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CNS pharmacokinetics of diphenhydramine in sheep Yeung, Sam Au 2003

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CNS Pharmacokinetics of Diphenhydramine in Sheep by  Sam A u Yeung B.Sc.(Pharm.), The University of British Columbia, Vancouver, Canada, 1998  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) (Division of Pharmaceutics and Biopharmaceutics)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July, 2003 © Sam Au Yeung , 2003  Page 1 of 1  In  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t 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 f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d without my w r i t t e n p e r m i s s i o n .  Department The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada  Printed for "Dr. Wayne Riggs" <riggskw@unixg.ubc.ca>  7/7/03  11  Abstract The CNS pharmacokinetics of the Hrreceptor antagonist, diphenhydramine (DPHM), was studied in 100 d (103.5 ± 1.7 d) and 120 d (124.1 ± 1.4 d) fetuses, 10 cl (11.5 ± 1.6 d) and 30 d (33.8 ± 1.2 d) newborn lambs, and adult sheep (5.21 ± 2.83 years) using in vivo microdialysis.  The first study involved i.v. administration of DPHM at 5 infusion rates to the animals, with each step lasting 7 h. At all ages, CSF and ECF concentrations were very similar to each other, which suggested that the transfer of DPHM between these two compartments was by passive diffusion. Also, the brain-toplasma concentration ratios were 3 or higher in all age groups indicating the existence of an active transport process for DPHM into the brain. Both brain and plasma DPHM concentrations increased in a linear fashion over the dose range studied.  C l j was the lowest in adult sheep, likely due to active renal tubular  reabsorption of the drug. On the other hand, the factors  fcsF  and  fecF  decreased  with age, indicating that DPHM was more efficiently removed from the brain as age increased. The extent of plasma protein binding of the drug increased with age. V d  s s  dropped postnatally in the newborn lambs and increased significantly  in the adults.  In the second study, DPHM was infused for 8 h and propranolol (PRN) was coinfused from 4 - 8 h. The purpose was to examine the effects of PRN on blood-  Ill  brain C S F and blood-brain ECF relationships. Pharmacokinetic analysis showed that CIT was not significantly different from that in the 5-step infusion study but AUCbrain/AUCpiasma ratios  administration.  (fcsF  and  f cF) E  increased in all age groups after PRN co-  On the other hand, protein binding was not changed by PRN.  The observed increase in f ratios appears to be due to the inhibitory effects of PRN on a transporter-mediated mechanism for DPHM brain elimination.  PRN  also caused decreases in heart rate in all groups except the 100 d fetuses.  In summary, these two studies suggest the existence of transporter-mediated mechanisms for the influx and efflux of DPHM into and out of the brain. While postulations have been made as to the possible identities of these transporters, further work is needed in order to obtain a definitive answer.  iv  Table of Contents Title Abstract Table of Contents List of Tables List of Figures List of Abbreviations Acknowledgments  i ii iv vii ix xiv xviii  Chapter 1  1  Introduction  1  1.1 The Blood Brain Barrier (BBB) 1.1.1 Influx Transporters 1.1.2 1.1.3 1.1.4 1.1.5 1.1.6  Efflux Transporters Cerebral Metabolism Efflux Pathways Development of the BBB Implications of Development on Fetal Brain Exposure  1.2 Diphenhydramine  3 7 8 10 10 11 18 19  1.2.1 Pharmacology, Clinical Applications, Adverse Effects  20  1.2.2 DPHM Use in Pregnancy  21  1.3 Pharmacokinetics of DPHM  22  1.3.1 Absorption  22  1.3.2 Distribution 1.3.3 Metabolism 1.3.4 Excretion  22 23 25  1.4 Disposition and Fetal Effects of DPHM in Pregnant Sheep  26  1.5 Behavioral Effects of DPHM and Other Drugs in the Fetus  27  1.6 DPHM Levels in the Brain and CSF  35  1.7 Microdialysis  37  1.8 Rationale  38  1.9 Hypothesis and Objectives  40  Chapter 2  41  DPHM 5-Step Infusions  41  2.1 Methods  42  2.1.1 Animals and Surgical Preparation  42  2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.1.8  49 51 52 53 53 55 57  Experimental Protocols Retrodialysis Physiological Recording Determination of DPHM Plasma Protein Binding DPHM and [ Hio]-DPHM Extraction Procedure Pharmacokinetic Analysis Statistical Analysis 2  2.2 Results  57  2.2.1 Steady-state Concentrations of DPHM in CSF, ECF, and Plasma in Fetuses, Newborn Lambs, and Adult Sheep 57 2.2.2 CSF, ECF, and Plasma Pharmacokinetics of DPHM in Fetuses, Newborn Lambs, and Adult Sheep 64 2.2.3 Physiological Responses in Fetuses, Newborn Lambs, and Adult Sheep 88 2.3 Discussion 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6  MD Probe Recovery CSF, ECF, and Plasma Relationships in Relation to Age Prediction of DPHM Brain Levels Using Plasma Concentration DPHM Pharmacokinetics in CSF, ECF, and Plasma Physiological Responses CNS Effects of DPHM in Fetus  2.3.7 Summary of DPHM 5-Step Infusion Study  98 98 99 105 106 109 110 112  Chapter 3  113  DPHM-Propranolol Co-administration Study  113  3.1 Methods 3.1.1 Animals and Surgical Preparation 3.1.2 Experimental Protocols  114 114 114  vi 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 3.1.8  Retrodialysis Physiological Recording Determination of DPHM Plasma Protein Binding DPHM and [ H ]-DPHM Extraction Procedure Pharmacokinetic Analysis Statistical Analysis 2  10  3.2 Results 3.2.1 Changes in DPHM Concentrations with PRN Co-administration 3.2.2 Pharmacokinetics of DPHM in CSF, ECF, and Plasma in Fetal, Newborn, and Adult Sheep 3.2.3 Physiological Responses in Fetuses, Newborn Lambs, and Adult Sheep 3.3 Discussion 3.3.1 Comparison of DPHM Concentrations Before and After PRN Coadministration 3.3.2 Effect of PRN on CNS Pharmacokinetics of DPHM 3.3.3 Physiological Responses 3.3.4 Summary of DPHM-PRN Co-administration Study  116 116 117 117 117 118 118 118 128 133 142  142 144 148 150  Chapter 4 Overall Summary and Conclusions  151  References  158  Vll  List of Tables Table 2.1 Summary of MD probe recovery rates in the 5-step infusion experiment.  59  Table 2.2a Mean DPHM plasma concentration at steady-state for each infusion step. 66 Table 2.2b Mean DPHM CSF concentration at steady-state for each infusion step. 66 Table 2.2c Mean DPHM ECF concentration at steady-state for each infusion step. 67 Table 2.3a Mean CCSFSS/CP ratios for each infusion step.  68  Table 2.3b Mean C CFSS/CPSS ratios for each infusion step.  69  Table 2.4 Extent of protein binding in steady-state plasma samples for each infusion step.  70  Table 2.5a Mean CCSFSS/CP S ratios for each infusion step.  71  Table 2.5b Mean CECFSS/CP S ratios for each infusion step.  72  Table 2.6 Summary of slopes, y-intercepts, and regression coefficients for figures 2.7a,c - 2.11a,c).  74  ss  E  US  US  Table 2.7 Pharmacokinetic parameters for fetal (100 & 120d), lamb (10 & 30d), and adult sheep. 85 Table 2.8 Blood gas parameters during 5-step infusion of DPHM in the 100 d fetus group. 90 Table 2.9 Blood gas parameters during 5-step infusion of DPHM in the 120 d fetus group. 91 Table 2.10 Average arterial blood pressure and heart rate in each infusion step for fetal (100 & 120 d), lamb (10 & 30 d), and adult sheep. 92  Table 3.1 Summary of MD probe recovery rates in the DPHM-PRN coadministration experiment.  120  Vlll  Table 3.2 Mean DPHM concentrations before and after co-administration of propranolol. 124 Table 3.3 Pharmacokinetic parameters for fetal (100 & 120d), newborn (10 & 30d), and adult sheep from the DPHM-PRN co-administration study. 129 Table 3.4 Arterial blood pressure and heart rate values before and after PRN coadministration for fetal (100 & 120 d), lamb (10 & 30 d), and adult sheep. 134 Table 3.5 Blood gas parameters during DPHM-PRN co-administration in the 100 d fetus group. 140 Table 3.6 Blood gas parameters during DPHM-PRN co-administration in the 120 d fetus group. 141  ix  List of Figures Figure 1.1 Generalized physiological compartment model of the CNS showing factors (in italics) that govern the distribution of drugs among the different compartments.  5  Figure 1.2 Sites of the BBB. The BBB comprises tight junctions between: (a) the endothelial cells of brain capillaries; (b) epithelial cells of the choroid plexus; and (c) epithelial cells of the arachnoid membrane. The arrows depict the flow of CSF, which originates from the choroid plexus. 6 Figure 1.3 Chemical Structure of Diphenhydramine (DPHM)  19  Figure 1.4 Changes in the overall incidence and duration of low, high and intermediate voltage ECoG episodes and the percentages of breathing and electro-ocular activity in the fetus before, during and after infusion of DPHM to the a) ewe, and b) fetus (* p<0.05). 30 Figure 2.1a Cross-sectional diagram of the fetal brain. MD probes were implanted into the lateral ventricle (LV) and the cerebral tissue for CSF and ECF sampling, respectively. 45 Figure 2.1b Cross-sectional diagram of the adult brain-. MD probes were implanted into the lateral ventricle (LV) and the cerebral tissue for CSF and ECF sampling, respectively. 45 Figure 2.2 A diagrammatic illustration of the anchoring of a flexible, modified {i.e. with the addition of the polyvinyl sleeves and extension by FEP tubings) microdialysis probe into a skull. 46 Figure 2.3 Mean DPHM CSF, ECF, and plasma concentrations vs. time profiles in (A) 100 d fetuses, and (B) 120 d fetuses from the 5-step infusion study. 60 Figure 2.3 Mean DPHM CSF, ECF, and plasma concentrations vs. time profiles in (C) 10 d lambs, and (D) 30 d lambs from the 5-step infusion study. 61 Figure 2.3 Mean DPHM CSF, ECF, and plasma concentrations vs. time profile in (E) adult sheep from the 5-step infusion study. 62 Figure 2.4 Changes in overall CCSFSS/CP and CECFSS/CP ratios in relation to age. Groups with different numbers (1-3) and letters (a-c) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). 67 ss  ss  X  Figure 2.5 Changes in the overall extent of protein binding in relation to age. Groups with different numbers (1-4) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). 73 Figure 2.6 Changes in the overall CCSFSS/CP S and CECF S/CP S ratios in relation to age. Groups with different numbers (1-2) and letters (a-c) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). 73 US  S  US  Figure 2.7a Relationship between mean DPHM plasma concentrations and infusion rate at steady-states in the 100 d fetus group.  75  Figure 2.7b Relationships between mean DPHM brain concentrations and infusion rate at steady-states in the 100 d fetus group.  75  Figure 2.7c Relationships between mean DPHM brain concentrations and unbound plasma concentrations at steady-states in the 100 d fetus group.  76  Figure 2.7d Relationships between mean CCSFSS/CPUSS and CECFSS/CP S ratios and infusion rate at steady-states in the 100 d fetus group. 76 US  Figure 2.8a Relationship between mean DPHM plasma concentrations and infusion rate at steady-states in the 120 d fetus group.  77  Figure 2.8b Relationships between mean DPHM brain concentrations and infusion rate at steady-states in the 120 d fetus group.  77  Figure 2.8c Relationships between mean DPHM brain concentrations and unbound plasma concentrations at steady-states in the 120 d fetus group.  78  Figure 2.8d Relationships between mean CCSF S/CP S and C CFSS/CP S ratios and infusion rate at steady-states in the 120 d fetus group. 78 S  US  E  US  Figure 2.9a Relationship between mean DPHM plasma concentrations and infusion rate at steady-states in the 10 d lamb group.  79  Figure 2.9b Relationships between mean DPHM brain concentrations and infusion rate at steady-states in the 10 d lamb group.  79  Figure 2.9c Relationships between mean DPHM brain concentrations and unbound plasma concentrations at steady-states in the 10 d lamb group.  80  Figure 2.9d Relationships between mean CCSFSS/CP S and CECFSS/CP S ratios and infusion rate at steady-states in the 10 d lamb group. 80 US  US  xi Figure 2.10a Relationship between mean DPHM plasma concentrations and infusion rate at steady-states in the 30 d lamb group. 81 Figure 2.10b Relationships between mean DPHM brain concentrations and infusion rate at steady-states in the 30 d lamb group.  81  Figure 2.10c Relationships between mean DPHM brain concentrations and unbound plasma concentrations at steady-states in the 30 d lamb group.  82  Figure 2.10d Relationships between mean C SFSS/CP S and CECWCPUSS ratios and infusion rate at steady-states in the 30 d lamb group. 82 C  US  Figure 2.11a Relationship between mean DPHM plasma concentrations and infusion rate at steady-states in the adult group.  83  Figure 2.11b Relationships between mean DPHM brain concentrations and infusion rate at steady-states in the adult group.  83  Figure 2.11c Relationships between mean DPHM brain concentrations and unbound plasma concentrations at steady-states in the adult group.  84  Figure 2.11d Relationships between mean CCSFSS/CP S and CECFSS/CP S ratios and infusion rate at steady-states in the adult group. 84 US  US  Figure 2.12 Changes in C I in relation to age. Groups with different numbers ( 1 4) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). 86 T  Figure 2.13 Changes in f sF and f cF in relation to age. Groups with different numbers (1-2) and letters (a-c) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). 86 C  E  Figure 2.14 Changes in V d in relation to age. Groups with different numbers (1-2) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). 87 s s  Figure 2.15a Fetal arterial pressure vs. time in the 100 d fetus group. The numbers 1-5 represent the infusion steps.  93  Figure 2.15b Fetal heart rate vs. time in the 100 d fetus group. The numbers 1-5 represent the infusion steps. 93 Figure 2.16a Fetal arterial pressure vs. time in the 120 d fetus group. The numbers 1-5 represent the infusion steps.  94  Xll  Figure 2.16b Fetal heart rate vs. time in the 120 d fetus group. The numbers 1-5 represent the infusion steps. 94 Figure 2.17a Arterial pressure vs. time in the 10 d lamb group. The numbers 1-5 represent the infusion steps. 95 Figure 2.17b Heart rate vs. time in the 10 d lamb group. The numbers 1-5 represent the infusion steps.  95  Figure 2.18a Arterial pressure vs. time in the 30 d lamb group. The numbers 1-5 represent the infusion steps. 96 Figure 2.18b Heart rate vs. time in the 30 d lamb group. The numbers 1-5 represent the infusion steps.  96  Figure 2.19a Arterial pressure vs. time in the adult group. The numbers 1-5 represent the infusion steps.  97  Figure 2.19b Heart rate vs. time in the adult group. The numbers 1-5 represent the infusion steps. 97 Figure 3.1 Mean DPHM CSF, ECF, and plasma concentrations vs. time profiles in (A) 100 d, and (B) 120 d fetuses from the DPHM-PRN study. The arrow denotes start of PRN infusion. 121 Figure 3.1 Mean DPHM CSF, ECF, and plasma concentrations vs. time profiles in (C) 10 d , and (D) 30 d lambs from the DPHM-PRN study. The arrow denotes start of PRN infusion. 122 Figure 3.1 Mean DPHM CSF, ECF, and plasma concentrations vs. time profiles in (E) adults from the DPHM-PRN study. The arrow denotes start of PRN infusion. 123 Figure 3.2 Comparison of extent of protein binding before and after PRN coadministration in different ages. *denotes significant difference from the PrePRN value (Paired t-test; p<0.05). 125 Figure 3.3 Changes in CCSFSS/CP and CECFSS/CP ratios before and after PRN co-administration in different ages. *denotes significant difference from the corresponding Pre-PRN value (t-test, p<0.05). 126 ss  ss  Figure 3.4 Percentage increase in CCSF/CP and C C F / C P ratios at steady-state after PRN co-administration in different ages. 127 u  E  u  Figure 3.5 Comparison of C I obtained from the 5-step infusion and DPHM-PRN co-administration studies in different ages. 130 T  xm  Figure 3.6 Comparison of fcsF and fecF obtained from the 5-step infusion and DPHM-PRN co-administration studies. 130 Figure 3.7 Correlation plot between the percentage increase in C S F / C P and C  u  C E C F / C P ratios and post-conceptional age. u  131  Figure 3.8 V d from the 5-step infusion and DPHM-PRN studies at different ages. *denotes significant difference from the 5-step infusion value (p<0.05). 132 s s  Figure 3.9a Arterial pressure vs. time in the 100 d fetus group. The arrow denotes start of PRN infusion.  135  Figure 3.9b Heart rate vs. time in the 100 d fetus group. The arrow denotes start of PRN infusion. 135 Figure 3.10a Arterial pressure vs. time in the 120 d fetus group. The arrow denotes start of PRN infusion. 136  Figure 3.10b Heart rate vs. time in the 120 d fetus group. The arrow denotes start of PRN infusion. 136 Figure 3.11a Arterial pressure vs. time in the 10 d lamb group. The arrow denotes start of PRN infusion.  137  Figure 3.11b Heart rate vs. time in the 10 d lamb group. The arrow denotes start of PRN infusion. *denotes significant decrease from Pre-PRN heart rate (p<0.05). 137 Figure 3.12a Arterial pressure vs. time in the 30 d lamb group. The arrow denotes start of PRN infusion.  138  Figure 3.12b Heart rate vs. time in the 30 d lamb group. The arrow denotes start of PRN infusion. *denotes significant decrease from Pre-PRN heart rate (p<0.05). 138 Figure 3.13a Arterial pressure vs. time in the adult group. The arrow denotes start of PRN infusion. 139 Figure 3.12b Heart rate vs. time in the adult group. The arrow denotes start of PRN infusion. *denotes significant decrease from Pre-PRN heart rate (p<0.05). 141  xiv  List of Abbreviations approximately %  percentage  u  micron  ug  microgram  10 d  10 day old lambs  30 d  30 day old lambs  100 d  100 gestational day old fetus  120 d  120 gestational day old fetus  ANOVA  Analysis of Variance  AUC -^~  Area under the plasma concentration vs. time curve from zero to infinity  AUCCSFO-^-  Area under the C S F concentration vs. time curve from zero to infinity  AUCECFO^-  Area under the ECF concentration vs. time curve from zero  0  to infinity AUMCo^-  Area under the first moment curve  BBB  Blood-brain barrier  bpm  Beats per minute  °C  Degree Celsius  CCSF  Drug concentration in the CSF  CCSFSS  Steady-state drug concentration in the CSF  CECF  Drug concentration in the ECF  CECFSS  Steady-state drug concentration in the ECF  XV  CIcSF  CSF clearance of the drug  CIECF  ECF clearance of the drug  CI  Total body clearance of the drug  T  CNS  Central Nervous System  c  Total plasma drug concentration  P  Cp  u  Free plasma drug (unbound) concentration  Cpss  Steady-state concentration of total drug in plasma  Cpuss  Steady-state concentration of free (unbound) drug in plasma  CSF  Cerebrospinal Fluid  d  day  Da  Dalton  DPHM  Diphenhydramine  [DPHM]dialysate  Diphenhydramine concentration in the output dialysate  ECF  Extracellular Fluid  fcSF  Ratio of A U C C S F O - - to A U C ^ » {i.e. A U C F O - > ~ / A U C ^ - )  ^ECF  Ratio of A U C E C F O — to A U C _ - (i.e. A U C E C F O — / A U C - - )  g  Gram  GC  Gas Chromatography  H  Hour  0  0  [ H ]-DPHM  Deuterium-labeled DPHM  i.d.  Internal diameter  i.v.  Intravenous  kg  Kilogram  2  10  C S  0  0  xvi LOQ  Limit of quantitation  M  Molar (moles/liter)  MD  Microdialysis  mg  Milligram  min  Minute  mL  Milliliter  mmHg  Millimeter mercury  MS  Mass pectrometry  n  Number of subjects or animals  ng  Nanogram  pH  Negative logarithm of hydrogen ion concentration  PRN  Propranolol  RD o  The dose required to achieve 50% of R  Rmax  The maximal C SFSS/CP SS or CECFSS/CP  r  Coefficient of determination  5  2  C  s.c.  Subcutaneous  S.D.  Standard eviation  t  Time  t  1/2  U  m a x  uss  ratio  Half life  t a  Distributional half life for plasma in a 2-compartment model  ti/2p  Terminal elimination half life for plasma in a 2-compartment model  ti/2acsF  Distributional half life for CSF in a 2-compartment model  1/2  XVII  ti/ pcsF  Terminal elimination half life for CSF in a 2-compartment model  ti/2aECF  Distributional half life for ECF in a 2-compartment model  ti/2pECF  Terminal elimination half life for ECF in a 2-compartment model  Vd  Apparent volume of distribution  2  Vd xg  ss  Apparent steady-state volume of distribution of the total drug Times gravity (centrifugal force)  XVlll  Acknowledgments I would like to take this opportunity to thank a number of individuals. First of all, I would like to thank my research supervisors, Drs. K. Wayne Riggs and Dan W. Rurak for their support and guidance throughout my graduate training. In addition, Dr. Riggs has been an excellent mentor and Dr. Rurak's outstanding surgical skills made my microdialysis experiments possible. Also, I would like to thank members of my research committee, Drs. F.S. Abbott, MHH. Ensom, K.M. McErlane, and T. Oberlander, for their valuable time and thoughtful suggestions. Lab members including Mr. Caly Chien and Mr. Eddie Kwan made my training experience special and interesting. I will always remember the time we spent together inside and outside our lab. Ms. Nancy Gruber should be acknowledged for her assistance with sheep studies. Also, thanks to Drs. S. Kumar and H. Wong for their friendship and help. Finally, I would like to acknowledge the financial support received from PMAC/MRC, Canadian Institute of Health Research (CIHR), and the University of British Columbia during the course of my graduate studies. These studies were funded by the CIHR. This thesis is dedicated to my family: my mom and dad, and my brother Tom. Without your love, patience, and encouragement it would have been impossible for me to complete this very demanding and challenging project.  1  Chapter 1 Introduction  The period of development extending from the fetus to the adult is a time of rapid physiological and anatomical changes that can profoundly affect drug disposition. Therefore, caution should be exercised for drug use during pre- and postnatal development due to our poor understanding of age-related changes in drug response and pharmacokinetics (Piper et al., 1987). Of all the issues related to xenobiotic use in the developing fetus and newborn, the effect of drugs on CNS development probably deserves the most attention since exposure to exogenous substances during this dynamic period of neurological growth can potentially cause deleterious  results on the developing brain (Patrick et al., 1985).  Considering the fact that most drugs are lipophilic in nature, these compounds have the ability to cross biological membranes including the blood-brain barrier (BBB).  As a matter of fact, there are studies suggesting alteration of brain  function and changes in behavior in the fetus after exposure to drugs during gestation (McLeod et al., 1983; Rurak et al., 1988). While information on the general processes of maturation and disposition of endogenous substances in the developing brain is available (Saunders, 1977; Dziegielewska et al., 1980b; Adinolfi, 1985; Mollgard and Saunders, 1986; Vannucci et al., 1998; Saunders et al., 1999b; Saunders et al., 2000; Vannucci and Vannucci, 2000; Dziegielewska et al., 2001), no study has been published to date involving direct measurement of drug concentrations in the fetal and newborn brain to allow estimation of the  2  extent of drug disposition. The reason for the lack of such critical information is two-fold.  Firstly, technologically  it has been  impossible to obtain  drug  concentrations in the brain without tissue removal which involves sacrifice of the animal.  This limits the collection of drug concentration vs. time data, unless  identical animals are sacrificed at different time points. Recently, however, the development of the in vivo microdialysis (MD) technique allows the investigator to have access to the CNS compartments without sacrificing the animal, thereby allowing for serial determination of drug concentrations in brain tissue. However, the procedure of probe insertion requires considerable surgical skill and experience in order to successfully locate and implant the MD probes reliably. Secondly, ethical considerations have prevented such studies from being conducted in human fetuses and neonates. On the other hand, such studies are very difficult, if not impossible, to perform in common animal models (i.e. rats, guinea pigs, rabbits) due to the small size of fetuses and newborns from these species.  Thus, there is a need for studies that systemically investigate the  changes that occur in CNS drug disposition from a developmental point of view. This thesis is an investigation of the CNS pharmacokinetics of diphenhydramine (DPHM) in fetal, newborn, and adult sheep.  In the following sections, I will  summarize relevant information about the function and physiology of the BBB in fetus and adult, the pharmacokinetics of my study compound, DPHM, and also, the in vivo microdialysis (MD) technique.  It is hoped that the information  generated from these studies will provide a better understanding of the  3  processes of brain xenobiotic disposition and help to devise safe drug therapies especially to pregnant women and neonates.  1.1 The Blood Brain Barrier (BBB)  Ehrlich and Goldman (Ehrlich, 1885; Goldman, 1909) were the first to observe the existence of the BBB. They observed that when the hydrophilic compound trypan blue was injected systemically in a rat, it did not distribute into and out of the brain.  It is now known that the BBB is composed of several specialized  elements which act together to regulate the internal milieu of the brain (Smith, 1989; Farrell and Risau, 1994; Davson and Segal, 1996), by controlling the exchange of compounds between blood and brain ECF (blood/brain barrier), and blood and CSF (blood/CSF barrier, Figure 1.1). For the blood/brain barrier, the most important elements are the brain capillary endothelial cells (Figure 1.2a), which possess several unique characteristics. These include highly complex, tight junctions, a high electrical resistance, few pinocytotic vesicles, a continuous basal lamina, specific transporters for the influx or efflux of various compounds and a close association with surrounding pericytes and astrocytes (Balabanov and Dore-Duffy, 1998; Rubin and Staddon, 1999; Saunders et al., 1999a). The latter cells have cellular extensions (club feet) that are tightly apposed to the abluminal surface of the brain capillaries (Rubin and Staddon, 1999; Saunders et al., 1999a). This feature may be, in part, responsible for the some of the other unique characteristics of CNS endothelial cells, since co-culture of non-neural  4  endothelial cells with astrocytes confers BBB characteristics upon them when the 2 cell types are closely apposed (Hayashi et al., 1997; Rubin and Staddon, 1999). For the blood/CSF barriers (Figure 1.2b,c), the most important elements are the tight junctions between the epithelial cells of the choroid plexus and the arachnoid membrane (Saunders et al., 1999a; Taylor, 2002). There are several areas of the brain that effectively lie outside of the BBB. These are termed the circumventricular organs and comprise structures (median eminence, pineal gland,  subfornical  organ,  subcommissural  organ,  area  postrema,  neurohypophysis, choroid plexus) which are close to the ventricular system of the brain, particularly the third ventricle (Davson and Segal, 1996). Because their capillaries lack tight junctions, they can receive solutes from blood and can release their secretory products into the blood.  5  Cerebral arterial and venous blood  Protein binding, cerebral blood flow, effective capillary perfusion, physicochemical properties of the drug, passive arid active transport Metabolism  Blood-brain barrier  Blood CSF barrier  Extracellular compartments of the brain  Brain ventricular CSF  Intracellular compartments of the brain  Intra-extracellular exchange  Diffusion •4  Metabolism  CSF turnover  Ii Diffusion and flow Metabolism  Metabolism  IT 'Metabolism Lumbar spinal CSF  Figure 1.1 Generalized physiological compartment model of the CNS showing factors (in italics) that govern the distribution of drugs among the different compartments (de Lange and Danhof 2002).  6  Blood Fenestrated capillary endothelial cells ifc^jSK.? ^naasa^. fegsig^ (•>'•;.•?,  Blood  CSF j§ Capillary endothelial cells E§||  Tight junction  I ECF  Choroid plexus epithelial cells Tight junction - Ependyma  Lateral ventricle Third ventricle Fourth ventricle Central canal Arachnoid cells Tight junction  C3F I* s  Figure 1.2  Pial membrane  Sites of the BBB. The BBB comprises tight junctions between: (a)  the endothelial cells of brain capillaries; (b) epithelial cells of the choroid plexus; and (c) epithelial cells of the arachnoid membrane. The arrows depict the flow of CSF, which originates from the choroid plexus (Kandel et al., 2000).  7  1.1.1  Influx Transporters  The tight junctions in the brain capillary endothelial cells and in the choroid plexus epithelial cells primarily restrict the entry of proteins and other large, hydrophilic molecules into the CNS (Davson and Segal, 1996; Habgood et al., 2000). For these substances, BBB permeability is determined by molecular size. For lipophilic compounds, not significantly bound to plasma proteins, there is a good correlation between the BBB permeability coefficient and the octanol/water partition coefficient, provided the molecular weight is <400-600 Da (Levin, 1980). However, numerous transporters are present in brain endothelial cells, which transfer nutrient molecules and some drugs into the CNS at rates higher than could occur via simple diffusion. These include a glucose transporter (Glut-1 isoform) for facilitated transfer of glucose into the CNS (Boado and Pardridge, 1990; Pardridge et al., 1990; Bauer, 1998; El Messari et al., 2002; Mann et al., 2003); it may also assist the entry of glycoside-conjugated drugs such as Lserinyl-B-D-glycoside  analogues  of Met-5-enkaphalin  (Polt  et al., 1994).  Monocarboxylic acid transporters (MCT) carry endogenous short and medium chain monocarboxylic acids (Oldendorf, 1973; Spector, 1988a; Tamai and Tsuji, 2000; Pierre et al., 2002; Taylor, 2002) and may also transport xenobiotics such as salicylic acid (Terasaki et al., 1991), simvastatin acid (Tsuji et al., 1993) and valproic acid across the BBB. Sodium independent organic cation transporters (OCT) have also been identified in the BBB, which transport endogenous compounds such as choline and thiamine (Koepsell, 1998; Wu et al., 1998; Wu  8 et al., 2000; Kido et al., 2001; Lee et al., 2001; Sweet et al., 2001; Slitt et al., 2002) . There is also evidence for saturable transporter mechanisms in the BBB for  a number  amphetamine,  of lipophilic rimantadine,  amine  drugs  amantidine  including  propranolol,  and the histamine  lidocaine,  Hi-antagonist,  mepyramine (Pardridge and Connor, 1973; Pardridge et al., 1984; Spector, 1988b; Yamazaki et al., 1994a; Yamazaki et al., 1994b; Yamazaki et al., 1994c). A number of amino acid transporters have been identified in the membrane components of the BBB (Smith and Stoll, 1998; Mann et al., 2003; Sakai et al., 2003) .  Neutral amino acid transporters may be involved in the facilitated  transport of amphoteric drugs such as baclofen and gabapentin (Pardridge, 1995; Segawa et al., 1999).  1.1.2 Efflux Transporters  While the transporters mentioned above appear to mainly transport compounds from blood to brain/CSF, they may also be involved in efflux of solutes from the brain. However, the most conclusive evidence for this latter action exists for Pglycoprotein (Pgp), a product of the multi-drug resistance (mdr) gene family, which actively pumps a wide range of structurally unrelated, hydrophobic, amphiphatic compounds from the brain endothelial cells into the capillary lumen (Cordon-Cardo et al., 1989; Tatsuta et al., 1992; Schinkel et al., 1996; van Asperen et al., 1997; Tsuji and Tamai, 1998; Kusuhara and Sugiyama, 2001; Furuno et al., 2002; Seegers et al., 2002). Indeed, it appears that this action is at  9 least in part responsible for the 400-600 Da cutoff for brain uptake of lipophilic compounds mentioned above, since many hydrophobic molecules of this size are substrates  for  domperidone  Pgp,  for  example  vincristine,  etoposide,  loperamide  (van Asperen et al., 1997). Recently, in vivo  and  microdialysis  experiments in rats demonstrated that overexpression of Pgp in epileptic cerebral tissue was likely to limit brain access of anti-epileptics such as phenobarbital, lamotrigine, and felbamate (Potschka et al., 2002).  In addition, Pgp and the  multidrug resistance-associated protein (MRP) were identified in blood-brain and blood-CSF barriers (Rao et al., 1999; Ayrton and Morgan, 2001; Kusuhara and Sugiyama, 2002; Wijnholds, 2002). MRP is located on the basal cell surface and thereby restricts the entry of compounds into the CSF (i.e. by pumping them out). Furthermore, organic ion transporters such as organic anion  transporting  polypeptide 2 (oatp2) (Gao et al., 1999; Asaba et al., 2000; Gao et al., 2000; Ayrton and Morgan, 2001; Kusuhara and Sugiyama, 2001; Kusuhara and Sugiyama, 2002), are expressed in both brain capillary endothelial and choroid plexus epithelial cells. In general, they accept amphipathic organic anions such as estradiol-17(3-glucuronide as substrates (Kusuhara and Sugiyama, 2001; Kusuhara and Sugiyama, 2002; van Montfoort et al., 2002). There is also the monocarboxylic transporter (MCT) family which regulates the uptake and efflux of lactic acid, ketone bodies, and other monocarboxylic acid compounds such as acetic acid and salicyclic acid in the brain (Takanaga et al., 1995; Koehler-Stec et al., 1998; Tamai and Tsuji, 2000; Pierre et al., 2002).  A few drugs have been  shown to be actively transported from the brain ECF to the circulating blood via  10 the transporter-mediated efflux system at the BBB.  These drugs include  baclofen (Deguchi et al., 1995), 6-mercaptopurine (Deguchi et al., 2000) and probenecid (Deguchi et al., 1997).  1.1.3  Cerebral Metabolism  The influx of xenobiotics can be secondary to metabolic enzyme activities in the cerebral blood vessels and their associated astrocytes. Phase I enzymes such as cytochrome P450s and flavin-containing monooxygenase, and phase II enzymes such as UDP-glucuronosyltransferases and glutathione S-transferases have been detected in these regions (Ghersi-Egea et al., 1995).  The overall  metabolic capacity of these enzymes in the brain collectively is low, but there are regional variations in terms of enzyme activities. Thus, there may be regionspecific uptake/metabolism of drugs. For example, in rat choroid plexus, UDPglucuronosyltransferase and epoxide hydrolase weight-normalized activities are as high as in the liver (Ghersi-Egea et al., 1995).  Like the hepatic enzymes,  these choroidal enzymes appear to be inducible (Leininger-Muller et al., 1994).  1.1.4  Efflux Pathways  After crossing the BBB or the blood-CSF barrier into brain extracellular fluid or CSF, compounds will eventually exit the CNS. There are two possible paths from the CNS back to the systemic circulation. One comprises efflux (transporter-  11 mediated or not) via brain or choroidal blood. The second route involves efflux  via bulk flow of CSF draining into either the lymphatic system or venous blood through the arachnoid villi in the superior sagittal sinus (Figure 1.2) (Bradbury et al., 1972; Kandel et al., 2000). The latter phenomenon is responsible for what is termed the sink effect, whereby the brain concentrations of various compounds under steady-state conditions are different from each other and lower than the unbound concentration in blood (Davson and Segal, 1996), as a consequence of the continuous removal of the substances via the CSF.  1.1.5 Development of the BBB  Morphologic and functional studies of the development of the BBB have been conducted in a number of species including the mouse, rat, gerbil, rabbit, sheep, rhesus monkey and human (Bradbury et al., 1972; Amtorp and Sorensen, 1974; Saunders and Mollgard, 1984; Mollgard and Saunders,  1986; Risau and  Wolburg, 1990; Farrell and Risau, 1994; Saunders and Dzielgielewska, 1998; Saunders et al., 1999b). Although there has been some debate about the degree of maturity/immaturity of the BBB in the fetus (Risau and Wolburg, 1990; Saunders et al., 1999b), the differences found in various studies have been explained on the basis of variations in experimental approach, particularly in terms of the volumes of fluid that were administered (Saunders et al., 1999b). Taking this into account, BBB development seems similar in all of the species studied to date. However, as with other aspects of brain growth and maturation, such as the brain growth spurt (Dobbing and Sands, 1979) and the development  12 of sleep states (Jouvet-Mounier et al., 1970; Romijn et al., 1991), the timing of this process in relation to parturition varies. In precocial species [i.e. those born relatively mature, e.g. guinea pig, sheep, human, monkey), BBB development appears to be largely completed prior to birth (Saunders et al., 1999b). In altricial species (i.e. those born relatively immature, e.g. mouse, rat), BBB development and maturation start later in gestation and after birth (Jouvet-Mounier et al., 1970). As the current thesis deals with sheep, the following discussion will focus on this species and on the human.  In both the sheep and human, the key element of the BBB, i.e. tight junctions on capillary endothelial and choroid plexus epithelial cells, are present very early in gestation.  Using  transmission  electron  microscopy  and  freeze  fracture  techniques, Saunders and colleagues (Saunders and Mollgard, 1984; Mollgard and Saunders, 1986) have noted tight junctions in brain capillary endothelial and choroid plexus epithelial cells in the sheep embryo as early as 22 days gestation (0.15 of term, term - 1 4 5 d,), and in the human embryo at 7 weeks (0.18 of term). More recently, immunohistochemistry  and electron microscopy  have been  employed to examine BBB development in the telencephalon cortical plate in 12 and 18 week fetuses (Bertossi et al., 1999). There were junctional complexes in the endothelial cells at both ages, as well as laminin, a component of basal lamina, adjacent to the cells. There were also immature astroglial  cells  surrounding the developing vessels. Thus, several important features of the adult BBB are present in early gestation. In anesthetized sheep, there are also  13 estimates of blood-brain and blood-CSF permeabilities to hydrophilic compounds from ~50 days gestation to adulthood (Evans et al., 1972; Dziegielewska et al., 1979). At all ages, permeability decreases as molecular radius increases. There is also a fall in permeability with development. The largest decreases occur between 50 and - 6 5 d gestation, but there are further decreases from 65 d to term, particularly for the blood-CSF barrier. These large changes in permeability are not accompanied by noticeable changes in tight junction  morphology  (Millgard et al., 1976; Dziegielewska et al., 1979; Saunders and Mollgard, 1984). The decrease in permeability may be due to a decrease in the number of transcellular pores, rather than a change in pore diameter (Dziegielewska et al., 1979).  In terms of CSF dynamics, the rate of CSF secretion in fetal lambs decreases from 6.5 Lil/min/g at 59 d gestation to 1.5 ul/min/g at term, with little change thereafter (Evans et al., 1972). The largest decrease occurred between 59 and 91 d gestation. This research by Evans et al. involved acute study of anesthetized fetuses, and it is unclear to what extent this would have affected the measurements. However, perhaps the most striking feature of CSF during fetal life in all species examined to date is the very high protein concentrations present in CSF in early gestation (Dziegielewska et al., 1980b; Bell et al., 1991; Saunders and Dzielgielewska, 1998; Saunders et al., 1999b). This phenomenon results from 2 factors: specific mechanisms in the choroid plexus that transport proteins from blood to CSF (Dziegielewska et al., 1980b; Dziegielewska et al., 1991) and  14  a unique type of intercellular junction, termed strap junctions, between the cells of the neuorependma. These appear to restrict the movement of proteins in the CSF into brain tissue (Fossan et al., 1985). In the fetal lamb, 5 proteins account for the high concentrations in CSF: albumin, fetuin, a-feto-protein, transferrin and ai-antitrypsin, and the concentrations are highest in the youngest fetuses examined, i.e. at 30 d gestation (Cavanagh et al., 1983). Thereafter, the protein concentrations decline progressively until term and this is accompanied by loss of the strap junctions and an increase in CSF-brain permeability. Although it has been suggested that the high CSF protein levels in early gestation may play some role in neural development (Saunders et al., 1999b), definitive information and evidence are lacking.  Stonestreet et al. conducted a series of studies examining various aspects of BBB development in conscious, chronically-instrumented sheep from fetal to adult age (Stonestreet et al., 1996; Stonestreet et al., 1999; Stonestreet et al., 2000a; Stonestreet et al., 2000b; Sysyn et al., 2001; Stonestreet et al., 2002). Briefly, by injecting an inert hydrophilic amino acid, a-aminoisobutyric acid (AIB), the investigators observed ontogenic decreases in BBB permeability in ovine fetuses from 60% of gestation to maturity in the adult (Stonestreet et al., 1996). Consistent with this finding were similar postnatal reductions in permeability to AIB in rabbits (Tuor et al., 1992), and to inulin, sucrose, mannitol, urea, and potassium in rats (Parandoosh and Johanson, 1982; Smith et al., 1982; Fuglsang et al., 1986; Keep et al., 1995; Stander et al., 2002).  Taken together, the  15 findings from various species indicated that barrier permeability decreased to both cation and hydrophilic molecules from fetal to adult life. Also, because the BBB was relatively impermeable to AIB even at 60% gestation, it suggested that the functional barrier forms very early in gestation and serves to protect developing neurons from adverse systemic factors (Stonestreet et al., 1996). Moreover, in another study where fetal blood pressure was increased 40% during infusions of dopamine, permeability to AIB was not substantially increased, even by 60% gestation (Harris et al., 2001). Therefore, even at early gestation the BBB of fetal sheep is sufficiently robust to withstand hypertension induced by dopamine.  In another study, Stonestreet et al. found that endogenous increases in Cortisol concentrations were, in part, responsible for the ontogenic decreases in BBB permeability that occur during normal ovine fetal life (Stonestreet et al., 2000a). In other words, the pituitary-adrenal-cortical axis appears to modulate BBB permeability during development. The increases of plasma Cortisol concentration from late gestation to after birth are most likely responsible for the normal ontogenic decreases in barrier permeability that occur during fetal and neonatal maturation (Stonestreet et al., 1996).  However, maternal treatment with  exogenous corticosteroids (i.e. dexamethasone) was associated with decreases in BBB permeability at 60 and 80%, but not 90% of gestation (Stonestreet et al., 2000b).  The  corticosteroids  fact  reduced  that  maternally-administered  barrier  permeability  early  exogenous but  not  late  antenatal in  fetal  16  development and that increases in endogenous plasma Cortisol concentrations were associated with decreases in regional BBB permeability during fetal development implies that there is an age-related differential responsiveness of the fetal  BBB to exogenous  corticosteroids  (Stonestreet  et  al., 2000b).  Therefore, it appears that, because of exposure to elevations in endogenous increases  in  corticosteroids,  the  corticosteroids (Sysyn et al., 2001).  BBB  is  not  responsive  to  exogenous  Corticosteroids might cause reductions in  barrier permeability by accelerating vascular maturation (Stonestreet et al., 1999); however, the exact mechanisms require further investigation.  On the other hand, brain water regulation is impaired in most brain regions of ovine fetuses at 60 and 90% of gestation and premature lambs (Stonestreet et al., 2002).  Brain water regulation develops first in the cerebral cortex of the  fetuses at 90% of gestation and in the cerebral cortex and cerebellum of newborn lambs, and then in the medulla of the lambs at 20-30 days of age (Stonestreet et al., 2002). Brain volume regulation is present in the brain regions of young lambs and adult sheep (Stonestreet et al., 2002), indicating that the ability of the brain to regulate its volume develops in a region- and age-related fashion.  Possible damage to the BBB in newborns by artificial ventilation have also been investigated (Stonestreet et al., 2000a). Positive-pressure ventilation can cause disruption of the BBB in premature lambs by increasing microvascular venous pressures (Stonestreet et al., 2000a).  Therefore, ventilation may place the  17  premature infant at risk for intraventricular hemorrhage because of endothelial rupture as a result of increases in cerebral microvascular venous pressures.  There is very limited information on brain transporters during fetal and postnatal development, with the exception of glucose transporters, for which there are data from several species (Bauer, 1998), including the human (Bertossi et al., 1999) and sheep (Das et al., 1999; Anderson et al., 2001). Also, a sodium-dependent amino acid transporter (ASCT1) that transports primarily zwitterionic amino acids at physiological pH in the developing brain was detectable as early as 14 days of gestation in rats (Weiss et al., 2001).  However, as glucose and amino acid  transporters are not involved in the current thesis, they will not be discussed further. Data on other transporters are very limited. In the human, Pgp has been detected immunohistochemically in brain microvessel endothelial cells by 8 weeks gestation (0.2 of term) (Schumacher and Mollgard, 1997). In fetal lambs at 132-139 days gestation, administration of probenecid (an organic anion transporter inhibitor) increases the CSF concentration of kynurenic acid, an endogenous antagonist of excitatory amino acids, which is synthesized primarily by astrocytes (Walker et al., 1999). This suggests the presence of an outwardly directed, probenecid-sensitive organic anion transporter in the fetal lamb brain, at least in late gestation.  However, information on the developmental aspects of  this transporter is lacking.  18 1.1.6 Implications of Development on Fetal Brain Exposure  Although there is considerable information on developmental changes in BBB permeability to lipid insoluble compounds, there are very limited data on lipophilic molecules, and most drugs are lipophilic in nature. This is also the case for the BBB transporters that are responsible for the brain influx/efflux of many drugs. However, several of the known features of blood-brain and blood-CSF barrier development likely will impact on pharmacokinetics in the CNS. One is the fall in CSF formation rate during gestation, which results in a decrease in the CSF sink effect of removing drugs from the CNS. However, this factor would be most significant in early gestation. A second factor, likely to be important throughout gestation, is the progressive fall in CSF protein concentration. Since fetal plasma protein concentration gradually increases during gestation (Dziegielewska et al., 1980b; Kwan et al., 1995), the reciprocal changes in CSF and plasma protein concentrations could potentially affect the relative ratio of free fraction of xenobiotics in both compartments and the drug clearance between the two compartments. A final feature that could affect CNS drug levels in late gestation and the newborn is age-related alterations in cerebral blood flow (Richardson et al., 1989; Bendeck and Langille, 1992). Brain blood flow in the fetal lamb increases by 137% between 120 and 140 d of pregnancy, then declines by 73% in the newborn, followed by a doubling of flow over the next 3 weeks. For drugs that are readily taken up by the brain or are extensively bound to plasma proteins, these alterations in brain perfusion could result in altered brain drug  19 levels via changes in the rate of drug delivery to the brain or in the capillary transit time.  1.2  Diphenhydramine  Diphenhydramine  [2-(diphenylmethoxy)-N,N-dimethylethylamine,  DPHM  (MW  255)] is a potent, reversible histamine H-i-receptor antagonist of the ethanolamine class (Garrison, 1991). It is a weak base with a pKa of 9.0; therefore, at physiological pH (i.e., pH 7.4) the drug is almost completely ionized.  DPHM  (Figure 1.3) is also highly lipophilic with an octanol/water partition coefficient 1862. It is marketed in Canada as a single entity product (Benadryl® or Nytol®) or combined with other drugs in cough and cold preparations.  Figure 1.3 Chemical Structure of Diphenhydramine (DPHM)  20  1.2.1 Pharmacology, Clinical Applications, Adverse Effects  DPHM binds reversibly and competitively to both peripheral and central H i receptors with little or no binding to H -receptors (Garrison, 1991). As a result, it 2  inhibits many of the pharmacological actions of histamine at these receptors. This  includes  histamine-stimulated  smooth  muscle  contraction,  increased  capillary permeability, itch, edema, wheal, and flare formation (Garrison, 1991). Therefore, DPHM is used in the treatment of various allergy-mediated diseases. It is effective in the symptomatic relief of urticaria, hay fever, seasonal rhinitis, and  conjunctivitis,  and allergic  dermatoses  (Moscati  and Moore,  1990).  Furthermore, DPHM has been used as a hypnotic, and for the management of post-operative-, pregnancy-, and cancer chemotherapy-related  nausea and  vomiting and motion sickness (Grunberg et al., 1988; Roila et al., 1989; Garrison, 1991). In addition to the H-i-receptor antagonist activities, DPHM also possesses atropine-like anticholinergic, antitussive activities (Garrison, 1991; Packman et al., 1991). DPHM has also been used as a local anesthetic agent for the repair of minor lacerations and wounds (Ernst et al., 1993; Ernst et al., 1994). Common adverse effects resulting from DPHM administration include dizziness, nervousness, drowsiness and fatigue. However, at high or toxic doses [i.e. > 6 times the normal therapeutic dose), DPHM can provoke CNS stimulation, convulsions, cardiovascular and pulmonary collapse (Koppel et al., 1987; Garrison, 1991).  One study reported that prenatal DPHM exposure was  associated with joint weakness in rat fetuses; however, the relevance of this finding in humans is unknown (Sturman et al., 2002).  21 1.2.2  DPHM Use in Pregnancy  While the use of drugs during pregnancy is generally discouraged, certain circumstances arise where drug treatment becomes necessary (Berkovitch et al., 2002; Levichek et al., 2002). According to drug use surveys reported in different studies, the percentage of women who take at least one drug during pregnancy varies between 40 and 90% (Olesen et al., 1999; Donati et al., 2000). A wide overview of epidemiological drug utilization studies in 1990 found a median of 4.7 drugs per woman with a range of 2.9 - 5.5 in comparable studies (Bonati et al., 1990). Therefore, the use of medications during pregnancy is not uncommon. DPHM is used in pregnancy for conditions such as pregnancy-related urticaria, nausea and vomiting, insomnia, cough and colds, and allergy (Piper et al., 1987). Nausea and vomiting are common symptoms experienced in early pregnancy, with nausea affecting between 70 and 85% of women, and about half of pregnant women experience vomiting (Jewell and Young, 2002).  It follows that Hi-  antagonists (i.e. DPHM) are one of the most commonly used medications among the treatments available (Jewell, 2002; Magee et al., 2002; Schatz, 2002). These findings suggest that a significant number of human fetuses may be exposed to this drug at some time during their gestation. Also, secretion into breast milk represents a potential route of neonatal exposure to DPHM (Dostal and Schwetz, 1989; Simons et al., 1990; Vener et al., 1996; Schatz, 2002).  22  1.3 Pharmacokinetics of DPHM  1.3.1  Absorption  DPHM is rapidly absorbed in humans following oral administration, with maximal plasma concentrations attained between 1-4 hours following administration (Carruthers et al., 1978; Blyden et al., 1986; Luna et al., 1989; Simons et al., 1990). Following the administration of a 50 mg therapeutic dose, peak plasma concentrations between 40-100 ng/mL are achieved in healthy human adults (Carruthers et al., 1978; Luna et al., 1989). The drug undergoes a substantial first-pass effect following oral administration in humans, with bioavailability between 0.43 to 0.78 (Albert et al., 1975; Carruthers et al., 1978; Spector et al., 1980; Blyden et al., 1986).  1.3.2  Distribution  Plasma protein binding in humans has been reported to be ~ 78-85% (Spector et al., 1980; Meredith et al., 1984; Zhou et al., 1990). Ethnic differences in DPHM protein binding have also been demonstrated between Orientals and Caucasians (i.e., binding was 76% in Caucasians and 85% in Orientals) (Spector et al., 1980; Zhou et al., 1990).  Since binding of DPHM to human serum albumin is only  - 3 0 % (Drach et al., 1970), it is likely that other plasma proteins, such as ctracid glycoprotein which is known to bind basic drugs, may also play a role (Kremer et  23  al., 1988; Zhou et al., 1990).  The apparent volume of distribution of DPHM  ranges from -3-7 L/kg suggesting extensive distribution to tissues (Carruthers et al., 1978; Spector et al., 1980; Berlinger et al., 1982; Blyden et al., 1986). The tissue  distribution  pattern  following  oral,  intraperitoneal,  and  intravenous  administration to either rats or guinea pigs showed that the highest tissue drug concentrations were in the lung, followed by the spleen, brain, liver, muscle, and heart (Glazko et al., 1974b). The high tissue distribution of DPHM in the lung may be due to the high binding of this drug to monoamine oxidases, as demonstrated in perfused rat lung and in isolated rat lung mitochondria (Yoshida et al., 1989; Yoshida et al., 1990). Not surprisingly, in a case of fatal overdosage of DPHM in a human patient, the concentrations were found to be the highest in lung tissue (Hausmann et al., 1983).  1.3.3  Metabolism  In rat,  guinea  pig, and  rabbit,  DPHM  is extensively  degraded  in  liver  homogenates, and to a lesser extent in lung and kidney homogenates (Glazko et al., 1974b).  The large first-pass effect following oral administration and the  reduced clearance of DPHM in patients with chronic liver disease suggest that hepatic elimination of DPHM is an important route of elimination in humans (Albert et al., 1975; Carruthers et al., 1978; Meredith et al., 1984; Blyden et al., 1986).  High hepatic DPHM extraction (-95%) was also observed in sheep, in  addition to the significant contribution by gut (-50%) in DPHM  systemic  24  clearance (Kumar et al., 1999b).  In humans, rhesus monkeys, and dogs, the  metabolites identified in vivo suggest that DPHM undergoes successive Ndemethylations to give N-demethyl DPHM and N.N-didemethyl DPHM, followed by deamination to yield diphenylmethoxyacetic acid (Drach et al., 1970; Chang et al., 1974; Glazko et al., 1974a). Diphenylmethoxyacetic acid (DPMA) has been demonstrated to form glycine conjugates in dogs and glutamate conjugates in rhesus monkeys (Drach et al., 1970). DPMA and its conjugates are the major urinary metabolites in dogs (-42%), and rhesus monkeys (-60%) (Drach et al., 1970). In addition, significant quantities (-10-20%) of the N-oxide metabolite of DPHM were found in the urine in all species examined to date (Drach et al., 1970; Chang et al., 1974).  Recently, DPHM was shown to form a quaternary  ammonium glucuronide conjugate in human urine that may account for 2-15% of the administered dose (Luo et al., 1991; Breyer-Pfaff et al., 1997; Fischer and Breyer-Pfaff, 1997).  Evidence also suggests that this quaternary ammonium  glucuronide can be formed in human liver microsomes (Breyer-Pfaff et al., 1997). The in vivo  metabolism of DPHM in the rat has not yet been completely  established, and many of the metabolites in this species remain to be identified (Drach et al., 1970). In fact, it is still unclear which enzymes are responsible for the metabolism of DPHM. orphenadrine)  form  DPHM and some closely related analogs (i.e.  metabolic-intermediate  complexes  with  the  rat  liver  cytochrome P450 2B1 and 2C6 isoforms (Rekka et al., 1989; Bast et al., 1990). The formation of these complexes may inhibit the metabolism, and thus, the elimination of other drugs by these P450 isoforms.  Consistent with this  25  hypothesis is the observation that DPHM inhibits the clearance of diltiazem in isolated perfused rat liver (Hussain et al., 1994). Furthermore, DPHM has been observed to bind to monoamine oxidases in rat lung (Yoshida et al., 1989; Yoshida et al., 1990). The exact role of these enzymes to DPHM disposition is not known.  1.3.4  Excretion  DPHM is primarily excreted as metabolites in urine accounting for ~35% of the dose in rats, and - 4 9 % in humans (Drach et al., 1970; Glazko et al., 1974b). However, the role of biliary excretion of DPHM and/or its metabolites has not yet been reported in either laboratory animals or humans. In contrast, only a small fraction of the dose administered is excreted as intact drug in the urine of rats (-4-6%), rabbits (< 3%), and monkeys (-3%) (Drach et al., 1970; Glazko et al., 1974a).  Similarly, only a small portion (2-4%) of intact DPHM is excreted in  humans (Albert et al., 1975; Meredith et al., 1984). DPHM systemic clearance in humans ranges between 6-15 mL/min/kg with terminal elimination half-lives ranging from 3-9 h (Carruthers et al., 1978; Meredith et al., 1984; Blyden et al., 1986; Spector, 1988b).  Adult oral clearance of DPHM (20-30 mL/min/kg) is  substantially higher than systemic total body clearance (Luna et al., 1989; Simons et al., 1990; Scavone et al., 1998). This is a result of the substantial firstpass effect of the compound. Finally, DPHM clearance exhibits age-dependence being highest in children (-2 fold higher than adults) (Simons et al., 1990).  26 1.4 Disposition and Fetal Effects of DPHM in Pregnant Sheep  Numerous  studies  have been  previously conducted  in our laboratory to  investigate the disposition of DPHM in chronically instrumented pregnant sheep (Yoo et al., 1986b; Yoo et al., 1993; Tonn et al., 1996; Kumar et al., 1997; Wong et al., 2000).  Yoo et al. (Yoo et al., 1986a) demonstrated that DPHM undergoes  rapid placental transfer to the fetal lamb, with maximum fetal plasma levels occurring within 5 minutes following maternal i.v. bolus administration.  There  was also extensive fetal exposure to the drug following maternal i.v. bolus administration (i.e. AUC fetal/AUC maternal = 0.85). Time-separated fetal and maternal infusions to steady-state, utilizing a two-compartment open model (Szeto, 1982), demonstrated that both mother and fetus can eliminate DPHM via placental and non-placental pathways (Yoo et al., 1993; Kumar et al., 1999a). In the mother, non-placental clearance (Cl ) contributed ~95% of total maternal mo  clearance (Kumar et al., 1999a).  However, fetal non-placental clearance (Cl ) f0  accounted for only - 4 0 % of total fetal clearance (Kumar et al., 1999a).  We  also observed that the weight-corrected Clf was about three fold higher than that 0  observed in the mother (i.e. C l ) (Kumar et al., 1997). mo  Efficient hepatic  extraction of DPHM in the maternal and fetal liver would explain our estimates of Cl  m o  and Clfo, with C l  f0  2.5 times higher than C l  m o  (Kumar et al., 1997).  In  addition, efficient fetal hepatic uptake of the drug resulted in an estimated transplacental clearance of drug from the fetal to maternal compartment (Clf ) m  approximately 4 times higher than trans-placental clearance of drug from the maternal to fetal compartment  (Cl ) (Kumar et al., 1999c). mf  Moreover, after  27  maternal dosing, a significant fraction of DPHM (~45%) transferred across the placenta and into the umbilical vein is metabolized by the fetal liver due to hepatic first-pass metabolism and does not reach the fetal circulation (Kumar et al., 1997).  More recent data suggest that gut uptake may also play a role in  DPHM clearance in adult sheep (Kumar et al., 1999b). The contribution of the gut in fetal DPHM disposition, however, remains to be assessed. Post-natally, 15 d lamb DPHM renal clearance (1.80 ± 1.24 mL/min/kg) was similar to that of the fetus (2.06 + 0.24 mL/min/kg), and significantly greater than that for the 2 month old lambs (0.26 ± 0 . 1 7 mL/min/kg) and the adult (0.012 ± 0.005 mL/min/kg) However, postnatal DPMA renal clearance (15 d = 0.02 ± 0.02 mL/min/kg; 2 month = 0.05 ± 0.01 mL/min/kg) was significantly less than adult values (0.53 ± 0.19 mL/min/kg).  Alterations in the renal clearance of DPHM and DPMA are  likely related to differences in the rate of development of mechanisms (i.e. tubular secretion and reabsorption and glomerular filtration rate) involved in the urinary excretion of organic acids and bases (Wong et al., 2000).  1.5 Behavioral Effects of DPHM and Other Drugs in the Fetus  The effects of DPHM on fetal behavioral states and breathing activity have been investigated in the fetal lamb after maternal or fetal i.v. infusions to steady-state (Rurak et al., 1988). Briefly, after maternal administration, the maternal and fetal drug concentrations averaged 212 ± 24 and 36 ± 5 ng/mL, respectively.  In  humans, the therapeutic concentration is ~ 30 ng/mL, whereas levels of 50 ng/mL or above are associated with noticeable CNS effects (Carruthers et al.,  28  1978).  During the maternal infusions, there were significant reductions in fetal  low voltage electrocortical (ECoG) activity, the amount of breathing-like activity and rapid eye movements (REM) during low voltage ECoG activity, and in the overall incidence of fetal breathing-like movements. There were no alterations in the average durations of high and low ECoG episodes (Figure 1.4a). The fetal effects observed during the maternal infusions are qualitatively similar to those elicited by CNS depressants such as pentobarbitone in terms of the changes in breathing activity and ECoG patterns (Boddy et al., 1976).  However, DPHM  infusion to the fetus resulted in markedly different effects including a fall in the amount of low voltage ECoG activity and increase in an intermediate voltage pattern (Figure 1.4b). There was a large rise in the occurrence of REM and fetal breathing during intermediate voltage ECoG activity, although no change in the overall incidence of either variable.  A significant reduction in the average  duration of low voltage ECoG episodes also occurred and reflected the disruption in the normal cycling between high and low voltage ECoG episodes resulting from the marked increase in the intermediate voltage pattern.  Furthermore, in  the initial phases of the infusion there was vigorous breathing activity and transient fetal tachycardia and hypoxemia. The maternal and fetal DPHM levels averaged 31 ± 4 and 448 ± 66 ng/mL, respectively; the fetal concentration is well above the therapeutic range in humans but much lower than the level of 5 ug/mL found in a male subject who died from a DPHM overdose (Hausmann et al., 1983).  29  •  The two routes of DPHM infusion thus resulted in different patterns of fetal behavioral  effects,  concentrations.  which  were  associated  with  markedly  different  fetal  Fetal CNS effects were observed at levels below those  associated with sedation in the human and, in addition, there was no evidence of behavioral  alterations  in the ewes  during either  maternal  or fetal  drug  administration. However, prolonged use of DPHM in pregnancy could potentially lead to the development of fetal tolerance to the drug, as is seen with ethanol, methadone and other narcotics (Umans and Szeto, 1985; Smith et al., 1989a) It follows that the development of fetal tolerance could result in withdrawal symptoms when drug administration stops, as occurs with narcotics (Umans and Szeto, 1985).  This may also be the case for DPHM,  since  Parkin et al.  reported apparent withdrawal symptoms (i.e. seizures, diarrhea, drowsiness, and restlessness) in a newborn whose mother had received a therapeutic dose of the drug daily during pregnancy for the treatment of a pruritic rash (Parkin, 1974).  Besides DPHM, fetal behavioral effects of the opiates have also been studied in pregnant sheep. There is fairly abundant information available on the effects of morphine, and similar effects have also been reported for methadone (Olsen and Dawes, 1983; Olsen et al., 1983; Szeto, 1983; Umans and Szeto, 1983; Sheldon and Toubas, 1984; Toubas et al., 1985; Bennet et al., 1986; Hasan et al., 1988; Szeto et al., 1988; Hasan et al., 1990a; Hasan et al., 1990b).  After  administration of morphine, two different patterns of fetal CNS effects were observed. One involved a reduction of both quiet and REM sleep, and a marked  30  MATERNAL INFUSIONS ECoG PATTERN  LOW HIGH aSS INTERMEDIATE  Lit  CONTROL  "MTUSION  ECoG EPISODE DURATION  C T ! TOTAL X E o G ' mm* O F L O W WITH E o C E 3 X Of" H I G H WITH E o C eZQ X O F INTERMEDIATE WITH EoC  EoG ACTIVITY  a  "5 M  4 0  20 O  POST  T  CONTROL  INFUSION  1  CONTROL  INFUSION  FETAL INFUSIONS ECoG PATTERN  E D  TOTAL X BREATHING X O F LOW WITH B R E A T H I N G CSS X O F H I G H WTTH.BREATHING eZ3 X O F INTERMEDIATE" WITH BREATHING  FETAL BREATHING  a  LOW mm HIGH CSS INTERMEDIATE  1  POST  EoG ACTIVITY  a  LOW mM H I G H CSS I N T E R M E D I A T E  f  E Z 3 TOTAL X EoG mmx  80  i  6 0  i  L O W WITH E O C  B  "o *  of  C S X O F HIGH WITH E o C eZ3 X O F I N T E R M E D I A T E WITH EoG  40  .  20  0  ECoG EPISODE DURATION cn  •mm CT  LOW HIGH INTERMEDIATE  FETAL BREATHING  CZi  TOTAL X BREATHING  mm  x O F L O W WITH B R E A T H I N G  CSS X O F H I G H WITH B R E A T H I N G eZ3 X O F I N T E R M E D I A T E WITH BREATHING  I Figure 1.4 Changes in the overall incidence and duration of low, high and intermediate voltage ECoG episodes and the percentages of breathing and electro-ocular activity in the fetus before, during and after infusion of DPHM to the A) ewe, and B) fetus (* p<0.05) (Rurak et al., 1988).  31 increase in an aroused state, with low voltage movements, increased activity.  ECoG  activity,  rapid  eye  body movements, enhanced breathing, and swallowing  This aroused state appears indistinguishable from the brief periods of  arousal that occur normally in the fetal lamb in late gestation, and which are considered by some investigators to represent wakefulness (Ruckebusch, 1972; Dawes et al., 1983; Szeto and Hinman, 1985; Rigatto et al., 1986). The other pattern of effects observed with morphine comprises suppression of breathing. With fetal bolus i.v. injection or CSF infusion, apnea generally is the first effect observed, and this is followed by increased breathing activity and fetal arousal (Olsen et al., 1983; Sheldon and Toubas, 1984; Toubas et al., 1985; Bennet et al., 1986; Hasan et al., 1988). The dual actions of morphine on fetal breathing and behavior appear to result from different doses and hence different circulating levels of the drug (and/or metabolites) in the fetal circulation (Olsen and Dawes, 1983; Szeto et al., 1988). It should be mentioned that in rats, the fetal brain to maternal brain AUC ratio for morphine was 9.5, suggesting large differences in their blood-brain barrier permeability (DeVane et al., 1999).  Szeto et al.  examined the effects of fetal infusions of morphine ranging from 0.075 to 80 mg/h on fetal breathing.  Stimulation of fetal breathing was observed with the lower  infusion rates, up to 2.5 mg/h, and suppression of breathing occurred with infusion rates greater than this level (Szeto et al., 1988).  Besides effects on  breathing and behavior, a study in rats also indicated that prenatal exposure to morphine induces long-term impairment of host-defense mechanisms, which may render the offspring more susceptible to infectious diseases (Shavit et al., 1998).  32  In addition, the behavioral effects of opiates in the human fetus have been examined in pregnant women on methadone maintenance programs. Breathing incidence in the methadone-exposed fetuses (4.7%) was markedly reduced compared with that in the control groups (37%), and it was further reduced to 1.3% following the daily methadone maintenance dose (Richardson et al., 1984). Wittman et al. also found decreased fetal breathing and a further significant postmethadone decrease in breathing and body movements in patients receiving a single daily methadone maintenance dose (Wittmann and Segal, 1991). Overall, the fetal breathing results from both these studies suggest a depressant effect on fetal respiratory neurons, similar to the effects of maternally-administered DPHM and acute high- dose morphine infusion in fetal lambs (Rurak et al., 1988; Szeto etal.,1988).  Another drug that is subject to maternal substance abuse during pregnancy is ethanol.  It has been reported that in normal, near-term human pregnancies,  maternal consumption of a dose of ethanol equivalent to that present in a social drink markedly suppressed fetal breathing for 1 to 3 hours (Fox et al., 1978; Lewis and Boylan, 1979; McLeod et al., 1983).  Moreover, the duration of  breathing inhibition appears related to the dose of ethanol (Smith et al., 1991). Similar to humans, ethanol-induced suppression of fetal breathing was also shown in fetal lambs in a series of studies (Lewis and Boylan, 1979; Patrick et al., 1985; Richardson et al., 1985; Richardson et al., 1987; Vojcek et al., 1988; Smith et al., 1989a; Smith et al., 1989b; Smith et al., 1990a; Smith et al., 1990b).  33  The ethanol-induced suppression of fetal breathing seen in sheep is similar to the effects of maternally-administered DPHM (Rurak et al., 1988). A more recent study indicates that ovine maternal ethanol exposure during the third trimester increases fetal ACTH and Cortisol concentrations, hormonal responses that may play a role in mediating alcohol-related birth defects (Cudd et al., 2001).  Cocaine is an illicit drug that is subject to maternal substance abuse and is associated with the highest incidence of obstetric and perinatal complications (Little etal., 1989; Calhoun and Watson, 1991; Kunkoetal., 1993). Vathy(1995) suggested that fetal or early neonatal exposure to cocaine may cause an overall inhibition of brain growth and development due to inappropriate neural response to hormones and neurotrophic signals during this critical period of CNS development (Vathy, 1995).  In pregnant sheep, maternal bolus injection of  cocaine results in dose-dependent increases in maternal and fetal arterial pressure and heart rate and decreases in uterine blood flow and fetal vascular p 0 (Moore et al., 1986; Woods et al., 1987; Burchfield et al., 1991). However, 2  there is an absence of physiological tolerance to cocaine in both the ewe and fetus (Burchfield et al., 2001).  The behavioral effects of direct fetal cocaine  administration have also been examined in sheep (Burchfield et al., 1990; Burchfield et al., 1995). Intravenous cocaine infusion for 90 minutes resulted in a marked reduction in the incidence and duration of REM sleep episodes and their replacement with an intermediate voltage pattern (Burchfield et al., 1990). The authors also concluded that neurobehavioral abnormalities associated with in  34  utero  cocaine exposure may be caused by chronic disruption of rapid-eye-  movement sleep (Burchfield et al., 1995). In a human study, non-stress results were compared in cocaine-exposed fetuses and in a control group. In the former group, there was reduced short-term oscillation  in heart rate and fewer  accelerations. The authors report that this is suggestive of quiet sleep, implying depressive effects of the drug on the fetus; alternatively, cocaine could alter fetal heart rate regulation (Tabor et al., 1991).  As shown in the various examples above, the use of therapeutic, social and illicit drugs by pregnant women may have effects on fetal CNS, cardiovascular or metabolic functions.  Since current diagnostic tests of fetal well-being, such as  the non-stress test (Brown and Patrick, 1981) and the fetal biophysical score (Manning et al., 1980), monitor elements of fetal behavior,  drug-induced  alterations in fetal behavior could affect the test outcome and lead to a misdiagnosis of fetal distress, particularly if medical staff were unaware that the patient was taking such a drug.  This seems most likely with non-prescription  medications {i.e. DPHM) and illicit drugs.  A more serious consequence could  result from long-term drug administration, leading to fetal tolerance to the drug, withdrawal symptoms antenatally or after birth (Jones and Barr, 2000), increased perinatal mortality and morbidity and perhaps permanent neurologic deficits (Kaltenbach and Finnegan, 1989; Rurak et al., 1991; Kunko et al., 1993; Vathy, 1995). Finally, given the similarities in behavioral changes between humans and the sheep when exposed to the substances described above, sheep serves as a  35  useful study model for human fetal and neonatal exposure to these CNS-active compounds.  1.6  DPHM Levels in the Brain and CSF  As noted in section 1.2.1, DPHM exhibits a significant sedative effect presumably  via the occupation of central Hi-receptors (Nicholson, 1983; Garrison, 1991). However, there are limited data on the CNS levels of the drug or on the mechanisms of transfer involved. In early studies (Glazko and Dill, 1949a; Glazko and Dill, 1949b), the drug was detected in the brain of rats and guinea pigs after sub-cutaneous, intraperitoneal, or i.v. administration. However, there was much greater accumulation of the drug in lung, spleen and liver and these studies provided no detailed information on blood-brain or blood-CSF concentration relationships. In a more recent study (Goldberg et al., 1987), there was significant C-DPHM accumulation in isolated rabbit choroid plexus (13-18 times 14  that in the incubation medium), which was significantly inhibited by unlabeled DPHM and other basic drugs (nicotine, tolazoline) and metabolic inhibitors (dinitrophenol, iodoacetate). Conversely, probenecid had no effect. In rabbits, the steady-state CSF concentration of DPHM was slightly higher than the plasma concentration, even though the plasma protein binding of the drug was - 5 0 % . Thus, the DPHM CSF concentration was about twice the unbound DPHM plasma concentration, suggesting that the choroid plexus actively transports the drug from blood to CSF via an energy dependent transport system. In the same study,  36  the in situ  rat brain perfusion technique demonstrated rapid but saturable  clearance of DPHM from blood to brain. A similar phenomenon was observed with diazepam, which is completely cleared from the perfusate with this technique (Takasato et al., 1984). The results suggest that DPHM enters the brain tissue and CSF extremely rapidly. Since DPHM has a pKa of -9.0 and only - 2 . 5 % is unionized at the physiological pH, these results cannot be explained by the passive diffusion of unionized drug through the blood-brain and blood-CSF barriers. As noted in section 1.1.1, there is evidence for saturable  BBB  transporter mechanisms for lipophilic, amine drugs (Pardridge and Connor, 1973; Pardridge et al., 1984; Spector, 1988b; Yamazaki et al., 1994a; Yamazaki et al., 1994b; Yamazaki et al., 1994c). This includes mepyramine, a histamine Hiantagonist. The available data suggest that these compounds cross the bloodbrain and blood-CSF barriers by both simple diffusion of the unionized lipidsoluble form, and by carrier-mediated transport of the ionized form (Pardridge et al., 1984; Goldberg et al., 1987; Yamazaki et al., 1994a). In vivo (rat carotid injection technique), brain (Yamazaki et al., 1994a).  uptake of mepyramine  is inhibited  by  DPHM  Moreover, both in vivo and in vitro (bovine brain  capillary endothelial cells), propranolol inhibits mepyramine uptake (Yamazaki et al.,  1994a; Yamazaki et al., 1994b). Together, these data suggest that  propranolol could inhibit brain (and perhaps CSF) DPHM uptake.  37  1.7 Microdialysis  In vivo  microdialysis is a recently developed technique that permits serial  sampling from compartments with limited fluid volume (Stahle, 1993; Wang et al., 1993; Elmquist and Sawchuk, 1997; De Lange et al., 1998). It is thus ideal for determining drug levels in brain ECF and CSF. The technique involves inserting a microfiber, with a dialysis membrane covering the tip, into a sampling site. An appropriate dialysate is then infused through the fiber and the compound of interest is extracted by creating a concentration gradient between the site and the dialysate. The concentration at the sampling site is determined by multiplying the concentration of the compound in the dialysate by an estimated recovery rate of the compound by the probe. The recovery rate of the compound is determined by the rate of diffusion across the dialysis membrane. There are 2 principal methods for determining the recovery rate, an in vitro method (zero-net flux), or an in vivo method (retrodialysis) (Wang et al., 1993; De Lange et al., 1998). The former method estimates the recovery rate of the probe before and after the experiment in an in vitro setting.  The drawback of this method is that the  estimated recovery rate does not accurately reflect the diffusion path differences between an in vivo tissue environment and an in vitro test setting. In addition, this approach assumes that the in vivo diffusion path and the efficiency of the membrane remain constant during the entire experiment.  The retrodialysis  method estimates the recovery rate using an internal calibrator in the dialysis fluid (De Lange et al., 1998). The dialysate contains a fixed concentration of a  38  compound with similar chemical and physical properties to the compound of interest (an ideal calibrator would be an isotopic analog of the compound). The recovery rate of the compound of interest is equal to the diffusion rate of the internal calibrator into the sampling site. This method assumes 3 conditions: (1) transfer of the compound and the calibrator is by simple diffusion, (2) identical conditions for the loss of calibrator and the recovery of the compound in the sampling site, and (3) no interaction between the calibrator and the compound. When these conditions are met, retrodialysis provides an accurate in vivo recovery rate that reflects the efficiency of the microdialysis probe even if the efficiency of transfer varies during the experiment.  In our study, retrodialysis  calibration is the method of choice and this method was established for DPHM  via the use of a stable isotope-labeled form of the drug as the internal calibrator.  1.8  Rationale  Drug use in pregnancy and the postnatal period will continue for therapeutic and societal reasons, and DPHM is an example of drug that is used in pregnancy and in infants and children. The drug readily crosses the placenta, and this is the case with most xenobiotics used in pregnancy. It alters fetal behavioral state and may also do this in the newborn. Alterations in fetal behavior can confound the commonly used diagnostic tests of fetal well-being and may also have long term impacts on neurological development. The DPHM-elicited behavioral effects occur at plasma concentrations lower than those required in adults. This is  39 similar to the actions of other drugs, and while the reasons for this phenomenon are not known, it may be because of a greater exposure of the fetal and neonatal brain to the drugs.  However, while there is considerable information on the  development of blood-brain barrier permeability to lipid insoluble compounds, there are much less data on lipophilic substances, which includes most drugs. Based upon the available information, several features of BBB maturation (decreasing CSF protein concentrations, altered cerebral blood flow) may result in altered blood-brain drug concentration relationships, but further information is required. Because of ethical and technical constraints, study of this issue in humans is not possible. However, the fetal and newborn lamb are a useful model for the human fetus and infant, because of the similarities in the ontogenesis of both the BBB and behavioral functions in the 2 species. In addition, this model can overcome limitations in the available sampling volume of biological fluids associated with smaller animal models, and thus allows for more detailed studies. Therefore, the proposed studies will provide useful information on drug exposure in the fetal, newborn and adult brains. Finally, the study is of direct relevance to the issue of therapeutic drug use in the perinatal period.  40  1.9 Hypothesis and Objectives  The working hypotheses of this thesis were: a) The blood-brain ECF and blood-CSF concentration ratios for DPHM will be higher in fetal and postnatal sheep than in the adult, due to higher brain blood flows and increased BBB permeability. b) The blood-brain ECF and blood-CSF concentration ratio for DPHM will decrease with advancing fetal and postnatal age due to increases in the plasma protein binding of the drug. c) The blood-brain ECF and blood-CSF concentration ratio for DPHM will decrease during propranolol administration due to inhibition of an inwardly-directed BBB lipophilic amine transporter.  The objectives of this project are as follows:  1. To examine blood-brain ECF and blood-CSF concentration relationships for DPHM in fetal, newborn and adult sheep. 2. To examine the effect of propranolol administration on the blood-brain ECF and blood-CSF concentration relationships for DPHM. 3.  To investigate the developmental changes in the levels of plasma protein  binding and the extent of CNS and plasma disposition of DPHM.  41  Chapter 2 DPHM 5 - S t e p Infusions  The purpose of this study is to assess blood-brain ECF and blood-CSF drug concentration relationships as a function of pre- and postnatal age, and in relation to variations in drug dose and hence plasma drug levels. By applying in  vivo microdialysis (MD) in chronically-instrumented fetuses, newborn lambs, and adult ewes, we were able to collect serial samples from the lateral ventricle and cerebral cortex and therefore elucidate DPHM pharmacokinetics in the CNS from these animals at different ages.  42  2.1 Methods  2.1.1 Animals and Surgical Preparation  All studies were approved by the University of British Columbia Animal Care Committee, and the procedures performed on sheep conformed to the guidelines of the Canadian Council on Animal Care.  Fetuses:  Time-dated pregnant Dorset Suffolk cross-bred ewes (term, ~145  days) were operated on between 95 and 105 days for the 100 d group (n=3) and between 115 and 125 days of gestation for the 120 d group (n=6).  In total, 9  pregnant ewes (3 for 100 d fetal study and 6 for 120 d fetal study), with a maternal weight of 81.50 ± 9.32 kg were used. Food was withheld for —18 h prior to surgery, but the animals were allowed free access to water. Approximately 30 min before surgery, a 6 mg iv.  bolus dose of atropine (Glaxo Laboratories,  Montreal, Canada) was administered via the jugular vein to control salivation. Surgery was performed asceptically under isoflurane (1-2%) and nitrous oxide (60%) anesthesia (balance O2), after induction with iv. sodium pentothal (1 g) and intubation of the ewe. Access to the fetus was gained through a midline abdominal incision in the ewe and an incision through the uterine wall free from placentomes and major blood vessels. Silicone rubber catheters (Dow Corning Corp., Midland, Ml) were implanted in a fetal femoral artery and vein, fetal trachea, amniotic cavity, and a maternal femoral artery and vein.  In addition,  43  flexible MD probes (CMA 20, Stockholm, Sweden) were implanted in the lateral ventricle  and  ipsilateral  parietal cortex for  collection  of CSF  and  ECF,  respectively (Figure 2.1a). The drilling locations in the skull for the CSF and ECF MD probe implantation holes were determined previously from autopsy.  Briefly,  the CSF hole was determined first by locating the occipital crest which is close to the neck on the fetal skull, then the drilling location was 2 mm anterior and 5-6 mm lateral from the midline. By using a pin-vise hand drill with a drill bit length of 5 cm and a diameter of 1.5 mm, a hole was created approximately 1.6 cm deep into the brain. The correctness of the drilling location was assessed by three means.  First, if the hole was drilled correctly to reach the lateral ventricle,  leakage of CSF from the hole could usually be observed.  Second, upon  withdrawal using a syringe, CSF could be collected. Third, the flow of saline into the hole by inserting a needle connected to a syringe filled with saline after opening the stopcock would indicate correct access into the lateral ventricle. The reason is because saline would flow into an empty space (i.e. lateral ventricle) but not if the hole was drilled into tissue. Next, the hole for the ECF probe was drilled using the pin-vise hand drill, usually 1-1.5 cm anterior to the CSF hole, 0.8 - 1 cm deep. The lack of fluid flow and failure to withdraw fluid using a syringe from the hole would indicate correct location for the ECF hole.  Before probe  insertion, a plastic introducer was first placed into the hole. The probe was then placed through the introducer, which was subsequently removed. The probe was initially secured to the skull by applying tissue adhesive (VetBond®, 3M Animal Care Products, St. Paul, MN).  It was then further secured by covering the  44  assembly with dental cement (Poly-F Plus®, Densply, Konstanz, Germany) (Figure 2.2). The MD probe input and output tubings were extended with 50 cm fluoropolymer (FEP) catheters (CMA, Stockholm, Sweden) to allow for CSF and ECF sampling access once exteriorized through the flank of the ewe.  The 4  catheters from the 2 probes (i.e. inflow and outflow) were coiled into loops over the skull to allow for head growth and then anchored onto the scalp tissue to prevent tension on the implanted probes.  Finally, the catheters were tunneled  underneath the neck skin and through a small incision on the back of the neck before passing through the uterine wall incision. All catheters were tunneled s.c. in the maternal abdominal wall and exteriorized via a small incision on the flank of the ewe; they were stored in a Ziploc® bag with bandages when not in use. All vascular catheters were flushed daily with approximately 2 mL of sterile 0.9% sodium chloride solution containing 12 U heparin/mL to maintain catheter patency.  The MD probes were flushed with sterile, degassed lactated ringer  solution daily to avoid air bubble formation along the tubing. The lactated ringer solution was first placed inside an Erlenmeyer flask with a rubber stopcock sealing the top opening. Then the flask was connected to a sink aspirator which was turned on for 20 min for the degassing process. Two antibiotics - Trivetrin® (Schering Canada Inc., Pointe Claire, QC) and ampicillin, were administered to the ewe on the day of the surgery and for 3 days postoperatively. After surgery, animals were kept in holding pens with other sheep and were allowed free access to food and water. The ewes were allowed to recover for at least 3 days before experimentation.  45 Cerebral tissue (Parietal Cortex)  (d)  Cerebral  Figure 2.1a Cross-sectional diagram of the fetal brain. MD probes were implanted into the lateral ventricle (LV) and the cerebral tissue for CSF and ECF sampling, respectively (Saunders et al., 1999b).  Cerebral tissue (Parietal Cortex)  Figure 2.1b Cross-sectional diagram of the adult brain. MD probes were implanted into the lateral ventricle (LV) and the cerebral tissue for CSF and ECF sampling, respectively (Saunders et al., 1999a).  CO CD  >  CD  O  CO  o Q. CD  o c o TJ TJ CO CD  CD TJ CD  jt; TJ O  E <D  _Q  X _CD ZJ  CO  CO  o  O)  CD  o X! cz o CO  CL CO CO  0  o  CO TJ O  CO  o  c  L.  CO ZJ  CO CD c  co ! Q  E^ E o_  co LU CO TJ X I c g CO CM c oi CD CD I* l_ CD  <  .E?  c  LL co  47  Post-natal Lambs: A total of 10 Dorset Suffolk cross-bred newborn lambs were used. The lambs were divided into a 10 day old group (n=5, weight = 7.05 ± 1.52 kg) and a 30 day old group (n=5, weight = 12.16 ± 1.14 kg). performed  asceptically  under  isoflurane  (1-2%) and  anesthesia (balance O2) and intubation of the lamb.  Surgery was  nitrous oxide  (60%)  Silicone rubber catheters  (Dow Corning Corp., Midland, Ml) were implanted in a carotid artery and a jugular vein. In addition, flexible MD probes (CMA 20, Stockholm, Sweden) were implanted in the lateral ventricle and ipsilateral parietal cortex for collection of CSF and ECF respectively (Figure 2.1b), as described in the previous section. The MD probe input and output tubings were extended with FEP catheters (CMA, Stockholm, Sweden) and tunneled s.c. and exteriorized via a small incision on the back of the neck for access. All catheters were stored in a Ziploc® bag with bandages when not in use.  All vascular catheters were flushed daily with  approximately 2 mL of sterile 0.9% sodium chloride solution containing 12 U heparin/mL to maintain catheter patency.  The MD probes were flushed with  sterile, degassed lactated ringer solution daily to avoid air bubble formation along the tubing. Two antibiotics - Trivetrin® (Sobering Canada Inc., Pointe Claire, QC) and ampicillin, were administered to the lamb on the day of the surgery and for 3 days postoperatively: After surgery, animals were kept in holding pens with their mothers and allowed to recover for at least 3 days before experimentation.  Adult Sheep: Six non-pregnant Dorset Suffolk cross-bred ewes were employed. The sheep were 5.21 ± 2.83 years old with a body weight of 74.64 + 21.96 kg.  48 Food was withheld for ~18 h prior to surgery, but the animals were allowed free access to water. Approximately 30 min before surgery, a 6 mg iv. bolus dose of atropine (Glaxo Laboratories, Montreal, Canada) was administered via the jugular vein to control salivation.  Surgery was performed asceptically under  isoflurane (1-2%) and nitrous oxide (60%) anesthesia (balance 0 ) , 2  after  induction with iv. sodium pentothal (1 g) and intubation of the ewe. Polivinyl or silicone rubber catheters (Dow Corning Corp., Midland, Ml) were implanted in both a carotid artery and a jugular vein. In addition, flexible MD probes (CMA 20, Stockholm, Sweden) were implanted in the lateral ventricle and ipsilateral parietal cortex for collection of CSF and ECF respectively (Figure 2.1b). The procedure for probe implantation in the adults was basically the same as that in fetuses and lambs with some minor changes. First of all, since the skull in adult was harder and therefore more difficult to penetrate, a hand-crank drill was used instead of a pin-vise drill to create the holes for the CSF and ECF probes. In addition, before suturing the scalp, the MD probe catheters were anchored directly onto the inside of the scalp without first forming loops. The reason is that allowance for head growth was not needed in adults as compared to fetuses and lambs. The MD probe input and output tubes were extended with FEP catheters  (CMA,  Stockholm, Sweden) and tunneled s.c. and exteriorized via a small incision on the back of the neck for access. All catheters were stored in a Ziploc® bag with bandages when not in use.  All vascular catheters were flushed daily with  approximately 2 mL of sterile 0.9% sodium chloride solution containing 12 U heparin/mL to maintain catheter patency.  The MD probes were flushed with  49  sterile, degassed lactated ringer solution daily to avoid air bubble formation along the tubing. Two antibiotics - Trivetrin® (Schering Canada Inc., Pointe Claire, QC) and ampicillin, were administered to the ewe on the day of the surgery and for 3 days postoperatively. After surgery, animals were kept in holding pens with other sheep and were allowed free access to food and water. The ewes were allowed to recover for at least 3 days before experimentation.  2.1.2  Experimental Protocols  The protocol involved a bolus i.v. loading dose of DPHM before each step (to hasten the achievement of steady-state), followed by i.v. infusion of the drug at 5 different rates, with each infusion rate lasting 7 h. All DPHM (Diphenhydramine hydrochloride, Sigma Chemical Co., St. Louis, MO) doses were prepared in 0.9% sodium chloride solution and were sterilized by filtering through a 0.22 urn nylon syringe filter (MSI, Westboro, MA) into a capped empty sterile injection vial. For the 100 d fetuses the DPHM loading doses were 0.5 mg/kg and the 5 stepped infusion rates were 17, 76.5, 136, 195.5 and 255 Lig/kg/min. For the 120 d fetuses the DPHM loading doses were 0.5 mg/kg and the infusion rates were 13.6, 61.2, 108.8, 156.4 and 204 Lig/kg/min.  For the 10 d lambs the DPHM  loading doses were 0.7 mg/kg and the infusion rates were 5.25, 19.25, 33.25, 47.25 and 61.25 Lig/kg/min. For the 30 d lambs the DPHM loading doses were 0.7 mg/kg and the infusion rates were 6, 22, 38, 54 and 70 Lig/kg/min. In adult ewes, the DPHM loading doses were 0.15 mg/kg and the infusion rates were 1.5,  50  5.5, 9.5, 13.5 and 17.5 ng/kg/min. The various dosages were determined based on:  1) Results obtained from previous pharmacokinetic studies performed in  newborn lambs, pregnant and adult sheep (Yoo et al., 1986b; Yoo et al., 1990; Yoo et al., 1993; Kumar et al., 1997; Kumar et al., 1999b; Kumar et al., 1999c; Wong et al., 2000), and 2) Results obtained from preliminary stepped infusion studies.  The doses were adjusted so that they would target a plasma  concentration range of ~ 35 - 450 ng/mL. This particular plasma concentration range was used because it was associated with behavioral changes in fetal lambs in a study performed previously (Rurak et al., 1988).  During the infusions arterial blood samples (3 mL adult, 0.5 mL fetus and lamb) were collected at -5, 5, 15, 30 min, and 1, 2, 3, 4, 5, 6, 7, 7.083, 7.25, 7.5, 8, 9, 10, 11, 12, 13, 14, 14.083, 14.25, 14.5, 15, 16, 17, 18, 19, 20, 2 1 , 21.083, 21.25, 21.5, 22, 23, 24, 25, 26, 27, 28, 28.083, 28.25, 28.5, 29, 30, 31, 32, 33, 34, 35, 35.083, 35.25, 35.5, 36, 36.5, 38, 40, 43, 46, 49, and 53 h. Samples (0.5 mL) were collected from the fetus at intervals for assessment of blood gas and metabolic status. Due to the relatively small total blood volume in the fetus [-125 - 300 mL, (Kwan et al., 1995)], drug free maternal blood (8 mL) was infused into the fetus every 7 hours for replacement.  Microdialysis sampling began at the  onset of the infusion. The microdialysis pump infusion rate was 2 uL/min and 60min  cumulative  experiment.  samples  were  collected  throughout  the  duration  of  the  The dialysate was degassed, sterile lactated ringer solution  51 containing 400 ng/mL of [ Hi ]-DPHM. 2  0  Samples were also collected for up to 18  hours after the end of the last infusion.  All blood samples collected were placed into EDTA-containing Vacutainer® tubes (Becton-Dickinson, Rutherford, NJ) and centrifuged at 2000 x g for 10 min. The plasma supernatant layer was removed and placed into clean borosilicate test tubes with polytetrafluoroethylene-lined caps.  MD dialysate samples were  collected directly into clean borosilicate test tubes.  Plasma and MD samples  were stored frozen at -20°C until the time of analysis.  2.1.3 Retrodialysis  Microdialysis sampling began at the onset of the infusion.  The microdialysis  pump (Harvard Apparatus Inc., Holliston, MA) infusion rate was 2 uL/min and 60min cumulative samples of CSF and ECF were collected throughout the duration of the experiment.  MD probe recovery was determined using the retrodialysis  technique (De Lange et al., 1998; Xie et al., 2000; Bouw et al., 2001). The MD dialysate (degassed, sterile lactated ringer solution) contained a calibrator ([ Hi ]2  0  DPHM) at a concentration of 400 ng/mL.  The probe recovery rate was  determined by comparing the input and output concentrations of the calibrator as follows:  52  [Calibrator-input] - [Calibrator tput] ou  Recovery = [Calibrator put]  (Equation 1)  in  Free fraction drug concentration in the CSF (CCSF) or in the ECF (C CF) at the MD E  sampling site = [DPHM] iaiysate/Recovery Rate. d  2.1.4 Physiological Recording  Arterial pressure was measured using strain-gauge manometers (Ohmeda Inc, Madison, Wl) and heart rate from a cardiotachometer (Astro-Med, West Warwick, Rl) in all animals.  In addition, amniotic and fetal tracheal pressures were  recorded.in the pregnant ewes.  All variables were recorded on a Grass K2G  polygraph (Astro-Med, West Warwick, Rl) coupled to a computerized data acquisition system (PowerLab Chart® v4.2, ADInstruments, Grand Junction, CO). Blood p02, p C 0 and pH measurements were made with an IL 1306 pH/Blood 2  gas analyzer (Allied Instrumentation Laboratory, Milan, Italy), blood O2 saturation and hemoglobin concentrations with a Hemoximeter (Radiometer, Copenhagen, Denmark), and blood glucose and lactate levels with a 2300 STAT plus glucose/lactate analyzer (Y.S.I. Inc., Yellow Springs, OH) to ensure the wellbeing of the animals throughout the studies.  Briefly, blood samples (0.5 mL)  53  were taken before the start of the experiments, during the first and last 30 min of each infusion steps, and at intervals post-infusion for the above analyses.  2.1.5 Determination of DPHM Plasma Protein Binding  Protein binding of DPHM in the plasma samples was determined by an equilibrium dialysis procedure described by Yoo et al. (1993).  Briefly, plasma  samples collected at the last 3 hours (when steady-state had been achieved) of each infusion step were dialyzed against an equal volume (0.5 mL) of phosphate buffer solution (Sorensen's isotonic buffer, 0.067 M, pH = 7.4) for 3 h. The composition  of the Sorensen's  buffer  was: 1.79 g  KH P0 , 2  4  14.35 g  N a H P 0 ' 7 H 0 , and 3.90 g NaCl qs to 1.0 L with deionized water. Dialysis was 2  4  2  carried out at 39.0°C for maternal and lamb plasma and 39.5°C for fetal plasma in Plexglass® dialysis cells in which buffer and plasma were separated by a cellophane dialysis membrane (Sigma Chemical Co., St. Louis, MO) with a molecular cutoff of 12,000 Da. DPHM plasma unbound fraction was calculated by dividing the DPHM concentration of the buffer by the concentration of the plasma following dialysis.  2.1.6 DPHM and [ H ]-DPHM Extraction Procedure 2  10  DPHM and [ H ]-DPHM concentrations were measured using a previously 2  10  developed gas chromatographic mass spectrometric (GC-MS) analytical method (Tonn et al., 1993). Briefly, plasma samples (100 uL) and MD samples (120 uL)  54  were made up to volume (1.0 mL) with distilled water. Then, internal standard (orphenadrine, Sigma Chemical Co., St. Louis, MO) and 500 uL of 1 M NaOH were added to the test tube along with 7.0 mL of solvent (0.05 TEA in 2% isopropyl alcohol: 98% hexane). The extraction sample tubes were capped and mixed on a rotary mixer for 20 min. Following cooling for 10 min at -5°C in order to break any emulsion formed during mixing, they were centrifuged for 10 min at 3000 x g.  The organic phase was then separated and dried under a gentle  stream of nitrogen gas at 30°C. The dried samples were reconstituted with 150 uL of 0.05 M TEA in toluene. A 3 uL aliquot of the reconstituted sample was injected into the GC-MS using the splitless mode of sample introduction (purge time 1.5 min).  Chromatographic separation of DPHM, [ Hio]-DPHM, and 2  orphenadrine was achieved using a 30 m J&W DB-1701 0.25 mm i.d. capillary column (J&W Scientific, Folsom, CA). Helium was used as the carrier gas at a 15 psi column head pressure. Injection  The GC operating conditions were as follows:  port temperature was  180°C; the initial oven temperature  was  maintained at 140°C for 1 min, then increased at 30°C/min to 200°C. The oven temperature was further increased at 17.5°C/min to 265°C, where it was held for 5 min. This resulted in a total run time of 12.7 min. The mass spectrometer was operated in the electron impact ionization mode (electron ionization energy 70eV) with selective ion monitoring (EI-SIM) at transfer line temperature of 280°C. Ion fragments m/z 165 and 173 were used to monitor DPHM and [ H ]-DPHM, 2  10  respectively.  The internal standard, orphenadrine, was also monitored at m/z  165 but at a different retention time than DPHM. The dwell time was set at 50  55  milliseconds for each ion being monitored, to ensure adequate sampling of the chromatographic peak of interest (i.e., 15 scans/peak). The DPHM and [ H i ] 2  0  DPHM calibration curve concentration range for the assay is 2.0-200.0 ng/mL. Inter- and intra-day variability were <17% at the LOQ (2 ng/mL) and <10% at all other concentrations (Tonn et al., 1993).  Determination of LOQ and [ Hi ]2  0  DPHM synthesis were as previously described (Tonn et al., 1993).  2.1.7 Pharmacokinetic Analysis  Pharmacokinetic parameters were calculated by standard methods as described in Gibaldi and Perrier (Gibaldi and Perrier, 1982). The apparent distribution and elimination ti/2's of DPHM were obtained from a 2-compartment model fitting of the data using the nonlinear least-squares regression software, WinNonlin version 1.1 (Scientific Consulting Inc., Apex, NC). All model fitting was carried out using a weighting factor of 1 /predicted y since it provides more accurate 2  estimates at lower DPHM concentrations. samples were used for modeling.  Specifically, only post-infusion  Model selection between a one or two-  compartment model for elimination was based upon lower AIC (Akaike Information Criterion) and Schwarz Criterion values generated (Perrella, 1988). Area under the plasma and dialysate DPHM concentration-time curve (AUC ->~) 0  values from time zero to infinity were calculated by the trapezoidal rule with a correction made for extrapolation to infinity beyond the last time point: AUC _- = AUC _ + AUC ^» 0  0  t  t  (Equation 2)  56 where: A U C ^ - = CWP t  Where Cj t is the concentration of the last sample. as  The total body clearance  (CIT) was obtained from: CI = Dose/AUCo-.-  (Equation 3)  T  The total area under the first moment of the plasma concentration-time curve from time zero to infinity, A U M C ^ - , was calculated by the trapezoidal rule with a 0  correction made for extrapolation to infinity beyond the last concentration point: AUMCo^- = AUMCo^t + A U M C _ -  (Equation 4)  t  where: AUMCt-^- = Ci t" t/3 + Ci t/0 as  2  as  The volume of distribution at steady-state (Vd ) was calculated by a modelss  independent method: Vd  s s  = (Dose • AUMCo^«)/(AUC -*~)  (Equation 5)  2  0  The dose in Equation 5 was based on the total dose administered to the animals (i.e. the sum of infusion rate multiplied by the infusion duration for each step with the addition of the bolus doses). The extent of DPHM transfer into the brain was calculated by relating the CSF and ECF AUCo->» values to the plasma AUC -*~ 0  value to yield  fcsF  and f cF ratios. Specifically, using fcsF as an example, the CSF E  A U C ^ ~ was divided by the plasma AUCo->- as follows: 0  fcsF = A U C C S F o-»-/AUCo->-  (Equation 6)  The f cF value was calculated in the same manner using A U C C F O->~E  E  This  method of characterizing drug transfer across the BBB has been used in numerous other MD studies for different drugs including atenolol,  gabapentin,  zidovudine,  acetaminophen,  morphine-3-glucuronide,  morphine-6-  57  glucuronide, camptothecin, lamotrigine, phenobaribital, felbamate, alovudine, codeine, and carbamazepine (Wang et al., 1993; Wong et al., 1993; de Lange et al.,  1994; Wang and Sawchuk,  1995; Wang and Welty,  1996; Xie and  Hammarlund-Udenaes, 1998; Graumlich et al., 1999; Luer et al., 1999; Stahle and Borg, 2000; Walker et al., 2000; Xie et al., 2000; Bouw et al., 2001; Tsai et al., 2001; Potschka et al., 2002).  2.1.8 Statistical Analysis  All data are reported as mean ± S.D.  Pharmacokinetic parameters and  concentrations were compared using one-way ANOVA followed by a Duncan's multiple comparison test. In occasions (see Table 2.7) where normal distribution of data was not observed after performing a normality test, a Kruskal-Wallis test was used instead of ANOVA.  The significance level was p < 0.05 in all cases.  2.2 Results  2.2.1  Steady-state Concentrations of DPHM in CSF, ECF, and Plasma in  Fetuses, Newborn Lambs, and Adult Sheep  The average age of the fetuses on the day of their experiments was 103.5 ± 1.7 days for the 100 d fetus group and 124.1 ± 1 . 4 days for the 120 d fetus group. For the lambs, the average age on the day of their experiments was 11.5 ± 1.6  58  days and 33.8 ± 1.2 days for the 10 d and 30 d lamb groups, respectively. For the adults, the average age on the day of their experiments was 5.21 ± 2.83 years. Estimated mean fetal body weight for the 100 d fetal group was 1.1 ± 0 . 1 kg, and for the 120 d fetal group 2.4 ± 0.3 kg.  Fetal weights in utero were  estimated from the weight at birth and the time between the experiment and birth as described by Gresham et al. (Gresham et al., 1972). Mean lamb body weight for the 10 d lamb group was 7.05 ± 1.52 kg and for the 30 d lamb group 12.16 ± 1.14 kg, and mean adult ewe body weight 74.64 ± 21.96 kg. MD probe recovery rates ranged between 40-50% across the different age groups (Table 2.1). However, the failure rate was high (~ 1/3 of the probes failed to work shortly after surgeries) and only animals that had functional probes are listed in Table 2.1. Most animals had one CSF and one ECF probe; however, 3 of the 30 d lambs had both probes implanted in the brain tissue (i.e. ECF) due to difficulties in locating the lateral ventricles for CSF probe insertion.  Figures 2.3a-e are  semilogarithmic plots of mean DPHM CSF, ECF, and plasma concentrations vs. time for all age groups. From the profiles, it can be seen that CSF, ECF, and plasma concentrations increased in proportion to increases in infusion rate. Also, both the CSF and ECF concentrations were very similar to each other. Steadystate concentrations were reached at the 4  t h  hour of each infusion step, as no  significant differences were observed among the concentrations in all three fluids beyond this point (ANOVA, p>0.05).  in IO 0)  (O  h-  CN  o  iri • +l T—  o  o  iri +l m CO  +i CO  T—•  o  +i CO in d  CO N-  oo  CD CN  CN  +1  iri +l o CN  +1  CN in CO  CN  CO CN CO  +1  00  T— ^ "  co  co  o  00  CD  +1  0) CD  > o u , d> I* «fo <D  CO  L_  O)  CN  O  CO m iri +l CD CO CN  00  iri  +1  (0  LO  o  00  0)  Q-  CN  CO  || o  +1  ^1" CM  CM  CN  "t  iri +l  Q-I +1  (0  al  CD CD  T—  T—  CD CO  CO in  CN CO CD  00  in in CN  +1  +1  oi co  CD  U-CN  o (0  TJ O  a>  <  II  LUcN  On  CO CO  +1  CO  00  4-1  o u.  +1  oo o  +1  +1 CN  m  m  CN +l CN  co +l CN CO CN  00  00 d  oo  CO CN  co CN  m in  CD in CN  +1  +1  in  a>  CN CO  co CO +i CN CO  •<t  co"  +1  CN  ^—•  CN  LLxf  LULO  T -  CN  in  CO  CO M-  m in CN +l CO CO  CN NCN  T—  CO CN oo  00  CO CN iri  00  ^JCN  o  CD  CD m  m CN  CO  CO •<*  +1  +1  LLLO  o  •o o  o  XI  CO  +1  +1  m CN CN +i CO  T—  CN m  o  E re  T—  T—  CD  t/>  <4->  m  hCD CO  LU^f  CA  CO  iri  oo  II  T—  Oi  +1  (0 n o n  II  00  co +l  CO  CD CN +1 in oo CN  +1  CO CD CO  00  CN +i CO  CD in CO +1  CO +i CO  CO CO co Ni-  +1  co  co  in  CD iri  in CO  CO +l o CO CO  o oo CO +i CO CO iri  CN  CN CO  +1  +1  m CO CN +l CO CN  co  ^-  CD CO  co CD CO  +1  +i in  oo  oi CO  CM CO 3  CO  co  co  CO +1  +1  m CO  CO co +l CD  00  U - ^ L U O T U - C D L U C O  (Oil  XI  E CO  On  (On  3  TJ <  Oil  60  0  10  20  30  40  50  60  Time (h)  Figure 2.3  Mean DPHM CSF, ECF, and plasma concentrations vs. time profiles  in (A) 100 d fetuses (n=2), and (B) 120 d fetuses (n=7) from the 5-step infusion study. Error bars are omitted for reasons of clarity.  61  10000  1000  ?5  100  10000  10  20  30  40  50  60  Time(h)  Figure 2.3  Mean DPHM CSF, ECF, and plasma concentrations vs. time profiles  in (C) 10 d lambs (n=4), and (D) 30 d lambs (n=4) from the 5-step infusion study. Error bars are omitted for reasons of clarity.  62  E  0  10  20  30  40  50  Time (h)  Figure 2.3 Mean DPHM CSF, ECF, and plasma concentrations vs. time profile in (E) adult sheep (n=8) from the 5-step infusion study. Error bars are omitted for reasons of clarity.  63 The  mean  steady-state  CSF  (CCSFSS),  ECF  (C FSS), EC  and  plasma  (C  Pss  )  concentrations in the different age groups are shown in Tables 2.2a-c. The high variability observed in some of the groups was likely due to growth of the animals. There was a trend where the brain concentrations started higher than plasma concentrations as seen in the fetal groups, then became roughly equal to each other in the newborn lamb groups, and eventually dropped to levels lower than the plasma concentrations in the adult group. This trend was also observed in the overall CCSFS /CP S  ss  and CECFSS/CP  ss  ratios which can be seen in the  diagrammatic representation shown in Figure 2.4. The ratios started at about 2-3 in the fetal groups and dropped to below 1 in the adult group (Tables 2.3a-b). Also, significant decreases were observed in both ratios after birth (Tables 2.3ab).  Equilibrium dialysis was performed on the steady-state plasma samples to  determine the extent of plasma protein binding (Table 2.4) and provide the plasma concentrations of unbound DPHM.  Since plasma protein binding  increased with advancing age and it is the free drug that crosses the BBB, it would be interesting to compare the steady-state CSF (CCSFSS) and ECF (C CFSS) E  concentrations with the unbound plasma levels (Cp ) to further examine the uss  brain/blood concentration relationships at each infusion step (Tables 2.5a-b). Results from equilibrium dialysis indicated that following an initial fall from the 100 d fetuses, there was a significant increase in the extent of protein binding between the 120 d fetus and 10 d lamb groups (Table 2.4).  After birth, a  significant increase in the level of plasma protein binding occurred from 30 d lambs to adults (Table 2.4; Figure 2.5). In contrast to the decreasing trend in the  64 overall  CCSFSS/C S PS  and C C F S S / C P E  ss  illustrated  in Figure  2.4,  the overall  CCSFSS/CPUSS and CECFSS/CP SS increased after birth (i.e. relative to the 120 d fetus U  values) before dropping to adult values (Figure 2.6). The relationships between infusion rate and plasma, CSF, and ECF concentrations were examined in all age groups (Figures 2.7-2.11).  First, it appeared that plasma, CSF, and ECF  concentrations increased in a linear fashion with the infusion rates used in the experiments without any signs of saturation (Figures 2.7a,b - 2.11a,b). A similar linear trend was also observed between the brain concentrations and unbound DPHM plasma concentrations.  In addition, the CCSF  VS.  C p and CECF vs. C p u  regression lines in all groups are very similar to each other in terms of their slope values (Figures 2.7c - 2.11c, Table 2.6). Relationships between the C C S F / C P  U  and CECF/CP ratios and infusion rate have also been examined. The profiles in u  all age groups did not show any specific patterns with levels remaining relatively constant over the infusion rates studied (Figures 2.7d - 2.11d).  2.2.2  CSF, E C F , and Plasma Pharmacokinetics  of DPHM in Fetuses,  Newborn Lambs, and Adult Sheep  As shown in Figures 2.3a-e, DPHM CSF, ECF, and plasma profiles appeared to follow two-compartmental pharmacokinetics following discontinuation of the final stepped infusion. Table 2.7 provides a summary of pharmacokinetic parameters. Total body (Cl-r) clearance increased significantly after birth until 30 d then dropped significantly to adult levels (Figure 2.12).  On the other hand, f sF and C  u  65 f c F values decreased significantly after birth, with both values at their lowest in E  adulthood. This is more evident upon examination of Figure 2.13, which shows a decreasing trend for f sF and f c F with respect to increasing age. Decreases in E  C  Vd  s s  were observed with age until 30 d postnatally then increased significantly (~  5.5 fold) in adults (Figure 2.14). (ti/2pcsF) and ECF  (U^ECF)  existed for plasma ( t into account.  1/2p  Increasing trends were observed for CSF  elimination half-lives with age. A similar trend also  ) half-life when the 100 d fetal group value is not taken  66 Table 2.2a Mean DPHM plasma concentration at steady-state for each infusion step. Age  Step 1  Step 2  Step 3  Step 4  Step 5  Group 100d  (ng/mL)  (ng/mL)  (ng/mL)  (ng/mL)  (ng/mL)  Fetus (n=3) 120d  13.2 + 6.6  36.7 ± 7 . 8  73.3 + 20.5  127.4 ± 1 3 . 4  161.5 ± 1 5 . 6  Fetus (n=7) 10d  2 6 . 9 ± 15.2  79.4 ± 30.2  138.3±48.9  205.4 ± 72.5  297.6 ±111.4  Lamb (n=5) 30d  43.5 ± 2 0 . 0  133.8 ±73.3  340.9 ±240.0  600.9 ± 337.2  1004.2 ±499.3  Lamb (n=5)  47.4 ± 9 . 2  168.1 ± 7 1 . 7  311.8 ± 122.2  473.4 ±225.6  757.9 ±270.5  Adult  62.9 ± 1 8 . 8  169.3 ± 6 1 . 4  306.6 ± 108.7  453.8 ± 203.0  599.3 ± 276.9  m=n  Table 2.2b  Mean DPHM CSF concentration at steady-state for each infusion  step. Age Group  Step 1  Step 2  Step 3  Step 4  Step 5  (ng/mL)  (ng/mL)  (ng/mL)  (ng/mL)  (ng/mL)  40.3 ± 3 . 7  76.8 ± 1 2 . 4  194.9 ± 1 . 4  303.4 ± 34.3  368.2 ± 25.2  100d Fetus (n=2) 120d Fetus (n=4) 10d  43.1 ± 1 7 . 3 168.7 ±81.1  357.1 ± 127.4 520.9 ±199.7  741.5 ±300.3  Lamb (n=4) 30d  42.7 ± 1 4 . 3  252.5 ± 78.8  575.2 ±222.1  861.4 ±130.8  Lamb (n=2)  43.7 ± 18.2 147.3+ 19.9  279.5 ± 0 . 7  539.9 ± 1 . 8  854.5 ± 1 2 . 6  112.8 ±42.7  158.0 ±58.0  221.9 ±89.0  Adult (n=6)  20.0 ± 4 . 2  109.7 ± 4 5 . 0  54.4 ± 1 5 . 9  67  Mean DPHM ECF concentration at steady-state for each infusion  Table 2.2c step. Age Group 100d Fetus (n=2) 120d Fetus (n=5) 10d Lamb (n=5) 30d Lamb (n=8) Adult (n=6)  Step 1  Step 2  Step 3  Step 4  Step 5  (ng/mL)  (ng/mL)  (ng/mL)  (ng/mL)  (ng/mL)  34.3 ± 4.4  94.9 ±22.8  209.0 ±23.2  51.0 ± 16.8  174.6 ±73.2  345.5 ± 126.8  60.3 ±27.9  135.4 ±56.9  291.2 ± 106.8  53.1 ± 15.6 227.0 ± 65.4  413.8 ±93.8  20.2 ± 5 . 0  119.6 ±68.8  56.7 ±27.2  • CCSFss/CPss • CECFss/CPss  «05 2.5 CC  a. 2! <D  S  ¥ v  <  |  1.5 1  100d Fetus  120d Fetus  10d Lamb 30d Lamb  Adult  Age  Figure 2.4 Changes in overall C F s s / C and C C F S S / C P ratios in relation to age. Groups with different numbers (1-3) and letters (a-c) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). C S  P S S  E  ss  6£  . 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LO  LO CM  ro  45±0.  > O  48±1.  5a>  'oo  CD  ©  E o  o CD  c  CO  £  c g 00 3  C  a & co  o  CO  cu  o  CM  -4—'  a  CO (A  <n  o  +l  +l  o  LU  o  +l  CD  CD  -<t  CD  CD CD CM  CD CO CM  00  CD  CO CD  CD q  NCO  +l  CO CO d  CO CO d  co  CD CM CO  +l  +l  v+l  T—  CNJ CD  O  TJ _>>  xLO CO  +l  o co +l -si-  +l  cb  LO  h-  CM  LO  ©  « I-  co <  QJ 3 3 O  3 CM S  O 2  ii S  StOTJcuTTJcOO  -s  ii o  c ii o  = ii  to  S  | CO ^  +1  co LO co  c  ' o .00  w ro  a. E E o o 3  l-Q  CM  C  CD  c  .9-  +1  S?  CO  T5  o CM  .j» £ ro  JQ  CN  ^  2> to ro a; ^—•  CM CD  "ro  +O= LO o ^ro = ov  +1  LO  +l  o LO  CO  0)  CM  LO LO  CD  o c CO  00  T—  +l  CO  0-  ^—'  CO LO CO  00  O  00  CM  to  i 00  CD  g =  G  •i »  ro sz > +-  73  3 0.8  1  C  T3 C  0.6  CQ  CD  2  JL  0.4 0.2 0 _|—1_ 1 , 1 1—,—i 1 , 1—-i 100d Fetus 120d Fetus 10d Lamb 30d Lamb  ,  1 1 Adult  Age  Figure 2.5 Changes in the overall extent of protein binding in relation to age. Groups with different numbers (1-4) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05).  Figure 2.6 Changes in the overall C S F S S / C P S S and C C F S S / C P ratios in relation to age. Groups with different numbers (1-2) and letters (a-c) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). C  U  E  u s s  74  Table 2.6 Summary of slopes, y-intercepts, and regression coefficients for Figures 2.7a,c - 2.11a,c  2.7a2.8a2.9a2.10a 2.11a  y-lntercept (ng/mL)  r  0.65±0.09  6.08±2.81  0.98410.013  1.40±0.21  3.03±5.14  0.98710.016  17.06±3.18  -142.58±50.71  0.94510.040  10.79±1.96  -58.28±15.77  0.96910.025  33.93±7.52  -4.00±2.16  0.99610.003  1.48±0.26  -5.00±2.11  0.97810.014 0.99310.002  CCSF VS. IR  1.54±0.28 3.67±0.52  -3.09±1.56 -33.51111.20  0.99210.002  C E C F VS. IR  3.61+.0.49  -25.10±14.18  0.99410.001  C C S F VS. IR  15.02±2.18  -131.11±40.89  0.93610.022  C E C F VS. IR  15.16±2.25  -110.48±52.73  0.94210.031  C C S F VS. IR  12.59±0.56  -105.37±20.16  0.94810.016  C E C F VS. IR  16.92+2.14  -133.38±49.50  0.93910.020  CCSF VS. IR  12.68±2.87  -7.07±10.25  0.99210.003  CECF VS. IR  12.41±2.75  -2.68±8.31  0.99510.002  -14.76±4.08  0.97510.011  Figure  Relationship  100 d Fetus 120 d Fetus 10 d Lamb - 30 d Lamb -Adult  [DPHM] [DPHM] [DPHM] [DPHM] [DPHM]  2.7b- 100 d Fetus  vs. vs. vs. vs. vs.  IR IR IR IR IR  C C S F vs. IR C E C F VS. IR  2.8b- 120 d Fetus 2.9b- 10 d Lamb 2.10b - 3 0 d Lamb 2.11b - Adult  2.7c- 100 d Fetus  2.9c- 10 d Lamb 2.10c - 3 0 d Lamb 2.11c -Adult  a  u  6.74±0.25  C C F VS. Cp  u  6.99±0.47  -13.10±3.54  0.98910.005  CCSF VS. Cp  u  5.48+1.21  -21.66±15.77  0.99810.001  C E C F VS. Cp  u  5.38±1.30  -12.64±9.29  0.99510.002  C C S F VS. Cp  u  5.81±1.59  -3.4811.06  0.99410.002  CECF VS. Cp  u  5.86±1.46  18.72112.27  0.99810.001  CCSF VS. Cp  u  4.42±0.28  1.7910.46  0.99910.001  CECF VS. Cp  u  5.94±1.03  10.8714.25  0.98910.006  C C S F VS. Cp  u  2.37±1.02  17.04114.10  0.95110.018  u  2.27±1.10  22.94116.63  0.91410.055  C E C F VS. Cp  The slope unit is applicable only to Figures 2.7a,b - 2.11 a,b. Figures 2 . 7 c - 2 . 1 1 c have no units. IR = Infusion Rate  a  2  C C S F VS. Cp E  2.8c- 120 d Fetus  Slope (mg/ mL/kg min)  The slopes in  75  200 -,  Infusion Rate (ug/kg/min) Figure 2.7a Relationship between mean DPHM plasma concentration and infusion rate at steady-state in the 100 d fetus group.  500 -  Infusion Rate (ug/kg/min)  Figure 2.7b Relationships between mean DPHM brain concentration and infusion rate at steady-state in the 100 d fetus group.  76  UL  500  0  10  20  30  40  50  60  70  Mean Unbound DPHM Plasma Concentration (ng/mL)  Figure 2.7c Relationships between mean DPHM brain concentration and unbound plasma concentration at steady-state in the 100 d fetus group.  Figure 2.7d Relationships between mean C C S F S S / C S S and infusion rate at steady-state in the 100 d fetus group. PU  CECFSS/CP S US  ratio and  77  Figure 2.8a Relationship between mean DPHM plasma concentration and infusion rate at steady-state in the 120 d fetus group.  1200  -  0  50  100  150  200  Infusion Rate (ug/kg/min) Figure 2.8b Relationships between mean DPHM brain concentration and infusion rate at steady-state in the 120 d fetus group.  78  Figure 2.8c Relationships between mean DPHM brain concentration and unbound plasma concentration at steady-state in the 120 d fetus group.  Figure 2.8d Relationships between mean C C S F S S / C P S and C C F S S / C P S S ratio and infusion rate at steady-state in the 120 d fetus group. US  E  U  79  1400  •g  1200  S  1000  H  [DPHM] =  17.06(lnf.Rate) R  2  142.58  = 0.945  20  30  40  50  -200 Infusion R a t e (ug/kg/min)  Figure 2.9a Relationship between mean DPHM plasma concentration and infusion rate at steady-state in the 10 d lamb group.  Figure 2.9b Relationships between mean DPHM brain concentration and infusion rate at steady-state in the 10 d lamb group.  80  Figure 2.9c Relationships between mean DPHM brain concentration and unbound plasma concentration at steady-state in the 10 d lamb group.  10  20  30  40  50  60  Infusion Rate (ug/kg/min)  Figure 2.9d Relationships between mean C C S F S S / C P S and C C F S S / C P S S ratio and infusion rate at steady-state in the 10 d lamb group. US  E  U  81  Figure 2.10a Relationship between mean DPHM plasma concentration and infusion rate at steady-state in the 30 d lamb group.  Figure 2.10b Relationships between mean DPHM brain concentration and infusion rate at steady-state in the 30 d lamb group.  82  1500  0  50  100  150  200  Mean Unbound DPHM Plasma Concentration (ng/mL)  Figure 2.10c Relationships between mean DPHM brain concentration and unbound plasma concentration at steady-state in the 3 0 d lamb group.  15  Infusion Rate (ug/kg/min)  Figure 2.1 Od Relationships between mean C S F S S / C P S S and C C F S S / C P S S ratio and infusion rate at steady-state in the 3 0 d lamb group. C  U  E  U  83  900  [DPHM] = 33.93(lnf.Rate) - 4.00  750  R = 0.996 2  600 450  CD O  O  300 150  3  6  9  18  15  12  Infusion Rate (ug/kg/min)  Figure 2.11a Relationship between mean DPHM plasma concentration and infusion rate at steady-state in the adult group.  oLU ^j T3 t C CO CO  £  O o ^ to X -fc D_ O  350 300  ECF = 12.41 (Inf.Rate)-2.68  250  R' = 0.995  200 150  c  L J  c c  05 O CD O  100  CSF = 12.68(lnf.Rate)-7.07  50  FT = 0.992 0  3  6  9  12  15  18  Infusion Rate (ug/kg/min)  Figure 2.11b Relationships between mean DPHM brain concentration and infusion rate at steady-state in the adult group.  84  Figure 2.11c Relationships between mean DPHM brain concentration and unbound plasma concentration at steady-state in the adult group.  8 ,  CO  DT  O  4  CO  a  [CSF]/[CPu] [ECF]/[CPu]  2  6  9  12  18  15  Infusion Rate (ug/kg/min)  Figure 2.11d Relationships between mean C S F S S / C P and infusion rate at steady-state in the adult group. 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Groups with different numbers (1-4) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05).  Figure 2.13 Changes in fcsF and f cF in relation to age. Groups with different numbers (1-2) and letters (a-c) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). E  87  100d Fetus  120d Fetus  10d Lamb  30d Lamb  Adult  Age  Figure 2.14 Changes in V d in relation to age. Groups with different numbers (1-2) are statistically different from each other as determined by the Duncan's multiple comparison test (p<0.05). s s  88  2.2.3  Physiological Responses in Fetuses, Newborn Lambs, and Adult  Sheep  Tables 2.8 and 2.9 summarize the blood chemistry data in the 100 d and 120 d fetus groups during the 5-step infusions.  In the 100 d fetus group, no specific  changes from control were observed for pH, Pco2, BE, H C O 3 , T C O 2 , Hb, and lactate concentration. In contrast, fetal arterial P02 and O2 saturation increased slightly, but significantly from the baseline values during all infusion steps. There were also significant alterations in fetal glucose concentration. In the 120 d fetus group, O2 saturation fell slightly during steps 1-3 and in the post-infusion period and there was a rise in glucose levels during steps 3, 4 , 5, and post-infusion, while the other parameters remained unchanged. Arterial blood pressure and heart rates were recorded throughout the experiments for the purposes of monitoring the cardiovascular function of the animals and detecting any potential changes caused by drug administration (Table 2.10).  The Powerlab® data  collection system samples the heart rate and blood pressure variables at 100 Hz and calculates 1-min averages for each variable; however, the data shown in Figures 2.15-2.19 are 10-min average values.  In the lamb and adult groups,  pronounced agitation was observed for infusion steps 4 and 5 in almost all animals.  Symptoms included restlessness, tremor, excessive bleating, feet  stomping, and heavy breathing. The agitation symptoms usually disappeared by 1-2 hours post-infusion. Upon examination of the blood pressure and heart rate profiles (Figures 2.15-2.19, Table 2.10), both increased significantly at step 5 in  89  the 10d lamb group.  On the other hand, little change occurred to the blood  pressure in the 30d lambs; however, heart rate increased significantly from steps 2-5. Increases in heart rate were observed after step 3 in the fetal groups, with the increase in the 120 d fetuses being statistically significant.  No specific  changes were observed for blood pressure during steps 4 and 5 in the fetal groups.  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TJ.52 E E TJ £ O > ro O 5 O _Q cyj Q. to C  "s  CL CQ  ro >  E o  o c  O co  o c  93  .g 60 E E 50 3  CD  a 40 CO  oo  2 30 CL  £ 20 CD  5  10  "ro  o5  0  500  1000  1500  2000  2500  3000  3500  Time (min)  Figure 2.15a Fetal arterial pressure vs. time in the 100 d fetus group. The numbers 1-5 represent the infusion steps.  400  n  0  500  1000  1500  2000  2500  3000  3500  Time (min)  Figure 2.15b Fetal heart rate vs. time in the 100 d fetus group. The numbers 1-5 represent the infusion steps.  94  60 D)  ?  50  2> 40 2 a.  30  "ro  "S  20  < JS 10 (D  500  1000  1500  2000  2500  3000  3500  Time (min)  Figure 2.16a Fetal arterial pressure vs. time in the 120 d fetus group. The numbers 1-5 represent the infusion steps.  Figure 2.16b Fetal heart rate vs. time in the 120 d fetus group. The numbers 15 represent the infusion steps.  95  100  80  CD  60  ^—•  OO 00  Q_  40  to CD  5  20  0  500  1000  i500  2000  2500  3000  3500  Time (min)  Figure 2.17a Arterial pressure vs. time in the 10 d lamb group. The numbers 15 represent the infusion steps.  300  0  500  1000  1500  2000  2500  3000  3500  Time (min)  Figure 2.17b Heart rate vs. time in the 10 d lamb group. represent the infusion steps.  The numbers 1-5  96  200 i  T i m e (min)  Figure 2.18a Arterial pressure vs. time in the 30 d lamb group. The numbers 15 represent the infusion steps.  0  500  1000  1500  2000  2500  3000  3500  Time (min)  Figure 2.18b Heart rate vs. time in the 30 d lamb group. represent the infusion steps.  The numbers 1-5  97  Figure 2.19a Arterial pressure vs. time in the adult group. represent the infusion steps.  120  The numbers 1-5  n  20 0  J  0  ,  ,  ,  ,  ,  .  ,  500  1000  1500  2000  2500  3000  3500  Time (min)  Figure 2.19b Heart rate vs. time in the adult group. The numbers 1-5 represent the infusion steps.  98  2.3 Discussion  2.3.1 MD Probe Recovery  Although the failure rate of the microdialysis probes was relatively high (~ 1/3 of the probes failed to work within a few days after the surgeries), those that remained working provided very consistent recovery of the drug with low variabilities (43.40 ± 2 . 1 5 %, Table 2.1). In comparison, the recovery rate from our study is comparable to that of phenobarbital (44%) and lamotrigine (42%) in another MD study (Potschka et al., 2002). However, MD probe recovery rates for other compounds varied from 7.9 ± 3.0% for zidovudine and 8.7 ± 0.6% for camptothecin to close to unity for 6-mercaptopurine and morphine-3-glucuronide (Wong et al., 1993; Deguchi et al., 2000; Xie et al., 2000; Tsai et al., 2001). It is still not clear why such a high failure rate existed; however, pulling of the catheters by the animals and/or air bubble formation inside the MD catheter are thought to be the main causes. The failure rate in the current study is similar to that in another MD study conducted in our laboratory on valproic acid (-30%, personal communication with Eddie Kwan). As stated in section 1.6, the method of retrodialysis for probe calibration provides several advantages over in vitro methods (Wang et al., 1993; De Lange et al., 1998). Therefore, together with the high consistency in recovery, we believe that this technique provided reliable measures of DPHM concentrations in the CSF and ECF compartments.  99  2.3.2 CSF, ECF, and Plasma Relationships in Relation to Age  It can be seen that the CSF and ECF concentrations were very similar to each other in all age groups throughout the dose ranges studied (Figures 2.3a-e). This rapid equilibrium in concentrations suggested that transfer of DPHM between the 2 compartments was a passive diffusion process.  In fact, no  transporters have been identified to date on the neuroependyma which is the cell layer separating the ECF and CSF compartments (Davson and Segal, 1996). Also, this homogeneous distribution of the drug over the brain suggests the lack of local metabolism (Kerr et al., 1984).  A similarity in CSF and ECF  concentrations has also been observed for phenytoin (Scheyer et al., 1994). However, for most of the drugs studied using microdialysis (i.e. zidovudine, 6mercaptopurine, lamotrigine, norfloxacin, valproic acid, and alovudine) the two CNS concentrations are different from each other (Wang et al., 1993; Wong et al., 1993; Ooie et al., 1997; Deguchi et al., 2000; Seism et al., 2000; Stahle and Borg, 2000; Walker et al., 2000; Deguchi and Morimoto, 2001). Specifically, ECF concentrations are higher than CSF concentrations for valproic acid (-30%) lamotrigine (~50%), and 6-mercaptopurine (~ 5 fold) (Deguchi et al., 2000; Walker et al., 2000; Deguchi and Morimoto, 2001; Lindberger et al., 2001). On the other hand, CSF concentrations are higher than ECF concentrations for zidovudine (~1.5 fold), norfloxacin (~2 fold) , and alovudine (up to ~5 fold) (Wong et al., 1993; Wang and Sawchuk, 1995; Ooie et al., 1997; Stahle and Borg, 2000).  The difference in CSF and ECF concentrations could be due to local  100 metabolism (Kerr et al., 1984). However, a more likely explanation would be that different transport processes were operative in the CSF from those in the brain ECF, resulting in different steady-state concentrations in the two compartments (Hammarlund-Udenaes, 2000). The similarity in DPHM concentrations in CSF and ECF suggests that this is not the case for this drug.  Figure 2.6 shows the C SFSS/CP S and CECFSS/CP S ratios from the different age C  US  US  groups. The fact that all of the ratios were higher than 1 indicates that DPHM concentrations were higher in the CNS than in the plasma. Such an observation is not limited to DPHM as other lipophilic drugs such as codeine, carbamazepine, morphine, and phenytoin also have brain/plasma concentration ratios close to or higher than unity (Scheyer et al., 1994; Van Belle et al., 1995; Wu et al., 1997; Xie and Hammarlund-Udenaes, 1998).  More polar compounds, on the other  hand, including morphine-6-glucuronide, morphine-3-glucuronide, lamotrigine, 6mercaptopurine, zidovudine, valproic acid, and gabapentin all have brain/plasma concentration ratios below 0.6 (Wang et al., 1993; Wang and Welty, 1996; Deguchi et al., 2000; Seism et al., 2000; Walker et al., 2000; Xie et al., 2000; Bouw et al., 2001). Data from another MD study of valproic acid in our laboratory show  a brain/plasma  concentration  communication with Eddie Kwan).  ratio  of approximately  0.2  (personal  Therefore, lipophilicity seems to play an  important role in determining the extent of entry into the brain for a compound.  101 Both CNS and plasma concentrations showed corresponding increases with dose.  Furthermore, a significant trend existed in which CNS concentrations  started higher than plasma concentrations in the fetal groups, approximated each other in the lamb groups, and finally became lower than plasma concentrations in the adult group.  Examination of CCSFSS/CP  ss  and CECFSS/CP ratios also reveals ss  a similar trend (Figure 2.4). Since only the unbound drug in plasma can cross the BBB and serve as the active moiety to elicit pharmacologic effects, the relationships between brain and free plasma concentrations are more relevant than the relationship with total plasma concentration. Although C SFSS/CP S and C  CECFSS/CPUSS  US  ratios increased post-natally (Figure 2.6), the increases were caused  by changes in protein binding.  The extent of protein binding  increased  significantly after birth (Figure 2.5), resulting in decreases in free DPHM concentration.  Therefore, the increases seen in CCSFSS/CP S and CECFSS/CP US  USS  ratios for the postnatal lambs were due to division by smaller values (i.e. lower free DPHM concentrations), rather than an increase in transfer of DPHM into the CNS.  Although a significant increase in protein binding was also observed in the  adults, the CCSFSS/CP S and C CFSS/CP S ratios are lower than those in the 120 d US  E  US  fetal group due to large differences in the brain concentrations between the two groups. For example, step 5 CCSFSS was 70.08% and CECFSS was 70.58% lower in the adult group compared to the 120 d fetus group. Overall, trends from these ratios indicate that brain clearance of DPHM increased with age, which will be discussed further later with comparisons of pharmacokinetic parameters.  102  The high CCSFSS/CP S and CECFSS/CP S ratios also suggest that DPHM was US  US  transported into the CNS in sheep. If only passive dffusion was involved in the transfer, the ratios should approximate 1. The fact that all of the calculated ratios were at least 3 or above indicates that a concentration-dependent, passive diffusion process alone is not sufficient to explain the transfer pattern of DPHM. Transporter-mediated  influx of the drug must be involved to create such  differences in concentrations between the CNS and plasma compartments.  As  stated in section 1.1.1, many transporters are present at the BBB to deliver substances into the CNS. These include the glucose transporter for facilitated transfer of glucose into the CNS (Boado and Pardridge, 1990; Pardridge et al., 1990; Bauer, 1998; El Messari et al., 2002; Mann et al., 2003); it may also assist the  entry  of  glycoside-conjugated  drugs  such  as  analogues of Met-5-enkaphalin (Polt et al., 1994).  L-serinyl-B-D-glycoside  A number of amino acid  transporters have also been identified in the membrane components of the BBB (Smith and Stoll, 1998; Mann et al., 2003; Sakai et al., 2003).  Monocarboxylic  acid  medium  transporters  (MCT)  carry  endogenous  short  and  chain  monocarboxylic acids (Oldendorf, 1973; Spector, 1988a; Tamai and Tsuji, 2000; Pierre et al., 2002; Taylor, 2002) and may also transport xenobiotics such as salicylic acid (Terasaki et al., 1991), simvastatin acid (Tsuji et al., 1993) and valproic acid across the BBB.  Sodium independent organic cation transporters  (OCT) have also been identified in the BBB (Koepsell, 1998). classes of OCT systems have been defined:  Two distinct  a potential-sensitive transporter  usually involved in the influx of organic cations and an H  +  gradient-dependent  103 transporter, mediating efflux (Ullrich, 1994). At present, the OCT family includes three potential-sensitive (i.e. OCT1, OCT2, OCT3) and two H -driven systems +  (i.e. OCTN1 and OCTN2) (Lee et al., 2001). Of the different subtypes, OCT2, OCT3, and OCTN2 have been identified in the brain (Koepsell, 1998; Zhang et al., 1998; Lee et al., 2001).  A diverse group of organic cations, including  endogenous bioactive amines (i.e. acetylcholine, choline, dopamine, epinephrine, norepinephrine, guanidine, thiamine),  and therapeutic drugs (i.e. cimetidine,  amiloride, morphine, quinidine, verapamil) are actively transported by the OCT system (Koepsell, 1998; Wu et al., 1998; Zhang et al., 1998; Wu et al., 2000; Kido et al., 2001; Lee et al., 2001; Sweet et al., 2001; Slitt et al., 2002). There is also evidence for saturable transporter mechanisms in the BBB for a number of lipophilic  amine  rimantadine,  drugs  amantidine  including  propranolol,  and  histamine  the  lidocaine, H antagonist, r  amphetamine, mepyramine  (Pardridge and Connor, 1973; Pardridge et al., 1984; Spector, 1988b; Yamazaki et al., 1994a; Yamazaki et al., 1994b; Yamazaki et al., 1994c).  Based on the  above information, DPHM appeared to be most likely transported into the CNS by  the  lipophilic  amine  drug  transporter  since  another  Hi-antagonist,  mepyramine, had been shown to be transported by the same system. However, results from the DPHM-PRN co-administration study, which will be described in chapter 3, suggest that a transport system different from the lipophilic amine drug transporter might be responsible for DPHM transfer into the CNS.  104  Several comparisons can be made between the current study and a study performed by Goldberg et al. which examined transport of DPHM in the CNS using a brain perfusion technique in rats (Goldberg et al., 1987). In their study, the concentrations of DPHM in the CSF at steady state were approximately twice the unbound plasma concentration.  The authors concluded that other factors  besides pH partitioning (active transport, for example) must be operating. Since a carrier for weak bases (i.e. for choline) at the BBB had been documented (Cornford  et  al.,  1978),  the  investigators  simultaneously in their experiments.  perfused  DPHM  with  choline  The result was that choline did not  significantly inhibit DPHM transport throughthe BBB. In short, findings from this study are similar to ours - DPHM concentration was higher in the CSF than in plasma, but the mechanism responsible for DPHM transfer remains unknown.  Plasma concentrations increased linearly with infusion rates at all ages (Figures 2.7a - 2.11a). This indicates that even at the higher infusion steps, when plasma DPHM were much higher than the therapeutic range in the human (~50 ng/mL), there was no saturation of systemic DPHM metabolism. This is consistent with the results of a bolus dose ranging study conducted previously in the laboratory (Yoo et al., 1990).  Similar linear trends were observed in plots of brain  concentrations vs. infusion rate and brain concentrations vs. free plasma levels (Figures 2.7b/c - 2.11b/c). Although brain concentrations increased in a linear fashion with infusion rate and free DPHM concentrations, plots of brain to free plasma concentration ratios vs. infusion rate revealed a pattern that the ratios  105  stayed within relatively constant ranges (Figures 2.16 - 2.11d). In other words, their brain concentrations increased in proportion to the increase in plasma concentrations.  Overall, our data indicated that the plasma, CSF and ECF  compartments were not yet saturated at the infusion rates studied.  2.3.3 Prediction of DPHM Brain Levels Using Plasma Concentration  In  the clinical  setting,  the possibility  of direct  measurement  of  brain  concentrations of drugs is highly restricted due to the limited number of methods available and the risks involved (de Lange and Danhof, 2002). As an alternative, drug concentrations in lumbar or ventricular CSF are sometimes used as a substitute for drug concentrations at the target site within the brain. However, as stated in a review paper (de Lange and Danhof, 2002), CSF concentrations may be difficult to interpret and may have limited value for many reasons.  For  example, these authors suggested that CSF concentration may not provide useful information at brain target sites more distant from the ventricles.  Also,  CSF concentrations may not reflect regional differences within the brain that may result from conditions such as tumour growth or epileptic seizures (de Lange and Danhof, 2002).  However, CSF and ECF concentrations from our study  correlated well with each other (Tables 2.2b/c).  In addition, in our study, free  plasma concentrations showed excellent correlations with brain concentrations at all ages (Figures 2.7c - 2.11c, Table 2.6). Although the current study was performed in sheep and there are obvious physiological differences between  106  sheep and humans, the basic structures of the BBB are the same between the two species (Saunders et al., 1999b; Saunders et al., 1999a). Therefore, free plasma DPHM levels might provide reliable predictions of brain concentrations in humans.  Prediction of brain concentrations using plasma free levels provides  several advantages over other methods such as positron emission tomography and intracerebral microdialysis in terms of cost and invasiveness (de Lange and Danhof, 2002). While good correlations were observed in the current study, such relationships  might  be  compound-specific  and  therefore  require  further  investigation.  2.3.4 DPHM Pharmacokinetics in CSF, ECF, and Plasma  Significant increases in total body clearance (CIT) of DPHM were observed after birth, with a subsequent significant decrease in the adults (Figure 2.12).  This  observation is consistent with previous findings in our laboratory that C I in T  newborn lambs is much higher than in adult sheep, and is similar to fetal values (Wong et al., 2000). Wong et al. suggested that DPHM was actively secreted in renal tubules in fetal and newborn lambs, whereas active reabsorption of the drug was observed in the adults resulting in much lower CIT.  Evidence in rats  has shown that expression of organic cation transporters (OCTs) in renal tissue increases post-natally (Slitt et al., 2002).  Therefore, the authors in that study  concluded that the high level of renal OCT expression may explain why the kidney is a target organ for xenobiotics with cationic properties (Slitt et al., 2002).  107  In the current study, the low CIT for DPHM in adults may be due to the postnatal expression of OCTs in renal tubules as observed in rats. Among the different members within the OCT family, OCT1-3 function as influx  transporters  (Koepsell, 1998; Zhang et al., 1998) and they may be responsible for the renal active reabsorption of DPHM in adult sheep.  However, this requires further  investigation as expression of these transporters may be different between rats and sheep.  In contrast to CIT, both  fcsF  and  f cF E  decreased with age indicating that CNS drug  elimination became more efficient with the increase in age (Figure 2.13). This is most likely due to the development and maturation of efflux mechanisms with age (Minn et al., 1991; Saunders and Dzielgielewska, 1998; Saunders et al., 1999a; Saunders et al., 2000).  In terms of distributional parameters, protein binding increased with age (Figure 2.5) and this finding is consistent with results from previous studies in sheep (Kumar et al., 2000). On the other hand, V d fetal values (Figure 2.14, Table 2.7).  s s  dropped postnatally compared to  A decrease in V d  s s  following birth is  expected since drug administered post-natally cannot distribute to the maternal compartment that is available to the fetus. decreases in V d  s s  Wong et al. also observed similar  in their study in post-natal lambs (Wong et al., 2000). V d  s s  in  the adult is significantly higher than fetal and lamb values due to the buildup of body fat (Smith et al., 1987; Owens et al., 1993).  At the same time, C I is T  108  significantly lower in adults due to reasons described earlier in this section. Presumably, since ti/2p is calculated by the equation 0.639'Vd/Cli, a higher V d  s s  but lower CIT value relative to the fetal and lamb groups would lead to a significantly longer elimination half-life in the adult.  In fact, this is what we  observed in the adult ti/2p (Table 2.7).  The relatively short ti p values in CSF and ECF indicated that DPHM was rapidly /2  eliminated from these compartments. Goldberg et al. also observed a rapid rate of efflux of DPHM from rabbit choroid plexus determined using a brain homogenate method (Goldberg et al., 1987). Because DPHM is, to a great extent, ionized at physiologic pH, simple diffusion of the drug back to cerebral circulation alone does not seem adequate to explain this observation.  As  mentioned in section 1.1.4, besides diffusion, substances can leave the CNS by two other means. One comprises efflux (transporter-mediated or not) via brain or choroidal blood. The second route involves efflux via bulk flow of CSF draining into either the lymphatic system or venous blood through the arachnoid villi in the superior sagittal sinus (Bradbury et al., 1972). The latter phenomenon is responsible for what is termed the sink effect.  While CSF flow is a normal  physiological process that happens regardless of the substance, transportermediated efflux is a substrate-specific process.  In the following chapter, the  possibility of transporter involvement in the efflux of DPHM from the CNS will be examined.  109  2.3.5 Physiological Responses  Arterial  blood  pressure  and  heart  rate  were  monitored  throughout  the  experiments to detect any potential changes that might be caused by the drug. As stated earlier in section 2.2.3, pronounced agitation was observed for infusion steps 4 and 5 in the lamb and adult groups. Symptoms included restlessness, tremor, excessive bleating, feet stamping, and heavy breathing. The symptoms were most likely caused by the high levels of DPHM in the brain. It has been reported that at high or toxic doses, DPHM can provoke CNS stimulation, convulsions, cardiovascular and pulmonary collapse (Koppel et al., 1987; Garrison, 1991). Upon examination of the blood pressure and heart rate profiles (Figure 2.15 - 2.19), blood pressure actually dropped after DPHM administration in the fetal groups, with increases in heart rate at higher infusion steps (i.e. steps 4 and 5). In contrast, both blood pressure and heart rate increased at steps 4 and 5 in the newborn lambs. In the adults, except for a blood pressure drop in step 3, both blood pressure and heart rate did not show any specific changes from control during the infusion.  It is not clear why changes in blood pressure  and heart rate did not happen to all ages; however, comparisons can be made to a former study. In a study conducted by Rurak et al. (Rurak et al., 1988), DPHM was given to the pregnant ewe as separate maternal and fetal infusions. During the fetal infusions (n = 8), DPHM was infused for 90 min at 0.17 mg/min and this rate was comparable to step 4 of the current study.  There was a significant  increase in heart rate during the first 30 min of the infusion period, with a return  110 to control levels thereafter. Arterial pressure was not significantly changed. The rise in fetal heart rate could be due to anticholinergic actions of the drug. Alternatively, potentiation of the effects ofendogenous histamine by DPHM could be involved in the tachycardia. However, the cardiac effects of histamine and its antagonists are complex and further study is needed to understand the cardiac effects of DPHM in sheep (Haggstrom and Hirschowitz, 1984; Kang et al., 1987). In terms of blood gas values, most of the parameters showed little change throughout the experiment (Tables 2.9, 2.10). Decreases in O2 saturation were observed in both fetal groups during the infusion; however, whether or not such decreases were due to the effects of DPHM is unknown.  2.3.6 CNS Effects of DPHM in the Fetus  In a previous study of DPHM in pregnant sheep, it was observed that the percentage of low voltage electrocorticographic (ECoG) patterns and the overall incidence of fetal breathing decreased with DPHM maternal administration (Rurak et al., 1988). These sedative-like effects occurred at fetal plasma drug concentrations of ~ 36 ng/mL which were lower than those resulting in discernible central nervous system effects in adults (> 50 ng/mL) (Carruthers et al., 1978). As noted in the introduction, in both sheep and humans, other CNS drugs affect fetal behavioral state at plasma concentrations lower than those required for CNS effects in adults and these observations provided one of the rationales for the current study.  However, fetal behavioral parameters were not measured in  Ill our study.  This was because measurement of these variables  requires  implantation of bilateral electrodes in the parietal cortex to monitor electrocortical (ECoG) activity (Rurak et al., 1988). Since microdialysis probes were implanted into the parietal cortex (ECF) and the lateral ventricle (CSF), implantation of the ECoG electrodes was not feasible.  However, given the findings from the  previous study, several inferences can be drawn. As noted above, in the study of Rurak et al. (1988), CNS sedative effects were achieved in the fetus (131 d gestation) at a plasma concentration of 36 ng/mL. This is slightly higher than the plasma concentration of 26.9 ng/mL achieved during the step 1 infusion in the 120 d fetal group in the current study (Table 2.2a).  However, the brain ECF  concentration at step 1 was 51 ng/mL (Table 2.2c), and in adult ewes this ECF concentration was not reached until infusion step 2, when plasma DPHM levels were 162.3 ng/mL. This is considerably higher than the plasma concentration of the drug associated with CNS effects in adult humans.  Thus, the greater  exposure of the fetal brain to DPHM appears to explain its greater CNS effect in the fetus.  In terms of the relevance of these observations to humans, fetal exposure to DPHM following maternal bolus administration is extensive (i.e. AUC fetal/AUC maternal = 0.85) (Yoo et al., 1986b).  In humans, peak plasma concentrations  following a 50 mg oral dose are between 40 r 80 ng/mL (Carruthers et al., 1978; Luna et al., 1989). Assuming that placental transfer of DPHM in human is similar to sheep, a 50 mg oral dose to a pregnant woman would result in peak fetal  112  plasma concentrations of 34 - 68 ng/mL.  Assuming a brain/plasma ratio of  approximately 2 (Tables 2.3a/b) also applies to human, the estimated fetal brain concentration will range from ~ 70 - 140 ng/mL. Thus, alterations in the behavior of human fetus are likely to occur.  2.3.7 Summary of the DPHM 5-Step Infusion Study  In summary, the MD probes in our study provided consistent and reliable measurement of DPHM concentrations in the CNS. DPHM was transported into the brain, resulting in C S F / C C  P u  and C C F / C E  P u  ratios higher than 1. CSF and ECF  concentrations increased linearly with the dose ranges studied in all ages. and f cF decreased with age, suggesting increasing efficiency for E  f sF C  DPHM  elimination from the brain. On the other hand, CIT was the lowest in adults, most likely due to renal tubular reabsorption.  Transporter-mediated efflux of DPHM  might be involved in its rapid elimination from the brain. This possibility will be discussed further in chapter 3. good  prediction  of  Free plasma DPHM concentrations provided  brain concentrations.  However,  before  applying  this  observation in the clinical setting, further studies are warranted to examine if such correlations exist for other compounds.  Finally, the greater effects of  DPHM in altering behavior of the fetus compared to adults appears to be due to the greater exposure of the fetal brain to the drug.  113  Chapter 3 DPHM-Propranolol Co-administration Study  In the previous chapter, we have investigated the blood-brain ECF and bloodCSF drug concentration relationships as a function of pre- and postnatal age, and drug dose.  One of the most interesting findings from the 5-step infusion  studies was the possibility of a transporter-mediated efflux mechanism in the CSF  and ECF compartments  development.  for DPHM, whose  activity  increased  with  The following study involved co-administration of DPHM and  propranolol (PRN) to examine if PRN alters blood-brain CSF and blood-brain ECF DPHM relationships. The results from this study will provide information on how the CNS eliminates lipophilic, amine compounds such as DPHM.  114 3.1  Methods  3.1.1 Animals and Surgical Preparation  Fetuses:  In total, 9 pregnant Dorset Suffolk cross-bred ewes (n=3 for 100 d  fetus; n=6 for 120 d fetus) were surgically prepared at least three days prior to experimentation.  The surgical procedure, estimation of fetal weights, and pre-  experimental preparations are as described in chapter 2 (section 2.1.1).  Newborn Lambs: A total of 10 Dorset Suffolk cross-bred newborn lambs were used. The lambs were divided into a 10 day old group (n=4) and a 30 day old group (n=5).  Surgical procedures and pre-experimental preparations are as  described in chapter 2 (section 2.1.1).  Adult Sheep: Six non-pregnant Dorset Suffolk cross-bred ewes were employed. All sheep were surgically prepared and allowed to recover as described in chapter 2 (section 2.1.1).  3.1.2 Experimental Protocols  The protocol involved bolus i.v. loading doses of DPHM (to hasten the achievement of steady-state), followed by i.v. infusion of the drug using an infusion pump. Following the administration of an i.v. bolus dose (0.7 mg/kg in all ages), DPHM (Diphenhydramine hydrochloride, Sigma Chemical Co., St. Louis,  115 MO) was infused at step 4 for 8 h {i.e. 195.5 Lig/kg/min for 100 d fetus; 156.4 ug/kg/min for 120 d fetus; 47.25 Lig/kg/min for 10 d lamb; 54 Lig/kg/min for 30 d lamb; and 13.5 Lig/kg/min for adult).  Four hours after initiating the DPHM  infusion, PRN (Propranolol hydrochloride, Sigma Chemical Co., St. Louis, MO) was co-infused [1.5 mg/kg loading dose, 20 Lig/kg/min for all ages] for the remaining 4 h of the DPHM infusion. The PRN dose was to target a C  P s s  of ~100  ng/mL based on pharmacokinetic parameters obtained in sheep from other studies (Jones and Ritchie, 1978; Mihaly et al., 1982; Czuba et al., 1988). Blood and MD samples were collected as described in section 2.1.2 during the infusion and up to 12 h post-infusion. All doses were prepared in 0.9% sodium chloride solution and were sterilized by filtering through a 0.22 um nylon syringe filter (MSI, Westboro, MA) into a capped empty sterile injection vial.  During the  infusions arterial blood samples (3 mL adult, 0.5 mL fetus and lamb) were collected at -5, 5, 15, 30 min, and 1, 2, 3, 4, 4.083, 4.25, 4.5, 5, 6, 7, 8, 8.083, 8.25, 8.5, 9, 10, 11, 13, 16, and 20 h.  Maternal drug-free blood (10 mL) was  infused into the fetus every 6 h for volume replacement. Samples (0.5 mL) were collected from the fetus at intervals for assessment of blood gas and metabolic status.  The microdialysis pump infusion rate was 2 uL/min and 60-min  cumulative samples were collected throughout the duration of the experiment. The dialysate was degassed, sterile lactated ringer solution containing 400 ng/mL of [ H ]-DPHM. 2  10  All blood samples collected were placed into EDTA-  containing Vacutainer® tubes (Becton-Dickinson, Rutherford, NJ) and centrifuged at 2000 x g for 10 min. The plasma supernatant layer was removed and placed  116  into clean borosilicate test tubes with polytetrafluoroethylene-lined caps.  MD  dialysate samples were collected directly into clean borosilicate test tubes. Plasma and MD samples were stored frozen at -20°C until the time of analysis.  3.1.3  Retrodialysis  Microdialysis sampling began at the onset of the infusion and 60-min cumulative samples were collected throughout the duration of the experiment.  Probe  recovery rates were determined using the retrodialysis technique (De Lange et al., 1998) as described in section 2.1.3.  3.1.4 Physiological Recording  Arterial pressure and heart rate were recorded in all animals.  In addition,  amniotic and fetal tracheal pressures were recorded in the pregnant ewes using a computerized data acquisition system as described in section 2.1.4. p0 , pC0 2  2  Blood  and pH measurements were made with an IL 1306 pH/Blood gas  analyzer (Allied Instrumentation Laboratory, Milan, Italy), blood 0 saturation and 2  hemoglobin concentrations with a hemoximeter (Radiometer,  Copenhagen,  Denmark), and blood glucose and lactate levels with a 2300 STAT plus glucose/lactate analyzer (Y.S.I. Inc., Yellow Springs, OH, USA) to ensure the well-being of the animals throughout the studies.  117 3.1.5 Determination of DPHM Plasma Protein Binding  Protein binding of DPHM in the plasma samples was determined by an equilibrium dialysis procedure described by Yoo et al. (Yoo et al., 1993).  Free  drug levels were determined in plasma samples collected at the 4 and 8 hours t h  th  of the experiment (when steady-states pre- and post-PRN administration had been achieved).  The equilibrium dialysis procedure and calculation of free  fraction were detailed in section 2.1.5.  3.1.6 DPHM and [ H ]-DPHM Extraction Procedure 2  10  DPHM and [ Hio]-DPHM concentrations were measured using a previously 2  developed gas chromatographic mass spectrometric (GC-MS) analytical method (Tonn et al., 1993) as described in section 2.1.6.  3.1.7 Pharmacokinetic Analysis  Pharmacokinetic parameters were calculated by standard methods as described in Gibaldi and Perrier (Gibaldi and Perrier, 1982). The CSF and ECF clearance values in this study were calculated by relating the CSF and ECF AUC ->- values 0  to the plasma AUC ^.«» value. Details can be found in section 2.1.7. 0  118  3.1.8 Statistical Analysis  All data are reported as mean ± S.D. compared  Pharmacokinetic parameters were  using paired t-test for pre- and post-PRN comparisons.  For  comparisons of heart rate changes associated with PRN administration, hourly averages of heart rate were calculated from 0-4 h and from 4-8 h, and the values were compared using ANOVA followed by a Duncan's multiple comparison test. The combination of ANOVA and Duncan's multiple comparison test was also applied to the comparisons of percentage increase in ratios after PRN adminstration.  CCSF/CP  u  and  C CF/CP E  U  The significance level was p < 0.05 in all cases.  3.2 Results  3.2.1 Changes in DPHM Concentrations with Propranolol Coadministration  Average age of the fetuses on the day of their experiments was 105.8 + 1.7 days for the 100 d group and 126.3 ± 1.5 days for the 120 d group. For the lambs, the average age on the day of their experiments was 13.6 ± 1.7 days and 35.9 ± 1.2 days for the 10 d and 30 d lamb groups, respectively. Mean pregnant ewe body weight for the fetal experiments (100 and 120 d) was 81.50 ± 9.32 kg, mean estimated body weights for the 100 d fetus was 1.40 ± 0.20 kg, and for the 120 d fetus was 2.70 ± 0.40 kg. Estimation of fetal weights in utero is described in section 2.2.1. Mean body weights for the 10 d and 30 d lamb groups were 7.05 ±  119 1.52 kg and 12.16 ± 1.14 kg, respectively.  Mean adult ewe body weight was  74.64 ± 21.96 kg. MD probe recovery rates ranged between 40-50% across the different age groups (Table 3.1).  The semilogarithmic plots of mean DPHM  concentration vs. time for all the age groups are shown in Figures 3.1a-e.  As  seen in the plots, the CSF and ECF concentration profiles were very similar to each other within each group. General increases in the brain concentrations can also be seen after co-administration of PRN at 4 h.  Table 3.2 provides a  comparison of steady-state DPHM concentrations before (Pre-PRN) and after PRN (Post-PRN) co-administration for all three fluids. Except for CSF in 120 d fetuses and ECF in adults, both CSF and ECF concentrations significantly after PRN co-administration in all ages.  increased  Although no statistically  significant increases were observed in CSF in 120 d fetuses and ECF in adults, their post-PRN values were comparatively higher than the Pre-PRN values. The lack of statistical significance in both cases appeared to be due to high interindividual variability.  DPHM plasma concentrations remained the same in all  groups except in the 100 d fetuses, where a significant increase was observed. Figure 3.2 illustrates the extent of protein binding before and after PRN coadministration.  Except in the 100 d fetus group, no changes were observed.  Changes in CcsFss/Cp s and C CFSS/CP SS ratios before and after PRN coUS  E  U  administration in the different age groups are shown in Figure 3.3. Increases in the ratios were seen in all age groups. However, significant increases were not found in all cases due to high inter-individual variability.  Figure 3.4 shows the  percentage increase in the CCSFSS/C S and CECFSS/CP S ratios in different age PUS  US  120  groups. As illustrated, there was a trend for higher percentage increases in the ratios as the animals developed.  Table 3.1 Summary of MD probe recovery rates in the DPHM-PRN coadministration experiment. Percentage of Recovery ("/o) CSF Probe ECF Probe 1  Age Fetus (100 d)  39.68±6.17  a  40.98±6.15  b  Fetus (120 d)  40.07±3.28  c  41.68±4.10  d  Lamb (10 d)  42.84+3.52  42.53±2.46  f  Lamb (30 d)  42.18±6.04  9  45.03±3.27  h  Adult  44.54±3.96  j  43.97±3.41  j  e  V r o b e recovery rates were calculated using Equation 1. n=2; n=2; n=4; n=4; n=3; n=4; n=2; n=8; 'n=5; n=3  a  b  c  d  e  f  9  h  j  121  1000  0  5  10  15  20  Time (h)  0  5  10  15  20  Time (h)  Figure 3.1 Mean DPHM CSF, ECF, and plasma concentration vs. time profiles in (A) 100 d (n=2), and (B) 120 d (n=6) fetuses from the DPHM-PRN study. The arrow denotes the start of the PRN infusion. Error bars are omitted for reasons of clarity.  122  1000  o  100 <D —'  c  I  O D)  10 t  CL Q  1000  £=  o  o O  100 cn  S  I CL Q  10  Figure 3.1 Mean DPHM CSF, ECF, and plasma concentration vs. time profiles in (C) 10 d (n=4), and (D) 30 d (n=4) lambs from the DPHM-PRN study.  The  arrow denotes the start of the PRN infusion. Error bars are omitted for reasons of clarity.  123  ECF  0  5  10 Time (h)  15  20  Figure 3.1 Mean DPHM CSF, ECF, and plasma concentration vs. time profiles in (E) adults (n=6) from the DPHM-PRN study. The arrow denotes the start of the PRN infusion. Error bars are omitted for reasons of clarity.  CD  Ct  LO  NT  (A O CL  CO  +i  CL  +i  CO s  I co  o  CD  CO I -' LO s  +1  o CN O CD  O  Ct Q. £ 0_  CO CO  +1 CO LO Nt  CN  I s  cq co T—  +i  CO LO CO LO  LO  LO I CM s  CL  CM  o  I d  0.  co  I LO  ct  CD co  d CN CM  +i s  q  co s  h-  cn ci Nt  CN  +1  I CM I CO s  s  +1  CD  d o  cci CD  CD CO CD  CD d  CO  +1  +i  I -  co co  CN d CO  CM CM CD  o CO  CD  s s  ro  (0  Ct CL  £ 0.  CD 00 CN  +1  q  co (0 3  Qi CD <  T—  +1  +1  LO CD CO  LO CO Nt  00  CD  Nt  I -  h-  s  hCM  +1  Nt Nt  s  2±150.  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CD C  +1  CD  s  LL  CO O  T—  I -  CD cj  LU  Nt  CN LO  00 CO CN  CD CD CD  w  3 +* Qi  Xi  o  •o o o  T3 O CM  ns -I TS O  E  CD  s  Nt  co  CN  LO o CD  CO CD CO  E ra  3  _i T5 O CO  I CN CO TJ <  _ to |co c II Qi C  TJ  125  1  i  • Pre-PRN PB • Post-PRN PB  100d Fetus  120d Fetus  10d Lamb 30d Lamb  Adult  Age  Figure 3.2 Comparison of the extent of DPHM protein binding before and after PRN co-administration in different ages. *denotes a significant difference from the Pre-PRN value (Paired t-test; p<0.05).  126  • Pre-PRN [CSF]/[Plasma] • Pre-PRN [ECF]/[Plasma] • Post-PRN [CSF]/[Plasma] • Post-PRN [ECF]/[Plasma] 100d 120d Fetus Fetus  10d Lamb  30d Lamb  Adult  Age  Figure  3.3  Changes in C C S F S S / C S and C C F S S / C P PS  co-administration in different ages.  E  s s  ratios before and after PRN  *denotes a significant difference from the  corresponding Pre-PRN value (t-test, p<0.05).  127  250 , P  100d Fetus 120d Fetus  10d Lamb  30d Lamb  Adult  Age  Figure 3.4 Percentage increase in C S F / C C  P u  and C  E C  after PRN co-administration in different age groups.  F/C  P u  ratios at steady-state  128 3.2.2 Pharmacokinetics of DPHM in CSF, ECF, and Plasma in Fetal, Newborn, and Adult Sheep  As shown in the semilogarithmic plots of mean D P H M concentration vs. time (Figures 3.1a-e), the pharmacokinetic profiles of D P H M in CSF, ECF, and plasma in all ages were best described by a 2-compartment infusion model. Table 3.3 provides a summary of the pharmacokinetic parameters obtained from the P R N co-administration study.  Comparisons have also been made with  respect to the results obtained from the 5-step infusion study (Table 2.7). First of all, CIT remained unchanged relative to the 5-step infusion values in all ages after P R N co-administration (Figure 3.5). Although not statistically significant in all cases due to high inter-individual variability, fcsF and f cF increased in all ages E  with P R N administration. Significant increases were observed in f cF in the 120 d E  fetuses and adults (Table 3.3, Figure 3.6). When the percentage increase in CCSF/CPU  and C E C F / C P ratios were plotted with post-conceptional age, a highly u  significant correlation (r = 0.7787, p<0.001) was observed with an equation form of y = -13717/x + 146.2 (Figure 3.7).  Significant decreases in V d  s s  were  observed in the two fetal groups and in the adults (Table 3.3, Figure 3.8). 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CN CO  CO CD + J CD  E S  CO  CL  E  LL CO  c  s  CM  XJ  ^  O  '  to  co E co  CO  _J0 CL  c  ' o T  LL CO  o  LL W O 8  LL CO  o cz  O Z> <  ° 3  0)  a E  3  CO  CO O  o < o  111  o  o LU  K CM T  _  130  Figure 3.5 Comparison of C I obtained from the 5-step infusion and DPHM-PRN co-administration studies in different ages. T  • 5-step fCSF • 5-step fECF  5 -  • DPHM-PRN fCSF 4  • DPHM-PRN fECF  u_  (\ LU H —  O LL w o  3  2  1  0 100d Fetus  120d F e t u s  10dLamb  30d L a m b  Adult  Age  Figure 3.6 Comparison of f F and f F obtained from the 5-step infusion and DPHM-PRN co-administration studies. C S  E C  131  Figure 3.7 Correlation plot between the percentage increase in CCSF/CP and CECF/CPU ratios and post-conceptional age. u  132  100  n  75 • 5-Step Vdss • DPHM-PRN Vdss 50  > 25  0  LL_£k  100d Fetus  120d Fetus  *  10d Lamb  30d Lamb  Adult  Age  Figure 3.8 V d from the 5-step infusion and DPHM-PRN studies at different ages. *denotes significant difference from the 5-step infusion value (p<0.05). s s  133 3.2.3 Physiological Responses in Fetuses, Newborn Lambs, and Adult Sheep Similar to the 5-step infusion study, arterial blood pressure and heart rates were recorded throughout the experiments for the purposes of monitoring the cardiovascular function of the animals and detecting any potential changes caused by drug administration.  In addition, we were interested in the potential  effects that the B-blocker PRN, might elicit on the cardiovascular function of the animals. The Powerlab® system samples each variable at 100 Hz and calculates consecutive 1-min averages; however, the data shown in Figures 3.9-3.13 are 10-min average values. The addition of PRN did not seem to cause any specific changes in blood pressure across all ages (Table 3.4).  On the other hand,  significant decreases in heart rate were observed in the 10 d lambs, 30 d lambs, and adult after the start of PRN infusion (Table 3.4). Such decreases in heart rate also occurred in the 120 d fetus group at the initial phase of the coadministration, but returned to pre-PRN levels in about 150 min.  Finally, no  specific changes were observed in the 100 d fetal heart rate after PRN coadministration.  Tables 3.5 and 3.6 provide a summary of the blood gas status in the fetal groups during the DPHM-PRN co-administration.  In general, most of the parameters  remained unchanged with PRN co-infusion except P02 and O2 saturation. Specifically, there was a significant increase in P02 in the 100 d fetus during DPHM infusion, but this returned to baseline levels after PRN co-infusion. In contrast, post-infusion O2 saturation dropped significantly compared to the level  134 during DPHM infusion (Table 3.5). In the 120 d fetus, both P 2 and 0 saturation 2  0  decreased with time during the experiment, with 0 saturation ending at a level 2  significantly lower than baseline (Table 3.6).  Table 3.4 .Arterial blood pressure and heart rate values before and after PRN coadministration for fetal (100 & 120 d), lamb (10 & 30 d), and adult sheep.  Pre-PRN  Age  Post-PRN  BP (mmHg)  HR (bpm)  BP (mmHg)  HR (bpm)  100d Fetus  3  35.6 ± 1.9  169.2 ± 2 1 . 6  35.8 ± 1.6  154.7 ± 14.4  120d Fetus  b  45.9 ± 5 . 2  139.6 ± 11.7  44.0 ± 4 . 5  124.3 ± 10.8  10d Lamb  0  55.9 ± 3.4  161.3±7.0  57.9 ± 2 . 8  113.8 ± 3 . 7 *  30d Lamb  d  63.6 ± 5.6  130.5 ± 4 . 5  68.4 ± 3.2  106.6 ± 4 . 1 *  101.9 ± 2 . 1  95.5 ± 5 . 8  100.5 ± 3 . 6  77.7 ± 8.5*  Adult  6  BP = blood pressure; HR = heart rate; mmHg = millimeter Mercury; bpm = beats per minute. n=3; n=6; n=4; n=4; n=6 *denotes significant difference from the Pre-PRN values (paired t-test; p<0.05) a  b  c  d  e  135  60 50 Z3  Ui in  40  0  Q_ O) 1" E 30 co E  •c  i  "53  20  10  200  400  600  800  1000  1200  Time (min)  Figure 3.9a Arterial pressure vs. time in the 100 d fetus group. T h e arrow d e n o t e s the start of the P R N infusion.  400.0  200  400  600  800  1000  1200  Time (min)  Figure 3.9b Heart rate vs. time in the 100 d fetus group. T h e arrow d e n o t e s the start of the P R N infusion.  136  100  80 -I  |  2 w  60  a. «  2  0  0  200  400  600  800  1000  1200  Time (min)  Figure 3.10a Arterial pressure vs. time in the 120 d fetus group. The arrow denotes the start of the PRN infusion.  200  _ 150 E Q.  € 100 or I  t  50  200  400  600  800  1000  1200  Time (min)  Figure 3.10b Heart rate vs. time in the 120 d fetus group. The arrow denotes the start of the PRN infusion.  137  100  80 8> 3 0) 05 ^ _  60  c —  40  CU  •tz <  20  200  400  600  800  1000  1200  Time (min)  Figure 3.11a Arterial pressure vs. time in the 10 d lamb group. The arrow denotes the start of the PRN infusion.  200.0  n  x 50.0  -  0.0  -I 0  1  200  ;  ,  400  ,  -600  1  800  ,  1000  ,  .  1200  Time (min)  Figure 3.11b Heart rate vs. time in the 10 d lamb group. The arrow denotes the start of the PRN infusion. *denotes significant decrease from Pre-PRN heart rate (p<0.05).  138  100  80 -  -  t  40  20  200  400  600  800  1000  1200  Time (min)  Figure 3.12a Arterial pressure vs. time in the 30 d lamb group. The arrow denotes the start of the PRN infusion.  200 -,  0  200  400  600  800  1000  1200  Time (min)  Figure 3.12b Heart rate vs. time in the 30 d lamb group. The arrow denotes the start of the PRN infusion. *denotes significant decrease from Pre-PRN heart rate (p<0.05).  139  400  600  800  1000  1200  Time (min)  Figure 3.13a Arterial pressure vs. time in the adult group. The arrow denotes the start of the PRN infusion.  120  n  110 100 <>  90 E CO  cu X  CL 80  -  70 60 50 40 -200  400  600  800  1000  1200  Time (min)  Figure 3.13b Heart rate vs. time in the adult group. The arrow denotes the start of the PRN infusion. *denotes significant decrease from Pre-PRN heart rate (p<0.05).  140 co (0  o o Is  d °  •  d^ ° d °  o  •  ^  0 o X LU 0  +'co d^ d °  0  CO CO  Q.  CQ II  3  E  LU CQ +1  + ' L O  CO II c  CM  + L  o ^ d  oo o  CM5  O  Q.  u o  O) CO  «-2:  w >  .a  o oT—  ox  1  o o  c g  -*—<  'E  TJ  LO'CM 00 LO  <  cri  i^. cq CD d d r-J  1  E  co cq LO d CM  CM CO  "  +1  I - CM S  d oq d d CM  +1  +i  NJ;  CO CM CM  oo oo ^ o  CM  CO  co cq d  S  +1  • o  E  x  +1 CM  CO CM  +1  +1  O)  c D TJ  OL  E E  +1  o"  CO CO  CM  co  {2  LO CM  CO L_  CO  Q_  +1  8E  CO CO O)  TJ O O  CQ LO CO  o  re  co  X  co ^ LO d +i CD CO  o~ -  +i TCM  o co  CJ) CO  CM O^  +l  oo f"T-' N t  CM  LO  o co co  E  X  II CM £ > £ O T J 0= O ™ —  CL •D  -O  xi  -£j  +i Nt Nt  .2  = P oS 5  03  a. DQ  Q  c  z  CO cfl) X^  CL  «  •- o o iS  CO CO  0  co  CL2  a> ^ D) II CM CM  '  5^0  |v. 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DO  II LU CQ +1  +1  co/o d r-i  b  CT)  d  CD  CO  CO CD  d +i  ^  CN d co +i  o~ £ O Et 0. ,, 0  CN +i  +i  d CN  CD CN  +i  +1  CD  coc/D£  - STJ O  T—  +i  co  "c  °- ^ 0 0 ro *^  co +i co +i co o+1 co CN q d d d d d +i  E  _C0  oro_ C O c CO O 3  c^™  ..  "5  23>  +l  ^ CCNN CD CO ^ d d  i_  CO +ZJ -*  X 0  +l  +1  co cq d— CN +1  T— LO CN LO q co CM CO d — LO ~ in in CN CN CN CN T  II  3  co ^  0  LJ  co ^ to co A ro  0 -a o TJ _0 'x  co-g  •2 o co TJ E 0 c 0 c o  0_  LU oi CQ CD  E  X D_ Q CO  c  ZJ TJ CO  CM  O  I  c  CL E  L—  Q) '  CD E  CO  C N ^  si  °- E  1." CN ~ T  +1  +i o  +i  C\i  +1 h~  CD  +1  +1 CO CJ)  +1  M" CO LO CO CN d LO dNLO co LO 1 ^ co C CN CN +1  CO o co cb cb •Sf CN  +1 *t CO  O £  0  9>  CM 0  CO CO CO TJ O O CO CO co"  a>  xi n  TJ TJ " X .> CL O TJ "° ro oo oc o CO  s -e  "ro  •- o CO rz — o iS ro S i c\T c  2  II co  0  N  d cb LO CN  §LO -I—»  co CL  0= c  O  CO  +1  O  -S ii II  cn  t_  X QJ  CN LO'S co z; co o^ CO  o  (flco  r  QQ  •—  I  a. Q  x Q  £  L 'CZO 0 c0 ? co 2 {jf o 0 *co 0 -2 fc TJ^ > ro C— C •JOXI5±3CZ CO OD o 0 II *iz C cS cO i CL  2° c ^ o g co 0.-  co  "I  O  x o >ro oo C L X  142 3.3 Discussion  3.3.1 Comparison of DPHM Concentrations Before and After PRN Coadministration  First of all, probe recovery was comparable to that in the 5-step infusion study (Table 3.1), ranging from - 4 0 - 45%. Figures 3.1a-e are semilogarithmic plots of mean concentration vs. time in different age groups. From visual inspection, it can be seen from the plots that elimination of all 3 fluids followed 2compartmental kinetics.  Also, fitting the data with WinNonlin® using a 2-  compartment infusion model provided the lowest AIC values, which suggested the most suitable fit. Plasma DPHM concentrations remained relatively constant but CSF and ECF concentrations increased following PRN co-administration. Table 3.2 shows the actual steady-state DPHM concentrations in all 3 fluids. In all cases, brain concentrations increased after PRN co-administration. addition, C S F S S / C P . and CECFSS/CP C  ss  ss  In  ratios indicate the same trend (Figure 3.3).  Except in the 100 d fetus group, no significant changes in protein binding levels were observed (Figure 3.2). This means the increases seen in brain DPHM concentrations were not caused by increases in the amount of unbound drug in plasma. It is not clear why the level of DPHM protein binding decreased in the 100 d fetuses. However, since PRN is a basic drug, it might compete for binding on the plasma protein a-i-acid-glycoprotein, which is known to bind DPHM (Kremer et al., 1988; Zhou et al., 1990). Considering the low levels of plasma proteins in the fetus (Dziegielewska et al., 1980a; Kwan et al., 1995), they may  143 be more susceptible to competition for binding by other drugs, particularly at earlier gestational ages. This might explain why such a significant fall in protein binding was observed in 100 d fetuses but not in the older groups.  The increase in the CCSFSS/CP unexpected result.  ss  and CECFSS/CP  ss  ratios (Figure 3.3) was a rather  As mentioned in section 1.1.1, evidence is available for a  saturable influx transporter mechanism in the BBB for a number of lipophilic amine drugs including propranolol and the histamine Hi-antagonist mepyramine (Yamazaki et al., 1994a; Yamazaki et al., 1994b; Yamazaki et al., 1994c). W e expected that the co-administration of the lipophilic amine compounds DPHM and PRN would result in competition for the transporter. This was expected to decrease the uptake of DPHM, reflected by a decrease in the CCSFSS/CP CECFSS/CPSS  ss  and  ratios. The fact that we observed an increase in these ratios might  suggest that the two drugs are transported by different influx transporters and as a result, competition was not observed.  Of all the influx transporters listed in  section 1.1.1, the organic cation transporter (OCT) in the BBB would be the most likely one responsible for DPHM transport. No studies have been conducted to assess if DPHM is a substrate for this transporter; however, such a possibility exists  since  many  methylnicotinamide,  organic choline,  cations  including  procainamide,  tetraethylammonium,  cimetidine  and  morphine  Nare  reported to be transported by OCT (Takano et al., 1984; Takano et al., 1985; Ullrich et al., 1991; Koepsell, 1998). Unfortunately, no information is available to indicate whether PRN is also a substrate for this transporter. Thus, further work is needed to confirm this assumption.  Finally, it should be noted that some  144  substrates can be transported by more than one transport system. The influx of morphine, for example, is mediated by OCT while the efflux is mediated by Pgp from the brain (Koepsell, 1998; Letrent et al., 1999; Aquilante et al., 2000; Crowe, 2002; Kastin et al., 2002; Upton, 2002; Wandel et al., 2002).  3.3.2 Effect of PRN on the CNS Pharmacokinetics of DPHM  Table 3.3 provides a summary of pharmacokinetic parameters obtained from the DPHM-PRN co-administration study.  First of all, no significant changes were  observed in CIT of DPHM compared to 5-step infusion values (Figure 3.5). However, f sF and f cF increased in all age groups after PRN co-administration C  E  (Table 3.3, Figure 3.6). Furthermore, even though not statistically significant in all cases, both ti pcsF and t i /2  /2pE  c F increased after PRN co-administration across  all age groups. Together these findings reflect lower DPHM clearances from the CNS after PRN co-administration. As mentioned earlier, besides the bulk flow of CSF, efflux of substances (transporter-mediated or not) can occur via brain or choroidal blood back to the systemic circulation. To date there is no information showing that PRN lowers CSF formation or its secretion rates or that it has any interference with the passive diffusion process of substances back to the cerebral circulation. It has also been shown that besides a slight decrease in heart rate, PRN causes no systemic or cerebral physiologic changes in sheep (O'Brien et al., 1999).  In another study PRN infusion did not significantly change choroid  plexus blood flow in sheep (Townsend et al., 1984).  Therefore, the lowered  rates of drug removal can only be explained by lowered rates of transporter-  145  mediated efflux of the drug. Propranolol could be involved in this process in one or both of the following manners - by directly inhibiting the efflux mechanism and/or by competing with DPHM for the efflux process.  Both of these actions  could account for the observed lowered rates of CNS clearances; however, the exact mechanism or transporter(s) responsible for this observation cannot be elucidated or identified by our current experiments.  Nevertheless, postulations  can be made as to the potential transporter that was responsible for the efflux of DPHM.  As stated in section 1.1.2, efflux transporters such as P-glycoprotein  (Pgp) (Cordon-Cardo et al., 1989; Tatsuta et al., 1992; Schinkel et al., 1996; van Asperen et al., 1997; Tsuji and Tamai, 1998; Rao et al., 1999; Ayrton and Morgan, 2001; Kusuhara and Sugiyama, 2002), multidrug resistance associated protein  (MRP)  (Rao et al., 1999; Wijnholds, 2002), the  monocarboxylic  transporter (MCT) family (Takanaga et al., 1995; Koehler-Stec et al., 1998; Tamai and Tsuji, 2000; Pierre et al., 2002), and organic anion transporters (i.e. oatp2) (Asaba et al., 2000; Gao et al., 2000; Ayrton and Morgan, 2001; Kusuhara and Sugiyama, 2001; Kusuhara and Sugiyama, 2002) have been identified in the BBB. The MRPs are considered amphipathic anion efflux pumps which play a major role in the elimination of amphipathic anions (many of them being phase II metabolites) from the brain (Borst et al., 1999).  It is well established that the  substrate specificity of Pgp is quite broad with respect to both chemical structure and size. The structural diversity of Pgp substrates (and inhibitors) is so broad that it is difficult to define specific structural features that are required for the substrates/inhibitors of Pgp. However, some of the properties that are shared by  146 many Pgp substrates include the presence of a nitrogen group, aromatic moieties, planar domains, large molecular size (>300), often the presence of a positive charge at physiological pH, amphipathicity, and lipophilicity (Ford et al., 1989; Bain and LeBlanc, 1996; Litman et al., 1997b; Litman et al., 1997a; Scala et al., 1997; Etievant et al., 1998; Seelig, 1998; Seelig et al., 2000; Hochman et al., 2002; Pajeva and Wiese, 2002).  Based on these general properties, one  would speculate that DPHM, which is a cation at physiological pH, would most likely be transported out of the brain by Pgp.  Results from in vitro studies,  however, showed that DPHM is not a substrate of Pgp (Mizuuchi et al., 2000; Chishty et al., 2001). The relevance and applicability of these in vitro results to our observations are difficult to assess, since experimental conditions are obviously different in vitro and in vivo. Evidence is available, on the other hand, to show that propranolol is a substrate of Pgp (Eneroth et al., 2001; Hamilton et al., 2001). It is apparent then that further work is needed to identify the specific transporters responsible for DPHM brain efflux.  There is also the possibility that the observed lowered brain clearances were due to inhibition of cerebral metabolism of DPHM. Although metabolite data are not available in the current study, such a possibility is very unlikely due to the following reasons. First, if inhibition of DPHM metabolism by PRN was involved, decreases in CIT and ti/2p should also be observed. However, both parameters remained unchanged after PRN co-administration (Table 3.3 and Figure 3.5). Moreover, previous DPHM studies by Kumar et al. (Kumar et al., 1999a; Kumar et al., 1999b) and Wong etal. (Wong et al., 2000) indicated that besides the liver,  147  the gut and lungs were likely the major organs of D P H M elimination. In addition, no studies in the literature have reported the inhibition of diphenhydramine metabolism by propranolol. Finally, the overall metabolic activity of the brain as a unit is low (Ghersi-Egea et al., 1995).  When taken together, this means that  even if cerebral metabolism of D P H M existed in the brain, it only played a very minor role in its elimination and that the observed decreases in brain clearances of D P H M were very unlikely due to inhibition of its metabolism in the brain.  Overall results from the co-administration study are consistent with the findings from the 5-step infusion study. The stepped infusion study suggested increased activity of brain clearance for D P H M in both the CSF and ECF compartments with age, and the current study showed a progressive increase in the percentage increase in C C S F S S / C P  uss  and CECFSS/CPUSS ratios after P R N administration with  advancing age (Figure 3.7).  In fact, the correlation between the percentage  increase in those ratios with age was highly significant (r = 0.7787, p < 0.001; Figure 3.7). In other words as the animals became older, the efflux mechanism played an increasingly  important role in D P H M  removal from the brain.  Therefore, interference with this mechanism by P R N caused greater increases in the brain-to-blood D P H M ratios in older animals.  Finally, significantly lower values of V d  ss  in the fetal and adult groups were  observed in the D P H M - P R N co-administration study compared to 5-step infusion values (Table 3.3, Figure 3.8). Except in 100 d fetuses, there was no change in the extent of protein binding with P R N co-administration (Figure 3.2).  Since  148  changes in Vd are a function of both changes in plasma and tissue binding, with no change in protein binding the lower V d  ss  was likely due to decreased tissue  drug uptake or binding. It should be noted that the total dose administered in the 5-step study was approximately three times higher than that in the DPHM-PRN study in all ages. A DPHM dose-ranging study performed by Yoo et al. (Yoo et al., 1990) also observed similar findings in which there was a trend towards a decrease in mean V d  ss  as the dose was decreased.  It is not clear why such  decreases were not observed in the 10 d and 30 d lamb groups (Tables 2.7 and 3.3). However, since levels of protein binding increased significantly after birth in lambs (Figure 2.5), it might have balanced out the lowered tissue binding associated with lower doses in the PRN study so that the V d  s s  values were not  significantly different in the two studies.  3.3.3 Physiological Responses  Since PRN is a (3-blocker, one would expect lowered blood pressure and heart rate with its administration.  In our experiments, it appeared that PRN did not  cause any specific effects on arterial blood pressures (Table 3.4, Figures 3.9 3.13).  However, significant decreases in heart rate were observed in 10 d and  30 d lambs and adults. The lack of physiologic effect except for changes in heart rate by PRN is consistent with similar findings in another study (O'Brien et al., 1999).  There is evidence to indicate that nerve growth along the coronary  arteries and to the myocardium starts between 75-110 days of gestation (Lebowitz et al., 1972), resulting in a progressive increase in autonomic  149  stimulation after 100 days of gestation (Vapaavouri et al., 1971; Vapaavouri et al., 1973).  This may account for our observed effect of PRN on the fetal  cardiovascular system. Furthermore, there is an increase in baroreceptor reflex sensitivity and response in the fetal lamb during the latter half of gestation (Shinebourne et al., 1971). Another study demonstrated that between 60 and 130 days gestation, the cholinergic system seems to exert little, if any, effect on the intrinsic heart rate and on the systemic and pulmonary vascular pressures and flows (Nuwayhid et al., 1975b).  In terms of the receptors involved, the fetal  heart is under the control of (3-adrenergic receptors in a manner qualitatively similar to that of the adult heart. On the other hand, both the fetal systemic and pulmonary vascular beds are under a-adrenergic receptor control (Nuwayhid et al., 1975a). Therefore, by administering a (3-adrenergic blocker (i.e. PRN), it is expected that the fetal heart rate will be more affected than the blood pressure. For example in a study conducted by Joelsson et al., PRN administration reduced fetal heart rate by a mean value of 15%, while the reduction in blood pressure amounted to only 5% (Joelsson et al., 1972).  Another study that  involved PRN administration to the fetus also observed a reduction in heart rate without effects on blood pressure (Barrett et al., 1972). When taken together, this evidence suggests that at gestational ages of 100 and 120 days, the autonomic pathways are still being developed. The administration of PRN, therefore, exerted effects on fetal heart rate only in more mature fetuses without affecting blood pressure. Overall, our results are consistent with other reported findings.  150 In terms of blood gas values, most of the parameters remained unchanged after PRN co-administration (Tables 3.5, 3.6).  A significant decrease in P02 was  observed in the 100 d fetus after PRN co-infusion; however, whether such decrease was due to the effects of DPHM is unknown.  3.3.4 Summary of the DPHM-PRN Co-administration Study  In summary, data from this study suggest that transporter-mediated efflux mechanisms exist in sheep for the CNS elimination of DPHM and that this activity appears to increase during development.  The role played by this efflux  mechanism in DPHM removal becomes increasingly more important with age; therefore, any interference with the efflux mechanism in the older animals would cause faster accumulation of the drug in the CNS compared to younger ones. Administration of PRN caused significant decreases in heart rate in the 10 d and 30 d lambs and adults.  Such an effect was not seen in the 100 d and 120 d  fetuses, however, apparently due to immaturity of their autonomic pathways. Finally, the lower V d  s s  values observed in this study compared to the 5-step  infusion study appears to be due to the administration of much smaller doses, resulting in decreases in tissue uptake or binding of the drug.  151  Chapter 4 Overall Summary and Conclusions  Although the use of xenobiotics is generally not encouraged during pregnancy, drug use by pregnant women and newborn children will continue for therapeutic and societal reasons.  DPHM, a common antihistamine, is an example of drug  that is used in pregnancy and in infants and children.  Due to its lipophilicity,  DPHM can cross the BBB and cause alterations in fetal behavioral state and may also do this in the newborn (Rurak et al., 1988). Furthermore, based on available information, several features of BBB maturation (decreasing CSF protein concentrations, altered cerebral blood flow) may result in altered blood-brain drug concentration relationships. By applying the technique of microdialysis, we were able to obtain estimates of drug concentrations in the lateral ventricle (CSF) and cerebral cortex (ECF) in sheep.  As there is limited information on the CNS  pharmacokinetics of this drug, the current studies provide information on the blood-brain relationships as a function of pre- and postnatal age.  The first study in my thesis involved i.v. administration of DPHM at 5 infusion rates in 100 d fetuses, 120 d fetuses, 10 d lambs, 30 d lambs, and adult sheep. Each step was infused for 7 h.  Semilogarithmic plots of mean  concentration vs. time for CSF, ECF, and plasma revealed  DPHM  bi-exponential  pharmacokinetics in DPHM elimination in all 3 fluids. At all ages, CSF and ECF concentrations were very similar to each other, which suggested that the transfer  152 of DPHM between these two compartments was by passive diffusion and there was a lack of local metabolism (Kerr et al., 1984). A trend existed where CNS concentrations started higher than plasma concentrations in the fetal groups, approximated each other in the lamb groups, and finally became lower than plasma concentrations in the adult group. CCSFSS/CP SS U  However, examination of the  and CECFSS/CP S ratios provided additional information. At all ages, US  the ratios were 3 or higher, indicating the existence of a transport process for DPHM into the brain.  One significance of this finding is that since drug  concentrations are higher in the fetal brain than in plasma, alterations in fetal behavioral effects may occur even at low fetal plasma concentrations. Although many transporters such as the glucose transporter (Boado and Pardridge, 1990; Pardridge  et  al.,  1990;  Bauer,  1998),  monocarboxylic  acid  transporters  (Oldendorf, 1973; Spector, 1988a), organic cation transporters (Koepsell, 1998), and amino acid transporters (Smith and Stoll, 1998) have been identified to date, DPHM was most likely delivered into the CNS by a transporter mechanism for a number of lipophilic amine drugs including propranolol, lidocaine, and the antihistamine, mepyramine (Pardridge and Connor, 1973; Pardridge et al., 1984; Yamazaki et al., 1994a; Yamazaki et al., 1994b; Yamazaki et al., 1994c). However, results from the DPHM-PRN co-administration study do not seem to support this hypothesis, indicating that further work is needed to identify the specific transporter(s) involved.  Overall, CSF, ECF, and free plasma concentrations increased linearly with dose at all ages without any indication of saturation.  In addition, the excellent  153 correlations between brain concentrations vs. free plasma levels in this study provided possibilities of predicting drug concentrations in the brain by utilizing free plasma levels in the clinical setting. Such correlations might be compoundspecific, however, again suggesting that further investigations are needed prior to application in the clinical setting.  Distributional parameters, such as protein binding and V d , changed with age. ss  Specifically, protein binding increased with age and this is consistent with results from previous studies in sheep (Kumar et al., 2000). Moreover, this is consistent with the rise in plasma protein concentration with age (Dziegielewska et al., 1980a; Kwan et al., 1995). In contrast, V d Vd  s s  s s  dropped after birth. A decrease in  following birth is expected since drug administered postnatally cannot  distribute to the maternal compartment that is available to the fetus. Wong et al. also observed similar decreases in V d  s s  in their postnatal lambs study (Wong et  al., 2000).  Although symptoms of agitation were observed at higher DPHM concentrations in the newborn lamb and adult groups, arterial blood pressure and heart rate data did not show any substantial changes in most animals.  However, obvious  increases in blood pressure in the 10 d lamb and adult groups and in heart rate in the newborn lambs at higher doses were observed.  Again, further study is  needed to obtain a better understanding of the cardiac effects of DPHM in sheep.  In terms of pharmacokinetics, CIT was lower in the adults than in the fetal and newborn lambs.  This result is consistent with previous findings (Wong et al.,  154 2000) which suggested that DPHM was actively secreted in renal tubules in fetal and newborn lambs whereas active reabsorption of the drug was observed in the adults.  On the other hand, the factors  f sF C  and  f cF E  decreased with age,  indicating that DPHM was more efficiently removed from the brain as age increased.  This is most likely due to development or maturation of efflux  mechanisms with age. The relatively short  ti/2p  values in CSF and ECF indicated  that DPHM was rapidly eliminated from these compartments.  Passive diffusion  alone appears to be inadequate to explain such a rapid elimination. In addition to the sink effect  caused  by CSF bulk flow, a transporter-mediated  mechanism might be responsible for this observation.  efflux  Such a possibility was  investigated in the DPHM-PRN co-administration study.  In the DPHM-PRN study, DPHM was infused for 8 h and PRN was co-infused from 4 - 8 h. The purpose was to examine the effects of PRN on blood-brain CSF and blood-brain ECF relationships. increased following  Both CSF and ECF concentrations  PRN co-administration, whereas plasma  concentration  remained relatively constant at all ages. Pharmacokinetic analysis showed that CIT was not significantly different from that in the 5-step infusion study but brain exposure to DPHM (fcsF and f cF) increased in all age groups after PRN coE  administration.  The higher brain exposure was unlikely due to inhibition of  DPHM metabolism by PRN, mainly because C I did not change with PRN coT  administration and also the overall metabolic activity of the brain as a unit is low (Ghersi-Egea etal., 1995).  155  PRN had been shown to have a lack of effect on cerebral physiology and choroid plexus blood flow in sheep (Townsend e t a l . , 1984; O'Brien et al., 1999). Since there is no information showing that PRN lowers CSF formation or its secretion rates or that it has any interference with the passive diffusion process of substances  back to the cerebral circulation, the observed  lowered  brain  clearances of DPHM could only be explained by lowered rates of transporter; mediated efflux.  PRN could be involved by direct inhibition of the active efflux  mechanism and/or competition with DPHM for the efflux process. Although from a physicochemical point of view, Pgp appears to be the most likely transporter for this efflux process, such a postulation requires further investigation due to the presence of some opposing in vitro results (Mizuuchi et al., 2000; Chishty et al., 2001).  In short, the exact mechanism of this active efflux process cannot be  elucidated by our current studies.  Protein binding remained unchanged after PRN co-administration in all groups except in the 100 d fetus. In the young fetus, low levels of plasma proteins might be more susceptible to competition for binding by other drugs (Dziegielewska et al., 1980a; Kwan et al., 1995). On the other hand, V d  ss  values in the fetal and  adult groups were lower than those in the 5-step infusion study. Since there was no change in plasma protein binding, the observed decrease in V d likely due to decrease in tissue drug uptake or binding.  s s  was most  The total dose  administered in the DPHM-PRN study was approximately 1/3 of that in the 5-step infusion study.  A DPHM dose-ranging study performed by Yoo et al. also  observed similar decreases in mean V d  ss  as the dose was decreased (Yoo et al.,  156  1990).  Since levels of protein binding increased significantly after birth in  newborn lambs, it might have balanced out the lowered tissue binding associated with lower doses in the DPHM-PRN study so that the V d  ss  values were not  significantly different in the two studies.  Except in 100 d fetus, PRN caused obvious decreases in heart rates in all age groups after its co-administration.  However, blood pressure appeared to be  unaffected. This observation is consistent with findings from other investigators (O'Brien etal., 1999).  The findings from the two studies are consistent to each other. The step infusion study suggested increased activity of brain clearance for DPHM in both the CSF and ECF compartments with age, and the co-administration study showed a progressive increase in the percentage increase in C C S F S S / C P ratios after PRN administration with advancing age.  uss  and C E C F S S / C P  USS  This implies that as the  efflux mechanism becomes more developed with age, any interference with this efflux process would cause greater accumulation of drug in the CNS of older animals.  In conclusion, this project provided information on the CNS disposition of a basic, amine drug which is highly lipophilic in nature.  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