniotic f l u i d . These r e s u l t s suggesting that a m p i c i l l i n i s l a r g e l y t r a n s f e r r e d i n d i r e c t l y from the maternal c i r c u l a t i o n to the amniotic f l u i d via the fe tus and to a l e s s e r extent by d i r e c t d i f f u s i o n across the p lacenta are cons is ten t with those obtained from the present diphenhydramine i n t r a -amniot ic i n j e c t i o n study. In one animal (ewe no. 287), uptake of diphenhydramine by c h o r i o - a l 1 a n t o i c membranes was c a l c u l a t e d by the fo l lowing equat ion: Amount of drug taken up by c h o r i o - a l 1 a n t o i c membranes = 2 ^ t 7 - + l - t 7 - ) [ (UVt 7 - + 1 + UVt f ) - ( F A t y + 1 + FAti)]Qum/2 (eq.31) where UVt7- and FAt7- are the drug concentrat ions in umbi l ica l venous and f e t a l a r t e r i a l plasma at sampling time t7- and Qum i s umbi l ica l blood f low. With a f e t a l body weight of 1.4 kg (Table 29) and Qum of 230 mL/min'kg (Cohn et al., 1982), the to ta l uptake by the membrane (eq. 31) was 14.2 mg. Fur ther , the percentage of to ta l drug l o s s from amniotic f l u i d due to c h o r i o - a l l a n t o i c uptake (eq. 32) c a l c u l a t e d in t h i s animal was 33%. Percent of to ta l drug l o s s from amniotic f l u i d due to c h o r i o - a l l a n t o i c uptake = amount taken up by c h o r i o - a l l a n t o i c membrane*100/(administered dose -amount remaining in amniotic f l u i d at the end of the sampling time) (eq. 32) where the amount of the drug taken up by c h o r i o - a l l a n t o i c membranes was 14.2 mg (ca lcu la ted by eq. 31) , the amount of the drug administered i s 43.8 mg as a f ree base (equivalent to 50 mg as hydrochlor ide s a l t ) and the amount of the drug remaining in the amniotic f l u i d was 0.2 mg, c a l c u l a t e d by m u l t i p l y i n g the drug concentrat ion in the amniotic f l u i d at the l a s t sampling time 28 hr (214.3 ng/mL) and an assumed amniotic f l u i d volume of 1,000 mL (Tomoda et al., 1985). The higher drug concentrat ion observed in umbi l ica l venous plasma than in f e t a l a r t e r i a l plasma i s cons is ten t with uptake of the drug from amniotic f l u i d by the c h o r i o - a l l a n t o i s and d e l i v e r y to the fetus via the umbi l ica l venous c i r c u l a t i o n . O v e r a l l , the data suggest p r e f e r e n t i a l uptake of 171 diphenhydramine from amniotic f l u i d by the f e t u s , r e s u l t i n g in a much greater degree of f e t a l drug exposure than maternal exposure. 4 .6 . Pulmonary D i s p o s i t i o n of Diphenhydramine in the Fetal Lamb Fol lowing Maternal or Fetal Infusions to Steady-State In both sheep and humans, the pattern of blood c i r c u l a t i o n during f e t a l l i f e i s d i f f e r e n t from that a f t e r b i r t h . In a d u l t s , systemic venous blood proceeds from the r i g h t atrium to the r i g h t v e n t r i c l e and to the lungs , and the oxygenated blood from the lungs c i r c u l a t e s to the l e f t atrium and v e n t r i c l e and i s then d i s t r i b u t e d in the body. In c o n t r a s t , f e ta l blood c i r c u l a t i o n i s charac te r i zed by the presence of blood shunts, viz, the ductus venosus, foramen ovale and ductus a r t e r i o s u s . In the fe ta l lamb, only a por t ion (-45%) of the oxygenated umbi l ica l venous blood passes through the l i v e r and the remainder (-55%) bypasses the hepat ic c i r c u l a t i o n via the ductus venosus (Reuss and Rudolph, 1980). Approximately 37% of umbi l ica l v e n o u s / i n f e r i o r vena caval blood bypasses the r i g h t heart and flows into the l e f t atrium via the foramen ova le , whereas only a small por t ion (-2%) of super ior vena caval blood enter ing the heart i s d e l i v e r e d to the l e f t atrium via the foramen ovale (Reuss and Rudolph, 1980; Rudolph, 1985). Th is blood flow pattern appears to favor greater oxygen supply to the f e t a l myocardium and the b r a i n . A l s o , in the f e t a l lamb, r i g h t v e n t r i c u l a r output i s -67% of combined v e n t r i c u l a r output . But only -12% of the r i g h t v e n t r i c u l a r output (-7% of the combined v e n t r i c u l a r output) i s d e l i v e r e d to the f e t a l lungs, the res t i s d i r e c t e d via the ductus a r t e r i o s u s to the descending aorta (Rudolph, 1985). 172 In the present study, uptake of diphenhydramine by the fe ta l lamb lung has been assessed by measuring pulmonary ar ter io -venous concentrat ions at s teady-s ta te fo l lowing maternal or f e t a l drug i n f u s i o n . During maternal i n f u s i o n , the drug concentrat ion in c a r o t i d a r t e r i a l plasma was higher than in pulmonary a r t e r i a l or venous plasma due to p r e f e r e n t i a l d i s t r i b u t i o n of d r u g - r i c h i n f e r i o r vena caval blood through the foramen ovale into the l e f t heart and ascending aor ta , and the drug concentrat ion in the pulmonary a r t e r i a l plasma was higher than in pulmonary venous plasma throughout the sampling p e r i o d . ( F i g . 26, Table 33). In c o n t r a s t , during the f e t a l i n f u s i o n , drug concentrat ion in pulmonary ar tery was higher than in c a r o t i d ar tery as the drug was d e l i v e r e d from the jugu la r vein to the r i g h t heart and pulmonary ar tery with the res t of super ior vena caval blood return ( F i g . 27, Table 33). But aga in , diphenhydramine concentrat ion in pulmonary ar tery was higher than in pulmonary v e i n . Fol lowing cessat ion of the fe ta l drug i n f u s i o n , there was a reversa l of the drug concent ra t ion , i n d i c a t i n g net re lease of i n t a c t diphenhydramine from the lung into the f e t a l c i r c u l a t i o n (Table 33). As descr ibed in the preceeding s e c t i o n s , we have found marked accumulation of diphenhydramine in the f e t a l t racheal f l u i d fo l lowing maternal i . v . bolus i n j e c t i o n or i n f u s i o n , f e t a l i n f u s i o n or i n t r a -amniotic i n j e c t i o n in pregnant sheep, and have suggested that the excre t ion of drug via t h i s route may be an important mechanism of drug e l im ina t ion in the f e t u s . O v e r a l l , the d e l i v e r y of diphenhydramine to the lungs via the pulmonary c i r c u l a t i o n would appear to be s u f f i c i e n t to account fo r the l e v e l s observed in t racheal f l u i d . This argument i s 173 based on rough c a l c u l a t i o n s done in the animals employed. It has been assumed that the lung f l u i d volume i s 30 mL/kg (Kit terman, 1984), lung f l u i d product ion rate 4.5 mL/hr 'kg (Mescher, 1975) and a lung blood flow of 38.5 mL/min'kg (Rudolph and Heymann, 1970). Using the measured drug concentrat ions in pulmonary a r t e r i a l and venous plasma and t racheal f l u i d , the to ta l amount of drug taken up by the lungs over the in fus ion per iod [(PA-PV) x pulmonary blood flow x in fus ion time] averaged 293.9 / ig . The amount of drug present in t racheal f l u i d at s teady-s ta te (87.8 fig) was c a l c u l a t e d as the product of to ta l lung f l u i d volume times t racheal drug concent ra t ion , and the amount excreted from the lungs into amniotic c a v i t y (19.7 /xg) as the product of lung f l u i d product ion rate x i n f u s i o n time x t racheal drug concent ra t ion . The drug taken up by the lungs (293.9 /ig) may be e i t h e r present in t racheal f l u i d (87.8 /ig) or excreted into the amniotic c a v i t y (19.7 / ig) , and the remainder (186.4 /ig) i s bound/metabolized by lung t i s s u e . Therefore , i t appears that dur ing the i n f u s i o n p e r i o d , roughly -30% of the to ta l drug d e l i v e r e d to the lungs i s accounted fo r by drug accumulation in the t racheal f l u i d , -7% by f l u i d secre t ion out of the lungs and -63% by c e l l u l a r b inding/metabol i sm. Total drug c learance in the maternal (CLmm, 4402.1 mL/min) and f e t a l (CLff, 357.7 mL/min) compartments obtained in the present study are wi th in the range observed prev ious ly (Table 18), e . g . , 1865.4-4808.0 mL/min f o r CLmm and 240.1-888.4 mL/min fo r CLff. The f e t a l pulmonary ex t rac t ion r a t i o of diphenhydramine averaged 0.08 ± 0.06. Assuming that f e t a l lungs rece ive -7% of the fe ta l combined v e n t r i c u l a r output (-550 mL/min/kg) in near-term sheep (Rudolph and Heymann, 1970), f e t a l 174 pulmonary blood flow i s c a l c u l a t e d to be 77 mL/min (mean f e t a l body weight, 2.0 kg) . Fetal pulmonary c learance of diphenhydramine c a l c u l a t e d as the product of pulmonary blood flow times pulmonary drug ex t rac t ion r a t i o averages 6.2 mL/min at s t e a d y - s t a t e , which comprises -2% of the f e t a l to ta l c learance (mean CLf f = 357.7 mL/min, Table 33) or -3% of the f e t a l nonplacental c learance (mean C L f 0 = 208.4 mL/min, Table 17). The drug secreted in to the t racheal f l u i d appears to be e l iminated from the fetus into the amniotic f l u i d or r e c i r c u l a t e d in to the f e t a l c i r c u l a t i o n via back ex t rac t ion of the drug from the lungs and/or by f e t a l swallowing of the t racheal and amniotic f l u i d s . Pulmonary ex t rac t ion r a t i o of drugs determined a f t e r i . v . bolus i n j e c t i o n could be overestimated i f drug concentrat ions are measured during the d i s t r i b u t i o n phase. A s teady-s ta te approach may be favored fo r assess ing the lung drug c learance . In summary, the pulmonary ex t rac t ion r a t i o of diphenhydramine averaged 0.08 in the f e t a l lamb in utero. The drug c learance by the f e t a l lung accounts fo r -2% of the to ta l drug c learance from the f e t u s . Even with the low rate of pulmonary blood flow in the f e t u s , the d e l i v e r y of the drug to the lung via the pulmonary c i r c u l a t i o n can account f o r the accumulation of the drug in the fe ta l lung f l u i d . 175 5. SUMMARY AND CONCLUSIONS A nitrogen-phosphorus detec t ion-gas chromatographic assay method (GC/NPD), which provides improved s e n s i t i v i t y and s e l e c t i v i t y fo r diphenhydramine, i s repor ted . A 25 m x 0.31 mm c r o s s - l i n k e d , 5% phenylmethyl s i l i c o n e - c o a t e d f u s e d - s i l i c a c a p i l l a r y column ( f i lm th ickness , 0.52 /im) was used fo r a l l ana lyses . The assay procedure involved a s ing le ex t rac t ion s tep , with no sample d e r i v a t i z a t i o n . The s p l i t l e s s i n j e c t i o n mode was employed in the assay, with a 2 /iL sample introduced by an automatic l i q u i d sampler. Standard curves , using orphenadrine as an in te rna l s tandard, were l i n e a r in the range of 2-320 ng/mL of diphenhydramine. This represents an amount of diphenhydramine from -40 pg to 6.4 ng at the de tec tor . Chromatographic separat ion of diphenhydramine and orphenadrine was e x c e l l e n t , with no in te r fe rence from endogenous c o n s t i t u e n t s . The assay method was used in the subsequent s tudies of the pharmacokinetics of diphenhydramine in the nonpregnant and pregnant sheep. Pharmacokinetics of diphenhydramine were charac te r i zed in nonpregnant sheep (n = 6) a f t e r i . v . bolus i n j e c t i o n of 25, 50, 100 and 200 mg doses of diphenhydramine hydrochlor ide on a crossover b a s i s . Plasma drug concentra t ion vs. time p r o f i l e s exh ib i ted mult iexponent ia l c h a r a c t e r i s t i c s . The average d i s t r i b u t i o n h a l f - l i f e ( t ^ a ) increased from 5 to 9 min and the e l im ina t ion h a l f - l i f e (t^ ^) from 34 to 68 min, as the dose was increased . There was a lso an increase in the volume of d i s t r i b u t i o n (l^cfss) from 3 to 6 L /kg , with increas ing dose. The e l im ina t ion h a l f - l i f e and the volume of d i s t r i b u t i o n a f t e r a 200 mg dose 176 were s i g n i f i c a n t l y greater than those obtained a f t e r a 25 mg dose. There was, however, a l i n e a r increase in the area under the plasma drug concentra t ion- t ime curves (AUC), as dose was increased . The average to ta l body c learance (C / .-rg, ~5 L /hr 'kg) remained unchanged regard less of dose. The f ree f r a c t i o n of the drug determined in the nonpregnant sheep plasma by e q u i l i b r i u m d i a l y s i s averaged 0.229 ± 0.080, and the extent of drug binding to plasma pro te in was independent of the drug concentrat ion encountered (30-780 ng/mL) in the nonpregnant sheep in vivo. Concentrat ion- independent b inding of the drug was a lso confirmed by in vitro b inding studies over the drug concentrat ion range of 10-2,000 ng/mL. There fore , i t appears that changes in the volume of d i s t r i b u t i o n are l i k e l y to be a r e s u l t of changes in t i s s u e uptake or binding of the drug, as a funct ion of dose. The p lacenta l t r a n s f e r and pharmacokinetics of diphenhydramine were studied in the c h r o n i c a l l y ca the ter i zed pregnant ewe and fetus fo l low ing i . v . bolus adminis t ra t ion of a 100 mg dose to the ewe (n = 4) over the gesta t iona l age range of 122- 129 days (term 145 days) . S e r i a l samples of the maternal and f e t a l a r t e r i a l b lood, f e t a l t racheal and amniotic f l u i d s were c o l l e c t e d fo r drug a n a l y s i s . Rapid p lacenta l t r a n s f e r was observed with peak fe ta l plasma concentrat ions occurr ing wi th in 5 min a f t e r i n j e c t i o n . The fe ta l to maternal AUC r a t i o averaged 0.85, i n d i c a t i n g s i g n i f i c a n t f e t a l exposure to the drug. The average e l im ina t ion h a l f - l i f e in the ewe (52 min) was not s i g n i f i c a n t l y d i f f e r e n t from that obtained in the fetus (46 min) . The maternal to ta l body c learance and the volume of d i s t r i b u t i o n were 3.6 L /hr -kg and 3.2 L /kg , r e s p e c t i v e l y . There was no s i g n i f i c a n t d i f f e r e n c e in any of these pharmacokinetic parameters between the pregnant and nonpregnant sheep. Approximately -95% of the dose 177 i n j e c t e d to the ewe appeared to be e l iminated by the mother via maternal nonplacental pathways, with only a small por t ion of the dose (-5%) e l iminated by the f e t u s . There was a s i g n i f i c a n t accumulation of the drug in the f e t a l t racheal f l u i d , with the mean r a t i o of the f e t a l t racheal to plasma drug concentrat ions of 2 .8 . Diphenhydramine was a lso accumulated in the amniotic f l u i d , but to a l e s s e r extent than in t racheal f l u i d (amniotic f l u i d to f e t a l plasma drug concentrat ion r a t i o , - 0 . 3 ) . In the f e t u s , diphenhydramine that i s accumulated in the t racheal f l u i d could be e i t h e r swallowed by the fetus or t rans fe r red in to the amniotic c a v i t y fo r fur ther r e c i r c u l a t i o n of the drug wi th in the f e t u s . Transplacenta l and nonplacental c learances of diphenhydramine were determined by use of a general two-compartment open model, with drug e l i m i n a t i o n occur r ing from both compartments. The drug was given by simultaneous i . v . bolus i n j e c t i o n and in fus ion to s teady-s ta te to the c h r o n i c a l l y ca the te r i zed pregnant ewe and, in a separate experiment, to the fetus (n = 8 ) , approximately 48 hrs apar t . Average s teady-s ta te plasma drug concentrat ions a f te r maternal in fus ion was 212.1 ± 67.8 ng/mL in the mother and 36.3 ± 14.4 ng/mL in the f e t u s , r e s u l t i n g in a f e t a l to maternal concentrat ion r a t i o of 0.19 ± 0.10. Fol lowing fe ta l i n f u s i o n s , maternal and f e t a l s teady-s ta te drug concentrat ions were 31.1 ± 11.6 ng/mL and 447.6 ± 185.2 ng/mL, r e s p e c t i v e l y . The plasma f ree f r a c t i o n of diphenhydramine in the pregnant ewe (0.141 ± 0.079) i s not s i g n i f i c a n t l y d i f f e r e n t from that determined in the nonpregnant sheep (0.229 ± 0.080), but i s s i g n i f i c a n t l y smal ler than that in the fetus (0.277 ± 0.087). The to ta l f e t a l c learance (CLf f , 472.7 ± 215.7 mL/min) was r e l a t i v e l y small as compared to the to ta l maternal c learance ( C L m m , 3426.1 ± 905.8 mL/min). 178 The t ransp lacenta l c learance from fetus to mother ( C L f m , 264.4 ± 138.7 mL/min) was ~3 f o l d higher than that from mother to fe tus ( C L m f , 82.4 ± 40.5 mL/min). Maternal nonplacental c learance ( C L m o , 3343.8 ± 890.7 mL/min) accounted f o r 97.8 ± 1.1% of the to ta l maternal c learance whereas the f e t a l nonplacental c learance ( C L f 0 , 208.4 ± 80.4 mL/min) accounted for only 45.2 ± 4.8% of the to ta l f e ta l c learance . There fore , diphenhydramine administered to the ewe appears to be e l iminated mainly by nonplacental pathways, viz, hepa t ic , renal and pulmonary metabolism in the mother. However, in the case where the drug i s given to the f e t u s , both the fe ta l nonplacental and p lacenta l c learances may be important f o r e l im ina t ion of the drug from the f e t u s . The maternal and f e t a l e l im ina t ion h a l f - l i f e of the drug obtained a f t e r maternal in fus ion (51 ± 12 min and 63 ± 32 min, r e s p e c t i v e l y ) are s i m i l a r to those a f t e r f e t a l in fus ions (53 ± 53 and 73 ± 25 min, r e s p e c t i v e l y ) . Diphenhydramine was accumulated in the f e t a l t racheal f l u i d , with an average f e t a l t racheal to plasma drug concentrat ion r a t i o of 3.4 ± 3.6 f o r maternal in fus ions and 5.4 ± 4.5 f o r f e t a l i n f u s i o n s . The e l i m i n a t i o n h a l f - l i f e of the drug in t racheal f l u i d averaged 49 ± 17 min a f t e r maternal in fus ion and 56 ± 16 min a f te r f e t a l i n f u s i o n . These values are s i g n i f i c a n t l y shor ter than that c a l c u l a t e d in the amniotic f l u i d (123 ± 32 min) . A l l these e l im ina t ion h a l f - l i v e s were comparable to those obtained a f t e r maternal i . v . bolus i n j e c t i o n . Diphenhydramine concentrat ions in the tracheal f l u i d were higher than the corresponding amniotic f l u i d drug concent ra t ions . It i s probable that the excret ion of drug in t racheal f l u i d in part accounts fo r the accumulation of the drug in the amniotic f l u i d s ince the t racheal f l u i d i s cont inuously produced at a 179 constant rate from the lungs and flows out , and i s e i t h e r swallowed by the fetus or secreted in to the amniotic sac . The e f f e c t s of diphenhydramine on f e t a l behavioural s t a t e s , i.e., f e t a l e l e c t r o c o r t i c a l (ECoG), e l e c t r o - o c u l a r (EoG) and breathing a c t i v i t i e s , blood gas s t a t u s , a r t e r i a l pressure and heart rate were inves t iga ted in the f e t a l lamb fo l lowing maternal and f e t a l drug in fus ions to s t e a d y - s t a t e . During maternal drug i n f u s i o n , there were dec l ines in the percentage of low voltage ECoG pattern (from 55 to 46%), the percentage of low vol tage a c t i v i t y conta in ing rap id eye movements (from 80 to 55%), the o v e r a l l inc idence of f e ta l breathing (from 42 to 21%) and in the amount of breathing during low voltage ECoG a c t i v i t y (from 67 to 36%). These s e d a t i v e - l i k e e f f e c t s occurred at f e ta l plasma drug concentrat ions (-36 ng/mL) lower than those r e s u l t i n g in d i s c e r n i b l e cent ra l nervous system e f f e c t s in a d u l t s . Drug in fus ion to fetus achieved higher f e t a l plasma drug l e v e l s (-448 ng/mL) and resu l ted in a t rans ien t d e c l i n e in a r t e r i a l P02 and pH, associa ted with t rans ien t tachycard ia and vigorous breathing movements during the i n i t i a l por t ion of the i n f u s i o n . There was a lso a s i g n i f i c a n t f a l l in the amount of low vol tage ECoG pattern (from 51 to 26%) and marked increases in the amount of intermediate vol tage pattern (from 8 to 46%). The two routes of diphenhydramine a d m i n i s t r a t i o n , t h e r e f o r e , r e s u l t e d in two d i s t i n c t patterns of f e t a l behavioural e f f e c t s , which could be a t t r i b u t e d to the markedly d i f f e r e n t f e t a l plasma drug concentrat ions achieved. Given the high use of diphenhydramine during pregnancy, fur ther examination of the e f f e c t s of the drug on the f e t u s , in both animals and humans, i s warranted. 180 The p o s s i b i l i t y of drug r e c i r c u l a t i o n from f e t a l t racheal and amniotic f l u i d s back to the fetus via several p o s s i b l e rou tes , i.e., swallowing of amniotic or lung f l u i d s and uptake from amniotic f l u i d by the f e t a l and maternal membranes, was examined. A 50 mg dose of diphenhydramine hydrochlor ide was in jec ted into the amniotic c a v i t y in pregnant sheep (n = 5) over the gesta t iona l age of 122-137 days. The peak plasma drug concentrat ion obtained in the fetus (228.0 ± 101.5 ng/mL) i s s i g n i f i c a n t l y higher than that in the ewe (43.5 ± 15.6 ng/mL). A lso the plasma drug concentrat ion in fetus was c o n s i s t e n t l y higher than the corresponding value in the ewe throughout the sampling p e r i o d , with the mean maternal to f e t a l r a t i o of 0.23 ± 0.10. The e l i m i n a t i o n h a l f - l i f e of the drug in the amniotic f l u i d (11.0 ± 4.5 hr) was comparable to that in the f e t a l t racheal f l u i d (10.8 ± 3.8 hr) but was s i g n i f i c a n t l y longer than that observed in fe ta l (7.7 ± 1.2 hr) or maternal (1.7 ± 0.5 hr) plasma. These e l im ina t ion h a l f - l i v e s were -2-10 times greater than those observed a f t e r maternal or f e t a l i . v . admin is t ra t ion . The e l im ina t ion h a l f - l i v e s fo l lowing the in t ra -amnio t i c i n j e c t i o n would be expected to be in f luenced p r i m a r i l y by the absorpt ion of drug across fe ta l and maternal membranes, 7 . e . , absorpt ion i s the r a t e - l i m i t i n g s tep . In the f e t u s , the t racheal to plasma drug concentrat ion r a t i o a f t e r i n t r a - a m n i o t i c i n j e c t i o n averaged 16.4 ± 18.0 (range 4 .2 -45 .9 ) . This r a t i o was greater than that obtained a f te r e i t h e r the maternal i . v . bolus i n j e c t i o n (2.7 ± 2 .4 ) , maternal i . v . i n fus ion (3.4 ± 3.6) or f e t a l i . v . i n f u s i o n (5.4 ± 4 .5 ) . Therefore , when the drug was in jec ted into the amniotic c a v i t y , i t appeared to be taken up by d i r e c t d i f f u s i o n from the amniotic f l u i d to fe ta l t racheal f l u i d due to a large concentrat ion 181 gradient between the two f l u i d s . The percentage of t o t a l amount of the drug a v a i l a b l e in the materna l - fe ta l sheep that was e l iminated by the fetus (37.1%) was greater than that a f t e r maternal i . v . bolus i n j e c t i o n (5.0%) or maternal i . v . i n fus ion (1.4%) and was somewhat l e s s than, but comparable t o , that a f te r d i r e c t i n f u s i o n of the drug to the fetus (45.0%). It appears that 63 ± 8% of the to ta l drug a v a i l a b l e in the materna l - fe ta l sheep a f t e r i n t r a - a m n i o t i c i n j e c t i o n was e l iminated by the mother, of which, an average of 47% was rece ived from the fetus and the res t (16%) from the amniotic f l u i d via d i r e c t d i f f u s i o n across the u terus . D i rec t d i f f u s i o n of the drug across the u terus , the re fo re , appears to play a r e l a t i v e l y minor r o l e in the t r a n s f e r of the drug from the amniotic f l u i d to the mother. O v e r a l l , the data suggest p r e f e r e n t i a l uptake of the drug from amniotic f l u i d by the f e t u s , r e s u l t i n g in a much greater degree of f e t a l drug exposure than maternal exposure. The pulmonary d i s p o s i t i o n of diphenhydramine in the f e t a l lamb was studied in the c h r o n i c a l l y instrumented pregnant sheep (n = 3) over the ges ta t iona l age of 128-133 days. The drug was given to the ewe via the maternal femoral catheter or to the fetus via the f e t a l jugu la r vein by simultaneous i . v . bolus i n j e c t i o n and in fus ion to s t e a d y - s t a t e . During maternal i n f u s i o n , the s teady-s ta te drug concentrat ion in c a r o t i d a r t e r i a l plasma was higher than that in pulmonary a r t e r i a l plasma due to p r e f e r e n t i a l d i s t r i b u t i o n of d r u g - r i c h i n f e r i o r vena caval blood through the foramen ovale in to the l e f t heart and ascending aor ta . Drug concentra t ion in the pulmonary a r t e r i a l plasma was c o n s i s t e n t l y higher than in the pulmonary venous plasma throughout the maternal i n f u s i o n experiment. In c o n t r a s t , dur ing f e t a l i n f u s i o n , the s teady-s ta te drug concentrat ion in 182 f e t a l pulmonary a r t e r i a l plasma (598.4 ng/mL) was higher than in c a r o t i d a r t e r i a l (479.0 ng/mL) or pulmonary venous plasma (536.1 ng/mL) as the drug was d e l i v e r e d from the jugu la r vein to the r igh t heart and pulmonary artery with the r e s t of super ior vena caval blood r e t u r n . Fol lowing cessat ion of the f e t a l drug i n f u s i o n , however, there was a reversa l of the drug concent ra t ions , r e s u l t i n g in higher drug concentrat ion in pulmonary venous plasma than in pulmonary a r t e r i a l plasma, which ind ica ted net re lease of the i n t a c t diphenhydramine from the lung into f e t a l c i r c u l a t i o n . The average percent ex t rac t ion by the l e f t f e ta l lamb lung , c a l c u l a t e d by use of the s teady-s ta te pulmonary a r t e r i a l and venous concent ra t ions , averaged 0. 08 ± 0.06 (range, 0.04-0.14) in utero. Thus, despi te the low rate of pulmonary blood flow in the f e t u s , the d e l i v e r y of the drug to the lungs via the pulmonary c i r c u l a t i o n would appear to be s u f f i c i e n t to account for the drug accumulation observed in the fe ta l t racheal f l u i d . B r i e f l y , the f ind ings from the present study may be summarized as f o l l o w s : 1. A c a p i l l a r y GC/NPD assay method with improved s e n s i t i v i t y developed fo r diphenhydramine (lower s e n s i t i v i t y l i m i t , -40 pg at the detector) was found to be s u i t a b l e f o r the pharmacokinetic assessment of the drug in sheep. 2. Dose dependency of the pharmacokinetics of diphenhydramine was examined in the nonpregnant sheep over the dose range of 25-200 mg: a) There were s i g n i f i c a n t increases in the e l im ina t ion h a l f - l i f e (34 to 68 min) and the volume of d i s t r i b u t i o n (3 to 6 L /kg ) , with increas ing dose, b) There was, however, a proport ional increase in AUC, with no change in CZ . T B (5 183 L / h r / k g ) . c) The average f ree f r a c t i o n of diphenhydramine unbound to plasma pro te in (0.229) was independent of plasma drug concentrat ions in vivo. 3. D i s p o s i t i o n of diphenhydramine in the materna l - fe ta l sheep was studied a f t e r a 100 mg maternal i . v . bolus dose: a) There was a rap id and extensive p lacenta l t r a n s f e r of the drug from the ewe to the f e t u s , as r e f l e c t e d by the average fe ta l to maternal AUC r a t i o of 0.85. b) There were no s i g n i f i c a n t d i f f e r e n c e s in the d i s t r i b u t i o n and e l im ina t ion h a l f -l i v e s of the drug between the pregnant ewe and f e t a l lamb, c) There was no s i g n i f i c a n t d i f f e r e n c e in the pharmacokinetics of the drug between pregnant and nonpregnant ewes, d) Diphenhydramine accumulated in amniotic f l u i d and, to a greater extent , in fe ta l t racheal f l u i d . Accumulation of drug in these f l u i d s may play a r o l e in f e t a l drug e l im ina t ion and/or r e c i r c u l a t i o n in the pregnant sheep. 4. Transplacenta l and nonplacental c learances of diphenhydramine were studied fo l lowing separate maternal and fe ta l in fus ions to s t e a d y - s t a t e : a) The to ta l body c learance of the drug in the ewe ( C L m m , 3426.1 mL/min) was s i g n i f i c a n t l y greater than that in the fetus (472.7 mL/min), but when normalized to body weight, the f e t a l c learance value was higher (223.9 vs. 44.3 mL/min). b) The fe ta l to maternal t ransplacenta l c learance ( C L f m , 264.4 mL/min) was -3 f o l d greater than the maternal to f e t a l t ransplacenta l c learance ( C L m f , 82.4 mL/min). c) The average con t r ibu t ion of the p lacenta l c learance to the to ta l drug c learance accounted fo r -2% in the ewe and -55% in the f e t u s , d) Therefore , the drug administered to the ewe appears to be c leared mainly via maternal nonplacental pathways, whereas in 184 the f e t u s , both the f e t a l nonplacental as well as f e t a l to maternal t ransp lacenta l c learances are important. 5. Fetal e f f e c t s of diphenhydramine were studied fo l lowing maternal and f e t a l i n f u s i o n s to s t e a d y - s t a t e : a) With low fe ta l drug concentrat ions (-40 ng/mL), s e d a t i v e - l i k e e f f e c t s were observed in the f e t u s , comprising reduct ion in the amounts of REM sleep and fe ta l breathing a c t i v i t y , b) With higher drug l e v e l s (-450 ng/mL), there was t r a n s i e n t vigorous breathing a c t i v i t y and a d i s r u p t i o n in the normal c y c l i n g of ECoG a c t i v i t y , as r e f l e c t e d in a marked increase in the amount of a t r a n s i t i o n a l ECoG pa t te rn . 6. The p o s s i b i l i t y of r e c i r c u l a t i o n of diphenhydramine from the amniotic f l u i d to the ewe and fetus was studied a f t e r in t ra -amnio t i c drug a d m i n i s t r a t i o n : a) The drug given into the amniotic c a v i t y appears to be taken p r e f e r e n t i a l l y into the fe ta l c i r c u l a t i o n (-84% of the to ta l drug) via the c h o r i o a l l a n t o i s and/or f e ta l swallowing, of which -37% i s e l iminated via f e t a l nonplacental pathways and -47% el iminated by the fe ta l to maternal t ransp lacenta l pathway, b) Approximately 63% of the to ta l drug was e l iminated by the ewe, of which -16% was rece ived from the amniotic f l u i d via d i r e c t d i f f u s i o n across the u terus; the remaining por t ion (-47%) was rece ived from the fetus via the fe ta l to maternal p lacenta l pathway. 7. The pulmonary ex t rac t ion of diphenhydramine in the f e t a l lamb was studied fo l lowing maternal or f e t a l i n f u s i o n to s t e a d y - s t a t e : a) The pulmonary ex t rac t ion r a t i o averaged 0.08 in the f e t a l lamb in utero. b) Even with the low rate of pulmonary blood flow in the f e t u s , d e l i v e r y of 185 the drug to the lung via the pulmonary c i r c u l a t i o n can account fo r the accumulation of the drug in the fe ta l t racheal f l u i d . The p lacenta l permeabi l i ty to l i p i d so lub le compounds does not d i f f e r g r e a t l y in d i f f e r e n t species and, the re fo re , diphenhydramine i s l i k e l y to be t r a n s f e r r e d from mother to fetus in human pregnancy. Whether s i m i l a r pat terns of the d i s p o s i t i o n of the drug would occur in humans i s not known. 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