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Interaction of griseofulvin with metoclopramide, propantheline and phenobarbital in the rat Jamali, Fakhredin 1976

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INTERACTION OF GRISEOFULVIN WITH METOCLOPRAMIDE, PROPANTHELINE AND PHENOBARBITAL IN THE RAT by FAKHREDIN JAMALI Pharm.D., University of Tehran, 1969 M.Sc, University of British Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1976 FakhreSih Jamali, 1976 DOCTOR OF PHILOSOPHY in the Faculty of Pharmaceutical Sciences Division of Pharmaceutics In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of th i s thesis for sc h o l a r l y purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or pu b l i c a t i o n of th i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Faculty of Pharmaceutical Sciences The University of B r i t i s h Columbia Vancouver, B.C. V6T 1W5 Date: October £L / fqj4 i ABSTRACT Phenobarbital pretreatment, in man and rat, has been observed to decrease the plasma levels attained after administration of solid dosage forms of griseofulvin. Various mechanisms including induced metabolism, increased gastrointestinal motility, diminished dissolution rate or a complex mechanism involving enzyme induction, first-pass metabolism and distribution rate-limited elimination of griseofulvin have been suggested. This present investigation was initiated in an attempt to cl a r i f y the mechanism of this interaction. Intravenous administration of griseofulvin in control and phenobarbital dosed rats confirmed that no obvious differences existed in griseofulvin metabolism under the experimental conditions. The influence of gut motility on the absorption of griseofulvin administered as single oral doses of either 100 mg/kg in 0.5% Tween 80 (suspension) or 50 mg/kg in 100% polyethylene glycol (PEG) 600 (solution) was evaluated by pretreating rats with single intraperitoneal doses of either 10 mg/kg metoclopramide hydrochloride or 5 mg/kg propantheline bromide two hours prior to griseofulvin administration. Metoclopramide pretreatment accelerated the absorption rate of griseofulvin suspension (the time of peak-plasma level, T was shortened from 6 to 3 hours) but max reduced i t s total absorption by 50%. Conversely, this treatment regimen increased the total absorption of griseofulvin by 230% when administered in solution form. Propantheline preadministration, however, retarded the absorption rate ( T m a x prolonged from 6 to 19 hours) but increased the total absorption of a suspension of griseofulvin by 50% The propantheline treatment regimen elicited a sharply contrasting effect by decreasing the total absorption (23%) of griseofulvin when given in solution form. i i I t was therefore, concluded that a) the i n t e r a c t i o n of g r i s e o f u l v i n with metoclopramide and propantheline i s formulation-dependent and b) the gut m o t i l i t y has a s i g n i f i c a n t influence on the absorption of g r i s e o f u l v i n . The i n t e r a c t i o n between g r i s e o f u l v i n and phenobarbital was evaluated by administering s i n g l e o r a l doses of a) suspension of 100 mg/kg g r i s e o f u l v i n i n either 0.5% or 2% Tween 80, b) suspensions of 20 or 100 mg/kg g r i s e o f u l v i n i n 70% PEG 300, or c) solutions of 50 mg/kg g r i s e o f u l v i n i n 100% PEG 600. Test r a t s received si n g l e o r a l doses of 15 mg/kg sodium phenobarbital 24 hours p r i o r to g r i s e o f u l v i n administration. The t r e a t -ment reduced the t o t a l absorption of g r i s e o f u l v i n administered i n 0.5% Tween 80 by 50%; The extent of the i n t e r a c t i o n was reduced to an i n -s i g n i f i c a n t l e v e l on increasing the concentration of Tween 80 from 0.5% to 2%. Phenobarbital did not a l t e r the apparent rate of g r i s e o f u l v i n absorption since the peak plasma concentrations were coincident between control and t e s t animals. Phenobarbital treatment did not a f f e c t the a v a i l a b i l i t y of g r i s e o f u l v i n when administered as suspensions or solutions i n PEG as r e f l e c t e d by equivalent areas under the plasma curves at the same dose l e v e l i n test and control animals. It can therefore be concluded that the i n t e r a c t i o n between g r i s e o f u l v i n and phenobarbital i n the r a t i s a) a formulation-dependent phenomenon and b) not caused by either enzyme induction or an increased gut m o t i l i t y . I t i s concluded that when g r i s e o f u l v i n i s administered as a suspension i n 0.5% Tween 80 the phenobarbital-griseofulvin i n t e r a c t i o n i n the rat i s caused by a complex mechanism r e s u l t i n g i n a diminished d i s s o l u t i o n rate and subsequent decreased absorption. Supervisor, i i i TABLE OF CONTENTS Page 1. INTRODUCTION 1 2. LITERATURE SURVEY 4 Pharmacokinetics of Griseofulvin 8 Pharmacokinetics of Griseofulvin Following Intravenous Administration 8 Pharmacokinetics of Griseofulvin Following Oral Administration ; . . . . 11 Distribution of Griseofulvin 19 Elimination of Griseofulvin ; 21 Griseofulvin-Phenobarbital Interaction 24 3. EXPERIMENTAL • 30 Assay Procedure 30 Calibration Curves 30 a. Standard Solutions after Dissolving Griseofulvin in Benzene 30 b. Standard Solutions after Extraction , 31 Gas-Liquid Chromatography of Griseofulvin 35 Internal Standard Solution 40 Extraction of Griseofulvin from Plasma . 40 Dosage Forms 42 Identification of the Materials 43 Equilibrium Solubility of Griseofulvin in the Presence of Polyethylene Glycol 300 and Tween 80 44 Animals and Treatments 44 Plasma Level Studies . . . . 47 Gastric Emptying Experiments 48 Determination of 4-Demethyl Griseofulvin 49 a. Silanization of the Column 49 b. Utilization of Different Columns 51 c. Derivatization of 4-Demethyl griseofulvin j 55 Treatment of Data 57 Continued... iv TABLE OF CONTENTS (Continued) Page 4. RESULTS AND DISCUSSION 60 Determination of Griseofulvin 60 Gas-Liquid Chromatography of Griseofulvin 60 Extraction Procedure 61 Equilibrium Solubility of Griseofulvin in the Presence of PEG- 300 and Tween 80 . . . . 61 Absorption of Griseofulvin Following Oral Administration of Different Dosage Forms 64 Effect of Motility Modifiers on Gastric Emptying 69 Effect of Motility Modifiers on Griseofulvin Plasma Levels . . . . 75 Single Oral Dose of Griseofulvin Suspended in 0.5% Tween 80 . . 75 Single Oral Dose of Griseofulvin Dissolved in 100% PEG 600 . . . 83 Multiple Dose and Long Term Treatment with Propantheline . . . . 90 Intravenous Studies 99: Griseofulvin-Phenobarbital Interaction . . . . . . 109 Griseofulvin Suspensions in Tween 80 110 Griseofulvin in PEG . . . . . . . . 120 5. SUMMARY AND CONCLUSION 137 6. REFERENCES . 140 7. APPENDICES 143 Appendix I, Identification of the Materials Appendix II, Computer Programme V LIST OF TABLES Table Page 3-1 Griseofulvin (Gris) Concentration, and the corresponding Peak Height Ratios (Gris/D) of the Standard Solutions 34 3-II Griseofulvin Recovery Following its Extraction from Aqueous Solutions Containing Rat Plasma 36 3- III Administered Dosage Forms and Different Treatments Applied to the Rat 46 4- 1 Percent Griseofulvin Remaining in Stomach and the Corresponding Plasma Levels in the Rat following Oral Administration of Single Doses of 100 mg/kg Griseofulvin in 0.5% Tween 80 71 4-II Percent Griseofulvin remaining in Stomach following Oral Administration of Single Doses of 50 mg/kg Griseofulvin in Polyethylene Glycol 600 to the Control, Metoclopramide Hydrochloride Pretreated Rats (10 mg/kg) and Propantheline Bromide i Pretreated Rats (5 mg/kg) 73 4-III Plasma Levels of Griseofulvin (Micrograms per Milliliter) following Oral Administration of 100 mg/kg in 0.5% Tween 80 to Control and Test Animals , 77 4-IV Plasma Levels of Griseofulvin (Micrograms per Milliliter) following Oral Administration of 100 mg/kg in 0.5% Tween 80 to Rats Receiving Propantheline (5 mg/kg, i.p.), two hours prior to Griseofulvin Administration . 78 4-V Peak Plasma Levels (Cmax) and its Time of Occurrence (Tmax) Following Administration of 100 mg/kg Griseofulvin in 0.5% Tween 80 to the Control, Metoclopramide Hydrochloride (10 mg/kg) and Propantheline Bromide (5 mg/kg) Pretreated Rats 80 4-VI Area Under Plasma Concentration-Time (y.;g.hr.ml •"?") of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control, Metoclopramide Hydrochloride Pretreated and Propantheline Bromide Pretreated Rats . . . 81 Continued... vx Table 4-VII 4-VIII 4-IX 4-X 4-XI 4-XII LIST OF TABLES (Continued) Plasma Levels of Griseofulvin (Microgram per Milliliter) following its Oral Administration in a Single 50 mg/kg in PEG 600 Dose in Control and Test Rats Plasma Levels of Griseofulvin (Micrograms per Milliliter) after a Single Oral Dose (50 mg/kg) in PEG 600 in Control and Test Rats . . . . . . . . Area Under Plasma Concentration-Time Curves, AUC (yg.hr.ml--*-) and the Peak Plasma Concentrations, Cmax (yg/ml) Following Oral Administration of Single Doses of Griseofulvin (50 mg/kg) in Polyethylene Glycol 600 (solution) to the Control, Metoclopramide Hydrochloride Pretreated (10 mg/kg) and Propantheline Bromide Pretreated (5 mg/kg) Rats Plasma Levels of Griseofulvin (y g/ml) following its Oral Administration (100 mg/kgiin 0.5% Tween 80) to Animals receiving either Single or Multiple Doses of Propantheline Plasma Levels of Griseofulvin (y g/ml) following its Oral Administration (100 mg/kg in 0.5% Tween 80) to the Control and Test Rats. Control Animals were treated Intraperitbneally with 1 ml of Distilled Water while Test Rats received 5 mg/kg Propantheline Bromide Intraperitoneally Twice Daily 7 days Prior to and During the Plasma Studies Biological Half-Life of the Post-Distribution Phases (0.693/g) and Area Under the Griseofulvin-Plasma-Time Curves (AUC) in the Rat after Intravenous Administration of Single Doses of the Drug in Poly-ethylene Glycol 600. Test Rats received Single Doses of Metoclopramide Hydrochloride (10 mg/kg), Propantheline Bromide (5 mg/kg) or Sodium Pheno-barbital (15 mg/kg) prior to the Griseofulvin Administration Page 85 86 88 93 97 105 4-XIII Plasma Levels of Griseofulvin (Micrograms per Milliliter) following Oral Administration of 100 mg/kg in 0.5% Tween 80 in the Control and Test Rats. Phenobarbital (15 mg/mg) was given Orally to the Test Rats, 24 hours prior to Griseofulvin Administration 112 Continued... v i i LIST OF TABLES (Continued) Table Page 4-XIV Area Under Plasma Concentration-Time Curves, AUC (yg.hr.ml - 1) of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control and Phenobarbital Pretreated Rats 113 4-XV Peak Plasma Levels, Cmax and their Time of Occurrence, Tmax Following Oral Administration of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control and Phenobarbital Pretreated Rats . . . . . 114 4rrXVI Griseofulvin-Plasma Levels <y g/ml) following Oral Administration of Single Doses of 100 mg/kg Griseofulvin in 2% Tween 80 to the Control Rats and Rats Pretreated orally with Single Doses of 15 mg/kg Phenobarbital Sodium 24 Hours Prior to the Griseofulvin Administration . . . . 117 4-XVII 4-XVIII 4-XIX 4-XX Area Under Plasma Concentration-Time Curves, AUC (ug.hr.ml~l), Peak Plasma Concentrations, Cmax (yg/ml) and their Time of Occurrence, Tmax (hr) of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 2% Tween 80 to the Control and Phenobarbital Pretreated Rats. . . 118 Plasma Levels of Griseofulvin (Microgram per M i l l i l i t e r ) following i t s Oral Administration of a Single 50 mg/kg in PEG 600 Dose i n Control and Test Rats. Phenobarbital Sodium (15 mg/kg) was given Orally to the Test Animals, 24 hours Prior to Griseofulvin Administration 122 Area Under Plasma Concentration-Time Curves, AUC (pg.hr.ml - 1) and the Peak Plasma Concentrations, Gmax (yg/ml) Following Oral Administration of Griseofulvin (50 mg/kg) in Polyethylene Glycol (solution) to the Control and Phenobarbital Pretreated Rats . . . . 123 Griseofulvin-Plasma Levels (yg/ml) following Oral Administration of Single Doses of 20 mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control Rats and Rats Pretreated Orally with Single Doses of 15 mg/kg Phenobarbital Sodium 24 hours Prior to the Griseofulvin Administration 127 Continued. v i i i LIST OF TABLES (Continued) Table Page 4-XXI Area Under Plasma Concentration-Time Curves, AUC (yg.hr-.ml""-") and the Peak Plasma Concentrations, Cmax (yg/ml) Following Oral Administration of 20 mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control and Phenobarbital Pretreated Rats 128 4-XXII Griseofulvin-Plasma Levels (yg/ml) following Oral Administration of Single Doses of 100 mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control Rats and Rats Pretreated Orally with Single Doses of 15 mg/kg Phenobarbital Sodium 24 Hours Prior to the Griseofulvin Administration 4-XXIII Area Under Plasma Concentration-Time Curves, AUC (yg.hr .ml--*•) and the Peak Plasma Concentrations, Cmax (yg/ml) Following Oral Administration of 100 mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control and Phenobarbital Pretreated Rats . . 5-1 Summary of the Observed Effects of Pretreatment with Single Doses of Metoclopramide, Propantheline or Phenobarbital on the Maximum Plasma Level, Cmax, and its Time of Attainment, Tmax, and the Area Under the Plasma Level-Time Curve, AUC, of Griseofulvin Following its Oral Administration in Preparations containing Tween 80 or Polyethylene Glycol (PEG) to the Rat ix LIST OF FIGURES Figure Page 2-1 Biliary flow, biliary bile acid concentration and biliary acid excretion in control rats and rats treated with phenobarbital (75 mg/kg) once daily for four days. From CD. Klaassen, J. Pharmacol. Exp. Ther., 176, 743, 1971 17 2-2 Solubility of griseofulvin as a function of simulated intestinal bile salt mixture concentration at 37° (Reference 52) 20 2-3 Blood-levels of griseofulvin in human volunteers given a single oral dose (500 mg). Control group received griseofulvin only; test group received phenobarbitone before griseofulvin (Reference 16) 25 2-4 Effect of phenobarbitone on blood levels of griseofulvin in male and female rats (Reference 17) 25 2-5 Griseofulvin plasma levels as a function of time of one human subject in presence and absence of phenobarbital treatment (Reference 20) 27 Continued LIST OF FIGURES (Continued) Figure Page 3-1,a Standard curves for griseofulvin (860 peg of internal standard) 32 3-1,b Standard curve for griseofulvin (860 peg internal standard) in concentration range from 4 to 40 ng (direct solutions) 33 3-2 Gas chromatograph of griseofulvin and diazepam (internal standard) following injection of 5 u l of an extracted sample of plasma 37 3-3 Gas chromatographs obtained following injection of 5 ul,iof samples containing extracts of the solutions of metoclopramide hydrochloride, propantheline bromide or sodium phenobarbital after extracting under identical conditions to those for griseofulvin, and gas chromatogram following injection of 5 y l of internal solution containing 860 peg diazepam . . . . 41 3-4 Gas chromatogram of 4-DMG following injection of 4 ng of the metabolite (in 1 yl) into a column packed with 3% OV 25 50 3-5 Gas chromatogram of 4-DMG following injection of 8 ng of the metabolite (in 2 yl) into a column packed with 3% OV 25 and treated with S i l y l 8 . . . . 52 3-6 Gas chromatogram of 4-DMG following injection of 4 ng of the metabolite (in 1 yl) into a column packed with 3% OV 17 53 3-7 Gas chromatogram obtained following injection of 4 ng 4-DMG (in 1 yl) into a column packed with 10% OV 101 (column temperature, 285°) . 54 3- 8 Gas chromatograms obtained following injection of 200 and 400 peg methylated.. 4-DMG (in 5 yl) into a column packed with 3% OV 25 '<. 56 4- 1 Equilibrium solubility of griseofulvin in aqueous solutions containing different concen-trations of polyethylene glycol 300 at room temperature and 37° 62 Continued... x i LIST OF FIGURES (Continued) Figure Page 4-2 Griseofulvin-plasma levels vs time following oral administration of single doses in 0.5% Tween 80 (100 mg/kg), 2% Tween 80 (100 mg/kg), 70% polyethy-lene glycol 300 (20 mg/kg), 70% polyethylene glycol 300 (100 mg/kg), and 100% polyethylene glycol 600 (50 mg/kg) 66 4-3 Percent griseofulvin remaining in stomach' in the rat following administration of a single oral: dose ^ of 100 mg/kg griseofulvin in 0.5% Tween 80 . . 70 4-4 Percent griseofulvin remaining in stomach in the rat following administration of a single oral dose of 50 mg/kg griseofulvin in polyethylene glycol 600 72 4-5 Plasma concentrations of griseofulvin as a function of time following oral administration of a 100 mg/kg dose of a suspension of micronized griseofulvin in 0.5% Tween 80 to the control rats, and rats pretreated with either 10 mg/kg metoclopramide hydrochloride or 5 mg/kg propantheline bromide . . . . . . . . 76 4-6 Plasma concentration of griseofulvin as a function of time following oral administration o f i a 1single dose of 50 mg/kg griseofulvin in PEG 600 'to the control, and rats pretreated with either 10 mg/kg metoclopramide hydrochloride or 5 mg/kg propantheline bromide 84 4-7 Griseofulvin-plasma concentrations following oral administration of single doses of 100 mg/kg griseofulvin in 0.5% Tween 80 to the rats pretreated iritraperitoneally with Either-a^singlehdbseiofxprcbpantheline^Xrafcs d & e) .orc.multapiesdoses, (rats .,a, -b & c), ' . •'. V 91 4-8 Griseofulvin-plasma concentrations following oral administration of single doses of 100 mg/kg griseo-fulvin in 0.5% Tween 80 to the rats treated intra-.. peritoneally with 5 mg/kg propantheline bromide twice daily, 7 days prior to and during the plasma studies . . . . 94 4-9 Computer fitt e d griseofulvin-plasma concentrations vs time following intravenous administration of a single dose of 26 mg/kg griseofulvin dissolved in polyethylene glycol 600 to the rat . . 101 Continued... x i i LIST OF FIGURES (Continued) Figure Page 4-10 Computer fi t t e d logarithm of plasma concentrations vs time curve following intravenous administration of a single dose of 26 mg/kg griseofulvin dissolved in polyethylene glycol 600 to the rat 102 4-11 Computer fitt e d curves of logarithm of plasma concentrations vs time and residual concentrations vs time following intravenous administration of single doses of griseofulvin/polyethylene glycol 600 to a control rat, and rats pretreated with single doses of metoclopramide, propantheline or phenobarbital . . . . 104 4-12 Mean griseofulvin-plasma levels vs time following oral administration of single doses of 100 mg/kg in 0.5% Tween 80 to the control, and phenobarbital pretreated rats .: I l l 4-13 Mean plasma levels vs time following oral administration of 100 mg/kg griseofulvin in 2% Tween 80 to the control, and phenobarbital pretreated rats . . . . 116 4-14 Mean griseofulvin-plasma levels-time following oral administration of single doses of 50 mg/kg in,100% polyethylene glycol 600 to the control, and phenobarbital pretreated rats 121 4-15 Mean griseofulvin plasma levels vs time following oral administration of 20 mg/kg of the drug in 70% polyethylene glycol to the control, and phenobarbital pretreated rats 126 4-16 Plasma levels of griseofulvin vs_ time following administration of 100 mg/kg of the drug in 70% poly-ethylene glycol 300 to the control, and phenobarbital pretreated rats 129 x i i i ACKNOWLEDGMENTS I wish to thank Dr. J. E. Axelson, thesis supervisor, Drs. F. S. Abbott, G. D. Bellward, D. V. Godin, A. G. Mitchell, B. E. Riedel and B. D. Roufogalis, committee members, and Dr. W. L. Hayton for their guidance during the course of this work. xiv to Elaheh and Vafa 1 1. INTRODUCT ION T h e r e i s i n c r e a s i n g i n t e r e s t i n d r u g - d r u g i n t e r a c t i o n s a n d a n e e d f o r m o r e k n o w l e d g e o f t h e m e c h a n i s m s i n v o l v e d . A d r u g i n t e r a c t i o n may b e d e f i n e d a s a n y r e a c t i o n b e t w e e n o n e d r u g a n d a n o t h e r s u b s t a n c e w i t h i n o r o u t o f t h e b o d y . I n t e r a c t i o n s may t a k e p l a c e a t d i f f e r e n t l e v e l s s u c h a s a b s o r p t i o n , p l a s m a p r o t e i n b i n d i n g a n d m e t a b o l i s m . G r i s e o f u l v i n i s a n o r a l l y a v a i l a b l e a n t i f u n g a l a g e n t w h o s e e r r a t i c a n d i n c o m p l e t e a b s o r p t i o n h a s b e e n s h o w n t o b e d i s s o l u t i o n - r a t e l i m i t e d . T h e l o w s o l u b i l i t y o f g r i s e o f u l v i n i n b i o l o g i c a l f l u i d s (15 mg/1, a t 37°) a n d i t s e x t e n s i v e m e t a b o l i s m may m a k e t h e d r u g v u l n e r a b l e t o i n t e r -a c t i o n i f o t h e r d r u g s a r e a d m i n i s t e r e d c o n c o m i t a n t l y . T h e s e p r o p e r t i e s a l s o m a k e g r i s e o f u l v i n a g o o d m o d e l f o r d r u g - d r u g i n t e r a c t i o n s t u d i e s . T h e i n f o r m a t i o n o b t a i n e d f r o m s u c h s t u d i e s may b e u s e f u l i n c l a r i f y i n g o t h e r s i m i l a r i n t e r a c t i o n s . Many d r u g s h a v e b e e n s h o w n t o e f f e c t i v e l y a l t e r t h e m e t a b o l i s m a n d a b s o r p t i o n o f o t h e r d r u g s a d m i n i s t e r e d c o n c u r r e n t l y . T h e w e l l - k n o w n m e t a b o l i s m i n d u c i n g e f f e c t o f p h e n o b a r b i t a l h a s b e e n t h e s u b j e c t o f many s c i e n t i f i c w o r k s . R e c e n t l y t h e i n f l u e n c e o f g u t m o t i l i t y m o d i f i e r s o n t h e a b s o r p t i o n o f some d r u g s h a s b e e n d i s c u s s e d b y many a u t h o r s . T h e r e i s e v i d e n c e i n t h e l i t e r a t u r e t o s u g g e s t t h a t g r i s e o f u l v i n i s s u s c e p t i b l e t o b o t h t y p e s o f d r u g i n t e r a c t i o n . I n 1963 B u s f i e l d e_t a i r e p o r t e d a g r i s e o f u l v i n - p h e n o b a r b i t a l i n t e r -a c t i o n i n man a n d c o n f i r m e d t h i s i n 1964 i n t h e r a t . T h e b a r b i t u r a t e r e d u c e d t h e p l a s m a l e v e l s o f o r a l l y a d m i n i s t e r e d g r i s e o f u l v i n s i g n i f i c a n t l y i n b o t h s p e c i e s . T h e y s u g g e s t e d t h a t t h e i n t e r a c t i o n w a s d u e t o a n i n d u c e d m e t a b o l i s m 2 of g r i s e o f u l v i n caused by pretreatment with phenobarbital. However, f a i l u r e to provide convincing evidence to support the hypothesis l e f t the subject open f o r further studies. In 1970, Riegelman et_ al observed the same i n t e r a c t i o n when g r i s e o f u l v i n was administered o r a l l y to man i n t a b l e t form. However, following intravenous administration of the drug i n the presence and absence of phenobarbital, they noticed no s i g n i f i c a n t e f f e c t on the griseofulvin-plasma concentrations. No change i n the slope of the p o s t - d i s t r i b u t i o n portion of the plasma l e v e l s vs_ time curves was observed with e i t h e r route of administration. Therefore, Riegelman et^ a l suggested that the observed griseofulvin-phenobarbital i n t e r a c t i o n could not be at the l e v e l of metabolism and might r e s u l t from a reduced absorption. They offered two p o t e n t i a l mechanisms: phenobarbital might "decrease the degree of dispersion of the drug from the tablet granules," or decrease "the t r a n s i t time i n the upper i n t e s t i n e , where g r i s e o f u l v i n appeared to be absorbed at a maximal ra t e . " These proposals were based on the a b i l i t y of phenobarbital to enhance the b i l e flow. In 1972 Axelson postulated another possible mechanism, namely, that i f the elimination of g r i s e o f u l v i n was r a t e - l i m i t e d by i t s access to the l i v e r rather than by i t s metabolism per se, enzyme induction would have no e f f e c t on the slope of the p o s t - d i s t r i b u t i o n phase of the drug. However, i t might s i g n i f i c a n t l y increase the f r a c t i o n of the dose metabolized on the f i r s t pass through the l i v e r upon o r a l administration. In view of t h i s p o s s i b i l i t y i t was of i n t e r e s t to examine the g r i s e o f u l v i n i n t e r a c t i o n with phenobarbital more c l o s e l y . The purpose of t h i s work was twofold: f i r s t l y , to examine i n 3 detail the griseofulvin-phenobarbital interaction to ascertain the precise mechanism responsible for the reduction in apparent availability of griseofulvin after phenobarbital preadministration. Secondly, to assess the characteristic(s) , i f any, of any interaction between the mp^ del poorly absorbed drug, griseofulvin, and drugs capable of altering gastric emptying and motility. Administration ofdsolid and solution dosage forms of griseofulvin in the presence of phenobarbital has provided conclusive evidence that the mechanism of their interaction exists at the level of absorption. Specifically, phenobarbital has been shown to interfere indirectly with the dissolution of the poorly soluble and poorly absorbed drug griseofulvin. Finally^ this project has clarified the effect of motility modifying drugs on the absorption of a poorly absorbed drug. 4 2. LITERATURE SURVEY Griseofulvin (7-chloro-2',4,6 trimethoxy-6'-methylspiro benzofuran-2(3H), l'-(2)cyclohexene-3,4' dione), a chemically neutral systemic antifungal antibiotic, is commonly used in the treatment of dermatophyte infections in human (1-4) and domestic animals (5). As a result of inherently poor aqueous solubility (approximately 15 mg/1 at 37°), the drug is slowly, erratically and incompletely absorbed from the gastrointestinal (Gl) tract of humans (6), Despite inherently poor absorption, griseofulvin is an exceptionally effective antifungal agent after oral administration. Griseofulvin has been shown to e l i c i t i t s effect after incorporation into the cells of the skin, preventing the downward cellular proliferation of topical fungal infections. In recent years the very fact that griseofulvin is poorly absorbed has resulted in the use of this compound as an investigational model to examine the various parameters affecting drug absorption. The slow and erratic absorption characteristics of this drug are a direct effect of unusually slow dissolution after oral administration as a solid dosage form. Early experimentation with griseofulvin led to o^b.sercv.ations;eabo.ut.othe aboi.1 L ' effect of particle size reduction (7), surfactants(8) and-dietary l i p i d s (9) on the absorption of a drug with low solubility and poor I availability. The effects of particle size reduction (7), micellar solubilization (10), molecular dispersion (11), and emulsification (12) on improving the absorp-tion of poorly absorbed drugs are well recognized. However, far less i s known about the effect of gastrointestinal motility on the absorption of poorly absorbed 5 drugs (13-15 ) . When the i n t e r a c t i o n between g r i s e o f u l v i n and phenobarbital was f i r s t reported (16-18) i t appeared to be the r e s u l t of a drug metabolism induction caused by phenobarbital. This f i n d i n g would be consistent with the usual e f f e c t of phenobarbital i n inducing the l i v e r microsomal metabol-i z i n g enzymes (19). Later speculation by Riegelman et a l (20) clouded t h i s view considerably and raised doubts as to whether the drug i n t e r a c t i o n was the r e s u l t of a metabolism change at a l l . Riegelman e_t a l provided evidence suggesting that the griseofulvin-plasma l e v e l s were diminished a f t e r pheno-b a r b i t a l administration as a r e s u l t of reduced absorption. They speculated that the mechanism f o r t h i s e f f e c t was mediated either by an increase i n Gl m o t i l i t y or by a diminished d i s s o l u t i o n rate caused by b i l e flow a l t e r a t i o n i n the presence of phenobarbital. The barbiturate has been shown to stimulate b i l e flow (21-23). Riegelman e_C a l suggested that the increase i n the volume of the b i l e , i n turn, accelerates the m o t i l i t y of the proximal segment of the small i n t e s t i n e although no supporting evidence was given by the authors. An a l t e r n a t i v e suggestion (;2:4):;':feo*:;thcaliltefigRiregelman 'et• a l was that, i f the metabolism of g r i s e o f u l v i n were l i m i t e d by i t s access to the l i v e r , then the rate of blood flow would be the c o n t r o l l i n g step i n the me.tabolism, i . e . , blood flow-rate l i m i t e d metabolism (30). Therefore, the h a l f - l i f e of the disappearance of the drug from plasma would not i n d i c a t e the true f u n c t i o n a l capacity of the enzyme system due to the blood flow rate l i m i t e d supply of the substrate to the metabolizing organ. Phenobarbital pretreatment has been shown (1 7) to induce the metabolism of g r i s e o f u l v i n by rat l i v e r s l i c e s . In v i v o , however, t h i s might not be the case. I f the metabolism of g r i s e o f u l v i n were blood-flow rate l i m i t e d then an enzyme induction would not influence the rate 6 of metabolism, i . e . , the rate of metabolism i s independent of the e f f i c i e n c y of the metabolizing enzymes. i A change i n the blood flow-^rate, however, may r e s u l t i n a '! consequent a l t e r a t i o n of the rate of metabolism. Branch e_t al (30) reported that intravenous administration of propranolol s i g n i f i c a n t l y prolonged the h a l f - l i f e of l i d o c a i n e . Since the prolonged h a l f - l i f e was accompanied by a reduction i n the concentration of l i d o c a i n e i n the hepatic vein they suggested that the e f f e c t of propranolol was to decrease the blood flow-rate i n the hepatic p o r t a l vein and hence reduce the rate of d e l i v e r y of l i d o c a i n e to i t s major s i t e of elimination i n the l i v e r . Ohnhaus et a l (31 ) measured the blood flow by implanting a thermo-couple i n the r i g h t lobe of the r a t l i v e r . They observed that treatment with phenab;acb.ita'>.h(3.0ifcmg)/l'kg±daliIy (f-olr n^/daysj^iincgejase'd cggiQ)l±gg£. blood flow by, 3!7.-ab7*5%-2inv the 3r7at.,75%. Although i n a system of blood flow-rate l i m i t e d metabolism, enzyme induction would not a f f e c t the rate of metabolism, i t may very well increase the extent of metabolism. However, t h i s seems u n l i k e l y , because g r i s e o f u l v i n i s extensively metabolized following i t s administration (,25-29). Only trace q u a n t i t i e s of i n t a c t g r i s e o f u l v i n have been recovered from the urine of r a t s (25),-mice (26) and man (27). The recovered quantity of the i n t a c t g r i s e o f u l v i n i n rabbits (28) and dogs (,29) has been n e g l i g i b l e . This suggests that the extent of g r i s e o f u l v i n metabolism cannot be increased.cany f£ur.ther by pre-administration of phenobarbital. A f t e r c a r e f u l study of the reported i n t e r -a ction between g r i s e o f u l v i n and phenobarbital ( 16.-20^), i t became apparent that the evidence accumulated was i n s u f f i c i e n t to point to the p r i n c i p a l 7 mechanism involved in this interaction. Further examination of the griseofulvin-phenobarbital interaction was, therefore, necessary. 8 Pharmacokinetics of Griseofulvin The kinetics of absorption, distribution and elimination of griseofulvin have been studied in man ( 3 2 ) , rabbit ( 3 3 ) , dog ( 3 4 ) and to a limited extent in the rat ( 35 . ) . Pharmacokinetics of Griseofulvin Following Intravenous Admin- istration The absorption kinetics of any drug are best studied when a complete understanding of its distribution and elimination is obtained after intravenous (iv) administration. Before initiation of an absorption study, as in this case to look for a possible interaction, i t is necessary to evaluate the pharmacokinetics of a drug following iv dosing. This rules out any unusual gut metabolism or" first-pass metabolism. These phenomena may likely be overlooked after oral administration of the drug unless suitable comparisons are made to the iv route. A known amount of drug is introduced into the body intravenously and the basic pharmacokinetic parameters describing distribution and elimination are estimated by following the time-course of the drug. The pharmacokinetic parameters for distribution and elimination are then estimated in the absence of incomplete absorption, gut metabolism or first-pass metabolism. In the case of poorly soluble drugs, such as griseofulvin, iv administration presents certain problems, e.g., a dose of 200 mg of griseo-fulvin would require approximately 20 1 of saline to dissolve the drug. Different solvents have been used to overcome the problem. Bedford et al (:35) dissolved griseofulvin in 75% N,N-dimethylformamide in water while Rowland et al ( 3 2 ) used polyethylene glycol (PEG) 300 to prepare iv preparations of griseofulvin. 9 Nevertheless, after iv administration i t has been reported that the decline of griseofulvin plasma levels in man, dog and rabbit can be described by a two-compartmental open model (page 107). The following equation defines such a^ .model: . . -at . „ - pt s Cp = A e + Be H (1) r o o where Cp represents the plasma concentration at any time, t, A and B are the ordinate concentration intercepts of the distribution and post-distribution phases at t = 0. The rate constants of the distribution and post-distribution phases are a and 3. The half-life (tJg) of the distribution phase (^ **^ "*) has a been reported to be approximately 4, 6, and 60 min in the dog ('3.4), rabbit (.33) and man ( 3 2 ) respectively. The post-distribution phase has a longer th (°' 6 9 3) ; 0;83^hrminedpgC >1328„..hr;in rabbit andbfromQ9.;5ihrrrto 21.0 "hr i n man. .... The amount of the drug in the central compartment at time t, Xg, can be calculated by multiplying the right hand stifdelqof lEqty ifcb.yytKe volume of distribution, Vd: X,, = Vd(A e" a t + B e" e t) (2) B o o The volume of distribution is an indication of the extent of distribution of the drug in the b o d y . In humans ( 3 2 ) and dogs (3.4) Vd has been reported to be approximately 56 and 10 1 respectively. In the rabbit (3. 3 ) , however, i t has been observed that the Vd for this drug is not constant. This was attributed to inconsistent plasma levels and consequently the area under the plasma concentration vs_ time curves (AUC) even at constant dosage. 'A ... twofold increase in the administered iv dose had no effect on the area under the plasma-level vs time curve (AUC). The mean AUC ± 95% confidence level after a single iv dose of 5 mg/kg of griseofulvin in 5 rabbits was observed to 10 be 350 ± 134 yg/min/ml, while i t is 306 ± 81 yg/min/ml following a single dose of 10 mg/kg in 10 rabbits. No adequate explanation for this unusual observation has been offered. The volume of distribution of the central compartment, Vd, is calculated using Eq. 3: Vd = dose/i».(A + +3B ) (3) C O 0 o From Equations 1 and 3 i t becomes obvious that inconsistent plasma levels (Cp) will give rise to inconsistent Vd, since A q and B q are proportional to Cp, and Vd is indirectly proportional to the former parameters. The area under the concentration v& time curve is usually accepted as being directly proportional to the amount of drug in the body. However, after iv administration to rabbits, i t does not seem to be a valid assumption. • / In the rat, Bedford et al ( 3 5 ) anticipated another difficulty. They suggested that after iv administration of griseofulvin more than 90% of.the drug dwasppe-lim-inated t'from...the! canimaltne-.c during the. first few minutes. No explanation was given for this phenomenon. Nevertheless, the rapid elimination of the drug in the first few minutes post-dosing introduces difficulties in estimating pharmacokinetic parameters of griseofulvin in the rat. Likewise and contrary to expectation, Chiou and Riegelman (through reference 3 6 •, Figs; 3 and 4), obtained an AUC following a single 50 mg iv dose of griseofulvin to a dog which seemed to be substantially lower than those of equal doses of orally administered solid dosage forms. This is inconsistent with the fact that cumulative urinary excretion of the major metabolite of griseofulvin following iv administration was higher than those of an orally administered dose ( 3 6 ) . 11 Pharmacokinetics of Griseofulvin Following Oral Administration -., Due to the low solubility properties (15 mg/1 at 37°C)t griseofulvin absorption from the gastrointestinal tract is erratic and incomplete. Katchen and Symchowicz (6) found a good correlation between dissolution rate of griseo-fulvin in simulated intestinal fluid and its absorption in man. They explained that, i f the gastrointestinal fluid is saturated with respect to griseofulvin when a dose of 500 mg is administered, a minimum of 33 1 of fluid is required to transfer the drug from the gastrointestinal tract into the systemic circulation, as shown in the following calculation: Dose _ 500 mg _ _„ Solubility 15 * mg/1 " Therefore, i t appears reasonable to suggest that the absorption of griseofulvin is dissolution rate limited. Twenty four hours after administration of griseofulvin Davis et_ al (2 8 ) recovered 55% of an oral dose of griseofulvin from the alimentary tract of the rat, of which 37.5% was in the faeces. Rowland et al ( 3 2 ) reported that, in man, from 27 to 72% of oral doses of micronized griseofulvin was absorbed, giving an indication of the extreme variability of the absorption process. In cats, Bedford e_t al ( 3 5 ) demonstrated that the major griseofulvin absorption site is the upper part of the small intestine. They ( 3 5 ) admin-istered griseofulvin directly into ligated segments of the gastrointestinal tract and studied the blood levels of the drug. The highest blood level was obtained when the drug was introduced into the duodenum, with the jejunum and ileum next. The absorption from the stomach was considerably lower andj- . blood 'levels '-dec-lined tbezerolSahourssafter.vtehewmaximumiplasma level was . attained. 12 Alternatively, Chiou and Riegelman (37) hypothesized that . there might be a limited residence time through a particular region of the gut where absorption is optimal. These reports suggest'.that: the absorption, of .griseofulvin:,: : from the gastrointestinal tract is limited by two major factors, viz., the dissolution rate and the gastrointestinal motility. Any factor influencing these two will consequently alter the absorption of an orally administered dosage form of griseofulvin. Several investigators have introduced different means to improve the absorption of griseofulvin through accelerating the dissolution rate. Kraml et al (7) suggested the use of micronized powder of griseofulvin in the preparations. They 0 ) noticed a substantial increase in the griseofulvin plasma level in the rat when the micronized powder was administered in an aqueous suspension as compared to the regular powder. The improved absorption is due to the fact that micronization will give rise to a higher surface area. This increases the rate of dissolution and consequently the absorption rate of the drug. Kraml e_t al (7) observed that,, addition of surfactant to aqueous and corn o i l suspensions failed to significantly affect griseofulvin-serum levels. "rr.These-authors used 0.5% Perminal BXN and 0.5% Nekal BX78 in aqueous suspensions and 0.077% Aerosol OT and 0.077% Tween 80 in corn o i l suspensions. In contrast to the observations of Kraml et al Duncan et. al (8) noticed. .•/.-/ that, addition of 0.5% lecithin Glidden R.G. or Perminal BXN into the suspension Of griseofulvin gave rise to higher blood levels in rats. They showed that the method of incorporation of the surfactant into the suspension influences 13 the absorption of the drug. When g r i s e o f u l v i n and the surface-active agents were mixed p r i o r to the addition of water, s i g n i f i c a n t l y higher g r i s e o f u l v i n blood l e v e l s were observed as compared to when the surfactant i s f i r s t dissolved into the water and the g r i s e o f u l v i n added afterwards. Chiou and Riegelman (36,37) administered g r i s e o f u l v i n dispersed i n a s o l i d matrix of polyethylene g l y c o l (PEG) 6000 to man and to dogs and noticed higher (approximately threefold) and more constant absorption as compared to that obtained a f t e r administration of tablets of micronized drug. I t was concluded that a f t e r the exposure of the griseofulvin-PEG preparation i n the g a s t r o i n t e s t i n a l f l u i d , the drug might p r e c i p i t a t e i n the c o l l o i d a l or submicron form which could Be. dissolved rapi'd-l'yVv .-'rr': Recently, a simple method of preparation was introduced (38) which has yielded g r i s e o f u l v i n dosage forms with higher r e l a t i v e b i o a v a i l a b i l i t y . In t h i s method g r i s e o f u l v i n formulations are prepared d i r e c t l y from the fermentation substance. The therapeutic e f f e c t of these preparations has been tested on patients with scalp i n f e c t i o n s and has been reported to be s i g n i f i c a n t l y higher than that achieved with g r i s e o f u l v i n t a b l e t s . These authors a t t r i b u t e d the improved effectiveness to the fa c t that,, i n these preparations, g r i s e o f u l v i n i s i n i t s "natural v e h i c l e " , and therefore i t can be absorbed more r e a d i l y and e l i c i t s i t s e f f e c t more completely. In man, the serum g r i s e o f u l v i n concentration has been observed to be almost doubled when the drug i s administered with a high f a t meal ( 9 ) . This observation was a t t r i b u t e d to an increase i n absorption due to e i t h e r a change i n m o t i l i t y or an increased e m u l s i f i c a t i o n caused by an increased b i l e s ecretion. Kraml et^ a_l (7) also observed two- to t h r e e f o l d higher serum l e v e l s following administration of g r i s e o f u l v i n suspended i n corn o i l . However, 14 Bloedow and Hayton (3.9,) have recently administered griseofulvin to the rat in a series of l i p i d and l i p i d - l i k e vehicles and noticed that only digestable • polar vehicles (e.g., Tween 80) caused an increase in griseofulvin plasma levels. These vehicles, they explained/,^rfo'rme^raBfstable-im'o'nolayer on" water ?and iwater,. da'ssbiv.edcan ifchem'.siThei bioavailability of. *ver-' i , griseofulvin in other classes of lipids was either lower or equal to 'that observed following administration of the drug in an aqueous suspension. Recently, Bates and co-workers studied the.absorption of griseofulvin from different liquid preparations in the rat (12) and man (.40). These authors (12) administered griseofulvin suspensions in corn o i l and griseofulvin emulsions in corn oil/water to the rat and compared the results with those of aqueous suspensions. The highest percent absorption with the smallest coefficient of variation was observed after the oil/water emulsion with the o i l suspension next. The aqueous suspension was reported to have the lowest bioavailability and exhibited the highest coefficient of variation. However, the authors (12) observed that the time to attain maximum plasma level was significantly longer when the drug was given in an o i l suspension or an oil/water suspension as compared to the aqueous suspension. In humans, also, Bates and Sequeira (40) have recently shown that - : griseofulvin was more rapidly, uniformly and completely absorbed when the drug was administered in an oil/water emulsion than when i t was administered in either tablet form or in an aqueous, suspension. These authors (40.) suggested that, the inhibitory effect of emulsified corn o i l on the gastric emptying process could be responsible for the increased absorption. The slower rate of gastric emptying coupled with the inhibitory effect of l i p i d on the motility 15 of the upper part of the small intestine causes a smaller amount of un-dissolved griseofulvin to be released at any particular time and become exposed to the absorptive s i t e . In other words, a smaller amount of micron-tzed griseofulvin with a longer residence time in the absorptive region has a better chance of being absorbed. As w i l l be discussed later, this confirms the observations of Jamali and Axelson (4.1,42) regarding the significance of gut motility on the absorption of griseofulvin in the rat. To further support their hypothesis, Bates and Sequeira (140) pretreated -a human subject with propantheline, an agent capable of retarding the gastrointestinal motility, prior to the administration of a single oral dose of micronized griseofulvin in an aqueous suspension. As a result, an increase in the maximum urinary excretion rate along with an increase in total recovery of the major metabolite in the urine was noticed. Since upon administration griseofulvin i s metabolized almost completely, an increase in the recovery of the metabolite indicates an increase in the absorption of the. drug. The stomach has a limited capacity for absorption of many drugs, while the proximal segment of the small intestine has been noticed to be the major site of absorption for many drugs, such as griseofulvin ( 3 5 ). This fact is attributed to the large surface area of this segment of the gut relative to that of the stomach. Consequently, a change in the residence time of a drug in the gut may influence absorption. Manyc drugs have been observed to influence the gut motility and consequently the residence time of a co-administered drug. Metoclopramide ( 4 3 - 4 , 5 ) and propantheline (46) are two commonly used drugs to respectively increase and decrease motility of the gastrointestinal t(GI>)ttr?iet. It has been observed that an increase in the 16 g a s t r i c emptying rate caused by metoclopramide increases the rate of absorption of many drugs such as paracetamol (47), t e t r a c y c l i n e (48), p i v a m p i c i l l i n (48) and ethanol (4=)). On the other hand, a reduced m o t i l i t y caused by propantheline or atropine reduced the rate of absorption of digoxin (13), paracetamol (47), t e t r a c y c l i n e (4.8), p i v a m p i c i l l i n (4.8), ethanol (4.9), sulfamethoxazole (5,0), r i b o f l a v i n (14) and phenolsulfonphthalein (15). With regard to the t o t a l absorption, however, m o t i l i t y modifiers a f f e c t only the absorption of drugs with inherently poor absorption c h a r a c t e r i s t i c s such as digoxin (13), r i b o f l a v i n (14) and phenolsulfonphthalein (15). In these cases, metoclopramide generally decreased the t o t a l absorption of the drugs while a n t i c h o l i n e r g i c agents •'. -increased: c-the •. ^ d s r e l a t i v e a v a i l a b i l i t y . I t i s i n t e r e s t i n g to note t h a t A although propantheline treatment increased the blood l e v e l s of digoxin when the drug was given i n tablet form, i t f a i l e d to influence t h i s parameter following administration of a s o l u t i o n of the glycoside (13). It i s also worth mentioning that, among the above mentioned drugs r i b o f l a v i n possesses an exceptionally i n t e r e s t i n g absorption c h a r a c t e r i s t i c . I t i s absorbed by a s p e c i a l i z e d absorption process i n the human small i n t e s t i n e (50.yjjl).Therefore i t s absorption i s dependent upon the residence time i n the small i n t e s t i n e . Propantheline treatment prolonged the residence time, thereby increasing the t o t a l absorption of r i b o f l a v i n (14). Grounse (9) and Bates and Sequeira (j30)- pointed to another mechanism that might be involved i n the absorption of g r i s e o f u l v i n when administered with f a t t y meals or i n l i p i d v e h i c l e s . Administration of l i p i d s may stimulate the secretion of b i l e . B i l e contains p h y s i o l o g i c a l surface active agents which are known to have important r o l e s i n the absorption" process of Hours Figure '2-1/' . B i l i a r y flow, b i l i a r y b i l e acid concentration and b i l i a r y acid excretion in contrail rats and rats treated with "phenobarbital (75 mg/kg) once da i l y for four days. The soiid ; l i n e s represent the results from the control group, and the dashed l i n e s represent the results from the phenobarbital treated r a t s . From C.D. Klaassen, J. Pharmacol 176, 743, 1971. Exp. Ther. 18 many drugs (,52-59 ). Kimura elt a_l ('59) studied the e f f e c t of sodium taurocholate (STC), a major b i l e s a l t , on the absorption of a series of drugs i n the r a t . They noticed s i g n i f i c a n t but v a r i a b l e changes i n the absorption of the drugs, depending upon t h e i r physicochemic.al nature of absorption c h a r a c t e r i s t i c s . As a r e s u l t of simultaneous perfusion of 20 mM STC (the c r i t i c a l m i c e l l e concentration, CMC, 2.9 mM f o r STC) d i r e c t l y to d i f f e r e n t cannulated segments of the small i n t e s t i n e the percent absorption of sulfaguanidine increased while the opposite e f f e c t was noticed with imipramine and quinine (.59). These authors a t t r i b u t e d the increased absorption of sulfaguanidine to the e f f e c t of b i l e s a l t on the "structure of the absorption surface" and the decreased absorption of imipramine and quinine to the " l o s s of thermodynamic a c t i v i t y of the drugs due to the formation of the m i c e l l a r complexes with the b i l e s a l t " . Thus, an increase i n the concentration of the physiologic surface-active agents caused by stimulation of b i l e secretion may a l t e r the d i s s o l u t i o n rate a i v i r1and</or %Lt«::\. the absorption of g r i s e o f u l v i n . A s i m i l a r mechanism may also be involved i n the griseofulvin-phenobarbital i n t e r a c t i o n . Phenobarbital treatment has been shown (.21-23.) to s i g n i f i c a n t l y stimulate the secretion of b i l e i n the r a t . However, t h i s increase i s a t t r i b u t e d to an increase i n the secretion of b i l e - s a l t independent f r a c t i o n of the b i l e (22) . Klaassen (22) treated rats with 75 mg/kg phenobarbital once d a i l y for four days and noticed a 50% increase i n the b i l i a r y flow. As i s shown i n F i g . 2-1, he also measured the concentration of four b i l e acids and. 1 9 reported that, with the exception of taurochenodesoxycholic acid, a l l of the acids showed a s i g n i f i c a n t decline i n t h e i r concentrations. An increase i n the b i l e flow r e s u l t i n g from phenobarbital treatment, without a simultaneous increase i n the secretion of the b i l e s a l t s , i s responsible, for the decreased concentration of the s a l t s i n the b i l e . Bates e_t a i (52) have shown (Fig. 2-2) that the s o l u b i l i t y of g r i s e o f u l v i n i n an,-aqueous i s o l u t i o n sharply increases a f t e r an increase i n the concentration of the simulated i n t e s t i n a l b i l e s a l t mixture above the CMC: . s o l u b i l i t y of g r i s e o f u l v i n . mg/100 ml concentration of simulated i n t e s t i n a l b i l e s a l t s „ _-2 M x 10 115 = TTTTT i n t n e range of 0.004 to 0.06 M of simulated i n t e s t i n a l b i l e s a l t s ) . This i s a t t r i b u t e d to a m i c e l l a r s o l u b i l i z a t i o n phenomenon. The observed reduction i n the concentration of the b i l e s a l t s ('2.2) r e s u l t i n g from phenobarbital treatment may reduce the s o l u b i l i t y and hence the absorption of poorly soluble drugs such as g r i s e o f u l v i n . • D i s t r i b u t i o n of G r i s e o f u l v i n - Bedford e_C a l (35) noticed that g r i s e o f u l v i n was d i s t r i b u t e d throughout most t i s s u e s . Following an i v dose, the concentration i n the lung and skin was found to be higher than i n the rest of the t i s s u e s . However, the g r i s e o f u l v i n l e v e l i n f a t was also quite high but declined to undetectable l e v e l s i n a short time. The r e l a t i v e l y high s k i n l e v e l i s of p a r t i c u l a r i n t e r e s t . G r i s e o f u l v i n i s an antifungal agent u t i l i z e d f o r the treatment of s u p e r f i c i a l skin i n f e c t i o n s . Therefore, the skin i s i t s , main site, of a c t i o n . A f t e r administration, i t 20 10 1 ' ' 4 • ' 1 1 —H. •1 2 3 4 5 6 7 8 Molar concentration of simulated intestinal bile salt mixture (X l b 2 ) Figure 2-2. Solubility of griseofulvin as a function of simulated intestinal b i l e salt mixture concentration at 37°. (reference 52 .) 21 selectively concentrates in the skin and elicits its action. Shah e_t al (.600 reported the influence of sweat as a carrier in the distribution of griseofulvin into the skin. They explained that, since griseofulvin is a lipid soluble neutral drug, i t readily passes through the sweat gland epithelium and concentrates in the sweat. Griseofulvin is 85% bound to the plasma proteins, therefore in the usual plasma levels of 1-3 yg/ml the concentration of the unbound fraction of the drug should be 150 to 450 ng/ml. Considering the high partition coefficient of griseofulvin, this concentration range of the free drug can serve as an adequate driving force for passive diffusion into the sweat. These authors also showed that the sweat concentration of griseofulvin is (150-450 ng/ml) when the plasma water level of the latter is in the range of 200 to 340 ng/ml. This indicates that the sweat and plasma water levels of griseofulvin'may equilibrate to the same concentration range. This suggests that there may exist a correlation between the plasma concentration of griseofulvin and its level in the skin and consequently its pharmacological response. Elimination of Griseofulvin - Metabolism is the principal path-way o£a elimination of griseofulvin. The metabolism of griseofulvin has. been studied in man ( :6 ' l ) , rats and rabbits ( $ 2 ) , mice ($3.0 and dogs (33). In rabbit ( .62), dog (330 and human (16.10 urine, 6-demethyl griseofulvin (6-DMG) has been identified as the major metabolite (95,1'l'OO and 86% respectively). 2 2 In the rat ( 6 2 ) and mouse ( 6 3 ) , however, two derivatives have been found to be the major products of griseofulvin metabolism: 4-demethyl griseofulvin 27% and 39% respectively; 6-DMG 52% and 27% respectively. In a l l species a negligible amount of the intact drug has been found in the urine. This is an indication of the extensive metabolism of the drug. It also reveals that the elimination of griseofulvin i s taking place mainly via metabolism. Symchowicz et a l (65) reported that, in the b i l i a r y cannulated rat, about 77% of a given iv dose was found in the bile and only 12% in the urine. They isolatedaa large quantity of 4-demethyl griseofulvin and a relatively small amount of 6-demethyl griseofulvin in the rat b i l e . The former metabolite was extensively conjugated in both bile and urine while the latter was excreted in urine mainly in the intact form. However, in normal rats the amount of metabolite excreted in the urine is greater due to the entero-hepatic circulation which gives rise to the absorption of the metabolites after i i n i t i a l ; b i l i a r y excretion. Symchowicz et a l ( . ' 6 2 ) also detected small quantities of some unidentified metabolites of griseofulvin in the b i l e of the rat and the rabbit. Symchowicz and Wong ( 6 4 * ) and Bedford et_ a l ( 3 5 ' , ) reported that, in vitro, the major site of griseofulvin metabolism in the rat was the l i v e r . No appreciable metabolism took place in the heart, kidney, lung or skin slices. Kaplan et a l (165) presented evidence suggesting that in rabbits the microsomal enzyme system is responsible for the O-dealkylation of griseofulvin. They studied the effect of potential inhibitors of griseofulvin metabolism on the rateiqf •fdri'g :-j disappearance in the Krebs-Ringer bicarbonate liver s l i c e 23 system. These authors selected three aromatic ethers known to be metabolized by oxidative O-dealkylation (p-ethoxyacetanilide, p-methoxy-benzylamide and codeine) and a nonspecific metabolic i n h i b i t o r (SKF 525-A). The r e s u l t s indicated that, while p-ethoxyacetanilide and p-methoxybenzylamide decreased the rate of g r i s e o f u l v i n disappearance, codeine and SKF 525-A f a i l e d to a f f e c t the metabolism of the drug. The conclusion was that g r i s e o f u l v i n appeared to be metabolized by the same system that metabolizes p-ethoxyacetanilide and p-methoxybenzylamide ( i . e . , the oxidative O-dealkylation system of the microsomal f r a c t i o n ) and not by the system known to metabolize codeine. I t has been observed that some drugs, such as phenobarbital, are capable of inducing the oxidative O-dealkylation enzyme system of the microsomal f r a c t i o n (19,). L i n je_t a l (66) observed that, i n v i t r o , phenobarbital treatment (80 mg/kg i . p . d a i l y f o r 4 days) was capable of increasing the maximum v e l o c i t y , Vmax, value f o r metabolism of g r i s e o f u l v i n i n the rat and mouse. The treatment, however, did not a l t e r the enzyme-•substrate d i s s o c i a t i o n constant, Km, for the reactions. This i n v i t r o observation would suggest that the observed phenobarbital-griseofulvin i n t e r a c t i o n i n the.rat was due, perhaps, to an induction mechanism. However, the observation of Riegelmah et a l (20) suggests otherwise, as discussed below. 24 Griseofulvin-Phenobarbital Interaction Pretreatment with phenobarbital has been observed to reduce blood l e v e l s of g r i s e o f u l v i n i n man (1,6,18,20) and i n rats (17). B u s f i e l d et a l (16) observed that i n man the peak griseofulvin-blood l e v e l declined by 37% following o r a l treatment with 7 doses of 30 mg phenobarbital (Fig. 2-3). In the rat al s o , B u s f i e l d &t_ a l (17) studied the griseofulvin-phenobarbital i n t e r a c t i o n and observed the same e f f e c t . To sel e c t optimal experimental conditions, they treated rats with d i f f e r e n t doses of the barbiturate, ranging from 1.88 to 60.00 mg/kg and noticed that the highest dose of pheno-b a r b i t a l which did not produce apparent sedation but s i g n i f i c a n t l y reduced the blood l e v e l was 15 mg/kg. The e f f e c t on g r i s e o f u l v i n plasma l e v e l s was maximal i f the drug was given between 12 and 48 hours a f t e r a dose of phenobarbital. Thus, they (17) pretreated rats with a s i n g l e o r a l dose of 15 mg/kg pheno-b a r b i t a l . Twenty four hours a f t e r phenobarbital, a s i n g l e o r a l dose of 100 mg/kg g r i s e o f u l v i n suspended i n an aqueous s o l u t i o n of 0.5% Tween 80 yiel d e d s i g n i f i c a n t l y lower blood l e v e l s (approximately 50% and 62% reduction i n the peak plasma l e v e l , Cmax, i n male and female rats respectively) as compared to co n t r o l r a t s (Fig. 2-4). In t e r e s t i n g l y , the g r i s e o f u l v i n blood l e v e l was s i g n i f i c a n t l y higher i n the female than i n the male r a t s . However, the extent of the i n t e r a c t i o n was approximately the same i n both sexes (Fig. 2-4). The authors also pretreated female rats with repeated doses of pheno-b a r b i t a l (15 mg/kg once d a i l y for up to 14 days) and noticed an even more profound e f f e c t on the griseofulvin-blood l e v e l . Due to the lack of a highly s e n s i t i v e assay and consequently the need f o r a large volume of blood, B u s f i e l d et_ a l c o l l e c t e d blood samples from the ra t by cardiac puncture. Therefore, each r a t was bled only once. This l i m i t a t i o n would not permit a 25 Hours Figure 2-3. Blood-levels of griseofulvin in human volunteers given a single oral dose (500;mg). Each point i s the mean for eight volun-teers. Vertical lines indicate standard errors. Control group received griseofulvin only; test gruop received phenobarbitone(7 doses of 30 mg.) before griseofulvin.(Reference 16 •;) Figure 2-4. Effect of phenobarbitone on blood levels of griseofulvin in male and female rats. Phenobarbitone sodium (15 mg/kg) was adminis-tered orally 24 hours before oral administration of griseofulvin (100 mg/kg). Blood antibiotic levels were measured 2, 4, 6, 8 or 16 hours later. A , Control female (n = 12); A , phenobarbi-tone-dosed female (n = 6); 9— , rcontrol male ( n = 12); and O -—> phenobarbitone-dosed males (n = 12). Standard errors are between 0.3 and 0.1. (Reference 17 .) 26 follow-up of the time course of the drug i n the same rat and provides only an estimation of the curve of the blood-level vs_ time. Furthermore, i n th e i r repeated phenobarbital treatment study the griseofulvin-blood l e v e l was measured only at one point (4 hours post-administration). To explain the p o t e n t i a l mechanism involved i n g r i s e o f u l v i n -phenobarbital i n t e r a c t i o n , B u s f i e l d and co-workers suggested that pheno-b a r b i t a l reduces the griseofulvin-blood l e v e l due, perhaps, to i t s a b i l i t y to induce the metabolism of g r i s e o f u l v i n . Further substantiation f o r t h i s was t h e i r observation that, i n v i t r o , the barbiturate accelerated the metabolism of g r i s e o f u l v i n by the rat l i v e r s l i c e s . This i s consistent with the observation of L i n et_ a l (66.) which was discussed e a r l i e r . However, l a t e r , Riegelman et a l (2'0) disputed t h i s hypothesis. They administered g r i s e o f u l v i n intravenously as w e l l as o r a l l y to man pretreated with repeated o r a l doses of 30 mg/kg (three times d a i l y f o r four days) and studied the griseofulvin-plasma l e v e l s i n the subjects. Riegelman et a l observed that phenobarbital treatment reduced the griseofulvin-plasma l e v e l s when griseo-f u l v i n was administered o r a l l y . However, the treatment f a i l e d to a l t e r the plasma l e v e l s of an intravenously administered dose of g r i s e o f u l v i n (Fig. 2-5). Furthermore, phenobarbital pretreatment did not influence the k i n e t i c s of the elimi n a t i o n of g r i s e o f u l v i n regardless of the route of administration; the slopes of the p o s t - d i s t r i b u t i o n phase of the griseofulvin-plasma level-time curves f e l l into the same range i n both control and test subjects (Fig. 2-5). This i s of p a r t i c u l a r importance. The slope of the p o s t - d i s t r i b u t i o n phase fo r a two-compartment open model i s described by Equation 4: ^ e l ^ l ' ? l o p e ="a(2.303) ( 4 ) 27 Figure 2-5. Griseofulvin plasma levels as a function of time of one human subject in presence and absence of phenobarbital treat-ment. -In; control experiment ? the'subjectr received^' either a single intravenous dose (100 mg in PEG 300) .or a single tablet of 500 mg: orally; in the test experiment the subject received 30 mg phenobarbital orally three times .daily for four days prior to the griseofulvin administration. (Reference 20 .) 28 Thus i t is directly proportional to the overall elimination rate constant, k^^. As mentioned before, the elimination of griseofulvin is mainly associated with its metabolism. Therefore any changes in the rate constant for metabolism of the drug would be expected to influence the slope of the post-distribution phase of the plasma level vs time curve. Stimulation and inhibition of the metabolism of a drug would result in a greater or smaller slope of the post-distribution phase respectively. The observed parallel slopes in the control and test subjects coupled with the phenobarbital's failure to reduce the griseofulvin-plasma levels following administration of an iv dose.suggested that the observed interaction was unlikely to occur at the level of metabolism (20). Since the interaction has been observed only following oral administration, Riegelman et al suggested that phenobarbital decreased the absorption of griseofulvin rather than inducing its metabolism. They proposed two potential mechanisms to explain this reduced absorption: phenobarbital either reduced the degree of dispersion of the tablet granules or increased the motility of the proximal segment of the small intestine where griseofulvin absorption appears to be maximal. The first mechanism causes a reduction of the surface area, therefore, dissolution of griseofulvin would be retarded. The second mechanism results in a reduced residence time of the poorly absorbed drug in the absorptive site, thereby decreasing its total absorption. Both mechanisms 6f-fered:jw.er-el based on the ability of phenobarbital to enhance the flow of bile. An increase in the volume of the bile may accelerate the motility of the gut and force the drug past the absorptive site before i t can be absorbed. Alternatively, considering the effect of bile salts on the dispersion and dissolution of poorly soluble drugs, Riegelman et al suggested that the change in the bile flow may be responsible for the reduced absorption of griseofulvin following the administration of phenobarbital. Other mechanisms have also been proposed in an attempt to explain this interaction. Axelson (24) hypothesized that the reduced griseofulvin-plasma level might be due to a complex mechanism involving enzyme induction, first pass metabolism and distribution rate-limited elimination of the drug. For example, i f the elimination of griseofulvin was rate-limited by its access to the liver rather than by its metabolism per se, enzyme induction would have no effect on the half-life of the drug but may significantly increase the fraction of the dose which is metabolized on the first pass through the liver upon oral administration. In such a case the slope of the post-distribution of the plasma level-time curve would not be an indication of the rate of metabolism but would rather be an indication of the rate of blood flow to the metabolizing organ (blood flow-rate limited elimination). However, as mentioned before, since in control rats as well as in other examined species griseofulvin is extensively metabolized (more than 9 9 % of ah<?iritrayenbus dose) this suggestion seems unlikely. The purpose of this investigation has been to study the mechanism of the observed griseofulvin-phenobarbital interaction. Administration of several different dosage forms of griseofulvin with different dissolution properties provided information which permitted a detailed evaluation of the observed interaction. The effect of stimulated and retarded gastrointestinal motility (caused by metoclopramide and propantheline) and the influence of utilization of different vehicles on the absorption of griseofulvin has also been studied. 30 3. EXPERIMENTAL Assay Procedure The electron capture-gas liquid chromatographic(EC-GLC)method of Shah, Riegelman and Epstein (67) was employed with some modification to determine griseofulvin levels in plasma, stomach.-and various .dosage :fqrms'.~^ Calibration Curves - To study the efficiency of the method of extraction, different series of standard solutions were prepared and their griseofulvin contents were quantitated; one by directly dissolving griseo-fulvin in benzene, and the others by dissolving the drug in d i s t i l l e d water and then extracting the drug into ether in the presence of rat plasma, polyethylene glycol (PEG), Tween 80 or ethanol. a. Standard Solutions after Dissolving Griseofulvin in Benzene - Eight mg of griseofulvin was accurately weighed and dissolved in 50 ml benzene.''' One ml of this solution was transferred into a 10 ml volumetric flask and benzene was added to volume. Volumes of 0.5, 1, 2, 5 and 6 ml of the latter were separately diluted by tenfold to obtain solutions of 4, 8, 16, 40 and 48 ng/5 ul respectively. Aliquots of 0.5, 1, 2 and 5 ml of the solutions containing 4 ng/5 pi were transferred into separate 10 ml volumetric flasks and solutions of 0.2, 0.4, 0.8 and 2 ng/5 ul were prepared respectively by adding benzene to volume. Aliquots of l l ml cand '2.-5: mlf of the solution containing 0.8 ng/5 yl were diluted separately to 50 ml to obtain solutions with respective concentrations of 16 and 40 pcg/5 yl . Finally, 1 ml of the solution Unless otherwise stated Nanograde benzene was used in a l l studies; Mallinkrodt. 3 1 containing 0.8 ng/5 y l was diluted by tenfold to prepare a solution of 80 pcg/5 yl of griseofulvin. One ml of the internal standard solution containing 1.72 rn;g 2 diazepam was transferred into the volumetric flasks prior to the f i n a l dilution. Five y l of the f i n a l solutions containing a constant amount of 860 peg internal standard were injected into a Hewlett Packard model 5713A electron capture gas chromatograph and the griseofulvin content was determined using the peak height ratio (griseofulvin/diazepam) method (Fig. -3-li a n d Table 3-1). b. Standard Solutions after Extraction - A 3.66 mg sample of griseo-fulvin was weighed accurately and dissolved in 10 ml of absolute ethanol in a 100 ml beaker. Approximately 50 ml d i s t i l l e d water was added to the solution gradually while i t was being stirred using a magnetic st i r r e r . The solution was then transferred into a one l i t e r volumetric flask and water was added to volume. Aliquots of 0, 0.1, 0.5, 1, 2, 3, 4 and 5 ml of the solution were transferred into teflon-stoppered 45 ml centrifuge tubes. Sufficient volumes of d i s t i l l e d water were added to the tube content to make up the volume to 5 ml. One half .ml ofu, rat plasma, 5 ml of 0.1 N. hydro-3 chloric acid and 10 ml of anhydrous ether were added to the solutions. The 4 tubes were shaken for 30 min using a Wrist-Action shaker. Two centrifuge Hoffman-La Roche Ltd., Montreal, Lot No. R-6658. i Unless otherwise stated ether absolute was used in this study; ether absolute, analytical reagent. Burrell Corp., Pittsburg, Pa. ro F i g u r e 3-l£,a. Standard curves f o r g r i s e o f u l v i n (860- p.cggpf diazepam as" internal standard). K e y : — % — d i r e c t l y -dissolved in benzene;" , extracted solutions dissolved in benzene. 1.0 20 30 40 50 G r i s e o f u l v i n , ng F i g u r e 3 r l j , b . Standard curve f o r g r i s e o f u l v i n (860.pcg:fof diazepam, as internal) standard) in c o n c e n t r a t i o n range from 4 to 40 ng t directly dissolved iii benzene). 34 Table 3-1 Gris e o f u l v i n ( G r i s ) Concentration, and the corresponding Peak Height Ratios (Gris/D) of the Standard Solutions. (('griseofulvin was d i r e c t l y dissolved i n benzene and constant concentrations of diazepam (D)' were added to a l l of the solutions.) Gris Concentration pcg/5 u 1 16 40 80 200 400 800 2000 4000 8000 16000 40000 Slope 0.000854 Intercept 0.013 Cor r e l a t i o n J of c o e f f i c i e n t 0.999 Standard e r r o r 0.046 Ratio Gris/D 0.014 0.035 0.067 0.173 0.343 0.686 1.716 3.412 6.864 13.810 34.109 35 racks were specially prepared for this purpose and were assembled on the shaker. Five ml of the ether layer were removed and transferred into 10 ml volumetric flasks containing internal standard (1.72 1*g/ml diazepam/ benzene) and were evaporated to dryness under nitrogen flow. The residue was dissolved in 10 ml benzene and 5 ul of the solutions containing 91.5, 457.5, 915, 1830, 2748, -3660 and 4575 peg griseofulvin respectively and 860 peg internal standard were injected into the gas chromatograph and the griseofulvin content was determined (Fig. .3-;l and Table 3-II). The same procedurerwas followed to determine the.efficiency of the extraction in the presence of other compounds. In these experiments, instead of rat plasma, aliquots of 0.1 ml PEG 300, PEG 600, aqueous solutions of 0.5%, 2% and 5% Tween 80 or 1 ml of 50% ethanol were added to the aqueous phase prior to the extraction. These compounds were usedLeither as vehicles in ddosag.esprepara,tionsior^as: ins the: case witlu.ethanol as,; solvent - in different experiments. Gas-Liquid Chromatography of Griseofulvin - A Hewlett Packard 63 model 5713A gas-liquid chromatograph equipped with a Ni electron-capture detector was used in this study. The following conditions were found satis-factory for the assay: column, 1.83 m x'0.315cm (6 f t x 0.12-5in) glass tube packed with 3% 0V-25 on 80-100. mesh.Chromosorb W; column temperature, 285°; injection port temperature, 250°; detector temperature, 350°; and carrier gas (5% methane, 95% argon) flow, 40 ml/min. The observed retention times were 6.24 and 2.08 minutes for griseofulvin and diazepam respectively. Previously, Shah £t a l (67) reported the assay for griseofulvin in skin and sweat using a glassircpl-umn..with :-an internal' diameter of '. 0;31 cm .packed with 3%: .OV'-slTin) . The appropriate temperatures reported by 0 36 Table 3-II Griseofulvin Recovery Following i t s Extraction from Aqeous Solutions Containing Rat Plasma. Griseofulvin Added, Griseofulvin Recovered^" pcg/5 u l , pcg/5.y1 91.5 92.2(4.4) 100.7(4.8) 457.5 455.2(14.5) 100.3(3.2) 915.0 888.9(20.1) 97.1(2.2) 1830.0 1752.3(22.3) 95.7(1.2) 2747.0 2790.1(2.9) 101.5(0.1) 3660.0 3600.1(101.8) 98.3(2.7). 4575..0 4607.8(3.5) 100.7(0.1) Mean Recovery, 99.2(2.8) 1. Average of two determinations; standard deviation of the mean in parenthesis. 37 1 i 1 -4 1 1 -4 8 12 16 Minutes Figure 3-2. Gas chromatogram of griseofulvin and diazepam (internal standard) following injection of 5 n l of an ethercextractedtsample of plasma dissolved in benzene; attenuation, 32X. Dotted lines depict the peak-height ratio method; calculations are as follows: cd 13.8 cm Ratio = = " ' . i l - = 1.10 l b 12.5 c m 3 8 these authors (67) were 310°, 270° and 340° for injection port, column and detector respectively. The resulting retention times reported by Shah et a l ( 67 ) were 3.00 minutes for diazepam "arid _8.25 minutes for /griseofulvin. High temperature blue Silicone rubber septums^ were used for the injection port and were replaced daily. Either Teflon^ or Vespel^ ferrules were used to plumb the column. Teflon ferrules were found to be unsatisfactory due to their deformation at high temperatures while the Vespel ferrules had long l i f e and were reusable. The peak height ratio (griseofulvin/diazepam) method was used to quantitate griseofulvin and construct standard curves. As demonstrated in Fig. 3-2j the peak baseline was determined by drawing a line through the baseline. A vertical line was drawn from the top of the peak to the baseline. The length of this line was considered as the peak height. The height of the peaks obtained after injection of a l l solutions were measured and the peak height ratio (griseofulvin/diazepam) was calculated and plotted as a function of concentration (Fig. 3-1 and Table 3-1). Linear regression analysis was employed to f i t the data. Using a HT-9 Septurns, Applied Science Laboratories Inc. 'Teflon R (PTEE) ferrules, Alltech Assoc., Arlington Hts., 111. R Vespel ferrules, Alltech Assoc., Arlington Hts., 111. 39 g programmable calculator the least square estimates of the slope, b, and the intercept, a, to the line y = a + bx were computed, based on the following equations: b = igxy - (Ex) (Ey), ^ nEx 2 - (Ex) 2 Ey - bEx n (6) where, x is the concentration of griseofulvin and y is the griseofulvin/ diazepam ratio. The correlation coefficient, r, and standard error of the estimate, Sy.x, were also calculated as follows: r = nZxy - (Zx)(Ey) , ^ 'nix 2 - (Ex) 2 nEy 2 - (Ey) 2 1 2 . .2 (nZxy -ExEy) 2 (8) sy- x = n V ^ y - 0 1 y) \—-f-^ nEx - (Ex) The percent griseofulvin recovery after extraction was calculated by comparing the ratios of extracted samples with those of solutions of griseofulvin directly dissolved in benzene (Table 3-II)). Prior to and during each experiment, the ratios of standard solutions were re-examined by injecting three standard solutions with concentrations of 457.5, 915.0, 1830.0 pcg/5 y 1 benzene --into the GLC. Some Wang 600, Programme Verify No. 2133. 4 0 occasional changes in the slope.of the curve due to minor tai l i n g of the peaks was noticed after heavy usage of the column. The tai l i n g problem could be overcome by replacing the silanized glass wool plug in the injection port end of the column. Internal Standard Solution - Diazepam was used as the internal standard in this experiment. A 17.2 mg sample of diazepam was accurately weighed and dissolved in 100 ml benzene. One ml of this solution was transferred into a 100 ml volumetric flask and benzene was added to volume. Aliquots of 0.1 and 1 ml of this solution were added to calibration solutions and plasma samples respectively to obtain a constant concentration of 860 pcg/5 y l of the internal standard in the solutions." Five y l of a solution containing 860 peg diazepam in benzene were injectredeintd ithe GLC under the conditions lis t e d above and the chromatogram shown in Fig. ,3-3,d was obtained. Extraction of Griseofulvin from Plasma - An aliquot of 0.01 to 0.1 ml of the plasma sample was accurately transferred into a 45 ml teflon-stoppered centrifuge tube containing 5 ml of.0.1 N HC1. Ten ml anhydrous ether was added to each tube and the tube was shaken for 30 min. Five to 8 ml of the ether layer was transferred into 10 ml volumetric flasks containing one ml of solution of 17.2 yg/100 ml diazepam in benzene (860 pcg/5 y l ) . The solvent was evaporated to dryness under nitrogen, then one ml of Nanograde benzene was added and five y l of the solution was injected into the gas-chromatograph. In order to ascertain the specificity df the assay 1 ml of either 41 10 8 12 16 20 Minutes 10 12 16". 20 8 12 16 4 8 12 16 20 20 Minutes Figure 3"—3.- Gas chromatograms obtained following injection of 5 u 1 samples containing extracts of the solutions of metoclopramide hydrochloride (a), propantheline bromide (b) or sodium phenobarbital (c) after extracting under identical conditions to those for griseofulvin.^and gas chromatogram following injection of 5 U 1 of internal standard solution containing 860 peg diazepam (d)'; attenuation, 32X. Aqueous solutions, of metoclopramide hydrochloride(.2.5 mg/ml) ... • j 12 and propantheline bromide (1.25 mg/ml) were freshly prepared for intraperitoneal administration by dissolving the drugs in d i s t i l l e d water. 13 Sodium phenobarbital was dissolved in d i s t i l l e d water (3.75 mg/ml) for oral administration. 4 2 aqueous solutions of metoclopramide hydrochloride, propantheline bromide or sodium phenobarbital containing a constant amount of 0.4 ug of the drugs were transferred into 45 ml centrifuge tubes containing approximately 0.5 ml rat plasma, and 5 ml of 0.1 N hydrochloric acid. Ten ml ether was added and the tubes were shaken for 30 min. Five ml of the ether layer was transferred into 15 ml.centrifuge tubes, and evaporated to dryness under nitrogen flow, 10 ml benzene was added and the tubes were shaken for 30 seconds. Five y l aliquots of these solutions were injected into the GLC under the conditions list e d above. No interfering peaks were detected following the injections (Fig. 3-4). 9 Dosage Forms - Griseofulvin suspensions were prepared by suspending micronized powder in 0.5% Tween 80 1 0 (25 mg/ml) or in 70% PEG 300 1 0 (5. mg/ml and 25 mg/ml). Griseofulvin was suspended into previously prepared aqueous solutions containing Tween 80 or PEG 300. The Tween 80 suspensions were stirred for one hour and the PEG suspensions for 24 hours. At the time of administration, samples of the preparations were assayed using the technique explained in the equilibrium solubility measurement. Pure PEG 600^ was used as the vehicle to prepare oral and parenteral solutions of griseofulvin.. The concentration of the drug was 12.5 mg/ml for the oral solution and either 5 or 10 mg/ml for the parenteral solutions. The total griseofulvin concentration in the dosage forms was also measured using the technique described in the procedure for extraction of griseofulvin from plasma. Specific surface area 1.32 m /gm; supplied by Dr. Milo Gibaldi, SUNY at Buffalo, Buffalo, N.Y. Baker grade, J.T. Baker Chemicals. 4 8 5 in a clinical centrifuge^ and the resulting plasma was assayed for intact drug. Animals were lightly anaesthetized with ether*^ to facilitate drug administration and blood sampling. ,In all&experiments:} the-anaesthetizing • feMeawaB*keptat'o«§^|^:cfe!iiJnB ofmSsAaiminut'esis time was as&~ ly 2—4 t Gastric Emptying Experiments - In order to assess the effect of GT .motility modifiers on the gastric emptying rate, separate groups of rats were treat;edrihtraperit'oneaMy either with a single dose of 10 mg/kg meto-clopramide or with a single dose of 5 mg/kg propantheline 2 hours prior to the oral administration of the appropriate dosage form of griseofulvin. Subsequent to the griseofulvin dose, the animals were sacrificed at either 0, 2, 4, 6 or 11 hours and, after an abdominal incision,the stomach was 'seaiedd at the pyloric and cardiac sphincters with two hemostats. The stomach was then removed and transferred into a stainless steel cylinder containing approximately 35 ml of distilled water. The organ was then minced 18 and homogenized using an Omini-Mixer homogenizer for one minute at the top speed. The homogenate was transferred into a 100 ml volumetric flask, and the cylinder was rinsed 3 times with 10 ml aliquots of absolute ethanol.• The alcoholic aliquots were added to the homogenate and the volume was made to 100 ml with distilled water. One ml of the homogenate was transferred into another 100 ml volumetric flask and a solution of 50% ethanol in water (v/v) Damon/IEC Division, Needham Hts., Mass. Ether Solvent, U.S.P. Ivan Sorvall Inc., Newtown, Connecticut. 49 was added to volume. The homogenate was vigorously stirred during the dilution using a magnetic s t i r r e r . One ml of the hbmpgenate'Was transferred to a teflon-stoppered 45 ml centrifuge tube containing 5 ml of 0.1 N hydrochloric acid and the griseofulvin content was extracted and measured using the technique described in the extraction of griseofulvin from plasma. Determination of 4-Demethyl Griseofulvin - An attempt was made to determine one of the major griseofulvin metabolites in the rat urine. 19 Ten mg of 4-demethyl griseofulvin (4-DMG) was- accurately weighed and dissolved in benzene in a 50 ml volumetric flask. One ml of this solution was then transferred into another 50 ml volumetric flask and benzene was added to volume. One u 1 of this solution containing 4 ng of 4-DMG was injected into the GLC with the same conditions as described in the griseofulvin assay. At antattenuafcion of• 3.2mar.minprap.eakswasto,btained. However, after repetitive injections of the same concentrations of 4-DMG the peak increased in height. Nevertheless, the obtained peak tended to t a i l for a long length of time after the injection (Fig. 3-4). Ahjlmpurity due to the presence of griseofulvin in the sample of 4-DMG was also noticed (Fig. .3-4). In order to improve the assay and the peak resolution different means were employed. They were as follows: 20 a. Silanization of the Column - The column was treated with S i l y l 8 Glaxo, Allenbury, England. I Pierce Chemical Co., Rockford, 111. 50 F i g u r e 3-4X-Gas chromatogram of 4-DMG f o l l o w i n g i n j e c t i o n of 4 ng of the m e t a b o l i t e ( i n I'-ul) i n t o a column packed with 3% OV 2 5 on •Chr.omos.orbJ;W.;r;attehuation 32X. 51 to reduce the polarity and thereby i t s adsorption capability. The column was disconnected from the detector and the oven temperature was reduced to 180°. Seven consecutive injections of 10 V'-l of S i l y l 8 with a 10 minute interval between each injection were made. The column was then connected to the detector and the oven temperature was raised to 285°. After attainment of a stable baseline, 2 p l of a solution containing 8 ng 4-DMG in benzene was injected into the GLC, with the same conditions described above. The magnitude of the peak height increased as a result of silanization but extensive ta i l i n g (Fig. 3-5) of the peak occurred and the peak height was found to be inconsistant and irreproducible. b. Utilization of Different Columns - In order to improve the 4-DMG peak resolution, different columns packed with less polar liquid phases were employed. It was reasoned that.,, the metabolite was of sufficient polarity that a less polar liquid phase may improve the technique and provide better resolution. As i t can be seen in Fig. 3-6, r-.esu'Iitsr similar^, to those obtained using a column packed with 3% 0V-25 were observed when 3% OV-17 was employed since the peak due to 4-DMG had a tendency to t a i l and the results were not reproducible. The following GLC conditions were used in this experiment: column 1.83 m x 0.315. cm(6 f t x 0.125 in) glass tube packed with 3% 0V-17 on mesh 80-100 m Chromosorb W; column temperature, 285°; injection port temperature, 250°; detector temperature, 350°; and carrier gas (5% methane, 95% argon) flow, 40 ml/min. Another column packed with 10% OV-101 was also employed. The 52 15* V. •W w • 15+ Ok 4-DMG G r i s e o f u l v i n "to" Minutes F i g u r e 3-5;. Gas chromatogram of 4-DMG f o l l o w i n g i n j e c t i o n of 8^  ,.ng of the m e t a b o l i t e ( i n 2 j l ) i n t o a column packed wftfth 3%$OV0V.25!5; oh Chromosorb W and.treated with.Silyl 8; attenuation, 32X. 53 F i g u r e 3-6'. Gas chromatogram of 4-DMG f o l l o w i n g i n j e c t i o n of 4 ng of the m e t a b o l i t e ( i n £ p l ) i n t o a column packed wi t h 3% OV 17 on^hromosorb -W; attenuation, 32X. 54 5 ; 10-4. ., to 4 8 12 16 Minutes Figure 3-.?. Gas chromatogram obtained following i n j e c t i o n of 4 ng 4-DMG C i n j d y l ) into a column packed with 10% OV 101 (column temperature, 285°) on Chrompsorb W;. attenuation, 8X. metabolite was injected into this column at different oven temperatures (220"> 260°, and 280°). The GLC conditions were as follows: column, 1.83 m x 0.6A cm (6 f t x 0.25 in) glass tube packed with 10% on 80-100 mesh Chromosorb W; column temperature, as mentioned above; injection port temperature, 250°; detector temperature, 350°; carrier gas (5% methane, 95% argon) flow, 40 ml/min. As can be seen in Fig. 3-7, the result obtained using a column packed with OV-101 also cannot be considered satisfactory. In fact, no peak due to 4-DMG was observed even after an injection of 20 ng of the metabolite. This is contrary to expectations. Due to the lesser polarity ascribed to OV-101, i t was expected that reduced absorption would be observed and as a consequence improved peak resolution would result after using this stationary phase. The loss of the peak, however, may have resulted from a firm binding between the stationary phase and the metabolite. c. Derivatization of 4-Demethylgriseofulvin - The griseofulvin metabolite, 4-DMG, differs from i t s parent compound by lacking a methyl group in the 4 position. To improve the assay, attempts were made to convert 4-DMG to griseofulvin. The metabolite was darivatized to i t s methylated form, R 21 griseofulvin, using MethElute , an effective methylating agent. The procedure uti l i z e d in the derivatization was as follows: to each of 0.5 ml solutions containing 200, 400 or 600 pcg/5 y 1 4-DMG in benzene, in 15 ml teflon-stoppered centrifuge tubes 25 y1 of MethElute (0.2 M in methanol) was added. The contents of the tubes were shaken for 30 sec and Trimethyl anilinum hydroxide, Pierce Chemical Co., Rockford, 111. 10 _• H 1 M 1 1 -1 >"4 8 12 16 20 24 28 Minutes Fig u r e 3-8.Gasz-chromatpgrams^ obtained f o l l o w i n g i n j e c t i o n of 200 and 400 peg •methylated 4-DMG ( i n 5« yl) i n t o a column packed with 3% OV 25 on"Chromosorb:W; attenuation 8X. 5 yl of each of the solutions were separately injected into the GLC using the same gas chromatograph parameters as described for the griseofulvin determination. Figure 3-8 depicts chromatograms obtained from injections of 5 yl of solutions of derivatized 4-DMG containing 200 and 400 peg of the metabolite. Although derivatization occurred, the resulting peak heights were extremely variable and Inconsistent after injection of the different solutions. The unfortunate extreme variability of the derivatization plus the lack of availability of pure 4-DMG metabolite resulted in the discontinuance of this portion of the project. In some instances, addition of the reagent resulted in the total disappearance of the peak. This might have resulted from destruction of the compound unde'rathenconditions'.of'the experiment. Treatment of Data - Mean plasma levels of griseofulvin and the deviation from the mean were calculated for each experiment. The plasma levels were plotted as a function of time and the area under the curves (AUC) were measured using a planimeter. The Student's t-test was used to statistically measure the significance of the differences at a = 0.05. 22 A digital computer programme was developed to best-fit the experimental data points and also evaluate pharmacokinetic parameters following a single intravenous injection of a given drug. The programme is based on the Equations ('!)-:"-. ""-I.B.M. 370/168. 58 Cp = Ae" a t + Be" e t ( 1 ) which describes a two compartment open model. The computer f i r s t f i t s - (& the terminal part of the curve based on Cp = Be and then using the method — c t t residualsal i t f i t s the curve describing the distribution phase (a = Ae ). Addition of these two exponential curves, i.e., the distribution and post-distribution curves, results in a single bi-exponential curve.which defines the time-course of the disappearance of the drug from the central compart-ment. The computer computes the calculated slopes of both exponential processes with their correlation of coefficient and also the intercepts of each curve with Y-axis (A and B respectively). In the next step the following parameters are computed: biological half-lives of the distribution Tj (ALPHA) and post-distribution, Tt (BETA) phases; the plasma concentration at zero time, Cpo; the volume of distribution of the central compartment, vc; the f i r s t order rate constant for the distribution to the second compartment, k^; the f i r s t order rate constant for the distribution back to the central compartment, k^^; the f i r s t order rate constant for the overall elimination of the drug from the central compartment, kel; the area A B under the plasma concentration vs time curve, AUC = — + -j-; and, clearance CL (Appendix I"i) . 59 4. RESULTS AND DISCUSSION Determination of Griseofulvin Gas-Liquid Chromatography of Griseofulvin - The pulsed-mode linear electron-capture detector used for the griseofulvin assay provided the necessary sensitivity and specificity desired for plasmaSlevel studies. Excellent correlation (r = 0.999, Table 3-1) was found between peak height ratio and the amount of the drug i n the range from 16 peg to 48 ng of griseofulvin , (Fig. 3-1). This analytical procedure for the detection of griseofulvin in plasma samples represents a substantial improvement over the procedure described by Shah et a l (67). Under their experimental.. . tffondift-iQhsjjrit Shahse eiuttejLrs found linearity from 100 peg to 7 ng. Further, the technique described herein detects only those compounds with highly electron-iwA^hxtfc^^ i s , therefore, selective. The resulting chromatograph therefore contains only peaks due to griseofulvin, diazepam and occasionally some minor non-interfering compounds. The resolution of the peak is excellent, allowing precise quantitation using the peak-height ratio method (Fig. 3-2); Under, such conditions, the retention time was 2.08 and 6.24 minutes for diazepam and griseofulvin respectively. Diazepam was found to be a suitable internal standard. The diazepam peak appeared 2.08 minutes after the injection which i s 4.16 minutes apart from the griseofulvin peak. The resultant peak was sharp and repro-ducible. However, the electron-capture response to diazepam has been observed to be linear only in a limited concentration range (68 ). Since the internal standard technique requires the diazepam to be present in a constant concentration, this factor was of no consequence. Extraction Procedure - The applied method of extraction was simple and efficient. The mean percent griseofulvin recovery after extraction was 9 9 . 2 ± 2 . 8 (Table 3-II). This level of efficiency permits determination of trace quantities of griseofulvin in the biological samples. This is of particular importance when the time course of the drug is being studied. The griseofulvin plasma concentration in some rats declined to very low levels 48 hours after administration of griseofulvin. This concentration ranged from 0 . 0 0 1 to 0 . 9 6 ug/ml of griseofulvin in plasma (Tables 4-HI, 4-IV, 4-VII, 4-VIII, 4-X, 4-XIII, 4-XV, 4-XVI, 4-XVIII, 4-XX and 4-XXII) and for this reason, large volumes of blood were subject to analysis. In these cases animals were sacrificed and 0 . 5 to 1 ml of blood wase collected via a cardiac puncture. Nevertheless, attempts were made to maintain the lower limit of the assay at an approximate concentration of 40 pcg/5 ,ji 1 (.vl"5'•,ng/ml ' plasma), because lower griseofulvin levels would result -in large variations in the determinations of peak height. Addition of 0 . 1 ml of PEG 3 0 0 , PEG 6 0 0 , 0 . 5 % and 2% Tween 8 0 , or 1 ml of 50% ethanol to the aqueous phase prior to the extraction had no effect on the percent griseofulvin recovery. Equilibrium Solubility of Griseofulvin in the Presence of PEG 3 0 0 and Tween 80 Since the griseofulvin-phenobarbital interact ion has i3es?r. fmay;ula:}.o:cturj oca.tir at the level of dissolution, several formulations (suspension and solution) were prepared and administered to control and phenobarbital treated rats. Prior to tt'hese;pexp.erfimentaji, i t was necessary to determine the solubility of griseofulvin in the suspensions. Therefore the 61 equilibrium solubility of the drug in various solutions containing different concentrations of PEG 300 and Tween 80 was measured. Fig. 4-1 depicts the concentration of griseofulvin in the supernatant vs_ percent PEG in d i s t i l l e d water at room temperature and 37°. Each data point represents the mean of two determinations. Preliminary determinations showed that the supernatant was saturated with griseofulvin prior to 48 hours. As can be seen the solubility of griseofulvin increases with an increase in percent PEG 300. This is particularly profound when PEG content exceeds 60%. At room temperature, 3.20 mg/ml griseofulvin i s dissolved in an aqueous solution containing 70% PEG 300. After administration of 1 ml of this suspension, because of the l i p i d soluble nature of griseofulvin, a high plasma peak level should be observed in a short period of time. Further absorption, however, as w i l l be discussed later, may depend on the dissolution rate of griseofulvin in the surrounding f l u i d . Griseofulvin equilibrium solubility was determined also in the supernatant of preparations containing 0.5, 2 and 5% Tween 80 at room temperature. The griseofulvin concentration in the supernatant of a l l three preparations was 0.015 mg/ml. This is the same as griseofulvin solubility in water. Two explanations could be given for this observation: 1) In the range of 0.5 to 5%, Tween 80 might act only as a wetting agent without any solubilizing effect; or 2) Griseofulvin and Tween 80 might yield a complex phase which in turn sediments along with the solid phase in the process of centrifugation. Since the c r i t i c a l micelle concentration of Tween 80 is approximately 0.05%, i t i s unlikely that in the range of 0.5 to 5% that solubilization or some other interaction does not occur. Visual examinations revealed that an emulsion-like phase existed in the suspensions containing Tween 80. However, after centrifugation of the suspensions, a clear supernatant was obtained. It is possible for griseofulyin to be incorporated into this phase and to sediment 20 40 60 80 100 % PEG 300 Figure 4-1 . Equilibrium solubility of griseofulvin in aqueous solution's containing different concentrations of polyethelene glycol 300 at room temperature '( A ) and 37° (, # )• Each point represents average of 2 measure-ments . 63 during the process of centrifugation. Lack of a complete griseofulvin-Tween 80 phase diagram does not permit further interpretation of this observation. 64 Absorption of Griseofulvin Following Oral Administration of Different Dosage Forms The griseofulvin-phenobarbital interaction in the rat was previously reported (17) after a single dose of 100 mg/kg griseofulvin suspension (in 0.5% Tween 80) was administered orally to the rat. To i n i t i a t e this study on the mechanism of the interaction and also to ascertain the reproducibility of the previous observation the same dosage form was adopted in this work. Furthermore, since there have been questions as to whether the phenobarbital effect on the griseofulvin-plasma levels In the rat^.is due to a reduced absorption or induced metabolism, different dosage forms with improved dissolution properties were administered in an attempt to obviate the problem of dissolution rate. Fig. 4-2 depicts the griseofulvin-plasma concentrations vs time plots following the oral administration of single doses of griseofulvin in 0.5% Tween 80 (100 mg/kg), 2% Tween 80 (100 mg/kg), 70% PEG 300 (20 and 100 mg/kg) and 100% PEG 600 (50 mg/kg) to the rat. Each curve represents data from one rat with closest griseofulvin-plasma levels to the mean (n = 4 to 8). The absorption properties of griseofulvin were found to be highly dependent upon the dosage form. With regard to the attainment of the maximum griseofulvin-plasma level (Tmax) which i s an indication of the rate of absorption, the administered preparations f e l l into two distinctly different categories: a) suspensions and solutions containing PEG with very rapid absorption rates (Tmax, 15 to 30 min), and b) suspensions containing Tween 80 with slow absorption rates (Tmax, approximately 6 hr). The very short onset of absorption observed following administration of the drug in PEG can be explained in terms of the high avai l a b i l i t y of dissolved griseofulvin. Griseofulvin is a highly l i p i d soluble 65 drug. Once dissolved in PEG, griseofulvin readily crosses the gut membrane and appears in the plasma. The observation that the administration of 1 ml of suspensions containing either 5 or 25 mg drug resulted in the same Cmax (4.2 and 4.8^ig/ml respectively, Tables 4-XXI and 4-XXIII) supports this auggestion. Both suspensions contained 3.2 mg griseofulvin in the dissolved form (Fig. 4-1). This relatively small quantity of the available griseofulvin appears in the plasma as a sharp peak shortly after the administration (Fig. 4-2). Further absorption, however, requires a longer time, because the solid griseofulvin must f i r s t dissolve and then be absorbed. Dissolution of griseofulvin even in PEG is very slow, e.g., to dissolve a sample of 5 mg griseofulvin in pure PEG 300 or 600 required approximately 1 hour stirring with a magnetic s t i r r e r . A second griseofulvin-plasma peak also appeared at 6 to 8 hours post-dosing. This peak was of small magnitude (Fig. 4-2). An increase of fivefold in the administered dose (from 5 to 25 mg) resulted in only a small increase in AUC (from 11.2 to 18.5 j^g/ml/hr, Tables 4-XXI and 4-XXIII). This observation suggests that the absorption of solid griseofulvin is possibly dissolution rate limited. It is interesting to note that when griseofulvin i s administered in the PEG vehicle, absorption commences from the time immediately following administration (the f i r s t blood samples usually contained the highest Cmax, i.e., Tmax ( 15-20 min). This observation seems consistent with a significant absorption of the readily available dissolved griseofulvin in the stomach. The gastric emptying studies (Fig. 4-4) revealed that after one hour, approximately 84% of the griseofulvin-PEG 600*osolution could be recovered from the stomach, i.e., following administration of griseofulvin-8. Hours Figure 4-2. Griseofulvin-plasma level vs time following oral administration of single doses in 0.5%,;Tween 80(100 mg/kg), Q , 2% Tween 80 (100 mg/kg), • , 70% polyethylene glycol 300 (20 mg/kg), • , 70% polyethylene glycol 300 (100 mg/kg), O ,and 100% polyethylene glycol 600 (50 mg/kg), e • Each curve represents data from one rat closest to the mean. 6 7 PEG preparation, Tmax occurs when the drug is mainly in the stomach. Alternatively, i t appears more likely that a portion of the dissolved griseofulvin may be emptied from the stomach and absorbed quite rapidly even though the majority of the dose may s t i l l be found in the stomach at one hour. Among the administered dosage forms, griseofulvin-PEG 600 (solution) showed the highest Cmax (10.98^.g/ml, Table 4-IX) and, considering the dose (50 mg/kg), the highest AUC (37.85 i^g/ml/hr, Table 4-IX). This observation is consistent with that of Chiou and Riegelman ( 3 6 ) who admini-stered different preparations of griseofulvin to dogs and found that the absorption from a griseofulvin-PEG 400 solution was much faster and more complete. However, the absorption of griseofulvin even when administered in solution form, is by no means complete, because, as will be discussed later, the extent of absorption following administration of griseofulvin in solution to metoclopramide pretreated rats is threefold higher than in control rats. The relative bioavailability (AUC) of the preparations containing 70% PEG 300 (11.2 and 18.5^g/ml/hr for 20 and 100 mg/kg doses respectively) was significantly lower than that of pure PEG 600. This is likely due to the fact that in griseofulvin-PEG 600 preparations a l l of the drug (12.5 mg) was in solution while in 70% PEG 300 preparations only 3.20 mg of the administered doses (5 or 25 mg) was in the dissolved form. Absorption of griseofulvin from preparations containing Tween 80 was also erratic and incomplete. The mean Cmax and mean AUC following administration of 100 mg/kg griseofulvin in 0.5% Tween 80 was 6.69M g/ml and 68 43.28 ug/ml/hr respectively. Addition of 2% Tween 80, however, reduced the Cmax to 3.64 yg/ml and AUC to 30.00 yg/ml/hr, i.e., increase in Tween 80 resulted in a significant decrease in absorption. Recently Bloedow and Hayton (39;) administered 50 mg/kg griseofulvin in pure Tween 80 (solution) to the rat and noticed a Cmax of 1.53 yg/ml, Tmax of 8 hr and AUC of 15.8 yg.hr ml \ i.e., a solution of griseofulvin in pure Tween 80 has lower relative availability than aqueous suspensions containing 0.5 or 2% Tween 80. The critical micelle concentration (CMC) of Tween 80 is approximately 0.05%. Concentrations used in this research were well above the CMC (0.5 and 2%). In this concentration range Tween 80 is expected to act as a solubilizing agent as well as a wetting agent. Griseofulvin, in a suspension containing Tween 80, seems to have been incorporated into a complex phase with a very low apparent availability. The significantly lower AUC observed following administration of 100 mg/kg griseofulvin in 2% Tween 80 as compared to the same dose in 0.5% Tween 80 supports this hypothesis. A slow release would give rise to a long Tmax and, due to the removal of the drug from the absorptive sites before complete absorption, a lower AUC would be obtained. As can be seen in Tables 4-III and 4-VII the griseofulvin concen-tration in the terminal part of the plasma-level vs time curves fluctuates considerably and makes an accurate evaluation of the biological half-life of the drug virtually impossible. This is perhaps because of the continuous absorption of griseofulvin throughout the gastrointestinal tract. This observation was consistent in almost a l l of the curves regardless of the received treatments or administered dosage forms. 69 Effect of Motility Modifiers on Gastric Emptying The principal griseofulvin absorption site has been suggested to be the proximal segment of the small intestine (35). Absorption of the drug from the stomach when administered in solid dosage forms is very limited. Thus, i t appears reasonable to suggest that the gastric emptying rate affects the rate of absorption of griseofulvin. The longer the drug remains in the stomach the longer w i l l be the time required for attainment of Cmax. Meto-clopramide and propantheline have been used in this study to increase and decrease respectively the gastric emptying rate and gastrointestinal motility. In order to ascertain the influence of these agents on the gastric emptying, their effects were studied by determining the percent of'amr^s' ; • administered dose of griseofulvin remaining in the stomach as a function of time. The results of gastric emptying experiments are detailed in Tables 4-1 and 4-II and their mean values are shown in Figs. 4-3 and 4^ -4 follow-ing the administration of suspension (0.5% Tween 80) and solution forms (PEG 600) of griseofulvin respectively. Since the gastric absorption of griseofulvin (particularly following the administration of suspension forms) is limited,, the disappearance of the drug from the stomach can be considered to be mainly due to the gastric emptying process rather than the absorption process. The stimulating effect of metoclopramide on the gastric emptying rate is clearly demonstrated by the fact that, one hour after administration, only 24% of griseofulvin in suspension and 62% of griseofulvin in solution remained in the stomach as compared to 76% and 83% of suspension and solution respectively in the control rats (Figs. 4-3 and 4-4). rc o rd fi o -H CO c •H W) C •H •H fi QJ > rH 4H O 0) CO •H CD -H C 0) O <P PH 80 60 40 2 0 1 n l 4 6 Hours -fi-l l Figure4~3.- Percent g r i s e o f u l v i n remaining i n stomach i n the r a t f o l l o w i n g a d m i n i s t r a t i o n o f a s i n g l e o r a l dose o f 100 mg/kg g r i s e o f u l v i n i n 0.5% Tween 80. T e s t animals r e c e i v e d a s i n g l e i n t r a p e r i t o n e a l dose of 10.'mg/kg metoclopramide h y d r o c h l o r i d e or 5 mg/kg pr o p a n t h e l i n e bromide 2 hours p r i o r to g r i s e o f u l v i n a d m i n i s t r a t i o n . Key: • c o n t r o l , 0 metoclopramide p r e t r e a t e d , | p r o p a n t h e l i n e p r e t r e a t e d , E r r o r bars r e p r e s e n t standard d e v i a t i o n of the mean ( n=2 ). Table 4-1 Percent Griseofulvin remaining in Stomach and the corresponding Plasma Levels in the Rat following Oral Administration of Single Doses of 100 nig/kg Griseofulvin in 0.5% Tween 80. Test Rats received Single Doses of either 10 mg/kg Metoclopramide Hydrochloride or 5 mg/kg P.ropan the line Bromide 2 Hours Prior to the Griseofulvin Administration. Control Metoclopramide Propantheline Hour, [ pretreated, pretreated,  post-dosing, % in Plasma level, % in Plasma level, % in Plasma level, stomach, yg/ml, stomach, yg/ml, stomach, yg/ml, 0 98.4 - 99.3 - 96.0 -96.8 - 100.4 - 98.1 -1 74.2 1.02 20.1 ... 2.74 84.4 • 0.73 77.7 1.02 27.7 3.85 89.2 0.83 2 69.6 1.61 32.7 1.44 60.9 1.93 57.7 0.83 • 16.4 3.13 89.7 0.99 4 18.7 2.85 •: 7.8 2.07 32.1 4.85 31.1 3.11 1,6 3.91 31.9 0.89 6 1.9 5.75 0.9 1.01 4.3 0.94 9.2 2.78 0.5 2.18 28.0 0.75 11 0.2 0.08 0.5 0.01 25.2 2.12 1.8 0.04 1T4 0.02 47.6 0.21 o 1 2 4 6 11 Hours F i g u r e 4-4.Percent g r i s e o f u l v i n remaining i n stomach i n the r a t f o l l o w i n g a d m i n i s t r a t i o n of a s i n g l e o r a l dose of 50 mg/kg g r i s e o f u l v i n i n p o l y e t h y l e n e g l y c o l 600. Tes t animals r e c e i v e d a s i n g l e i n t r a p e r i t o n e a l dose of 10 mg/kg metoclopramide hydro-c h l o r i d e or 5 mg/kg p r o p a n t h e l i n e bromide 2 hours p r i o r t o g r i s e o f u l v i n a d m i n i s r r a t i o n . Key: Q c o n t r o l , ^ metoclopramide p r e t r e a t e d , | propan-t h e l i n e p r e t r e a t e d . E r r o r bars r e p r e s e n t standard d e v i a t i o n of the mean (n=2-3). Table 4-II Percent Griseofulvin remaining in Stomach following Oral Administration of Single Doses of 50 mg/kg Griseofulvin in Polyethylene Glycol 600 to the Control, Metoclopramide Hydrochloride Pretreated Rats(10 mg/kg) and Propantheline Bromide Pretreated Rats(5 mg/kg). Hour Percent remaining in stomach, .-dosing, Control, Metoclopramide Propantheline c * ' 'pretreated, - pretreated. 0 98.1 96.5 103.3 101.3 95.9 96.4 1 86.3 46.7 96.1 87.8 88.6 97.1 77.6 50.9 -2 89.4 44.8 104.3 65.6 49.0 81.5 82.9 30.8 96.4 4 56.5 37.5 102.4 46.9 :.7.3 74.6 49.7 41.7 73.0 6 31.4 5.5 68.7 - 2.6 52.1 24.6 22.6 -11 0.0 0.4 49.4 - - 54.4 2.3 1.4 35.1 74 This observation confirms the previous reports ( 43 - 45 ) that metoclopramide accelerates the g a s t r i c emptying rate. In both man (43) and dog (45) metoclopramide has been e f f e c t i v e i n reducing t r a n s i t time i n the stomach as w e l l as i n the small i n t e s t i n e . The e f f e c t of propantheline was found to sharply contrast with that of metoclopramide (Figs. 4-3 and 4-4). Propantheline s i g n i f i c a n t l y retarded the g a s t r i c emptying rate i n the r a t . The magnitude of th i s e f f e c t was i n d i r e c t l y indicated by the amount of g r i s e o f u l v i n remaining i n the stomach at various times post-dosing. As a r e s u l t of propantheline treatment, 36% of g r i s e o f u l v i n i n suspension and 46% i n s o l u t i o n was recovered from the stomach 11 hours a f t e r g r i s e o f u l v i n administration. A l t e r n a t i v e l y , i n the cont r o l and metoclopramide treated r a t s , at 11 hours post-dosing only a n e g l i g i b l e amount of g r i s e o f u l v i n was recovered from the stomach. The e f f e c t of propantheline on the g a s t r i c emptying rate, l i k e a t r o p i n e , l i s due to i t s i n h i b i t o r y influence on the motor a c t i v i t y of the stomach (.46). Such an i n h i b i t o r y e f f e c t would give r i s e to a longer residence time i n the gut. Table 4-1 also contains the griseofulvin-plasma l e v e l s at the time of determining the g a s t r i c emptying content. These l e v e l s are i n agreement with those observed i n the plasma l e v e l studies (Tables 4-III and 4-IV). I t i s worth noting that administration of g r i s e o f u l v i n i n PEG 600 caused a noticeable change i n the appearance of the gut along with an apparent tendency to r e t a i n water. Despite a large i n t e r animal v a r i a t i o n , the r e s u l t s c l e a r l y i n d i c a t e that s i n g l e i n t r a p e r i t o n e a l doses of 10 mg/kg metoclopramide hydrochloride and 5 mg/kg propantheline bromide are s i g n i f i c a n t l y e f f e c t i v e i n stimulating and retarding r e s p e c t i v e l y the g a s t r i c emptying rate i n the r a t . Effect of Motility Modifiers on Griseofulvin Plasma levels The principal absorption site of the drug administered as a solid form has been suggested to be the proximal segment of the small intestine (35). Any changes in gastrointestinal motility and hence the residence time of the drug in the gut would be expected to significantly influence the absorption of this incompletely absorbed drug. A recent report by Riegelman et al (20) pointed to another important factor which may influence griseofulvin absorption. In the course of examining the griseofulvin-phenobarbital interaction they postulated that this interaction may be triggered by an increase in the gut motility (20). In order to rationally examine the griseofulvin-phenobarbital interaction in greater detail i t was important to f i r s t c l a r i f y precisely the influence of gut motility on the absorption of griseofulvin. Through preadministration of selected motility modifying agents, the influence of stimulated and retarded gastric emptying and motility on griseofulvin absorption was evaluated. Therefore, a suspension of 100 mg/kg griseofulvin in 0.5% Tween 80 and a solution of 50 mg/kg griseofulvin in 100% PEG 600 were administered in the presence and absence of either metoclopramide or propantheline pretreatments. Single Oral Dose of Griseofulvin Suspended in 0.5% Tween 80 -Figure 4-5 depicts mean plasma concentration vs_ time plots obtained following a single dose administration of 100 mg/kg griseofulvin in 0.5% Tween 80 in the presence and absence of single doses of metoclopramide and propantheline. Data points represent means of 7 to 9 experimental observations (Tables 4-III ft 8 12 16" 20 2ft 28 ft8 Hours Fi g u r e 4-5-. Plasma concentrations of g r i s e o f u l v i n as a f u n c t i o n of time f o l l o w i n g o r a l a d m i n i s t r a t i o n of a 100 mg/kg dose of a suspension of micronized g r i s e o f u l v i n i n 0.5% Tween 80 to the c o n t r o l r a t s ( A ) , and r a t s p r e t r e a t e d with e i t h e r 10 mg/kg metoclopramide h y d r o c h l o r i d e ( O ) , or 5mg/kg pr o p a n t h e l i n e b r o m i d e ( Q ) . E r r o r bars represent standard e r r o r of the mean (n=7-9). Table Plasma Levels of Griseofulvin (Micrograms per M i l l i l i t e r ) following Oral Administration of 100 mg/kg i n 0.5% Tween 80 to Control and Test Animals. Metoclopramide (10 mg./Kg.) was given Intraperitoneally to the Test Animals, two hours prior to Griseofulvin Administration. Rat Hours 2 4 5 6 8 12 : 24 48 Griseofulvin 1 1.88 2.67 5.96 7.35 2.25 0.52 0.24 0.06 Control 2 1.66 6.01 7.68 7.06 4.53 1.51 0.28 0.12 3 1.48 2.53 6.01 6.92 3.74 0. 21 0.12 — 4 0.63 5.56 6.61 7.02 3.25 0.04 0.15 — 5 1.38 5.68 — 6.02 1. 23 0.43 0.81 — 6 3.84 5.22 — 6.05 3.82 2.01 0.16 0.17 7 1.99 5.62 — 6.60 — 1.16 0.31 0.62 8 1.42 2.53 5.3 6.24 0.52 0.82 1.10 0.11 9 1.46 5.18 6.32 — 1.30 0.28 0.34 0.12 Mean^ 1.75 4.55 6.31 6.58 2.58 0.78 0.39 0.20 (S.E.) (0.29) (0.50) (0.33) (0. 20) (0.52) (0.22) (0.11) (0.08) Metoclopramide 1 — 3.12 2.73 2.31 2.36 — 0.45 0.02 (10 mg./Kg. , 2' 2. 26 4.01 3.82 3.99 1.40 0.32 0.47 0.16 i.p.) 3 1.26 2.50 2.02 1.78 1.33 0.28 0.39 0.08 predosing 4 1.31 0.88 0.61 0.43 0.43 0.17 0.21 0.02 5 2.63 2.42 — 0.43 0.62 0.34 0.42 0.10 6 1.85 1.90 — 0.69 1.83 1.02 0.81 — 7 3 - 8 2 * * ** 0-73^ 0.36 0.42 — Mean^ 2.19 2.34 2.29 1.67 1.26 0.41 0.45 0.08 (S.E.) (0.39) (0.39) (0.67) (0.45) (0.26) (0.12) (0.07) (0.03) *) Standard error of the mean in parentheses. **) Significantly different at a=0.05. Table 4-IV Plasma Levels of Griseofulvin (Micrograms per M i l l i l i t e r ) following Oral Administration of 100 mg/kg i n 0.5% Tween 80 to Rats Receiving Propantheline (5 mg./Kg., i. p . ) , two hours prior to Griseofulvin Administration. Rat Hours 2 4 6 8 12 14 17 20 23 26 29 : 48 Propantheline 1 0.577 1.150 1.231 1.181 1.293 1.782 1.782 1.081 — 1.252 1.311 0.621 (5 mg./Kg., i.p.) 2 0.925 0.555 0.822 0.950 0.955 0.933 1.364 3.165 — .2.300 0.497 0.202 predosing- 3 1.263 1.311 1.421 1.372 1.491 1.420 1.850 3.235 7.622 3.477 1.610 0.727 4 1.611 1.778 1.835 1.525 1.573 1.532 1.662 1.410 -- 0.554 0.401 0.212 5 1.792 1.400 1.773 2.002 2.322 2.302 2.434 3.535 5.422 2.629 2.555 0.437 6 1.820 1.900 2.031 2.333 2.352 2.080 3.650 6.277 — 1.400 1.010 0.370 .7 2.210 2.088 2.457 2.530 2.433 4.781 2.807 2.131 1.901 1.632 1.107 0.913 8 2.584 2.611 2.832 3.308 3.500 3.531 3.443 3.872 6.630 4.307 1.922 0.770 9 3.350 1.707 1.005 0.791 0.660 0.701 0.888 1.250 3.374 2.073 0.815 0.920 Mean *1.792 1.611 1.712 1.777 1.842 2.118 2.209 2.884 4.990 2.180 1.248 0.554 (S.E.) (.283) (0.198) (0.222) (. 277) (0. 295) (0. 433) (.314) (. 550) (1. 048) (3. 88) (0. 231) (0.105) *) Standard error of the mean i n parentheses 79 and 4-IV) and error bars are the standard error of the mean. Pretreatment with metoclopramide, an agent capable of stimulating the gastric emptying reflex and gastrointestinal motility (43), decreased the Cmax by 59% while at the same time i t reduced Tmax by 2.7 hours (Table 4-V). The area under the griseofulvin plasma level vs_ time curve was also decreased by 50.6% (Table 4-VI) as compared to controls. Alternatively, predosing with propantheline delayed the time for maximum plasma concentration from 5.8 hours to 19 hours (Table 4-V) while at the same time increasing the AUC by 50.4% (Table 4-VI). These alterations were accompanied by a 30% reduction in the Cmax for plasma griseofulvin (Table 4-V). These observations are consistent with previously observed influences of gastrointestinal motility modifiers on drug absorption. Meto-clopramide has been shown to effectively shorten the time for attainment of peak levels of paracetamol (47,), sulfamethoxole (50i), tetracycline and pivampicillin (|8) with l i t t l e or no effect on the total absorption of these drugs. The unchanged total absorption in the presence and absence of meto-clopramide is due, perhaps, to the high availability of these drugs, i.e., the readily available drugs are absorbed completely despite the reduced transit time caused by metoclopramide treatment. With griseofulvin a dramatically different situation is encountered since the absorption of this drug is incomplete and erratic (6). Metoclopramide, through i t s stimulatory influence on the gastrointestinal tract, has forced the drug out of the stomach to the primary site of absorption, namely the small intestine (shorter Tmax), and decreased the residence time of griseofulvin in the small intestine, Table 4-V Peak Plasma Levels (Cmax) and its Time of Occurrence (Tmax) Following Administration of 100 mg/kg Griseofulvin in 0.5% Tween 80 to the Control, Metoclopramide Hydrochloride (lOmg/kg)?and Propan-theline Bromide (5 mg/kg) Pretreated Rats. Control Cmax, y g/ml. Tmax, hr. Metoclopramide pretreated, Cmaxj^ g/ml.,, Tmax, hr. Propantheline pretreated, Cmax, yg/ml Tmax, hr. 7.35 6 3.12 4 3.37 23 7.68 5 4.01 4 1.78 20 6.92 6 2.50 4 7.62 23 7.02 6 1.31 2 1.83 6 6.02 6 2.63 2 5.42 23 6.05 6 1.90 4 6.28 20 6.60 6 3.82 2 4.78 14 6.24 6 6.63 23 6.32 5 Mean, 6.69 5.8 2.75 3.1 4.71 19.0 Standard error, 0.20 0.15 0.37 0.40 0.78 2.15 Coefficientof 8.9 7.8 35.6 33.7 46.6 32.1 variation, %, Statistical difference,"'' s s s s 1. Determined by the Student's t-test at a=0.05 (2 tailed); s, significant. 81 Table 4-VI Area Under Plasma Concentration-Time (jig.hr.ml "'") of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control, Metoclopramide Hydrochloride Pretreated and Propantheline Bromide Pretreated Rats. Control, Metoclopramide Propantheline pretreated, pretreated, 37.5 24.9 50.4 53.0 30.9 41.6 37.3 19.9 42.3 37.8 10.5 76.0 52.9 15.8 42.8 50.6 27.2 85.7 45.8 23.5 73.2 34.1 65.9 40.4 105.7 Mean, 43.3 4 21.8 65.1 Standard error, 2.46 2.62 7.64 Mean AUC/dose, hr.ml - 1.10 3 1.73 0.87 2.61 Coefficient of 17.1 32.0 35.3 variation, %, St a t i s t i c a l difference^, s s 1. Determined by the Student's t-test; a=0.05 (2 tailed); s, significant. 82 resulting in a substantial decrease in total absorption. When propantheline was administered prior to the administration of griseofulvin suspension, a distinct contrast with the influence of meto-clopramide was observed. Propantheline preadministration markedly increased the relative availability of griseofulvin suspension from controls presumably through a -mechanism similar to that reported with digoxin tablets (13) and phenolsulf onphthalein solution (15)', i.e., an increased residence time in the gastrointestinal tract thereby allowing more time for these poorly absorbed drugs to be absorbed. In the case of griseofulvin, the increase ' in residence time allows more time for the dissolution process thereby enhancing the extent of absorption. It i s interesting to note that propantheline pretreatment has been observed to delay Tmax of many drugs regardless of their degree of availability, while i t s effect on the total absorption, similar to that of metoclopramide, is limited only to those drugs with low availability. The anticholinergic drug delayed the Tmax for sulfamethoxole (50) and paracetamol (•4,7) with no significant effect on their relative a v a i l a b i l i t i e s . It is worth noticing that the inter animal variation which i s indicated as the percent coefficient of variation (Tables 4-VI: and 4-VI.) was observed to be greatest in the test animals pretreated with propantheline. s This may be attributed to the inter animal variation in pharmacological response to the propantheline treatment. 83 Single Oral Dose of Griseofulvin Dissolved in 100% PEG 600 - An interesting contrasting interaction between the motility modifiers and griseofulvin was observed when griseofulvin was administered in solution. Figure 4-6 represents mean plasma-griseofulvin concentration vs time plots measured after oral administration of griseofulvin dissolved in PEG 600 in control and test rats. Pretreatment with metoclopramide showed i t s effect by increasing, Cmax by 140% (Table 4-VII) and AUC by 230% (Table 4-IX). The effect of propantheline predbsing was in sharp contrast to that observed after metoclopramide administration. Propantheline reduced the relative bio-availability of griseofulvin solution by 50.4% and decreased the plasma levels significantly (Table 4-IX). Absorption of griseofulvin in a l l cases was found to be rapid. The maximum plasma concentrations were attained between 20 and 30 mins (Table 4-VII and 4-VIII) after griseofulvin administration. This is due to the fact that when the drug is given in solution form, absorption is not limited by the dissolution process, in contrast to the undissolved fraction of suspension, but takes place as soon as the dosage form reaches the absorption site. In the control rats, although the administered dose of griseofulvin in the solution form was one-half of that in the suspension form, the observed AUCs were approximately of the same magnitude, indicating higher relative bioavailability of the solution form (Tables 4-VI and 4-IX). Lower percent coefficient of variation in the rats treated with metoclopramide (Table 4-IX) is an indication of a more uniform relative availability due, perhaps, to a more complete absorption in the absence of/ the dissolution problem inherent in the use of solid dosage forms of griseofulvin. Hours Fig u r e 4-6\. Plasma c o n c e n t r a t i o n of g r i s e o f u l v i n as a f u n c t i o n of time f o l l o w i n g o r a l a d m i n i s t r a t i o n of a s i n g l e dose of 50 mg/kg g r i s e o f u l v i n i n PEG 600 to the c o n t r o l ( A ) , and r a t s p r e t r e a t e d with e i t h e r 10 mg/kg metoclopramide h y d r o c h l o r i d e ( O ) , or 5mg/kg propantheline bromide ( • ) . E r r o r bars r e p r e s e t standard e r r o r of the mean( n=7-8). Table 4-VII": Plasma L e v e l s o f G r i s e o f u l v i n (Microgram p e r M i l l i l i t e r ) f o l l o w i n g i t s O r a l A d m i n i s t r a t i o n i n a S i n g l e 50 mg/kg i n PEG 600 Dose i n C o n t r o l and T e s t R a t s , M e t o c l o p r a m i d e (10-mg/kg) was g i v e n I n t r a p e r i t o n e a l l y t o t h e T e s t A n i m a l s 2 h o u r s p r i o r to G r i s e o f u l v i n A d m i n i s t r a t i o n , HOURS 0.33 0.5 1 2 3 4 . 6 8 12 24 48 Rat G r i s e o f u l v i n 1 — 15.321 8.120 5. .440 3.280 1.613 0.856 0.451 0.854 0.500 0.030 C o n t r o l 2 — 13.098 7.946 4. .403 2.554 1.304 0.487 1.344 0.751 0.032 0.024 3 — 12.008 8.704 5, .512 4.531 5.227 5.781 2.607 0.308 0.148 0.005 4 — 6.093 3.671 1. .921 2.416 1.526 2.099 1.523 0.473 0.233 0.018 5. — • 11.677 6.896 3, .000 1.337 • 1.021 2.251 2.486 0.508 0.290 0.036 6. .12.331 — 8.120 4. .915 — . 1.280 0.675 0.471 0.816 — — 7. 8.124 — — 3. .466 — 1.191 0.450 0.623 0.983 0.310 0.001 8. — 9.233 — 4, .204 — 1.380 0.724 0.591 0.814 0.204 0.028 Mean 10.2275 11.238 7.243 4. .108 2.824 1.818 1.665 1.262 0.688 0.208 0.020 ( S E ) 3 (2.100) (1.309) (0.754) (0.4415) (0.528) (0.491) (0.634) (0.314) (0.082) (0.062) (0.005) Meto- 1. — 20.341 — 14.644 — 9.416 12.322 7.817 — 0.506 0.096 c l o p r a m i d e 2. — 25.416 22.240 14.960 10.311 10.107 12.100 9.554 1.310 0.130 0.008 (10 mg/kg, 3. -- 22.026 18.466 11.681 8.504 9.622 7.983 3.751 0.556 0.038 0.013 i . p . ) 4. — 34.813 30.323 18.477 13.138 13.887 12.807 6.763 0.432 0.083 0.009 p r e d o s i n g 5. 33.034 16.046 12.040 10.546 11.354 7.613 5.278 1.358 0.410 0.016 6. — 25.254 . 19.654 15.703 — 12.116 5.722 7.800 2.964 0.267 0.014 7. — 17.600 — 13.051 — 8.564 7.870 7.506 — 0.484 0.307 < 8. — 37.204 — 25.484 — — 9.117 5.870 — 0.310 0.080 Mean 26.9605 21.346 15.8175 10.625 10.724 9.442 6.792 1.324 0.2785 0.068 ( S E ) 3 (2.551) (2.456) (1.645) (0.954) (0.695) (0.931) (0.635) (0.451) (0.064) (0.036) S t a t i s t i c a l S i g n i f i c a n c e s s s s s s s ns ns ns a) Standard e r r o r o f t h e mean i n p a r e n t h e s e s b) Determined by s t u d e n t ' s t - t e s t , (2 T a i l e d ) ; P<0.05 / Table 4-i.V.III P l a s m a . L e v e l s o f G r i s e o f u l v i n (.Micrograms p e r M i l l i l i t e r ) a f t e r a S i n g l e O r a l Dose (50 mg./Kg.) i n PEG 600 i n C o n t r o l and T e s t R a t s . P r o p a n t h e l i n e (5 mg./Kg.) was g i v e n I n t r a p e r i t o n e a l l y to t h e T e s t A n i m a l s , two h o u r s p r i o r t o G r i s e o f u l v i n A d m i n i s t r a t i o n . Rats Hours 0.33 0.5 2 4 6 8 10 12 17 20 24 48 G r i s e o f u l v i n 1 —• 15.321 5.440 1.613 0.856 0.451 0.854 0.500 0.030 c o n t r o l 2 — 13.098 4.403 1.304 0.487 1.344 — 0.751 0.210 0.081 0.032 0.024 3 — 12.008 5.512 5.227 5.781 2.607 — 0.308 — — 0.148 0.005 4 — 6.093 1.921 1.526 2.099 1.523 — 0.473 — — 0.233 0.018 5 — 11.677 3.000 1.021 2.251 2.486 — 0.508 — 0.103 0.029 0.036 6 12.331 — 4.915 1.280 0.675 0.471 — 0.816 — — — — 7 8.124 — 3.466 1.191 0.450 0.623 — 0.983 0.787 — 0.310 0.001 8 — 9.233 4.204 1.380 0.724 0.591 0.814 0.691 0.311 0.204 0.028 Mean. 10.2275 11.238 4.108 1.818 1.665 1.262 0.688 0.563 0.165 0.208 0.020 • ( S . E . ) 3 (2.100) (1.309) (0.4415)(0.491) (0.639) (0.314) — (0.082) (0.178) (0.073) (0.062) (0.005) P r o p a n t h e l i n e 1 5.831 — — . 0.851 0.819 0.932 1.031 0.985 1.115 2.311 0.517 0.005 C5 mg/kg 2 3.874 — 1.474 1.038 1.042 2.840 1.415 1.071 0.603 0.400 0.201 0.060 i p ) 3 3.514 — — 0.699 0.337 0.211 0.347 1.310 0.188 0.102 0.101 0.031 p r e d o s i n g 4 — 1.731 3.547 0.532 0.709 0.988 0.901 0.980 0.832 0.747 0.531 0.006 5 — 1.701 1.684 ' 2.610 0.484 0.125 0.044 0.029 0.030 0.419 0.394 0.010 6 ~ 3.401 3.393 2.133 2.112 0.589 0.053 0.173 0.132 -- 0.101 0.000 7 3.405 — 1.196 0.756 0.602 2.488 0.552 0.831 0.525 — 0.110 0.000 Mean 4.156 2.278 2.259 1.231 0.872 1.168 0.620 0.768 0.489 0.795 0.279 0.016 (S.E.)^0.567) (0.562) (0.501) (0.304) (0.224) (0.407) (0.196)(0.181) (0.151) (0.392) (0.074) (0.008) S t a t i s t i c a l ^ s s s ns ns ns ns ns ns ns ns ns s i g n i f i c a n c e a) Standard e r r o r o f t h e mean i n p a r e n t h e s i s b) Determined by S t u d e n t ' s t - T e s t , (2 t a i l e d ) ; P < 0 . 0 5 87 Since metoclopramide shortens the gastric emptying time and stimulates gastrointestinal motility (4$), the absorption of the readily available dissolved drug w i l l be enhanced by rapid exposure of the drug to the proximal portion of the intestine. Consequently, i f griseofulvin is presented to this region more quickly in a readily available dosage form, the rate of absorption w i l l be increased. Conversely, i f griseo-fulvin i s administered in a less readily absorbed form, such as a suspension, then the increased absorption rate w i l l be accompanied by a decreased relative availability due to the decreased residence time at the absorption site.- Shorter residence time at the absorption site may cause the undissolved fraction of the drug to bypass the. region before absorption can take place. These observations suggest that the absorption of griseofulvin suspension i s limited to two major factors, v i z t h e gastric emptying rate and the dissolution process. The diminished time for attainment of the peak plasma level following administration of griseofulvin suspension in metoclopramide pretreated rats (Figure 4-5) is due to a more rapid gastric emptying rate, as clearly illustrated by the emptying experiments (Fig. 4-3 and Table 4-1) which show that one hour following administration of griseofulvin suspension only 24% of the drug i s recovered from the stomachs of the metoclopramide pretreated , rats. The lower relative availability may be due to the passage of the suspension from the absorption site prior to complete dissolution. On the other hand, the delayed Tmax following propantheline pretreatment i s lik e l y due to a very low gastric emptying rate (as shown in Fig. 4-3, 36% of griseofulvin was recovered from the stomach 11 hours after administration). Table 4-IX Area Under Plasma Concentration-Time Curves, AUC ( ug.hr.ml ^) and the Peak Plasma Concentrations, Cmax ( ug/ml) Following Oral Administration of Griseofulvin (50 mg/kg) in 100% Polyethylene Glycol 600 to Control and Metoclopramide and Propantheline Pretreated Rats. Control, Metoclopramide pretreated, Propantheline pretreated, • AUC, Cmax, r > r- AUG, Cmax, AUC, Cmax, 37.6- 15.32 126.6 20.34 3784 5.83 38.8 13.10 138.6 25.42 27.5 .3.87 55'. 5- 12.01 84.6 22.03 16.4 3.51 30.1 6.09 142.4 34.81 :-2«:2 :3.40 37.9 11. 63 115.0 33.03 17.8 3.40 41.3 12.33 126.4 25.25 22.6 28.9 8.12 119.4 17.60 13.5 3 2.6 9.23 158*2 37.20 6.1 Mean, 37.8 10.98 126.4 26.96 21.2 4.00 Standard error, 2.96 1.051 7.73 2.551 3.46 0.48 Mean AUC/dose, hr.ml~ 1.10 3 ' 3?03 10.11 1.69 Coefficient of 22.1 27.1 17.3 26.8fe 46.3 27.0. variation, %, Statistical difference, s s s , s 1. Determined by the Student's t-test; a=0.05 (2 tailed); s, significant. 89 Consequently, the undissolved drug travels through the gastrointestinal tract very slowly, permitting more drug dissolution and subsequent absorption. The oral solution studies support the preceding explanation. With an oral solution of griseofulvin, the drug i s readily available and a dissolution process i s less significant; the absorption is more rapid and begins from the moment that the dosage form is in contact with the absorptive site. Some precipitation of the dissolved fraction may occur but i t s consequence on the absorption w i l l be less significant than that of a suspension form as i s evidenced by the observed AUC. Metoclopramide pretreat-ment markedly increased the bioavailability of the solution. This is because the transit time in the gastrointestinal tract has been shortened, and there is less chance for precipitation of griseofulvin in the stomach. Therefore there i s no dissolution process to delay absorption, and the drug is readily absorbed. However, propantheline pretreatment induces a longer residence time of the drug in the stomach where the absorption of griseofulvin is limited. This delayed release of the drug to the absorption site may result in either precipitation of the drug or in degradation of griseofulvin. Davis et a l (28) noticed a continuous disappearance of griseofulvin from the alimentary canal of the rat corresponding with the f a l l in blood level. They attributed this observation to griseofulvin destruction in the gut. However, in this work addition of 1 ml d i s t i l l e d water to 1 ml of griseo-fulvin in PEG 600 solution in a test tube caused the drug to gradually pre-cipitate into fine crystals. This observation, coupled with the fact that a large percent of unchanged griseofulvin was recovered from the stomach of the propantheline pretreated rats 11 hours after griseofulvin administration, 90 suggests that precipitation rather than destruction of the drug may be responsible for the poor relative bioavailability. In contrast' to the observed effect of propantheline on griseofulvin solution, Manninen e_t a l ( . 1 3 ) noticed that the absorption properties of digoxin, when given in solution form, were unaffected by the treatment with the anticholinergic drug. This difference can perhaps be explained by the different solubility properties of these two drugs, i.e., following admin-istration, upon longer residence time in the stomach griseofulvin may precipitate and yield a lower relative availability while digoxin may remain in so'Sufei6*nita"ndi s.ub'setq.uently. b.e ;abIs,oj-Aed>., Multiple Dose and Long Term Treatment with Propantheline - The results of multiple dose treatment with propantheline are detailed in Table 4-X. Figure 4-7 a,b,c depicts plasma levels of griseofulvin following administration of a single dose of 100 mg/kg griseofulvin in 0.5% Tween 80 to rats treated intraperitoneally with 5 mg/kg propantheline bromide at 2 hours prior to and at 6, 14 and 26 hours post-griseofulvin dosing. Table 4-X and Figs. 4-7 d and e show the results of an oral administration of a single dose of 100 mg/kg griseofulvin to rats pretreated with a single intraperitoneal dose of 5 mg/kg propantheline bromide. The latter group of rats was used to simultaneously study the effect of single and multiple treatment of propantheline on the plasma levels of griseofulvin suspension. The results, D f long-term treatment with propantheline on,vgriseofulvin-ipl?asmaofle,vel!S:ofuiyin are detailed in Table 4-XI and are plotted as griseo-•-' fulvin plasma concentration vs_ time in Fig'i( 4-8. Test animals received doses of 5 mg/kg propantheline bromide twice a day for seven days prior to and during the experiment. 9 1 -Figure 4-7, ,(a,b,and c).. . For details see next page -* i g u * 4- • f.;Ca'D% ana •£) . .tor a e u u see neict 6 t page 9 2 4 12 20 28 36 44 52 Hours Fi g u r e 4 - 7 : . ^ G r i s e o f u l v i n - p l a s m a c o n c e n t r a t i o n s f f bilowinsgr-oral a d m i n i s t r a t i o n of s"±tt|iie^ iii^ o6e'i3j;.••. of 100 mg/kg g r i s e o f u l v i n i n 0 i 5% Tween 80 to the r a t s p r e t r e a t e d i n t r a p e r i t o n e a l l y with either, a .single, dose/•".of ^ propantheline (2 hours prior to jgriseofulyin;' "rats d and ~e) :or multi^^ at' 6,.. 14..and 24 hour's. after' gri-seofulvin administration;' rats, a, ;b and -c. in.- preceding" page) -~\ Arrows'indicate the" time of propanthelineadministration. Table 4-XJ Plasma Levels of Griseofulvin (jjgY ml).M-olt^liig-it s lOMl'i A4minlsEratiofiO(jbgQlqgg/kgOiri%QT5%-TwSen 80) to Animals Receiving either a.SingleiposeopjfwJ>.r.opantKeiine',26H6iirs'nP.r$ort'to, Griseofulvin Administration .or. Multiple. Doses at. 2^-Hours -Prioretc ka'h(ia*aty6, 14 and 24 Hours After Griseofulvin Administration. latRat Hours 10 14 17 20 26 30 48 72 Propantheline a 0.398 1.102 1.878 1.180 1.149 3.129 1.800 0.622 0.361 — — Pretreated (5 mg/kg, i.p., b — 0.461 8.91 1.295 3.525 3.260 1.726 0.548 0.584 1.020 0.000 at -2, 6, 14 and 26 hours). c 0.844 1.777 3.882 5.245 2.910 4.669 1.082 0.375 0.237 — — Propantheline d — 0.334 0.726 4.235 2.609 1.694 0.854 0.241 0.282 Pretreated (5 mg/kg, i.p. e 0.565 0.440 2.534 1.920 1.955 5.232 1.604 0.945 — at -2 hours) 94 12 20 28 36 44 52 Hours Fig u r e 4 -8" ( a, b, and c ). iFor. i e t a i l s s!ee3p§fel^96. ho G r i s e o f u l v i n - P l a s m a L e v e l s , ug/m-L -i i r o c l-i CO K3 N3 O N3 oo Lo 4> N3 H i 96 Figure 4-8 . Griseofulvin-plasma concentrations following oral administration of single doses of 100 mg/kg g r i s e o f u l v i n in 0.5% Tween 80 to the rats treated i n t r a p e r i t o n e a l l y with 5 mg/kg propantheline bromide twice d a i l y , 7 days prior to and during the plasma: studies, • :.'(ffats ^  $hrQHgh:hj):-X*see last, two preceding pages), (h=l). ' . Table 4-XIT. Plasma Levels of Griseofulvin (ug.^l)1' f o l a o w i n ^ Tween;.80) to the Control and Test Rats. Control Animals were treated Intraperitoneally with 1 ml of Di s t i l l e d Water while Tests Rats received 5 mg/kg Propantheline Bromide Intraperitoneally Twice Daily 7 Days Prior to and During the Plasma Studies. Rat Hours ConfrcIs 2 4 6 11 14 17 20 24 28 48 Controls 1 1.188 5.982 3.843 0.517 0.523 0.291 0.235 0.030 0.036 0.010 2 1.637 3.565 5.143 0.547 0.450 0.432 0.588 0.501 0.082 0.000 Propantheline a 1.015 2.481 1.156 4.093 3.139 5.082 1.375 Long-term Pretreated b 0.647 1.021 1.200 1.892 3.715 — 1.874 1.458 0.479 0.019 c 2.190 1.383 0.729 1.907 2.280 1.208 2.799 2.156 — — d 1.785 0.568 1.041 1.973 1.819 0.928 1.507 2.264 0.000 0.015 e 2.595 1.468 1.730 1.078 0.641 — 0.429 0.328 0.146 ' — f 0.875 0.795 0.819 3.401 — 2.954 2.960 0.340 0.453 — g 1.136 8.588 4.855 3.290 2.035 1.837 0.787 0.000 0.378 0.000 h 1.681 3.544 4.505 2.445 4.149 5.063 2.858 0.902 0.282 0.000 98 Following repetitive treatment with propantheline, a l l of the griseofulvin-plasma levels-time curves share the unusual characteristic of multiple peaks" and-.4, demonstrated f tendency to fluctuate. This observation was also noticed to a lesser degree in some^of the single treatment studies which were described previously (Table 4-V). Attention should be paid to the fact that Fig. 4-4, which, depicts the results obtained after treatment with single doses of propantheline, is expressed in terms of mean plasma levels vs time. As a result of averaging the data points, the fluctuations are obscured and the resulting curve appears like a smooth curve. No correlation between the time of propantheline treatment and frequency of occurrence of the peak plasma levels was found. Further, no reasonable explanation for inconsistency of the plasma levels can be given except the possible inter animal variations in response to the pharmacologic effect of propantheline. Nevertheless, the ge'lafeiveilbioavaiMhMity of griseo-fulvin'y] as a result of multiple treatments with propantheline is higher than that of controls. The explanation outlined in single propantheline treatment studies.is also valid in repetitive treatment studies, namely that propantheline causes retardation of gastrointestinal motility. As a result of>delayed' emptying ct^e^ratWo~P^ f&dmvit-heatstomalchr.mays .x*.- :. be reduced; the drug w i l l travel slowly through the small intestine and w i l l be exposed to the absorptive sites. These effects may respectively give rise to: a) reduced rate of absorption, reflected in a longer time for the peak plasma level to be reached; and b) an increase in the extent of absorption, indicated by a larger AUC. Nevertheless, these two experiments, which are conducted in a manner 99 analogous to c l i n i c a l use ( i . e . , therapeutic administration of propantheline), c l e a r l y demonstrate the unexpected g r i s e o f u l v i n absorption p r o f i l e one may observe i f g r i s e o f u l v i n and propantheline are administered concomitantly. These observations also point to the need f or follow-up experimentation i n humans to examine t h i s p o t e n t i a l i n t e r a c t i o n . G r i s e o f u l v i n represents a unique model drug due to i t s exceptionally e r r a t i c d i s s o l u t i o n and absorption c h a r a c t e r i s t i c s . Thus the observed findings with g r i s e o f u l v i n may lead to further experimentation^;' with other poorly absorbed drugs such as dicoumarol. Intravenous Studies - Although there i s no evidence i n the l i t e r a t u r e to suggest that e i t h e r metoclopramide or propantheline influence the metabolism of any drug, i t appeared necessary to investigate whether they have any e f f e c t on the k i n e t i c s of g r i s e o f u l v i n metabolism. Therefore g r i s e o f u l v i n was administered intravenously to the cont r o l r a t s and to those pretreated i n t r a -p e r i t o n e a l l y with s i n g l e doses of eit h e r metoclopramide (10 mg/kg) or propan-th e l i n e (5 mg/kg) 2 hours p r i o r to the g r i s e o f u l v i n administration. This experiment was also conductedoon rats pretreated with si n g l e o r a l doses of 15 mg/kg sodium phenobarbital 24 hours p r i o r to the griseofuLvin. administration. Phenobarbital i s a known enzyme inducer. B u s f i e l d et a l ( 1 7 ) , who reported the griseofulvin-phenobarbital i n t e r a c t i o n i n the r a t , suggested that the i n t e r -action occurs at the l e v e l of metabolism. This hypothesis was questioned by Riegelman et a i ( 2 0 ) . In man, they (20 ) suggested that the g r i s e o f u l v i n -phenobarbital i n t e r a c t i o n r.eBul-.tejd' ea from a reduced absorption rather than induced metabolism. In t h i s study, the findings of the i v experiments,' coupled with those of o r a l studies, provide;.!, a better understanding of the mechanism of the i n t e r a c t i o n . 100 Figures 4-9 and 4-10 depict plots of plasma concentration and logarithm of plasma concentration of griseofulvin vs time following a single iv dose of 26 mg/kg of the drug on a control rat. The curves are fitt e d by computer, based on the assumption that the pharmacokinetics of griseofulvin in the rat follows a biexponential equation: C P = A.e- a t + B . e - S t £1, The calculated parameters for each set of data are given in: Appendix II.. The biological half-lives of the post-distribution and the AUCs of griseofulvin are shown in Table 4-XII. The biological h a l f - l i f e of the post-distribution phase was observed to be reproducible and ranged from 1.9 to 2.4 hours in both control and test animals, i.e., pretreatment with phenobarbital, meto-clopramide and propantheline had no significant effect on the slope of the post-distribution phase of griseofulvin. This is of particular interest; the slope of the elimination (post-distribution) phase after iv administration is defined by the following equation: . Hi k21  s l o p e = = ~ a(2.303) where aais the rate constant of the distribution phase and the rest of the terms are defined on pager1072.v In the case of griseofulvin, since"less than •U%.tof. an iv dose is excreted unchanged '(iii arine.a(66.);? t-he^rate constant of elimination, k is almost exclusively associated with the metabolism of the drug. Therefore, since there is a direct relation between the slope of 101 ( 3 D C0NT.02 I 15.0 TIME(HR) - 1 — 27.0 0.0 18.0 21.0 24.0 30.0 F i g u r e 4-9. Computer ^fi«ed-.^ c o n c e n t r a t i o n s vs time f o l l o w i n g intravenous • a d m i n i s t r a t i o n of a s i n g l e dose of 26 mg/kg g r i s e o f u l v i n d i s s o l v e d i n p o l y e t h y l e n e g l y c o l 600 to the rat,(h=l). 1 0 2 o CD 0.0 . C0NT.02 3.0 6.0 15.0 TIME(HR) 30.0 4-1:0- Computer C • f3!ttfcdV l o g a r i t h m of plasma c o n c e n t r a t i o n s " I S/5» a d m i n i s t r a t i o n of a s i n g l e F i g u r e vs time curve f o l l o w i n g intravenous., ifse of 26 mg/kg g r i s e o f u l v i n d i s s o l v e d "in p o l y e t h y l e n e g l y c o l 600 to the rat„.( n=l ) . 103 the terminal part of the curve and k ^ , any changes i n the g r i s e o f u l v i n metabolism rate constant must be r e f l e c t e d i n the slope of the post-d i s t r i b u t i o n phase and consequently, the h a l f - l i f e of the drug. The fa c t that, i n both control and test animals, the terminal part of the curves are p a r a l l e l ( F ig. 4-11) leads to the conclusion that pretreatment with pheno-b a r b i t a l , metoclopramide and propantheline has not s i g n i f i c a n t l y influenced the metabolism of g r i s e o f u l v i n i n the r a t . Despite the reproducible b i o l o g i c a l h a l f - l i v e s , large deviations were observed among other calculated pharmacokinetic parameters. These v a r i a t i o n s were p a r t i c u l a r l y noticed i n the AUC values (Table 4-XII). Further-more, the calculated AUCs following i v administration of g r i s e o f u l v i n were found to be generally smaller than expected, because the mean AUC/dose obtained following o r a l administration of g r i s e o f u l v i n i n PEG 600 to the metoclopramide -1 3 pretreated rats was 10 hr.ml .10 (Table 4-IX), while t h i s value following i v -1 3 i n j e c t i o n of g r i s e o f u l v i n was approximately 6 hr.ml .10 (Table 4-XII). These r e s u l t s cannot be r e a d i l y interpreted due to the l i m i t e d number of blood samples taken during the d i s t r i b u t i o n phase. In j e c t i o n of the g r i s e o f u l v i n -PEG 600 preparation into the t a i l vein caused noticeable paling of the t a i l during the f i r s t hour, making the blood sampling extremely d i f f i c u l t . Never-theless, the t t of the p o s t - d i s t r i b u t i o n phase was found to be consistent throughout the experimentation. The d i s t r i b u t i o n phase plays an important r o l e i n the evaluation of pharmacokinetic parameters. Bedford e_t a l (35) suggested that, i n the r a t , following a single i v dose of 20 mg/kg g r i s e o f u l v i n more than 90% of the drug disappeared from the blood wi t h i n f i v e minutes. Due to the experimental design, the frequency of blood sampling during the 104 3 9 15 21 27 3 9 15 21 27 Hour s Hours F i g u r e 4-.ifi. Computer .fitted* " l curves of l o g a r i t h m of plasma c o n c e n t r a t i o n s vs time and r e s i d u a l c o n c e n t r a t i o n s vs time f o l l o w i n g i n t r a v e n <3us.o a d m i n i s t r a t i o n of s i n g l e doses of g r i s e o f u l v i n / p o l y e t h y l e h e g l y c o l 600 to a c o n t r o l r a t X a ), and r a t s p r e t r e a t e d with s i n g l e doses of meto-clopramide ( b ) , p r o p a n t h e l i n e ( c ), or p h e n o b a r b i t a l ( d ),.,( n = 1) . 1 0 5 Table 4-XII Biological Half-Life of the Post-Distribution Phases(0.693/- B) and Area^. Under the Griseofulvin-Plasma-Time Curves(AUC) in the Rat after Intravenous Administration of Single Doses of the Drug in Polyethylene Glycol 600. Test Rats received Single Doses of Metoclopramide Hydro-chloride(10 mg/kg), Propantheline Bromide(5 mg/kg) or Sodium Pheno-barbital(15 mg/kg) prior to.the Griseofulvin Administration. Dose, 0.693/B, AUC,_1 AUC/Dose. mg/kg. hr. u g.hr.ml . hr.ml--'-. lO-^  t Controls, 20 20 T w Phenobarbital 40, j pretreated, 20 , 16 T t Metoclopramide 20 ^  pretreated, 20"; t Propantheline 20 ^  pretreated, 20 2.4 27.0 5.40 2.0 22.2 2.4 29.6 5.92 2.3 42.5 4". 25 2.4 33.1 6.62 2.1 25.2 6.30 2.1 25.2 5.04 2.1 25.8 5.16 2.1 61.2 12,24 2.1 "<25..2 '5.04 Y) Solution of 10 mg/kg griseofulvin in 100% PEG 600. t) Solution of 5 mg/kg griseofulvin in 100% PEG 600. 106 f i r s t few minutes of experimentation was limited. Similar results have been observed by Chiou and Riegelman ( 3 6 ) . In Fig. 3 of their paper, the plasma concentrations of griseofulvin obtained after administration of various dosage forms ( a l l corrected to a 50 mg dose) to a dog are presented with another curve obtained following iv administration of a single dose of 50 mg griseofulvin. The AUCs of a l l of the oral administrations seem to be substantially larger than those of the intravenous. No explanation appears to have been given for this observation by Chiou and Riegelman. Two possible mechanisms may be responsible for the observed low AUC after iv administration: (a) the drug may precipitate in the systemic circulation immediately after the injection due to i t s very low water solubility; or (b) i t may be eliminated at a significantly higher rate during the f i r s t few minutes. The f i r s t hypothesis seems unlikely; reproducible half-lives of the post-distribution phase would seem to indicate that no re-distribution of the drug due to dissolution of the precipitated griseofulvin has taken place. If the drug were precipitated in the systemic circulation one would expect to observe a gradual dissolution of the drug in that region which in turn would give rise to irreproducible post-distribution half-lives. The second mechanism, i.e., higher rate of elimination in the 1 0 7 f i r s t few minutes, could easily be responsible for the observation. The disappearance of a given drug from the central compartment takes place according to the following rate equation: dc 'dt k e l C - ~ k12 C + k21 T (-9) dc where - is the rate of disappearance of the drug from the central compartment, C is the drug ccajnQiiat-jitxor.,. k ^ is the rate constant for the elimination of the drug from the central compartment, k ^ and k2^ are the ratecconstants for the distribution of the drug into the other compart-ment (s)and for the redistribution of the drug back into the central compartment respectively, and T is the drug con aniount ci O I 1 i n other compart-ment (s) as shown in the following diagram: iv 12 k21 k ^ and k^^ can be considered as hybrid rate constants/where more than two compartments are involved. 108 As shown in Eq. 9 the rate of disappearance ( - -^ ~) is directly proportional to the drug amount in the central compartment. If k^, k^ a n <^ k.^ remain constant (Eq. 9), only the amount terms will be variable and any increase in the amount will give rise to an increase in the rate of disappearance. After injection of a dose of 5 mg griseofulvin into a 250 g rat the amount of the drug in the systemic circulation will rise sharply. Therefore, either the rate of disappearance increases and a large portion of the drug crosses the central compartment and distributes into other compartments or i t will be eliminated in the very first few minutes after administration. The main objective of the iv experiments was to show that the pre-treatments with phenobarbital, metoclopramide and propantheline did not influence the biological half-life and hence metabolism of griseofulvin. The results of absorption studies also support the hypothesis that these treatments did not influence the rate of griseofulvin metabolism. Since alternative experimentation served to clarify the interaction between griseofulvin and phenobarbital, continued study of the pharmacokinetics of griseofulvin in the rat was not performed. An induced metabolic system for a drug would be expected to result in reduced plasma levels. Conversely, a metabolic inhibition would give rise to an increase in the plasma levels. These alterations in the metabolism of a drug should be observed irrespective of the dosage form or the route of administration. Formulation-dependent griseofulvin-plasma levels observed in the rats pretreated with metoclopramide and propantheline (as discussed earlier) imply that these motility modifiers do not affect the rate of griseofulvin metabolism in the rat. As will be discussed later, the effect of phenobarbital on the griseofulvin-plasma levels was also formulation-dependent . 1 09 Griseofulvin-Phenobarbital Interaction The significance of gut motility on the absorption of griseo-fulvin has been ishpwn>earli-e.ri and sthis part vof." thejsthesis i s devoted to clarifying the mechanism of the observed interaction between griseofulvin and phenobarbital. The griseofulvin-phenobarbital interaction has been observed when griseofulvin i s given orally (;16,1 7,1.8>,203). It is also interesting to note that, in previous reports (ll'6 s J 17,«18» 20") regarding this observation, griseo-fulvin had been administered in solid dosage forms, i.e., tablets in man and suspension (in 0.5% Tween 80) in the rat. Therefore, i t appeared necessary to investigate the griseofulvin-phenobarbital interaction after administration of solutions and more rapidly dissolving suspensions. Unless precipitation occurs following administration, the effect of the dissolution process should be minimal, and more information regarding the mechanism of the interaction may be provided. Thus, in this work, the griseofulvin-phenobarbital interaction was studied following administration of single doses of the following preparations: 1) 100 mg/kg griseofulvin in 0.5% Tween 80 (Fig. 4-12) 2) 100 mg/kg griseofulvin in 2% Tween 80 (Fig. 4-13) 3) 50 mg/kg griseofulvin in 100% PEG 600 (Fig. 4-14) 4) 20 mg/kg griseofulvin in .70% PEG 300 (Fig. 4-15) 5) 100 mg/kg griseofulvin in 70% PEG 300 (Eig. 4-16). A l l preparations except griseofulvin-PEG 600 are in suspensions. Griseo-fulvin concentrations in the supernatants of the formulations were measured 110 and reported in the equilibrium solubility measurement section. Griseofulvin-PEG 600 formulation was a clear solution of the drug. A suspension of griseofulvin in 0.5% Tween 80 was administered to examine the reproducibility of the reported phenobarbital interaction ( 1 6 ) . The suspension of griseo-fulvin in 2% Tween 80 was used to study the effect of an increase in the surfactant concentration and hence to simulate the influence of bile salts on the observed interaction. Because of the relatively high solubility of griseofulvin in PEG, this vehicle was used in an attempt to prepare more rapidly dissolving preparations. However, as mentioned earlier, administration of griseofulvin in pure PEG 600 caused noticeable change in the appearance of the gut along with an apparent tendency to retain water. Since such changes might influence the absorption of griseofulvin, suspensions of the drug in 70% PEG 300 were used to further study the interaction. This solvent produced no noticeable effect on the gut appearance. The observed changes in Cmax, Tmax and AUC caused by phenobarbital pretreatments following administration of different griseofulvin preparations are summarized and compared with those of motility modifiers in Table 5-T... -Griseofulvin Suspensions in Tween 80 - Plasma levels following oral administration of a single dose containing 100 mg/kg griseofulvin in 0>5% Tween 80 to the control rats and those pretreated orally with single doses of sodium phenobarbital are shown in Table 4-XIII. The mean plasma-griseofulvin levels vs time plots obtained from control and test animals are shown in Fig. 4-12. It has been hypothesized (20) that the griseofulvin-phenobarbital interaction may be due to the a b i l i t y of the barbiturate to increase gut motility. In this work, phenobarbital pretreatment, like Hours Figure 4-12. Mean griseofulvin -plasma levels vs time following oral administration of single doses of 100 mg/kg in 0.5% Tween 80 to the control, A (n=6) and phenobarbital pretreated,O (n=6) rats. Error bars represent standard error of the mean. Table 4-XIII Plasma Levels of Griseofulvin (Micrograms per M i l l i l i t e r ) following Oral Administration of 100 mg/kg i n 0.5% Tween 80 i n the Control and Test Rats . Phenobarbital (15 mg/kg) was given Orally to the Test Rats, 24 hours prior to Griseofulvin Administration. Hours Rat 12 24 48 Griseofulvin 1 suspension 2 control 3 4 5 6 Mean (SE) a 1.881 2.670 1.656 6.009 1.482 2.533 0.633 5.557 1.378 5.680 3.841 5.224 1.812 4.592 (0.441) (0.664) 5.962 7.236 7.679 7.061 6.013 6.920 6.613 7.018 6.020 6.048' 6.567 6.717 (0.399) (0.22Q) 2.251 . 0.520 4.528 1.508 3.743 0.214 3.249 0.036 1.234 0.432 3.817 2.014 3.137 0.787 (0.489) (0.322) 0.238 0.058 0.280 0.120 0.120 0.147 0.813 0.161 0.174 0.293 0.117 (0.107) (0.024) Phenobarbital 1 1.251 4.236 4.822 3.372 1.177 0.326 0.087 0.003 sodium 2 2.162 2.835 3.926 4.073 0.740 0.389 0.062 0.012 (15 mg/kg, 3 1.256 1.745 2.391 3.640 0.836 0.219 0.008 0.001 Orally) 4 1.620 2.984 3.301 1.815 0.673 0.136. 0.012 0.000 predosing 5 2.483 2.720 2.035 1.362 0.564 0.093 0.030 0.003 6 1.420 2.033 2.811 3.203 0.815 0.535 0.071 0.010 Mean 1.699 2.759 3.214 2.911 0.801 0.283 0.045 0.005 (SE) a (0.209) (0.355) (0.421) (0.439) (0.085) (0.068) (0.013) (0.002) S t a t i s t i c a l significance ns . s s s s ns s s a) Standard error of the mean in parentheses b) Determined by Student's t-Test (2 t a i l e d ) ; P<0.05 1 1 3 TableT4-XIV Area Under Plasma Concentration-Time Curves, AUC (ugrhr.ml ^) of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control and Phenobarbital Pretreated Rats. Control, Phenobarbital pretreated, 37^ .5 26.98 53.0 25.8 37.3 19.3 37.8 19.6 40.4 19.2 52.96 22.2 Mean, 43.2 22.1 Standard error, 3.13 1. 39 Mean AUC/dose, hr.ml 1.73 0.87 103 Coefficient of 17.8 15.5 variation, %, ^ St a t i s t i c a l difference » s 1. Determined by the Student's t-test; a=0.05 (2 tailed); s, significant. 114 Table 4-XV Peak Plasma Levels, Cmax and their Time of Occurence, Tmax Following Oral Administration of 100 mg/kg of the Drug in 0.5% Tween 80 to the Control and Phenobarbital Pretreated Rats. .Phenob arb i t al 1 Control > pretreated, Cmax, u g/ml. Tmax, hr. Cmax, ru g/ml. Tmax, hr. 7c. 35 6 4.82 5 7.68 5 4.10 6 6.92 6 3.64 6 7.02 6 3.30 5 6.02 6 2.72 5 6.05 6 3.20 6 i Mean, 6.84 5.8 3.63 5.5 Standard error, 0.28 0.17 0.30 • 0.22 ;Coefficient of 9.9 7.0 20.4 10.0 variation, %, i Statistical difference . s ns 1. Determined by the Student's t-test; a=0.05 (2 tailed) s,significant. 1 1 5 metoclopramide pretreatment, reduced both griseofulvin plasma levels and the area under the griseofulvin-plasma concentration-time curve (AUC). The observed effects of phenobarbital are in agreement with the report of Busfield et a l (16). Although both drugs decreased the AUC to the same extent, i.e., by about.50% (Tables 4-VI and 4-XIV), the effect of metoclopramide preadministration on the mean peak plasma level (Cmax) was more profound than that of phenobarbital pretreatment (Tables 4-V and 4-XV). The former drug reduced the mean Cmax by 62% while the latter lowered this parameter by 47%. A very significant difference between these observed interactions was that phenobarbital, unlike metoclopramide, did not shorten Tmax (Table 4-XV). This might be an indication that the barbiturate did not influence gastric emptying. Metoclopramide has two distinct effects on the Gl tract. It shortens the residence time in the stomach, where absorption is limited, and i t increases the motility of the proximal part of the intestine, i.e., the principal absorption site for griseofulvin. The former effect results in a more rapid absorption and is mostcobviouswwhentthe drug is given in suspension form. The latter effect of metoclopramide, i.e., increased motility of the proximal part of the intestine, however, causes the drug to be forced past the absorption site and gives rise to a decreased absorption. This effect is clearly shown in the suspension studies, where, as a result of shorter residence time at the absorption site, the AUC is reduced significantly (Table 4-VI). Therefore, the observed effect of phenobarbital on the plasma levels of griseofulvin (in 0 .5% Tween 80) seems to suggest that the barbiturate mimics the second effect of metoclopramide, i.e., decreases the transit time in the region of the absorption site. But at this stage one Hours Figure 4-13. Mean plasma levels vs_ time following oral administration of .1.00 mg/kg griseofulvin in 2% Tween 80 to the control, O (n=4) and phenobarbital pretreated, # (n=5) rats. Error bars represent standard error of the mean. • ' Table 4-XVT; Griseofulvin-Plasma Levels (au g/ml) following Oral Administration, of Single Doses of 100 mg/kg Griseofulvin in 2% Tween 80 to the Control Rats and Rats Pretreated orally with Single Doses of 15 mg/kg Phenobarbital Sodium 24 Hours Prior to the Griseofulvin Administration. Hours Rats 1.5 4 6 8 12 24 Controls, 1 1.097 1.208 2.723 1.960 0.436 0.314 2 1.119 3.884 4.597 2.652 1.095 0.353 3 1.123 3,501 3.332 1.781 0.911 -4 3.068 4.222 3.930 2.852 0.880 -Mean, 1.602 3.204 3.645 2.311 0.830 0.333 Standard error, 0.488 0.681 0.401 0.260 0.140 0.019 Phenobarb i t a l pretreated, 1 2 0.434 1.034 0.967 0.812 2.620 2.416 1.063 0.700 0.721 0.952 0.457 0.295 3 3.480 2.447 3.447 2.599 1.010 -4 1.340 2.321 1.961 1.765 - 0.369 5 - . 0.873 3.151 2.186 ,„„2.394 _ _ J ) . 921 . 0.192 _ .. • ^ ~ "- • -• Mean, 1.432 1.940 2.526 1.707 0.901 0.328 Standard error, 0.532 0.452 0.255 0.322- 0.125 0.056 Stati s t i c a l significance,! ns2 ns S3 ns ns ns 1. Determined by Student's t-test ; a=0.05 . 2. Not significant. 3. Significant. Table 4-XVII Area Under Plasma Concentrations-Time Curves, AUC (uu g.hr.ml j) ,Peak Plasma Concentrations, Cmax ( y g/ml) and their Time of Occurence, Tmax (hr) of Griseofulvin Following Oral Administration of Single Doses of 100 mg/kg of the Drug in 2% Tween 80 to the Control and Phenobarbital Pretreated Rats. Control, Phenobarbital pretreated, AUC Cmax Tmax AUC Cmax Tmax 19.6 2.72 6 17.1 2.62 6 35.2 4.60 6 15.7 2.42 6 27.4 3.50 4 32.6 3.45 6 37.6 4.22 4 22.1 2.32 4 21.8 3.15 4 Mean, Standard error, 30.0 4.09 Mean AUC/dose, hr.ml - 1.10 3 1.20 Coefficient of variation, %, Stati s t i c a l difference 27.3 3.76 0.41 22.0 5.0 0.58 23.1 21.9 2.79 5.5 1.64 0.22 0.39 0.88 16.8 17.5 15.7 ns p<0.1 ns 1. Determined by the Student's t-test;;(2 tailed); ns, not significant. 11 9 cannot rule out the influence of other possible mechanisms (e.g., reduced dissolution due to a reduction in bi l e salt concentration or blood flow rate limited elimination). To c l a r i f y the problem, a suspension containing 2% Tween 80 was prepared and administered to the rat in the presence and absence of pheno-barbital. It was expected that a fourfold increase in the concentration of the surfactant should mask the surfactant effects of the bile salts. Fig. 4-13 and Table 4-XVI depict the griseofulvin-plasma levels vs time curves following administration of single doses of the suspension of griseofulvin in 2% Tween 80 in the presence and absence of phenobarbital. Although pheno-barbital pretreatment reduced the plasma levels of griseofulvin i t s effect was significant only at Cmax (a = 0.1). The treatment also failed to significantly influence AUC and Tmax (Table 4-XVII). These observations indicate that a fourfold increase in the concentration of Tween 80 has significantly reduced the extent of the interaction. Phenobarbital pretreat-ment followed by the administration of a suspension of griseofulvin in 0.5% Tween 80 reduced the mean Cmax by $0%, while under identical conditions but using 2% Tween 80 the barbiturate lowered the mean Cmax by only 31%. Therefore, i t i s reasonable to suggest that a decreased dissolution rate may be mainly responsible for the griseofulvin-phenobarbital interaction. TablesA^XIV and 4-XVII show that an increase in the Tween 80 concentration caused a decrease in the relative bioavailability of griseo-fulvin. Therefore a shorter residence time in the region of the absorptive sites would be expected to decrease the availability of the preparations with higher Tween 80 concentrations s t i l l further.- Thus, the griseofulvin-1 20 phenobarbital interaction is unlikely to result from an increase in motility. • Addition of 2% Tween 80 to the griseofulvin suspension has perhaps reduced the relative bioavailability of the drug to such an extent that phenobarbital treatment cannot reduce i t further. Phenobarbital treatment and an increased concentration of Tween 80 may influence the absorption of griseofulvin by the same mechanism. It is feasible to hypothesize that the pretreatment with phenobarbital followed by the administration of griseofulvin in 0.5% Tween 80 might have retarded the dissolution of griseofulvin by reducing the concentration of the b i l e salts. The mechanism of such an effect is not clear . because the change in the bile constituent as a result of pheno-barbital treatment has not yet been f u l l y studied. Furthermore, since the addition of extra surfactant to the preparation did not completely eliminate the effect of phenobarbital on the plasma levels of griseofulvin other mechanisms could also be involved in the interaction. However, preparations containing PEG provide more information regarding the mechanism of the interaction. Griseofulvin in PEG - The plasma levels following a single oral dose of 50 mg/kg griseofulvin in PEG 600 (solution) in the control and phenobarbital pretreated animals are shown in Table 4-XVIII and their means are plotted as functions of time in Fig. 4-14. Phenobarbital predosing failed to significantly affect the plasma levels of griseofulvin when the drug was administered in the solution form (Fig. 4-14, Tables 4-XVIII and 4-XX). This further suggests that, despite the similarity in the result from 0.5% Tween 80 suspension studies, the effects of phenobarbital and metoclopramide on the plasma levels of griseofulvin may not be due to the same mechanism. 10 12 24 Hours Figure 4-14. Mean griseofulvin-plasma level-vs time- following oral administration..of single doses of 50 mg/kg in 100% polyethylene glycol^.600 to the control, a (n=8) and phenobarbital pretreated, • (n=6) rats. Error bars represent standard error of the mean. Table" 4-XVI-II Plasma L e v e l s o f G r i s e o f u l v i n (Microgram p e r M i l l i l i t e r ) f o l l o w i n g i t s O r a l A d m i n i s t r a t i o n o f a S i n g l e 50 mg/kg i n PEG 600 Dose i n C o n t r o l and T e s t R a t s . P h e n o b a r b i t a l Sodium ( 1 5 mg/kg) was g i v e n O r a l l y t o t h e T e s t A n i m a l s , 24 h o u r s p r i o r t o G r i s e o f u l v i n A d m i n i s t r a t i o n : HOURS R a t s 0.33 0.5 1 2 3 4 6 8 12 24 48 G r i s e o f u l v i n 1 15.321 8.120 5.440 3.280 1.613 0.856 0.451 0.854 0.500 0.030 c o n t r o l 2 — 13.098 7.946 4.403 2.554 1.304 0.4'87 1.344 0.751 0.032 0.024 3 — 12.008 8.704 5.512 4.531 5.227 5.781 2.607 0.308 0.148 0.005 4 — 6.093 3.671 1.921 2.416 1.526 2.099 1.523 0.473 0.233 0.018 5 — 11.677 6.896 3.000 1.337 1.021 2.251 2.486 0.508 0.290 0.036 6 12.331 — 8.120 4.915 — 1.280 0.675 0.471 0.816 • — — 7 8.124 — 3.466 — 1.191 0.450 0.623 0.983 0.310 0.001 8 — 9.233 — 4.204 — 1.380 0.724 0.591 0.814 0.204 0.028 Mean 10.2275 11.238 7.243 4.108 2.824 1.818 1.665 1.262 0.688 0.208 0.020 ( S E ) a (2.100) (1.309) (0.754) (0.4415) (0.528) (0.491) (0.639) (0.314 (0.082) (0.062) (0.005) P h e n o b a r b i - 1 t a i sodium 2 (15 mg/kg, 3 o r a l ) 4 p r e d o s i n g 5 6 Mean ( S E ) a S t a t i s t i c a l b s i g n i f i c a n c e 15.301 8, .739 5.461 4.318 3.900 1.125 1.356 0.840 0.391 0.057 8.530 4 .779 1.685 0.871 0.737 0.183 0.233 0.736 0.314 0.014 18.246 6. .821 3.893 2.393 1.304 1.000 1.140 0.411 0.556 0.026 7.677 — 4.008 — • 1.411 2.010 0.528 0.724 0.380 0.018 14.464 — 8.263 — 2.560 0.594 0.560 0.803 0.318 — 9.360 — 5.440 — 2.850 0.487 0.911 0.798 0.210 0.021 12.283 6. .779 4.792 2.527 2.127 0.900 0.788 0.719 0.362 0.027 (1.763) (1. 143) (0.894) (0.997) (0.482) (0.263) (0.172) (0.064) (0.047) (0.008) ns ns ns ns ns ns ns ns ns ns a) S t a n d a r d e r r o r o f the mean i n p a r e n t h e s e s b) Determined by S t u d e n t ' s t - t e s t , (2 T a i l e d ) ; P ^ 0 . 0 5 Table 4-XIX Area Under Plasma Concentration-Time Curves, AUC (y g.hr.ml ) and the Peak Plasma Concentrations, Cmax (yg/ml) Following Oral Administration of Griseofulvin(50;mg/kg) in Polyethylene Glycol 600' ( solution ) to the Control and Phenobarbital Pretreated Rats. Phenobarbital Control, pretreated, AUC, Cmax, AUC, Cmax, 37.6 15.32 43.8 15.30 55.5 12.01 24.4 8.53 30.1 6.09 35.7 18.25 41.3 12.33 35.3 7.68 28.9. 8.12 41.4 14.46 32.6" 9.23 35.2 9.36 Mean, 37.7 10.52 36.0 12.26 Standard error, 4.05 1.36 2.75 1.76 -1 3 , - - • Mean AUC/dose, hr.ml .10 3.01 — 2.88 — Coefficient of variation, %, 26.3 . 31.7 18.7 35.2 Stati s t i c a l difference!, • ns ns 1. Determined by the Student's t-test; a=0.05 (2 tailed); ns, not significant. 1 24 Riegelman ejt a_l ( 2 0 ) hypothesized that phenobarbital might increase the motility of the proximal part of the intestine through i t s effect on bile .flow -since the stimulating effect of phenobarbital on bil e flow has been observed by several investigators ( 21 '-t2 3 ). Nevertheless, the present evidence implies that the influence of phenobarbital on the absorption of griseofulvin is unlikely to result from an increase^ in motility. If the observed effect of phenobarbital on the plasma levels of griseo-fulvin were due to an enhanced motility, a change in the AUC would be expected after preadministration of phenobarbital regardless of the form in which griseofulvin is administered. A shorter residence time in the intestine resulting from increased motility, without a simultaneously enhanced gastric emptying, would be expected to cause a decreased total absorption of griseofulvin irrespective of the form in which i t is administered. In fact the same AUCs were observed with or without pheno-barbital pretreatment when griseofulvin was administered i n solution (Table 4-XIX). AUC for the controls and phenobarbital pretreated rats were significantly lox^er than those pretreated with metoclopramide (Tables 4-IX and 4-XIX). This i s perhaps due to precipitation of the drug i n the stomach as a result of a longer residence time in this region where absorption is limited. Following precipitation, the limiting effect of dissolution rate w i l l be introduced into the absorption process. Therefore, although some of the readily available drug is absorbed prior to precipitation, a major fraction of the administered griseofulvin may be in the form of undissolved crystals (precipitated) which w i l l be absorbed only i f sufficient time at the absorptive site i s allowed for dissolution. This is additional support for the suggestion 1 2 5 that an increase in peristalsis ( i f any) caused by phenobarbital has no significant role in the griseofulvin-phenobarbital interaction. If the transit time in the upper part of the small intestine were to be significantly decreased by phenobarbital, the AUC in phenobarbital treated rats following administration of the solution form should have been decreased. t However, since griseofulvin in PEG 600 preparation caused a noticeable change in the gut appearance, PEG 600 i t s e l f might have an effect on peristalsis. It is possible that the absorption properties of griseo-fulvin might have been altered by the vehicle in both control and test rats. In an attempt to c l a r i f y this point, two different griseofulvin dosage forms containing 100 mg/kg drug in either 70% PEG 600 or 70% PEG 300 in d i s t i l l e d water were prepared and their effects on the gut were tested. Single doses of 100 mg/kg griseofulvin in either 70% PEG 300 or 70% PEG 600 (1 ml) were administered to two separate groups of 5 rats. One rat from each group was sacrificed at either 1, 3, 5, 8 or 12 hours post-dosing and after an abdominal incision the gut was examined for any abnormalities. While the 70% PEG 600 preparation showed a noticeable increase in the volume of gut f l u i d , the 70% PEG 300 preparation exerted no effect on the gross appearance of the gut. Therefore, 70% PEG 300 was chosen as a vehicle to administer griseofulvin orally at two different concentrations (20 and 100 mg/kg). In addition the effect of pretreatment with phenobarbital on the griseofulvin-plasma levels was studied. The griseofulvin-plasma levels following administrat ion of 20 and 100 mg/kg griseofulvin in 70% PEG 300 are shown in Tables 4-XX and 4-XXII and their means are plotted as a function of Hours Figure 4-15. Mean griseofulvin plasma levels vs time following oral administration of 20 mg/kg of the drug in 70% polyethylene glycol to the control, A and phenobarbital pretreated, A rats (n=4). Error bars represent standard error of the mean. Table 45XX Griseofulvin-Plasma Levels C'U g/mL) following Oral Administration of Single Doses of 2© mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control Rats and Rats Pretreated Orally r with Single Doses of 15 mg/kg Phenobarbital Sodium 24 Hours Prior to the Grd:sedr;ulv;inMdm'in'istration. Hours . Rats 0.25 1 3 5.16 6.5 8 13 24 Controls, 1 3. 768 3.044 0.420 0.250 0.191 0.111 0.430 0.002 2, 4. 466 2.037 0.585 0.578 1.542 0.626 0.362 0.041 3 5. 318 3,763 0.624 0.468 0.354 0.292 0.255 0.030 4 3. 303 1.180 0.411 0.321 1.871 0.470 - -Mean, ' 4. 214 2.506 0.510 0.404 0.989 0.375 0.349 0.024 Standard 0. 439 0.566 0.055 0.073 0.421 0.111 0.051 0.012 error, Phenobarbital 1 9-955 6.938 1.320 0.287 0.467 0.132 0.079 0.016 pretreated, 2 6. 760 3.080 0.287 0.063 0.095 0.123 0.161 0.028 3 4. 631 2.619 0.188 1.085 0.485 0.399 0.252 0.038 4. 3. 718 1.317 0.718 0.550 1.417 0.425 - 0.003 Mean, 6. 266 3.488 0.628 0.496 0.616 0.270 0.164 0.021 Standard 1. 385 1.209 0.258 0.220 0.282 0.082 0.050 0.007 error, Statistical''' .2 ns ns ns ns ns ns ns ns significance, 1. Determined by Student's t-test; a=0.05. 2. Not significant. Table 4-XXI Area Under Plasma Concentrations-Time Curves, AUC (u g.hr.ml ) and the Peak Plasma Concentrations, Cmax ( u g/ml) Following Oral Administration of 20 mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control and Phenobarbital Pretreated Rats. Control, AUC, . 9.3 .13.1 12.3 .10.-3 Cmax, 3.77 4.47 5.32 3.30 Phenobarbital pretreated, AUC, 16.8 8.6. 11.4 10.4 Cmax, 9.95 6.76 4.63 3.72 Mean, Standard error, Mean AUC/dose, hr.ml-1.103 Coefficient of variation, %, 11.2 0.88 2.24 15.7 4.21 0.44 20.8 11.8 6.27 1.76 1.38 2.36 29.8 44.2 Statistical difference,,, ns ns 1. Determined by the Student's t-test (2 tailed); a=0.05; ns, not significant. ho CO Figure 4-16, Plasma levels of griseofulvin vs r time following administration of 100 mg/kg of the drug in 70% polyethylene glycol 300 to the control and phenobarbital pretreated rats. Key, Q t control° (n=4) ; #2,stest (n=4) ; error bars represent standard error of the mean. Table 4-XXII Griseofulvin-Plasma Levels ( yg/ml) following Oral Administration of Single Doses of 100 mg/kg Griseofulvin in 70% Polyethylene Glycol 300 to the Control Rats and Rats Pretreated Orally with Single Doses of 15 mg/kg Phenobarbital Sodium 24 Hours Prior to the Griseofulvin Adminis-tration . Hours Rats 0.25 1 2.5 5.25 6.5 8 13 24 Controls, 1 4.988 1.415 0.264 0.765 0. 230 1.016 1.008 0.011 2 3.735 1.013 2.097 0.825 0. 932 1.294 0.339 0.082 3 : 7.536 - 2.089 0.512 0. 425 1.256 1.372 0.130 4 3.115 .0.903 0.703 0.611 1. 938 . 0.832 0.711 — Mean, 4.843 1.110 1.288 0.678 0. 881 1.099 0.857 0.074 Standard 0.978 0.155 0.473 0.143 0. 382 0.108 0.219 0.034 error, Phenobarbital 1. 3.436 1.630 0.400 0.155 0. 279 1.148 0.668 0.033 pretreated, 2,: 4.372 0.905 1.660 1.447 3. 164 1.992 1.008 0,020 3 3.144 1.047 0.457 0.474 0. 486 0.500 0.764 0.004 4 5.325 2.130 1.681 0.813 2. 371 0.911 0.373 0.011 Mean, 4.069 1.428 1.049 0.722 1. 575 1.138 0.837 0.017 Standard 0.494 0.282 0.359 0.276 0. 708 0.315 0.076 0.006 error, Statistical significace,! ns2 iis ns ' ns ris ns ns ns" 1. Determined by Student's t-test; ct=0.05 . 2. Not significant Table 4-XXIII Area Under Plasma Concentrations-Time Curves, AUC (pg.hr.ml ) and the Peak Plasma Concentrations, Cmax ( u g/ml) Following Oral Administration of 100 mg/kg Griseofulvin in. 70% Polyethylene Glycol 300 to the Control and Phenobarbital Pretreated Rats. Control, AUC Cmax Phenobarbital pretreated, AUC Cmax 15.8 4.99 12.2 3.44 17.8 3.73 25.6 4.37 26.4 7.54 11.7 3.14 14.2 3.11 18.3 5.32 Mean, 18.5 4.84 16.9 4.07 Standard error 2.73 0.98 3.26 0.49 -1 3 Mean AUC/dose, hr.ml .10 0.72 - 0.61 -Coefficient of variation, %, 29.5 40.4 38.5 24.3 Statistical difference^, ns ns 1. Determined by the Student's t-test (2 tailed); a=0.05; ns, not significant. 132 time in Figs. 4-15 and 4-16 respectively. Pretreatment with phenobarbital did not reduce the griseofulvin-plasma concentrations of either of the 70% PEG 300 preparations. This is due, perhaps, to the wetting and solvent properties of PEG. Administration of 20 and 100 mg/kg griseofulvin in 70% PEG 300, like griseofulvin in PEG 600, yielded a rapid rise in the plasma concentration. It i s suggested that this i s due to the absorption of the dissolved fraction of drug in the dosage form. Attainment of approximately the same Cmax following administration of both preparations (4.2 to 6.3 and 3.1 to 5.3 jj'gV'ml for 20 and 100 mg/kg doses respectively) supports this suggestion (Tables 4-XXI and 4-XXIII). The remaining fraction of the drug (undissolved fraction), however, was absorbed slowly and incompletely. Both preparations had the same concentration of the drug in the supernatant (3.2 ug/ml) but were different in their total concentration of griseofulvin (5 or 25 mg/ml). Therefore, the increase in AUC from 11 to 17-18 ug/ml/hr following administration of the higher dose, although small, may be related to the absorption of the undissolved fraction in the two dosage forms. The appearance of a second plasma-concentration peak after 6 to 8 hours (Tables 4-XX and 4-XXII) may also result from absorption of drug from the previously undissolved fraction of griseofulvin in the dosage form. Phenobarbital seemed to f a i l to reduce the absorption of either the dissolved or the undissolved fraction of griseofulvin when given in 70% PEG 3Q0. These observations further suggest that the observed griseofulvin-phenobarbital interaction may result from a reduced dissolution rather than an increased gut motility. If the gut motility had increased following pretreatment with phenobarbital, i t should have affected the absorption of the undissolved fraction of the dose. This fraction of the dose, due to very slow dissolution rate, requires a longer residence time in the absorptive sites to be dissolved 133 and subsequently absorbed. Therefore, a reduced residence time i s li k e l y to reduce the total absorption of griseofulvin (i.e., as reflected by AUC). Absorption studies in this investigation have revealed that the availability of griseofulvin in PEG, unlike that of griseofulvin in Tween 80, is high. Once the drug i s dissolved in PEG i t w i l l be absorbed very rapidly (Tmax 20 min), while the griseofulvin absorption from Tween 80 is relatively slow. Bloedow and Hayton (39) reported a Tmax of eight hours following administration of griseofulvin in pure Tween 80 (solution). In this investigation values of Tmax obtained after administration of a suspension of griseofulvin in 0.5 or 2% Tween 80 were about 6 hours. A delayed Tmax coupled with the observed reduced AUC on increasing the concentration of Tween 80 imply that the absorption rate of griseofulvin decreases with increasing concentration of Tween 80. Phenobarbital pretreatment reduces the plasma levels of griseofulvin when the drug is administered either in tablet (in man) or in suspension forms containing Tween 80. These dosage forms, unlike PEG preparations, share the property of being very slowly absorbed. It is possible that bile salts accelerate the griseo-fulvin release rate from these preparations. Preparations containing PEG, however, may not, due to the solvent effect of the 1 3 4 vehicle, be affected by this change. Therefore, the griseofulvin-plasma levels remain unchanged after pretreatment with phenobarbital. The mechanism of this effect, however, is not completely clear. An in vitro dissolution study of griseofulvin in different dosage forms and in the presence of b i l e from control and phenobarbital pretreated rats may c l a r i f y the problem. Originally i t was hypothesized ('2.4,) that the reduced plasma levels might be related to a complex mechanism involving enzyme induction, first-pass metabolism and distribution rate-limited elimination of griseofulvin. The fact that phenobarbital pretreatment had no effect on the plasma levels of griseofulvin when the drug was administered in PEG ruled out these p o s s i b i l i t i e s , because such effects are expected to be independent of the dosage forms, i.e., the interaction would have taken place regardless of the; availability of the 1 3 5 dosage form. Furthermore, phenobarbital's failure to reduce the griseofulvin plasma levels following administration of the drug in PEG coupled with the observed identical biological half-lives in control and test rats (Table 4-1) support the suggestion ( 2 0 ) that the enzyme induction had no significant role in the griseofulvin-phenobarbital interaction. Therefore i t i s suggested that the griseof ulvin-phenobarbital interaction results r from reduced availability and/or reduced dissolution rather than induced metabolism or decreased transit time in the region of absorption. Phenobarbital has been observed to reduce plasma, concentration of" many other drugs, such as dilantin, dicumarol ( 6 9 ) and digoxin ( 7 0 ). A l l these drugs, lik e griseofulvin, show low solubility in aqueous media, but unlike the latter lack h,hlgh>ali%©.ph4jiic .-pr.o.p^ rMes."' rT-he'tshoBtefJ^-bi'ological half-l i f e observed in phenobarbital pretreated subjects suggests that induced metabolism; is responsible for the reduced dicumarol-plasma levels.'' However, the reducedidicumarolrplasma levelpmaynalsodbeupartlyi'duehtc5 reduced-' absorpV <tiph(?redo Considering the low solubility of dicumarol, it" i s possible that a more complex mechanism consisting of simultaneous reduced metabolism and reduced absorption may be involved in this interaction. Aggeler and O'Reilly ( 7 ' 1 ) reported a dicumarol-heptobarbital interaction in man. As a result of heptobarbital pretreatment both plasma levels and the biological h a l f - l i f e of the anticoagulant drugs were reduced. However, they ( 7 1 ) noticed that the barbiturate effect was greater when the drug was given orally as compared to intravenous administration. The presence of greater recovery of unchanged dicumarol in the stool of the heptobarbital treated subjects might explain the difference. The authors ( 7 1 ) suggested that a reduced absorption might also 136 be involved in the dicumarol-heptobarbital interaction. Barbiturates have been shown to interact with many drugs. This effect is usually attributed to the acknowledged ab i l i t y of these compounds to induce metabolism of other drugs. Lesser importance, however, has been given to another possible mechanism by which barbiturate administration may alter drug disposition, e.g., increase in bile flow with simultaneous decrease in bile salts concentration. In this work the influence of these changes was not directly studied. However, the evidence suggests that these mechanisms could very well be involved in the griseofulvin-phenobarbital interactions. Other poorly soluble drugs may also be susceptible to this effect. More detailed studies on the barbiturate influence in reducing absorption of poorly soluble drugs, particularly the proposed effect of reduced bile salts concentration, appear to be necessary to totally c l a r i f y this problem. 137 5. SUMMARY AND CONCLUSION 1. It has been shown that griseofulvin absorption in the rat is altered by treatment with drugs which affect gut motility (Table 5-1): (a) An increase in gastrointestinal motility caused by metoclopramide pretreatment increased the rate of absorption but decreased the total absorption of griseofulvin when administered as a suspension in 0.5% Tween 80. In contrast, the treatment markedly increased the total absorption of griseofulvin administered in a solution of 100% PEG 600. (b) A reduction in gastrointestinal motility caused by pretreatment with propantheline decreased the rate of absorption but increased the total absorption of griseofulvin when administered as a suspension in 0.5% Tween 80. In contrast, propantheline pretreatment significantly reduced the total absorption of griseofulvin when administered as a solution in 100% PEG 600. It can be concluded that gastrointestinal motility has a significant influence on the absorption of griseofulvin. 2. The interaction of griseofulvin with phenobarbital in the rat is a formulation-dependent phenomenon (Table 5-1): (a) Phenobarbital pretreatment significantly decreased the plasma levels of griseofulvin when administered as a suspension in 0.5% Tween 80. However, the treatment did not affect the rate of appearance of griseofulvin in the plasma. (b) The extent of interaction decreased on increasing the concentration of Tween 80 from 0.5% to 2%. S u mm a r y; of : the Table 5-1. ... Observed Effects of Pretreatment with Single Doses of Metoclopramide, Propantheline or Phenobarbital on the Maximumm Plasma Level, C , and i t s Time of Attainment, T , max max and the Area Under the Plasma Level-Time Curve, AUC, of Griseofulvin Following i t s Oral Administration Of . Preparations. containing Tween-80 or Polyethylene Glycol (PEG) to the Rat. Metoclopramide pretreated, Tween 80" PEG Propantheline pretreated, Tween 80 PEG ' ' Phenobarbital pretreated, . Tween-8.0 0.5%" 2% PEG max max AUC 139 (c) Phenobarbital pretreatment failed to significantly alter the plasma levels of griseofulvin administered either as a solution in 100% PEG 600 or as suspensions in 70% PEG 300. It is concluded that phenobarbital decreases plasma levels of griseofulvin through a mechanism involving a diminished dissolution rate rather than a stimulated gut motility, an induced metabolism or an increased hepatic blood flow. It is suggested that the diminished dissolution rate of griseofulvin is due to a reduction in the b i l e salts concentration caused by pretreatment with phenobarbital. 140 6. REFERENCES 1. G. Riehl, Klin. Med., 3_» 1 3 1 (1959). 2. D.I. Williams, R.H. Marten and I. Sarkany, Lancet, 2, 1216 (1958). 3. H. Blank and F.J. Roth, A.M.A. Arch. Dermatol., 79, 259 (1959). 4. H. Blank, Am. J. Med.', 39, 831 (1956). 5. i.M. Lauder and'j.G. O'Sullivan, Vet. Rec, _70, 949 (1958). 6. c. Bedford, D. Busfield, K.J. Child, I. MacGregor, P. Sutherland and E.G. Tomich, A.M.A. Arch. Dermatol., 81_, 735 (1960). 7. M. Kraml, J. Dubuc and D. Beall, Can. J. Biochem. Physiol., 40, 1449 (1962). 8. W.A. Duncan, G. Macdonald and M.J. Thornton, J. Pharm. Pharmacol., 14, 217 (1962). 9. R.G. Crounse, J. Invest. Dermatol., 3_7, 529 (1961). 10. T.R. Bates, M. Gibaldi and J.L. Kanig, J. Pharm. Sci., j>5, 191 (1966). 11. W.L. Chiou and S. Riegelman, ibid., 59, 937 (1970). 12. P.J. Carrigan and T.R. 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C. Lin, R. Chang, J. Magat and S. Symchowicz, J. Pharm. Pharmacol., 24, 911 (1972). 141 27. C. Lin, J. Magat, R. Chang, J. McGlotten and S. Symchowicz, J. Pharmacol. Exp. Ther., 187, 415 (1973). 28. B. Davis, K.J. Child and E.G. Tomich, J. Pharm. Pharmacol., 13, 166 (1961). 29. P.A. Harris and S. Riegelman, J. Pharm. Sci., 5_8, 93 (1969). 30. R.A. Branch, D.G. Shand, R.W. Wilkinson and A.S. Nies, J. Pharmacol. Exp. Ther., 184, 515 (1973). 31. E.E. Ohnhaus. S.S. Thorgeirsson, D.S. Davies and A. Breckenridge, Biochem. Pharmacol., 20, 2561 (1971). 32. M. Rowland, S. Riegelman and W.L. Epstein, J. Pharm. Sci., 5_7, 984 (1968). 33. L.J. Fischer and S. Riegelman, ibid., 54, 1571 (1965). 34. W.L. Chiou and S. Riegelman, ibid., 58, 1500 (1969). 35. C. Bedford, D. Busfield, K.J. Child, I. MacGregor, P. Sutherland and E.G. Tomich, A.M.A. Arch. Dermatol., jU, 137 (1960). 36. W.L. Chiou and S. Riegelman, J. Pharm. Sci., 59_, 937 (1970). 37. W.L. Chiou and S. Riegelman, ibid., 60, 1376 (1971). 38. Antitinea Capitis Team, Institute of Dermatology, Kiangsu, Chin. Med. J., (9) 163 (1973). 39. D.C. Bloedow and W.L. Hayton, J. Pharm. Sci., _65, 328 (1976). 40. T.R. Bates and J.A. Sequeira, ibid., 64, 793 (1975). 41. J.E. Axelson and F. Jamali, presented to the Research Symposium and Research Conference of the Association of Faculties of Pharmacy of Canada, Ottawa, Canada, May 20, 1974. 42. F. Jamali and J.E. Axelson, presented to the Academy of Pharmaceutical Sciences, Pharmaceutics Section, San Francisco meeting, April, 1975. 43. A.G. Johnson, Gut, _12, 421 (1971). 44. T.H. Howells, T. Khanam, L. Kreel, C. Seymour, B. Oliver and J.A. Davies, Brit. Med. J., 2, 558 (1971). 45. H.I. Jacoby and D.A. Brodie, Fed. Proc, 25_, 200 (1966). 46. L.S. Goodman and A. Gilman, "The Pharmacological Basis of Therapeutics", MacMillan Publishing Co., New York, f i f t h edition, 1975, p.523. 47. J. Nimmo, R.C. Heading, P. T o t h i l l and L.F. Pres'cott, Brit. Med. J., 1_, 587 (1973). 48. G. 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Staub and K.K. Wong, Biochem. Pharmacol., 16, 2405 (1967). 63. C. Lin, R. Chang, J. Magat and S. Symchowicz, J. Pharm. Pharmacol., 24, 911 (1972). 64. S. Symchowicz and K.K. Wong, Biochem. Pharmacol., 1_5, 1601 (1966). 65. S.A. Kaplan, S. Riegelman and K.H. Lee, J. Pharm. Sci. ^  55^ , 14 (1966). 66. C Lin and S. Symchowicz, Drug Metab. Rev., j4, 75 (1975). 67. V.P. Shah, S. Riegelman and W.L. Epstein, J. Pharm. Sci., 61, 634 (1972). 68. J.E. Axelson, unpublished work. 69. S.A. Cucinell, A.H. Conney, M. Sansur and J.J. Burns, Pharmacol. Ther., 6, 420 (1965). 70. A. Kaldor, G. Somogyi, L.A. Debreczeni and B. Gachalyi, Int. J. Clin. Pharmacol., 1_2, 403 (1975). 71. P. Aggeler and R. O'Reilly, J. Lab. Clin. Med., 74, 229 (1969). 1 4 3 7. APPENDICES 144 Appendix I' Identification of the Materials Identity of griseofulvin>.and propantheline bromide was verified by determining their melting points'!". Sodium phenobarbital was identified 2 mass spectrometrically . The-mass speetras of griseofulvin, 4- demethyl-griseofulvin, metoclopramide ihyjir.plch^^ propantheline bromide were also obtained.-The results"are as followes: Griseofulvin, Molecular weight, 352.:3 . '3"' • ° Melting point , ,(220) 219.6-' Mass spectra, Mass/charge, 138 69 214 310 215 140 321 123 352 139 Relative 100 89 64 63 61 44 34 32 31 30 intensity, Sodium Phenobarbital, Molecular weight,of 232.2-the acid, 3 .Massf spectra* , Mass/charge, 204 l i 7 146 ;17.4 91 143- 232. Relative 100 43 20 12 10 8 7 intensity, ^Determined 'using a Mettler FP2-FP11 Capillary' Melting Point 'Apparatus:. 21.Determined using a Finnigan 3200 with 6100 Data System; direct solid probe; electron energy of 30 electron volts. 3.-.Verified, Atlas of Spectral Data and Physical Constants for Organic Compounds, J.Gv.Grasselli,. C.R.C. Press, Cleveland,,Ohio, 1975, (reported value- in parentheses). 145 Metoclopramide - hydrochloride.monohydrate Molecular weight of the base, 299..8 ' =Melting point, 141° . with decompositidn. Mass spectra, Mass/charge, 86 99 58 87 184 10 16 299 Relative intensity, 100 75 38 28 22 20 16 4 Propantheline Bromide, Molecular weight of the base, 353.0 Melting range, 4 (156- 159.0° 3 162) Mass spectra, - . -» 'J ; Jo2 / ' ' Mass/charge,. 181 114 86 310 23 338 21 182 7 24 253 311 353 Relative intensity, 100 89 67 53 52 42 31 16 15:14 13 11 4 4-Demethyl griseofulvin, Molecular weight, 337.8 Mass spectra, Mass/charge, 152 138 69 153 296 201 338 123 200 323 Relative intensity, 100 91 61 57 50 47 43 37 36 31 Verified, U.S.P. XIX, 1975; reported value in parentheses. 1 4 6 Appendix II A Computer Programme The following programme was developed to evaluate pharmacokinetic parameters following intravenous administration of a single dose of a given drug based on the Eq. Cp = Ae~ a t + Be" ^ Z; M I C H I G A N TERMINAL SYSTEM FORTRAN G l 4 1 3 3 6 ) 00 0 1 C0C2 0C0 3 CCC5 0 0 0 6 _coo7_ O J oa 0 0 0 9 r-i' 0 0 1 1 0 0 1 2 -0.013-0 0 1 4 C J 1 5 3 0 1 7 C02G 00 2 1 0 0 2 3 0 0 2 4 1 :',•> ? KCRRE 1 0 - 2 5 - 7 5 1 5 : 2 8 : 55 10 100 SL'CRCUTINE K 0 R R E I T , Z , N , A, B, R, » ) DIMENSION T i l ) ,111) SUMSCT=0. Si;VT = r  SUMTZ-0. SUMZ=0. _£LNSCZ=C : DC 100 I = l , . \ SUMr=Sl,'MT + T I I I •SI f'S<,'T..= S m S . QTrT l l ! » n i | TZ = S I :MTZ + T ! I I - - 2 I I ] S I . M Z = S U M Z + Z ( i) ~SLK.SC2=.SLM_S C i * . l U . U U . U J Crr-.T INUt SL'M2=SU>'TtSo,''T -IXEJ-i= ii.«S CZ.'..Si=Sl_KX2 I F { C f N . L E . l . c - 1 5 ) R E T U R N AA = .\wSUMTZ-SUM.T*SUMZ i / 0 E > i _ K= ( S U M S C T * S U M Z - S U M T Z » S U M T ) / D E N tip. = 0 E N- ( N « S U M SC Z-S UM Z » S U M Z ! F = ftf /;o3 TLSHX-1000 10 .»<= i;. V.R I TE ( 6 , 1 ) N ,A,:',B,R " 6 . 5 X , ' R = < , 6 6 . 3 ) RE TURN .ii£-i.^4Jl.<.LJJiT.£&CE£X=i.^£J.a. »j?Tir,\s vQPT ICNS — i S J . A J . i S X NO ERR'JRS E F F E C T * ID,EBCDIC,SOURCE,NOL 1ST , NiJDFCK . L O A C N O M A P EFFECT'.' NAME = KCRRfc , L I N E C N T = 57 SXU8££_SiAI£>l£;,.tS_= 2J.^PR PGR A'.- , A T I S T I C S * NC O I A S N C S T I C S GENERATED I N IN _1C0.4_ 1. 0 0 0 2 . 0 0 0 3 . 0 0 0 4. OilO 5. 0 0 0 6 . 0 0 0 -7_ooa_ 3 . 0 0 0 9 . 0 0 0 l l . G C O 1 2 . 0 0 0 - a s . O G O _ 1 4 . 0 0 0 1 5 . 0G0 _ i 6 . . 0 i i a _ 1 7 . 0 0 0 1 8 . 0 0 0 — 1 9 . 0 0 0 — 2 0 . 0 0 0 2 1 . 0C0 _ 2 2 - O 0 a _ 2 3 . 0 0 0 2 4 . 0 0 0 - 2 5 . 0 0 0 — 2 6 . 000 2 7 . 0 0 0 _ 2 & - a Q G _ IN KCR.PE *) IBM 370/168 MICHIGAN TERMINAL SYSTEM FORTRAN G( 41326) CAIN 10-25-75 15:28: 55 C001 0002 0003 0004 CCC5 0006 0003 0009 0010 0011 0012 0014 0015 .001 6 0017 0013 ___13_ 0020 0021 J023 0024 -.002 5-0026 0027 no ? ? 0029 0030 0032 0033 0034 0035 _fl.0_3.6_ 00 37 0038 0040 0041 nn4-> 0043 0044 on 4 5 0046 0 04 7 0049 0050 0051 0052 CIMENS ION 2(100 ),T (100),ZL!100! 10GICAL*1 CASEN0.8) EQUIVALENCE { C PM AX , CP SCL ) ,ITMAX,TSCL) I r . G t f Al El FT I — 1234 N=L _L3_ READ15,13,END=400)CASENO __QRMA_(-8.A.U_ READ r5,12,EN0 = 400) TMAX , CPMI N ,CPMAX 12 FORMAT ( 3 F 1 0 . 2 ) PI p T j ^ T ^ r i i g n PR r o s r . i . I F . n . 10 READl5,1,END=100)T(N),2(N) N = N + 1 c-.__io_i.c_ F0PMAT(2F10.3) 100 N = N-1 TF(M . r-o n i r.r TO 40n KRITE(6,14)CASENG 14 FORMAT!1H1,/20X,8A1//I 11 FORMAT (5X,- TIME- ,10X,'CP' / 5X,4( ' + ' ) ,9X,4(' +' )/ 1/3X,F8.3,5X, F8 . 3 i CO 110 1=1, N 71 1 M - ? i T 1 — _ 110 CONTINUE REAC(5,2)NBETA _2 EJ3£i_AT_U--__ FORMAT(1 HI ) !..DEX=N-NBETA + 1 nn ? P 0 I=TM,-.FY,M 200 2 ( I ) - A LOG10(Z(I)) CALL K.ORREI Tl I NO EX) ,Z( INDEX) ,N6ETA,A,B,R1 ,£3 70) _F_LX£J_-,-4..J - — : — 4 F0F.MATI/1X,20.•*•>//) • 6=10.**B _ r P C T A T<- M f ^ i i q P F T A : -BETA=ALOG(10.)*A INf.EX=INDEX—1 K_J DO 300 1 = 1 , INDEX BT=BETA*T< I ) 250 15 I F ( Z ( K ) .LE. 0.) GO TO 250 Z ( K) = Al CG10( 21 K) ) K = Kt 1 — • — GO TO 300 WRITE16,15 )T( I ) CPgt . , . .T . / / * Y , I T H F W M I ' F GIVEN F f " T=' F8.2, DELETFI.')-300 CONTINUE INDEX=K-1 CAU ' T l f l T i M NT'" • A P , R P , P ? . E 3 7 0 I A=10 .**BP C ALPHA IS fINUS ALPHA .M or.i i n . i c A P _ 29. 000 3C. 000 31. 000 3 2. 0OQ. 33.000 34.000 _35.-00.0_ 36.000 37.000 _3_-0_0-V.R IT E (6 , 5 ) FORMAT!///, r o 7 - A + R 5X, 'FINAL RESULTS:•// 1 39.000 4 0. 000 _4.1-000_ 42.000 43.000 __4—OO0-45.000 46.000 _A.7.-000-48.000 4y. 000 51.000 52. 000 _53~000_ 54. 000 55.000 56. 000-57. 000 58.000 -5.9-0.0 0-60. 000 61.000 63.000 64.000 __.5-0_i.a_ 66.000 67. 000 ___,a—aao-69.000 7 0. 000 __J_l-.-0.OC--72.000 73.000 —IA-aao_ 75.000 76. 000 ..77. OOQ-78.000 79. 000 o n . n i . n PAGE P00 1 81. 000 82. 000 63.000 _-MICHIGAN TERMINAL SYSTEM FORTRAN G(41336) MAIN 10-25-75 15: 28 : 55 PAGE P002 0054 G055 0056 on s 7 RFAD15, 1 )XBZ VC=XPZ/CPZ AK21 = A*VC*(ALPHA-F3ETA)/XBZ— ALPHA AKFI = AI PHA*8FTA/AK -, 1 84.000 85. 000 8 6 . 0 0 0 R 7 AO fl 005S 0059 CiOM A K12=-[ALPFA + BETA + AK21 + AKEL) *AUC=—A/ALPHA-3/BETA C1.=XI,2/AUC ... 88. 000 8 9 . 0 0 0 9 0 000 0061 0062 6 TAL=—AL0GI2. )/ALPHA VJR IT E ( 6 , 6 ) A .ALPHA , R.2, TAL EliaaAIi//5X, ' A=' . F l 1 , f i t . 5 X . ' -A l PHA= • . F l 1 . A. ? Y . I I • RF*. 3 , > I t , s y , 9 1 . 000 9 2 . 0 0 0 q -3 f; f) 0 0064 C C 6 5 ** T 1/2 (ALPHAi =• ,F11.6> T6E=-AL0G(2. l/BETA klP-LLEi 6-. J . JJ3.. Jj r J A. R! .TRF 9 4 . 0 0 0 9 5 . 0 0 0 0066 • nr-A7 7 FORMAT<//5X,«B=',F11.6,5X,«-BETA = • , F11 .6 , 2X , •(«,F6.3,•)•,5X, * 'T 1/2 (BETA ) =•,F 11.6) liiU_TFJ.A..B ) CP 7 , y P7 , \ir 96 .-000 9 7 . 0 0 0 9 8 . 0 0 0 C G A •*•""! 0068. 0069 ran 6 9 F O R M A T I / / 5 X , > C P 0 = > , F 1 1 . 6 , 5 X , • X B 0 = ' , F l 1 . 6 , 5 X , • V C = • , F l 1 . 6 ) WP IT E 16 , 9 I AK 1 2, AK 2 1, AK EL , AUC ,C L EJJZtiAXI./y_5X J , ;02=' .F_Ll_i>.5X. 'K?l-t f F] l fr, sx , .K Fl = ' ,F1 1 , A / / q x , i j - 3 * - U j _ l U 100.000 101. 000 A 11 r — i n ? A n n C071 nr. 7 5 * ' , F U . 6 , 1 0 X , ' C L = ' , F 1 1 . 6 ) I FI PLOT I) GO TO 1234 nx = T<:ri / i n . — i i i — i i ; / . n u n 103.000 104 .000 0073 CC74 r 7 CALL A X I S I O . , 0 . , "TIME! HR)',-6,10., C . , C . , D X ) DY=CPSCL/10. — C A LI—A.X I_S-(XU.^JD—.UiiiCfA'' K f M f. G / MI | , t p , i n ,QH , , .I , , PY ) L-Q 5 *. Q.Q-.Q 106 .000 107. 000 1 r\ p n A A 0076 00 77 "07M 00 120 I=1,N XX = T ( I l /DX YY = 7I i n / n y . I U-C-* -U U U 109 .000 11C. 000 i l l Ann 0079 0C30 0,18 1 i ?n 2L( I ) = ALOG10(ZL ( I ) ) CALL SYMBOL (XX ,YY , . 1 , 3 , 0 . ,-1) I » u U Li 112 .000 113. 000 • 1 1 L. A A A 0CS2' 0083 n n a 4 X=T(1) XX=X/DX ... Y = A*PXPf Al P H l * X l + 3 « F » B r S F T » « » ) i~i , it».uU.U 115. 000 116. ocd CC35 C C 86 •ir,a YY=Y/DY CALL PL0T (XX ,YY ,3 ) ..f.AII P! OT( y y , Y Y r ?> 1-LX.-0 0 0 118. 000 119 .000 1 A A A C083 CC39 r-r.or, 320 XX=XX+.01 X=XX*0X i = M E J i P l l l P H A * V U R * F V O l n F T A i Y ] X £\J »-U U J 121 .000 122. 000 CC91 C092 YY=Y/DY CALL P L0T !XX ,YY ,1 ) —LFJ.X.. . I F . n m i m m 1.2-3- QG Q 124. 000 125. 000 0094 0095 CALL S Y M B O L ( 6 . , 5 . , . 1 , C A S END ,0 . , 8) CALL A X I S ( 0 . , 1 4 . ,< TIME(HR)> , - 8 , 1 0 . ,0.,0.,DX) CAM P i n T i n . , 1 4 . , 1 1 ji.26-.-G-G 0 127. 000 128. 000 17C A A A CC97 0098 nnQQ LL L O T ( 0 . , 2 4 . , 2 )CPMAX = AL0G10(CPMAX) —CP'-'Ui-uu r.r-. l n (r P u i M J L-*Cx . . .U .LJJJ 13 0. 000 131.000 0100 0101 23 C 0Y=10./ICPMAX-CPMI N) YY=14. . VY=YV+nv 1-32-.-000 133. 000 134.000 0103 0 104 n i .-,<; CALL SYNBOHO. ,YY , . 1, 1 5 , 0 . ,-1 ) IF (YY . L T . 24 . ) GO TO 330 —CAJJ—<iiLMP.Pi I-.7S T i « , , . i L, , or, np rnnr.N ( " f G/Ml ) , s n 13 5. Q 0 0 136.000 137.000 1 3 fl A A A —• • i J O . J J U oo MICHIGAN TERMINAL SYSTEM FORTRAN G.41336) MAIN 1 0 - 2 5 - 7 5 010 6 0107 0108 OLIO 0111 - O i l ? 0113 0114 O l l S 0116. 0117 _xu.ia_ 0119 0120 0122 C123 _ilL2.4_ Oi25 0126 PI ?7 012B 0129 0131 0132 0 1 3 3 0134 C135 _X.L3.6_ 0137 0138 PI 39 0140 .. 0! 4 i . 0142 0143 PI 44 l b : 28:55 335 340 YY=i4. AA=C PMI N CALL NUMBER(-.2,YY,.07,AA,0. ,-YY=YY+OY 1) 3 4 S A A =A A .-1. IFIYY .LE. 24.1 GO TO 335 _Q CL.3_45_I_1-,.M YY=!ZL(I)-CPMINj*DY+14. X X = T( I ) / D X r.Al l S Y ^ P I IYY r v v , . i , i i , n . XX=T( 1) /DX X=T(1) __'=.AiEXJL(jlX£HAiXJ_t.8*EX.R.l.B.£XA*X.l_ YY=( AIOG10! Yl-CPMl N)*DY+14. CALL PL0T(XX,YY,3) _CALJ PI PT I XX ,YY ,?) 350 XX-XX + .01 X=XX*DX YY=IALOG10IY l-CPMIN )*DY+14. CALL PLOTIXX.YY.l) IXXX - I F . T IM ) i m Tn YY=IALCG10 (A l-CPMIN)*OY+14. CALL PLOT!0.,YY,3) _Y__=CmiiN_U X=AL0G!10.)*(AL0G10(A)-YY)/(-ALPHA) X X = X / C X Y Y - 1 5 . . CALL PLOT ( X X,YY ,2) CALL SYMBOL t 6. , 19. , . 1, C A SE NO , 0. , 8) _ C A . L I _ P _ L C r u _ 4 ^ , £ _ ^ _ J J 370 16 GO TO 1234 F.EA0I5, 1 )X6Z WR 1 T F I A , IA ) 139.000 14C. 000 141.000 -1A2-XUIQ 143.000 144.000 —1.45..00Q 146.000 147.000 _l-4iL.X>_0 149. 000 15 0.000 —15.1..000 152.000 153.000 —15-4 ..000 155.000 156. 000 -1.57...000 158.000 159.000 -16-0.-0-00 161. 000 162.000 -163.000 164. 000 16 5.000 _16£_X«H)_ 400 FORMAT!//5X,'***# ERROR RETURN FRCK KCRRE'//5X ,•**** PROCESS * I NAT ED CUE TO PAD CATA 1) —GI1_TX 1?34  WR!TF(6,3) CALL PLOTND STOP :  167. 000 168.000 _16.9..0U__ 17C. 000 171.000 _L72._0.Qa TERM 173.000 174.000 -113 ..000 176.000 177.000 • 173. 003-179.000 0145 'OPTIONS * P P T_I.L'1-1S_ Ef-i D IN EFFECT* IN—f_E£_LCJj__ *STAT1ST ICS* *STATI STI CS* Nn =apoj<: IM M.. jfi ID,EECD1C,SOURCE,NOLI ST,NODECK.LOAD,NOMAP -NA.MF = M-AiN r I T M F r f . ' T = SOURCE STATEMENTS NO DIAGNOSTICS GENERATED 145,PROGRAM SIZE 5696 NO STATEMENTS FLAGGED IN THE F X F f t l T T P . N T F R M I M g T F I - 1B0VE COMPILATIONS. $R IV.2 EXECUTION BEGINS 1 5 0 In order to run the programme the following information should be punched in the computer cards: Card number Column Input 1 1-8 Lable,ee,g., CONT. 02 2 1—7 Scale orl the time-axis, e.g., 30. 8-15 Lower limit on* the plasma level-axis, e.g., 0.001 16-23 Upper limit on the plasma level-axis, e.g., 20. 3-n 1 Time of sampling, e.g., 1. 11 Plasma level, e.g., 6.309 Note, one card for each plasma level and the corresponding time of sampling; n, number of data cards following card number 3 n+_ 1 $END n+2 4 Number of data points in the $-phase, e.g., 4 n+3 1 Administered dose, e.g., 6.5 n+4 1 $END n+5 1 $R PL0T:Q PAR= , commanding plotting n+6 1 $SIG 151 The print-out containes the data (time of sampling and plasma levels), calculated parameters and f i t t e d plasma level vs time and logarithm of plasma level vjs time curves. Each data set i s labeled as follows: CONT,control rats PHEN, phenobarbital pretreated rats MET, metoclopramide pretreated rats PROP, propantheline pretreated rats A number following the label indicates the rat number i n the category, e.g., CONT 02 indicates rat number 2 of the controls. The resulted plasma level-time and the logarithem of plasma l e v e l -time curves and the calculated parameters are presented i n following pages. 152 CONT.Ol TIME CP 4-.+-. 0.860 8.235 "2 ."860 2.85V' 4.280 1.158 11.660 0.162 22.620 0.006 3 POINTS: SLOPE* -0.123473 INTEPCEPT= 4.106844 P=-1.000 2 POINTS: SLOPE* -0.343314 INTERCEPT* 9.905205 R*-1.000 FINAL RESULTS: A= 9.905205 -ALPHA- -0.790510 (-1.000) T 1/2 (ALPHA) = 0.876836 B= 4.106844 -BETA * -0.284307 (-1.000) T 1/2 (BETA) = 2.438022 CP0= 14.012049 XH0= 5.CC0000 VC = 0.356836 K12 = 0.122704 AUC* 26.975235 K21* 0.432672 KEL= 0.519441 CL- 0.185355 154 155 CONT.02 TIME CP >. — 1.000 6.3C9 3.000 2.100 /. n.m 1 . 1 1 S 9.000 0.253 12.000 C.09C 23.000 0.002 <, POINTS: SLOPE* -0.151248 INTERCEPT* 5.214C19 R=-0.999 2 P0IN1S: .m.f_ - f . M 7 « 7 INTERCEPT* 8.26381 2 R=-1.000 FINAL RESULTS: A* 8.263E12 -AlPhA* -1 . 145529 l - l . C O & l T 1/2 (ALPHA) * 0.605089 B" 5.214C19 -BETA = -0.348262 (-0.999) T 1/2 (BETA) * 1.990301 CPO- 1 3 - 4 7 7 8 3 1 X80= 6.5C000C VC* 0.482273 r...5'.« K?l = C.656692 KEL* 0.607506 AUC* 22.185486 1 CL* 0.292984 : _= : — : • — — 156 CONT,03 TIKE CP 1.170 t.119 2.500 2.450 4.000 1.213 5.17C 0.752 12.000 C.1CC 23.000 0.004 3 POINTS: SLCPE= -0.127503 INTERCEPT* 3.414751 R*-1.000 3 POINTS: SI OPE* -0. 548774 INTERCEPT* 22.763e70 R*-0.996 FINAL RESUL IS: A* 22.71.3870 ALPHA* -1.2635S7 l - C . 5 . 1 T 1/2 I ALPHA ) = 0.548551 B* 3.41H751 BETA = -C. 293587 l-l.COO) T 1/2 (BETA) = 2.360963 CPO* 26.17E619 XBO* 5.CC0OOC VC= 0.190996 K12* 0.254035 K21= 0.420116 K E l * 0.883031 AUC* 29.646271 CL* 0.168655 138 159 PHEN.Ol TIME CP + »f + H H 1.000 11.376 <:.-.0 4.984 6.000 1.097 12.000 C.267 24.000 0.006 — — 3 POINTS: ~ — SLOPE* -0.128666 INTERCEPT* 7.507994 R* -0.997 2 POINTS: SLOPE* -0 .628743 INTERCEPT* 25.214798 R*-1.000 FINAL RESLLTS: — *->.21WStl -ALPHA* -1 .47076C ( -1.C00) T 1/2 (ALPHA) = 0.471285 B- 7.5C7S94 -BETA = -0 .296265 ( -0.997) T 1/2 (BETA ) = 2.339622 CP0= 32.72.778 XBO* 10.000000 VC* 0.305597 K12- 0.431083 K21= 0.565743 KEL* 0.770197 AUC* H2.486221 CL= 0. 2353 7C • ~ : t 160 161 162 PHEN ,02 TIKE CP **** • + + + v _ ____ l . ooo 9.37C 3.170 3.021 4. 000 1.4 19 5.500 0.711 9. 170 C.263 12.000 0.103 23.000 C. C05 4 POINTS: SLOPE = -C.123299 INTERCEPT* 3.364924 R*-1.000 3 POINTS: SLCPE* -0.404743 INTERCEPT* 19.783463 R=-0.960 F1NAL RESULTS: A* 19.763463 -ALPHA* -0.931956 (-0.960) T 1/2 (ALPHA) = 0.743756 B= 3.364924 - B E T A * -0.283906 (-1 .000) T 1/2 (BETA) * 2.441465 CP0= 23.148376 X&0= 5.C00OC0 VC* 0.215998 K12* 0.137986 K21* C.378109 KEL* 0.699767 TuC = 33.0CC124 CL* 0.151148" 163 1 64 165 PHEN.03 TIME CP 1.170 t.COl 3.000 2.440 4.000 1.591 5. SOC C.75C 9.000 0.373 12.200 0. 155 23.000 0.003 4 POINTS: SLOPE* -0.14CG49 INTERCEPT* 6.006315 R*-0.994 THE VALUE GIVEN FOR T * 4.00 OELETEO 2 POINTS: SLCFE* -0.572000 INTERCEPT* 8.830572 R=-1.000 FINAL RESULTS: A = 8.83C572 -ALPHA* -1.317078 ( -1 . c o o t T 1/2 (ALPHA) = 0.526277 B = 6.C06315 —BETA = -C.324317 ( -C.994I T 1/2 (BETA) * 2.137249 CP0= 14.836887 XBO* 4.000000 VC* 0.269598 K12* 0.326992 K21* 0.726210 KEL = 0.588192 AUC* 25.224533 CL* 0.158576 166 O o PHEN.03 167 168 / TIME + f f f METfl. Cl CP t+ »<• > 1 .OOC 7. 103 3.200 2.233 4.330 1.245 5.5CC 0.932 9.COO 0.299 12.170 C.C85 .3.000 0.003 4 POINTS: SLOPE= -0.142361 INTERCEPT^ 5.3B169C R--0.999 THE VALUE GIVEN FCR T = 4.33 DELETED 2 POINTS: SLOPE- -0.439533 INTERCEPT- 8.874506 R--1.0C0 F I N AL RESULTS: A = 6.874506 ALPHA = -1.012061 (-1.C00) T 1/2 (ALPHA) = 0.684887 8 = 5.3-165C BE TA = -C.327643 (-0.999) T 1/2 I BETA) = 2.114263 CPO- 14.25-19- >B0= 5.00000C VC= 0 .350725 K12 = C.167692 K21 = C.566135- KEL = 0.566077 AuC = 25.1-4158 CL= 0.LS8637 169 170 171 c ' TIME •. fr f MET0.02 CP > 0.660 11.006 -.000 3.60C 4.170 1.600 5. 5CC C.682 12.000 0.C96 23.OCC C.002 3 POINTS: SLOPE- -C.144298 INTERCEPT* 4.57SC74 R=-0.999 3 P O I N T S : SLCPE* -C.331641 INTERCEPT* 9.169435 R = -0.957 F I N A L RESULTS: A* 9.169435 -ALPHA * -0.763631 ( -0.9571 T 1/2 ( ALPHA) * 0.907699 6 = 4.579C74 - e E T A * -0.332258 1 -0.999) T 1/2 (BETA) = 2.086173 CPU* 13.7465C6 X80* 5.CC000C VC = 0.3o3b76 K12 = 0.08665C K21* C.475931 KEL* 0.533107 AUC* 25.789368 CL* 0.19387U 172 173 174 PROP,01 TIME CP 0.167 16.711 0.670 14.085 4.067 4 .4 76 5.720 2.760 a.830 1.215 23.OCO C.013 3 POINTS: SLOPE- -0.142024 INTERCEPT- 23.2C1E74 R- -1 .000 THE VALUE GIVEN FOR T= 0.17 DELE1 ED THE VALUE GIVEN FOR T= 0.67 DEL ET EC THE VALUE GIVEN FCP T - 4 .07 'OEL ET ED * * * * ERROR RETURN FROM K.ORRF * * * * PROCESS TERMINATED CUE TC BAD DATA 175 r V T I ME • t+ + PROP,C3 CP *i + + /• 0. 3 70 12.589 2.450 3.B2C 5.78'. 3.858 8.700 1.34 1 23.200 29.200 0.012 0.003 3 POINTS: SLCPE= -0 .129626 INIEBCEPT= 16.634018 R=-0.997 THE VALUE GIVEN FOR T= 0.37 OELETED THE V'-LUE GIVEN FOR T= 2.45 DELETED THE VALUE GIVEN FOR T= 3.82 OELETED . * * * * ERROR RETURN FROM KORRE *<•*« PROCESS TERMINATED OLE TC BAD LATA 

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