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Effects of acute moderate hypoxemia on the pharmacokinetics of metoclopramide and its metabolites in… Kim, John 1995

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EFFECTS OF ACUTE MODERATE HYPOXEMIA ON T H E PHARMACOKINETICS OF METOCLOPRAMIDE AND ITS METABOLITES IN CHRONICALLY INSTRUMENTED SHEEP by J O H N K I M B.Sc, Simon Fraser University, A THESIS SUBMITTED  IN PARTIAL  1991  FULFILLMENT  T H E R E Q U I R E M E N T S F O R T H E D E G R E E M A S T E R  O F  O F S C I E N C E in  T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Faculty of Pharmaceutical Sciences) (Division of Biopharmaceutics and Pharmacokinetics) W e a c c e p t this thesis as c o n f o r m i n g to the r e q u i r e d s t a n d a r d  T H E U N I V E R S I T Y O F B R I T I S H January  1995  © John K i m ,  1995  C O L U M B I A  O F  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his or  her  representatives.  It  is understood that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  The University of British Columbia Vancouver, Canada  DE-6  (2/88)  A B S T R A C T  Hypoxemia is known to induce various physiological changes which can result in pharmacokinetic changes.  To examine the effect of acute, moderate hypoxemia in  metoclopramide (MCP) pharmacokinetics, a continuous infusion [14 hours] of M C P was administered during pre-hypoxemia (2hr), hypoxemia (6hr) and post-hypoxemia (6hr) in non-pregnant sheep.  Hypoxemia was achieved by lowering the ewe's inspired O 2  concentration. During the experiment, arterial blood and urine samples were collected. M C P and its mono- and di-deethylated metabolites were measured in these fluid samples using a gas chromatography-mass selective detector (GC-MSD) method.  Steady-state  concentrations of M C P were achieved in each of the three periods. During hypoxemia, M C P plasma steady-state concentration increased significantly from 50.72 ± 1.06 to 63.62 ± 1.79 ng/mL, and later decreased to 55.83 ± 1.15 ng/mL during the posthypoxemic recovery period.  Plasma mdMCP concentration (32.78 ± 1.73 ng/mL) also  increased, compared to the control group (21.20 ± 1.39 ng/mL), during hypoxemia and the subsequent normoxemic period.  Renal excretion of M C P and its metabolites  significantly decreased during hypoxemia.  Increased urine flow with decreased urine  osmolality was also observed. Thus the results indicate that acute, moderate hypoxemia affects M C P pharmacokinetics.  Ul  T A B L E O F  C O N T E N T S  ABSTRACT  ii  T A B L E OF CONTENTS  iii  LIST OF T A B L E S  vii  LIST OF FIGURES  viii  LIST OF ABBREVIATIONS  x  LIST OF SCHEMES  xiii  ACKNOWLEDGMENTS  xiv  1.  INTRODUCTION  1  1.1.  Pharmacology and Clinical Use  1  1.2.  Pharmacokinetics of Metoclopramide  3  1.3.  Hypoxemia and Associated Physiological Changes  7  1.3.1  Hypoxemia and hypoxia  7  1.3.2  Physiological changes during hypoxemia  8  1.4.  Respiratory Disorder and Drug Disposition and Metabolism  13  1.4.1.  Effects of acute and chronic hypoxemia on drug metabolism  14  1.4.2.  Effects of hypoxemia on hepatic drug metabolism  15  1.4.3.  Effects of hypoxemia on drug disposition and protein binding  16  1.4.4.  Digoxin kinetics in hypoxemia  18  1.4.5.  Theophylline kinetics and respiratory disorders  19  1.4.6.  Lidocaine kinetics/metabolism during hypoxemia and similarity with M C P  21  1.5.  Rationale  23  1.6.  Objectives  24  iv  2.  EXPERIMENTAL  25  2.1.  25  Materials and Supplies 2.1.1.  Chemicals  25  2.1.2.  Reagents  25  2.1.3.  Enzymes  26  2.1.4.  Solvents  26  2.1.5.  Gases  26  2.1.6.  Supplies for animal studies  27  2.2.  Stock and Reagent Solutions  27  2.3.  Sample Preparation, Extraction and Derivatization  28  2.3.1.  Sample extraction  28  2.3.2.  Analysis of glucuronide and sulphate conjugates  31  2.4.  Standard Curve Preparation  32  2.5.  Instrument and Equipment  32  2.6.  2.5.1.  Gas chromatography  32  2.5.2.  Operating conditions for the G C and MSD  33  Animal Preparation  34  2.6.1.  Animal handling  34  2.6.2.  Surgical preparation  34  2.7.  Experiment Protocol  36  2.8.  Recording Procedures and Analysis of Physiological Parameters  39  2.9.  Data Analysis  40  2.9.1.  Determination of steady-state drug concentration  40  2.9.2.  Calculation of pharmacokinetic parameters  41  2.9.3.  Statistical tests  42  V  3.  RESULTS 3.1.  43  Quantitative Analytical Assay Methods for Metoclopramide and Selective Metabolites  43  3.1.1. GC-MSD methods for the quantitative analysis of M C P and selective metabolites  3.2.  43  3.1.2. Calibration (standard) curve  46  3.1.3. Enzyme incubation  46  Physiological Changes Associated with Hypoxemia 3.2.1  Arterial blood gas status and pH  3.2.2. Arterial blood lactate and glucose concentration  51 52 56  3.2.3. Plasma electrolyte concentration, urine flow, osmolality and pH and renal osmolal excretion rate 3.2.4. Mean arterial blood pressure and heart rate 3.3  60 66  Metoclopramide Pharmacokinetics following i.v. Infusion to Steady-State and Induction of Hypoxemia  67  3.3.1. Steady-state plasma M C P concentration and total body clearance (TBC) during normoxemia and hypoxemia  67  3.3.2. Plasma mdMCP concentration during hypoxemic and normoxemic M C P steady-state 3.4.  75  Renal Excretion of M C P and its Metabolites Following the i.v. Infusion to Steady-state and Induction of Hypoxemia  77  3.4.1. Renal clearance of M C P and its metabolites during normoxemia and hypoxemia 3.4.2. Fractional renal excretion constants of M C P and its metabolites  4.  DISCUSSION 4.1.  4.2.  77 82  87  G C - M S D Method of Analysis of Metoclopramide and Selected Metabolites  87  Physiological Changes associated with Hypoxemia  89  4.2.1. Induction of hypoxemia and blood gas changes  89  4.2.2. Blood pH and lactate concentration during hypoxemia  91  vi  4.2.3. Adrenosympathetic system and blood glucose level during hypoxemia 4.2.4. Urine flow and osmolality during hypoxemia and M C P infusion 4.3.  Statistical and Practical Aspects of Experiment Design 4.3.1. Designof experimental protocol  93 95 99 99  4.3.2. Statistical and practical considerations for determining the steady-state drug concentration 4.3.3. Theoretical aspects of infusion with loading dose 4.4.  103 106  Metoclopramide Pharmacokinetics following i.v. Infusion to Steady-State and Induction of Hypoxemia  111  4.4.1. Steady-state plasma MCP concentratioxi and total body clearance (TBC) during normoxemia and hypoxemia 4.4.2. Plasma mdMCP concentration during normoxemia and hypoxemia  111 114  4.4.3. Renal excretion of M C P and its metabolites during normoxemia and hypoxemia  116  5.  S U M M A R Y A N D CONCLUSIONS  120  6.  REFERENCES  121  APPENDIX A  137  LIST O F T A B L E S  Table 1.  Table 2.  Table 3.  Table 4.  Weighted calibration curve data (mean peak area ratio ± SD) for ddMCP, mdMCP and MCP in plasma  49  Weighted calibration curve data (slope, intercept and r ) for ddMCP, mdMCP and MCP of enzyme incubation study in urine  50  Identification and weight of ewes, samples collected and nitrogen /MCP infusion.  51  2  Mean (± SEM) arterial blood pH, gas partial pressure (PaCCte and PaCte), bicarbonate (HCCb") concentration and base excess.  54  Table 5.  Mean (± SEM) blood lactate concentration (mmol/L).  59  Table 6.  Mean (± SEM) blood glucose concentration (mmol/L).  59  Table 7.  Mean (± SEM) plasma electrolyte concentration, haemoglobin content and urine flow, pH and osmolality.  65  Table 8.  Mean (± SEM) heart rate (beats/min).  66  Table 9.  Mean (± SEM) arterial blood pressure (mm Hg).  67  Table 10.  Mean (± SEM) steady-state plasma MCP concentration (ng/mL).  69  Table 11.  Plasma MCP total body clearance (TBC).  74  Table 12.  Mean (± SEM) plasma mdMCP concentrations during the MCP infusion (ng/mL).  76  Table 13a.  MCP renal clearance (L/h) [CL i cp)]  80  Table 13b.  MCP renal clearance (L/h) [CL  80  Table 14a.  mdMCP renal clearance (L/h) [CL i cp)]  81  Table 14b.  mdMCP renal clearance (L/h) [CL i( dMCP)]  81  Table 15.  MCP fractional renal excretion constants \f (MCP)]  85  Table 16.  mdMCP fractional renal excretion constants \f (mdMCP)]  86  Table 17.  ddMCP fractional renal excretion constants \f (ddMCP)]  86  .  1  rena (M  p)]  2  renamC  1  rena (mdM  2  rena  m  u  U  U  viii  LIST OF FIGURES Figure 1.  Figure 2.  Representative total ion chromatograms (SIM mode) of 1 ng/mL of ddMCP, mdMCP and M C P , and 33.3 ng/mL of B M Y spiked in 0.3 mL of plasma. Superimposed chromatograms of blank plasma and urine are also shown.  44  Mass spectra of ddMCP (A), mdMCP (B), M C P (C) and the internal standard B M Y (D) obtained in S C A N mode, showing the molecular ions (M ) and the ions used for quantitative analysis.  45  Representative calibration (weighted) curves for M C P , mdMCP and ddMCP from spiked plasma, [mean ± SD]  48  Mean (± SEM) arterial blood pH and gas partial pressure ( P a C 0 2 and P a 0 2 ) over the duration of the experiment [experimental group].  55  Mean (± SEM) arterial blood pH and gas partial pressure ( P a C 0 2 and P a 0 2 ) over the duration of the experiment [control group].  56  Mean (± SEM) arterial blood lactate and glucose (mmol/L) over the duration of the experiment [experimental group].  58  Mean (± SEM) arterial blood lactate and glucose (mmol/L) over the duration of the experiment [control group].  58  Mean (± SEM) urine flow (mL/h) and osmolality (mOsm/Kg) over the duration of the experiment [experimental group]  62  Mean (± SEM) urine flow (mL/h) and osmolality (mOsm/Kg) over the duration of the experiment [control group]  62  Correlation of osmolality (mOsmol/Kg) and urine flow (mL/h). Correlation coefficient (r)=0.80  63  Mean (± SEM) plasma M C P and mdMCP concentration (ng/mL) over the duration of the experiment [experimental group].  70  Mean (± SEM) plasma M C P and mdMCP concentration (ng/mL) over the duration of the experiment [control group].  71  Representative plot of the accumulated drug (or metabolite) in urine (ZDu) versus A U C . The slope of the curve represents the renal clearance [L/h]  79  +  Figure 3.  Figure 4 a .  Figure 4 b .  Figure 5a.  Figure 5b.  Figure 6a.  Figure 6b.  Figure 7.  Figure 8a.  Figure 8b.  Figure 9.  Figure 10.  Figure 11.  Figure 12.  Figure 13.  Representative plot of the accumulated drug (or metabolite) in urine (ZDu) versus time (h). The slope of the curve represents the product of the fractional renal excretion rate constant for M C P (or metabolite) and the infusion rate constant of MCP.  84  Simulation of Cp after simple i.v. infusion without loading dose using pharmacokinetic parameters from Riggs et al. (1989).  108  Simulation of Cp after i.v. infusion with loading dose (one-compartment model).  108  Simulation of Cp after i.v. infusion with loading dose (twocompartment model) using pharmacokinetic parameters from Riggs et al. (1989).  109  LIST OF ABBREVIATIONS  <> |  determining power  rj  population variance  2  A.R.E.  amount remained to be excreted  ACS  American Chemical Society  ANF  atrial natriuretic factor  ANOVA  analysis of variance  AUC  area under the curve  AVP  arginine vasopressin  BMY  4-amino-5-chloro-2(2-butanone-3-yl)-oxy-N,N-diethylaminoethyl benzamide  BSP  sufobromophthalein  Cl  total body clearance  COPD  chronic obstructive pulmonary disease  C  apparent arterial steady-state concentration  CV  coefficient of variation  ddMCP  N,N-dideethyl-metoclopramide  D  accumulated drug/metabolite in urine  EI  electron impact  f  fractional renal metabolite elimination constant  f  metabolite formation fraction constant  t  ss  u  u  m  f  renal metabolite elimination fraction constant  /„  fractional renal elimination constant  GC-ECD  gas chromatography-electron capture detector  GC-MSD  gas chromatography-mass selective detector  GFR  glomerular filtration rate  GX  glycinexylidide  HC1  hydrochloric acid  HCO3"  bicarbonate  HFB  heptafluorobutyryl  mu  HFBA  heptafluorobutyric anhydride  I.D.  inner diameter  ICG  Indocyanine Green  kio  apparent drug elimination rate constant from the central compartment  ki2, k2i  apparent first order inter-compartmental distribution rate  KE  apparent first order drug elimination rate constant  kf  metabolite formation rate constant  K O*2  Michaelis affinity constant of oxygen  k  renal metabolite elimination rate constant  k  infusion rate  k  renal elimination rate constant  LD  i.v. bolus loading dose  LOQ  limit of quantitation  MA  maternal arterial blood sample  MB  amount of metabolite in the body  MCP  metoclopramide  mdMCP  N-monodeethyl-metoclopramide  MEGX  N-monoethyl-glycinexylidide  MSD  mass selective detector  NaOH  sodium hydroxide  PaCG*2  arterial carbon dioxide partial pressure  PaG"2  arterial oxygen partial pressure  PO2  oxygen partial pressure  PTFE  polytetrafluorethylene  r  coefficient of determination  m  mu  a  u  r  2  coefficient of correlation  SCAN  mass scanning mode  SD  standard deviation  SEM  standard error of the mean  SIM  selective ion monitoring mode  SMZ  sulphamethazine  TBC  total body clearance  TEA  triethylamine  TRIS  2-amino-2-hydroxymethyl-1,3-propanediol  UHP  ultra-high purity  UR  urine total collection  V  c  apparent volume of distribution of the central compartment  v  d  apparent volume of distribution  X  B  amount of drug in the body  xiii  LIST O F  S C H E M E S  Scheme I  Comparison of N-deethylation reactions of MCP and lidocaine.  22  Scheme II  Extraction procedure for MCP and its metabolites.  30  Scheme LTJ  Schematic diagrams of the experimental protocol  37  Scheme IV  MCP infusion-hypoxemia sampling protocol  38  Scheme V  Diagram of metabolic and renal elimination of a drug  82  Scheme VI . Diagrams of the compartmental model used for computer simulation.  107  XIV  ACKNOWLEDGMENTS  I would like to express my sincere appreciation and thanks to my research supervisors Drs. James E. Axelson and Dan W. Rurak, for their guidance, encouragement and support. Special thanks to Dr. Wayne Riggs for his warm support and friendship throughout the program. Thanks to Drs. Frank Abbott, Joan Douglas, Don Lyster, Keith McErlane for their advice and guidance throughout my study. I would like to thank to Mr. Eddie Kwan and Ms. Caroline Hall for their technical assistance in animal studies. I would also like to thank to Mr. George Tonn and Sanjeev Kumar for their critical evaluation, support and friendship. Thanks to all the graduate students in the laboratory, particularly Dr. Matthew Wright, Dr. Swamy Yeleswaram, Ms. Judit Orbay for their friendship and help. A special thanks to my family for their support and understanding. I greatly appreciate the financial support by Medical Research Council of Canada and British Columbia Medical Services Summer Scholarship.  1  1. 1.1  INTRODUCTION  Pharmacology and Clinical Use Metoclopramide  (MCP),  4-amino-2-methoxy-5-chloro-N-(2-diethylaminoethyl)  b e n z a m i d e , is ap o t e n t a n t i e m e t i c a n d g a s t r i c m o t i l i t y m o d i f i e r t h a t h a s b e e n u s e d t o treat n a u s e a a n d v o m i t i n g associated w i t h u r e m i a a n d to reduce the incidence o f v o m i t i n g pulmonary aspiration associated with emergency anaesthesia (Davies and Howells, D u n d e e et al, 1 9 7 4 ) .  and 1973;  M C P is s t r u c t u r a l l y r e l a t e d to p r o c a i n a m i d e , b u t is d e v o i d  of  a n t i a r r h y t h m i c o r l o c a l a n a e s t h e t i c a c t i v i t y a t a n t i e m e t i c c l i n i c a l d o s e s ( H a r r i n g t o n et al, 1983).  However,  MCP-elicited  prolactin  secretion  increase  and  extrapyramidal  s y m p t o m s h a v e b e e n r e p o r t e d ( P i n d e r et al, 1 9 7 6 ) . T h e p h a r m a c o l o g i c a l a c t i o n s o f M C P are m o s t p r o n o u n c e d in the gastrointestinal tract w h e r e generalized increases in motility a r e s e e n a f t e r o r a l a n d i . v . a d m i n i s t r a t i o n ( P i n d e r et al, 1 9 7 6 ; S c h u l t z e - D e l r i e u ,  /  H N 2  >f X  J— =  <  CO - N H - C H  OCH  2  - C H  2  - N  C  2  H  5  N  C H 2  5  C H  5  3  Metoclopramide  H N—if 2  V— CO  - N H - C H  W  2  - C H  2  - N  X  2  Procainamide  1981).  2  Metoclopramide Breivik, 1970).  is  effective  in reducing  postoperative  vomiting  (Lind  and  A t h i g h d o s e s (5 d o s e s o f 2 m g / K g a d m i n i s t e r e d i n t r a v e n o u s l y o v e r  h o u r s ) , M C P is effective i n r e d u c i n g the n a u s e a a n d v o m i t i n g a s s o c i a t e d w i t h the u s e c i s p l a t i n u m ( G r a l l a et al, MCP  1 9 8 1 ) a n d o t h e r c h e m o t h e r a p e u t i c a g e n t s ( S t r u m et al,  8 of  1984).  also increases lower oesophageal sphincter tone, oesophageal peristalsis a n d gastric  emptying, all o f w h i c h w o u l d relieve patients with gastro-oesophageal A s a r e a n d E l - B a s s o u s s i , 1 9 8 0 ; W i n n a n et al,  reflux  (Bright-  1980). M C P reduces the nausea  associated  with m i g r a i n e a n d increases the rate o f absorption o f oral analgesic agents,  including  aspirin (Matts, 1974; Volans, 1978).  M e t o c l o p r a m i d e also significantly reduces the emesis of early pregnancy a n d L e a n , 1 9 7 0 ) a n d d u r i n g l a b o u r ( M c G a r r y , 1 9 7 1 ; V e l l a et al,  1985).  (Singh  It s i g n i f i c a n t l y  increases the gastric e m p t y i n g rate o f w o m e n in labour ( H o w a r d a n d S h a r p , 1973)  and  i n c r e a s e s the t o n e o f the l o w e r e s o p h a g e a l s p h i n c t e r , w h i c h is i m p a i r e d d u r i n g p r e g n a n c y a n d l a b o u r ( B r o c k - U t n e et  al,  1978).  T h e s e actions a n d the ability to d i m i n i s h  the  f r e q u e n c y o f v o m i t i n g d u r i n g l a b o u r h a v e l e d to its m o r e f r e q u e n t u s e i n e l e c t i v e  and  e m e r g e n c y o b s t e t r i c a l a n a e s t h e s i a ( C o h e n et al,  also  prevents  nausea  and vomiting  C a e s a r e a n s e c t i o n ( C h e s t n u t et al,  associated  with  1984; Shaughnessy, epidural anaesthesia  1985).  M C P  during  elective  1987). T h e antiemetic effects of M C P are thought  be mediated through both peripheral (gastrointestinal)  a n d central sites.  believed to raise the threshold o f the c h e m o r e c e p t o r trigger z o n e  f  to  The drug  is  hrough antagonism  of  d o p a m i n e receptors, a n d to decrease the sensitivity o f visceral nerves w h i c h  transmit  3  afferent i m p u l s e s f r o m the gastrointestinal tract to the e m e t i c centre in the lateral reticular f o r m a t i o n ( P i n d e r et al,  A  1976).  decrease in renal plasma flow of about 2 0 %  patients receiving high doses of M C P (1-2.5 mg/kg)  has been reported in ( I s r a e l et al,  oncology  1986), and also a  d e c r e a s e i n h e p a t i c b l o o d f l o w w a s n o t e d i n the rat at M C P d o s e s o f > 2 5 m g / k g , r e s u l t i n g i n d o s e - d e p e n d e n t k i n e t i c s ( T a r n et al,  1981a).  various hormones in m a n and in animals.  M C P also stimulates the release  Prolactin release was observed in the  of rat,  h e a l t h y a d u l t s , p r e g n a n t w o m e n , c h i l d r e n ( H a r r i n g t o n et al, 1 9 8 3 ; A r v e l a et al., 1 9 8 3 ) , n e o n a t e s ( R u p p e r t et al, 1 9 8 6 ) a n d p r e g n a n t e w e s ( F i t z g e r a l d a n d C u n n i n g h a m , 1 9 8 2 ) . T h i s e f f e c t is l i k e l y m e d i a t e d b y a n t a g o n i s m o f the d o p a m i n e - m e d i a t e d i n h i b i t i o n prolactin secretion b y the pituitary or hypothalamus.  A n increased level of  of  plasma  a l d o s t e r o n e w a s r e p o r t e d i n r a t s , a n d m a n ( H a r r i n g t o n et al, 1 9 8 3 ; S o m m e r s et al,  1988;  V o n m o o s et al, 1 9 9 0 ) a s a r e s u l t o f i n h i b i t i o n o f c e n t r a l d o p a m i n e r e c e p t o r s ( A l b i b i a n d McCallum,  1983)  or  acetylcholine  release  from  post-ganglionic  t e r m i n a l s w i t h i n t h e a d r e n a l c o r t e x ( S o m m e r s et al, 1 9 8 8 ) .  cholinergic  Stimulation of  s e c r e t i o n h a s a l s o b e e n r e p o r t e d i n h e a l t h y h u m a n v o l u n t e e r s ( N o r b i a t o et al,  Pharmacokinetics  1.2.  A  two-compartment  of  model  nerve  vasopressin 1986).  Metoclopramide  adequately  describes  the  f o l l o w i n g i . v . a d m i n i s t r a t i o n i n h u m a n s ( B a t e m a n et al, 1 9 8 0 ) .  disposition  of  M C P  T h e d r u g is r a p i d l y  distributed w i t h a relatively h i g h v o l u m e o f distribution in h u m a n s (2.2 to 3.4 L / K g ) indicating extensive extravascular distribution, w h i c h m a y be expected for a lipid-soluble,  b a s i c d r u g . R a p i d p l a c e n t a l t r a n s f e r o c c u r s i n m a n ( B y l s m a - H o w e l l et al, al,  1 9 8 4 ) a n d i n s h e e p ( R i g g s et al,  1988; 1989).  M C Pis e x t e n s i v e l y m e t a b o l i s e d  s u l p h a t e a n d g l u c u r o n i d e c o n j u g a t i o n i n r a b b i t a n d h u m a n s ( A r i t a et al, al,  1 9 7 6 ; B a t e m a n et al,  via  1970; C o w a n  et  1970; Bakke a n d Segura, 1976; C o w a n  1 9 7 6 ) . T h e e l i m i n a t i o n h a l f - l i f e i s = 1 2 0 m i n i n t h e d o g ( B a t e m a n et al,  et  1980) a n d =  5 0 m i n i n t h e r a t w i t h a d o s e d e p e n d e n t i n c r e a s e a t d o s e s e x c e e d i n g 1 5 m g / k g ( T a r n et 1981a).  et  1980), a n d b y O-demethylation, N-deethylation a n d amide  h y d r o l y s i s i n r a b b i t , r a t , a n d d o g ( A r i t a et al, al,  1983;Cohen  T h e d o s e dependent kinetics at higher doses a r e likely d u e to altered  al,  hepatic  b l o o d f l o w ( T a r n et al,  1981a), a n observation confirmed using the Indocyanine  Green  (ICG) clearance method.  T h e p l a s m a elimination half-life i n non-pregnant, maternal, a n d  fetal sheep is 6 3 ± 14, 8 8 ± 17, a n d 113 ± 2 9 m i n ,respectively ( m e a n ± S E M ) , after a n i . v . b o l u s d o s e ( R i g g s et al,  1988). T h e v o l u m e o f distribution i n sheep is higher (5.5 to  7 . 0 L / K g ) t h a n i n h u m a n s ( 2 . 2 t o 4 . 0 L / K g ) ( R i g g s et al,  Hepatic clearance and  1988).  metabolism : W i t h i n 2 4 h o u r s o f i . v . b o l u s M C P a d m i n i s t r a t i o n i n  h u m a n s , about 8 0 % o f the dose w a s excreted i n the urine as free drug ( = 2 5 % o f the dose), g l u c u r o n i d e ( = 2 - 5 % o f t h e d o s e ) a n d s u l p h a t e ( = 5 0 % o f t h e d o s e ) c o n j u g a t e s ( T e n g et 1 9 7 7 ; B a t e m a n et al,  1980). T h e ratio o f conjugates excreted f r o m h u m a n s a n d a variety  o f animals is significantly different, suggesting metabolism.  al,  significant species differences i n M C P  Sulphate conjugation a n d glucuronic acid conjugation are the t w o major  m e t a b o l i c p a t h w a y s f o r M C P i n h u m a n s a n d t h e r a b b i t ( C o w a n et al,  1976).  However,  the sulphate a n d glucuronide conjugates o f M C P have n o t been f o u n d in the d o g a n d the r a t , w h e r e t h e d e e t h y l a t i o n o f M C P i s t h e d o m i n a n t m e t a b o l i c p a t h w a y ( T e n g et  al,  5  1977).  In h u m a n s , the total b o d y clearance approximates hepatic p l a s m a flow  mL/min/Kg),  w i t h renal clearance (2.6 m L / m i n / K g )  accounting for =20%  c l e a r a n c e , s u g g e s t i n g the c l e a r a n c e is f l o w - l i m i t e d r a t h e r t h a n m e t a b o l i c ( B a t e m a n et al, mL/min/Kg)  1980).  (11.61  o f total  body  capacity-limited  I n s h e e p , the total b o d y c l e a r a n c e is c o n s i d e r a b l y h i g h e r  (86.7  c o m p a r e d to h u m a n s , w h i c h coincides w i t h the higher hepatic b l o o d f l o w in  s h e e p ( 0 . 5 - 3 . 0 L / m i n ) t h a n h u m a n s ( 0 . 5 - 1 . 5 L / m i n ) . T h e l i v e r is t h o u g h t to b e t h e  major  site o f M C P e l i m i n a t i o n ( D e s m o n d a n d W a t s o n , 1986). T h e elimination half-life o f M C P i n h u m a n s i s 2 . 6 t o 4 . 6 h o u r s ( B a t e m a n et al,  1 9 8 0 ; G r a f f n e r et al,  s h e e p , i t i s m u c h s h o r t e r ( 1 . 1 - 1 . 6 h o u r s ) ( R i g g s et al,  1979), whereas  1988). A high total b o d y  clearance  i n s h e e p h a s a l s o b e e n o b s e r v e d w i t h d r u g s s u c h a s d i p h e n h y d r a m i n e ( Y o o et al, m e p e r i d i n e ( S z e t o et al,  1 9 7 8 ) , a n d l i d o c a i n e ( B l o e d o w et al,  in  1986),  1980).  Renal failure : S t u d i e s o f M C P k i n e t i c s i n r e n a l f a i l u r e s h o w e d a s i g n i f i c a n t d e c r e a s e i n t o t a l b o d y c l e a r a n c e ( B a t e m a n et al,  1 9 8 1 ; T a r n et al,  1 9 8 1 b ; W r i g h t et al,  1988)  with  a n i n c r e a s e d t e r m i n a l half-life i n b o t h h u m a n s a n d rat. H o w e v e r , the c o n t r i b u t i o n o f r e n a l c l e a r a n c e o f M C P as intact d r u g as a f r a c t i o n o f total b o d y c l e a r a n c e is = 2 0 % ,  t h u s , it is  not e a s y to explain the o b s e r v e d 2-4 fold decrease in total b o d y clearance n o t e d in renal failure b o t h i n h u m a n s a n d the rats.  A  renal failure-induced change in  extrahepatic  m e t a b o l i s m w a s p r o p o s e d t o e x p l a i n t h e d e c r e a s e d t o t a l b o d y c l e a r a n c e ( T a r n et 1 9 8 1 b ) , h o w e v e r , K a p i l et al.  al,  (1984) ruled out significant extrahepatic metabolism  using  t i s s u e m e t a b o l i s m s t u d i e s in vitro. D i m i n i s h e d h e p a t i c M C P m e t a b o l i s m s e c o n d a r y r e n a l f a i l u r e w a s a l s o s u g g e s t e d ( B a t e m a n . , 1 9 8 1 ; K a p i l et al, W r i g h t et al.  1 9 8 4 ; W r i g h t et al,  to  1988).  (1988) speculated the presence o f an unidentified substance in the p l a s m a of  6  uremic patients w h i c h might inhibit M C P metabolism. could be  that the  conjugates,  might  accumulation of metabolites, reduce  the  activity  of  the  Another possible  explanation  including glucuronide and metabolic  enzymes  sulphate  involved  in  the  elimination of the d r u g through a negative feedback m e c h a n i s m (Stryer, 1988).  Hepatic cirrhosis :  Increased  c i r r h o s i s p a t i e n t s ( H e l l s t e r n et al.,  M C P plasma concentrations 1 9 8 7 ; M a g u e u r et al,  are observed  in  1 9 9 1 ; A l b a n i et ai,  hepatic 1991).  A  r e d u c t i o n o f f u n c t i o n a l h e p a t i c b l o o d f l o w d u e to i n t r a - a n d e x t r a - h e p a t i c s h u n t i n g is the l i k e l y c a u s e o f t h e a l t e r e d M C P k i n e t i c s w i t h t h i s c o n d i t i o n ( M a g u e u r et  al,  1991),  Plasma concentrations of  ox-  a c i d g l y c o p r o t e i n , t h e m a j o r b i n d i n g p r o t e i n o f M C P , i s s t a b l e i n c i r r h o s i s ( K r e m e r et  al,  although r e d u c e d hepatic m e t a b o l i s m cannot be ruled out.  1 9 8 8 ) a n d t h e p l a s m a b i n d i n g o f M C P i s r e l a t i v e l y l o w , i.e. T h e r e f o r e , t h e p r o t e i n b i n d i n g (i.e. unlikely to b e affected.  =40%  ( W e b b et al,  1986).  free fraction) a n d tissue distribution o f M C P  P h a s e J J p a t h w a y s o f m e t a b o l i s m i.e.  conjugation  w h i c h account for the majority of M C P elimination in h u m a n s , are not a f f e c t e d b y l i v e r d y s f u n c t i o n ( M a g u e u r et al,  reactions, significantly  1991).  Therefore, f r o m the data describing M C P kinetics associated with renal h e p a t i c d y s f u n c t i o n , it is s u g g e s t e d t h a t d i s e a s e - i n d u c e d c h a n g e s i n M C P k i n e t i c s m a y due  to  altered blood  metabolism.  flow  are  distribution and changes  in intrinsic capacity  for  and be drug  7  1.3.  Hypoxemia and  1.3.1. Hypoxemia and  Associated Physiological Changes  hypoxia  H y p o x e m i a is a r e d u c t i o n i n b l o o d P 0 , w h e r e a s h y p o x i a is a fall i n t i s s u e 2  utilization w h e n demands.  tissue oxygen  d e l i v e r y is n o t sufficient  Hypoxia can be a consequence  of severe  to m e e t  normal  0  2  metabolic  hypoxemia, but can also  occur  w i t h o u t a r e d u c t i o n i n b l o o d P Q , as in the case o f c a r b o n m o n o x i d e p o i s o n i n g or  severe  2  anemia.  H y p o x i a is u s u a l l y a c c o m p a n i e d b y m e t a b o l i c / l a c t i c a c i d o s i s d u e to  metabolism.  W h e n hypoxemia causes P 0  maintain the o x y g e n acidosis  will  to d r o p b e l o w the l e v e l that is r e q u i r e d to  diffusion gradient f r o m capillary b l o o d to m i t o c h o n d r i a ,  occur  due  phosphorylation accounts (Weibel, 1984).  2  to  anaerobic  for about 85  glycolysis  1986).  Oxidative  per cent o f total b o d y  oxygen  consumption  H o w e v e r , o x i d a t i v e p h o s p h o r y l a t i o n is n o t l i k e l y a f f e c t e d b y l o c a l P 0 , 2  2  1 m m Hg.  widely differing Km0 , 2  more  3  oxidase  vulnerable  there  such  is as  a n d oxidases ( e x c e p t c y t o c h r o m e a ), h a v e m u c h h i g h e r  and  r a n g i n g f r o m 5 to 2 5 0 m m H g , therefore these processes  are  3  to  hypoxemia.  W h e n  the  body  is  deprived  progressively, quantitatively minor but qualitatively important processes before  for oxygen  O t h e r e n z y m e s involved in the o x y g e n - c o n s u m i n g processes  hydroxylases, oxygenases,  m u c h  lactic  (Denison,  s i n c e t h e M i c h a e l i s a f f i n i t y c o n s t a n t (Km0 ) o f c y t o c h r o m e a below  anaerobic  is a n o t i c e a b l e  i m p a i r m e n t o f total b o d y  phosphorylation (Denison, 1986).  oxygen  1981).  oxygen  will fail  uptake  Thus, for example, some oxidative drug  m a y be affected in relative m i l d h y p o x i a / h y p o x e m i a (Jones,  of  or  long  oxidative metabolism  8  1.3.2. Physiological changes during hypoxemia  C o m p e n s a t o r y r e s p o n s e m e c h a n i s m s d u r i n g h y p o x e m i a consist of: 1.  increased oxygen extraction and  decreased oxygen demand in tissues.  2.  increased cardiac output.  3.  preferential redistribution of available oxygen supply.  T h e rate at w h i c h o x y g e n is d e l i v e r e d to the tissues b y the arterial b l o o d is the p r o d u c t o f cardiac output a n d the arterial o x y g e n concentration.  W h e n arterial 0  2  concentration  r e d u c e d as i n h y p o x e m i a , o x y g e n d e l i v e r y c a n b e m a i n t a i n e d , o r at l e a s t its fall c a n  is be  limited, by: A.  increasing oxygen extraction and  Chapler  (1979) examined  the  oxygen  decreasing oxygen demand in extraction  (i.e.  increasing the  tissues:  arterial-venous  o x y g e n concentration difference) b y the canine h i n d l i m b during hypoxic h y p o x i a h y p o x i a d u e to r e d u c e d inspired o x y g e n  content).  e x t r a c t i o n d u r i n g h y p o x e m i a w a s o b s e r v e d (i.e. 60%  in m o d e r a t e a n d to = 8 0 %  A  significant increase in  ranging from =20%  oxygen  in n o r m o x e m i a to  =  O x y g e n uptake b y tissue  was  maintained during moderate hypoxemia, therefore no metabolic vasodilatory effect  was  developed.  in severe hypoxemia).  (i.e.  H o w e v e r , during severe hypoxemia, a significant decrease in oxygen  uptake,  an increase in cardiac output a n d l i m b b l o o d flow, a n d decrease in total a n d peripheral resistance were observed (Cain and Chapler, 1979).  T h i s suggests that p r o l o n g e d  severe  hypoxemia (>20 min) causes an increased blood flow by local metabolic vasodilation in s p i t e o f a n y c e n t r a l l y m e d i a t e d c o n s t r i c t o r a c t i o n [ a u t o r e g u l a t o r y e s c a p e ] ( G r a n g e r et 1975; 1976).  A n increase in 0  2  extraction causes a fall in vascular P 0 . 2  al,  Therefore, this  Cain a n d  9  c o m p e n s a t o r y m e c h a n i s m will likely be limited b y the onset o f autoregulatory escape local metabolic vasodilation, since a critical arterial o x y g e n level ( P a 0 H g ) i n t i s s u e s is n e e d e d to m a i n t a i n a n a d e q u a t e 0 m i t o c h o n d r i a ( C o n n e t t et al, B.  2  2  = -10-12  m m  diffusion gradient f r o m blood  to  1990).  increasing cardiac output: A n e l e v a t e d a o r t i c b l o o d f l o w i s a s s o c i a t e d p r i m a r i l y w i t h  a n i n c r e a s e i n h e a r t rate w h i c h is m e d i a t e d i n l a r g e p a r t b y e n h a n c e d  sympathoadrenal  a c t i v i t y ( K r a s n e y , 1 9 6 7 ) , a n d a f a l l i n s y s t e m i c v a s c u l a r r e s i s t a n c e ( K o n t o s et al, S y l v e s t e r et al, al,  i.e.  1979).  1965;  A net increase in cardiac output a n d arterial pressure (Kontos  et  1 9 6 7 ) is m e d i a t e d b y the p e r i p h e r a l c h e m o r e c e p t o r interactions, s u c h as a p u l m o n a r y  vasodilator reflex ( D a l y a n d R o b i n s o n , 1968), the local vasodilating action o f ( D a u g h e r t y et al,  hypoxia  1967), a n d in addition, b y the direct central nervous system action  h y p o x i a ( D o w n i n g et al,  of  1963).  T h e preferential redistribution of cardiac output during hypoxia was reported in t h e d o g ( K r a s n e y , 1 9 7 1 ; A d a c h i et al,  1 9 7 6 ) a n d i n t h e s h e e p ( N e s a r a j a h et al,  R e d i s t r i b u t i o n o f b l o o d f l o w is a c c o m p l i s h e d b y l o c a l effects o f h y p o x i a , w h i c h vasodilation in coronary and cerebral vessels, and by chemoreceptor  1983). produces  reflex,  which  produces vasoconstriction in skeletal muscle  a n d the splanchnic b e d a n d dilation  in  coronary vessels (Heistad and A b b o u d , 1980).  S u p e r i o r c a v a l f l o w w a s e n h a n c e d at the  e x p e n s e o f inferior caval flow during hypoxia, but this h y p o x i c redistribution o f systemic f l o w w a s a b o l i s h e d b y c h e m o r e c e p t o r d e n e r v a t i o n ( K r a s n e y , 1 9 7 1 ; M a l o et al, Carotid a n d aortic chemoreceptors  provide for reflex vasoconstrictive  c i r c u l a t i o n d u r i n g s y s t e m i c h y p o x i a ( C h a l m e r et al,  1984).  support of  1965; D a l y a n d Scott, 1964),  the  which  10  leads to preferential redistribution o f b l o o d f l o w to the c e p h a l i c r e g i o n d u r i n g h y p o x i a , with a decline in splanchnic blood flow.  Local  f l o w is a l t e r e d v a r i a b l y i n  hypoxia  depending u p o n the relative balance of vasoconstrictor a n d vasodilator  components,  w h i c h i s i n f l u e n c e d b y t h e l e v e l o f o x y g e n i n t h e b l o o d ( A d a c h i et al.,  1976).  e x a m p l e , t h e p u l m o n a r y c i r c u l a t i o n is v e r y s e n s i t i v e to t h e d e c r e a s e i n t h e P 0 ,  which  2  alters  the  regional  vasoconstriction]  distribution  of  pulmonary  arterial  (Benjamin and Gorlin, 1952; West, 1988).  blood  flow  For  [hypoxic  In contrast, the  coronary  circulation exhibits a v e r y sensitive vasodilator response to the local decrease in ( G r e g g , 1 9 5 0 ; A d a c h i et ai,  P 0  2  1976). T h e m a i n determinants of the distribution o f cardiac  output are the relative arterial flow resistance a n d v e n o u s pressures (Mitzre a n d G o l d b e r g , 1975), a n d these changes in the distribution of cardiac output reflect alteration in a r t e r i a l p r e s s u r e - f l o w r e l a t i o n s h i p o f e a c h v a s c u l a r b e d ( M a l o et al,  1984).  T h e preferential redistribution of cardiac output and reduced oxygen d e m a n d non-essential  tissues  (e.g.  digestive  system  and  skeletal  the  muscles)  will  c o m p e n s a t e for a r e d u c e d o x y g e n s u p p l y u p to a certain limit. H o w e v e r , this  in  temporarily mechanism  is a l s o l i m i t e d b y the d e v e l o p m e n t o f s y s t e m i c a n d l o c a l lactic a c i d o s i s as a c o n s e q u e n c e o f the increased anaerobic glycolysis in tissues, w h i c h in turn causes local  vasodilation  ( N e s a r a j a h et al,  hemoglobin  1983).  Furthermore, acidosis will cause a decrease in the  G*2 s a t u r a t i o n b y t h e B o h r e f f e c t , w h i c h i s f o l l o w e d b y r e d u c e d o x y g e n d e l i v e r y a n d further d r o p in p H , ultimately c o m p r o m i s i n g the o x y g e n ( N e s a r a j a h et al,  1983)  d e l i v c y to the vital  a  organs  11 Hepatic haemodynamics and a n d i n d o g s ( H u g h e s et al, alter either hepatic  1979; Scholtholt and Shiraishi, 1970) does not  arterial or portal v e n o u s  h y p o x e m i a in the d o g hepatic  1979).  conductance.  However,  indicating increased  hepatic  more  severe  and  reduces  arterial vascular  Portal v e n o u s b l o o d f l o w s e e m s to be unaffected b y  ( S c h o l t h o l t a n d S h i r a i s h i , 1 9 7 0 ; L a r s e n et al,  1 9 7 6 , I s h i k a w a et al,  1976)  significantly  significantly increases systemic arterial pressure  arterial b l o o d flow,  ( H u g h e s et al,  function: M o d e r a t e h y p o x e m i a i n c a t s ( L a r s e n et al,  resistance hypoxemia  1 9 7 4 ; H u g h e s et  al,  1979), but M a t h i e and Blumgart (1983) showed a small but significant increase in portal venous flow. measurement  These different observations  are likely related to the precise t i m i n g  a n d the level of h y p o x e m i a chosen for the investigation.  of  Overall, the  effects o f h y p o x e m i a o n hepatic h a e m o d y n a m i c s s e e m to b e m i n o r .  Alternatively, Roth and R u b i n (1976) showed  a significant decrease in  total  hepatic a n d portal v e n o u s flow, with a slight increase in hepatic arterial flow in a study hypoxic hypoxia.  T h i s d i s c r e p a n c y is l i k e l y d u e to the h y p o c a p n i a w h i c h results  hyperventilation.  H y p e r c a p n i a a n d h y p o c a p n i a have b e e n s h o w n to affect total  f l o w s i g n i f i c a n t l y ( M a t h i e a n d B l u m g a r t , 1 9 8 3 ; H u g h e s et al,  1979).  of  from hepatic  Hypercapnia tends  to increase total hepatic a n d portal v e n o u s flow, d u e to decreased mesenteric  vascular  resistance (Mathie and Blumgart, 1983).  It h a s b e e n s u g g e s t e d t h a t h e p a t i c f u n c t i o n is m o r e s e n s i t i v e t o h y p o x e m i a other organs in spite of m i n o r alteration in h a e m o d y n a m i c s , because = 7 0 % b l o o d s u p p l y i s v e n o u s i.e.  l o w b l o o d o x y g e n t e n s i o n ( P r e i s i g et al,  1972).  of O n the  than  hepatic other  12  hand, the liver receives nearly 2 5 % e x t r a c t i o n r a t i o i.e. ( R o w e l l et al, 0  2  = 1 5 - 2 0 % ( R o w e l l et al,  1984).  oxygen  Studies on humans and other species  1968; 1984) s h o w e d that the liver c a n extract nearly 1 0 0 % o f the  available  a n d this i n c r e a s e i n o x y g e n e x t r a c t i o n is the m a j o r a d j u s t m e n t o b s e r v e d i n r e s p o n s e to  reduced hepatic 0 efficient 0 al,  o f the total cardiac output a n d has a l o w  2  2  d e l i v e r y ( L a r s e n et al,  1 9 7 6 ; T a s h k i n et  al,  1972).  Despite  this  extraction, abnormalities in sulfobromophthalein ( B S P ) retention (Shorey  1 9 6 9 ) a n d b i l e f l o w ( L a r s e n et al,  1976) were observed during moderate  hypoxemia.  T h e extraction of Indocyanine G r e e n was also reduced during both moderate and h y p o x e m i a ( B l a c k m o n a n d R o w e l l , 1 9 8 6 ; M a r l e a u et al,  1987).  However,  severe Blackmon  a n d R o w e l l (1986) s h o w e d that acute h y p o x e m i a in h u m a n s d i d not affect splanchnic uptake in spite o f a fall in hepatic v e n o u s 0  2  content.  et  0  2  In addition, hepatic glucose release  a n d lactate uptake w e r e not affected b y acute h y p o x e m i a in this study.  Renal function : H y p o x e m i a i n f l u e n c e s t h e r e n a l e x c r e t i o n o f s o d i u m a n d w a t e r . b e e n l o n g k n o w n that acute moderate h y p o x e m i a ( P a 0 in urine volume  (diuresis)  v o l u n t e e r s ( B u r r i l l et al,  and renal excretion 1 9 4 5 ; B e r g e r et al,  2  > 40 m m Hg) causes an  of sodium  increase  and chloride in  1 9 4 9 ; K i l b u r n et al,  significant  moderate  resistance.  T h u s , m e a n r e n a l b l o o d f l o w is m a i n t a i n e d d e s p i t e r e d u c e d m e a n a r t e r i a l p r e s s u r e . without  healthy  1971). Acute  hypoxemia also causes renal vasodilation, thereby decreasing renal vascular  are significant changes in intrarenal h a e m o d y n a m i c s  It h a s  There  changes  in  e x c r e t o r y f u n c t i o n s s u c h a s g l o m e r u l a r f i l t r a t i o n r a t e a n d s o d i u m e x c r e t i o n ( Z i l l i g et 1978).  Diuresis d u r i n g m o d e r a t e h y p o x e m i a s e e m s to b e related to decreased  a l d o s t e r o n e ( C o l i c e a n d R a m i r e z , 1 9 8 5 ) a n d a n g i o t e n s i n I I ( V o n m o o s et al,  1990)  plasma levels,  al,  13  a n d i n c r e a s e d a t r i a l n a t r i u r e t i c f a c t o r c o n c e n t r a t i o n s ( d u S o u i c h et al, 1 9 8 7 ) . B a s i c r e n a l metabolism  is a l s o m a i n t a i n e d i n m o d e r a t e  delivery (Sinagowitz  et al,  1976).  hypoxemia  in spite o f reduced  In patients with chronic obstructive  oxygen  pulmonary  disease, r e n a l p h y s i o l o g i c a l functions s u c h as r e n a l p e r f u s i o n , g l o m e r u l a r filtration a n d sodium excretion were reduced abruptly during severe hypoxemia (Pa0 hypercapnia ( P a C 0  1.4.  2  2  <40 m m Hg) or  > 6 0 m m H g ) ( K i l b u r n et al, 1 9 7 1 ; B r u n s , 1 9 7 8 ) .  Respiratory  Disorders  and Drug Disposition  and  Metabolism  T h e effects of respiratory disorders including h y p o x e m i a o n d r u g disposition m e t a b o l i s m a r e t h e s u b j e c t o f s e v e r a l r e v i e w s ( d u S o u i c h et al, 1 9 7 8 ; F a r r e l l , 1 9 8 7 ; et al, 1 9 8 9 ; a n d T a b u r e t et al, 1 9 9 0 ) .  T h e o n s e t o f h y p o x e m i a is the c o m m o n  associated with hypoxemia.  Changes in PaC0  2  chronic  and blood p H are  In severe pneumonia, pulmonary e d e m a  and  acidosis.  T h e increased  h y p o x e m i a is a s s o c i a t e d w i t h h y p e r c a p n i a a n d adverse  effects a n d toxicity  associated with  also  pulmonary  e m b o l i s m , h y p o x e m i a is a s s o c i a t e d w i t h h y p o c a p n i a a n d r e s p i r a t o r y a l k a l o s i s 1988), whereas in COPD,  Jones  pathway  o f m o s t o f the frequently o b s e r v e d respiratory disorders s u c h as a s t h m a a n d obstructive pulmonary disease (COPD).  and  (West,  respiratory  clinical  dose  a d m i n i s t r a t i o n o f d i g o x i n ( S m i t h , 1 9 7 5 ; D o h e r t y et al, 1 9 7 7 ) a n d t h e o p h y l l i n e ( Z w i l l i c h et al, 1 9 7 5 ; H e n d e l e s et al, 1 9 7 7 ) t o p a t i e n t s w i t h p u l m o n a r y d i s o r d e r s s u g g e s t s c h a n g e s i n d r u g c l e a r a n c e is o f m a j o r s i g n i f i c a n c e .  that  14  1.4.1. Effects of acute and  chronic hypoxemia on drug metabolism  M e r r i t t a n d M e d i n a (1968) o b s e r v e d that hexobarbital sleeping time in m i c e r e d u c e d at altitude. i n the study.  A l o w p l a s m a c o n c e n t r a t i o n o f h e x o b a r b i t a l at altitude w a s  was  reported  T h e in vitro m e t a b o l i s m o f h e x o b a r b i t a l i n h e p a t i c m i c r o s o m e s w a s  higher  i n the p r e p a r a t i o n f r o m the rats at altitude t h a n t h o s e f r o m the rats at g r o u n d  level.  Similar results w e r e s h o w n in m a n y drugs undergoing oxidative m e t a b o l i s m such  as  zoxazolamine, phenobarbital, and phenylbutazone ( M e d i n a and Merritt, 1970). In contrast, C u m m i n g a n d M a n n e r i n g (1970) reported increased hexobarbital halflife at l o w P 0  ( = 4 5 r n m H g ) i n b o t h i s o l a t e d l i v e r a n d in vivo, a n d p r o v e d r e d u c e d h e p a t i c  2  m e t a b o l i s m at l o w e r P 0 . 2  R o t h and R u b i n (1976) further e x a m i n e d the  relationship  between hepatic blood flow and hexobarbital metabolism in hypoxic-hypoxemia carbon monoxide (CO) induced hypoxemia. decreased  P a 0  2  and oxygen  Both CO-induced and hypoxic  saturation, whereas PaC0  h y p o x e m i a ( h y p o c a p n i a d u e to hyperventilation). i n d u c e d b y h y p o x e m i a (i.e.  33%  A  2  hypoxemia  is r e d u c e d o n l y i n  redistribution of cardiac  reduction in hepatic blood flow) was  hypoxic hypoxia/hypocapnia, resulting in reduced hepatic metabolism of  and  hypoxic output  observed  in  hexobarbital.  T h e s e experiments s e e m e d to give contradictory results f r o m the previous studies (Merritt a n d M e d i n a , 1968; M e d i n a a n d M e r r i t t , 1 9 7 0 ) , h o w e v e r , w e s h o u l d c o n s i d e r that rats at a l t i t u d e i n t h e s t u d i e s o f M e d i n a a n d M e r r i t t w e r e e x p o s e d t o c h r o n i c h y p o x e m i a (i.e. days), whereas the studies of C u m m i n g a n d M a n n e r i n g (1970) a n d R o t h a n d (1976) involved acute hypoxemia.  5  Rubin  15  T h e s e studies demonstrated the different effects of acute a n d chronic  hypoxemia.  A c u t e h y p o x i a is u s u a l l y a s s o c i a t e d w i t h i m p a i r e d h e p a t i c d r u g m e t a b o l i s m 1981), whereas chronic hypoxia m a y  actually stimulate  e n z y m e s y s t e m s [ i n d u c t i o n ] ( d u S o u i c h et al,  1978).  oxidative  drug  Therefore, the  (Jones,  metabolizing  pharmacokinetic  study during h y p o x e m i a m u s t take into account the different effects o f acute a n d chronic h y p o x e m i a o n d r u g metabolism and, in turn, drug kinetics.  1.4.2. Effects of hypoxemia on hepatic drug metabolism It is v a l u a b l e t o e x a m i n e t h e s t u d i e s o n a n t i p y r i n e k i n e t i c s d u r i n g since  antipyrine elimination  microsomal enzyme  activity  seems  to  be  proportional to  ( K o l m o d i n e et  al,  1969).  the  hypoxemia,  non-specific  hepatic  In addition, antipyrine  is  distributed evenly throughout b o d y water, a n d metabolized in the liver, without extensive excretion b y the kidney in a n u n c h a n g e d C u m m i n g  (1976) o b s e r v e d that patients w i t h l o w P a 0  f o r m . ( L a y b o u m et 2  (i.e.  2  were  (1986) also found lower antipyrine  ==18%) in patients with p u l m o n a r y disease than in healthy volunteers.  A g n i h o t r i et al  1986).  (less than 55 m m H g ) h a d  prolonged antipyrine half-life (18.4 hr) c o m p a r e d with those w h o s e P a 0 t h a n 5 5 m m H g ( 8 . 4 h r ) . L a y b o u m et al.  al,  In  a  greater clearance contrast,  (1978) observed enhanced antipyrine clearance in patients with C O P D .  S o m e patients showed hypercapnia, which could cause an increase in hepatic blood ( H u g h e s et al,  any  1979) and, in turn, hepatic drug clearance.  flow  Patients with C O P D are also  likely to suffer c h r o n i c h y p o x e m i a , thus l e a d i n g to the i n d u c t i o n o f hepatic  enzyme  16  s y s t e m s ( d u S o u i c h et al, 1 9 7 8 ) .  Therefore, these contradicting observations m a y  have  resulted f r o m different clinical conditions in these studies.  1.4.3. Effects of hypoxemia on drug disposition and protein binding  D u S o u i c h et al.  (1978) suggested that the effects o f respiratory disease o n  e f f i c a c y o f m o s t l o n g t e r m antibiotic u s a g e is l i k e l y to b e m i n i m a l , s i n c e the indices are wide.  H o w e v e r , h y p o x e m i a causes significant alteration in  s e r u m p h a r m a c o k i n e t i c s . M y e r s et al. amikacin in hypoxemic (Pa0 infants (4.8 hr). blood  flow,  with  2  the  therapeutic  aminoglycoside  (1977) s h o w e d a significant increase in half-life of  < 5 0 m m H g ) infants (7.3 hr) c o m p a r e d to  normoxemic  T h e d e c r e a s e d c l e a r a n c e i n h y p o x e m i a is l i k e l y d u e to ar e d u c e d r e n a l a concomitant  vasoconstriction (Bruns, 1978).  decrease  in glomerular  filtration rate  from  renal  H y p o x i c stimulation o f aortic chemoreceptors results in  r e n a l v a s o c o n s t r i c t i o n ( K o m e r , 1 9 6 3 ) . Ar e d u c t i o n i n a m i k a c i n a n d g e n t a m i c i n d u r i n g h y p o x e m i a w a s o b s e r v e d i n r a t a n d r a b b i t ( M i r h i j et al, 1 9 7 8 ) .  clearance  The  apparent  v o l u m e o f distribution was not affected b y h y p o x e m i a , thus the change in d r u g clearance is d i r e c t l y r e l a t e d to a c h a n g e i n d r u g e l i m i n a t i o n .  Since aminoglycoside excretion  a l m o s t e n t i r e l y m e d i a t e d b y g l o m e r u l a r f i l t r a t i o n ( L e v y et al.  is  1975), the alteration in renal  excretion during h y p o x e m i a could account for the change in a m i k a c i n elimination. Very  different results  Souich  and  Significant increases in the apparent v o l u m e o f distribution a n d  non-  renal clearance were observed in rabbits during h y p o x e m i a and metabolic acidosis.  A n  Couteau, 1984).  were  observed  with  sulphamethazine  (du  i n c r e a s e i n the a p p a r e n t v o l u m e o f d i s t r i b u t i o n d u r i n g h y p o x e m i a is p r o b a b l y r e l a t e d to  17  decreased protein b i n d i n g o f this drug, thus leading to a n increase in  sulphamethazine  f r e e f r a c t i o n . S i n c e the p K a o f s u l p h a m e t h a z i n e is 7.4, a c h a n g e i n p l a s m a p H a s s o c i a t e d with  pulmonary  disorders  hypercapnia and metabolic  will  affect  acidosis,  the  ionization  of  sulphamethazine.  the proportion of non-ionized  With  sulphamethazine  increased (the ratio o f i o n i z e d to n o n - i o n i z e d s u l p h a m e t h a z i n e d e c r e a s e d b y 4 2 % ) , this  subsequently  may  affect  the  distribution  and  clearance  of  and  sulphamethazine.  H o w e v e r , the changes in b l o o d p H a n d subsequent alteration in renal elimination  will  h a v e various results, since the increase in non-ionized d r u g m a y not only e n h a n c e  drug  diffusion (increasing volume  o f distribution, glomerular filtration rate a n d  non-renal  clearance), but also increase tubular re-absorption (decreasing renal clearance).  Since  these c h a n g e s in disposition c a n counter balance each other, the c o n s e q u e n c e appears that the changes in p H associated with hypercapnia only slightly affects  sulphamethazine  kinetics.  I n contrast, h y p o x e m i a , w h i c h h a s little effect o n b l o o d p H ,  increased  sulphamethazine  free  fraction, thereby  significantly  increasing  c l e a r a n c e a n d e x e r t i n g a g r e a t e r e f f e c t o n d r u g k i n e t i c s ( d u S o u i c h et al, and Couteau, 1984).  an  increased  hypoxemia.  non-renal  1978; d u Souich  D r u g s s u c h as p h e n o b a r b i t a l ( W a d d e l l a n d B u t l e r , 1 9 5 7 ) ,  s a l i c y l a t e s ( H i l l , 1 9 7 1 ) a n d s u l p h a e t h i d o l e ( K o s t e n b a u d e r et al, show  significantly  volume  of distribution and decreased  1 9 6 2 ; D e t t l i et al,  renal elimination  the 1967)  during  18  1.4.4. Digoxin kinetics in hypoxemia  A n increased sensitivity to digitalis in patients w i t h C O P D / h y p o x e m i a ( B a u m al.,  1 9 5 6 ; B a u m et al.,  and Smith,  1972;  1959; M o r r i s o n and K i l l i p , 1971) and in dogs with hypoxia (Beller  H a r r i s o n et  al.  1968)  has been reported.  It w a s  suggested  hypoxemia decreases digoxin plasma concentration, but increases myocardial l e v e l s i n a n a e s t h e t i z e d a n d a r t i f i c i a l l y v e n t i l a t e d d o g s ( H a r r o n et al, observation (increased myocardial digoxin level) was 1981;  du  Souich  concentrations  et  al,  of digoxin  1985a,b).  D u  were  in hypoxemic  lower  Souich  et  total b o d y clearance  ( - 4 5 % ) a n d the v o l u m e  compartments.  Digoxin  tissue  that  digoxin  1978), however,  this  l a t e r c o n t r a d i c t e d ( S a i t o et  al,  al,  (1985a)  showed  and hypercapnic  plasma  dogs.  d e c r e a s e d d i g o x i n p l a s m a c o n c e n t r a t i o n is p r o b a b l y d u e to a n i n c r e a s e i n the  peripheral  et  This apparent  of distribution (-36%), mostly concentrations  were  increased  in  the  during  h y p o x e m i a - h y p e r c a p n i a , especially in liver ( - 1 5 % ) , w h i c h suggests that the increase  in  total b o d y c l e a r a n c e is m a i n l y d u e to a n i n c r e a s e i n h e p a t i c c l e a r a n c e .  A n increase  in  1976; H u g h e s  et  h e p a t i c b l o o d f l o w h a s b e e n r e p o r t e d d u r i n g h y p e r c a p n i a ( D u t t o n et al, al,  1979), further supporting the speculation of increased hepatic clearance, since  many  d r u g s exhibit b l o o d f l o w - l i m i t e d m e t a b o l i c rate. Increased d i g o x i n b i n d i n g to erythrocyte m e m b r a n e s w a s a l s o r e p o r t e d d u r i n g h y p o x e m i a / h y p e r c a p n i a ( d u S o u i c h et al,  1985a),  a n d there s e e m s to b e a direct relationship b e t w e e n erythrocyte d i g o x i n receptor  and  cardiac glycoside  and  receptor (Akera, 1977)  and volume  of distribution (Aronson  G r a h a m - S m i t h 1 9 7 6 ; 1 9 7 7 ) . T h e r e f o r e , it is s p e c u l a t e d t h a t t h e c h a n g e i n t h e d i s t r i b u t i o n p a t t e r n is p a r t l y d u e to the c h a n g e i n r e c e p t o r b i n d i n g d u r i n g h y p o x e m i a a n d h y p e r c a p n i a .  19  T h e influence  of h y p o x e m i a on digoxin kinetics a n d tissue distribution was  e x a m i n e d u s i n g t r i t i a t e d d i g o x i n b y d u S o u i c h et al. hypercapnia  study  (du  Souich  et  al,  1985a),  (1985b). a  S i m i l a r to the  significant  d i s t r i b u t i o n w a s o b s e r v e d d u r i n g h y p o x e m i a ( d u S o u i c h et al,  further  hypoxemia-  change  in  volume  1985b).  However,  this  increase in the v o l u m e o f distribution w a s largely d u e to a n increase in the size o f central c o m p a r t m e n t rather than the peripheral compartment.  of  Therefore, the increase  the in  the v o l u m e o f d i s t r i b u t i o n is l i k e l y d u e to a s i g n i f i c a n t i n c r e a s e i n p l a s m a p r o t e i n b i n d i n g of digoxin during hypoxemia.  Total body clearance of digoxin was not  significantly  changed, but an increase in renal clearance and a decrease in non-renal clearance occurred d u r i n g h y p o x e m i a ( d u S o u i c h et s t u d y ( d u S o u i c h et al,  al.  1985b).  U n l i k e with the  hypoxemia-hypercapnia  1985a), no hepatic accumulation of digoxin was observed.  These  results o n hepatic digoxin a c c u m u l a t i o n are consistent with the findings regarding hepatic b l o o d f l o w i n h y p o x e m i a ( L a r s e n et al, h y p o x e m i a / h y p e r c a p n i a ( H u g h e et  al,  1976; Richardson and Withrington, 1981) 1979; Mathie and Blumgart, 1983).  and  Increased  hepatic blood flow during hypoxemia-hypercapnia resulted in hepatic accumulation digoxin, w h e r e a s h y p o x e m i a alone d i d not alter hepatic b l o o d flow or hepatic  of  digoxin  accumulation.  1.4.5. Theophylline kinetics and  There  has  been  extensive  respiratory disorders  study  of theophylline  kinetics  during  respiratory  d i s o r d e r s , b e c a u s e t h e o p h y l l i n e is a p o t e n t b r o n c h o d i l a t o r u s e d i n t h e m a n a g e m e n t asthma  and chronic obstructive  airway  diseases  (Hendeles  et  al,  of  1983,1985,1986;  20  Ogilvie,  1978).  K o l b e c k et  al  (1979) examined  the influence  of acute  acidosis a n d alkalosis o n the v o l u m e of distribution a n d half-life in dogs. volume  o f distribution were noted, but there was  respiratory  N o changes in  a r e d u c e d h a l f - l i f e (i.e.  increased  elimination rate) d u r i n g respiratory acidosis. Since o n l y 8 % o f a d o s e o f theophylline  was  r e c o v e r e d in urine as the parent c o m p o u n d (Ogilvie, 1978), the c h a n g e in clearance  was  likely d u e to the c h a n g e in metabolic clearance.  (1981) found  no  significant change in theophylline kinetics during respiratory acidosis in dogs, w h i c h  is  c o n t r a r y t o t h e f i n d i n g o f K o l b e c k et al.  (1979).  H o w e v e r , C l o z e l et al.  A reduced theophylline clearance  o b s e r v e d i n h e p a t i c c i r r h o s i s p a t i e n t s ( M a n g i o n e et al. t o h a v e i n c r e a s e d c l e a r a n c e ( P o w e l l et al,  1978), while smokers were  and  du  Souich  found  1977; 1978). T h e s e results further suggest that  t h e o p h y l l i n e is m a i n l y e l i m i n a t e d b y h e p a t i c m i c r o s o m a l o x i d a t i v e  Letarte  was  (1984)  observed  enzymes.  increased  theophylline  serum  concentrations during hypoxemia and/or hypercapnia, but not with metabolic acidosis  in  rabbits. T h e d e c r e a s e d clearance w a s m a i n l y d u e to a r e d u c t i o n in n o n - r e n a l t h e o p h y l l i n e clearance, thus suggesting a reduction in theophylline biotransformation.  A  decreased  v o l u m e o f distribution w a s also o b s e r v e d w i t h h y p o x e m i a , therefore the elimination rate constant was not significantly affected during h y p o x e m i a unlike with hypercapnia  and  hypoxemia/hypercapnia.  and  S a u n i e r et al.  (1987) further e x a m i n e d the effects of acute  chronic hypoxemia on theophylline disposition in conscious  dogs.  In contrast to  the  previous studies, no changes in theophylline disposition were observed.  F u r t h e r m o r e , the  r e c o v e r y o f t h e o p h y l l i n e a n d its m e t a b o l i t e s , 1 , 3 - d i m e t h y l u r i c a c i d a n d  3-methylxanthine,  in urine was not significantly different f r o m control.  21  1.4.6. Lidocaine kinetics/metabolism during hypoxemia and  The  available  data o n lidocaine kinetics  similarity with  MCP  during hypoxemia/hypercapnia  may  provide s o m e insight about the effects o f these perturbations o n M C P kinetics, since these two  drugs  share  some  similar  chemical  structure  and  in vivo m e t a b o l i s m  and  pharmacokinetic properties, despite their rather different pharmacological effects. M a r l e a u et al.  (1987) e x a m i n e d the effects o f h y p o x e m i a and/or h y p e r c a p n i a o n  l i d o c a i n e a n d I n d o c y a n i n e G r e e n k i n e t i c s i n rabbits. I n h u m a n s , l i d o c a i n e is e x t e n s i v e l y a n d r a p i d l y m e t a b o l i s e d i n t h e l i v e r i.e. 70%  it is c h a r a c t e r i z e d b y ah e p a t i c e x t r a c t i o n r a t i o o f  ( S t e n s o n et al, 1 9 7 1 ) , t h u s t h e h e p a t i c c l e a r a n c e i s l i k e l y t o b e a f l o w - l i m i t e d  process, just as w i t h I C G a n d M C P . T h e apparent v o l u m e o f distribution a n d the  total  b o d y c l e a r a n c e o f l i d o c a i n e w e r e n o t s i g n i f i c a n t l y a f f e c t e d at l o w e r l i d o c a i n e d o s e s  (130  m g / m i n / K g , infusion).  T h e s e r u m to cerebrospinal fluid ratio o f lidocaine w a s  not  m o d i f i e d with a l o w lidocaine dose, but slight decreases in lidocaine clearance  were  observed with higher doses (infusion, 260 m g / m i n / K g ) . A n increased s e r u m level of  one  metabolite, N-monoethyl-glycinexylidide (MEGX),  and  hypoxemia-hypercapnia  (Marleau  et al,  1987).  was observed with hypercapnia The  increased  plasma  c o n c e n t r a t i o n is m o r e l i k e l y d u e to ad e c r e a s e i n the 3 - h y d r o x y l a t i o n o f M E G X , t h a n d u e to ad e c r e a s e i n the d e e t h y l a t i o n o f M E G X al, 1 9 8 4 ) ,  therefore  the  elimination  of MEGX  M E G X rather  t o g l y c i n e x y l i d i d e ( G X ) ( S u z u k i et is r e d u c e d  while  concentration was similar in hypercapnia and hypoxemia-hypercapnia.  the  G X  plasma  22  Scheme I  Comparison of the N-deethylation reactions of MCP and lidocaine  ci  /CH  H N—^  /  - N H - iC H - C H - N ^  \ — CO  2  2  C  2  H  5  C H2" 9  /  ^  2  OCH  3  C,H 2 "  5  /CH CO - N H - C H - C H - N H - C H 2  OCH  2  2  ^  5  ,  \  H N—ft  V - C O -NH - C H - C H - N H  9  2  OCH  3  2  5  ^  2  3  ^—NH-CO -CHj-NH-C^j CH  3  .CH  3  monodeethyl M C P  Cl  5  Lidocaine  Cl 2  H  2  Metoclopramide  H N—^  2  C  NH - C O - C H - N ^  monomethylglycinexylidide (MEGX)  ^ — N H - C O - C H - NH CH  dideethyl M C P  2  3  glycinexylidide (GX)  T h i s b i o t r a n s f o r m a t i o n i.e. d e e t h y l a t i o n o f l i d o c a i n e t o M E G X a n d G X m a y  share  similar characteristics w i t h the m e t a b o l i s m o f M C P to m o n o - a n d di-deethyl M C P . S o u i c h et al. ( 1 9 9 2 ) o b s e r v e d i n c r e a s e d p l a s m a M E G X a n d G X c o n c e n t r a t i o n s h y p o x e m i a i n al i d o c a i n e i n f u s i o n s t u d y i n d o g s , w h e r e a s p l a s m a l i d o c a i n e remained unchanged.  T h e radioactive microsphere m e t h o d w a s used to estimate hepatic,  moderate h y p o x e m i a , there was an increase in cerebral b l o o d flow, but renal a n d flows  decreased  the  were rate  during  concentrations  renal a n d cerebral b l o o d f l o w s three hours after the e n d o f lidocaine infusion. With  blood  D u  not  affected.  of elimination  I tw a s  concluded  of both  active  that  acute  metabolites  moderate  acute hepatic  hypoxemia  of lidocaine  without  23  m o d i f y i n g t h e p e r f u s i o n t o t h e o r g a n s r e s p o n s i b l e f o r t h e i r e l i m i n a t i o n ( d u S o u i c h et  al,  1992).  Rationale  1.5.  T h e r e is s u b s t a n t i a l e v i d e n c e that p u l m o n a r y d y s f u n c t i o n c a u s i n g influences drug disposition and metabolism.  hypoxemia  These changes are likely either a  c o n s e q u e n c e o f the perturbations in b l o o d gas  and acid-base  s t a t u s (e.g.  direct  respiratory  alkalosis d u e to hyperventilation a n d lactic acidosis) or a result o f the c a r d i o v a s c u l a r a n d m e t a b o l i c r e s p o n s e s t o h y p o x e m i a (e.g.  redistribution of cardiac output).  T h e r e is a l s o a  possibility that therapeutic agents administered will interfere w i t h the r e s p o n s e to h y p o x e m i a . hypoxemic conditions. chemical  A  were altered  in  H o w e v e r , the changes w e r e highly variable a n d dependent o n  the  characteristics  of  number of pharmacokinetic parameters  the  specific  drug,  the  prevailing  conditions, protein b i n d i n g a n d the species used in the experiments. likely caused  by  described in Section  compensatory  various  physiological  changes  induced  by  blood  gas/acid-base  These changes  hypoxemia/acidosis  were as  1.4.  T h e study of M C P kinetics a n d p h a r m a c o d y n a m i c s during h y p o x e m i a will result in an i m p r o v e m e n t o f our understanding in the following areas: 1.  A pharmacokinetic study during h y p o x e m i a will provide information o n the changes in both M C P disposition and metabolism.  I  24  2.  A  study  of metabolite  kinetics  metabolism during hypoxemia.  will provide information on  alteration in  drug  T h e a s s e s s m e n t o f d r u g m e t a b o l i s m is i m p o r t a n t ,  s i n c e t h e r e i s i n c r e a s i n g e v i d e n c e t h a t t h e m e t a b o l i t e s o f s o m e d r u g s (e.g.  morphine  glucuronides (Mulder, 1992) and m o n o - and di-deethylated lidocaine, MEGX G X ( N a r a n g et ai,  1978)) have their o w n pharmacological activity (agonistic  and and/or  antagonistic) and, in selected instances, toxicity. T h i s toxicological response m a y  be  altered during h y p o x e m i a and hypoxia. T h e past studies o f M C P have provided a solid base of pharmacokinetic, metabolic  and  p h a r m a c o d y n a m i c information in n o r m o x e m i c non-pregnant and pregnant sheep.  The  present  study  parameters  will examine  o f the effect  of hypoxemia/hypoxia  characterizing M C P in non-pregnant  sheep.  on  all the  Such a study  will  salient provide  essential g r o u n d w o r k for future studies o f the effects o f h y p o x e m i a o n m a t e r n a l a n d fetal MCP  disposition and metabolism during pregnancy.  1.6.  Objectives  T h e objectives of the present study are : 1.  to c o n f i r m the applicability in a h y p o x e m i a study o f a n existing GC-MSD  assay  m e t h o d f o r M C P a n d its m e t a b o l i t e s , m o n o - a n d d i - d e e t h y l M C P . 2.  to d e t e r m i n e t h e e x t e n t to w h i c h t h e p h a r m a c o k i n e t i c s o f M C P a n d its are altered b y reduced oxygen  3.  metabolites  supply.  to e x a m i n e physiological changes i n d u c e d b y h y p o x e m i a d u r i n g steady-state administration.  drug  25  2.  2.1 2.1.1  EXPERIMENTAL  Materials and Supplies Chemicals 4-armno-5-chloro-2-m  monohydro-  chloride monohydrate (MCP»HC1»H 0) 2  and MCP'HCL  5 m g / m L (Reglan® Injectable, 2  and 5m L ampoules) were obtained from A . H .Robins Research Laboratories (Richmond, V A , U S A ) a n d A . H . R o b i n s C a n a d a Inc. (Montreal, P Q ) . N-ethyl-aminoethyl-benzamide MCP»HC1»H 0)  and  hydrochloride  monohydrate  2  monohydrochloride  yl)-oxy-N,N-diethylaminoethyl 2  2.1.2  monohydrate  (monodeethyl  4-amino-5-chloro-2-methoxy-arninoethyl-benzamide (dideethyl  MCP»HC1»H 0) 2  l a b o r a t o r y ( R i g g s et al, 1 9 9 4 ) . T h e i n t e r n a l s t a n d a r d ,  (BMY«HC1»H 0)  4-amino-5-chloro-2-methoxy-  benzamide  were  synthesized  monoi n our  4-amino-5-chloro-2(2-butanone-3-  monohydrochloride  monohydrate  was supplied by Bristol-Myers Squibb Co. (Wallingford, C T , U S A ) .  Reagents S o d i u m acetate a n d 2-amino-2-hydroxymethyl-l,3-propanediol ( T R I S free  were obtained from B D H Chemicals, Toronto, O N . American Chemical Society  base) ( A C S )  reagent grade S o d i u m H y d r o x i d e pellets were obtained f r o m Fisher Scientific C o . , Fair Lawn, NJ.  A C S reagent grade Hydrochloric acid 3 7 %  was obtained from American  Scientific a n d C h e m i c a l , Seattle, W A . A m m o n i a Solution 2 5 % was obtained f r o m Fisher  26  Scientific C o . Heptafluorobutyric anhydride (HFBA)  a n dtriethylamine ( T E A ) sequanal  Grade were obtained from Pierce Chemical Co., Rockford, IL, U S A .  2.1.3  Enzymes  G l u c u r o n i d a s e ( G l u c u r a s e ® : P - D - G l u c u r o n i d e g l u c u r o n o s o h y d r o l a s e : E C 3.2.1.3 (bovine liver) approx. 5 0 0 0 S i g m a units/mL, acetate buffered to p H 5.0 at 25°C) a n d sulfatase  (Arylsulfatase,  aryl-sulfate  sulfohydrolase,  phenolsulfatase;  E C 3.1.6.1 1 9  units/mL partially purified e n z y m e i n 5 0 % glycerol-0.01 M Tris solution, p H 7.5 [ L o t 42H6814]) were purchased from Sigma Chemical Co., St.Louis, M O , U S A .  2.1.4  Solvents  Toluene, a n d dichloromethane (distilled i n glass) w e r e purchased f r o m  Caledon  Laboratory. Inc., G e o r g e t o w n , Ont.. D e i o n i z e d w a t e r w a s p r o d u c e d o n site u s i n g a MilliQ ® s y s t e m , M i l l i p o r e C o r p . , B e d f o r d , M A . . A C Sr e a g e n t g r a d e m e t h a n o l a n d a c e t o n e were obtained from B D H Chemicals, Toronto, O N .  2.1.5  Gases  Hydrogen Ultra H i g h Purity ( U H P ) , helium U H P a n dargon/methane (95:5) were o b t a i n e d f r o m M a t h e s o n G a sP r o d u c t s C a n a d a L t d . , E d m o n t o n , A B .  27  2.1.6. Supplies for  animal studies  The  supplies  following  was  used in the a n i m a l studies: needles a n d  disposable Lure-Lok® syringes for drug administration Dickinson  Canada,  Mississauga,  O N ) ;  heparinized  and sample collection Vacutainer®  tubes  works,  C o m i n g , N Y , U S A ) ; polytetrafluorethylene  (PTFE)  (Becton-  (Vacutainer  Systems, R u t h e r f o r d , N J , U S A ) ; 15 m L P y r e x ® disposable glass culture tubes Glass  plastic  (Coming  lined screw  caps  (Canlab, V a n c o u v e r , B C ) ; silicone rubber tubing for catheter preparation ( D o w C o r n i n g , Midland, M I , U S A ) .  Stock and  2.2.  Reagent Solutions  Metoclopramide»HCl»H 0 (=11.82 m g of MCP«HC1»H 0  is e q u i v a l e n t to = 1 0  m g  (=12.00 m g of mdMCP«HCl»H 0  is  2  2  o f M C P free base), m o n o d e e t h y l M C P » H C 1 » H 0 2  equivalent to =10 m g o f m d M C P ddMCP»HCl»H 0 2  2  free base) a n d dideethyl M C P » H C 1 » H 0 2  is e q u i v a l e n t to = 1 0 m g o f d d M C P  and dissolved in HPLC  (=12.24 m g  free base) w e r e accurately  of  weighed  grade water in individual volumetric flasks. U s i n g serial dilution,  these stock solutions w e r e a d d e d to a c o m b i n e d stock solution o f final concentration o f = 0.04 jig/rnL each.  T h e internal standard, BMY»HC1»H 0 2  (=11.03 m g of  BMY*HC1»H 0 2  is e q u i v a l e n t to = 1 0 m g o f B M Y free b a s e ) w a s a c c u r a t e l y w e i g h e d a n d d i s s o l v e d d e i o n i z e d water using serial dilution to a final concentration o f =0.2 |ig/mL. T h e s e s o l u t i o n s w e r e s t o r e d at 4 ° C f o r u p to t w o  pellets in deionized water.  stock  months.  S o d i u m h y d r o x i d e ( N a O H ) , as I M a n d 5 M solutions, w a s p r e p a r e d b y NaOH  in  A q u e o u s a m m o n i a (4%)  dissolving  solution was prepared  by  28  diluting a m m o n i a solution strong (27%) in deionized water. Triethylamine 0.0125 M in toluene was prepared by diluting triethylamine with toluene and subsequently adding four or five NaOH A  0.2  pellets to the resulting solution. M ( p H 5.0)  s o d i u m acetate buffer, for glucuronidase  incubation,  was  p r e p a r e d b y dissolving s o d i u m acetate in deionized water a n d adjusting this solution to a final p H o f 5.0 using glacial acetic acid.  TRIS  buffer, 0.05 M( p H 7.5), for  incubation w a s prepared b y dissolving TRIS free base in deionized water a n d  sulphatase adjusting  the final p H o f this solution to 7.5 u s i n g a 1 M H C 1 solution.  2.3  Sample Preparation, Extraction and  Derivatization  T h e procedure for sample extraction a n d derivatization r e m a i n e d the s a m e p u b l i s h e d b y R i g g s et al.  as  (1994).  All glassware used in the preparation of stock solutions a n d extraction was  pre-  washed with detergent in.an automatic-dishwasher and then soaked in chromic acid for a m i n i m u m of six hours.  T h e r e a f t e r , it w a s t h o r o u g h l y r i n s e d w i t h t a p w a t e r o v e r 6 h o u r s  using amechanical rinser and finally with distilled/deionized water before being  dried  overnight.  2.3.1  Sample extraction  A l l extractions w e r e carried out in 15 m L a n d 10 r n L glass culture tubes PTFE-lined screw caps. deionized  with  Plasma (100-200 u L ) and urine (100-200 jiL of 50x dilution in  water) obtained  f r o m animal studies were  a d d e d to clean  15  m L  tubes  29  containing 0.5 m L I M N a O H 0.2 jig/mL).  ( p H =14) a n d 0.3 m L o f B M Y internal s t a n d a r d solution  T h e a q u e o u s p h a s e w a s then adjusted to a final v o l u m e o f 2.2 m L  deionized water.  (=  with  S i x m L o f d i c h l o r o m e t h a n e w a s a d d e d , t u b e s c a p p e d , a n d M C P a n d its  selective metabolites a n d B M Y w e r e extracted into the organic layer b y shaking for  20  m i n o n a rotary shaker (Labquake® m o d e l 415-110, L a b Industries, Berkeley, C A , U S A ) . After  shaking, tubes were  stored  in a freezer  (=  -20°C)  for  10  min.  Following  c e n t r i f u g a t i o n at 2 5 0 0 r p m f o r 5 m i n , the t u b e s w e r e r e m o v e d a n d s h a k e n lightly to b r e a k a n y e m u l s i o n that m a y have f o r m e d d u r i n g the extraction process. m i n o f centrifugation, the aqueous layer w a s  Following a further 5  v a c u u m aspirated and discarded.  The  r e m a i n i n g organic layer w a s transferred to clean 10 m L tubes using Pasteur pipettes a n d taken to dryness using a n A S 2 9 0 A u t o m a t i c S p e e d V a c ® concentrator (Savant Inc.,  Farmingdale, N Y ,  U S A )  under  controlled  vacuum.  reconstituted w i t h 0.8 m L o f toluene containing 0.0125 M heptafluorobutyric anhydride (HFBA)  The T E A . A  Instruments  dried residue  was  20 u L volume  was added, tubes capped, vortex-mixed  (Vortex-  G e n i e ® , Fisher Scientific Industries, Springfield, M A , U S A ) a n d these mixtures p l a c e d i n a n o v e n at 5 5 ° C f o r 6 0 m i n . derivatizing agent (HFBA)  After c o o l i n g to r o o m temperature, the  for 10 sec).  were excess  w a s r e m o v e d b y hydrolysis w i t h 0.5 m L o f d e i o n i z e d  (vortex-mix for 10 sec) a n d neutralized with 0.5 m L o f 4 % a m m o n i a solution  of  water  (vortex-mix  F o l l o w i n g c e n t r i f u g a t i o n o f the t u b e s at 2 5 0 0 r p m f o r 1 m i n , the  toluene  layer w a s i m m e d i a t e l y transferred to a clean a u t o s a m p l e r vial w i t h a glass insert  and  c a p p e d with a PTFE-lined  the  GC-MSD  for the final assay  a l u m i n u m seal. measurement.  A 2 u L v o l u m e aliquot was injected into  30  Scheme II  Extraction procedure for MCP and its metabolites  Plasma and urine samples (0.1-0.2 mL)  Blank plasma or urine samples spiked with MCP/metabolite standard  1M NaOH, 0.5 mL BMY (I.S.), 0.3 mL Water q.s. 2.0 mL Dichloromethane, 6 mL Shake, 20 min Centrifuge, 2 X 5 min  Organic layer Aspirate aqueous layer j  Evaporate to dryness under reduced pressure at 40 C 9  Dried extract 0.0125 M TEA in toluene, 0.8 mL HFBA, 20 uL 1 hour at 55 C 9  Cool to room temperature Water, 0.5 mL, vortex 10 sec 4% aq. ammonia, 0.5 mL, vortex 10 sec Centrifuge, 1 min Discard aqueous layer Inject into GC/MSD (2 uL)  31  2.3.2  Analysis of glucuronide and  sulphate conjugates  The presence of glucuronide and sulphate ddMCP  conjugates of M C P , mdMCP  and  in urine was determined by enzymatic hydrolysis. T h e hydrolysis procedure  i n t h e p r e s e n t s t u d y w a s a m o d i f i e d v e r s i o n o f B r a s h e r et al. of ritodrine conjugates in humans. to d e t e r m i n e  (1988) for the  used  determination  U r i n e s a m p l e s w e r e d i v i d e d i n t o 3 a l i q u o t s o f 0.1  the concentrations  o f the c o m p o u n d s  in non-conjugated  form  m L  [Set  I],  g l u c u r o n i d e - c o n j u g a t e d f o r m [ S e t II] a n d s u l p h a t e - c o n j u g a t e d f o r m [ S e t III].  Non-conjugated [Set  I] : A v o l u m e o f 0 . 9 m L o f d e i o n i z e d w a t e r w a s a d d e d t o e a c h  aliquot. Glucuronide-conjugated [Set ( p H 5.0)  a n d 0.5  II]  : V o l u m e s o f 0.4 m L 0.2 M s o d i u m acetate buffer  m L o f glucuronidase w e r e a d d e d to e a c h aliquot  to  provide a glucuronidase activity of 2 5 0 0 U / m L . Sulphate-conjugated [Set in 0.05 M  TRIS  III]  : A v o l u m e o f 0.9 m L o f diluted sulphatase  solution  buffer ( p H 7.5) w a s a d d e d e a c h aliquot to g i v e a  final  sulphatase activity of 0.25 U / m L .  A l l the a l i q u o t s w e r e i n c u b a t e d o v e r n i g h t at 3 7 ° C  in a water bath.  Following  i n c u b a t i o n , the s a m p l e s w e r e c o o l e d to r o o m t e m p e r a t u r e a n d extracted as d e s c r i b e d in Section 2.3.1. i n S e t I.  Non-conjugated MCP/mdMCP/ddMCP  T h e concentrations  w e r e m e a s u r e d f r o m the  of glucuronide-conjugated MCP/mdMCP/ddMCP  samples were  32  c a l c u l a t e d b y d e d u c t i n g n o n - c o n j u g a t e d c o n c e n t r a t i o n s f r o m t h e s a m p l e s i n S e t II.  The  c o n c e n t r a t i o n o f s u l p h a t e - c o n j u g a t e s w a s c a l c u l a t e d s i m i l a r l y f r o m t h e s a m p l e s i n S e t III.  2.4  Standard Curve Preparation V o l u m e s o f 0.02, 0.04, 0.08, 0.16, 0.32, 0.64 a n d 0.80 m L o f m i x e d stock  of M C P , mdMCP NaOH,  and ddMCP  solution  w e r e pipetted into 15 m L tubes containing 0.5 m L 1  0.3 m L o f internal s t a n d a r d B M Y solution (0.3 u , g / m L ) a n d c o r r e s p o n d i n g b l a n k  biological fluids.  T h e a q u e o u s p h a s e w a s adjusted to a final v o l u m e o f 2.2 m L  with  d e i o n i z e d water a n d then extracted a n d derivatized as d e s c r i b e d in S e c t i o n 2.3. standard curve for each of M C P , mdMCP  and ddMCP  was calculated and plotted by  p e a k area ratio of heptafluorobutyryl ( H F B ) derivatives of M C P , m d M C P over B M Y versus the k n o w n concentration of M C P , m d M C P  and ddMCP  and  a c c o r d i n g t o t h e . m e t h o d u s e d i n R i g g s et al.  2.5  the  A  curves  (1994).  Instruments and Equipment  Gas  chromatography (GC-MSD)  A Model 5971A  A  ddMCP  free base.  weighting factor 1/Y(area ratio) w a s used for linear regression o f the standard  2.5.1  M  Mass  Windows®  5 8 9 0 S e r i e s II H e w l e t t - P a c k a r d g a s c h r o m a t o g r a p h e q u i p p e d w i t h a H P Selective Detector  (MSD),  split-splitless  capillary inlet system,  and  a  G C ChemStation on a H P Vectra® 25T 486 computer (Hewlett-Packard Co.,  A v o n d a l e , P A ) was used for the M S D assay. phenylmethylsilicone  (Ultra-2®) 0.33  A 25 m x 0.2 m m I.D. cross-linked  u\m film thickness  fused-silica capillary  5%  column  33  ( H e w l e t t - P a c k a r d C o . , A v o n d a l e , P A ) , 78 m m x 2 m m I D . borosilicate glass inlet liners (Hewlett-Packard Co., Avondale, PA), and Thermogreen®  L B - 2 silicone rubber  septa  were also used. 2.5.2  Operating conditions for the GC and MSD The operating conditions for the GC/MSD  s y s t e m w e r e as follows: injection port  temperature, 260°C;  p u r g e time, 30 sec; split vent flow, 30 mL/rnin; initial  column  temperature, 100°C;  c o l u m n h e a d pressure, 10 p.s.i.; carrier gas (helium) f l o w rate, 3  m L / m i n ; t e m p e r a t u r e p r o g r a m : 1 0 0 ° C h o l d f o r 0.8 m i n , t e m p e r a t u r e r a m p at 4 0 ° C / m i n 270°C  t h e n h o l d c o n s t a n t f o r 5 m i n , a n d t h e n af u r t h e r i n c r e a s e to 2 9 0 ° C  h o l d for 3 m i n , f o l l o w e d b y afurther increase to 300°C  at 70°C/min  at 7 0 ° C / m i n  to and  a n d a final h o l d f o r  3.5 m i n to clean out the c o l u m n after e a c h r u n ; T o t a l r u n time, 16.98 m i n ; M S D transfer line a n d source temperature, 310°C.  Aliquots o f 2u L are injected onto the c o l u m n .  A H P 5 9 7 1 A M a s s Selective Detector was used in electron impact (EI) ionization mode  with -70  manually  tuned  e V  ionization energy  t othe  perfluorotributylamine.  ions  of  and 300uA  m/e  100,  emission  264  and  current. T h e M S D 414  using  the  standard  Chromatograms were generated in both mass scanning  and selective ion monitoring ( S I M ) m o d e s in the study.  was  (SCAN)  F o r the quantitative assay, M C P  a n d its m e t a b o l i t e s w e r e o p t i m a l l y d e t e c t e d i n S I M m o d e u s i n g t h e total i o n c u r r e n t o f m/e 3 8 0 f o r d d M C P a n d m d M C P ( G r o u p 1 ) , m/e 8 6 a n d 3 8 0 f o r M C P ( G r o u p 2 ) a n d m/e 8 6 a n d 3 6 6 for B M Y ( G r o u p 3).  T h e d w e l l t i m e w a s set at 7 0 0 m s e c , p e r g r o u p ,  which  p r o v i d e d 1.37 s c a n c y c l e / s e c f o r G r o u p 1a n d 0 . 6 9 7 s c a n c y c l e / s e c f o r G r o u p s 2 a n d 3.  34  2.6 2.6.1  Animal preparation Animal handling T h e e w e s (Dorset, Suffolk, or crossbred) w e r e b r o u g h t to the A n i m a l  U n i t in the  Children's V a r i e t y R e s e a r c h C e n t r e a b o u t o n e w e e k p r i o r to surgery to a l l o w t h e m  to  b e c o m e acclimatized. T h e ewes were fasted overnight before surgery a n d each received a 3 - m g i.v. injection o f atropine sulfate ( A s t r a P h a r m a c e t i c a l s Inc., M i s s i s s a u g a , O N )  10-15  m i n p r i o r to i n d u c t i o n o f a n a e s t h e s i a w i t h P e n t o t h a l (1 m g / k g , i.v.; A b b o t t L a b o r a t o r y , Montreal, Que.).  Following endotracheal intubation, anaesthesia was  maintained  by  ventilating the e w e s throughout the surgery with 1.0-1.5 % halothane (Ayerst Laboratory, Montreal, Que.) and 60-70% N  2  0 in oxygen.  2.6.2. Surgical Preparation Aseptic techniques were employed throughout surgery. catheters ( D o w  C o r n i n g , C o m i n g , N Y ) , filled with  i m p l a n t e d in the femoral vein a n d artery.  Sterile silicone  heparinized 0.9%  In s o m e animals, an ultrasound  rubber  saline,  were  transit-time  f l o w p r o b e ( T r a n s o n i c S y s t e m s Inc., Ithaca, N Y , U S A ) w a s i m p l a n t e d o n the left f e m o r a l artery. T h e n , a n oblique incision w a s m a d e in the a b d o m e n to the right o f the u m b i l i c u s , a n d the liver a n d b o w e l w e r e retracted to e x p o s e the gall bladder. T h e gall b l a d d e r held steady with an artery forceps, a n d a small incision was m a d e in the apex,  was  through  w h i c h asilicone r u b b e r catheter w a s inserted. T h e catheter w a s s e c u r e d b y ap u r s e string suture w h i c h also closed the gall bladder incision.  Except during experiments,  this  35  catheter w a s k e p t sealed to a l l o w for the n o r m a l d r a i n a g e o f bile t h r o u g h the bile duct. H o w e v e r , these flow probes a n d bile catheters w e r e used in the other studies. All  catheters  were  filled  with  heparinized  saline  (12  U/mL),  tunneled  subcutaneously, a n d exteriorized through a small incision in the right flank of the where they w e r e stored in a cloth pouch. T h e a b d o m i n a l incision was closed in layers.  ewe A  catheter for nitrogen infusion (10.0 m m o.d.) (Fisher Scientific, C a m b r i d g e , M A , U S A ) was then implanted in the trachea, b e t w e e n adjacent tracheal rings, 5-6 c m b e l o w  the  larynx, a n d inserted for 4-5 c m ; this catheter d i d not affect n o r m a l breathing b y the  ewe  ( G l e e d et  m L ;  Baxter  al.,  1986).  Canada,  D u r i n g s u r g e r y , a n i.v. d r i po f 5 %  Toronto, O N )  was  administered.  dextrose solution (500  Immediately  following  surgery,  ampicillin (500 m g ; N o v o p h a r m Ltd.,Toronto, O N ) and gentamicin (40 m g ; Garamycin®; Schering, Pointe Claire, Q u e . ) w e r e administered i.m. to the ewe.  F o l l o w i n g surgery, the  e w e s w e r e kept in h o l d i n g p e n s w i t h other s h e e p a n d g i v e n free access to f o o d a n d water. A m p i c i l l i n (500 m g ) a n d g e n t a m i c i n (40 m g ) i.m. w e r e also g i v e n prophylactically to the e w e for 5 days f o l l o w i n g surgery. A l l animals w e r e a l l o w e d to recover for a m i n i m u m o f 3 days before  experiments.  36  2.7  Experimental Protocol On e x p e r i m e n t a l days, the e w e w a s p l a c e d in a m o n i t o r i n g c a g e adjacent to  the  h o l d i n g p e n in full v i e w o f c o m p a n i o n e w e s a n d w i t h free access to f o o d a n d water.  A  F o l e y ® catheter ( B a r d U r o l o g i c a l D i v . , C R B a r d Inc., C o n i n g t o n , G A , U S A ) w a s via  the urethra for continuous urine collection.  injectable 5 mg/mL, to 3 mg/mL.  inserted  Metoclopramide hydrochloride (Reglan®  A . H . R o b i n s , M o n t r e a l , Q u e . ) w a s diluted w i t h sterile isotonic  saline  T h e i n f u s i o n [rate = 0.21 m g (0.07 m L ) / m i n ] w a s p r e c e d e d b y a l o a d i n g  d o s e o f 1 5 m g ( 5 m L ) g i v e n via  the femoral vein.  After reaching steady-state during  n o r m o x i a (2 hrs.), h y p o x e m i a w a s i n d u c e d b y g i v i n g 7 L / m i n i m p l a n t e d tracheal catheter.  of nitrogen through  the  T h e b l o o d gas status w a s m o n i t o r e d b y taking arterial b l o o d  s a m p l e s at the intervals d e s c r i b e d in the s a m p l i n g schedule.  T h e nitrogen flow rate  a d j u s t e d to m a i n t a i n the h y p o x e m i a at the d e s i r e d l e v e l o f P a 0 hypoxemic period was continued for 6 hours.  2  (i.e.  was  55-60 m m Hg). The  Then, the nitrogen flow was stopped  and  the infusion w a s continued for another 6 hours. T h e total infusion time o f M C P w a s  thus  14 hours.  A r t e r i a lb l o o d (2.5 m L ) s a m p l e s for M C P d e t e r m i n a t i o n a l o n g w i t h b l o o d  gas  s a m p l e s ( ~ 0 . 7 - 1 . 0 m L ) w e r e t a k e n as s h o w n in the p r o t o c o l [ S c h e m e I V ] .  The  samples  heparinized  for  M C P  determination  were  transferred  immediately  to  a  blood  V a c u t a i n e r ® ( B e c t o n - D i c k i n s o n , R u t h e r f o r d , N J ) a n d c e n t r i f u g e d at 3 5 0 0 r p m f o r 10 m i n . U r i n e s a m p l e s w e r e collected a c c o r d i n g to the protocol.  T h e volume and p H of urine  were measured with a graduated cylinder and a Fisher Accumet®  p H meter model  (Fisher  urine  Scientific  Inc.,  Cambridge, M A , U S A ) .  Plasma  and  samples  620 were  37  transferred to disposable borosilicate glass culture tubes ( C o r n i n g Glass, C o r n i n g , N Y ) w i t h T e f l o n o r P T F E - l i n e d s c r e w c a p s a n d s t o r e d a t -20°C u n t i l t h e t i m e o f a s s a y . Control experiments  were also carried out, using the s a m e M C P infusion  sampling protocol, but without hypoxemia.  T h e following are the schematic diagrams  experimental protocol used in the present study.  Scheme III  Schematic diagrams of the experimental protocol  1. Experimental Group  Pre-hypoxemic (normoxemic)  2.  8  2  HourO  14  Post-hypoxemic (normoxemic)  Hypoxemic  Control Group  HourO  8  2  Pre-hypoxemic . , (normoxemic) 3V  Normoxemic  14  Post-hypoxemic , ' . . (normoxemic)  and of  38  S c h e m e IV  M C P infusion-hypoxemia sampling protocol  [Pre-hypoxemic peroid (2 hours)] BG  0:05  BL  0:00  Loading dose (15 mg)/lnfusion (rate  blood controls U  0:05  BL  0:15  BL  0:30  BL  0:45  BL  1:00  BL  1:15  BL  1:30  BL  2:00  BL  n.) fluid controls  BG BG  U  BG  U  BG  U  : hypoxemia starts  [Hypoxemic period (6 hours)] * rate of N2 flow = 7 L/min. initially and increased to 11 L/min. 2:05  BL  BG BG  2:30 3:00  BL  BG  U  4:00  BL  BG  U  5:00  BL  BG  U  6:00  BL  BG  U  7:00  BL  BG  U  7:15  BL  BG  7:30  BL  BG  8:00  BL  BG  U  hypoxemia ends  [Post-hypoxemic period (6 hours)] 8:05  BL  BG BG  8:30 9:00  BL  BG  U  10:00  BL  BG  U  11:00  BL  BG  U  12:00  BL  BG  U  13:00  BL  BG  U  13:15  BL  BG  13:30  BL  BG  14:00  BL  BG  U  : infusion ends  * B L : plasma sample, B G : blood gas sample, U : urine sample.  39  2.8  Recording Procedures and  Blood Gas  Analysis  F o r 24 hours before, during a n d 24 hours following M C P administration, arterial pressure was measured using a disposable strain-gauge D T X transducer (Statham P 2 3 D b , G o u l d Inc., O x n a r d , C A ) .  H e a r t rate was  m e a s u r e d f r o m the arterial  pressure using a cardiotachometer (Model 9857, Sensormedic These  variables  were  recorded on  model pulse  Corp., Anaheim, C A ) .  a polygraph recorder (Beckman  R612  recorder,  B e c k m a n , S c h i k k e r P a r k , I L or G o u l d T A 4 0 0 0 thermal array recorder, G o u l d Inc., V a l l e y View,  O H ) .  T h e analog signals o f arterial pressure a n d heart rate w e r e  converted  simultaneously to digital data using a n on-line c o m p u t e r s y s t e m consisting o f a n A p p l e He® computer, Interactive systems (Daisy Electronics, N e w t o n Square, P A ) , a n d  A - D  converter a n d clock card ( M o u n t a i n Software, Scott's Valley, C A ) with a s a m p l i n g rate of 15 H z a n d a v e r a g e d over 1 m i n . T h e recordings w e r e analysed to p r o v i d e a n estimate o f arterial pressure a n d heart rate averaged over 1 m i n intervals. S a m p l e s (= 1 m L ) for b l o o d gas analysis a n d glucose/lactate m e a s u r e m e n t taken simultaneously with those for drug analysis. ArterialP a 0 , PaC0 , 2  pH  were measured with an J L 1306  p H / b l o o d gas  base excess and  2  analyzer (Allied  Instrumentation  L a b o r a t o r y , M i l a n , Italy) set at a t e m p e r a t u r e o f 3 7 . 0 ° C a n d c o r r e c t e d to 3 9 . 0 ° C . saturation  and  Hemoximeter® concentrations  hemoglobin (Radiometer,  content  were  Copenhagen,  measured, Denmark).  in duplicate, Blood  were  using  glucose  Oxygen an  OSM2  and  lactate  w e r e measured, in triplicate, using a glucose/lactate 2 3 0 6 STAT  analyzer ( Y S I Inc., Y e l l o w S p r i n g , O H , U S A ) .  Osmolality of urine was measured  plus by  40  freezing-point depression method using A d v a n c e d DigiMatic® Osmometer Model  3D2  (Advanced Instruments, N o r w o o d , M A , U S A ) .  2.9  2.9.1.  Data Analysis  Determination of steady-state drug concentration  Steady-state M C P concentration  was  determined  a c c o r d i n g to the  following  procedures and criteria: 1.  Visual inspection :  t h e p l o t o f p l a s m a c o n c e n t r a t i o n versus t i m e w a s  visually  inspected for a plateau portion using a straight e d g e ruler. 2.  Coefficient of variance  : the coefficient o f v a r i a t i o n f o r the d a t a set o f M C P  concentrations in the plateau portion w a s calculated. o f C V = 1 0 %, 3.  With  am a x i m u m criterion  all e x t r e m e outlier(s) w e r e e l i m i n a t e d f r o m t h e d a t a set.  Student's t-test/Analysis of Variance (ANOVA) for the regression :  the slope  of  l i n e a r r e g r e s s i o n line f r o m the d a t a set o f p l a s m a M C P c o n c e n t r a t i o n i n  the  plateau portions was  the  a n a l y z e d u s i n g a two-tailed t-test a n d A N O V A  correlation with the null hypothesis h y p o t h e s i s (HA)  (Ho)  : slope = 0 against the  :s l o p e * 0 w i t h a l p h a = 0 . 0 5 .  for  alternative  In these tests, the rejection o f  H o  suggests that the p l a s m a d r u g concentration tends to either increase or decrease in a g i v e n p e r i o d (i.e.  a steady-state was not  achieved).  41  2.9.2. Calculation of pharmacokinetic parameters  T o t a l b o d y c l e a r a n c e (Ch) w a s e s t i m a t e d a s : CU  =  ko /  Css  w h e r e ko i s t h e i n f u s i o n r a t e a n d Css  is the a p p a r e n t a r t e r i a l s t e a d y - s t a t e c o n c e n t r a t i o n .  R e n a l c l e a r a n c e v a l u e s o f M C P a n d m d M C P w e r e c a l c u l a t e d f r o m 1) d i v i d i n g t h e accumulated  drug/metabolite  recovered  in  urine  (Du) b y  area  under  the  d r u g / m e t a b o l i t e c o n c e n t r a t i o n s c u r v e ( A U C ) as af u n c t i o n o f t i m e d u r i n g the  plasma  hypoxemic  a n d n o r m o x e m i c p e r i o d s [Du(t -t )/AUC(t -t )] a n d 2) c a l c u l a t i o n u s i n g t h e s l o p e o f 2  1  2  a c c u m u l a t e d d r u g / m e t a b o l i t e i n u r i n e (Du)  1  versus A U C (see A p p e n d i x Afor the  the  equation  derivation). T h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t o f M C P (f  u  =k / u  was calculated  dividing the slope o f the asymptote o f the a c c u m u l a t e d M C P in urine versus time  by  curve  w i t h t h e i n f u s i o n r a t e , ko. Du  =  (k*ko/K )t  -  E  the slope of the asymptote =  (k *k„/K ) 2  u  E  k *ko/K u  E  w h e r e k i s t h e r e n a l e x c r e t i o n r a t e , a n d Du i s t h e c u m u l a t i v e a m o u n t o f i n t a c t d r u g i n u  urine.  S i n c e ko i s g i v e n , t h e r a t i o (k / K ) c a n b e c a l c u l a t e d .  T h e detailed  equation  d e r i v a t i o n s are s h o w n i n a p p e n d i x Aa n d t h e o r e t i c a l a n d p r a c t i c a l d i s c u s s i o n is  described  u  E  i n t h e s e c t i o n 4.4.2. The  fractional renal metabolite excretion  conjugates w e r e also calculated in the study.  constants for mdMCP,  ddMCP  T h e s e parameters are the product of  and two  42  fractional  c o n s t a n t fm(metaboiite) a n d fmu(metaboiites).  The  metabolite  formation  fraction  c o n s t a n t fm(metaboiue) i s t h e f r a c t i o n o f kflmetaboiue)/ K.E( arent drug), w h i c h r e p r e s e n t s P  metabolic  e l i m i n a t i o n p r o p o r t i o n (to a specific  elimination.  The second  fractional constant,  metabolite)  o f the total parent  the renal metabolite  excretion  t  drug  fraction  fmu(metaboiite) i s t h e f r a c t i o n o f kmu(metabolite)/ Km(metabolite), w h i c h r e p r e s e n t s t h e r e n a l e x c r proportion o f the constant,  the  total metabolite fractional  renal  elimination. metabolite  Therefore the excretion  composite  constant  fractional  fu(metaboiite)  fm(metaboiite)fmu(metaboiite), r e p r e s e n t s t h e p r o p o r t i o n o f r e n a l e x c r e t i o n o f a s p e c i f i c f r o m the total d r u g elimination.  2.9.3  Statistical tests  Statistical physiological ANOVA  evaluations  parameters  were  using  performed  either  Student's  on t-test  various (paired  pharmacokinetic or  unpaired),  and F-test,  ( A n a l y s i s o f V a r i a n c e ) a n d T u k e y test. T h e l e v e l o f s i g n i f i c a n c e w a s c h o s e n p <  0.05. T h e o r y a n d formulae used for statistical-analysis w e r e obtained f r o m Zar- (1984), and Microsoft E x c e l ® for W i n d o w s ®  program (Microsoft Corp., Redmond, W A , U S A )  with Analysis Tools® and SlideWrite Plus® for Windows® Inc.,  Carlsbad, C A , U S A )  were  used  for  data  (Advanced Graphics Software  processing/analysis  and  graphical  presentation. T h e m e a n values in the text a n d tables are presented as the m e a n ± s t a n d a r d error of the m e a n (SEM), unless otherwise described.  metabolite  43  3.  3.1.  RESULTS  Quantitative Analytical Assay Methods for  Metoclopramide and Selected  Metabolites.  3.1.1.  GC-MSD  method for the quantitative analysis of MCP and selected metabolites.  F o r the quantitative analysis o f M C P , m o n o d e e t h y l M C P ( m d M C P ) a n d M C P ( d d M C P ) , a G C - M S D m e t h o d o f R i g g s et al. ( 1 9 9 4 ) w a s u s e d f o r i t s h i g h selectivity and reproducibility. ddMCP  Representative  chromatograms  of M C P ,  dideethyl sensitivity,  mdMCP  and  (1 n g / m L e a c h ) i n p l a s m a a n d b l a n k e x t r a c t s f r o m p l a s m a a n d u r i n e a r e s h o w n  F i g u r e 1. T h e s t e p - l i k e s h i f t i n g o f t h e b a s e l i n e o f t h e c h r o m a t o g r a m s a t 1 0 . 2 5 m i n  in  represents  a c h a n g e i n t h e b a c k g r o u n d i o n c u r r e n t a t t h e S I M g r o u p s w i t c h i n g (i.e. f r o m G r o u p 1 [m/e 3 8 0 ] t o G r o u p 2 [m/e 8 6 a n d 3 8 0 ] ) . A n a d d i t i o n a l g r o u p s w i t c h i n g o c c u r s w i t h G r o u p 3 [m/e 8 6 & 366] at 11.50 m i n w i t h n o visible c h a n g e i n baseline.  In spite o f these c h a n g e s in the  selective  stable  ion  monitoring  group,  the  baseline  remained  throughout  C h r o m a t o g r a m s f r o m urine a n d bile are similar to those f r o m p l a s m a . retention times of the H F B - d e r i v a t i v e s of d d M C P , BMY  were 9.29  m i n , 10.13  m i n , 10.38  mdMCP,  m i n a n d 12.33  The  the  assay.  individual  M C P a n d the internal standard min, respectively.  N o  sign  of  significant interference f r o m the e n d o g e n o u s c o m p o u n d s was s h o w n in any o f the biological fluids collected in the study. MCP  (mdMCP),  Figure 2( A - D ) s h o w s the m a s s spectra o f M C P , m o n o d e e t h y l  dideethyl M C P ( d d M C P ) a n d the internal standard, B M Y ,  respectively.  B o t h M C P a n d B M Y u n d e r g o e x t e n s i v e f r a g m e n t a t i o n r e s u l t i n g i n a b a s e p e a k o f m/e 8 6 ( F i g u r e 2 , C a n d D ) . I n a d d i t i o n t o t h e i o n o f m/e 8 6 , a l e s s a b u n d a n t i o n o f m/e 3 8 0 a n d  366,  44  respectively, w a s also m o n i t o r e d for M C P a n d B M Y , t oe n h a n c e the selectivity. T h e  base  p e a k o f i o n m/e 3 8 0 w a s u s e d f o r d d M C P a n d m d M C P .  [Abundance 7000 6000 12 . 3 3  5000 4000  BMY  3000 MCP 2000  ddMCP  mdMCP 10.38  1000 0 (Time ->9.  A -  9 . 29 A  10.13  T 1 1 1 1 1 1 1 1~| 1 1 1 I J 1 00  9.50  10.00  10.50  1  1  1 1 1 1  11.00  I I i 11.50 12.00 1  1  1  1  1  1  1  '  1  12.50  2000 A Blank plasma 1000 i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i  time ->9.00  9.50  10.00  10.50  11.00  11.50  i ' i i  1  1  12.00  1  i  1  12.50  2000  Blank urine  1000  [Time  i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i ->9.00 9.50 10.00 10.50 11.00 11.50 12.00  Figure 1.  '  ' i i 12.50 1  Representative total ion c h r o m a t o g r a m s ( S I M m o d e ) o f1 n g / m L ddMCP, m d M C P , M C P a n d 33.3 n g / m L B M Y a d d e d t o0 . 3 m L o f p l a s m a . S u p e r i m p o s e d chromatograms of blank plasma a n d urine are also shown.  45  2 c S .2 5 J3  -5 — c c  c3  CD  C y  CQ  o cn  O  ed  e c  §< c  oo  < -o  u  c 3  o- .S  U 2 T3 15 •£> C O  ca  0)  U  DO  —  tn  C  C  B S  |  cn  CN  3 60  '  46  3.1.2. Calibration (standard) curve Calibration (standard) curves were obtained by analyzing blank plasma, urine or bile spiked with varying amounts of M C P ,mdMCP of  each)  and  M C P / B M Y , ddMCP  plotting  the  m d M C P / B M Y  area  ratio  of  and ddMCP the  and d d M C P / B M Y  (1,2,4,8,16, 24, 32 a n d 40  heptafluorobutyric  ( H F B ) derivatives  against the indicated M C P , m d M C P  of and  concentrations, respectively [ F i g u r e 3]. T h e d a t a for a representative calibration u s e d  i n the quantitation o f M C P , m d M C P  and ddMCP  f r o m plasma are presented in T a b l e  L i n e a r i t y is o b s e r v e d o v e r the c o n c e n t r a t i o n r a n g e s t u d i e d ( 1 - 4 0  ng) with acoefficient  variation ( C V ) < 1 5 % for all the data points c o m p o s i n g the curves o f ddMCP, MCP.  ng/mL  T h e limit of quantitation ( L O Q ) was  biological fluids collected in the study.  mdMCP  1 n g / m L for all three c o m p o u n d s  1. of and  in  T h e coefficient of variation was relatively lower  the in  the m i d - r a n g e o f the c a l i b r a t i o n c u r v e a n d h i g h e r at either the l o w - o r h i g h - e n d r a n g e o f the curve.  T h e coefficients o f regression a n d the line o f best-fit through the data points  was  determined f r o m linear regression with a weighting factor of 1/Y.  3.1.3. Enzyme incubation The  enzyme  activities o f glucuronidase a n d sulphatase  were tested following  the  standard procedures p r o v i d e d b y the supplier under the experimental conditions described in Section 2.3.  In order to evaluate the effect o f glucuronidase a n d sulphatase i n c u b a t i o n o n  q u a n t i t a t i o n o f M C P a n d its m e t a b o l i t e s , 3 sets o f c a l i b r a t i o n c u r v e s ( n o e n z y m e  incubation,  glucuronidase incubation and sulphatase incubation) were prepared and extracted using s a m e p r o c e d u r e s listed in S e c t i o n 2.3.  the  the  Since biological samples spiked with standard drugs  47  do not contain any conjugates of M C P , mdMCP  and ddMCP,  the calibration curves  from  e a c h set w o u l d not s h o w a n y difference unless e n z y m e i n c u b a t i o n s h a v e affected the  samples.  T a b l e 2 lists the results o f the w e i g h t e d l i n e a r r e g r e s s i o n o f 3 sets. T h e s l o p e s a n d  intercepts  from enzyme  incubation of glucuronidase and sulphatase  against Set I which was regression. N o calibration  incubated without any enzyme  significant difference  curves.  Therefore,  these  ( S e t J J a n d 131) w e r e using Student's  in slopes or intercepts results  demonstrate  was that  p r o c e d u r e does not affect the quantitation a n d linearity o f the assay  t-test for  observed the  enzyme  a n d sulphatate to determine the existence o f these conjugates o f M C P , m d M C P detected.  among  linear these  incubation  method.  P l a s m a a n d urine samples f r o m the experiments were incubated with  however, no significant amount of conjugate was  analyzed  glucuronidase and  ddMCP,  48  4.00  3.20 ddMCP 2.40  h  o 1.60  mdMCP MCP  0.80  0.00 16  32  24  40  Standard Drug (ng/mL)  F i g u r e 3.  Representative  calibration (weighted)  from spiked plasma, [mean ± SD]  curves for M C P , m d M C P  and  ddMCP  49  Table 1.  Weighted calibration curve data (mean peak area ratio ± SD) for ddMCP, mdMCP and MCP in plasma.*  Cone. (ng/mL)  ddMCP  mdMCP  MCP  40  3.95 ±0.32 (8.11%) 2.23 ±0.25 (11.03%) 1.65 ± 0.07 (4.48%)  32  2.86 ±0.21 (7.31%) 1.75 ±0.17 (9.69%)  1.26 ±0.04 (2.93%)  24  2.20 ± 0.20 (9.09%) 1.25 ± 0.08 (6.00%)  0.93 ± 0.05 (4.95%)  16  1.47 ±0.12 (8.09%) 0.85 ± 0.07 (8.32%)  0.62 ± 0.05 (8.39%)  8  0.70 ± 0.02 (3.50%) 0.38 ± 0.04 (9.58%)  0.32 ± 0.03 (7.78%)  4  0.34 ± 0.03 (8.99%) 0.18 ±0.02 (10.38%) 0.16 ±0.01 (4.43%)  2  0.18 ±0.01 (3.10%) 0.10 ±0.01 (12.07%) 0.08 ± 0.01 (11.48%)  1  0.10 ±0.01 (5.95%) 0.06 ± 0.01 (6.64%)  0.05 ±0.01 (12.38%)  slope  0.0934  0.0541  0.0397  intercept  -0.0068  -0.0099  0.0042  r2  0.9932  0.9930  0.9976  Average CV  6.77%  9.21%  7.10%  The values shown in the parenthesis are the coefficient of variance.  50  Table 2.  Weighted calibration curve data (slope, intercept and r ) for ddMCP, 2  mdMCP and MCP with enzyme incubation study in urine.  ddMCP  mdMCP  MCP  Setl  slope  0.0976  0.0539  0.0411  (no enzyme)  intercept  -0.0176  -0.0252  -0.0073  r  0.9969  0.9937  0.9995  2  Set II  slope  0.0903  0.0524  0.0393  (glucuronidase)  intercept  -0.0082  -0.0073  0.0001  r  0.9978  0.9941  0.9998  2  Set III  slope  0.0937  0.0489  0.0396  (sulphatase)  intercept  -0.0071  -0.0085  0.0030  r  0.9980  0.9914  0.9996  2  3.2  Physiological Changes Associated with Hypoxemia T a b l e 3 lists the i d e n t i f i c a t i o n a n d  the b o d y weight o f e w e s o n w h i c h  experiments  were p e r f o r m e d , a n d the types o f samples collected a n d the duration o f the infusion.  The  b o d y weight o f the experimental g r o u p averaged 61.4 ± 3.5 K g ( M e a n ± S E M ) a n d c o n t r o l g r o u p a v e r a g e d 61.3 ± 2.1 K g .  Arterial plasma samples for drug assay and  were collected according to the experiment  the  blood  gas/pH/lactate/glucose  measurement,  protocol  shown in appendix A .  In addition, urine a n d bile s a m p l e s w e r e also collected a c c o r d i n g to  the animal experimental protocol.  Table 3.  Identification and weight of ewes, samples collected and nitrogen/MCP infusion. Ewe  No.  Weight  Sample*  Nitrogen Infusion (L/min)  (Kg) Experimental  Infusion* (hr)  102  69.5  MA.UR  Initial  7 to 10 at hr 7.5  14  139  60.4  MA.UR  Initial  7 to  14  989  69.5  MA,UR  7  14  1154  53.1  MA,UR  7  14  1158  54.5  MA,UR  7  14  Mean  61.4  SEM  3.53  8 at hr 6  220  61.7  MA,  UR  0  14  327  55.4  MA,  UR  0  14  328  63.6  MA,  UR  0  15**  1159  64.5  MA,  UR  0  Mean  61.3  p > 0.05  SEM  2.05  Control  #  * MA: maternal arterial blood sample, UR: urine total collection. ** MCP *** MCP infusion was discontinued at hour 9. MCP infusion rate (0.21 mg/min) with 15 mg loading dose at hour 0. * weight compared between the two groups according to Student's t-test. A  Change  MCP  infusion was temporary disrupted.  52  3.2.1.  Arterial blood gas status and pH  T h e m e a n ( ± S E M ) arterial b l o o d gas partial pressure of C 0  2  and 0  ( P a C 0  2  and  2  P a 0 ) a n d b l o o d p H o f the experimental a n d control groups are s h o w n in Figure 4 a a n d  4b,  2  respectively.  M e a n ( ± S E M ) values for arterial b l o o d gas pressure, p H , o x y g e n  saturation,  b a s e e x c e s s a n d b i c a r b o n a t e c o n c e n t r a t i o n are p r e s e n t e d i n T a b l e 4.  Pa0  and 0 saturation  2  2  : T h e initiation of nitrogen infusion through the intra-tracheal  catheter at h o u r 2 i n the e x p e r i m e n t a l g r o u p d e c r e a s e d M e a n P a 0  2  arterial P a 0 , in less than 5 m i n . 2  w a s significantly d e c r e a s e d ( = 4 8 % ) f r o m 1 1 6 . 3 5 ±2.83 to 60.31  (p<0.0001) during the h y p o x e m i c period.  0  2  2  and 0  nos.  i n c r e a s e d slightly ( = 7 0 - 8 0 m m H g ) d u r i n g the late h y p o x e m i c p e r i o d , at 2  close to  A f t e r 6h o u r s o f h y p o x e m i a , t h e i n t r a - t r a c h e a l n i t r o g e n i n f u s i o n w a s s t o p p e d . s a t u r a t i o n s h a v e r e t u r n e d t o 1 1 6 . 3 8 ± 1 . 2 6 m m H g a n d 1 0 0 . 1 5 ± 0 . 2 0 %,  2  (=  In s o m e animals (ewe  w h i c h point the nitrogen f l o w w a s increased to 9-11 L / m i n to m a i n t a i n the P a 0 m m H g .  H g  saturation was also significantly decreased  1 8 % ) f r o m 1 0 0 . 1 6 ±0 . 4 0 % t o 8 2 . 7 8 ± 1 . 5 3 % d u r i n g h y p o x e m i a . 102 a n d 139), P a 0  ± 1.57 m m  levels w h i c h are similar to the p r e - h y p o x e m i c level (p>0.05).  60  P a 0  2  respectively,  N o changes in P a 0  or  2  0  2  saturation w a s observed throughout the periods in the control group.  PaC0 , 2  P a C 0  2  bicarbonate concentration and base excess  also significantly  (p<0.0005), areduction of  decreased  (=10%) f r o m 41.71  : D u r i n g h y p o x e m i a , the  ± 0.39  am u c h lesser degree than P a 0 . 2  to 37.35 ± 0.48  Base excess was  s i g n i f i c a n t l y f r o m 3.4 ±0 . 2 9 ( p r e - h y p o x e m i c ) to 2.5 ±0 . 2 9 m e q / L ( p o s t - h y p o x e m i c  mean m m H g  decreased period).  Initially, the b a s e e x c e s s w a s slightly i n c r e a s e d at the o n s e t o f h y p o x e m i a , f r o m 3.9 (at  hour  53  2) to 4.8 m e q / L (at 2 : 0 5 ) , a n d g r a d u a l l y d e c r e a s e d .  Blood bicarbonate ( H C 0 ~ ) concentration 3  w a s also decreased d u r i n g h y p o x e m i a f r o m 26.51 ± 0.32 m e q / L to 2 5 . 2 1 + 0.25 m e q / L , but w i t h o u t a n initial increase as s h o w n in b a s e excess. A f t e r nitrogen infusion w a s stopped, PaC0  2  returned to 4 0 . 8 2 ± 0.52 mrnHg,  the  a level similar to the p r e - h y p o x e m i c p e r i o d (p>0.05).  H o w e v e r , after cessation o f the nitrogen infusion, base excess a n d bicarbonate concentrations r e m a i n e d lower than the p r e - h y p o x e m i c levels.  (HC0 ~) 3  In the control group,  no  significant change was observed in PaC0 , bicarbonate concentration or base excess. 2  Blood pH apparent  and  hemoglobin concentration : A r t e r i a l b l o o d p H s h o w e d a v e r y  increasing  trend  from  7.424  ±  0.0051  during  the  pre-hypoxemic  period  ( n o r m o x e m i a ) to 7 . 4 4 2 ± 0 . 0 0 5 7 d u r i n g the h y p o x e m i c period, a l t h o u g h this d i d not statistical significance. 0.0053  (p <  reach  D u r i n g the p o s t - h y p o x e m i c period, b l o o d p H decreased to 7 . 4 0 9  0.0005), a level significantly lower than those of the p r e - h y p o x e m i c  hypoxemic periods.  T h i s d e l a y e d acidosis appears to coincide w i t h the d e l a y e d lactic  a c c u m u l a t i o n in the b l o o d , as s h o w n  i n T a b l e 5.  D u r i n g the h y p o x e m i c  slight  period,  ± and  acid the  h e m o g l o b i n concentration also increased slightly (statistically not significant; p = 0 . 0 5 8 ) f r o m 7.97 ± 0.23 g % ( p r e - h y p o x e m i c period) to 8.40 ± 0.15 g % ( h y p o x e m i c period), a n d restored to 7.83 ± 0.18 g % d u r i n g the p o s t - h y p o x e m i c period.  was  54  Table 4.  Mean (± SEM) arterial blood pH, gas partial pressure ( P a C 0 and Pa0 ), bicarbonate (HCGy) concentration and base excess. 2 1  2  hypoxemia  post-hypoxemia  Experimental 7.424 ± 0.0053  7.442 ± 0.0057  7.409 ± 0.0053 *  Control  7.495 ± 0.0068  7.492 ± 0.0063  37.35 ± 0.48 "  40.83 ± 0.58  37.70 ± 0.65  37.22 ± 0.50  pre-hypoxemia pH  PaCG-2  7.501 ± 0.0076  (mm Hg) Experimental 41.27 ±0.39 Control  37.14 ±0.41  (mmHg)  Pa02  Experimental 116.9 ± 1.15 Control  O2 sat  117.7 ± 1.90  60.7 ± 1.57 *"# 116.5 ±0.71 116.1 ±1.88  120.0 ±1.91  (%) Experimental 100.16 ±0.40 97.07 ±0.17  96.60 ± 0.22  96.59 ±0.14  Experimental 26.51 ±0.32  25.21 ±0.25  25.57 ±0.25  29.29 ± 0.38  28.86 ± 0.40  28.35 ± 0.46  Experimental  3.4 ± 0.27  2.5 ±0.29  2.1 ±0.28  Control  7.3 ±0.46  6.7 ± 0.42  6.3 ± 0.47  Experimental  7.95 ± 0.23  8.40 ±0.15  7.83 ±0.18  Control  7.71 ±0.13  7.76 ±0.13  7.75 ±0.17  Control HCO3-  82.78 ± 1.53 *"# 100.15 ±0.20  (meq/L) Control  Base Excess (meq/L)  Hemoglobin  * ** *** ****  (g %)  Significantly lower than pre-hypoxemia and hypoxemia according to A N O V A (p < 0.0025) and Tukey test (p < 0.005). Significantly lower than pre-hypoxemia and post-hypoxemia according to A N O V A ( p < 0.0005) and Tukey test (p < 0.001). Significantly lower than pre-hypoxemia and post-hypoxemia according to A N O V A (p < 0.0001) and Tukey test (p < 0.0005). Significantly higher than hypoxemia and post-hypoxemia according to A N O V A and Tukey test (p < 0.05).  #  Significantly lower than control group according to Student's t-test (p < 0.0001).  1  n = 5 for the experimental group, n = 4 for the control group  55  130  -£  104  8.00  JL TIL .T  [5f1T  T  Hypoxemia'  T5._  T  • o j_\ —o—2 0  i 7.86 x  GL  7.72  78  -o O O  T  X  T  oil  1  52  m  V IJ o -  J > O < 1 ^ - 0  7.58  — I i  < XT±-+  26  -±+  T -  T  T  T  -+ X  7.44  T  VX  7.30 10  12  14  Time (hr)  Figure 4a.  Mean ( ± S E M ) arterial blood p H and gas partial pressure ( P a C 0 and 2  P a 0 ) over the duration of the experiment [experimental group, n = 5]. 2  —H— p H , - o - P a Q , - • - P a C 0 . 2  2  56  140  112  -S X  8.00  7.88  h  E CD =J  7.76  O O  co to  m  CD  .5  7.64  co  CO  Q) <  co a.  Time  F i g u r e 4b.  (hr)  M e a n ( ± S E M ) arterial b l o o d p H a n d gas partial pressure (PaCC»2 a n d PaC«2) o v e r t h e d u r a t i o n o f t h e e x p e r i m e n t [ c o n t r o l g r o u p , n =  -I-  3.2.2.  p H , - o - P a Q  Arterial blood lactate and  2  )  -•- P a C Q  2  .  glucose concentration during hypoxemia  T a b l e s 5 a n d 6 list the m e a n ( ± S E M ) lactate a n d g l u c o s e experimental and control groups.  concentrations  V i s u a l inspection of the plots of arterial b l o o d  c o n c e n t r a t i o n v e r s u s t i m e f o r t h e e x p e r i m e n t a l g r o u p ( F i g u r e 5a) noticeable changes  4]  in lactate a n d glucose  s h o w s that there  concentration within the h y p o x e m i c  and  of  the  lactate were post-  57  hypoxemic periods.  E a c h o f the h y p o x e m i c a n d p o s t - h y p o x e m i c periods w e r e  into three 2 - h o u r intervals for the statistical analysis.  In the experimental group, the arterial  b l o o d lactate concentrations g r a d u a l l y i n c r e a s e d (= 6 0 % b y h o u r 8) d u r i n g the p e r i o d (statistically  significantly  subdivided  at the late h y p o x e m i c  p e r i o d i.e.  hypoxemic  hour 6:00-8:00)  and  r e m a i n e d elevated after cessation o f the nitrogen infusion. T h e lactate levels h a d returned to the p r e - h y p o x e m i c level b y the late p o s t - h y p o x e m i c period (hour 12:00-14:00). lactate concentrations  f r o m 6:00  to  12:00  (i.e.  d u r i n g late h y p o x e m i a  and  Thus,  the  subsequent  recovery period) w e r e significantly (p < 0.05) higher than the r e m a i n i n g experiment  periods  [baseline-level]. In the c o n t r o l g r o u p , lactate c o n c e n t r a t i o n r e m a i n e d at a level s i m i l a r to that during the p r e - h y p o x e m i c period through the entire experiment (p > 0.85) [Figure 5b]. Arterial b l o o d g l u c o s e concentration also increased, to a lesser d e g r e e (= 10 %  higher  at h o u r 8), i n the late h y p o x e m i c p e r i o d , i n a m a n n e r s i m i l a r to the i n c r e a s e s e e n i n the lactate concentration.  D u r i n g the post-hypoxemic period, the glucose level r e m a i n e d higher  than  that o f the p r e - h y p o x e m i c period in the experimental group, In contrast, in the control group, the glucose concentration remained relatively constant.  Figure 5b shows a decreasing  t h r o u g h the h y p o x e m i c a n d p o s t - h y p o x e m i c periods, but this decrease w a s not significant.  trend  statistically  58  1.35  3.25  1.15  if  0.95  0.75  3.00 2.75  h  o E E  2.50 o o  0.55  F i g u r e 5a.  M e a n ( ± S E M ) arterial b l o o d lactate a n d glucose concentration ( m m o l / L ) over the duration o f the experiment [ e x p e r i m e n t a l g r o u p , n = 5]. - o - b l o o d l a c t a t e a n d -•glucose.  F i g u r e 5b.  M e a n ( ± S E M ) arterial b l o o d lactate a n d glucose ( m m o l / L ) over the duration of the experiment [ c o n t r o l g r o u p , n = 4]. - o - b l o o d l a c t a t e a n d -•-  concentration glucose.  Table 5.  Mean (± SEM) blood lactate concentration (mmol/L). Experimental  Period  1  Control  Pre-hypoxemia 0:00-2:00  0.558 ± 0.024  0.524 ± 0.047  Hypoxemia  2:00-4:00  0.578 ± 0.035  0.476 ± 0.035  4:00-6:00  0.644 ± 0.054  0.504 ± 0.062  6:00-8:00  0.849 ± 0.044*  0.565 ± 0.052  0.879 ± 0.049*  0.614 ±0.043  10:00-12:00  0.804 ± 0.064*  0.533 ± 0.069  12:00-14:00  0.615 ±0.025  0.446 ± 0.029  Post-Hypoxemia 8:00-10:00  Significantly higher than baseline group (pre-hypoxemia, 2:00-6:00 and 12:00-14:00) according A N O V A (p < 1E-10) and Tukey test (p < 0.05). n = 5 for the experimental group and n = 4 for the control group.  Table 6.  Mean (± SEM) blood glucose concentration (mmol/L). Experimental  Period  1  Control  Pre-hypoxemia  0:00-2:00  2.74 ± 0.05  3.24 ±0.14  Hypoxemia  2:00-4:00  2.81 ±0.05  3.23 ±0.17  4:00-6:00  2.81 ±0.07  3.09 ± 0.23  6:00-8:00  2.91 ±0.04*  3.17±0.18  3.03 ± 0.04*  2.92 ±0.16  10:00-12:00  2.97 ± 0.09*  2.78 ±0.17  12:00-14:00  2.98 ±0.05*  2.80 ±0.11  Post-Hypoxemia 8:00-10:00  Significantly higher than baseline group (pre-hypoxemia and 2:00-6:00) according to A N O V A (p < 3E-05) and Tukey test (p < 0.05). n = 5 for the experimental group and n = 4 for the control group.  60  3.2.3. Plasma electrolyte concentration, urine flow, osmolality and  pH  and  renal osmolal  excretion rate. T a b l e 7 lists m e a n ( ± S E M ) p l a s m a electrolyte ( N a , K +  +  and Cl")  concentrations,  hemoglobin content and urine flow (volume collected per hour), osmolality and p H . m e a n urine flow in the experimental g r o u p was increased significantly d u r i n g  The  hypoxemia,  f r o m a n average o f 8 0 . 2 0 ± 12.65 mL/hr d u r i n g the p r e - h y p o x e m i c p e r i o d to 1 3 1 . 9 0 ±  19.53  mL/hr d u r i n g the h y p o x e m i c period, a n d later w a s restored to 7 8 . 3 0 ± 7.58 mL/hr d u r i n g the post-hypoxemic period (p < 0.025). A s s h o w n in Figure 6a, h y p o x e m i a - i n d u c e d diuresis  was  o b s e r v e d in the early to m i d d l e h y p o x e m i c p e r i o d ( b e t w e e n h o u r 2:00 to 6:00) a n d returned to the values close to the n o r m o x e m i c level d u r i n g the late h y p o x e m i c a n d  post-hypoxemic  periods. H o w e v e r , in t w o o f the animals in the experimental g r o u p (ewe no. 1154 a n d hypoxemia-induced diuresis was not observed.  In the control group, there w a s n o  1158),  significant  change in urine flow throughout the entire experimental period [Figure 6b].  In addition, urine osmolality was also significantly decreased f r o m 1141.15 ±  37.22  m O s m o l / K g ( p r e - h y p o x e m i c ) to 8 0 8 . 1 3 ± 9 2 . 9 9 m O s m o l / K g d u r i n g h y p o x e m i a , a n d restored to 1 1 7 2 . 0 3 ± 4 0 . 7 7 m O s m o l / K g after h y p o x e m i a . osmolality in ewe  no. 1154  and 1158  A g a i n , similar to urine flow, the  did not change during hypoxemia.  These  describing urine flow a n d osmolality suggest that there m a y be s o m e correlation these two parameters. s h o w n i n F i g u r e 7.  urine results  between  T h u s , the plot of urine osmolality versus urine flow w a s plotted A s s e e n i n the p l o t , t h e r e is a n a p p a r e n t i n v e r s e - r e l a t i o n s h i p  between  t h e s e t w o p a r a m e t e r s (r = 0.80). In addition, the r e n a l o s m o l a l e l i m i n a t i o n rate constant, product of urine osmolality and urine flow, was  as  also significantly decreased f r o m  the 88.23  61  m O s m o l / h in the p r e - h y p o x e m i c p e r i o d to 61.32 m O s m o l / h d u r i n g the h y p o x e m i c  period,  a n d later restored to 89.37 m O s m o l / h in the p o s t - h y p o x e m i c period. Therefore, the  negative  correlation between urine osmolality and flow and hypoxemia-induced reduction in osmolal e x c r e t i o n s u g g e s t s that t h e i n c r e a s e d u r i n e f l o w d u r i n g h y p o x e m i a is d u e to i n c r e a s e d excretion  rather than  a secondary  urine flow  e x c r e t i o n ( W a l k e r , 1 9 8 2 ; C o l i c e et al,  increase  by  elevated  electrolyte/osmolal  1991).  S i m i l a r to the findings w i t h urine v o l u m e a n d osmolality, the urine p H decreased  water  significantly  f r o m 7 . 5 3 2 ± 0 . 0 2 1 8 in the p r e - h y p o x e m i c p e r i o d to 7 . 4 6 8 ± 0 . 0 1 4 4 in  h y p o x e m i c period. Later, the urine p H w a s restored to 7.519 ± 0 . 0 1 0 2 in the  the  post-hypoxemic  p e r i o d , w h i c h is a l e v e l s i m i l a r to the p r e - h y p o x e m i c p e r i o d i n the e x p e r i m e n t a l  group.  Increased renal lactate excretion m a y b e related to the urinary acidosis d u r i n g h y p o x e m i a , but no  lactate  measurement  was  conducted  correlation c o u l d be m a d e in the present  during the  experiment.  remained  fairly constant  no  direct  study.  In the control group, the physiological parameters osmolality  Therefore,  throughout  the  such  as urine flow,  experimental  H o w e v e r , urine flow (p <0.05), urine p H a n d osmolality (p «  periods  0.05)  in the  (p  p H »  and 0.05).  experimental  g r o u p w e r e significantly different f r o m the control group during the h y p o x e m i c period (hour 2 to 8).  500  1500 Hypoxemia'  400  E  •T  T I  1200  T  300  900  O E  200  600  O  100  300  2  4  6  8  10  12  E  14  Time (hr)  Figure 6a.  E  Figure 6b.  Mean (± SEM) urine flow (mL/hr) and osmolality (mOsm/Kg) over the duration of the experiment [experimental group, n = 5]. - o - urine osmolality and - • - urine flow.  500  1500  400  1200  300  900  200  600  100  300  Mean (± SEM) urine flow (mL/hr) and osmolality (mOsm/Kg) over the duration of the experiment [control group, n = 4] - O - urine osmolality and - • - urine flow.  63  1800  1440 CD  2£  o  1080  E J5 o E  720  V)  o CD  c  "C  3  360  80  240  160  320  400  Urine flow (mL/hr)  F i g u r e 7.  Correlation of urine osmolality ( m O s m / K g ) and urine flow (mL/hr). C o r r e l a t i o n c o e f f i c i e n t (r) = 0.80.  P l a s m a s o d i u m (Na ) concentration was not significantly changed during hypoxemia. +  Na  +  concentration tends to increase slightly as the infusion proceeds, f r o m 146.6 ± 1.02  during  the p r e - h y p o x e m i c  p e r i o d to  150.1  ±  0.59  m M  during the  hypoxemic  m M  period.  H o w e v e r , this apparent tendency towards an increase was observed in both the  experimental  a n d control g r o u p , a n d yet, n o difference w a s f o u n d b e t w e e n the t w o groups.  T h e r e f o r e , it  suggests that h y p o x e m i a does not affect the p l a s m a N a  +  concentration, in spite o f changes in  urine flow and osmolality. However, plasma potassium (K ) concentration was increased significantly +  during  h y p o x e m i a f r o m 4.12 ± 0 . 0 3 0 m M ( p r e - h y p o x e m i c ) to 4.24 ± 0 . 0 2 2 m M ( h y p o x e m i c ) [p  <  64  0.05], a n d returned to 4.15 plasma K  +  ± 0.034 m M during the p o s t - h y p o x e m i c period.  concentration in the control group r e m a i n e d constant  during the  In  contrast, hypoxemic  period, 4.11 ± 0 . 0 3 0 m M a n d 4.17 ± 0 . 0 2 5 m M , a n d significantly increased to 4.41 ± mM  during the post-hypoxemic period. Plasma  chloride ion  (Cl") concentration  was  significantly  h y p o x e m i c period in the experimental group, f r o m 111.4 ± 0.48 114.1  0.037  ± 0.53  m M  (hypoxemic)  increased  during  m M (pre-hypoxemic)  , a n d r e m a i n e d elevated throughout the  the to  post-hypoxemic  period (113.4 ± 0.57 m M ) . In the control group, p l a s m a C l "concentration was also increased d u r i n g the h y p o x e m i c p e r i o d f r o m 110.7 ± 0.24 m M to 112.0 ± 0.18 m M , but to a lesser degree (significantly lower, p < 0.01) than the experimental group.  65  Table 7.  Mean (± SEM) plasma electrolyte concentration, hemoglobin content and urine flow, urine pH and osmolality. 1  pre-hypoxemia  hypoxemia  post-hypoxemia  (ml_/hr)  Urine Flow  Experimental  80.20 ± 12.65  131.90 ± 19.53**  78.30  ±7.58  Control  76.00 ± 15.38  92.58 ± 10.54  94.96  ± 10.52  Experimental  7.532 ± 0 . 0 2 1 8  7.468 ± 0 . 0 1 1 2 " * *  7.535  ±0.0148  Control  7.551  7.514  7.519  ±0.0102  Urine pH  Plasma K  +  Plasma Cl"  * ** #  ** ### #### A  A A  A A A  1  ±0.0144  (mOsmol/Kg)  Osmolality  Plasma N a  ±0.0134  +  Experimental  1141.15 ± 37.22  808.13  Control  1241.69 ± 63.64  1239.88  ± 92.99****  1172.03  ± 40.77  ± 46.28  1256.92  ± 29.08  (mmol/L) Experimental  146.6  ± 1.02  150.1  ±0.59  149.1  ±0.98  Control  148.4  ±0.25  150.2  ±0.27 •  150.1  ±0.18  4.15  ± 0.034  4.41  ± 0.037™***  (mmol/L) Experimental  4.12  ± 0.030  4.24  ± 0.022  Control  4.11  ± 0.030  4.17  ± 0.025  A  (mmol/L) Experimental  111.4  ± 0.48™  114.1  ± 0;53*  Control  110.7  ± 0.24™  112.0  ±0.18  ###  113.4  ±0.57  112.5  ±0.19  Significantly higher; p < 0.025 according to A N O V A and p < 0.025 from post-hypoxemia from pre-hypoxemia in Tukey test. Significantly lower; p < 0.001 according to A N O V A and p < 0.001 from post-hypoxemia from pre-hypoxemia in Tukey test. Significantly lower; p < 0.05 from control group according to Student's t-test. Significantly lower; p < 0.005 from control group according to Student's t-test. Significantly higher; p < 0.0001 from experiment group according to Student's t-test. Significantly higher; p < 0.01 from control group according to Student's t-test. Significantly higher; p < 0.05 from control group according to Student's t-test. Significantly higher; p < 0.0001 from pre-hypoxemic and hypoxemic period according to Tukey test. Significantly lower; p < 0.005 from hypoxemic and post-hypoxemic periods according to Tukey test. n = 5 for the experimental group and n = 4 for the control group.  and p < 0.10 and p < 0.06  A N O V A and A N O V A and  66  3.2.4. Mean arterial blood pressure and heart rate. M e a n  arterial b l o o d pressure a n d heart rate w e r e also m e a s u r e d in s o m e o f  animals in the study.  the  T a b l e s 8 a n d 9 list m e a n heart rate a n d arterial b l o o d p r e s s u r e d u r i n g  the pre-hypoxemic, h y p o x e m i c a n d post-hypoxemic periods.  D u r i n g the h y p o x e m i c  period,  m e a n heart rate a n d arterial b l o o d pressure w e r e significantly increased (p <0.05) to 122.49 ± 9.15 beats/min a n d 96.99 ± 10.26 m m H g f r o m 108.95 ± 6.61 beats/min a n d 9 0 . 3 2 ± 10.63 m m  H g (the p r e - h y p o x e m i c period) in the e x p e r i m e n t a l g r o u p .  After the cessation  of  nitrogen infusion, b o t h arterial pressure a n d heart rate returned to the levels close to those o f the p r e - h y p o x e m i c periods (p > 0.05).  M e a n arterial pressure in the control g r o u p  constant throughout the entire experiment.  M e a n heart rate in the control g r o u p a p p e a r e d to  d e c r e a s e g r a d u a l l y t h r o u g h t h e e x p e r i m e n t p e r i o d , b u t it w a s n o t s t a t i s t i c a l l y  Table 8.  significant.  Mean (± SEM) heart rate (beats/min) Ewe  Experimental  #  prehypoxemia  hypoxemia  posthypoxemia  139  100.0 ±1.3  109.3 ±0.7  89.3 ±0.9  989  105.0 ±1.0  118.1 ±0.5  93.4 ±0.4  1158  121.9 ±0.8  140.1 ±0.7  129.7 ±0.6  109.0 ±6.6  122.5 ±9.2*  104.2 ±12.8  327  112.7 ±1.7  110.6 ±0.8  96.4 ±0.6  220  119.6 ±0.7  112.1 ±0.4  101.1 ±0.4  1159  125.0 ±0.5  123.3 ±0.3  121.0 ±0.3  119.1 ±3.6  115.3 ±4.0  106.1 ±7.5  Mean ± SEM Control  remained  Mean ± SEM  Significantly higher than the pre-HO and post-HO periods according to paired t-tests (p < 0.05)  67  Table 9.  Mean (± SEM) arterial blood pressure (mm Hg)  Ewe #  Experimental  prehypoxemia  hypoxemia  posthypoxemia  139  70.8 ± 0 . 4  77.6 ± 0 . 2  72.5 ±0.25  989  107.3 ± 0 . 3  112.6 ± 0 . 3  96.0 ± 0 . 2 7  1158  92.8 ± 0 . 5  100.8 ± 0 . 3  100.3 ± 0 . 3 3  Mean ± S E M  90.3 ± 10.6  97.0 ±10.3*  89.6 ±8.62  327  85.9 ± 1 . 0  85.4 ± 0 . 6  73.5 ± 0 . 5  220  76.3 ± 0 . 3  78.2 ± 0 . 2  80.6 ± 0 . 3  1159  94.6 ± 0 . 3  95.3 ± 0 . 2  94.4 ± 0 . 2  Mean ± S E M  85.6 ± 5 . 3  86.3 ± 5 . 0  82.8 ±6.1  Control  Significantly higher than the pre-hypoxemic period according to paired t-tests (p < 0.01).  3.3  Metoclopramide Pharmacokinetics Following Lv. Infusion to Steady-state and  Induction of Hypoxemia.  3.3.1. Steady-state plasma MCP concentration and total body clearance (TBC) during normoxemia and hypoxemia. Infusion parameters for the study were chosen based o n results obtained during the previous metoclopramide study in sheep (Riggs, 1989).  S i m i l a r to the previous study, a  visual inspection o f p l a s m a M C P concentrations a n d statistical analysis indicated that MCP  steady-state concentrations were attained between 30 m i n a n d 75 m i n of the  the  infusion  d u r a t i o n ( i n f u s i o n rate at 0.21 m g / m i n ) after a n initial 15 m g i.v. b o l u s l o a d i n g d o s e i n 8 o f 9  68  a n i m a l s (4 in the e x p e r i m e n t a l g r o u p a n d 4 in the control g r o u p ) u s e d in the study.  One  a n i m a l in the experimental g r o u p ( E w e N o . 1154) d i d not reach the state-state concentration within 2 hours o f infusion, despite the administration o f a loading dose.  Data from  this  a n i m a l w a s e x c l u d e d f r o m the final pharmacokinetic data analysis, since the h y p o x e m i c  and  post-hypoxemic  pre-  steady-state  h y p o x e m i c steady-state  drug  concentrations  could  not  be  compared  to  the  concentrations.  Semi-logarithmic plots of m e a n ( ± S E M ) p l a s m a M C P a n d m d M C P  concentration  versus t i m e profiles o b t a i n e d f o l l o w i n g the initial i.v. l o a d i n g d o s e a n d 1 4 - h o u r i n f u s i o n are s h o w n in F i g u r e 8 a for the experimental g r o u p a n d F i g u r e 8b for the control group. plasma ddMCP  concentration w a s b e l o w the assay quantitation limit in the p l a s m a  samples  a s s a y e d , t h u s , it w a s n o t a p p r o p r i a t e t o a t t e m p t s t a t i s t i c a l a n a l y s i s a n d p l o t t i n g o f t h e ddMCP  The  plasma  data. Table  experimental  10  lists  mean  (±  and control groups.  S E M ) plasma  metoclopramide  Plasma metoclopramide  concentrations  reached  average  concentrations o f 5 0 . 7 2 ± 1.06 n g / m L for the e x p e r i m e n t a l g r o u p a n d 5 0 . 8 4 ±0.99 the control g r o u p in the p r e - h y p o x e m i c period (p > 0.05).  in  the  steady-state n g / m L for  Steady-state concentrations  were  o b t a i n e d 3 0 - 4 5 m i n after initiation o f infusion in 3 e w e s a n d b y 1 to 1.25 h o u r in 5  ewes.  During  the  hypoxemic  period,  the  mean  M C P  steady-state  concentration  significantly f r o m 5 0 . 7 2 ± 1.06 to 6 3 . 6 2 ± 1.79 n g / m L (p < 0 . 0 0 0 1 ) in the  increased experimental  group.  In the control group, p l a s m a M C P steady-state concentration r e m a i n e d constant (p  0.05).  T h e elevated M C P steady-state concentration during h y p o x e m i a in the  g r o u p is s i g n i f i c a n t l y h i g h e r t h a n that o f the c o n t r o l g r o u p (p < 0 . 0 0 5 ) .  >  experimental  69  Table 10.  Mean (± SEM) steady-state plasma MCP concentration (ng/mL) prehypoxemia  Ewe#  45.80 ±1.95*  65.39 ± 1 . 6 1 * *  56.58 ±1.79***  139  54.75 ±2.12*  73.31 ± 3 . 1 4  72.68 ± 1 . 8 1  989  68.26 ±2.68  74.71 ±4.03****  59.31 ± 0 . 9 1  1158  30.95 ± 1 . 0 3  43.69 ± 1.94**  33.64 ± 1 . 1 1  49.94 ±7.83  64.28 ± 7 . 1 6 *  55.55 ± 8 . 1 1  Mean ±SEM2  50.72 ± 1.06  63.62 ±1.79**#  55.83 ± 1 . 1 5  327  65.07 ±3.41  78.39 ±4.01  75.47 ±3.05  328  57.65 ±3.81  55.02 ± 1 . 1 9  59.99 ± 1 . 8 8  220  43.91 ± 1 . 8 6  38.48 ± 3.02  42.42 ± 1 . 5 8  1159  33.08 ± 1 . 5 0  44.41 ± 1 . 3 0  41.46 ±2.64  49.93 ± 7 . 1 2  54.08 ± 8 . 8 0  54.84 ±8.09  50.84 ±0.99  54.23 ± 2 . 1 5  51.74 ± 1 . 5 9  Mean ± SEM  Mean ± S E M  1  1  Mean ±SEM2  * ** *** **** # #  A  1 2  posthypoxemia  102  Experimental  Control  hypoxemia  ##  Significantly lower than hypoxemic and post-hypoxemic period according to ANOVA/Tukey test (p< 0.05). Significantly higher than pre- and post-hypoxemic period according to ANOVA/Tukey test (p < 0.05). Significantly higher than pre-hypoxemia and lower than hypoxemic period according to A N O V A and Tukey tes (p < 0.05). Significantly higher than post-hypoxemic period according to ANOVA/Tukey tests (p < 0.005). Significantly higher than control group according to Student's t-test (p < 0.005). Significantly higher than control group according to Student's t-test (p < 0.05). Significantly higher than pre- and post-hypoxemic periods according to paired t-tests (p < 0.05). mean ± sem calculated from the set of individual plasma M C P steady-state concentrations and analyzed by serie; of paired t-tests among the periods. ( n = 4) mean ± sem calculated from the mean plasma M C P concentration curve and analyzed by A N O V A and Tukey test.  70  Figure  8a.  M e a n (± S E M ) plasma M C P and m d M C P concentration (ng/mL) over t h e d u r a t i o n o f t h e e x p e r i m e n t [ e x p e r i m e n t a l g r o u p , n = 4]. - o - M C P a n d -•- m d M C P .  71  100 .0—6-  •°  „oo._;  •  T  l  /  1  10  10 Time  Figure 8b.  12  (hr)  M e a n (± S E M ) plasma M C P and m d M C P concentration (ng/mL) over the duration o f the experiment-[control group, n = 4]. - o - M C P a n d -•- m d M C P .  14  72  During  the  post-hypoxemic  period,  the  arterial  plasma  M C P  steady-state  c o n c e n t r a t i o n d e c r e a s e d to a concentration close to that s e e n in the p r e - h y p o x e m i c (55.83  ±  1.15  n g / m L ) in the  experimental  group.  However,  period  this concentration  was  significantly higher than that o f the equivalent p e r i o d in the control g r o u p (p = 0.04).  The  m e a n M C P steady-state concentrations in the control g r o u p r e m a i n e d the s a m e  throughout  the entire experimental period (p > 0.05).  T h e r e w a s s o m e individual variability in p h a r m a c o k i n e t i c response to the stress.  hypoxemic  In general, h y p o x e m i a appears to h a v e i n d u c e d a r e d u c t i o n in d r u g e l i m i n a t i o n  (i.e.  increased p l a s m a steady-state concentration), but there was a noticeable difference in M C P kinetics during the post-hypoxemic  recovery period in the experimental group.  In  n u m b e r 989 a n d 1158, the M C P steady-state concentrations increased significantly h y p o x e m i a a n d returned to the p r e - h y p o x e m i c level d u r i n g the s u b s e q u e n t recovery period.  during  post-hypoxemic  In e w e n u m b e r 102, the M C P concentration also increased  d u r i n g h y p o x e m i a , but d i d not return to a p r e - h y p o x e m i c level.  ewe  The M C P  significantly concentration  during the p o s t - h y p o x e m i c period in this a n i m a l r e m a i n e d b e t w e e n the p r e - h y p o x e m i c hypoxemic  level  (but  significantly  different  from  both).  Alternatively,  the  concentration increased significantly during h y p o x e m i a in ewe n u m b e r 139, a n d  and M C P  remained  high throughout the post-hypoxemic period.  T a b l e 11 lists the p l a s m a M C P total b o d y clearance ( T B C ) i n b o t h the  experimental  and control groups.  In the experimental group, T B C decreased significantly f r o m 274.22  47.99 L / h (4.47  1.04  ±  L/h/Kg)  to 2 0 5 . 4 0 ±  28.17  L / h (3.33  ±  0.66  L/h/Kg)  ±  during  73  h y p o x e m i a . D u r i n g the p o s t - h y p o x e m i c period, T B C w a s increased to 2 4 5 . 7 8 ± 4 4 . 2 4 L / h (4.00 ± 0.96 L/h/Kg).  In contrast, the T B C o f control g r o u p r e m a i n e d relatively  throughout the e x p e r i m e n t p e r i o d (p > 0.05). T h u s , these results f r o m cross experiments  constant  comparison  s u g g e s t that the total b o d y c l e a r a n c e o f m e t o c l o p r a m i d e is r e d u c e d  during  moderate h y p o x e m i a (PaO*2 = 60 rrimHg) a n d restored during the subsequent recovery period (post-hypoxemia).  74  Table 11.  Plasma MCP total body clearance (TBC) . §  Ewe No. pre-hypoxemia Experimental  102  139  989  1158  Mean ± SEM  (L/h)  Mean ± SEM  (L/h/Kg)  Control  327  328  1159  220  Mean ± SEM  (L/h)  Mean ± SEM  (L/h/Kg)  hypoxemia  post-hypoxemia  275.11  192.70  222.70  (3.96)  (2.77)  (3.20)  230.12  171.87  173.36  (3.81)  (2.84)  (2.87)  184.58  168.64  212.43  (2.66)  (2.42)  (3.06)  407.08  288.40  374.61  (7.47)  (5.29)  (6.87)  274.22 ± 47.99 205.40 ±28.17*  245.78 ± 44.24  4.47 ± 1.04  3.33 ± 0.66*  4.00 ± 0.96  193.64  160.73  166.96  (3.50)  (2.90)  (3.01)  218.57  228.76  210.04  (3.44)  (3.60)  (3.30)  380.87  283.70  303.94  (5.90)  (4.40)  (4.71)  286.95  327.45  297.03  (4.65)  (5.31)  (4.81)  270.01 ±41.89  250.16 ±36.00  244.49 ± 35.53  4.37 ± 0.58  4.05 ± 0.52  3.96 ± 0.47  change*  Numbers in parentheses show the total body clearance normalized to ewe body weight (L/h/Kg). [n = 4] Significantly lower than pre-hypoxemic period (p < 0.05) according to paired t-test. Relative change during hypoxemic period compared to pre-hypoxemic period.  -30%  -25%  -9%  -29%  -16%  +5%  -25%  +14%  75  3.3.2. Plasma mdMCP concentration during hypoxemic and  normoxemic MCP  T h e m e a n ( ± S E M ) arterial p l a s m a concentration o f m d M C P is listed i n T a b l e 12. P l a s m a m d M C P  steady-state.  during M C P steady-state  concentration (metabolite) increased significantly (p  <  0.005) f r o m the p r e - h y p o x e m i c to the h y p o x e m i c periods in b o t h e x p e r i m e n t a l a n d control groups.  This increase  in plasma mdMCP  concentration,  unlike that o f p l a s m a  concentration, d o e s not a p p e a r to b e related to the i n d u c t i o n o f h y p o x e m i a , elevation of plasma mdMCP  M C P  since  the  concentration was gradual throughout the pre-hypoxemic period  a n d o b s e r v e d i n b o t h e x p e r i m e n t a l a n d c o n t r o l g r o u p s . T h e r e f o r e , it is l i k e l y t o b e a r e s u l t o f gradual metabolite a c c u m u l a t i o n during the d r u g infusion. mdMCP  V i s u a l inspection of the  concentration-time profiles (Figures 8a a n d 8b) suggests that m d M C P  plasma  concentration  also reached a n apparent steady-state in the e w e s in both the experimental a n d control groups, h o w e v e r , the time to a n apparent steady-state w a s longer than for M C P , r a n g i n g f r o m 4 to 5 hours.  Plasma mdMCP  hypoxemic period.  concentration  The mean mdMCP  was  not  significantly  changed  concentrations d u r i n g the h y p o x e m i c (32.78 ±  n g / m L ) a n d p o s t - h y p o x e m i c p e r i o d s ( 3 3 . 0 7 ± 1.08 n g / m L ) w e r e s i m i l a r (p » However, when  during the  the p l a s m a m d M C P  concentrations  post1.73  0.05).  of the h y p o x e m i c  and  post-  h y p o x e m i c periods in the e x p e r i m e n t g r o u p w e r e c o m p a r e d to those o f the control group, mdMCP  concentrations  of experimental  suggesting a higher plasma mdMCP  group were  significantly  accumulation during hypoxemia.  higher (p <  0.0001),  76  Table 12.  Mean (± SEM) arterial plasma mdMCP concentrations during the MCP infusion (ng/mL) Ewe #  Experimental  Significantly Significantly Significantly Significantly  post-hypoxemia  22.99 ± 1.72  30.35 ±2.94  27.20 ±1.68  139  13.12 ±1.13*  26.63 ±0.51  30.20 ±1.13  989  21.89 ±1.37*  39.03 ±2.44  38.47 ±1.51  1158  18.33 ±3.36*  40.01 ±1.55  34.99 ±2.01  18.62 ±1.30*  32.78 ± 1.73*  33.07 ± 1.08  327  13.17 ±1.75*  23.18 ±1.97  25.59 ±1.41  328  24.01 ±0.61**  26.89 ±0.63  27.42 ± 1.23  220  13.38 ±0.53  13.72 ±0.39  16.37 ±0.59***  5.40 ±0.17*  19.31 ±1.94  26.84 ±1.12  14.26 ±0.68*  21.20 ±1.39  22.27 ±0.81  1159 Mean ± SEM  hypoxemia  102  Mean ± SEM Control  pre-hypoxemia  lower than hypoxemic and post-hypoxemic period according to ANOVA/Tukey test (p< 0.005) lower than hypoxemic and post-hypoxemic period according to ANOVA/Tukey test (p< 0.05). higher than pre-hypoxemia and hypoxemic period according to ANOVA/Tukey test (p < 0.05) higher than control group according to Student's t-test (p < 0.0001).  #  77  3.4.  Renal  Excretion  Infusion  of Metoclopramide  to Steady-state  and its Metabolites  and Induction  of  the Lv.  Following  Hypoxemia.  3.4.1. Renal clearance of MCP and its metabolites during hypoxemia and normoxemia. Renal clearance values of M C P and mdMCP  w e r e c a l c u l a t e d f r o m 1) d i v i d i n g  a m o u n t r e c o v e r e d (Dw) b y a r e a u n d e r d r u g p l a s m a c o n c e n t r a t i o n s  the  c u r v e (AUC„~) a s a  f u n c t i o n o f t i m e d u r i n g t h e h y p o x e m i c a n d n o r m o x e m i c p e r i o d s [Du(t -t )/AUC(t -t )] a n d 2  c a l c u l a t i o n u s i n g t h e s l o p e o f t h e a c c u m u l a t e d d r u g i n u r i n e (Du~) 9 a n d A p p e n d i x A for the equation derivation).  ]  2  l  v e r s u s (AUC„~) ( s e e F i g u r e  Since the p l a s m a concentration o f  a p p e a r s to b e b e l o w t h e q u a n t i t a t i o n l i m i t , its r e n a l c l e a r a n c e v a l u e c o u l d n o t b e  ddMCP  calculated.  A s s h o w n i n T a b l e s 13 a n d 14, the renal c l e a r a n c e v a l u e s o b t a i n e d b y b o t h m e t h o d s  were  relatively consistent.  were  Some  2)  discrepancies b e t w e e n the calculated clearance values  observed w h e n there w e r e data point(s) noticeably deviating f r o m the regression line in the p l o t o f Du"  v e r s u s AUC ". 0  T a b l e s 13a a n d 13b s h o w the M C P renal clearance calculated b y both m e t h o d 1a n d 2 during the h y p o x e m i c a n d post-hypoxemic periods.  D u e to the short duration o f the  pre-  h y p o x e m i c p e r i o d {i.e. 2 h o u r s w i t h 3 d a t a p o i n t s ) , t h e r e n a l c l e a r a n c e d u r i n g t h i s p e r i o d c o u l d only b e a p p r o x i m a t e d , a n d the value d u r i n g h y p o x e m i a w a s c o m p a r e d to the  post-  h y p o x e m i c values.  In the experimental group, renal M C P clearance during h y p o x e m i a  (2.75  ± 0.22 L / h ) w a s significantly lower (by = 64%) than during the p o s t - h y p o x e m i c period  (8.15  ± 1.86 L / h ) . In contrast, the renal clearance r e m a i n e d constant (7.01 ± 0.72 L / h a n d 6.73 ± 0.59 L / h respectively) d u r i n g the equivalent times in the control group.  In addition, renal  clearance in the experimental group was significantly lower than in the control g r o u p during  78  the h y p o x e m i c period, but not afterwards.  There was a considerable individual variation in  the reduction o f renal clearance during h y p o x e m i a .  F o r example, ewe number 1158  showed  an apparent 7 5 % reduction in the renal clearance during h y p o x e m i a , whereas e w e  number  139 showed a 3 3 %  decrease.  T a b l e s 1 4 a a n d 1 4 b list the m d M C P hypoxemia.  T h e values of mdMCP  than those of M C P . mdMCP  renal clearance during hypoxemia and  post-  renal clearance w e r e considerably higher (= 6-7  fold)  H i g h e r urinary a c c u m u l a t i o n and, to a lessor degree, l o w e r  concentration contributed to higher renal clearance values for m d M C P .  the M C P renal clearance in the experimental group, the m d M C P  S i m i l a r to  renal clearance  h y p o x e m i a (14.60 ± 2.56 L / h ) was significantly lower (= 6 2 % ) than d u r i n g  plasma  during  post-hypoxemia  (38.57 ± 9.09 L/h). In the control group, the renal clearance r e m a i n e d constant (43.90 ± L/h a n d 38.39 ± 7.12 L / h , respectively). value for mdMCP  C o n s e q u e n t l y , as w i t h M C P renal clearance,  the  in the experimental g r o u p w a s significantly l o w e r than that in the control  g r o u p d u r i n g the h y p o x e m i c period, but not during post-hypoxemia. considerable  9.41  individual variation in the  reduction  of  mdMCP  There was  renal clearance  also  a  during  h y p o x e m i a , although less than that seen for M C P renal clearance. F o r e x a m p l e , w i t h e w e  no.  1158, there was an apparent 6 9 % reduction in renal clearance during h y p o x e m i a while  ewe  no. 139 showed a 3 9 %  decrease.  79  6000  5000  HO  cn o  = 2.89  L/h;  post-HO = 8.99  L/h  /  E  g  4000  o. o  3000  •a to E  /  A  /'  /  /  /'+ ,. —  2000  O o  <  1000  „  +  /  +  / 180  /  ,  360 AUC  Figure 9  i  540  720  900  (mcg*h/L)  Representative plot of the accumulated d r u g (or metabolite) in urine ( Z D u ) versus A U C . Slope of the curve represents the renal clearance [L/h] (ewe no. 102 in the experimental group). HO = hypoxemia, p o s t - H O = post-hypoxemia.  Table 13a.  MCP renal clearance (L/h) [CLmnaifMCPJ] 1  CLrenal(MCP)  ewe no.  hypoxemia  post-hypoxemia  Experimental  102  3.20  8.25  Group  139  2.09  2.92  989  3.33  7.21  1158  2.94  11.07  Mean  2.89*  ±SEM  7.36  #  0.28  1.69  Control  220  6.58  6.97  Group  327  4.96  5.78  328  7.59  8.01  1159  7.73  7.41  Mean  6.72  7.04  0.64  0.47  ±SEM  * Significantly lower (p<0.03) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.001) than the control group according to t-test. renal clearance calculated using time-averaged urinary excretion [Du(t2-tl)/AUC(t2-tl)]. 1  Table 13b.  MCP renal clearance (L/h) [CLrenai(MCPJ]  CLrenal(MCP)  2  ewe no.  post-hypoxemia  hypoxemia  Experimental  102  2.89  8.99  Group  139  2.09  3.13  989  2.95  8.39  1158  3.05  12.08  2.75*  8.15  Mean ±SEM  #  0.22  1.86  Control  220  6.83  6.21  Group  327  5.11  5.44  328  7.66  7.06  1159  8.45  8.19  Mean  7.01  6.73  ±SEM  0.72  0.59  * Significantly lower (p<0.025) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.001) than the control group according to t-test. renal clearance calculated using the slope of Du(t-0)/AUC(t-0) plot. 2  Table 14a.  mdMCP renal clearance (L/h)  CLrenal(mdMCP)  ewe no.  1  hypoxemia  [CLrenai(mdMCP)] post-hypoxemia  Experimental  102  12.38  26.67  Group  139  12.14  20.90  989  21.79  53.68  1158  11.44  40.66  14.44**  35.48  2.46  7.35  Mean ±SEM  Control  220  65.96  59.97  Group  327  35.75  36.23  328  44.25  52.12  1159  30.88  22.03  44.21  42.59  7.76  8.45  Mean ±SEM  * Significantly lower (p<0.02) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.001) than the control group according to t-test. renal clearance calculated using time-averaged urinary excretion [Du(t2-tl)/AUC(t2-tl)]. 1  Table 14b.  mdMCP renal clearance (L/h)  CLrenal(mdMCP)  ewe no.  2  hypoxemia  [CLrenai(mdMCP)] post-hypoxemia  Experimental  102  9.84  26.72  Group  139  13.20  21.69  989  21.85  61.70  1158  13.49  44.17  14.60**  38.57  2.56  9.09  Mean ±SEM Control  220  70.91  55.85  Group  327  31.65  33.64  328  42.58  42.10  1159  30.44  21.99  Mean  43.90  38.39  ±SEM  9.41  7.12  * Significantly lower (p<0.02) than post-hypoxemia according to paired t-test. # Significantly lower (p<0.001) than the control group according to t-test. renal clearance calculated using the slope of Du(t-0)/AUC(t-0) plot. 2  82  3.4.2. Fractional renal excretion constants of MCP  and its metabolites  T h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t s w e r e a p p r o x i m a t e d f o r M C P a n d t w o o f its metabolites,  mdMCP  c o n s t a n t s (e.g.  fu(MCP)  and ddMCP,  in the present study.  T h e fractional renal  excretion  = ku/KE) w e r e c a l c u l a t e d b y d i v i d i n g t h e s l o p e o f t h e a s y m p t o t e o f t h e  a c c u m u l a t e d d r u g / m e t a b o l i t e s i n u r i n e v e r s u s t i m e c u r v e w i t h t h e i n f u s i o n r a t e , ko ( F i g u r e 10), a n d t h e s e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t s a r e d i m e n s i o n l e s s proportion o f the total excretion constant in terms o f a fraction).  (i.e. t h e y r e p r e s e n t a T h e detailed  equation  d e r i v a t i o n s a r e s h o w n i n a p p e n d i x A a n d t h e o r e t i c a l a n d p r a c t i c a l d i s c u s s i o n is d e s c r i b e d i n s e c t i o n 4.4. T h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t w a s u s e d t o d e t e r m i n e r e l a t i v e c h a n g e s i n t h e r e n a l e x c r e t i o n o f t h e p a r e n t d r u g a n d i t s m e t a b o l i t e (i.e. t h e p r o p o r t i o n o f t h e excretion to the total b o d y elimination o f the  Scheme V  renal  drug/metabolite).  Diagram of metabolic and renal elimination of a drug  kf  kmu  where X and M represent the amount of drug and metabolite in the body, respectively, X and M represent the drug and metabolite excreted in urine, respectively. k and K are renal and metabolite elimination rate constants and the sum of these two rate constants is equal to K , apparent first order drug elimination rate constant, kmu represents renal elimination rate constant of the metabolite. B  u  E  B  u  u  f  83  T a b l e 1 5 l i s t s t h e f r a c t i o n a l r e n a l M C P e x c r e t i o n c o n s t a n t (fu(incp) = ku/KE). D u e t h e s h o r t d u r a t i o n o f t h e p r e - h y p o x e m i c p e r i o d (i.e.  to  2 hours with 3 data points), the fractional  r e n a l e x c r e t i o n c o n s t a n t , fu(MCP), w a s n o t c a l c u l a t e d d u r i n g t h e p r e - h y p o x e m i c p e r i o d .  A s  with the M C P a n d m d M C P  was  r e n a l c l e a r a n c e v a l u e s , t h e /U(MCP> v a l u e d u r i n g h y p o x e m i a  c o m p a r e d to the p o s t - h y p o x e m i c significantly  lower by 58%,  estimate.  (0.0138 ±  D u r i n g the h y p o x e m i c  0.0019) than d u r i n g the p o s t - h y p o x e m i c  (0.0329 ± 0.0053) in the experimental group. constant  p e r i o d , t h e /U(MCP> i s period  I n c o n t r o l g r o u p , t h e /U(MCP> r e m a i n e d f a i r l y  throughout these periods, 0.0296 ± 0.0032 and 0.0282 ±  0.0040,  respectively.  C o n s e q u e n t l y , t h e fu(MCP) v a l u e d u r i n g t h e h y p o x e m i c p e r i o d i n t h e e x p e r i m e n t a l g r o u p  was  significantly lower than corresponding estimate in the control group. T h e fractional renal metabolite excretion constants for mdMCP calculated in the study.  and ddMCP  were also  B e c a u s e the i n d i v i d u a l parameters s u c h as metabolite f o r m a t i o n a n d  elimination rate constants cannot be determined with the present study design, unless metabolites are injected individually, these parameters are the product o f t w o  the  fractional  constants fmfmetaboiite) a n d fmu(metaboiites). T h e m e t a b o l i t e f o r m a t i o n f r a c t i o n " c o n s t a n t fm(metab i s t h e f r a c t i o n o f kflmetaboiite)/ K.E( arent drug), w h i c h r e p r e s e n t s t h e m e t a b o l i c p r o p o r t i o n ( t o P  specific metabolite) o f the total parent d r u g elimination.  T h e second fractional constant,  the  r e n a l m e t a b o l i t e e l i m i n a t i o n f r a c t i o n fmu(metaboiite) i s t h e f r a c t i o n o f kmu(metaboiite)/ Km(metabo which  represents  Therefore,  the  the  renal  composite  excretion fractional  proportion constant,  the  of  the  renal  total  metabolite  metabolite  elimination.  excretion  fraction  fu(metaboiite) = fm(metaboiite) xfmu(metaboiite), r e p r e s e n t s t h e p r o p o r t i o n o f r e n a l e x c r e t i o n o f a metabolite  f r o m the total d r u g elimination. F o r example,  i f t h e v a l u e o f f (metaboiite A> = U  84  fm(metaboiite A) x fmu(metaboiite A)  w a s 0 . 0 1 = 0 . 0 5 * 0.2, t h e n it w o u l d m e a n t h a t 5 % o f p a r e n t d r u g  w a s b i o - t r a n s f o r m e d i n t o t h e m e t a b o l i t e Aa n d 2 0 % o f t h e m e t a b o l i t e w a s e x c r e t e d i n u r i n e (i.e.  the  o v e r a l l 1 % o f the p a r e n t d r u g is e x c r e t e d i n the u r i n e i n the f o r m o f m e t a b o l i t e A ) .  4000  HO = 159, 58; post-HO = 320.20  3200 h  8> E  A-  C  2400 D-  o  -o •o TJ  1600  o  800  <  J  Z  L  6  10  8  12  14  Time (hr)  F i g u r e 10.  Representative plot of the accumulated d r u g (or metabolite) in urine ( S D u ) v e r s e s t i m e (h). T h e s l o p e o f the c u r v e represents the p r o d u c t o f the fractional renal excretion rate constant for M C P (or metabolite) a n d the infusion rate constant o f M C P . ( E w e no. 102 in the experimental group). H O =hypoxemia, p o s t - H O = post-hypoxemia.  T h e fractional renal metabolite excretion constants of mdMCP i n T a b l e s 16 a n d 17.  The  fu(mdMCP)  was  and ddMCP  significantly lower (-65%) during  are listed hypoxemia  ( 0 . 0 3 7 8 ±0 . 0 0 9 8 ) c o m p a r e d to that o f the p o s t - h y p o x e m i c p e r i o d ( 0 . 1 0 8 0 ±0 . 0 3 3 9 ) i n the experimental group.  In the control group, the  and 0.0711 ±0.0125, respectively.  fu(mdMCP)  T h u s , the  remained constant, 0.0719 ± 0.0085  fu(mdMCP)  value during hypoxemia in  the  85  e x p e r i m e n t a l g r o u p w a s also significantly l o w e r than that o f the control group. S i m i l a r to the r e n a l c l e a r a n c e v a l u e s f r o m t h e p r e v i o u s s e c t i o n , t h e fu(mdMCP) v a l u e s w e r e a b o u t 2 t o 4 f o l d h i g h e r t h a n t h o s e offu(MCP).  In s o m e cases (ewe n u m b e r s 989 a n d 1158 a n d 328), this process  has a c c o u n t e d for m o r e than 1 0 % o f the total d r u g elimination. The f* (ddMCP) w a s a l s o s i g n i f i c a n t l y l o w e r ( - 6 0 % ) d u r i n g h y p o x e m i a ( 0 . 0 2 2 3 ± 0 . 0 5 6 ) U  c o m p a r e d to that o f the p o s t - h y p o x e m i a (0.0562 ±0.0152) in the e x p e r i m e n t a l group. In the c o n t r o l g r o u p , t h e fr(mdMCP) r e m a i n e d c o n s t a n t , respectively.  0.0259 ± 0.0053 and 0.0259 ± 0.0052,  H o w e v e r , u n l i k e fu(MCP) andfu(mdMCP), t h e fu(ddMCP) v a l u e d u r i n g h y p o x e m i a i n  the experimental g r o u p w a s not significantly different f r o m that o f the control group.  Table 15.  MCP fractional renal excretion constants [fu(MCPJ\-  fu(MCP)  ewe no.  hypoxemia  post-hypoxemia  Experimental  102  0.0151  0.0401  Group  139  0.0124  0.0182  989  0.0183  0.0410  1158  0.0094  0.0325  0.0138**  0.0329  0.0019  0.0053  Mean ±SEM Control  220  0.0205  0.0214  Group  327  0.0312  0.0250  328  0.0349  0.0399  1159  0.0319  0.0266  Mean  0.0296  0.0282  ±SEM  0.0032  0.0040  * Significantly lower (p<0.02) than the post-hypoxemic period according to paired t-test. Significantly lower (p<0.005) than the control group according to t-test. Renal elimination fraction constant fu(MCP) denotes the ratio of ku/KE of M C P .  #  a  Table 16.  mdMCP fractional renal excretion constants*  f*u(mdMCP)  ewe no.  hypoxemia  [fu(mdMCPJ\-  post-hypoxemia  Experimental  102  0.0248  0.0560  Group  139  0.0273  0.0530  989  0.0668  0.1955  1158  0.0323  0.1274  0.0378**  0.1080  0.0098  0.0339  Mean ±SEM Control  220  0.0766  0.0758  Group  327  0.0638  0.0614  328  0.0933  0.1032  0.0539  0.0442  Mean  0.0719  0.0711  ±SEM  0.0085  0.0125  1159  * Significantly lower (p<0.05) than post-hypoxemia according to paired t-test. Significantly lower (p<0.05) than the control group according to t-test. f*u(mdMCP) denotes the composite fraction constant fm(mdMCP) x fmu(mdMCP), the  #  a  product O f the ratio o f kflmdMCP)/KE(MCP) and kmu(mdMCP)/Km(mdMCP).  Table 17.  ddMCP fractional renal excretion constants*  f*u(ddMCP)  ewe no.  hypoxemia  [fu(ddMCP)]-  post-hypoxemia  Experimental  102  0.0127  0.0262  Group  139  0.0188  0.0369  989  0.0384  0.0926  1158  0.0194  0.0693  Mean  0.0223*  0.0562  ±SEM  0.0056  0.0152  Control  220  0.0365  0.0377  Group  327  0.0153  0.0157  328  0.0181  0.0190  0.0335  0.0314  Mean  0.0259  0.0259  ±SEM  0.0053  0.0052  1159  * Significantly lower (p<0.02) than post-hypoxemia according to paired t-test. fu(ddMCP) denotes the composite fraction constant fm(ddMCP) x fmu(ddMCP), the  a  product of the ratio of kf(ddMCP)/KE(MCP) and kmu(ddMCP)/Km<ddMCP).  87  DISCUSSION  4. 4.1.  GC-MSD Method of Analysis of Metoclopramide and A  developed  GC-MSD  method  for the  assay  of M C P , mdMCP  a n d v a l i d a t e d i n o u r l a b o r a t o r y ( R i g g s et  al,  its Metabolites  and  ddMCP,  1994), was  chosen  f o r its  sensitivity a n d reproducibility for application to the present study. T h e G C - M S D a n u m b e r o f inherent advantages over the GC-ECD  previously high  method  has  method previously developed in  our  l a b o r a t o r y ( R i g g s et al,  1983; 1990), d u e to the selective nature o f m a s s spectrometry.  The  simultaneous  a n d q u a n t i f i c a t i o n o f M C P a n d its m e t a b o l i t e s at h i g h e r  (=2x)  detection  sensitivity was m a d e possible using M S D technology. are simpler than with the GC-ECD  M o r e o v e r , the extraction  procedures  m e t h o d , thereby reducing the s a m p l e preparation  time  significantly.  The use of n a r r o w bore fused silica capillary c o l u m n s a n d GC-MSD  with  i o n m o n i t o r i n g e n a b l e d us to separate a n d quantitatively a n a l y z e the deethylated  selective  metabolites  o f M C P in the presence of M C P in the biological fluids collected in the study. deethylated  metabolites  of  M C P , mdMCP  and  ddMCP,  were  analysed  from  T w o various  b i o l o g i c a l fluids. T h e use o f n a r r o w b o r e (0.2 m m I D . ) f u s e d silica capillary c o l u m n s in the p l a c e o f t h e c o n v e n t i o n a l 0 . 3 3 m m I . D . c o l u m n s a n d t h e s o l v e n t t r a p p i n g e f f e c t ( R i g g s et 1994)  has p r o v i d e d baseline separation a m o n g the metabolites a n d M C P .  separation, especially between M C P and mdMCP  This  increased  (a metabolite often present in  quantities  greater than the intact M C P ) was essential for the accurate quantitation o f these in urine. Significant amounts of mdMCP  al,  compounds  w e r e recovered in urine during the infusion  study,  t h e r e f o r e there is a p o s s i b i l i t y o f o v e r - e s t i m a t i o n o f M C P i n t h e s e s a m p l e s i f the provides insufficient separation, particularly w h e n GC-ECD contrast, the use o f selective ion monitoring m e t h o d in GC-MSD  analysis  is t h e m e t h o d o f c h o i c e .  In  has enabled the detection  the analytes with m i n i m a l interference f r o m endogenous c o m p o u n d s .  of  A s s h o w n i n F i g u r e 1,  the interference f r o m contaminants was m i n i m a l even with reduced extraction procedures.  Linearity over a 1-40 n g / m L concentration range w a s o b s e r v e d for M C P , m d M C P ddMCP  extracted from plasma and urine with individual standard curves from each  h a v i n g a coefficient o f d e t e r m i n a t i o n (r ) o f at least 0.98. 2  1 ng/mL.  variability  slightly  (i.e.  T h e standard curves higher  concentration b e l o w 4 ng/mL.  C V  values)  was  were  matrix  T h e m e t h o d has b e e n f o u n d to be  reliable with an overall average coefficient o f variation less than 10 % quantitation of  and  linear, however,  observed  in  the  a n d the limit slightly  greater  corresponding  S i n c e a slight variation i n the p e a k area ratio at the  drug higher  concentrations c a n greatly affect the y-intercept o f the linear regression curve, the lower concentrations in the curve will be affected m o r e b y this variation. T h e use o f the  drug  weighting  factor o f l / ( p e a k a r e a ratio) i m p r o v e d the linearity o v e r the w h o l e r a n g e , e s p e c i a l l y at lower concentration range. MSD  the  E v e n t h o u g h it w a s n o t c r u c i a l t o t h e i n f u s i o n s t u d y , t h e G C -  method permitted attainment of a m i n i m u m quantitation limit of 1 ng/mL,  representing  2.5 p g at the detector, a r e a s o n a b l e i m p r o v e m e n t o v e r the q u a n t i t a t i o n l i m i t o f 2 obtained with the previous G C - E C D  Thus, the GC-MSD  of  ng/mL  method.  method with selective ion monitoring provides a m o r e  straightforward and reliable m e a n s for measurement previous methods of M C P measurement.  o f M C P a n d its m e t a b o l i t e s  F u r t h e r m o r e , it is a n t i c i p a t e d that, w h e n t h e  stable, than present  89  w o r k is c o m p l e t e d , the G C - M S D  method will enable us further investigations  of  stable  isotope labeled M C P in pregnant hypoxemic animals under another animal research protocol.  4.2.  Physiological  Changes  Associated  with  Hypoxemia  4.2.1. Induction of hypoxemia and Blood gas changes Several experimental methods  of inducing hypoxemia have been previously  used,  s u c h a s r e d u c e d i n s p i r e d o x y g e n c o n t e n t a n d c a r b o n m o n o x i d e ( C O ) a d m i n i s t r a t i o n ( R o t h et al., 1 9 7 6 ) . T h e r e a r e s e v e r a l r e p o r t e d m e t h o d s f o r r e d u c i n g i n s p i r e d 0  2  content, including an  i s o l a t e d g a s c h a m b e r ( M e d i n a a n d M e r r i t t , 1 9 7 0 ) , a h y p o b a r i c c h a m b e r ( J a c o b s et ai, 1 9 8 8 ) , a P l e x i g l a s ® c h a m b e r e n c l o s i n g t h e h e a d a n d n e c k o f t h e e w e ( R u r a k et ai, 1 9 9 0 ) o r p l a s t i c b a g o v e r t h e h e a d ( d u S o u i c h et al., 1 9 8 4 ) i n t o w h i c h a g a s m i x t u r e c a n b e d e l i v e r e d t o c a u s e h y p o x e m i a , a n d l a s t l y , a n i n t r a - t r a c h e a l n i t r o g e n i n f u s i o n ( G l e e d et ai, 1 9 8 6 ) . method involves  i n f u s i o n o f n i t r o g e n ( = 4 - 1 0 L / m i n ) via a s m a l l n o n - o c c l u s i v e  chronically i m p l a n t e d in the trachea.  2  concentration.  method of lowering P a 0  2  2  catheter  Various degrees of hypoxemia  b e a c h i e v e d b y v a r y i n g the nitrogen f l o w rate. A r a p i d P a 0 be obtained after c h a n g i n g the N  latter  T h e nitrogen m i x e s with the air n o r m a l l y inspired by  the animal, resulting in alowering of 0  flow.  2  in the sheep. T h e lowered P a 0  appear to be disturbed b y nitrogen infusion.  2  can can  effective  was reached within 5m i n . of the M o r e o v e r , the animal did  T h e implantation of an intra-tracheal  a m i n o r s u r g i c a l p r o c e d u r e a n d p e r f o r m e d at the  procedures during the general anaesthesia.  e q u i l i b r a t i o n t i m e (=3 m i n )  Intra-tracheal nitrogen infusion was an  o n s e t o f h y p o x e m i a (first s a m p l i n g after n i t r o g e n infusion).  was  The  same  time  with  other  not  catheter surgical  Other methods of inducing hypoxemia in  the  90  e x p e r i m e n t a l a n i m a l , s u c h as a n isolation gas c h a m b e r a n d p l a c e m e n t o f the plastic b a g the head, c o u l d potentially c a u s e u n d u e restriction a n d stress to the animals.  over  The  tracheal infusion m e t h o d s e e m s to m i n i m i z e s o m e o f the side effects o f other  intra-  experimental  m e t h o d s used in the sheep. A l l animals u n d e r the study have free access to water a n d f o o d in their n o r m a l housing before a n d during experiments.  Furthermore, all e w e s are maintained in  a relatively close contact with other c o m p a n i o n ewes during each experimental period, and, thus,  environmental  alterations  during and between  experiments  were  minimized.  a d d i t i o n , t h e i n t r a - t r a c h e a l n i t r o g e n i n f u s i o n p r o c e d u r e c o n s u m e s l e s s n i t r o g e n (i.e. vs. >40 L/min for the c h a m b e r methods), thus resulting in a substantial cost  Intra-tracheal hypoxemia (Pa0  nitrogen  infusion  (7  to  11  L/min)  In  « 7 L/min  savings.  rapidly induced  a  moderate  ~ 60 m m H g ) in the sheep, within 5 m i n of the onset of nitrogen infusion.  2  T h i s is s i m i l a r to the result r e p o r t e d f o r p r e g n a n t e w e s i n the s t u d y that first d e s c r i b e d n i t r o g e n i n f u s i o n t e c h n i q u e ( G l e e d et al, PaC0  2  (hypocapnia)  and a tendency  1986).  the  A t the s a m e time, a significant decrease in  of increased blood p H (respiratory alkalosis)  was  observed in the experimental group. Infused nitrogen m i x e d with n o r m a l l y inspired r o o m air will result in a reduction o f o x y g e n content in the inspired gas mixture in the l u n g resulting in reduction of Pa0 . 2  I n a d d i t i o n , h y p o c a p n i a is c a u s e d b y h y p e r v e n t i l a t i o n , s i n c e a  typical r e s p o n s e to r e d u c e d P a 0 This lowered P a 0  2  2  is i n c r e a s e d v e n t i l a t i o n ( h y p e r v e n t i l a t i o n ) ( D a l y ,  w a s m a i n t a i n e d through intra-tracheal nitrogen infusion (rate = 7  H o w e v e r , in s o m e animals, nitrogen infusion w a s increased to m a i n t a i n P a 0 mmHg.  A  thus  variation in individual response to h y p o x e m i a w a s  2  observed, but  1963). L/min).  around  correlation  b e t w e e n h y p o x e m i c r e s p o n s e a n d p h y s i o l o g i c a l values s u c h as urine f l o w a n d lactate w a s obvious from a visual survey.  60  not  91  4.2.2. Blood pH  and  lactate concentration during hypoxemia  Increased b l o o d lactate concentration w a s o b s e r v e d i n the late h y p o x e m i c period i n the e x p e r i m e n t a l g r o u p a n d it r e m a i n e d relatively h i g h t h r o u g h t h e e a r l y a n d m i d d l e hypoxemic recovery periods.  Thus, there seems to b e a trend o f a gradual increase in the  b l o o d lactate concentration during hypoxemia.  H o w e v e r , this a c c u m u l a t i o n o f lactate is n o t  likely the result o f inadequate o x y g e n supply to the tissues, since the 0 h y p o x e m i a i n t h e s t u d y r e m a i n e d a r o u n d 7 0 t o 8 0 %. an indication o f tissue 0  2  post-  2  saturation during  In addition, the "excess lactate"  level,  a d e q u a c y , is estimated to b e close to z e r o at t h e level o f  P a 0  2  obtained in the study (= 6 0 m m H g ) (Cain, 1965), thus the change i n the lactate concentration in t h e present study is n o t likely d u e to 0  2  deficit o f body.  R a t h e r , it is m o r e l i k e l y r e l a t e d t o  hypocapnia a n d the resulting changes in acid-base balance during h y p o x e m i a  (Huckabee,  1958). T h e increased glucose level d u r i n g late h y p o x e m i a a n d h y p o c a p n i a d u r i n g h y p o x e m i c period m a y also, i n part, explain the lactate accumulation.  It h a s b e e n s u g g e s t e d t h a t t h e l a c t a t e p r o d u c t i o n i s n o t c o n t r o l l e d e x c l u s i v e l y b y t h e a d e q u a c y o f cellular oxygenation, a n d is affected to a very significant extent b y the pyruvate changes, hyperventilation o r p H alteration o f the b o d y concentration ( H u c k a b e e , 1957) a n d sympathetic activity.  and changes  Takano (1968) has  that p l a s m a lactate accumulation i n dogs w a s less f o r isocapnic hypocapnic hypoxia.  Cain  (1969) also showed  in blood  demonstrated  hypoxia compared  diminished lactate accumulation  h y p o x e m i a with P-adrenergic blockade a n d hypercapnia. Furthermore, Cain (1973) lactate a c c u m u l a t i o n i n d u c e d b y P-stimulation without o x y g e n deficit.  glucose  to  during observed  Zborowska-Sluis and  92 Dossetor (1967) observed increased b l o o d lactate concentration in hyperventilated dogs  with  n o increase in lactate production across m a j o r organ systems (no changes in the A - V lactate concentration difference across liver, muscle, a n d gut).  In this study, a small decrease  h e p a t i c l a c t a t e u t i l i z a t i o n w a s o b s e r v e d , b u t it w a s e s t i m a t e d to a c c o u n t f o r o n l y a  small  portion o f the lactate increase. A stimulation of red b l o o d cell glycolysis b y l o w PaCCte respiratory alkalosis was 1967).  With  also observed  (Murphy,  1960;  Zborowska-Sluis and  b l o o d cell is a l m o s t b y a n a e r o b i c g l y c o l y s i s , a n d t h u s i n d e p e n d e n t o f c h a n g e s i n  hypoxemia  Therefore, these studies suggest that lactate a c c u m u l a t i o n d u r i n g a is  associated  with  concentration and sympathetic  alteration  in  blood  acid-base  balance,  red  oxygen moderate  and/or  glucose  activation.  A c i d o s i s w a s also observed in the p o s t - h y p o x e m i c period, but not in the period.  or  Dossetor,  no effective aerobic metabolic system present, energy production in the  availability.  in  hypoxemic  T h i s d e l a y e d r e s p o n s e s u g g e s t s that a c i d o s i s is l i k e l y a s e c o n d a r y r e s p o n s e  hypoxemia, rather than a direct response.  T h e decrease in b l o o d p H s e e m s to  to  correspond  w i t h a n increase in lactate level in the late h y p o x e m i c a n d early/middle  post-hypoxemic  p e r i o d (hour 6:00 to 12:00).  acidosis during  A n i n c r e a s e i n h y d r o g e n i o n c o n c e n t r a t i o n (i.e.  p o s t - h y p o x e m i a ) i n the b l o o d is l i k e l y a s e c o n d a r y r e a c t i o n to t h e a c c u m u l a t i o n o f l a c t i c a c i d (Frommer,  1983).  93 4.2.3. Adrenosympathetic system and blood glucose during hypoxemia  Increased heart rate a n d m e a n arterial b l o o d pressure d u r i n g the h y p o x e m i c was observed in the present study.  period  S i n c e h y p o x e m i a is k n o w n to c a u s e a c t i v a t i o n o f  the  a d r e n o s y m p a t h e t i c s y s t e m and, also, to increase circulating c a t e c h o l a m i n e concentrations,  the  increased heart rate a n d arterial b l o o d pressure in the study m a y be associated with  this  adrenosympathetic  activation.  Stimulated  adrenosympathetic  activity  and  increased  plasma  catecholamine  concentration during hypoxia has been observed in m a n y previous studies. Since C a n n o n H o s k i n s (1911) demonstrated secretion of epinephrine into adrenal venous b l o o d  and  during  a s p h y x i a , it h a s b e e n g e n e r a l l y a c c e p t e d t h a t h y p o x i a s t i m u l a t e s a s y m p a t h o a d r e n a l d i s c h a r g e . C l a u s t r e a n d P e y r i n ( 1 9 8 2 ) a n d C l a u s t r e et al. ( 1 9 8 5 ) r e p o r t e d a m i l d activation  and  a significant  increase  in  plasma  free  adrenosympathetic  catecholamine  (epinephrine,  n o r e p i n e p h r i n e a n d d o p a m i n e ) concentration d u r i n g a m o d e r a t e h y p o x i a in rats a n d cats.  The  increase in plasma epinephrine level in these studies was especially profound during hypoxia, and  this increased  epinephrine  associated with hypoxia.  concentration  may  contribute  to  physiological  J o h n s o n et al. ( 1 9 8 3 ) a l s o o b s e r v e d e l e v a t e d a d r e n a l  changes medullary  activity a n d urinary epinephrine excretion during acute moderate h y p o x e m i a independent cardiac s y m p a t h e t i c activity in rats, thus speculating that the r e s p o n s e d u r i n g acute h y p o x e m i a is p r i m a r y b y a d r e n a l m e d u l l a r y a c t i v a t i o n .  of  moderate  In addition, the activation of  the  a d r e n o m e d u l l a r y s y s t e m b y h y p o x e m i a m a y h a v e s o m e residual effect e v e n after restoration o f P a C h . C r i t c h l e y et al.  (1980) s h o w e d that the release o f c a t e c h o l a m i n e f r o m the  m e d u l l a , in response to p r o l o n g e d carotid b o d y h y p o x e m i a , outlasted the stimulus b y  adrenal more  94 than 3 0 m i n . T h e y also f o u n d that a m o d e r a t e h y p o x e m i a w i t h carotid arterial PaCh o f 5 0 - 6 0 mmHg  w a s sufficient to evoke the release o f catecholamine.  A  small, b u t statistically  significant,  increase  h y p o x e m i a w a s also observed in the present study.  in glucose  concentration  during  T h e relatively small increase i n blood  glucose concentration i n the present study is likely d u e to a moderate level o f h y p o x e m i a . The d e v e l o p m e n t o f a m o d e s t degree o f h y p e r g l y c e m i a u n d e r stress s u c h as h y p o x i a a n d h y p o t h e r m i a m a y represent a n a d a p t i v e r e s p o n s e to stress, h e l p i n g to protect a n d m a i n t a i n t h e supply o f a n important fuel f o r the brain a n d other tissue ( B a u m a n d Porte, 1980).  Increased  blood  in  acute  Earlier studies have suggested that the elevated  blood  glucose  concentration  d u r i n g stress (stress h y p e r g l y c e m i a )  h y p o x e m i a ( B a u m a n d Porte, 1980). glucose  concentration w a s a result o f increased hepatic  is c o m m o n  glycogenolysis  h o r m o n a l e f f e c t s o f h y p o x i a ( B r i t t o n a n d K l i n e , 1 9 4 5 ; H i m w i c h et al,  a n d the  direct  1943; Shelley,  1961).  T h e r e is a n increased epinephrine secretion b y the adrenal m e d u l l a i n combination s e c o n d a r y g l u c a g o n release b y a-cells o f pancreatic islets d u r i n g h y p o x e m i a .  of  This, in turn,  stimulates glycogen b r e a k d o w n i n muscles a n d liver, thus resulting i n the elevation o f b l o o d sugar level.  C a t e c h o l a m i n e s c a n d i r e c t l y s t i m u l a t e g l u c o s e p r o d u c t i o n b y t h e l i v e r ( R i z z a et  al,  1 9 8 0 ) . I n a d d i t i o n , t h e y c a n i n t e r f e r e w i t h i n s u l i n - m e d i a t e d g l u c o s e u p t a k e ( C h i a s s o n et  al,  1981;  Deibert a n d DeFronzo, 1980).  Therefore, increased plasma catecholamine  during h y p o x e m i a result i n a n increased b l o o d glucose level a n d subsequent  levels  shunting of  b l o o d glucose to insulin-independent tissues, s u c h as t h e brain. T h e r e is also e v i d e n c e f o r a s t i m u l a t o r y e f f e c t o f c a t e c h o l a m i n e o n g l u c a g o n s e c r e t i o n m e d i a t e d via  a p-adrenergic a n d ,in  95  a l e s s o r d e g r e e , o c - a d r e n e r g i c m e c h a n i s m ( G e r i c h et al, 1 9 7 3 ; G e r i c h et al, 1 9 7 4 ; S a m o l s and Weir,  1979).  W h e n anaesthetized  dogs were  made  acutely hypoxic, a very  inhibition o f insulin secretion a n d increased plasma glucagon levels were observed  large (Baum  and Porte, 1980).  The  sympathetic nervous system,  activated at l o w o x y g e n  tension (Comline a n d  S i l v e r , 1 9 6 6 ; C u n n i n g h a m et al, 1 9 6 5 ) , h a s a n i m p o r t a n t r o l e i n m e d i a t i n g t h e h y p o x i c inhibition o f insulin release ( B a u m a n d Porte, 1969). a-adrenergic receptor  stimulation,  k n o w n to inhibit insulin secretion, is am a j o r factor i n this process ( B a u m a n d Porte,  1972).  A l t h o u g h b o t h a- a n d p - a d r e n e r g i c r e c e p t o r s a r e s t i m u l a t e d w i t h s y m p a t h e t i c a c t i v a t i o n , aadrenergic enhancement  suppression  of  insulin  is i m p a i r e d ( B a u m  predominates  a n d Porte,  combination with hepatic glycogenolysis  1976).  in  hypoxia This  p-adrenergic  because  suppression  o f insulin  mediated b y epinephrine a n d glucagon  in  during  hypoxia m a y explain the elevated level o f blood glucose during h y p o x e m i a a n d subsequent post-hypoxemia in the present study.  4.2.4. Urine flow and osmolality during hypoxemia and MCP infusion.  In the present study, urine flow w a s significantly increased during h y p o x e m i a , at the s a m e time, urine osmolality w a s significantly decreased i n the experimental group.  Diuresis  during acute h y p o x e m i a , either hypocapnic o r isocapnic, w a s demonstrated i n the conscious d o g ( W a l k e r , 1 9 8 2 ) a n d r a t ( C o l i c e et al, 1 9 9 1 ) .  Studies previous to these, however,  reported a variety o f urine flow responses (diuresis a n d antidiuresis) to acute  have  hypoxemia  ( S e l k u r t , 1 9 5 3 ; S t i c k n e y et al, 1 9 4 6 ; A n d e r s o n et al, 1 9 7 8 ) . H o w e v e r , m a n y o f t h e s e e a r l i e r  96  studies used anaesthetized and/or extensively manipulated animal preparations, w h i c h have complicated results a n d m a d e interpretation difficult. u s u a l l y h a s r e s u l t e d i n a d i u r e t i c r e s p o n s e ( B e r g e r et al,  Acute hypoxemia in  1 9 4 9 ; B u r r i l l et al,  may  humans  1945; Currie and  Ullman, 1961; U l l m a n , 1961).  C u r r e n t l y , t h e m e c h a n i s m o f d i u r e s i s d u r i n g h y p o x e m i a is n o t c l e a r l y u n d e r s t o o d .  A  n u m b e r o f m e c h a n i s m s s u c h as increased G F R s e c o n d a r y to increased renal b l o o d f l o w  and  arterial b l o o d pressure, a n d h y p o x e m i a - i n d u c e d h o r m o n a l c h a n g e s in p l a s m a atrial natriuretic factor (ANF), aldosterone and vasopressin levels have been proposed. T h e diuretic response d u r i n g h y p o x e m i a m a y be, in part, related to a alteration associated with hypoxemia. GFR  and renal blood flow  increase  rate  In dogs, hypocapnic h y p o x e m i a caused an increase in  secondary  to the arterial b l o o d pressure a n d cardiac  (with a lesser degree in isocapnic hypoxemia),  r e m a i n e d u n a l t e r e d ( K o e h l e r et al, ( G F R ) was  also  increased  hemodynamic  1980; Walker, 1982). during hypoxemia  while renal vascular  output  resistance  In addition, glomerular filtration  in these  studies.  However,  acute  h y p o x e m i a - i n d u c e d diuresis in the rat has b e e n associated with a fall in systemic arterial b l o o d p r e s s u r e a n d c a r d i a c o u t p u t ( C o l i c e et al,  1 9 9 1 ; O u et al,  the involvement o f other diuretic mechanism(s).  In the present study in sheep, there was  1989), thereby  suggesting a  small, but statistically significant, increase in m e a n arterial b l o o d pressure d u r i n g h y p o x e m i a i n t h e e x p e r i m e n t a l g r o u p . B u t , it is n o t c l e a r w h e t h e r t h e h y p o x e m i a - i n d u c e d d i u r e s i s is, i n fact, directly related to the increased m e a n arterial b l o o d pressure, d u e to lack o f i n f o r m a t i o n r e g a r d i n g r e n a l b l o o d f l o w a n d G F R . H o w e v e r , d i u r e s i s d u r i n g h y p o x e m i a m a y b e , at least in part, d u e to elevated g l o m e r u l a r filtration rate d u e to a n increase in renal b l o o d s e c o n d a r y to arterial b l o o d pressure  increase.  flow  97  Acute  hypoxia  in  anaesthetized  dogs  has  been  reported  to  stimulate  v a s o p r e s s i n ( A V P ) r e l e a s e a n d a n a n t i d i u r e t i c r e s p o n s e ( A n d e r s o n et al,  arginine  1978), but  acute  h y p o x i a i n c o n s c i o u s h u m a n s a n d r a t s d o e s n o t a f f e c t p l a s m a A V P l e v e l s ( d u S o u i c h et 1 9 8 7 ; A s h a c k et al,  1 9 8 5 ; J o n e s et al,  1981).  Aldosterone levels fall with acute h y p o x i a in  h u m a n s , but only after 3 0 to 6 0 m i n o f exposure ( C o l i c e a n d R a m i r e z , 1985). studies suggested induced diuresis. suggested  Thus, these  that A V P a n d aldosterone m a y not b e closely related to acute The involvement  ( C o l i c e et al,  of A N F in hypoxemia-induced  1 9 9 1 ; K o l l e r et al,  al,  hypoxia-  diuresis  was  1990; S h i m i z u and N a k a m u r a , 1986).  also It h a s  b e e n r e p o r t e d that a c u t e h y p o x e m i a is a p o t e n t s t i m u l u s f o r atrial n a t r i u r e t i c f a c t o r ( A N F ) r e l e a s e ( d u S o u i c h et al,  1 9 8 7 ; R a m i r e z et al,  i s o l a t e d r a t a n d r a b b i t h e a r t s ( B a e r t s c h i et al,  1 9 8 8 ; L a w r e n c e et al,  1990) in humans,  1 9 8 6 ) a n d i n r a b b i t s ( B a e r t s c h i et al,  1988).  A n i m m e d i a t e A N F release f r o m the heart at the o n s e t o f h y p o x i a w a s r e p o r t e d ( B a e r t s c h i al,  1986), w h i c h coincides with the transient diuresis in acute h y p o x e m i a .  A  1987).  H o w e v e r the increased p l a s m a A N F levels during h y p o x e m i a are m o r e  et  likely  a s s o c i a t e d w i t h r i g h t a t r i a l d i s t e n t i o n s e c o n d a r y t o p u l m o n a r y h y p e r t e n s i o n ( O u et al, a n d h y p e r v e n t i l a t i o n ( C o l i c e et al,  et  correlation  between p l a s m a A N F concentration a n d arterial b l o o d pressure was suggested (du S o u i c h al,  in  1986)  1 9 9 1 ) , s i n c e the d i s t e n t i o n o f c a r d i a c atria is the p r i m a r y  p h y s i o l o g i c a l s t i m u l u s f o r t h e r e l e a s e a n d s y n t h e s i s o f A N F ( L a n g e et al,  1987).  There was a significant decrease in urine osmolality during hypoxemia, unlike  the  a c u t e m o d e r a t e h y p o x e m i a - i n d u c e d d i u r e s i s i n t h e s t u d i e s o f W a l k e r ( 1 9 8 2 ) a n d C o l i c e et  al.  (1991) which showed no changes in urine osmolality during hypoxemia.  T h e average  o s m o l a l excretion rate, the p r o d u c t o f urine f l o w a n d urine osmolality, w a s also  renal  decreased  98  significantly during hypoxemia.  T h e osmolal excretion parameter was used in the study  and  c o u l d b e r e g a r d e d as a r o u g h indicator o f the quantity o f salt a n d other soluble  compounds  excreted in urine in a given time, since urine electrolyte analysis could not be  performed  satisfactorily in all the urine samples in the present study.  U r i n e electrolyte analysis o f the  t w o a n i m a l s in the e x p e r i m e n t a l g r o u p w a s p e r f o r m e d , a n d there a p p e a r to b e n o changes in electrolyte  excretion  (i.e.  no  natriuresis or kaliuresis)  c o n t r a s t t o t h e f i n d i n g o f W a l k e r ( 1 9 8 2 ) a n d C o l i c e et relationship between urine osmolality and urine flow. the coefficient  o f d e t e r m i n a t i o n (r) o f 0.80,  accompanied by lowered urine osmolality.  al.  (1991).  significant  during hypoxemia  in  Figure 7 shows  the  There was a negative correlation with  w h i c h m e a n s the increased urine flow  was  T h e s e relationships suggest that the  increased  u r i n e f l o w d u r i n g h y p o x e m i a is n o t l i k e l y the result o f the i n c r e a s e d u r i n e v o l u m e  excretion  s e c o n d a r y to increased electrolyte excretion b y the kidney, as s h o w n . i n the p r e v i o u s ( W a l k e r , 1 9 8 2 ; C o l i c e et al,  studies  1991). Therefore, the diuresis in the current study m a y be  to e n h a n c e d water excretion independent o f urinary electrolyte excretion.  Since  was i n d u c e d during the continuous M C P infusion, the presence of M C P c o u l d be  due  hypoxemia associated  with the low urine osmolality.  S i n c e b o t h the administration o f M C P a n d acute h y p o x e m i a are k n o w n to affect renal h a e m o d y n a m i c s a n d urine flow in a n o p p o s i n g direction, the present study w h i c h these two factors raises an interesting perspective. f l o w a n d u r i n e f l o w i n h u m a n s ( I s r a e l et al,  combines  M C P is r e p o r t e d to d e c r e a s e r e n a l b l o o d  1 9 8 6 ) . N o r b i a t o et al.  (1986) reported that M C P  decreased free water excretion in m a n b y increased vasopressin secretion.  During  m o d e r a t e h y p o x e m i a similar to the present study ( o x y g e n saturation = 7 5 - 8 0 % ) , the aldosterone  concentration  (either  normal  or  elevated  by  M C P  administration)  acute plasma was  99  s i g n i f i c a n t l y r e d u c e d a n d A N F l e v e l s w e r e s l i g h t l y i n c r e a s e d i n h u m a n s ( V o n m o o s et 1 9 9 0 ; L a w r e n c e et al,  al,  1990). T h e hypoxemia-induced increase in plasma A N F levels caused  a p r o m i n e n t s u p p r e s s i o n o f p l a s m a a l d o s t e r o n e w i t h o u t n a t r i u r e s i s ( L a w r e n c e et al,  1990;  L a w r e n c e a n d S h e n k e r , 1 9 9 1 ) . I n a d d i t i o n , a l o w - d o s e A N F i n f u s i o n , w h i c h is a s i m i l a r l e v e l to a m o d e r a t e (Shenker  et  hypoxemia-induced  al,  1992).  These  h y p o x e m i a in the present study. l e v e l s (e.g.  A N F increase,  results  may  has  little o r n o  explain the  absence  effect o n  natriuresis  of natriuresis  Unfortunately, the lack o f information o f p l a s m a  during hormone  A N F a n d aldosterone ) d o e s not allow us to address this subject in the  present  study, and thus further studies m a y be required.  Statistical and  4.3.  Practical Aspects of Experiment Design  4.3.1. Design of experimental protocol The infusion experimental protocol with 3 phases (pre-hypoxemic, hypoxemic p o s t - h y p o x e m i c periods) in the study w a s d e v e l o p e d in order to address s o m e o f c o m m o n l y associated with studying pharmacokinetics during hypoxemia.  Most  and  problems previous  p h a r m a c o k i n e t i c studies d u r i n g h y p o x e m i a h a v e b e e n b a s e d o n the i.v. b o l u s d e s i g n  with  d r u g administration to the separate h y p o x e m i c a n d control ( n o r m o x e m i c ) groups [Design ( d u S o u i c h et al,  1 9 8 5 a, b).  In s o m e studies, paired experiments were p e r f o r m e d o n  s a m e s u b j e c t s [ D e s i g n J J ] ( S a u n i e r et  al,  1987;  C l o z e l et  al,  1981).  Since there  I] the are  considerable individual variations in the physiological a n d p h a r m a c o k i n e t i c responses  to  h y p o x e m i a ( d u S o u i c h et al,  of  1 9 7 8 ; T a b u r e t et al,  1990), conducting a comparative study  p h a r m a c o k i n e t i c s o n s e p a r a t e h y p o x e m i c a n d c o n t r o l g r o u p s [ D e s i g n I] p o s e s s o m e problems.  potential  T h i s is p a r t i c u l a r l y t r u e w h e n the s a m p l e s i z e is s m a l l a n d t h e v a r i a n c e i n  the  100  g r o u p is large, as h a s b e e n e n c o u n t e r e d i n p r e v i o u s studies.  A larger sample size w o u l d  be  required to test a significant difference b e t w e e n t w o g r o u p s w i t h a higher p o p u l a t i o n variance and small m i n i m u m detectable difference between population means (Zar, 1984).  T h u s , this  design m a y be suitable for conducting experiments in a large population of small  animal  species s u c h as the rat a n d rabbit w h e r e m i n i m a l surgical preparations are i n v o l v e d . design  [Design  I] w a s  rejected  from  consideration  due  to  the  This  practical limitation  of  conducting a large n u m b e r of complicated surgeries and pharmacokinetic experiments  on  larger animals like sheep.  T w o experimental protocols w e r e initially considered. b o l u s s t u d y [ D e s i g n II],  O n e p r o t o c o l w a s a p a i r e d i.v.  w h e r e t w o separate i.v. b o l u s d o s e s o f M C P w o u l d b e g i v e n  on  s e p a r a t e d a y s u n d e r h y p o x e m i c a n d n o r m o x e m i c c o n d i t i o n s , s i m i l a r t o S a u n i e r et al.  (1987).  T h e other was continuous M C P infusion with normoxemic control and hypoxemic  periods  [ D e s i g n H I ] . T h e i.v. b o l u s m e t h o d offers c o m p l i m e n t a r y i n f o r m a t i o n to that o b t a i n e d the present distribution.  i.v. i n f u s i o n m e t h o d ,  i.e.  the elimination rate constant  a n d the  volume  with of  H o w e v e r , the p a i r e d i.v. b o l u s m e t h o d p o s e s several practical p r o b l e m s i n the  pharmacokinetic study of h y p o x e m i a . T h e present study requires the quantitative c o m p a r i s o n o f p h a r m a c o k i n e t i c parameters b e t w e e n h y p o x e m i c a n d n o r m o x e m i c conditions, in order to evaluate the impact of h y p o x e m i a o n drug disposition.  A c e r t a i n t i m e d e l a y (i.e.  at least  2-3  days) b e t w e e n p a i r e d experiments w o u l d b e required to ensure total elimination o f d r u g  and  related metabolites f r o m the b o d y b e t w e e n successive experiments  a n d all the  except inspired gas content should be identical for both experiments.  T h e i.v.  conditions infusion  m e t h o d [ D e s i g n III] h a s e l i m i n a t e d t h e s e r e q u i r e m e n t s o f a p a i r e d e x p e r i m e n t a n d r e d u c e s time for experiments.  Physiological and pharmacokinetic changes induced by  the  hypoxemia  101 could be demonstrated within a single experiment.  I n a d d i t i o n , it w o u l d b e p o s s i b l e  to  observe d y n a m i c physiological a n d p h a r m a c o k i n e t i c c h a n g e s d u r i n g a n d after h y p o x e m i a .  A n o t h e r reason for developing the infusion m e t h o d was  to establish  intra-subject  c o n t r o l s , s i n c e it h a s b e e n r e p o r t e d t h a t i n d i v i d u a l t o l e r a n c e t o w a r d h y p o x e m i c s t r e s s a n d t h e d e g r e e o f p h a r m a c o k i n e t i c c h a n g e s a r e h i g h l y v a r i a b l e ( d u S o u i c h et al, 1990).  T h e use  1 9 7 8 ; T a b u r e t et  of an intra-subject control m e t h o d in the present protocol has  al,  greatly  i m p r o v e d the statistical analysis o f p h a r m a c o k i n e t i c a n d physiological data. F o r e x a m p l e ,  the  d a t a set o f the p l a s m a M C P steady-state c o n c e n t r a t i o n f r o m p r e - h y p o x e m i c , h y p o x e m i c  and  p o s t - h y p o x e m i c periods f r o m a single e x p e r i m e n t w a s u s e d as the p o p u l a t i o n a n d the values for each time period were c o m p a r e d using analysis of variance (ANOVA). in other methods  mean  Whereas,  [ D e s i g n I a n d JJ], it w o u l d b e n e c e s s a r y to c o m p a r e t h e h y p o x e m i c  and  c o n t r o l g r o u p v a l u e s o f p h a r m a c o k i n e t i c p a r a m e t e r s s u c h as rate o f e l i m i n a t i o n , w h i c h  have  considerably higher variance. physiological parameters  This process of c o m p a r i s o n of the pharmacokinetic  f r o m the h y p o x e m i c  and normoxemic  periods  animal, instead o f the h y p o x e m i c a n d control groups, greatly decreases a  within a [the  and single  population  variance] a n d thus resulting in a h i g h e r § v a l u e [the d e t e r m i n i n g p o w e r ] o f the test i n a g i v e n n u m b e r o f e x p e r i m e n t s (Zar, 1984). T h i s m e a n s that the p o w e r o f the test in a g i v e n o f the experiment subjects was i m p r o v e d in the present study.  Therefore, the  number  intra-subject  c o m p a r i s o n s h o u l d provide a m o r e reliable interpretation o f the effect o f h y p o x e m i a a n d also reduce the n u m b e r of animals required for the study.  In addition to the intra-subject control periods  (Scheme  JJI), the  control  experiments w e r e c o n d u c t e d in four o f the ewes. In the control group, the infusion study  group was  102  c o n d u c t e d a c c o r d i n g to the s a m e p r o t o c o l as i n the e x p e r i m e n t a l g r o u p ( S c h e m e I V ) , e x c e p t for the nitrogen infusion-induced h y p o x e m i a . periods  between  experimental  statistically analysed.  and  control  T h e data sets c o r r e s p o n d i n g to the groups  were  qualitatively  same  compared  and  F o r example, a parallel comparison of changes in M C P steady-state  concentrations b e t w e e n the e x p e r i m e n t a l a n d control g r o u p s enables us to c o n f i r m that  any  c h a n g e s o b s e r v e d are not a n artifact o f the experiment, s u c h as m i g h t h a v e arisen d u e to the prolonged infusion. group eliminates  T h u s , the c o m p a r i s o n o f data f r o m the experimental with the  a n y possibility that the results w e r e affected b y factors not related  h y p o x e m i a , s u c h as obscure time-dependent  in s o m e parameters w i t h higher variation, these statistical analysis  groups,  could  a d e q u a t e l y p e r f o r m e d d u e t o l o w 0 v a l u e [ t h e d e t e r m i n i n g p o w e r o f t h e t e s t ] (i.e. variance and low  sample  number).  to  effects.  Statistical c o m p a r i s o n s u s i n g Student's t-test w e r e p e r f o r m e d b e t w e e n t w o however,  control  T h e s e c o m p a r i s o n s , in a sense, are similar to  not high the  c o m p a r i s o n in the D e s i g n I type experiment, w h i c h c o m p a r e s the data f r o m t w o data sets r a n d o m l y chosen f r o m the two populations. Therefore, the control g r o u p in the present  study  provides the additional c o m p a r i s o n with the physiological a n d p h a r m a c o k i n e t i c data gathered during the h y p o x e m i c period in the experiment group.  103  4.3.2. Statistical and practical considerations for determining the steady-state drug concentration. P r e v i o u s s t u d i e s o n m e t o c l o p r a m i d e p h a r m a c o k i n e t i c s i n s h e e p ( R i g g s et al,  1988:  1990) reported that the half-life o f M C P in non-pregnant sheep w a s about 50 m i n a n d that a s t e a d y - s t a t e c o n c e n t r a t i o n c o u l d b e a c h i e v e d w i t h i n 1h o u r u s i n g a n a p p r o p r i a t e l o a d i n g  dose.  T h e o r e t i c a l l y , it w o u l d r e q u i r e 4 - 5 h o u r s to r e a c h a s t e a d y - s t a t e  dose.  without a loading  H o w e v e r , the time to steady-state c a n b e greatly r e d u c e d w i t h a n appropriate l o a d i n g  dose  (see S e c t i o n 4.3.3.). Since the protocol used in the present study requires the determination of steady-state  drug concentrations within a single experiment (one for each  three  pre-hypoxemic,  h y p o x e m i c a n d p o s t - h y p o x e m i c periods in the experimental g r o u p a n d the equivalent  periods  i n t h e c o n t r o l g r o u p ) , it w a s e s s e n t i a l to set u p a n a p p r o p r i a t e a s s e s s m e n t p r o c e d u r e f o r t h e steady-state  drug concentrations.  criteria were used.  F o r the assessment o f steady-state,  the three  following  T h e s e criteria w e r e designed so that the steady-state concentration  be experimentally d e t e r m i n e d in a practical a n d statistically s o u n d m a n n e r .  could  I n a d d i t i o n , it  w a s d e s i g n e d to m i n i m i z e bias d u r i n g the data p r o c e s s i n g p r o c e d u r e : 1.  the p l a s m a M C P concentration versus time plot was visually inspected. i n the pre-hypoxemic, h y p o x e m i c a n d post-hypoxemic plasma statistical  2.  concentrations  were  remained  relatively  Plateau  sections  p e r i o d s (i.e. t h e s e c t i o n  constant)  were  chosen  for  where further  analysis.  the data sets f r o m these plateau sections w e r e analysed for the coefficient o f variation ( C V ) . T h e m a x i m u m C V limit o f 1 0 % w a s u s e d as criteria for the deviation f r o m steady-state concentration.  T h e d a t a set f r o m the p r e - h y p o x e m i c p e r i o d t e n d s to  mean exhibit  104 higher C V values d u e to the shorter time a l l o w e d to attain steady-state a n d the n u m b e r of data points gathered. in two of the animals.  smaller  C o n s e q u e n t l y , a C V value o f 1 5 % w a s u s e d as the limit  During the data processing procedure, the e x t r e m e outliers  e x t r e m e t h a n 2 S D f r o m the m e a n ) f r o m e a c h d a t a set w e r e e l i m i n a t e d .  (more  In this case  the  terminal data points f r o m the later time p e r i o d w e r e g i v e n preference to r e m a i n in the  set  o v e r e a r l i e r d a t a p o i n t s , s i n c e the s t e a d y - s t a t e d r u g c o n c e n t r a t i o n is l i k e l y a t t a i n e d d u r i n g the later time period.  In practice, any extreme outlier(s) w a s carefully e x a m i n e d  possible error(s) a n d in s o m e cases re-assayed.  for  After this step, a terminal data point f r o m  the earliest t i m e p e r i o d w a s r e m o v e d u n t i l the C V v a l u e is e q u a l o r s m a l l e r t h a n the limit, t h e s l o p e (P)  of the regression line was also examined.  T h e slope o f the regression  line  c o m p u t e d f r o m the sample data expressed quantitatively the straight-line d e p e n d e n c y Y o n X (i.e.  of  p l a s m a drug concentration o n time) in the s a m p l e (Zar, 1984). If the slope o f  t h e r e g r e s s i o n l i n e o f t h e p l a t e a u s e c t i o n is s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o , t h e n it w o u l d suggest that p l a s m a drug concentration has a trend o f either a n increase or decrease t h e g i v e n t i m e (i.e.  steady-state was not reached).  over  Therefore, the slope w a s analysed  to  d e t e r m i n e d w h e t h e r it w a s d i f f e r e n t f r o m z e r o u s i n g a n a l y s i s o f v a r i a n c e ( A N O V A )  and  t-test for linear regression.  In statistical terms, the linear r e g r e s s i o n o f the d a t a set  was  p e r f o r m e d w i t h the null-hypothesis ( H o : P = 0) that the slope o f the linear regression  line  : P ^ 0. T h e a n a l y s i s o f v a r i a n c e ( A N O V A )  for  i s z e r o a n d t h e a l t e r n a t e h y p o t h e s i s , HA  linear regression a n d Student's t-test w e r e u s e d w i t h the level o f significance, a =  0.05.  T h i s p r o c e d u r e w a s c h o s e n to e n s u r e that there is n o t e n d e n c y f o r a g r a d u a l i n c r e a s e decrease in the drug concentrations over time. w o u l d m e a n that steady-state w a s not reached.  T h e rejection of the null-hypothesis  or (Ho)  105  From  this  steady-state  assessment,  in most  of  animals,  the  M C P  steady-state  concentration w a s f o u n d to be r e a c h e d b e t w e e n 30 m i n a n d 75 m i n w i t h a 15 m g dose in the p r e - h y p o x e m i c period. steady-state  O n e animals (ewe  no. 1154)  within 2 hours of infusion with a loading dose.  loading  w a s f o u n d not to  reach  D a t a f r o m this a n i m a l  excluded f r o m the subsequent pharmacokinetic data analysis, since the h y p o x e m i c a n d  was post-  h y p o x e m i c steady-state concentration c o u l d not be c o m p a r e d to the p r e - h y p o x e m i c  steady-  state concentration. A f t e r onset o f h y p o x e m i a , another steady-state ( h y p o x e m i c ) w a s  reached  b e t w e e n 2 to 4 hours f r o m the onset o f h y p o x e m i a .  T h e post-hypoxemic steady-state  r e a c h e d b e t w e e n 2 to 3 hours after stopping nitrogen infusion. T h i s apparent shorter time  was to  steady-state d u r i n g the p o s t - h y p o x e m i c p e r i o d , as c o m p a r e d to that o f the h y p o x e m i c p e r i o d , is l i k e l y d u e to the fact that the half-life o f M C P d u r i n g the p o s t - h y p o x e m i c p e r i o d is s h o r t e r ( = 2 0 % ) than that o f the h y p o x e m i c period.  106  4.3.3.  Theoretical aspects of infusion with loading dose.  T h e s t e a d y - s t a t e d r u g c o n c e n t r a t i o n ( C p ° ° i f ) a f t e r ac o n t i n u o u s d r u g i n f u s i o n is  the  p o i n t w h e r e the rate o f i n p u t o f d r u g ( i n f u s i o n rate) is e q u a l to the rate o f d r u g l e a v i n g  the  n  b o d y (elimination rate).  S i n c e the i n f u s i o n rate is c o n s t a n t , t h e t i m e to r e a c h s t e a d y - s t a t e is  primarily dependent u p o n the elimination rate constant.  For the present study, the o n e - c o m p a r t m e n t o p e n m o d e l a n d the  two-compartment  o p e n m o d e l w i t h d r u g elimination f r o m the central or "well p e r f u s e d " c o m p a r t m e n t (see schematic d i a g r a m s o n the next page), w e r e used for c o m p u t e r - a i d e d simulation,  the  theoretical  interpretation a n d calculation o f p h a r m a c o k i n e t i c parameters. O t h e r c o n c e p t s p r o v i d e d utility in data interpretation, including physiological modeling, m o m e n t compartmental clearance concept. al.  analysis  a n d the  non-  U s i n g p h a r m a c o k i n e t i c p a r a m e t e r s d e t e r m i n e d b y R i g g s et  ( 1 9 8 9 ) i n n o n - p r e g n a n t sheep, the c o m p u t e r a i d e d s i m u l a t i o n s o f i.v. i n f u s i o n b a s e d o n the  o n e - a n d t w o - c o m p a r t m e n t m o d e l s w e r e p e r f o r m e d a s s h o w n i n t h e F i g u r e 1 1 . It w o u l d  take  a p p r o x i m a t e l y 3.4 h o u r s to r e a c h asteady-state (95%)  one-  c o m p a r t m e n t m o d e l without aloading dose.  a n d 5.2 hours for 9 9 % f r o m the  In the t w o - c o m p a r t m e n t m o d e l , the time  to  r e a c h steady-state w a s slightly longer (4.0 h o u r for 9 5 % a n d 6.4 h o u r for 9 9 % ) than that  of  the o n e - c o m p a r t m e n t model.  T h e l o n g e r t i m e to r e a c h steady-state in the  two-compartment  is l i k e l y to b e a c o n s e q u e n c e s o f the d e l a y e d t r a n s f e r o f d r u g b e t w e e n the c e n t r a l  and  peripheral  one-  compartments,  compartment model.  when  compared  to  immediate  drug  distribution in the  107  Scheme V  Diagrams of the compartment models used for computer simulation.  One-compartment Open Model  ko (+ LD)  Two-compartment Open Model with central drug elimination  ko  12  Central Compartment  (+ LD)  Peripheral Compartment  21 Vc  Vr  10  Nowhere ko represents the infusion rate constant of M C P , LD is the i.v. bolus loading dose, V d is the apparent volume of distribution in one-compartment model, V c and Vr is apparent volume of the central and peripheral compartment, respectively, Ke is the apparent first order M C P elimination rate constant, k and k i are the apparent first order inter-compartmental distribution rate constants for M C P and k is the apparent M C P elimination rate constant from the central compartment. 12  2  10  108  70  -r  60 - -  2-ccrrp - - - 1-ocrrp Ia\er (95%) upper (105%) 0  1 1 i i i i i I I 1 1 i i i i i i i i i i i i i 1 1 1 i 1 1 i i i i i i 1 1 1 i i i i 1 1 1 i i 1 1 1 1 i i i i i i i i i i i I I 1 1 1 i i i i i i i i i 1 1 O  T  —  C  N  C  O  T  L  £  >  <  O  I  i i i 11 i i i i i i i i i i i 111 i i i ^  C  O  O  O  T ime (hour)  F i g u r e 11.  S i m u l a t i o n o f C p after s i m p l e i.v. i n f u s i o n w i t h o u t p h a r m a c o k i n e t i c p a r a m e t e r s f r o m R i g g s et al. ( 1 9 8 9 )  loading dose  using  - NoLD - 100% O0.6 Q .  o  0.4  0.2  LD  - 90%  LD  - 80%  LD  -  110%  LD  -  120%  LD  — Lower (95%)  4- .  — Upper (105%)  i u  11111111  I I I I I I I I I I  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  CSI  T imeAidf-life  F i g u r e 12.  S i m u l a t i o n o f C p after i.v. i n f u s i o n w i t h l o a d i n g d o s e model)  (one-compartment  109  tS-30  T ime  F i g u r e 13.  (hour)  S i m u l a t i o n o f C p after i.v. i n f u s i o n w i t h l o a d i n g d o s e m o d e l ) u s i n g p h a r m a c o k i n e t i c p a r a m e t e r s f r o m R i g g s et al.  (two-compartment (1989)  In the present study, a loading dose w a s c o m b i n e d with the infusion to reduce required  to  reach  the  steady-state  drug  concentration.  Therefore,  further  time  computer  simulations o f the infusion with a loading dose were p e r f o r m e d using the one- a n d  two-  c o m p a r t m e n t m o d e l as s h o w n in F i g u r e s 12 a n d 13. A n ideal (optimal) l o a d i n g d o s e w i t h the o n e - c o m p a r t m e n t m o d e l i s LD and  maintenance  of  = C  ss  a steady-state  * Vd, as  w h i c h theoretically results in instantaneous a r r i v a l shown  in Figure  12.  However,  in the  actual  e x p e r i m e n t a l a n d t h e r a p e u t i c e n v i r o n m e n t , it is n e i t h e r p r a c t i c a l n o r p o s s i b l e t o c a l c u l a t e  and  adjust a loading dose for each individual subject, thus the loading dose calculated f r o m  the  m e a n values of C  this  ss  a n d Vd w o u l d b e u s e d f o r a l l t h e e x p e r i m e n t s u b j e c t s .  Therefore,  loading dose r e g i m e n c o u l d result in either over- or under-estimation o f the optimal loading dose.  F i g u r e 12 s h o w s a simulated plot of p l a s m a drug concentration (in terms of  plasma  110  steady-state d r u g concentration) versus time (in terms o f half-life), w h i c h b a s e d o n the  one-  c o m p a r t m e n t m o d e l with first-order kinetics. A loading dose of 9 0 % or 1 1 0 % o f the optimal d o s e will take a p p r o x i m a t e l y 1 half-life to reach the range o f steady-state. A l o a d i n g d o s e 8 0 % or 1 2 0 % of the optimal dose will take approximately 2 half-lives.  Therefore, even  s o m e deviation f r o m the o p t i m a l l o a d i n g dose, the time to r e a c h steady-state will b e  of  with greatly  reduced with an appropriate loading dose.  I n the t w o - c o m p a r t m e n t m o d e l , the u s e o f a l o a d i n g d o s e is m o r e c o m p l i c a t e d d u e the distribution phase to the peripheral c o m p a r t m e n t .  U n l i k e the o n e - c o m p a r t m e n t  two loading dose protocols are possible for drugs best described b y a p h a r m a c o k i n e t i c c h a r a c t e r i s t i c s . A l o a d i n g d o s e o f LD  = C  ss  to  model,  two-compartment  * V will result in an c  immediate  p l a s m a d r u g concentration equal to the steady-state concentration, h o w e v e r the p l a s m a d r u g concentration will be decreased b e l o w the steady-state concentration a n d gradually increased as s h o w n in F i g u r e 13.  A n a l t e r n a t i v e l o a d i n g d o s e o f LD  = C  ss  * V  ss  will initially give  higher p l a s m a concentration than the steady-state concentration, but very rapidly decreased the steady-state concentration.  T h e t i m e to steady-state u s i n g the s e c o n d r e g i m e n is  a to  usually  m u c h shorter than the former. Therefore, a loading dose o f 15 m g o f M C P w a s u s e d in the study as suggested b y R i g g s (1989), w h i c h represents the l o a d i n g d o s e close to the  second  protocol. T h e use o f a l o a d i n g d o s e also greatly r e d u c e d the time to attain steady-state in the two compartment model.  T h i s m a y be a better approximation for M C P , since the  p h a r m a c o k i n e t i c s f o l l o w i n g the i.v. b o l u s d o s e e x h i b i t e d t w o - c o m p a r t m e n t ( R i g g s et al,  1988).  W i t h a n o p t i m a l l o a d i n g d o s e o f LD  minutes to attain the range o f steady-state  = C  (95 - 1 0 5 % o f Css).  ss  * V, ss  M C P  characteristics  it w i l l t a k e a b o u t  A loading dose of 80%  1 2 0 % o f t h e o p t i m a l d o s e w i l l t a k e a p p r o x i m a t e l y 1.5 h o u r to r e a c h t h e r a n g e .  30 and  Ill  T h e r e f o r e , these c o m p u t e r - a i d e d simulations theoretically d e m o n s t r a t e that the t i m e to the steady-state c a n be greatly reduced b y an appropriate loading dose. f i n d i n g o f R i g g s et al.  This confirms  (1989) that steady-state conditions for M C P w e r e achieved  a p p r o x i m a t e l y 2half-lives using a l o a d i n g d o s e o f 15 m g .  the  within  In the present study, M C P steady-  state c o n c e n t r a t i o n w a s a c h i e v e d b e t w e e n 0.5 to 1.25 h o u r s after initiation o f the i n f u s i o n a n d a d m i n i s t r a t i o n o f a l o a d i n g d o s e , w h i c h is i n g o o d a g r e e m e n t w i t h the p r e s e n t  theoretical  derivation and computer-aided simulation.  4.4.  Metoclopramide Pharmacokinetics Following the state and  4.4.1.  Lv.  Infusion to Steady-  Induction of Hypoxemia  Steady-state plasma MCP concentration and total body clearance of MCP during normoxemia and hypoxemia  The  present  study  shows  that  acute  moderate  hypoxemia  affects  metoclopramide pharmacokinetics in chronically instrumented conscious sheep.  T h e steady-  state p l a s m a M C P concentration significantly increased a n d total b o d y clearance during acute moderate  hypoxemia.  During  the post-hypoxemic  period, the  plasma  decreased steady-state  p l a s m a M C P c o n c e n t r a t i o n d e c r e a s e d to al e v e l s i m i l a r to that o f t h e p r e - h y p o x e m i c p e r i o d . T h e m e a n p l a s m a M C P concentration during the post-hypoxemic period was slightly  higher  than the m e a n p l a s m a M C P concentration during the p r e - h y p o x e m i c period, but w a s significantly different (p > 0.05).  Therefore, these results suggested that acute  moderate  h y p o x e m i a r e d u c e s t o t a l b o d y c l e a r a n c e o f M C P (i.e. r e s u l t i n g i n a h i g h e r p l a s m a steady-state concentration).  not  M C P  112  A reduction in total b o d y d r u g clearance d u r i n g acute h y p o x e m i a w a s o b s e r v e d w i t h a n u m b e r o f d r u g s s u c h a s t h e o p h y l l i n e ( S a u n i e r et al,  1 9 8 7 ) , a m i n o g l y c o s i d e s ( M i r h i j et  1 9 7 8 ) , p h e n y t o i n ( B a b i n i a n d d u S o u i c h , 1 9 8 6 ) a n d p r o p o f o l ( A u d i b e r t et al, s u c h as hexobarbital a n d antipyrine, w h i c h u n d e r g o  oxidative  1992).  al,  Drugs  biotransformation by  the  hepatic m i x e d oxygenases, are directly affected by reduced oxygen tension (Jones,  1981).  Conjugation  major  reactions  such  as  glucuronidation  and  sulphation,  which  are  the  elimination pathway of m a n y drugs, are also affected b y l o w o x y g e n tension ( A w a n d Jones, 1981), but the f o r m a t i o n o f these conjugates d o e s not appear to b e a significant p r o p o r t i o n o f MCP  elimination in sheep.  In addition, acute h y p o x e m i a decreases the activity o f  i s o e n z y m e s o f t h e c y t o c h r o m e P - 4 5 0 ( S r i v a s t a v a et al, al,  1 9 8 0 ; J o n e s et al,  several  1989; d u Souich  et  1990). T h e r e are also observations that m o d e r a t e h y p o x i a c o u l d p r o m o t e the f o r m a t i o n o f  oxygen  free-radicals (Proulx and d u Souich, 1990;  B r a s s et  al,  1991)  that are able  d i m i n i s h the activity o f certain i s o z y m e s o f the c y t o c h r o m e P - 4 5 0 (Proulx a n d d u 1990).  Souich,  T h e s e reports suggest that acute h y p o x e m i a reduces d r u g elimination processes,  g e n e r a l , w h i c h is i n a g r e e m e n t w i t h the f i n d i n g s o f the p r e s e n t  In s o m e  c a s e s , t h e r e is a n i n c r e a s e  observed  1 9 8 5 ) a n d d i l t i a z e m ( d u S o u i c h et al,  in the apparent total b o d y  clearance  h y p o x e m i a w i t h s o m e d r u g s s u c h a s s u l p h a m e t h a z i n e ( d u S o u i c h et al, ( d u S o u i c h et al,  1993).  during  acute  1984) and  digoxin  1985a; 1985b). H o w e v e r , this increase in total b o d y clearance w a s  mainly  d u e t o a n i n c r e a s e i n t h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n (i.e.  increased tissue distribution and  c h a n g e s in p l a s m a protein binding), rather than to a n actual increase in d r u g elimination the body.  in  study.  Alternatively, no change in pharmacokinetics during acute hypoxemia was w i t h d r u g s s u c h a s f u r o s e m i d e ( d u S o u i c h et al,  to  from  113  N o studies o n the e n z y m a t i c p a t h w a y for the oxidative N - d e e t h y l a t i o n o f M C P to mdMCP  and ddMCP  have been reported, h o w e v e r the N-deethylation o f a structurally similar  c o m p o u n d , lidocaine, appears to be m e t a b o l i z e d b y the hepatic P - 4 5 0 e n z y m e s y s t e m ( S u z u k i et al,  1 9 8 4 ; B a r g e t z i et al,  1989) a n d there are m a n y reports that N - d e a k y l a t i o n  reactions  s u c h as demethylation, deethylation a n d depropylation are m e d i a t e d b y the hepatic  P-450  e n z y m e system (Testa and Jenner, 1976).  major  m e t a b o l i c s i t e f o r M C P ( K a p i l et al,  I n a d d i t i o n , the l i v e r is t h o u g h t to b e t h e  1984; D e s m o n d and W a t s o n , 1986).  T h e r e f o r e it is  p r o b a b l e that the N - d e e t h y l a t i o n o f M C P is a l s o m e d i a t e d b y the h e p a t i c P - 4 5 0 system. K  m  enzyme  In vitro s t u d i e s h a v e d e m o n s t r a t e d t h a t a l o w p a r t i a l p r e s s u r e o f o x y g e n r e d u c e d t h e  v a l u e o f t h e d e m e t h y l a t i o n o f e t h y l - m o r p h i n e ( H o l t z m a n et al,  1 9 8 2 ) a n d o f t h e h y d r o x y l a t i o n o f p h e n y t o i n ( T s u r u et al,  1 9 8 3 ; E r i c k s o n et  1982), indicating that  o x y g e n pressure m a y be essential for this route o f biotransformation.  al,  adequate  In conscious  rabbits,  h y p o x e m i a r e d u c e d t h e d e m e t h y l a t i o n a n d / o r t h e h y d r o x y l a t i o n o f t h e o p h y l l i n e ( L e t a r t e et 1984)  a n d t h e h y d r o x y l a t i o n o f p h e n y t o i n ( d u S o u i c h et  al,  1986).  In addition,  al, acute  h y p o x e m i a appears to decrease the activity o f the c y t o c h r o m e P - 4 5 0 in rat l u n g a n d ( S r i v a s t a v a et  al,  1980).  These  studies suggested that h y p o x e m i a  may  affect  liver several  c y t o c h r o m e P - 4 5 0 isozymes, a n d thus m a y explain, in part, the accumulation in p l a s m a  of  MCP  of  and mdMCP  due  to r e d u c e d m e t a b o l i c  hypoxemia on plasma ddMCP concentration of the  elimination o f the drug.  T h e effect  concentration w a s not determined, d u e to the l o w  plasma  metabolite.  Alternatively, changes in hepatic blood flow during acute  hypoxemia-hypocapnia  c o u l d explain the reduction o f M C P clearance. A m o d e r a t e h y p o x e m i a alone appears to exert o n l y m i n i m a l e f f e c t i n t o t a l h e p a t i c b l o o d f l o w ( L a s e n et al,  1 9 7 6 ; H u g h e s et al,  1979;  du  114 S o u i c h et al, 1 9 9 2 ) .  H o w e v e r , a h y p o x e m i a in combination with either hypocapnia  or  h y p e r c a p n i a m a y a f f e c t t o t a l h e p a t i c b l o o d f l o w m o r e t h a n h y p o x e m i a a l o n e ( H u g h e s et al, 1979;  Mathie and Blumgart,  1983).  Since  hepatic  elimination of M C P i s essentially  m o d u l a t e d b y t h e b l o o d f l o w t o t h e l i v e r [ f l o w - l i m i t e d ] ( B a t e m a n et al, 1 9 8 0 ) , t h e e f f e c t o f acute h y p o x e m i a - h y p o c a p n i a o n M C P clearance m a y be explained b y the reduction in total hepatic blood flow.  4.4.2. Plasma mdMCP concentration during normoxemia and hypoxemia  D e a l k y l a t i o n o f secondary a n d tertiary a m i n e g r o u p s to yield p r i m a r y a n d a m i n e g r o u p s , r e s p e c t i v e l y , is o n e o f the m o s t i m p o r t a n t a n d m o s t f r e q u e n t l y reactions in drug metabolism.  secondary encountered  Biological N-dealkylation occurs without apparent changes in  the state o f o x i d a t i o n o f the n i t r o g e n a t o m , b u t the r e m o v e d a l k y l g r o u p is o x i d i s e d and Jenner, 1976).  In the present study, considerable p l a s m a mdMCP  concentrations  average =30-50% of plasma M C P concentration) were found. Plasma m d M C P  (on  concentration  also a p p e a r e d to r e a c h a n apparent steady-state in b o t h e x p e r i m e n t a l a n d control H o w e v e r , it is n o t p o s s i b l e t o c o n f i r m t h a t s t e a d y - s t a t e , i n a c o n v e n t i o n a l s e n s e , w a s i n the study, since the input rate constant o f m d M C P  (Testa  groups. achieved  {i.e. a p r o d u c t t o i n f u s i o n r a t e o f M C P  a n d kf(MCP->mdMCP)) c o u l d n o t b e e v a l u a t e d i n t h e c u r r e n t i n f u s i o n d e s i g n .  Plasma mdMCP  concentration was significantly higher in the experimental group  c o m p a r e d to the control group.  T h e increased mdMCP  concentration in the  g r o u p appears to b e related to the induction o f h y p o x e m i a , h o w e v e r , m d M C P  experimental concentration  did not decrease during the post-hypoxemic period. T h e interpretation of metabolite mainly mdMCP  as  i n this s t u d y , is m o r e c o m p l i c a t e d t h a n the p a r e n t d r u g , s i n c e  kinetics, plasma  115  mdMCP  concentrations  mdMCP;  (2) the rate o f deethylation o f m d M C P  of mdMCP  d e p e n d o n : (1) the rate o f N - d e e t h y l a t i o n o f M C P that producing ddMCP;  a n d (4) other b i o t r a n s f o r m a t i o n rates for m d M C P .  yields  (3) r e n a l e l i m i n a t i o n rate  T h e input rate o f m d M C P  rate o f N-deethylation o f M C P ) c o u l d not be directly determined in the study.  (the  H o w e v e r , it is  unlikely increased d u r i n g h y p o x e m i a , since total b o d y clearance o f M C P w a s decreased,  and,  as m e n t i o n e d above, there are reports that the c y t o c h r o m e P - 4 5 0 s y s t e m w h i c h m e d i a t e s N d e e t h y l a t i o n r e a c t i o n i s r e d u c e d d u r i n g a c u t e h y p o x e m i a ( S r i v a s t a v a et al, 1 9 8 0 ) . the rates o f m d M C P  e l i m i n a t i o n processes, s u c h as renal excretion a n d m d M C P  are likely decreased during hypoxemia.  Renal elimination of mdMCP  d e c r e a s e d d u r i n g h y p o x e m i a as s h o w n i n S e c t i o n 3.4.  was  Therefore, metabolism, significantly  Similar accumulation of MEGX  and  G X m e t a b o l i t e s o f l i d o c a i n e w a s a l s o o b s e r v e d d u r i n g a c u t e h y p o x e m i a ( M a r l e a u et al, 1 9 8 7 ; d u S o u i c h etal,  1992).  4.4.3. Renal elimination of MCP  and its metabolites during normoxemia and hypoxemia  R e n a l e l i m i n a t i o n o f M C P a n d its m e t a b o l i t e s w a s e x a m i n e d i n t h e p r e s e n t Since the infusion protocol was  of determining  urinary  e x c r e t i o n p a r a m e t e r s s u c h a s t h e p l o t s o f A . R . E . ( a m o u n t r e m a i n e d t o b e e x c r e t e d ) vs.  time or  u r i n a r y e x c r e t i o n r a t e vs.  used, the conventional m e t h o d s  study.  time c o u l d not be applied in this study. In addition, c h a n g e s in d r u g  elimination during the h y p o x e m i c a n d post-hypoxemic periods complicated the estimation renal drug elimination parameters.  of  Therefore, two modified methods of determining urinary  d r u g e x c r e t i o n d u r i n g the p h a s e d i n f u s i o n w e r e d e v e l o p e d a n d a p p l i e d i n the study. T h e first  116  m e t h o d w a s based o n the estimation o f renal clearance f r o m urinary d r u g a c c u m u l a t i o n AUC  [Method la and lb].  and  T h e other m e t h o d was based o n the determination o f fractional  renal d r u g excretion constant, w h i c h was derived a n d m o d i f i e d f r o m the concept o f urinary excretion d e t e r m i n a t i o n d u r i n g infusion b y G i b a l d i a n d Perrier ( 1 9 8 2 ) [ M e t h o d 2]. Firstly, the renal clearance of M C P a n d m d M C P  was calculated in the study  f r o m a ) d i v i d i n g t h e a m o u n t o f d r u g / m e t a b o l i t e r e c o v e r e d i n u r i n e (Du)  either  b y the area  under  p l a s m a drug/metabolite concentration curve ( A U C ) in a given time period [ M e t h o d la], and b ) u s i n g t h e s l o p e o f t h e a c c u m u l a t e d d r u g / m e t a b o l i t e i n u r i n e (Du)  versus A U C [Method lb].  A s s e e n in Section 3.4.1., b o t h m e t h o d s appear to give a similar estimation o f renal clearance. T h e slope f r o m the p r e - h y p o x e m i c period c o u l d not be determined, since there w e r e only data points in the period, a n d also only 1 or 2 data points representing a urine during M C P steady-state.  collection  A s a result, in the experimental group, the clearance  values  obtained d u r i n g the h y p o x e m i c period w e r e c o m p a r e d with those determined d u r i n g subsequent p o s t - h y p o x e m i c s e g m e n t (totaling 12 o f the 14 h o u r total experiment  3  the  duration).  In addition, renal clearance parameters f r o m the equivalent period in the control g r o u p  were  also c o m p a r e d with the h y p o x e m i c period. Since n o significant change in renal clearance in t h e c o n t r o l g r o u p w a s o b s e r v e d , it is s u p p o r t e d t h a t t h i s a p p r o a c h is v a l i d .  Secondly, the fractional renal excretion constants for M C P , m d M C P w e r e c a l c u l a t e d b y d i v i d i n g t h e s l o p e o f t h e a s y m p t o t e o f t h e Du  and  ddMCP,  versus time curve with  the  i n f u s i o n r a t e o f M C P . I n t h e c a s e o f p a r e n t d r u g , t h e f r a c t i o n a l r e n a l e x c r e t i o n c o n s t a n t (fu)  is  the proportion o f the renal elimination rate constant over the apparent total d r u g elimination r a t e c o n s t a n t (ku/KE), w h i c h i n d i c a t e s t h e f r a c t i o n o f t o t a l d r u g e l i m i n a t i o n d u e t o r e n a l d r u g  117  excretion.  In addition, the fractional renal excretion constants for metabolites w e r e  calculated.  T h e theoretical derivation o f these parameters w a s similar to those o f the  drug.  H o w e v e r , the fractional renal excretion parameters determined f r o m the  plot represent  h y b r i d f r a c t i o n a l c o n s t a n t s (i.e.  also parent  accumulation  a product of fractional renal  elimination  constant of a metabolite a n d fractional metabolic elimination constant of the parent drug)  [see  Section 3.4.2. a n d A p p e n d i x A ] . T h e s e parameters m a y a p p e a r to b e highly c o m p l i c a t e d , but, in simple terms, represent the fraction/proportion o f renal excretion in the f o r m o f a metabolite out o f the total d r u g elimination.  D u e to the constraints i m p o s e d b y the  design,  absolute  it w a s  not feasible to d e t e r m i n e d  (i.e.  specific infusion  not fractional constants)  renal  elimination a n d metabolic parameters in the study.  S i n c e the present study w a s designed to detect the c h a n g e s in the d r u g kinetics d u r i n g h y p o x e m i a , it is c r i t i c a l to d e t e r m i n e t h e r e n a l d r u g e l i m i n a t i o n p a r a m e t e r s d u r i n g n o r m o x e m i c and hypoxemic period within a single experiment (phased infusion).  A s  both shown  in S e c t i o n 4.3., a n y infusion c a n be theoretically c o n s i d e r e d as a series o f infusions appropriate loading doses.  Therefore, each pre-hypoxemic, hypoxemic and  post-hypoxemic  p e r i o d i n the study c o u l d b e c o n s i d e r e d as "separate" infusions w i t h appropriate doses, a n d renal excretion parameters f r o m each periods could be treated  with  loading  independently.  Previous studies on renal clearance during acute h y p o x e m i a have reported  various  responses. N o changes in renal clearance during acute hypoxemia were observed with  many  d r u g s s u c h as theophylline (Letarte a n d d u S o u i c h , 1984), f u r o s e m i d e ( B a b i n i a n d d u S o u i c h , 1 9 8 6 ) , l i d o c a i n e ( d u S o u i c h et  al,  1992)  a n d d i l t i a z e m ( d u S o u i c h et  al,  1993).  clearance o f p h e n y t o i n decreased d u r i n g acute h y p o x e m i a ( f r o m 0.2 to 0.03 m L / m i n / K g ) ,  Renal but  118  i t a p p a r e n t l y w a s n o t s t a t i s t i c a l l y s i g n i f i c a n t ( d u S o u i c h et al,  1986). Alternatively, renal  clearance o f sulfamethazine appears to b e increased, but statistically not significant, acute hypoxemia  (du  Souich  and  Courteau,  1984).  Renal clearance  s i g n i f i c a n t l y i n c r e a s e d d u r i n g a c u t e h y p o x e m i a ( d u S o u i c h et studies, renal clearance of metabolites was  measured.  N o  al,  of digoxin  1985).  significant changes in  There was significant reduction in renal clearance of M C P a n d mdMCP h y p o x e m i c p e r i o d i n the e x p e r i m e n t a l g r o u p as s h o w n i n S e c t i o n 3.4.1.  was  In one of  c l e a r a n c e s o f l i d o c a i n e m e t a b o l i t e s , M E G X a n d G X , w e r e o b s e r v e d ( d u S o u i c h et al,  MCP  during  the renal  1992).  d u r i n g the  T h e reduction in  renal clearance (about 2.75 L / h during h y p o x e m i a a n d 8.15 L / h during  normoxemia)  are m u c h m o r e extensive c o m p a r e to the reduction in total b o d y clearance (205 L / h d u r i n g hypoxemia and 245 L / h during normoxemia).  E v e n t h o u g h the renal clearance o f M C P  accounts for only about 5 % o f total b o d y clearance d u r i n g n o r m o x e m i a (about 8 L / h out  of  245 L/h), the reduction of renal clearance during h y p o x e m i a accounts for about 1 5 % o f the c h a n g e i n total b o d y clearance d u r i n g h y p o x e m i a ( c h a n g e o f about 5.5 L / h out o f 4 0 L / h ) . Therefore, the reduction in renal clearance o f M C P contributes significantly to the reduction o f total b o d y clearance o f M C P .  Similarly, the renal clearance of mdMCP  was significantly affected by  (14.6 L / h during h y p o x e m i a a n d 38.6 L / h during normoxemia). clearance of mdMCP  evaluated.  H o w e v e r , the total  body  c o u l d not be determined in this study, thus quantitative assessment  the contribution o f the m d M C P be  hypoxemia  renal elimination to total b o d y clearance o f M C P c o u l d  Renal elimination of ddMCP  was  also significantly  affected  by  of not  acute  119  hypoxemia. MCP  Therefore, acute h y p o x e m i a in the study appears to r e d u c e renal elimination o f  a n d its d e e t h y l  metabolites.  Overall, the present metabolite, mdMCP  s t u d y o f the p h a r m a c o k i n e t i c s o f M C P a n d its  and ddMCP,  deethylated  d u r i n g a c u t e h y p o x e m i a h a s s h o w n that t h e r e is a r e d u c e d  e l i m i n a t i o n o f M C P a n d its m e t a b o l i t e s d u r i n g a c u t e h y p o x e m i a , a n d this r e d u c t i o n s y s t e m i c c l e a r a n c e is a s s o c i a t e d w i t h a r e d u c e d r e n a l e l i m i n a t i o n o f the  compounds.  in  120  5.  SUMMARY AND CONCLUSIONS  A c a p i l l a r y G C - M S D m e t h o d o f R i g g s et al. ( 1 9 9 4 ) f o r M C P , m d M C P a n d d d M C P was  adapted  and  applied  pharmacokinetics of  to  study  the  effect  of  acute  moderate  hypoxemia  on  the  M C P a n d its d e e t h y l a t e d m e t a b o l i t e s i n p l a s m a a n d u r i n e f r o m  non-  p r e g n a n t e w e s f o l l o w i n g i . v . i n f u s i o n t o s t e a d y - s t a t e a n d i n d u c t i o n o f h y p o x e m i a via i n t r a tracheal nitrogen infusion.  A.  Acute h y p o x e m i a induces several physiological changes in non-pregnant ewes during continuous M C P infusion: 1.  R e d u c e s a r t e r i a l b l o o d PaC>2 ( h y p o x e m i a ) a n d P a C 0  2  (hypocapnia) d u r i n g the  h y p o x e m i c period in the experimental group. 2.  Elevates arterial b l o o d lactate a n d glucose level d u r i n g the late  hypoxemic  p e r i o d a n d f o r at i m e a f t e r r e s t o r a t i o n o f n o r m o x i a . 3.  Increases urine flow  and decreases urine osmolality  and p H  during  the  hypoxemic period. B.  H y p o x e m i a a f f e c t s t h e p h a r m a c o k i n e t i c s o f M C P a n d its d e e t h y l a t e d m d M C P 1.  metabolites,  and d d M C P . Total body clearance ( T B C ) was decreased during hypoxemia, and  restored  during the post-hypoxemic recovery period. 2.  Plasma mdMCP  concentration was increased during the h y p o x e m i c a n d post-  h y p o x e m i c p e r i o d as c o m p a r e d to the c o n t r o l g r o u p . 3.  The  renal elimination  hypoxemic period. mdMCP  and ddMCP  of M C P and  mdMCP  was  decreased  during  the  T h e fractional renal elimination rate constants o f M C P , were also lower during hypoxemia.  121  6.  REFERENCES  Adachi H , Strauss H W et al. 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Theoretical Derivation of Pharmacokinetic Equations used in the  study  Part I: One-compartment O p e n M o d e l T h e following are schematic representation of one-compartment  open model  metabolism.  Parent Drug Compartment  ko (+  Metabolite Compartment  LD) Xp,  oth  Cp,  Xm,  Vp  Cm,  Vm  six  T h e abbreviation used in the following equation derivation a n d the m a i n text are :  kf  = metabolite/conjugate formation rate constant for the parent drug koth = non-metabolic elimination rate constant for the parent drug kr = renal elimination rate constant of the parent drug kmr = renal elimination rate constant of the metabolite kmnr - non-renal elimination rate of the metabolite KE = kf+ koth = apparent elimination rate of the parent drug km — kmr + kmnr = apparent elimination rate of the metabolite fm = kf/KE = metabolic fraction of the parent drug elimination fmr = kmr/km = renal fraction of the metabolite elimination fr = kr/ KE"= renal fraction of the parent drug elimination ko = infusion rate constant Do = loading dose Xp = amount of the parent drug in the body Cp = concentration of the parent drug in the body Vp = apparent volume of distribution of the parent drug in the body Xm = amount of the metabolite in the body Cm = concentration of the metabolite in the body Vm = apparent volume of distribution of the metabolite in the body ti = Do/ko = infusion time equivalent loading dose  with  138  Theoretical Derivation : T h e rate o f d r u g disposition d e f i n e d as  in the parent d r u g c o m p a r t m e n t  during an  infusion  below dXp/dt = ko - KE * X  (1)  P  w i t h L a p l a c e t r a n s f o r m a t i o n (£(X) = X), E q . 1 sX  becomes  - X (0) = ko/s - KeXp  P  (2)  P  Case 1 : a simple infusion without loading dose s i n c e X (0)  = 0, E q 2 .  P  becomes  sXp + KE* Xp X  = ko/s  (3)  = ko/(s(s + KE))  P  by reverse Laplace  (4)  transformation,  X = ko/KE * ( 1 - exp(-KE * t))  (5)  C = ko/KE/Vp *( 1- exp(-KE * t))  (6)  P  P  Case 2: an infusion with a loading dose s i n c e X (0)  = Do,  P  sXp X  P  + KE*  E q 2. X  becomes = ko  P  /s  +Do  (7)  = ko/ (s(s + KE)) + DO/(S + KE)  by reverse Laplace  transformation,  X = ko/KE * ( 1 - exp(-KE *t)) + Do*( P  C  P  Urinary  (8)  1- exp(-KE * t))  = ko/KE/Vp * ( 1 - exp(-KE * t)) + Do/V *(lP  (9)  exp(-KE * t))  (10)  excretion dXu/dt = kr*X  (11)  P  w i t h L a p l a c e t r a n s f o r m a t i o n , E q . 11  becomes  sXu-Xu(0) = kr*Xp  Case 1 : a simple infusion without loading dose (Gibaldi and Perrier, s i n c e Xu(0)  = 0, E q . 1 2  1984)  becomes  sXu =kr*ko/ /(s+KE) S  (12)  (13)  139  Xu  =kr*ko/s /(s+KE)  (14)  2  by reverse Laplace transformation, Xu = kr* ko * t/KE - kr* ko/KE  * (1 - exp(-KE * t))  2  F r o m the the t e r m  plot  of  Xu vs. t, w h e n  exp(-KE * t)  the  drug  approaches zero. T h e asymptote  is  infused  (15)  to  becomes  Xu = kr* ko * t/KE - kr* ko/KE  (16)  2  T h e urinary excretion fraction constant asymptote,  kr* ko/KE,  by dividing  a steady-state,  kr/KE  c a n be determined f r o m the slope  of  ko.  C a s e 2: an infusion with a loading dose s i n c e X (0) P  = Do  sXu Xu  , E q . 12  becomes  = kr* ko/s/(s+KE) + DO/(S+KE) =kr*ko/s /(s+KE) 2  (17)  +DO/S/(S+KE)  (18)  b y reverse Laplace transformation, Xu = h * ko * t/KE + Do /KE*(  F r o m t h e p l o t o f Xu vs.  - kr * ko/KE  2  1 - exp(  * ( 1 - exp( - KE * t))  (19)  - KE * t))  t, w h e n t h e d r u g i s i n f u s e d t o a s t e a d y - s t a t e , t h e t e r m exp(  * t) a p p r o a c h e s z e r o . T h e a s y m p t o t e  becomes  Xu = kr* ko * t/KE + kr* ko /KE + DO/KE  (20)  2  T h e urinary excretion fraction constant a s y m p t o t e , kr*  By  ko /KE,  by dividing  - KE  kr/KE  can be determined f r o m the slope  of  ko.  u s i n g t h e t - i n t e r c e p t , KE c a n b e d e t e r m i n e d , h o w e v e r  the error associated with  the  i n t e r c e p t i s a c c u m u l a t i v e i.e. a n y e r r o r i n e a c h d a t a p o i n t w i l l b e d i r e c t l y r e f l e c t e d i n t h e t i n t e r c e p t o f t h e a s y m p t o t e u n l i k e to t h e s l o p e , t h u s d o e s n o t w a r r a n t its u s e .  T h e rate o f metaobolite disposition in the metabolite c o m p a r t m e n t d u r i n g an infusion defined as  below  140  dXm/dt = kf*Xp-Km*Xm  (21)  w i t h L a p l a c e t r a n s f o r m a t i o n (£(X) = X), E q . 2 1  becomes  sXm - Xm( 0) = kf *Xp - Km Jim  (22)  C a s e 1: as i m p l e i n f u s i o n w i t h o u t l o a d i n g d o s e s i n c e X (0) = 0, E q 2 2 . m  becomes  sXm = kf* ko/( s (s +KE)) - km  * Xm  (23)  Xm = kf* ko /(s (s +KE)(S + km))  by reverse Laplace  (24)  transformation,  Xm = kfkof 1/km /KE+exp(-Km t)/km/(km-KE)+exp(-kE t)/kE/(kE-Km)]  (25)  Cm = kfko/Vm [ 1/km /KE+exp(-Km t)/km/(km-KE) +exp(-kEt)AE/(kE-Km)]  (26)  C a s e 2 : a n i n f u s i o n w i t h al o a d i n g d o s e sXm = kfko/(s(s+KE)) - kmXm + kf Do/(s + K ) E  Xm = kf ko /(s(s +KE)(S + km))+ kf Do /((s +KE)(S + km))  b y reverse Laplace Xm =  kf*ko [1/km /KE+exp(-Km *t)/km/(km-KE) (29)  kf*ko/Vm [ 1/km /KE+exp(-Km *t)/km/(km-KE) +exp(-kE*t)/kE/(kE-Km)] + kfDo /Vm/(s+KE)/(s+km)  Urinary  (28)  transformation,  +exp(-kE*t)/kE/(kE-Km) ] + kfDo /(s+KE)/(s+km) Cm =  (27)  (30)  excretion dXmu/dt = kmr * Xm  with L a p l a c e transformation, E q . 31  (31)  becomes  sXmu = kmr Xm  (32)  C a s e 1: as i m p l e i n f u s i o n w i t h o u t l o a d i n g d o s e sXmu — kmr Xmu — kmr  by reverse Laplace  ko kf/s/(s+KE)/(s+k )  (33)  ko kf/s /(s+KE)/(s+k )  (34)  m  2  m  transformation,  141  Xmu  —  k kfko[t/kJKE -(K +krm)/k /KE 2  mr  E  2  m  + C * exp(-K t)+ D * exp(-km t))]  (35)  E  where C = (k +2K )/K /k 2  m  D  E  = - K /K /k 2  E  F r o m t h e p l o t o f Xmu exp(-KE t)  Xmu  E  E  - 1/(K k (K + kj)  2 m  E  m  E  + 1/(K k (K + k ))  2 m  E  m  E  m  vs. t, w h e n t h e d r u g i s i n f u s e d t o a s t e a d y - s t a t e ,  a n d exp(-km t) a p p r o a c h z e r o . T h e a s y m p t o t e = k  E  terms  becomes  kfko[t /kJKE -(K +krm)/k /KE J 2  mr  the  (36)  2  m  T h e m e t a b o l i t e u r i n a r y e x c r e t i o n f r a c t i o n c o n s t a n t kmr  kr/KE/km c a n b e  f r o m t h e s l o p e o f a s y m p t o t e , ko kmr kr/KE/km, b y d i v i d i n g  determined  ko.  Case 2 : an infusion with a loading dose sJXmu — kmr  ko  kf/s/(s+KE)/(s+k )+ k  Xmu — kmr  kf/s /(s+K )/(s+k ) + k  m  k D /(s+K )/(s+k )  mr  f  0  E  m  (37) ko  k D /s/(s+K )/(s+k )  2  E  m  mr  f  Q  E  m  (38)  b y reverse Laplace transformation, Xmu  = k  kf ko [t /kJKE -(K +krm)/k /KE + C exp(-K t)+ D * exp(-km t))] 2  mr  E  + k  E  k (D K k - k (K + k )) /K /k 2  mr  f  0  E  m  0  where C = (k +2K )/K /k 2  m  D  2  m  E  E  = - K /K /k 2  E  F r o m t h e p l o t o f Xmu  E  E  m  E  (39)  2 m  - 1/(K k (K + k ))  2 m  E  m  E  m  + 1/(K k (K + kj)  2 m  E  m  E  vs. t, w h e n t h e d r u g i s i n f u s e d t o a s t e a d y - s t a t e ,  exp(-KE t) a n d exp(-km t) a p p r o a c h z e r o . T h e a s y m p t o t e Xmu  = k  mr  kfko[t AJKE  +k  mr  kf  the  terms  becomes  -(K +krm)/k /KE ] 2  E  2  m  (D K a  E  km  " k (K + k ))/K A 2  0  E  m  E  (40)  2 M  T h e m e t a b o l i t e [ u r i n a r y e x c r e t i o n f r a c t i o n c o n s t a n t kmr f r o m t h e s l o p e o f a s y m p t o t e , ko kmr kr/KE/km, b y d i v i d i n g  kr/KE/km c a n b e ko.  determined  

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