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Protein binding displacement interactions between propafenone, 5-hydroxypropafenone and other antiarrhythmic… Wang, Jing 1990

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P R O T E I N B I N D I N G D I S P L A C E M E N T I N T E R A C T I O N S BETWEEN PROPAFENONE, 5-HYDROXYPROPAFENONE AND OTHER A N T I A R R H Y T H M I C D R U G S ; H P L C A N A L Y S I S INDOCYANINE GREEN  by JING WANG B.Sc. (Pharm), B e i j i n g M e d i c a l U n i v e r s i t y , 1987  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Faculty o f Pharmaceutical Science D i v i s i o n o f P h a r m a c e u t i c s and B i o p h a r m a c e u t i c s )  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA MAY, 1990 (5) C o p y r i g h t b y J i n g Wang, 1990  OF  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 department or by  his or her  representatives.  be granted by the head of  It is understood that copying or  publication of this thesis for financial gain shall not be allowed without my permission.  Department The University of British Columbia Vancouver, Canada  DE-6  (2/88)  my  written  ii ABSTRACT  Propafenone (PF) is a class I antiarrhythmic agent used to treat ventricular  and  superventricular  tachyarrhythmias.  administration, PF undergoes extensive metabolism.  After  oral  5-hydroxypropafenone  is one of the major active metabolites of PF.  In this thesis, the displacement of PF and 5-hydroxypropafenone in purified  human aj-acid glycoprotein  (AAG),  albumin, serum and whole  blood was examined using equilibrium dialysis. hydroxypropafenone concentrations  Free and bound PF and 5-  were analyzed by gas-chromatography  with electron-capture detection (GC-ECD).  Several antiarrhythmic agents  (e.g.  phenytoin and quinidine) and  propranolol, verapamil, lidocaine,  acetyl salicylic displacing  acid  agents  (ASA) were used  were  chosen  as  as  displacing  specific  probe  agents. for  These  binding  to  recognized sites of the plasma protein molecules.  The  binding  of  5-hydroxypropafenone  to  isolated  human AAG  phosphate buffer solution was studied over the concentration range of 50-20,000 ng/ml.  One class of binding site was found.  Saturation of  binding sites occurred at concentrations of 5-hydroxypropafenone greater than 2,000 ng/ml.  In  purified  solutions  of  human AAG or  albumin, propranolol,  verapamil, ASA and quinidine did not displace PF from its binding sites to a statistically statistically  significant  degree.  Phenytoin displaced PF to a  significant degree from its binding sites in albumin but  not  in AAG solution.  Lidocaine caused  a statistically  significant  increase in PF free fraction in solution of either AAG or albumin.  5-  Hydroxypropafenone free fraction was not affected by verapamil ASA and propranolol, but increased with the addition of lidocaine in purified human AAG solution.  In purified human albumin solutions, ASA caused a  significant increase in 5-hydroxypropafenone, while no displacing effect was observed with either lidocaine, propranolol or verapamil.  In  human serum,  statistically  quinidine,  significant  phenytoin  increased  and lidocaine  PF free  fraction.  caused  a  Neither ASA,  quinidine nor phenytoin caused a statistically significant displacement of 5-hydroxypropafenone from its binding site in serum.  Quinidine also  displaced PF from its binding sites in whole blood while no displacing effect was observed with phenytoin.  Quinidine and lidocaine were the most potent displacing agents for PF protein binding.  They exhibited the displacement through competitive  inhibition in purified human AAG.  None of the displacing agents used  seemed to be a potent inhibitor of 5-hydroxypropafenone protein binding in serum.  Since PF is a drug known to exhibit very significant metabolism,  we wished  interventions clearance.  to  which might  prepare alter  to  hepatic  certain pharmacological  blood flow,  and,  hence PF  Before such a study of PF could be conducted, we required a  reliable measure of hepatic blood flow. affords  study  first-pass  such an estimate.  The dye indocyanine green (ICG)  We, therefore,  set  out to duplicate and  iv refine on existing HPLC ICG assay.  In early attempts, the detection  limit of our HPLC analysis of ICG in human serum was 0.80 //g/ml.  The  analysis method of ICG in rat plasma gave an apparently good on-column detection limit (3.8 ng) using current detection technology available in our  laboratory.  However,  we  still  concentration ICG samples as reported.  could  not  detect  the  low  This is, in part, due to the low  injection volume that can be accommodated by our microbore HPLC system and  more  available chemical  importantly the (600  nm).  nature  of  limitation  of  Fluorometric of this  maximum detection wavelength ICG is  compound limit  extraction method for sample preparation.  the  not use  feasible of  and  the  solvent-solvent  With the manipulation of the  sample extraction procedure, such as the use of solid phase extraction, or modification of our current liquid sample introduction configuration devices,  it might be possible to develop a better sample preparation  procedure in order to detect the low concentration which most likely will  be  encountered  during  experimentation with ICG.  the  course  of  animal  and/or  human  V  TABLE OF CONTENTS page ii  ABSTRACT LIST OF TABLES  x  LIST OF FIGURES  xi  LIST OF ABBREVIATIONS  xiv  ACKNOWLEDGEMENTS  xvi  1. INTRODUCTION  1  1.1  Pharmacology of Propafenone  1  1.2  Therapeutic Uses  2  1.3  Pharmacokinetics of Propafenone 1.3.1 Absorption 1.3.2 Distribution 1.3.3 Metabolism and Elimination 1.3.4 Protein Binding of Propafenone  2 2 3 3 8  ,  1.4  Pharmacological Effect of 5-Hydroxypropafenone  9  1.5  Protein Binding of 5-Hydroxypropafenone  10  1.6  Propafenone and Food Interaction  10  1.7  Effect of Alteration of Protein Binding and Blood Flow on Total Body Clearance 11 1.7.1 Effect of Binding Displacement on High Clearance Drugs 13 1.7.1.1 Displacement Mechanisms: Drugs and Endogenous Substrate 14 1.7.1.2 Selection of Protein Solutions as Drug Binding Displacement Media 15 1.7.1.3 Quantitative Analysis of Drug Protein Binding 16 1.7.2 Effects of an Increase in Liver Blood Flow on the Bioavailability of High Clearance Drugs 18  1.8  Indocyanine Green Clearance as an Indicator of Hepatic Blood Flow  1.9  1.10  19  Rationale 1.9.1 Rationale for Protein Binding Displacement Study 1.9.2 Rationale for Reproduction of the HPLC Analysis Method of ICG in Serum Samples  23 23  Objectives  26  26  vi  2. EXPERIMENTAL  28  2.1  Materials and Supplies 2.1.1 Drugs, Metabolites and Internal Standards 2.1.2 Chemicals and Reagents 2.1.3 Proteins 2.1.4 Solvents 2.1.5 Gases 2.1.6 Equil ibrium Dialysis Device 2.1.7 Other Supplies  28 28 28 29 29 29 29 30  2.2  Columns 2.2.1 GLC Column 2.2.2 HPLC Column  30 30 30  2.3  Equipment 2.3.1 Gas-Liquid Chromatography 2.3.2 High-Performance Liquid Chromatography 2.3.3 Spectrometer 2.3.4 Miscellaneous  31 31 31 31 32  2.4  Preparation of Stock and Reagent Solutions 2.4.1 Drug, Metabolites and Internal Standards for GLC Analysis 2.4.2 Drugs for Equilibrium Dialysis 2.4.3 Purified Protein Solutions 2.4.4 Drug and Internal Standard Solutions for HPLC Analysis 2.4.5 Reagent Solutions  32  35 36  2.5  Equilibrium Dialysis Procedure  37  2.6  Protein Binding of 5-Hydroxypropafenone to Purified Human AAG  38  Rosenthal Analysis of the Binding of 5-Hydroxypropafenone to Purified Human AAG  38  2.8  Scatchard Plot of 5-Hydroxypropafenone Binding to AAG  38  2.9  Free Fraction vs Total Concentration Plot of 5-Hydroxypropafenone Binding to AAG  39  2.10  Protein Binding Displacement Studies 2.10.1 Effect of Buffer Strength on the Protein Binding of Propafenone and 5-Hydroxypropafenone 2.10.2 Effect of Anticoagulant on the Protein Binding of Propafenone and 5-Hydroxypropafenone 2.10.3 Protein Binding Displacement Study Using Human AAG and Albumin as the Biological Fluids 2.10.4 Protein Binding Displacement Study Using Human Serum and Whole Blood as the Biological Fluids 2.10.5 Characterization of the Nature of Binding  2.7  32 33 35  39 39 40 40 41  vii  2.10.6  Displacement Interaction between PF and Lidocaine and PF and Quinidine in Purified Human AAG 2.10.5.1 Binding of PF to AAG over the Therapeutic Concentration Range of PF 2.10.5.2 Characterization of the Nature of the Displacement Interaction: Competitive or Non-competitive Measurement of Albumin Concentration in Purified Albumin Solution  41 41 42 42  2.11  Analysis of Propafenone and 5-Hydroxypropafenone  42  2.12  HPLC Analysis of Indocyanine Green (ICG) 2.12.1 Reproduction of Reported HPLC ICG Assay Methods 2.12.2 Modification of the Analysis Method of Indocyanine Green of C h r i s t i e (1986) and Burns (1989) 2.12.3 Comparison of the Chromatogram of Serum Spiked with Indocyanine Green with That of Serum Blank 2.12.4 Modification of the Chromatographic Conditions to Minimize the Blank Interference 2.12.5 Extraction Efficiency 2.12.6 Post Extraction S t a b i l i t y 2.12.7 Animal Preparation 2.12.8 Analysis of Plasma Samples 2.12.9 The Potential to Detect Low Concentration Samples with the New Method 2.12.10 Solid Phase Extraction Method  43 43  2.13  Data Analysis 2.13.1 S t a t i s t i c a l Analysis 2.13.2 Protein Binding Data 2.13.3 Indocyanine Green Plasma Clearance  3. RESULTS 3.1 3.2  3.3  Volume Shift and pH Change of Equilibrium Dialysis Samples Binding of 5-Hydroxypropafenone to Purified Human AAG 3.2.1 Rosenthal Analysis 3.2.2 Scatchard Plot of 5-Hydroxypropafenone Binding to Human AAG 3.2.3 Free Fraction versus Total Concentration Plot Protein Binding Displacement Effect 3.3.1 Effect of Buffer Strength on the Binding of PF and 5-Hydroxypropafenone 3.3.2 Effect of Anticoagulant on the Protein Binding of PF and 5-Hydroxypropafenone 3.3.3 Protein Binding Displacement Studies using Purified Human Protein Solutions (AAG and Albumin) as the Biological Fluids 3.3.3.1 Protein Binding Displacement of Various Drugs on PF Binding to Purified Human  44 46 46 46 47 47 48 49 49 50 50 50 50 52 52 52 52 52 54 54 54 58 63  viii AAG Protein Binding Displacement Effect of Various Drugs on the Binding of PF to Purified Human Albumin 3.3.3.3 Protein Binding Displacement Effect of Various Drugs on the Binding of 5-Hydroxypropafenone to Human AAG 3.3.3.4 Protein Binding Displacement Effect of Various Drugs on the Binding of 5-Hydroxypropafenone to Human Albumin Protein Binding Displacement Effect Using Human Serum as the Biological Fluid Protein Binding Displacement Effect on PF Free Fraction Using Human Whole blood as the Biological Fluid The Nature of the Displacement Interaction between PF and Quinindine and PF and Lidocaine Using Purified Human AAG as the Biological Fluid 3.3.6.1 Rosenthal Plot of the Binding of PF to Purified AAG over the Therapeutic Concentration Range of PF 3.3.6.2 The Nature of the Displacement of PF from Human AAG by Quinidine and Lidocaine 3.3.3.2  3.3.4 3.3.5 3.3.6  3.4  HPLC Analysis of Indocyanine Green (ICG) 3.4.1 HPLC Analysis of Indocyanine Green According to the Method of Rappaport and Thiessen 3.4.2 HPLC Analysis of Indocyanine Green with Fluorometric Detection 3.4.3 HPLC Analysis of ICG with Diode Array Detection 3.4.4 Comparison of Sample and Blank Chromatograms Using a Modified Method of Christie (1986) and Burns (1989) 3.4.5 ICG Extraction Efficiency 3.4.6 Post Extraction Stability 3.4.7 Plasma Data of ICG in Rat 3.4.8 The Potential to Detect ICG in Low Concentration Samples with the New Method 3.4.9 Solid Phase Extraction  4. Discussion  63 63 68 68 73 73 77 77 77 80 80 80 81 87 87 90 90 94 94 96  4.1  Protein Binding Technique  96  4.2  Protein Binding of 5-Hydroxypropafenone  99  4.3  Displacing Effect of Verapamil, Propranolol, ASA, Phenytoin, Lidocaine and Quinidine 4.3.1 Verapamil 4.3.2 Propranolol 4.3.3 Acetyl salicylic Acid (ASA) 4.3.4 Phenytoin 4.3.5 Quinidine  101 101 102 103 104 106  ix  4.3.6 4.4 4.5  Lidocaine  107  Clinical Significance of the Protein Binding Displacement Effect HPLC Analysis of ICG 4.5.1 HPLC Analysis of ICG with Fluorometric Detection 4.5.2 HPLC Analysis of ICG with Diode Array Detection 4.5.2.1 Sample Preparation 4.5.2.2 HPLC Analysis of ICG Plasma Concentration in Rat 4.5.2.3  Summary  108 109 109 110 110 111 112  5. Summary and Conclusions  115  6. References  119  X  LIST OF TABLES page 1.  Methods for the determination of hepatic blood flow.  20  2.  Free fraction of PF in a solution of AAG or albumin dissolved in 0.067 M or.0.1 M phosphate buffer.  57  Free fraction of 50HPF in a solution of AAG or albumin dissolved in 0.067 M or 0.1 M phosphate buffer.  59  4.  Free fraction of PF in whole blood using heparin or EDTA as the anticoagulant.  60  5.  Free fraction of 50HPF in whole blood using heparin or EDTA as the anticoagulant.  62  6.  Extraction efficiency of ICG in serum.  87  7.  Peak height ratios of ICG/diazepam before and after serum extraction.  90  Free fraction of 50HPF and PF in AAG, albumin, serum and whole blood.  100  PF free fraction before and after treatment with differing displacing agents  116  10. 50HPF free fraction before and after treatment with differing displacing agents  117  3.  8. 9.  xi LIST OF FIGURES Page 1.  Chemical structure of propafenone.  1  2.  Relationship between steady-state mean plasma concentration and total daily dose. The dashed line indicates the response expected if the relationships were linear. Points and bars indicate mean and SEM.  4  3.  Chemical structure of PF major metabolites:  5-hydroxypropafenone  and N-depropylpropafenone  6  4.  Chemical structure of indocyanine green (ICG).  22  5.  A scheme of extraction procedure of ICG in human serum.  45  6. 7.  A scheme of extraction procedure of ICG in rat serum. Relationship between the ratio of bound concentration/free concentration and bound concentration of 5-hydroxypropafenone in purified human AAG (Rosenthal plot), the inserted graph shows the relationship between bound and total concentration of 5-hydroxypropafenone.  49  53  Relationship between [r'/bound concentration] and r' of 5-hydroxypropafenone in purified human AAG (Scatchard plot); concentration of AAG was used in the calculation of [ r ' ] .  55  Relationship between the ratio of free concentration/total concentration (free fraction) and total concentration of 5-hydroxypropafenone over the concentration of 20-20,000 ng/ml.  56  8.  9.  10. Protein binding displacing effect ASA on the free fraction of PF in in 0.067 M phosphate buffer. The 7.4, the value is the mean + s.d. replicates.  of propranolol, verapamil and purified human AAG dissolved pH in each treatment group is and n is the number of  64  11. Protein binding displacing effect of phenytoin, quinidine and lidocaine on the free fraction of PF in purified human AAG dissolved in 0.1 M phosphate buffer. The pH in each treatment group is 7.4 and the value is the mean ± s.d., * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05, n is the number of replicates.  65  12. Protein binding displacing effect of propranolol, verapamil and ASA on the free fraction of PF in purified human albumin dissolved in 0.067 M phosphate buffer. The pH in each treatment group is 7.4, the value is the mean ± s.d. and n is the number of replicates.  66  xii  13. Protein binding displacing effect of phenytoin, quinidine and lidocaine on the free fraction of PF in purified human albumin dissolved in 0.1 M phosphate buffer. The pH in each treatment group is 7.4 and the value is the mean ± s.d., * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05, n is the number of replicates.  67  14. Protein binding displacing effect o f propranolol, verapamil and ASA on the free fraction of 5-hydroxypropafenone in purified human AAG dissolved in 0.067 M phosphate buffer. The pH in each treatment group is 7.4, the value is the mean ± s.d. and n is the number of replicates.  69  15. Protein binding displacing effect of lidocaine o n the free fraction of 5-hydropropafenone in purified human AAG dissolved in 0.1 M phosphate buffer. The pH in each treatment group is 7.4 and the value is the mean ± s.d., * indicates statistically significant difference with two sample t-test at p < 0.05, n is the number of replicates. 70 16. Protein binding displacing effect of verapamil, ASA and propranolol on the free fraction of 5-hydropropafenone in purified human albumin dissolved in 0.067 M phosphate buffer. The pH in each treatment group is 7.4 and the value is the mean ± s.d., * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05, n is the number of replicates. 17. Protein binding displacing effect of 5-hydropropafenone in purified phosphate buffer. The pH in each value is the mean ± s.d. and n is  71  o f lidocaine on the free fraction human albumin dissolved in 0.1 M treatment group is 7.4 , the the number of replicates. 72  18. Protein binding displacing effect o f DPH (phenytoin), quinidine and lidocaine on the free fraction o f PF in human serum. The pH in each treatment group is 7.4 and the value is the mean ± s.d., * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05, n is the number of replicates.  74  19. Protein binding displacing effect of phenytoin, ASA and quinidine on the free fraction of 5-hydropropafenone in human serum. The pH in each treatment group is 7.4, the value is the mean ± s.d. and n is the number of replicates.  75  20. Protein binding displacing effect of quinidine and phenytoin on the free fraction of PF in human whole. The pH in each treatment group is 7.4 and the value is the mean ± s.d., * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05, n is the number o f replicates.  76  21. Relationship between the ratio of bound concentration/free  xiii concentration and bound concentration of PF in purified human AAG (Rosenthal plot), the inserted graph indicates the relationship between bound and total concentration of PF.  78  22. Relationship between the reciprocal of the moles of PF bound per mole of AAG and the reciprocal of unbound PF without displacing agent ; in the presence of quinidine ; in the presence of lidocaine .  79  23. Excitation and emission (at excitation wavelength of 260 nm) spectra of ICG using pH 6.0 phosphate buffer as part of the mobile phase.  82  24. A representative chromatogram that compares fluorometric detection with UV detection.  83  25. Excitation spectrum of ICG using pH 4.0 citrate buffer as part of the mobile phase.  84  26. A representative chromatogram obtained using the modification method (section 2.12.2).  85  27. A representative standard curve obtained using the modification method (section 2.12.2).  86  28. Representative chromatograms of serum (0.25 ml) spiked with ICG and serum blank.  88  29. Representative standard curve obtained using 0.25 ml serum.  89  30. A representative chromatogram in rat plasma.  91  31. A representative standard curve obtained in rat plasma.  92  32. Plasma concentration-time curve of ICG in rat plasma. 33. A representative chromatogram obtained using solid phase extraction method.  93 95  34. Ultraviolet/visible spectrum of ICG in distilled water (10 /zg/ml).  113  xiv  L I S T OF A B B R E V I A I T I O N S  AAG  a p a c i d glycoprotein  AUC  a r e a u n d e r t h e c o n c e n t r a t i o n versus  time  a r e a u n d e r t h e c o n c e n t r a t i o n versus  time curve a f t e r o r a l  AUC  o r a  i  curve  administration AV C  atrioventricular  m a x  CI  peak c o n c e n t r a t i o n o f drug i n serum clearance  Cli t  intrinsic  CV.  coefficient of variation  D  dose  [D]  f r e e drug  [DP]  drug-protein  E  extraction ratio  ECD  electron-capture detector  F  systemic  GLC  g a s - l i q u i d chromatography  GLC-ECD  gas-liquid  n  clearance  complex  availability  chromatography-electron-capture  detection HDL HP  high density l i p o p r o t e i n Hewlett-Packard  HPLC  high-performance  ICG  indocyanine  I.D.  internal diameter  i.v. LDL  l i q u i d chromatography  green  intravenous low d e n s i t y l i p o p r o t e i n  X V  M.W.  molecular weight  n  number of homogeneous binding sites  50HPF  5-hydroxypropafenone  [P]  free protein (unbound)  PF  propafenone  PTFE  polytetrafluoroethylene  r  correlation coefficient  rpm  revolutions per minute  s.d.  standard deviation  t  time  t h a l f - l i f e t  m a x  time to reach peak concentration  T  dosing interval  TEA  triethyl amine  UV  ultraviolet  VLDL  very low density lipoprotein  xvi  ACKNOWLEDGEMENTS  I would like to sincerely thank Dr. James Axel son for his supervision, guidance and financial support. I would like to thank my committee members Drs. Frank Abbott, Jack Diamond and John Sinclair for their advice and support through out my study. Thanks to Johannes Schenk and Zhen Yu for their help in animal experiments. Special thanks to George Tonn, Mathew Wright, Swamy Yeleswaram, Sue Panesar and Grace Chan for their c r i t i c a l advice in my experiments and in the writing of the thesis and their constant support and encouragement. My appreciation to Judit Orbay and Andras Seitz for their help in experiments and friendship. Thanks to Sonia Chan for helping me through the days when I was a teaching assistant. Thanks to my fellow graduate students Marion Wong, Seema Gadkari, Heyi Liu and Yongjiang Hei for their friendship and help. Special thanks to my husband, Futong Cui, and my family, for their support and understanding. This project was supported by The Canadian Heart and Stroke Foundation.  I would finally like to gratefully thank the B.C. and Yukon  Heart and Stroke Foundation for the financial Traineeship support.  XVI 1  This thesis is dedicated to father, for giving me the courage to believe . my dear s i s t e r ,  t  in my c a p a b i l i t i e s ,  o  for her continuous support and encouragement, to  husband, Futong, for his unfailing encouragement and understanding and to the memory of my mother.  1  INTRODUCTION  1.  1.1 Pharmacology of  Propafenone  Propafenone  (PF) (Figure 1, marketed as a racemate)  antiarrhythmic agent with weak B-blocking a c t i v i t y blocking  activity  (Harron and  Brogden,  1987).  is  a type IC  and calcium channel Like  other  type  IC  antiarrhythmic agents, PF exerts i t s pharmacological effects through the depression  of sodium influx  dose-dependent conduction  decrease  velocity  (Vaughan Williams, 1970).  in  the  in tissue  maximum  depolarized  (Dukes and Vaughan Williams, 1984).  of  by the  depolarization fast  sodium  and  channel  Compared with propranolol, the B-  blocking a c t i v i t y  of PF is r e l a t i v e l y  patients  ventricular  with  rate  PF also causes a  small  arrrhythmia,  prolongation of PR and QRS i n t e r v a l s ;  (McLeod et PF  al.,  caused  1984).  In  significant  increases in atroventricular nodal  conduction time and His-Purkinje conduction time (Hartel, 1985; Nauardla et  al.,  1984;  Connolly et al.,  change mean a r t e r i a l  1983a,b).  pressure  or heart  from 956 to 1564 /zg/ml (Shen et al.,  rate  2  6  5  0-CH -CH-CH -NH-C H 2  2  3  PF did not  at concentrations  1984).  -CO-CH -CH -C H 2  Haemodynamical l y ,  7  Figure 1. Chemical structure of propafenone (PF)  ranging  2  1.2 Therapeutic Uses  Propafenone has been used in the treatment of chronic ventricular arrhythmias, atrial and AV nodal junctional re-entrant tachycardias, and Wolff-Parkinson-White tachycardias (Harron and Brogden, 1987).  It  is  effective for the acute management of atrial f i b r i l l a t i o n (Vita et  al.,  1989)  al.,  1988).  and prevention of  recurrent atrial  f i b r i l l a t i o n (Kerr et  It is more effective than the traditional antiarrhythmic agents:  quinidine, flecainide,  disopyramide,  tocainide  and metoprolol  and  is  in efficacy  comparable  and has  to  a low  lidocaine,  incidence  of  adverse effects (Harron and Brogden, 1987).  1.3 Pharmacokinetics of Propafenone  1.3.1 Absorption  The bioavailability of PF after oral administration is quite low (4.8% with a 150 mg dose and 12% with a 300 mg dose ) due to extensive first-pass metabolism (Hollmann et al., after  oral  1983).  administration and dose-dependent  It is rapidly absorbed increases  in both peak  plasma concentration and AUC have been reported (Hollman et al., Connolly et  al.,  1983b; Frabetti et  al.,  1986;  Zoble et  al.,  1983; 1989).  After administration of three different doses of PF (150, 300, 450 mg) to 19 healthy subjects, Hollmann (1983) found disproportional in peak plasma concentration ( 139.3, 384.0, 827.0 /ig/ml). relationship was found in arrhythmia patients A 3 fold increase  increases A similar  (Connolly et al.,  in dose from 300 to 900 mg resulted  1983).  in a 10 fold  3  elevation of steady-state PF concentrations (Figure 2).  This non-linear  relationship between dose and plasma concentration was most likely due to the saturation of the cytochrome P-450 isoenzyme(s) which was the same cytochrome  P-450 responsible  (Siddoway et a / . , 1987).  for debrisoquine's  4-hydroxylation  Further study using human kidney donor livers  indicated that 5-hydroxypropafenone was formed by the cytochrome P-450 isozyme which participated in the polymorphic oxidation of bufuralol (Kroemer et al.,  1989).  1.3.2 Distribution  The tissue distribution of  PF and 5-hydroxypropafenone  in one  patient was determined in autopsy samples 24 hours after death (Latini, 1987).  The highest concentration of PF was found to be in the lung (12  ug/q wet tissue), followed by the heart (6.2 fig/g) The metabolite,  5-hydroxypropafenone,  tissue  and liver (4  concentration  was  than the parent compound in most organs except for the liver (7.2  fig/g). lower fig/g).  However, the metabolite/parent drug ratio was close to 1 in the heart (compare with 0.1  - 0.4  in plasma)  partitioning of 5-hydroxypropafenone  indicating that there was greater into the myocardium than for the  parent drug, propafenone, and thus that antiarrhythmic activity of this metabolite was possible.  1.3.3 Metabolism and Elimination  Propafenone undergoes extensive first-pass administration (Harron and Brogden, 1987).  metabolism after oral  In humans, less than 1% of  4  CONNOLLY et al.  E  0  300  600  900  DAILY DOSE PROPAFENONE (mg) Figure 2. Relationship between steady-state mean plasma concentration and total d a i l y dose. The dashed l i n e indicates the response expected i f the relationships were l i n e a r . Points and bars indicate mean and SEM.  5 the parent drug was excreted  in urine or feces after  deuterium-labelled PF (Hege et dose  was  recovered  in  the  al.,  1984).  feces  as  administration of  F i f t y three percent of  metabolites.  In  patients  the with  frequent ventricular ectopy who underwent chronic treatment with PF (300 mg, b . i . d . for 2 weeks), the major metabolites were 5-hydroxypropafenone and N-depropylpropafenone (the structures are shown in Figure 3)  (Kates  et  of  al.,  1985).  At  steady-state,  the  plasma  concentrations  5-  hydroxypropafenone and N-depropylpropafenone are about 23% and 17% of the corresponding propafenone concentration,  The effect its  of enzyme induction on the pharmacokinetics of PF and  major metabolite  Phenobarbital hepatic  cigarette significant observed.  5-hydroxypropafenone  treatment  microsomal  metabolism of  respectively.  was  studied  by Chan (1989).  (100 mg d a i l y at bedtime for 23 days)  enzymes and enhanced  the  extent  of  the  first-pass  PF in eight healthy non-smoking and eight healthy  smoking  Caucasian  increase  males  in C I o f  (age  20-45  PF after  y),  elimination,  which  heavy  respectively.  phenobarbital treatment  On the other hand, phenobarbital treatment  5-hydroxypropafenone  indicates  that  to enzyme induction caused by phenobarbital.  A was  has no effect on the  responsible for the metabolization of PF to 5-hydroxypropafenone subject  induced  enzyme is not  When compared to  the non-smokers, heavy cigarette smokers appeared to show an increase in the clearance of PF, however, the  metabolism  concluded  that  of  PF was  while  a general conclusion that smoking induced  difficult  phenobarbital  to  address.  induced  the  It  was,  metabolism  therefore, of  PF in  smokers, there was no effect of smoking per se on the metabolism of PF.  6  <^~\-CO-CH -CH -C H 2  2  6  5  O-CHo-CH-CHo-NH-CoH-7 OH propafenone  <QKO-CH-CH-CH 0-CH-CH-CH-NH OH 2  2  2  6  ?  N-depropyl propafenone  5  C  ?  HO  ^J^-CO-CH -CH -C H 2  2  6  5  O-CH0-CH-CH0-NH-C0H7  OH  * '  5-hydroxy propafenone  Figure 3. Chemical structures of PF major metabolites: 5-hydroxypropafenone and N-depropylpropafenone  7  The pharmacokinetics of PF is time-dependent as studied by Giani et al.  (1988).  Giani et al.  (1988) compared the ratio of the plasma  AUCs of 5-hydroxypropafenone and PF after a single dose of PF to that seen after one month multiple dose therapy of PF (300 mg t . i . d . ) patients with stable, frequent, premature ventricular beats.  in 10  They found  that the AUC ratio of metabolite/PF decreased from 0.63 after the single dose  experiment  to  elimination half-life  0.32  following  one  month therapy  was almost doubled (6.7  therapy and 3.5 h after single dose).  and also  the  h following one month  They suggested that this decrease  in PF clearance was due partly to a drop in the rate of metabolism of PF to 5-hydroxypropafenone, as 5-hydroxypropafenone did not accumulate in the same magnitude as PF.  The mean total body clearance of PF after single i.v.  dose of 70  mg to 8 healthy subjects before and after 12 day's application of PF 150 mg b . i . d . were 1.14 L/min and 1.10 L/min, respectively (Hollmann et a / . , 1983).  In 10 patients with superventricular tachycardia given 2.3 ± 0.2  mg/kg i.v.,  total body clearance was 1.03 L/min (Arboix et al.,  The volume of distribution of PF after i.v. L/kg  in healthy  patients greater  (Hollmann et  subjects  with arrhythmias (Arboix et than the  actual  body volume  al.,  al.,  1985).  administration was « 3 1983)  1985).  suggesting  and 1.6  L/kg in  These values considerable  are  tissue  binding.  The mean  °f  ^  i " healthy  volunteers  was  found to  be  approximately 4.6 h (range 2.3-9.5 h) after an oral single dose of 300  8  mg, and 2.8 h (range 2.1-4.1 h) after a 70 mg i.v. 1983).  (Hollmann et  a/.,  In patients displaying episodes of paroxysmal supraventricular  tachycardias, the elimination half-life  (ti/2) of PF s  approximately  wa  80 min (range 41-148 min) after a single intravenous dose of 2.3 ± 0.2 mg/kg (Arboix et al.,  1985).  In patients with chronic, stable cardiac  dysrhythmias, the t]/2 of PF was 3.6 h after a single 900 mg oral dose (Keller, 1978).  In another study, the tj/2 of" PF  w  a  s  3  -  8  n  (range 1.9-  7.9 h) and 3.75 h (range 1.65-7.8 h) after a single dose of 150 mg or 300 mg, respectively (Frabetti et al.,  1986).  Individual differences in  drug metabolism may contributed to the variation seen in the tj/2 of PF.  The effective  plasma concentration of PF at steady-state shows  great intersubject variability.  The clinical therapeutic range for this  drug is 0.06-1 //g/ml (Latini et al., suppression  of  ventricular  1990).  ectopic  To obtain greater than 70%  depolarizations,  the  plasma  concentration ranges from 42 to 1801 ng/ml between subjects (Siddoway et al.,  1987).  Changes in PR and QRS in the ECG are related to plasma  steady-state concentrations (Frabetti et a7.,1986; Zoble et al.,  1989).  Taking the non-linear relationship between dose and plasma concentration into account, it is obvious that there is a disproportional relationship between dose and pharmacological response with PF.  1.3.4 Protein Binding of Propafenone  Propafenone binds extensively to both plasma and tissue proteins. More than 95% of PF binds to serum proteins 1980),  and the  binding  is  proportional to  (Seipel  the  and Breithardt,  concentration  of AAG  9  (Gill is et al.,  1985).  Gill is et al.  (1985) have studied the binding of  PF and other antiarrhythmic agents to purified human AAG. labelled  drugs,  two  classes  of  binding  sites  for  concentration range of 0.009-81 ug/m\ were demonstrated. to a greater extent than lidocaine, In vitro,  Using radioPF over  the  PF also bound  verapamil and propranolol to AAG.  in normal serum PF bound independent of concentration within  the range of 0.5-1.5 ^g/ml and the binding was concentration dependent at PF concentration greater than 0.2 free fraction of PF decreased  uq/m\ (Chan et  al.,  1989).  The  approximately 50% in uraemic serum.  A  good correlation (r=0.8302) between AAG concentration and the PF binding ratio was also reported. protein  solutions  of  The binding of PF in whole serum and isolated  AAG, albumin, HDL, LDL and VLDL was  extensively by Tonn (1990).  The binding of  examined  PF was found to be pH  dependent in serum and in solutions of albumin and AAG, respectively. Moderate binding to albumin and extensive binding to AAG were found. The binding to AAG correlated well with the binding observed in serum. The  degree of  deficient  binding was similar  serum.  in control  serum and lipoprotein  The binding of PF to HDL and LDL was greater than  serum and non-saturable binding was found in solutions of LDL and VLDL.  1.4 Pharmacological Effect of 5-Hydroxypropafenone  5-Hydroxypropafenone, the major metabolite of PF, has been found to be pharmacologically active in animal models (von Philipsborn et 1984; Valenzuela et al.,  1987; Thompson et al.,  1988).  In the  al.,  intact  animals (aconitine induced arrhythmia rats and coronary ligation induced infarction arrhythmic dogs), 5-hydroxypropafenone was more potent as an  10  antiarrhythmic agent than the parent compound (von Philipsborn et 1984).  al.,  This phenomenon was also observed by Malfatto et a7.(1989) in  coronary ligation induced infarction arrhythmic dogs.  1.5 Protein Binding of 5-Hydroxypropafenone  The in vitro studied  protein binding of the active metabolite  extensively  in  our  hydroxypropafenone displays  laboratory.  concentration  In  human  independent  has been  serum,  5-  binding with a  mean free fraction of 0.184 over the concentration range of 0.12 to 35.3 jug/ml (Tonn, 1990).  The binding of 5-hydroxypropafenone was found to be  pH dependent in serum and in a solution of AAG, but not in a solution of albumin.  Moderate binding  observed.  of  5-hydroxypropafenone  to  albumin was  A significant decrease in 5-hydroxypropafenone binding to AAG  was observed upon the addition of PF.  Decreased binding was also found  in albumin upon the addition of a physiological concentration of AAG. Removal  of  fraction  lipoprotein  in serum resulted  of 5-hydroxypropafenone  in  increases  in the  free  as compared to control  serum.  The  binding of 5-hydroxypropafenone to HDL, LDL, VLDL was similar to PF.  1.6 Propafenone and Food Interaction  Food induced changes in the pharmacokinetics of high clearance drugs such as propranolol (Mclean et al.,  1981) and metoprolol (Melander  et al.,  This food effect was partially  1977) have been well documented.  explained  by  a food  induced  change  in  intrinsic drug metabolism (Mclean et al.,  splanchnic  blood  flow  1978; Svensson et al.,  and 1983;  11 Svensson et of  al.,  1984).  When Axelson et  (1987) studied the  effect  food on the pharmacokinetics of propafenone given as a single  dose,  they  found  that  b i o a v a i l a b i l i t y of this states, t  a/.  m a x  a  highly  drug (> « 150% increase)  characterized by increased C  in most  partially induced  subjects  contributed  upon food  significant  studied. by  a  intake  m a x  and A U C  The effect  transient  and the  of  increase  possible  increase  in  in the fed vs o r a  i,  the fasted  with no change  food on this in  oral  liver  interference  in  drug was  blood with  flow  hepatic  clearance induced by food components.  1.7 Effect of A l t e r a t i o n of Protein Binding and Blood Flow on Total Body Clearance  Hepatic blood flow, clearance  are  the  plasma protein binding and hepatic  three  important  factors  which  disposition of drugs that undergo extensive f i r s t - p a s s  intrinsic  determine  the  metabolism.  The  elimination of most drugs from the body involves  the processes of both  metabolism  For many drugs  undergo  (biotransformation)  extensive  clearance  and  metabolism,  is n e g l i g i b l e ,  excretion.  i.e.,  high  and the total  clearance  drugs,  body clearance  is  the  that renal  approximately  equal to hepatic clearance: C l j g « C l ^ . When the l i v e r is considered as a w e l l - s t i r r e d compartment with concentration of drug equilibrium clearance,  with  the  effluent  blood,  protein binding and hepatic  the  relationship  in the among  C1  H  = Q  int  f  B  (1)  H  %  +  c l  int  f  B  in  hepatic  blood can be described by  following equation (Pang and Rowland, 1977; Pang, 1980):  c 1  liver  the  12  where CI^ is hepatic clearance;  is hepatic blood flow; C l  is the  i n t  intrinsic clearance which is used to describe the ability of the liver to remove drug in the absence of flow limitations and it reflects the inherent ability of the  liver to  irreversibly remove drug by all  pathways; fg is the free fraction of the drug.  For high clearance drugs, (fy « C I j  nt  fg, therefore CI^ « Q^. In  this case, drugs are so avidly extracted by the liver that binding to plasma proteins does not limit their removal, thus total body clearance is not affected by alterations in plasma protein binding. hepatic  However, the  clearance is hepatic blood flow dependent (Gibaldi and Perrier,  1982).  For low clearance drugs,  » Cl^  n t  fg, and CI^ « C l ^ fg.  This  indicates that the intrinsic elimination processes responsible for the removal of these drugs are very weak, such that only unbound drug in the plasma can be cleared. clearance  is  Hepatic extraction and consequent  hepatic  limited by the extent of plasma protein binding and  independent of hepatic blood flow (Gibaldi and Perrier, 1982).  Steady-state plasma concentrations,  volume of distribution and  elimination half-life are affected by protein binding displacement from blood and/or tissue  proteins  Mackichan, 1984 and 1989).  (D'arcy and McElany,  1982  and 1983;  The relationship between clearance and  steady-state plasma concentration can be described by the following equations:  13  C  FD = ------  s s  T  C1  (2) T B  Where T is the dosing interval and CIjg is the total body clearance.  1.7.1  Effect of Binding Displacement on High Clearance Drugs  As  mentioned  clearance  before,  for  high  clearance  is approximately equal to total  drugs,  the  body clearance.  Also, the  hepatic clearance is limited by hepatic blood flow and is not by the degree of protein binding.  affected  Therefore, the total body clearance  of high clearance drugs would remain the  same upon a change  degree of protein binding induced by displacement. state total  hepatic  plasma drug concentration  in the  Since the steady-  is determined by the total body  clearance under fixed dosing regimen (Equation 2), changes in the degree of protein binding as a result of binding displacement has no effect on steady-state total  drug concentration.  drug concentration  is  equation:  = fg C  increase  C  s s  f  r e e  in free  concentration.  affected  fraction This  is  s s  .  by protein  binding according  Binding displacement  and the of  However, the steady-state free  consequent  clinical  will  increase  significance  for  to  the  lead to an in free drug a number of  reasons.  It is well believed that only the free drug is the pharmacological active species as free drug can pass through cell membrane, reach the receptor  sites  Accordingly,  and exert pharmacological  the  pharmacological  activity  response  is  (Goldstein,  likely  to  be  1949). better  14  correlated with free drug concentration than total concentration. drugs  with  narrow therapeutic  indices,  fluctuations  in  free  For drug  concentration can lead to either toxic effects or subtherapeutic drug levels depending upon circumstances. is  based  on  total  drug  concentration may well drugs.  When therapeutic drug monitoring  concentration,  be undetected  the  in the  change case of  in  free  drug  high clearance  Polydrug therapy is frequently used in the treatment of disease,  therefore, instances,  drug  binding  displacement  interactions  be important (Mackichan, 1989).  It is,  can,  thus,  in  some  important to  study the displacement effect on free drug concentration in order to modify dosage regimens.  It  is  also  important to  characterize drug  plasma protein binding in order to predict the possible alteration in binding and, hence the effect,  in the presence of coadministered drugs,  of various disease states which alter plasma protein concentration.  1.7.1.1 Displacement Mechanisms: Drugs and Endogenous Substrate  Displacement competitive.  If  involving two  drugs  drugs  can  be  are  bound  at  competitive the  same  and site  nonon  a  macromolecule, there will be a simple competition between the drugs for these sites; compounds with a high affinity will displace drugs with a lower affinity at the same binding sites. by  another  will  depend  competitive displacement  on  the  The displacement of one drug  displacer's  can occur if  concentration.  one drug changes  Non-  the physical  chemistry of the macromolecule to which it binds and induces tertiary conformational changes in the macromolecule itself.  15  Endogenous substances which can be elevated during certain disease states may compete with drugs for plasma binding sites. shown  that  administration  phenytoin (Craig et propranolol level  al.,  (Wood et  as a result  of  alters  the  plasma  binding of  1976), quinidine (Kessler et a?.,  al.,  of  heparin  It has been  1979 a,b).  in vitro  1979) and  The increased free fatty  acids  hydrolysis of plasma triglycerides by  lipases after in vivo administration of heparin is responsible for the observed changes in binding.  Although such an interaction is unlikely  to give rise to adverse effects in patients, such changes in binding may distort  pharmacokinetic data derived from free  drug serum levels  in  subjects using a heparin lock for blood sampling.  1.7.1.2 Selection  of  Protein Solutions  as  Drug Binding Displacement  Media  Albumin and AAG are, quantitatively speaking, the most important proteins in drug binding in human blood. give  detailed  information  about  capacity and binding sites.  Isolated protein solutions can  binding,  such  as  binding  Compared with isolated protein  affinity, solutions,  serum and whole blood contain more ligand binding macromolecules and, therefore,  possess  higher  buffer  capacity  upon binding  particularly when the ligand binding is nonspecific. better  to  displacement  use  isolated  AAG and  albumin  interaction mechanism(s)  Therefore, it  solutions  fraction of high clearance drug is  agent in serum or whole blood, will  to  define  is the  and using serum as well as whole  blood to find the c l i n i c a l l y important displacing agents. free  displacement  the  increased,  Only when the  by the displacing  interaction be of  potential  clinical  significance,  since drug and/or its  metabolite(s) exists  in  whole blood in physiological conditions.  1.7.1.3 Quantitative Analysis of Drug Protein Binding  Drug protein binding is one type of a 1igand-receptor interaction. For reversible drug protein binding, equilibrium exists among free drug [D], unbound protein [P] and drug protein complex [DP] as shown below:  [D] + [P] = * : [ D P ]  K2  (3)  Where KI and K2 are the absolute association and dissociation constants, respectively.  At equilibrium, the relationship between [D],  [P] and  [DP] can be described by the following equation:  Ka = K1/K2 = [DP]/([D][P])  (4)  Where Ka is the apparent or intrinsic association constant.  Using r as  the molar ratio of drug bound to total protein, 7.e. r = [DP]/([DP] + [P]), substituting [P] with ([Pt] - [DP]) ( [Pt] is the total protein concentration), the following equation is derived: Ka[D][Pt] [DP] =  1 + Ka[D]  (5)  If more than one number of binding sites exist per protein molecule, the following equation is used:  17  nKa[D][P] [DP] =  (6)  1 + Ka[D]  This is the equation to describe the simplest one ligand binding to one group of mutually noninteracting binding sites.  With the mathematical  manipulation of Equation 6, a few equations can be derived: 1 — - =  r  1  n[D]Ka  1 + —  (7)  n  r/[D] = nKa - rKa  (8)  [DP]/[D] = n[Pt]Ka - [DP]Ka  (9)  Equation 7 describes the Klotz reciprocal plot (Svensson et al., Klotz,  1983), while equation 8 is  (Scatchard,  1949), equation 9 is  (Rosenthal, 1967).  1986;  used to create the Scatchard plot used to produce the Rosenthal  From these plots,  plot  the number of binding sites and  the intrinsic association constant can be determined. The Klotz reciprocal plot is similar to the Lineweaver-Burke plot (Svensson et al.,  1986; Klotz,  1983)  used in enzyme kinetics.  In the  presence of a pure competitive inhibitor for protein binding, the slope of this plot will be altered by a factor of (1 + [I]/Ki).  Where [I]  the concentration of inhibitor and Ki is the inhibitory constant. Ki value can thus be determined from the following equation:  is The  18  (10)  Slope = (Uninhibited Slope) ( 1 + [I]/Ki) 7  .e. Ki =  PHSlope) (11)  Slope - Uninhibited Slope  Where slope and uninhibited slope are the slopes in the Klotz reciprocal plots  in the presence and absence of  inhibitor, respectively (Engel,  1988).  1.7.2  Effect of an Increase in Liver Blood Flow on the Bioavailability  of High Clearance Drugs  Increases  in blood flow can be classified  constant increases and transient increases.  into two  categories:  The pharmacokinetic changes  as a consequence of constant increase in liver blood flow includes: a) ,  reduction in tj/2 due *° the augmentation in total body clearance (Cljg) (assuming the volume of distribution does not change);  b) .  alteration in AUC ^ due to the following reasons: ora  Equation  12  describes  the  relationship  between  oral  bioavailability (AUC -|) and systemic availability (F) and total body ora  clearance (Clyg), where the systemic availability can be expressed by Equation 13  AUC  oral  =  (  F  (12)  °ose)/Cl  F = 1-E = 1 - C l / ( Q + C l int  i n t  ).  (13)  19  As  the  Cljg  increases,  F increases,  depending on the  magnitude of  changes, the AUC -| may increase, decrease, or not change (Gibaldi and ora  Perrier, 1982; McLean et al.,  If food  1978).  the increase in liver blood flow is transient, as seen after  intake,  the magnitude of  increase  in systemic  availability  is  greater than total body clearance, and a net increase in AUC -| will be ora  observed (Gibaldi and Perrier, 1982; McLean et al.,  1978).  1.8 Indocyanine Green Clearance as an Indicator of Hepatic Blood Flow  One possible partial explanation for the interaction between food and  PF has been a transient  increase  suggested that food components well (Axelson et al., this study.  1987).  in Q^, although  it  also was  interferred with hepatic clearance,  However, no measurement of  as  was made in  There are two main obstacles in the measurement of liver  blood flow in human: the dual nature of hepatic supply: one by hepatic artery,  one by portal vein,  liver circulation.  and the relative  inaccessibility of  the  Before 1945, all the hepatic blood flow measurements  made were highly invasive.  In 1945, Bradley and his colleagues employed  the Fick principle to measure liver blood flow in man using the dye: bromosulphthalein (BSP), a substance which is into the bile. The  methods  techniques,  removed by  hepatocytes  Thereafter, a series of techniques has been developed. can  be  such as  classified  into  indocyanine green  three  categories:  clearance,  clearance  indicator dilution  techniques, such as injection of radio labelled ( C r ) red blood cells, 15  and  Doppler techniques  which using ultrasound to measure blood flow  20  (Bradley et a l . , 1974; Ohnhaus, 1979; Baker, 1978).  Table 1 l i s t s the  available methods. Table 1.  Methods for the determination of hepatic blood flow  CLEARANCE TECHNIQUES Hepatocyte excretory clearance Bromsulthphalein Indocyanine green I-Rose Bengal Hepatocyte metabolic clearance Ethanol Galactose Reticuloendothelial particle clearance •^P-chromic colloid 198 S^Fe-saccharate 99 Tc-sulfur colloid ^^I-heat denatured serum albumin Inert gas 133 131  Au  m  Xe  INDICATOR DILUTION TECHNIQUES serum albumin ^^Cr-red blood cells ULTRASOUND TECHNIQUE (Adapted from Bradley et a l . , 1974)  The clearance technique is one of the most convenient methods to measure blood flow.  In this technique, there are several  for the test substance or indicator.  requirements  It should:  1.  distribute only within the vascular space,  2.  only be removed from the body by the liver with an extraction  21  ratio ~1, 3.  not participate in enterohepatic circulation,  4.  not be toxic at the doses employed,  5.  and  its  concentration  in  blood  should  be measurable  with  sufficient accuracy (Paumgartner, 1975).  Indocyanine green (ICG), a tricarbocyanine dye (Figure 4),  is one  of the substances that can be used for measurement of blood flow.  It  was introduced into clinical medicine by Fox et al.(1957a; 1957b; 1960). Theoretically, based on its molecular weight of 775 and its two polar sulphonic acid groups,  it was seemed very likely that  biliary excretion into feces (Levine, 1978). al.  ICG undergoes  Experimentally, Wheeler et  in 1958 found that more than 97% of the administered dose of ICG can  be recovered in unaltered form in the bile of dog. uncomplicated gall  In patients with  bladder disease undergoing cholecystectomy,  Meijer  found that the recovery of unchanged ICG in bile over 18 h was 80% of the dose after i.v. al.,  1988).  period  Extrapolation of biliary excretion rate curves after that  indicated  subsequently,  injection of either 0.5, 1.0 or 2.0 mg/kg (Meijer et  that  at  least  which means that  these experimental circumstances,  5% of  the  dose would  be  excreted  recovery was almost complete.  Under  urinary excretion and metabolism did  not occur to a significant extent with biliary excretion accounting for the largest  proportion of the elimination process  for the dye.  The  volume of distribution was close to plasma volume indicating that the distribution of ICG was primarily within the vascular space. appears to be eliminated from blood almost exclusively  Thus, ICG  by the liver.  Uptake of ICG by peripheral tissue, kidneys, or lungs is negligible in  22  Figure 4. Chemical structure of indocyanine green (ICG).  23  either human or experimental animals (Cherrick et al., a7.,1960; Ketterer et al., 1959; Reubi et al., relatively low.  1959; Leevy et al.,  1966; Wheeler et al.,  1960; Humton et  1963; Rapaport et  1958).  The toxicity of ICG is  Given in high doses up to 5 mg/kg body weight,  significant toxic effects were observed in human (Leevy et al., Therefore,  it  al.,  has been concluded that  the disappearance of  no  1967). ICG from  blood is a reflection of hepatic removal and the dye has been determined to be safe for use in the evaluation of liver function and blood flow.  1.9 Rationale  1.9.1. Rationale for protein binding displacement study  Hypothesis:  Displacement of PF and 5-hydroxypropafenone from plasma proteins such as AAG and albumin by other highly bound antiarrhythmic drugs and ASA may result  in  an  increase  in  free  drug  concentration  with a  resulting increase in therapeutic effect and/or toxicity.  Propafenone volunteers  is  highly protein bound in serum of normal healthy  (Chan et al.,  1989)  and the binding is proportional to the  concentration of AAG (Gill is et al., drugs,  PF undergoes  extensive  1985).  hepatic  Like most antiarrhythmic  metabolism with  excreted unchanged in urine and feces (Hege et total  systemic  clearance  (1.14  L/min  al.,  less  1984).  in healthy volunteers  than 1% The high and  1.03  L/min in arrhythmic patients) numerically approaches liver blood flow  24  (1.5  L/min  ) (Hollmann et  al.,  1983; Arboix et  a/.,  1985  ), which,  together with the extensive metabolism, results in the characterization of  PF as  a liver  clearance drug.  blood  that  highly  extracted  and high  Moreover, PF appears to show a steep dose-response  relationship (Connolly et indicates  flow-dependent,  a small  al.,  1983b; Siddoway et al.,  change  1984a).  in drug concentration will  This  result  in  greater change in response when compared with drugs that display normal dose-response relationships. concentration  To date,  and therapeutic  good correlation between serum  response  of  propafenone  has  not  been  developed and large individual variations in therapeutic concentration of PF exist in patients (Harron and Brogden, 1987). free  drug  concentration  and  the  Fluctuation in PF  antiarrhythmic  activity  of  5-  hydroxypropafenone and possible other metabolites most likely contribute to the observed variation in therapeutic drug concentrations.  The antiarrhythmic activity experimental  models has been well  of  5-hydroxypropafenone  documented.  in various  These models  include  isolated cardiac strips and coronary ligation in dogs (Von Philipsborn et al.,  1984; Thompson et al.,  1985; Delgado et al.,  rat heart preparation (Oti-Amoako et a7.,1990).  1987), and isolated  In human, comparison of  the pharmacodynamic effects of intravenous and oral propafenone showed that the presence of relatively large amounts of 5-hydroxypropafenone after oral PF administration prolonged further the PQ interval and QRS duration  than  intravenous  PF administration where the  hydroxypropafenone was negligible  (Haefeli  et  al.,  amount of  1990).  5-  Moreover,  poor metabolizer patients who are not able to form substantial amounts of  5-hydroxypropafenone require higher propafenone concentrations  for  25 arrhythmia  suppression  than  extensive metabolizers  levels of this metabolite (Siddoway et a / . , 1987).  with  high plasma  Although there is no  data available to display the direct electrophysiologic  effect of  hydroxypropafenone  the  in  human,  pharmacological effect.  it  contributes  to  5-  observed  Therefore, it is important to study its protein  binding behavior to obtain a better understanding of the dose-response relationship observed after PF administration.  A fluctuation  in PF free drug concentration could be induced by  dose-dependent protein binding of the drug itself, interactions with coadmininstered drugs.  Quite frequently,  is used in clinical treatment of dysrhythmias. drugs  coadministered  with  PF,  or by displacement  phenytoin,  polytherapy  Among all the lidocaine,  possible  propranolol,  quinidine and verapamil are the most likely potent displacers due to the high plasma protein binding characteristics of these drugs. is  approximately  25-50% bound  to  AAG, phenytoin  Lidocaine  93% to  albumin,  propranolol 55% to albumin and 58% to AAG, quinidine 60-90% to albumin and AAG, and verapamil is 90% bound to plasma protein (Edcardsson and Olsson, 1987; Evans et al., permanent acetylation  1986).  ASA binds to albumin and results in  of the albumin molecule  (Hawkins et al.,  1968).  Also, since ASA is such a commonly used drug, it seemed appropriate to study the displacement effect of this drug on the binding of both PF and 5-hydroxypropafenone. /zg/ml  are:  1.4-6.0  The therapeutic concentrations for these drugs in for  lidocaine,  10-18  for  phenytoin,  0.05-1  propranolol, 2-5 for quinidine, 0.06-0.2 for verapamil (Latini et 1990) and 50-400 for ASA (Evans et al., concentrations  1986).  for al.,  The maximum therapeutic  for these displacers will be used.  Purified human AAG  26 and albumin, serum and whole blood will be used as equilibrium dialysis media  due  to  the  fact  that  different  media  exhibits  different  "buffering" capability with respect to displacement.  1.9.2. Rationale for Reproduction of the HPLC Analysis Method of ICG in Serum Samples  Several HPLC methods for the measurement of ICG concentrations in serum are  available  with  relative  Thiessen, 1982; Hollins et al., 1984;  Christie et  objective suitable  to for  al.,  1986;  establish the  sensitivity  1987; Dorr et al., Burns et  al.,  of  (Rappaport and  1989; Donn et  1989).  an HPLC measurement  measurement  course of experiments  high  It will  method  which  ICG serum concentration  al.,  be our  would be during the  designed to determine hepatic blood flow under  different experimental conditions.  1.10 OBJECTIVES 1.  To study the effect of buffer strength on the free fraction of PF and 5-hydroxypropafenone in solutions of purified human albumin, AAG and whole blood.  2.  To study the effect of heparin and EDTA on the free fraction of PF and 5-hydroxypropafenone.  3.  To  characterize  the  binding  of  5-hydroxypropafenone  to  purified human AAG. 4.  To study  the  propranolol,  displacing quinidine,  effects verapamil  of  phenytoin, and  human AAG, albumin, serum and whole blood.  ASA  in  lidocaine, purified  To  characterize  the  nature  of  the  protein  binding  displacement in purified human AAG solution. To reproduce and attempt to improve upon an HPLC assay method for ICG.  28  2. EXPERIMENTAL  2.1 Materials and Supplies  2.1.1 Drugs, Metabolites and Internal Standards  Propafenone 1115  hydrochloride,  hydrochloride  (internal  (internal  standard for  5-hydroxypropafenone  standard  5-hydroxypropafenone  hydrochloride were the kind gifts (Markham,  Ontario,  quinidine sulphate,  for  Canada).  of Knoll  hydrochloride, L i -  PF quantitation), quantitation)  L i-1548  and verapamil  Pharmaceuticals Canada Inc.  ASA, lidocaine,  phenytoin  sodium  salt,  propranolol hydrochloride and indocyanine green (ICG)  (90% pure) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Diazepam was obtained from the Vitamin and Chemical Divison, Hoffmann-La Roche Limited (Montreal, Canada).  2.1.2 Chemicals and Reagents  Triethyl amine  (TEA)  (Sequanal™  grade)  and  heptafluorobutyric  anhydride  (HFBA) were obtained from Pierce Chemical Co. (Rockford, IL,  U.S.A.).  ACS reagent  Scientific  Co. (Fair Lawn, NJ, U.S.A).  phosphate,  disodium  grade sodium hydroxide was purchased from Fisher  phosphate,  sodium  ACS reagent grade monopotassium carbonate,  sodium  chloride  and  potassium carbonate were purchased from BDH Chemicals (Toronto, Ontario, Canada)  while  ACS reagent  grade  hydrochloric acid  American Scientific and Chemical (Seattle, WA, U.S.A.).  was  obtained  from  29 2.1.3 Proteins  Purified human AAG, and albumin were purchased from Sigma chemical Co. (St. Louis, MO, U.S.A.).  2.1.4 Solvents  Toluene, acetonitrile Laboratories  isopropyl  (ultraviolet Ltd.  alcohol  (distilled  (UV) cutoff  (Georgetown,  in  glass)  and HPLC grade  190 nm) were obtained from Caledon  Ontario,  Canada).  HPLC  grade  dichloromethane and methanol were obtained from BDH Chemicals (Toronto. Ontario,  Canada).  obtained  via  Deionized d i s t i l l e d water and HPLC grade water were  a Milli-R0^  Water System  (Millipore Corp.,  Bedford, MA,  U.S.A.).  2.1.5 Gases  Ultra purchased Canada).  high  purity  (UHP) hydrogen  from Matheson Gas Products  and argon/methane  Canada  Ltd.  (95:5) were  (Edmonton,  Alberta,  Nitrogen, U.S.P. and medical air (breathing grade) were obtained  from Union Carbide Canada Ltd. (Toronto, Ontario, Canada).  2.1.6 Equilibrium Dialysis Device  Plexi-Glass^ dialysis dialysis.  cells  (1.0  ml)  were  used  for equilibrium  Cellophane dialysis membrane "sacks" (molecular weight cutoff of  12,000 Daltons) were obtained from Sigma Chemical Co. (St. Louis, U.S.A.).  30  2.1.7 Other Supplies  Pyrex^  disposable  screw-top  glass  culture  tubes  (10  ml),,  with  polytetrafluoroethylene (PTFE) lined screw caps were purchased from Canlab (Vancouver,  B . C . , Canada).  Veinsystems™ Butterfly^-19  purchased from Abbott Laboratories, blood collection (Mississauga,  Ltd. (Montreal, Canada).  tubes were obtained  Ontario, Canada).  INT Cannula were  from Becton  Vacutainer^  Dickinson Canada Inc.  Syringe sample f i l t e r and C-18 extraction  columns (100 mg/1 ml) were purchased from Phenomenex (Torrance, California, U.S.A.).  2.2 Columns  2.2.1 GLC Column  A bonded-phase fused-silica used for all GLC analyses.  capillary column, 25 X 0.31 mm I.D. was  The stationary phase of the column was cross-  linked 5% phenylmethylsilicone with a film thickness of 0.52 fim and a phase ratio of 150 (Hewlett-Packard, Palo Alto, CA, U.S.A.).  2.2.2 HPLC Column  An ODS-Hypersil reverse phase column,  100 mm X 2.1  mm I.D.  with  s i l i c a particle size of 5 fim (Hewlett Packard, Toronto, Ontario, Canada), was used for HPLC analyses of ICG.  31  2.3  Equipment  2.3.1  Gas-Liquid  GLC  a n a l y s e s were performed  gas-liquid capillary  Chromatography  chromatograph,  equipped  on a Model with  5830A H e w l e t t - P a c k a r d ( H P )  a Model  18835B  i n l e t s y s t e m , a ^ N i e l e c t r o n - c a p t u r e d e t e c t o r , a Model 18850A GC  t e r m i n a l f o r c h r o m a t o g r a p h i c p e a k i n t e g r a t i o n a n d a Model sampler.  split/splitless  A splitless  employed.  7671A a u t o m a t i c  i n j e c t i o n mode u s i n g a f u s e d - s i l i c a i n l e t l i n e r was  Thermogreen ^ T  LB-2 s e p t a ( S u p e l c o , I n c . , B e l e f o n t e , PA, U.S.A.)  c h a r a c t e r i z e d by v e r y l o w - b l e e d a t h i g h i n l e t t e m p e r a t u r e s were used. septum  was  changed  routinely  to  prevent  leakage  following  The  repeated  puncturing during automatic injections.  2.3.2  High-Performance Liquid  A HP Model  Chromatography  1090 l i q u i d c h r o m a t o g r a p h  d i o d e - a r r a y UV d e t e c t o r a n d a Model  e q u i p p e d w i t h a HP Model  1040A  310 HP c o m p u t e r f o r d a t a a n a l y s i s was  used.  2.3.3  Spectrophotometer  A H e w l e t t P a c k a r d 8 4 5 2 A d i o d e a r r a y s p e c t r o p h o t o m e t e r was u s e d f o r quantitation o f albumin.  32  2.3.4 Miscellaneous  Other equipment used were: Eppendorf micropipettes, (type  37600,  Sybron),  an  incubation  oven  (isotemp,  a vortex mixer  model  350,  Fisher  Scientific Industries, Springfield, MA, U.S.A.), an IEC model 2K centrifuge (Damon/IEC Division,  Needham Hts.,  MA, U.S.A.),  and a METTLER AE 163  balance (Mettler Instrumetente AG, CH-8606 Greifensee, Switzerland).  2.4 Preparation of Stock and Reagent Solutions  2.4.1 Drug, Metabolites and Internal Standards for GLC Analysis  Propafenone hydrochloride was accurately weighed deionized  distilled  water  using  sequential  and dissolved  dilution  to  a  in  final  concentration of 100 ng/ml (11.07 mg of PF hydrochloride is equivalent to 10 mg of PF free base).  5-Hydroxypropafenone dissolved dilution  hydrochloride  in methanol:deionized to  a  final  hydroxypropafenone  distilled  concentration  hydrochloride  is  of  was  accurately  water 100  (1:9)  ng/ml  equivalent  to  weighed  using  sequential  (11.02 10  and  mg mg  of  5-  of  5-  dissolved  in  hydroxypropafenone free base).  Li-1115 deionized  hydrochloride  distilled  was  water  accurately  using  weighed  sequential  and  dilution  to  a  final  concentration of 200 mg/ml (11.11 mg of Li-1115 hydrochloride is equivalent to 10 mg of Li-1115 free base).  33  Li-1548 deionized  hydrochloride  distilled  was  water  accurately  using  weighed  sequential  and  dilution  dissolved to  a  in  final  concentration of 200"ng/ml (11.06 mg of Li-1548 hydrochloride is equivalent to 10 mg of Li-1548 free base).  All  stock  and working solutions  were protected  wrapping the glass containers with aluminum f o i l .  against  light by  Solutions were stored at  4°C after preparation for up to a maximum of four months.  2.4.2 Drugs for Equilibrium Dialysis  Propafenone  and 5-hydroxypropafenone  hydrochloride were accurately  weighed and dissolved in pH 7.4 isotonic phosphate buffer (0.067 M and 0.1 M) to produce a concentration of 10 //g/ml for use in the protein binding displacement study.  The solutions were stored at 4°C after preparation for  up to 4 months.  5-Hydroxypropafenone hydrochloride (4.96 mg) was accurately weighed and dissolved  in pH 7.4  isotonic  stock solution of 45 //g/ml. obtained  from this  stock  phosphate buffer (0.1  M) to produce a  Solutions ranging from 50-40,000 ng/ml were solution  using  sequential  dilution.  These  solutions were used for characterization of binding of 5-hydroxypropafenone to purified human AAG.  Verapamil hydrochloride was accurately weighed and dissolved  in pH  7.4 isotonic phosphate buffer to produce a concentration of 10 //g/ml (1.09  34  mg of  verapamil hydrochloride is equivalent  to  1 mg of verapamil  free  base).  Propranolol hydrochloride was accurately weighed and dissolved in pH 7.4  isotonic phosphate buffer to a concentration of 10 //g/ml (1.14 mg of  propranolol hydrochloride is equivalent to 1 mg of propranolol free base).  Lidocaine (6 mg) was accurately weighed and dissolved in a few drops of  1 N HCl and diluted  with  pH 7.4  isotonic  phosphate  buffer  to a  concentration of 60 //g/ml.  Quinidine sulfate was accurately weighed and dissolved 7.4  in 0.1 M pH  isotonic phosphate buffer to a concentration of 50 //g/ml (5.755 mg of  quinidine sulfate is equivalent to 5 mg of quinidine free base).  These solutions  (verapamil,  propranolol, lidocaine  and quinidine)  were stored in light-tight containers at 4°C for up to 6 weeks.  Aspirin was phosphate  buffer  accurately weighed  and dissolved  (containing  PF or  either  in pH 7.4  isotonic  5-hydroxypropafenone)  concentration of 400 //g/ml (20 mg aspirin was weighed).  to  a  Solutions were  prepared fresh prior to dialysis.  Phenytoin sodium was accurately weighed isotonic  phosphate  buffer  containing  PF or  and dissolved  in pH 7.4  5-hydroxypropafenone  to  a  concentration of 18 //g/ml (1.956 mg of phenytoin sodium salt is equivalent to 1.8 mg of phenytoin free acid).  35  The  final  solutions  for the displacement  studies were prepared by  diluting the respective stock solutions with pH 7.4 isotonic buffer. solutions  include PF combined with propranolol; 5-hydroxypropafenone with  propranolol;  PF with lidocaine;  5-hydroxypropafenone with lidocaine; PF  with quinidine; 5-hydroxypropafenone with quinidine. used  for  These  displacement  studies,  the  1 /zg/ml  and 0.5  In a l l the solutions  concentrations  PF and  respectively.  5-  hydroxypropafenone  were  concentrations  propranolol, verapamil, lidocaine and quinidine were  of  #g/ml,  of  The  1.0, 0.2, 6.0 and 5 //g/ml, respectively.  2.4.3 Purified Protein Solutions  AAG was accurately weighed and dissolved in pH 7.4 isotonic phosphate buffer to a final concentration of 90 mg/dL.  Albumin was accurately weighed  and dissolved  in pH 7.4  isotonic  phosphate buffer to a final concentration of 4.5 g/dL.  2.4.4 Drug and Internal Standard Solutions for HPLC analysis  Indocyanine Green was accurately weighed and dissolved in HPLC grade methanol to a final concentration of 1  ug/m].  Diazepam was accurately weighed and dissolved  in either HPLC grade  methanol (I.S.a) or HPLC grade acetonitrile (I.S.b) to a concentration of 10  ug/m].  36  2.4.5 Reagent Solutions  Triethylamine (0.03%, v/v) was prepared by diluting TEA (75 a]) with toluene (250 ml).  Four or five pellets of NaOH were added to this solution  to keep the solution dry.  Sodium hydroxide  (NaOH) 1 M and 5 M solutions  were prepared by  dissolving NaOH pellets in deionized d i s t i l l e d water.  Sodium carbonate  (Na2C03) 0.1  M solution  and potassium  carbonate  (K2CO3) 5 M solution were prepared by dissolving Na2C03 and K2CO3 powder in deionized d i s t i l l e d water.  Hydrochloric acid (HC1) 1 M was prepared by diluting concentrated (37%) ACS reagent grade HC1 in deionized d i s t i l l e d water.  Isotonic  phosphate  buffer  accurately weighing  1.80  disodium  (Na2HP04);  phosphate  (pH  7.4,  0.067  M) was  g of monopotassium phosphate 4.20  g  of  sodium  prepared by  (KH2PO4);  chloride  7.40 g  (NaCl) and  dissolving in deionized d i s t i l l e d water to a final volume of 1 l i t e r .  Isotonic phosphate buffer (pH 7.4, 0.1 M) was prepared by dissolving 1.8 g of NaCl, 11.4 g of Na HP0 and 2.7 g of KH P0 in deionized distilled 2  4  2  4  water to a final volume of 1 l i t e r .  Phosphate buffer (pH 6.0, 0.067 M) was prepared as follows: 4.70 g of  37  Na2HP04 and 4.60 g of KH2PO4 were accurately weighed  and dissolved  in  deionized d i s t i l l e d water to a final volume of 500 ml, respectively.  The  final  in a  buffer solution was obtained by combining these two solutions  ratio of 9:1 (KH P0 : Na HP0 ). 2  4  2  4  Phosphate buffer (pH 6.0, 0.005 M) was prepared by dissolving 0.605 g of KH P0 and 0.079 g of Na HP0 to one l i t e r of HPLC grade water. 2  4  2  4  Citrate buffer (pH 4.0, 0.005 M) was prepared by dissolving 0.596 g of c i t r i c acid and 0.270 g of disodium phosphate in one l i t e r of HPLC grade water.  Citrate buffer (pH 4.4, 0.005 M) was prepared by dissolving 0.52 g of c i t r i c acid and 0.62 g of disodium phosphate  in one l i t e r of HPLC grade  water.  2.5 Equilibrium Dialysis Procedure  The equilibrium dialysis was performed in the following manner: The cellophane dialysis membrane was boiled in distilled water for 1 hour and then soaked in phosphate buffer (pH 7.4) for a minimum of 1 hour, maximum of 2 days (kept at 4°C) before mounting into the dialysis cells.  Extreme  care was taken in order not to touch the surface of the membrane during the process.  Equal volumes (0.8 ml) of drug (in isotonic phosphate buffer) and  study fluid (protein solution, serum or blood) were added to the respective sides of the dialysis cells, unit  was dialysed  at 37°C  separated by the cellophane membrane. for 8 hour to  reach equilibrium.  The  During  38  equilibrium dialysis, the cell was rotated automatically by a motor rotator at a speed of 14 rpm.  Before and after equilibrium dialysis, both pH and  volume of the sample were measured in order to examine volume shift or pH change.  Aliquots of buffer and biological  fluid were then subjected  to  GLC-ECD analyses.  2.6 Protein Binding of 5-Hydroxypropafenone to Purified Human AAG  The characterization of the protein binding of 5-hydroxypropafenone to purified human AAG was studied by dialyzing various concentrations of 5hydroxypropafenone against the solution of AAG. AAG were dissolved  in pH 7.4  isotonic  phosphate  5-Hydroxypropafenone and buffer  (0.1  M).  The  concentrations of 5-hydroxypropafenone used were: 50, 100, 150, 200,  500,  1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 45,000 ng/ml.  Five replicate  measurements were made at each concentration.  2.7 Rosenthal Analysis of the Binding of 5-Hydroxypropafenone to Purified Human AAG  The  characterization  of  the  binding  parameters  hydroxypropafenone to purified AAG were examined using Rosenthal (Rosenthal, 1967).  of  5-  analysis  The molar concentration of bound 5-hydroxypropafenone  was plotted against the concentration ratio of bound to free.  2.8 Scatchard Plot of 5-Hydroxypropafenone Binding to AAG  A Scatchard plot was also used to interpret the binding data, where  39  the moles of drug bound per mole of [r'/free  drug  concentration]  protein  (Scatchard,  (r')  1949).  was plotted From this  against  plot,  the  classes of binding sites can be obtained.  2.9  Free Fraction  vs Total  Concentration Plot of 5-Hydroxypropafenone  Binding to AAG  The 5-hydroxypropafenone binding data was further treated by plotting the free fraction vs total concentration of 5-hydroxypropafenone. fraction is the concentration ratio of free drug to total drug. and  free  drug  concentration  hydroxypropafenone  in  the  were  obtained  AAG solution  and  by  The free Both total  GLC analysis  buffer  of  solution  5-  after  equilibrium dialysis.  2.10 Protein Binding Displacement Studies  In  the  protein  binding  hydroxypropafenone were dissolved  displacement  studies,  in phosphate buffer,  PF  and  5-  respectively,  and  spiked with various displacing agents or their phosphate buffer subsequently. biological  The combined solution was then dialysed against  fluids  (purified AAG, albumin, serum or whole  solutions various  blood).  The  dialysis was performed in either three or four replicates.  2.10.1 Effect Of Buffer Strength on the Protein Binding of Propafenone and 5-hydroxypropafenone  The influence of ionic strength of phosphate buffer on the binding of  40  PF and 5-hydroxypropafenone  to  human AAG, albumin, or whole blood was  studied at buffer concentration of 0.067 M and 0.1 M. The free fraction of PF and 5-hydroxypropafenone at each buffer concentration was determined and compared.  2.10.2 Effect of Anticoagulant on the Protein Binding of Propafenone and 5hydroxypropafenone  The  effect  of  anticoagulant  on the  protein binding of  hydroxypropafenone in whole blood was determined as follows: containing either phosphate  buffer  heparin or EDTA was dialysed against solutions  containing  Propafenone or 5-hydroxypropafenone analyses.  The  concentration  of  concentration.  free  fraction  was  PF  Whole blood  pH 7.4  isotonic  or  5-hydroxypropafenone.  concentrations  were measured by GLC  calculated  PF or 5-hydroxypropafenone The effect  PF and 5-  of anticoagulant  by  by the  dividing  the  free  corresponding  total  on the protein binding was  observed by comparing the free fraction of PF and 5-hydroxypropafenone in heparinized whole blood to those obtained by using EDTA.  2.10.3 Protein Binding Displacement Study Using Human AAG and Albumin as the Biological Fluids  The phenytoin,  displacing quinidine  agents  included:  and lidocaine.  ASA,  In those  propranolol, studies  verapamil,  involving ASA,  propranolol and verapamil, the concentration of phosphate buffer was 0.067 M, other displacement studies were conducted at a buffer concentration of 0.1  M.  The  free  fractions  of  PF  and  5-hydroxypropafenone  after  41  displacement  were compared to  those  obtained  without  addition  of  the  various displacing agent.  2.10.4 Protein Binding Displacement Study Using Human Serum and Whole Blood as the Biological Fluids  Blood  (the  respectively) volunteers.  blood  was  anticoagul ated  with  heparin  and  EDTA,  and serum were obtained from healthy, young, male Caucasian The heparinized whole blood was collected using the green top  Vacutainers^ which contain 143 USP units of Lithium heparin per tube.  The  whole blood  top  anticoagulated  with  EDTA 'was  collected  using  lavender  Vacutainers which contain 0.048 ml of 15% EDTA (K ) (7.2 mg) and 0.01 mg R  3  of potassium sorbate. the  displacers  When human serum was used as the biological  used  were:  phenytoin,  ASA  and  quinidine  fluid, for  hydroxypropafenone and phenytoin, quinidine and lidocaine for PF. whole blood was used as the biological quinidine and phenytoin for PF.  fluid,  the displacers  5When  used were  The free fractions were compared before  and after the addition of each displacing agent.  2.10.5 Characterization of the Nature of Binding Displacement between PF and Lidocaine and  Interaction  PF and Quinidine in Purified Human AAG.  2.10.5.1 Binding of PF to AAG over the Therapeutic Concentration Range of PF  This study was carried out at PF concentrations 0.6,  0.8,  1.0,  1.5  jug/ml.  The binding of  of 0.1,  PF alone,  0.2,  0.4,  PF spiked with  42  lidocaine (6 //g/ml) or quinidine (5 /zg/ml), were dialysed against an equal volume of AAG solution.  2.10.5.2 Characterization of the Nature of the Displacement  Interaction:  Competitive or Non-competitive  The binding data obtained from the above three studies were analyzed by plotting the reciprocal of the moles of bound drug per mole of protein versus the reciprocal of the concentration of unbound drug (moles).  The  nature of the displacement was then determined from the plot (Webb, 1963).  2.10.6 Measurement of Albumin Concentration in Purified Albumin Solution  The  concentration  of  albumin  in  measured by the method of Lowry, et al. as standard protein. concentration  of  The analytical  albumin  used  for  purified  albumin  solutions  was  (1951) using bovine serum albumin wavelength the  used was 650 nm.  determination  of  PF and  hydroxypropafenone free fraction in 0.067 M and 0.1 M isotonic buffer were 3.80 and 3.90 g/dL, respectively. used for the study of the displacement  The 5-  phosphate  The concentration of albumin  effects  of ASA, propranolol and  verapamil on the free fraction of PF and 5-hydroxypropafenone is 3.64 g/dL.  2.11 Analysis of Propafenone and 5-Hydroxypropafenone  The concentration of PF and 5-hydroxypropafenone in both buffer and biological  fluid compartments of the dialysis cell  GLC-ECD methods of Chan et a7., 1987; 1989.  were analyzed by the  43  2.12 HPLC Analysis of Indocyanine Green (ICG)  2.12.1 Reproduction of Reported HPLC ICG Assay Methods  Rappaport and Thiessen reported the f i r s t HPLC analysis method for ICG (1982). acetonitrile  The method involved precipitation of plasma samples with in a volume ratio of  1.6:1  (acetonitrile:plasma).  After  centrifugation at 1,700 X g for 2 min, the supernatant was injected into the HPLC and detected by UV absorption at 254 nm. 0.4 /xg/ml using 0.25 ml of plasma.  The detection limit was  However, there was no report about the  volume injected and the on-column detection limit was not known.  In order to increase the assay sensitivity, with  fluorescence  detection  fluorometric  determination  a7.(1987).  The sample  were method  also  tried  tried  was  two other HPLC methods on  our  system.  reported  by  one  Hoi 1 ins  et  preparation procedure and the chromatographic  conditions were the same as that of Rappaport and Thiessen.  The excitation  emission maxima were 780 nm and 810 nm, respectively.  An excitation  spectrum and an emission spectrum at an excitation wavelength of 260 nm were obtained within our fluorometric wavelength range.  The detection of  ICG was tried at various excitation and emission wavelength pairs. method of Dorr and Pollack (1989) was also tried on our system.  The They  utilized the same sample preparation procedure as that of Rappaport and Thiessen and the mobile phase was composed of pH 4.0  phosphate buffer  (containing 1% triethylamine) and acetonitrile in a ratio of 2:1).  The  excitation and emission wavelengths were 214 nm and 370 nm, respectively.  44  The on-column detection excitation detection  limit was 3 ng with  spectrum at  these  experimental  20-200 //l  conditions  injected.  was  An  obtained and  of ICG at the reported excitation and emission wavelength were  attempted.-  2.12.2 Modification of the Analysis Method of Indocyanine Green of Christie (1986) and Burns (1989)  The  phase  Christie et  al.  separation  method  (1986) was used  with  ammonium sulphate  in our f i r s t  assay of  modification of the procedure reported by Burns et al. in our sample  preparation.  The following  reported by  ICG.  A slight  (1989) was applied  procedure was  used for  the  0.4,  0.8  preparation of a standard curve:  Standard curve range was from 0.1 to 1 fig (0.1, and 1.0 ug).  0.6,  The corresponding volumes of standard ICG solution (1 /ig/ml)  were added to each tube. 40°C under  0.2,  The solution was evaporated in the water bath of  After evaporation, 100 /zl of serum was added to each tube  and set for half an hour with frequent vortexing to get complete protein binding. acetonitrile  Saline (0.35  (0.35  protein.  ammonium sulphate,  diazepam  solution  (75  ml) were then added to the samples  mixture was then vortexed precipitate  ml,  and centrifuged  The supernatant  fi\)  (I.S.a)  and  in sequence.  The  at 2,500 X g for 10 min to  was  transferred  and mixed with  the mixture was again vortexed and centrifuged.  The  organic phase was then passed through a syringe sample f i l t e r , the filtrate (100 fi\) was diluted with 100 u} of pH 6.0 phosphate buffer, and 80 /zl of this solution was injected into the HPLC.  The mobile phase contained 40 %  45  of acetonitrile and 60 % of pH 6.0 phosphate buffer and methanol in a ratio of 50:4.  The ICG and diazepam (internal standard)  array detection (DAD) at 216 nm.  was measured by diode  A scheme of the procedure is shown in Fig  5.  ICG methanol standard solution evaporation under No ,in water bath (30°C) ICG residue 0.1 ml serum vortexing for 30 min ICG serum solution 0.35 ml saline 75 a\ diazepam solution(I.S.a) 0.35 ml acetonitrile vortexing centrifuging (10 min) discard precipitant  solvent layer ammonium sulphate vortexing centrifuging  discard aqueous layer  organic layer | syringe sample filtration  filtrate I diluted with equal volume of pH 6.0 phosphate buffer  t  inject 80 /il to HPLC Figure 5.  A scheme of extraction procedure of ICG in human serum  46  2.12.3 Comparison of the Chromatograph of Serum Spiked with Indocyanine Green with that of Serum Blank  The chromatograph of serum spiked with ICG and serum blank (volume 0.25  ml)  were  obtained  following  the  same preparation procedure and  compared.  2.12.4 Modification of the Chromatographic Conditions to Minimize the Blank Interference  The modifications phase;  changing pH 6.0  citrate  buffer;  involved were: changing the composition of mobile phosphate buffer  changing the  flow  rate.  in the mobile phase to pH 4.0 None of  these changes could  completely eliminate interference peaks in the chromatogram, but changing the mobile phase composition from 40 % to 39 % of acetonitrile and flow rate from 0.5 ml/min to 0.2 ml/min gave the best minimization of blank interference.  These HPLC conditions were then used for studying extraction  efficiency and post extraction stability.  2.12.5 Extraction Efficiency  Different concentrations of ICG methanol solutions were made (40, 32, 24 and 16 /zg/ml). two sets of tubes.  Each of these solution (25 fi\) was added to each of the To one set,  0.25 ml serum was added to each tube  followed by extraction with acetonitrile. internal  Immediately before f i l t r a t i o n ,  standard solution (60 jul) was added to each tube.  devoid of serum, methanol (40 ji\)  To the  set  and internal standard solution (60 //l)  47  were  added.  procedure.  The samples The  amounts  were of  then  subjected  ICG recovered  to  was  the  same dilution  calculated  from  the  calibration curve of ICG in methanol and compared with the amount added.  2.12.6 Post Extraction Stability  Aliquots of the extracted  serum sample were stored  holders and injected at varies times (10 times, Peak height  ratios  48 h) after preparation.  (ICG/diazepam) and chrbmatograms obtained after  injection were used as indicator of ICG stability. ratio  and  in black vial  chromatogram  indicated  that  there  each  Constant peak height  were  no  ICG degradation  products and ICG was stable up to that injection time.  2.12.7 Animal Preparation  In order to test the u t i l i t y of the assay method to analyze  the  plasma concentration of ICG, a i.v.  injection was made into a rat (Sprague-  Dawley).  with methohexital  The rat was anesthetized  sodium ( i . p . ) .  Two  cannulas (PE50) were surgically implanted: jugular vein and carotid artery. After the rat regained consciousness, ICG solution (0.3 ml, 5 mg/ml) was injected  to the jugular vein followed  heparin saline (100 iu/ml).  by a catheter  flush with 0.3 ml  Blood (0.2-0.3 ml) was taken from the carotid  artery at 1, 3, 5, 8, 10, 15, 30, 45 and 60 min after injection.  The blood  samples were centrifuged at 2,000 X g for 10 min and plasma was immediately collected and ICG concentration analyzed the following day.  48  2.12.8 Analysis of Plasma Samples  Due to the small volume of plasma sample obtained from rat (100 fi]), the assay had to be carried out at microassay level. centrifuge tubes were used for sample preparation. our  laboratory did not have the  in  this  centrifuge  was  As the centrifuge in  appropriate rotor to hold these small  vials, another thermostatic centrifuge was used. force  SIickseal micro-  17,000  X g.  The maximum centrifugal The  deproteinized by the procedure described before.  plasma  sample  was  After centrifugation at  17,000 X g for 3 min, the supernatant was clear such that direct injection on  to  instead  the of  HPLC was possible. diluting  the  In order to detect low  sample  with  normal  concentrations,  saline  (followed  by  deproteinization with acetonitrile), as before, the sample was precipitated with acetonitrile directly. HPLC. ml/min. A  The supernatant (10 fi\) was injected into the  The mobile phase was the same as before, but with a flow rate of 0.5 A scheme of the sample preparation procedure is shown in Figure 6.  standard  curve  was  prepared  using  peak  height  ratio,  the  plasma  concentration of the samples were determined from this standard curve.  49  ICG standard methanol solution Jevaporate under N at room temp 2  ICG residue 50 fi\ blank rat plasma ICG plasma sample at various time after ICG injection  ICG plasma solution  /  80 fi\ acetonitrile contain diazepam (I.S.b) centrifuge at 17,000 X g, 3 min discard precipitant  Figure 6.  inject 10 fi\ supernatant to HPLC  A scheme of the extraction procedure of ICG in rat plasma  2.12.9 The Potential  to  Detect  Low Concentration Samples with the New  Method  One  possible  way  to  increase  sensitivity  concentration is to increase the injection volume. leads to  for  samples  of  low  This approach, however,  higher background contributions to the chromatogram which can  possibly interfere with drug measurement.  With this new method increasing  the injection volume from 10 to 50 /zl was tried.  2.12.10 Solid Phase Extraction Method  Donn et a7.(1984) used solid phase extraction to prepare samples for  50  HPLC  assay  acetonitrile,  of  ICG.  Plasma  samples  first  deproteinized  by  diluted with pH 4.4 citrate buffer, and then loaded onto a  C18 extraction column (100 mg/lml). water twice,  were  After washing with deionized distilled  ICG was eluted by methanol and injected into the HPLC.  The  same method was tried on our system.  2.13 Data Analysis  2.13.1 Statistical Analysis  The free fractions of PF and 5-hydroxypropafenone after spiked with various drugs were compared to  the  corresponding control  values.  For  comparison of two treatment groups, two sample t-test at a significance level  of 5% (p<0.05) was used (Zar, 1984).  For comparison of multiple  treatment groups, two sample t-test combined with Bonferroni inequality at p < 0.05 was used (Meddis, 1984).  2.13.2 Protein Binding Data  For both the Rosenthal  and Scatchard plots,  the computer programs  AUT0AN (Sedman and Wagner, 1976)  and N0NLIN (Metzler, 1974) were used to  obtain the best f i t of the data.  Linear regression was used to test the  linearity of the data.  2.13.3 Indocyanine Green Plasma Clearance  Indocyanine plasma concentration-time profile was analyzed by AUT0AN  51  and two compartment model was the best f i t calculated  by trapezoidal  method and the  for the data.  The AUC™ was  plasma clearance  was obtained  using the following equation: CI = Dose / AUC°°.  52  3.RESULTS  3.1 Volume Shift and pH Change of Equilibrium Dialysis Samples  Both volume and pH of samples on each side of the dialysis cell were measured before and after dialysis.  No significant  change was observed in the dialysate  using the two sample t-test at p <  0.05.  volume shift  and pH  The volume shift was within 5% of the original volume (0.80 ml, the  volume of the fluid on each side of the dialysis cell before dialysis) and the pH change was within 1.3% of the original pH (7.40).  3.2 Binding of 5-Hydroxypropafenone to Purified Human AAG  3.2.1 Rosenthal Analysis  The binding ratio hydroxypropafenone  (bound concentration/free  in purified human AAG was  concentration using the method of Rosenthal relationship was found with r = -0.951.  concentration)  plotted  of  5-  versus  the bound  (1967) (Figure 7).  A linear  A single class of binding sites  was observed with a intrinsic association or affinity constant of K = 1.614 X 10^ M~l.  The estimation of the affinity constant was also obtained by  using the computer program Enzfitter  R  (Leatherbarrow, 1986).  The affinity  constant obtained this way was 1.531 X 10^ M" . 1  3.2.2 Scatchard Plot of 5-Hydroxypropafenone Binding to Human AAG  The binding data plotted as r' (the moles of drug bound per mole of  3.00 15.00  2.50 2.00  CO  i  Linear regression Y=-0.161X---+ 2.209 r=-0.951  o c  8 6.00 h  Q)  C  Z3 m  1.50  .00.  3.00  0.00 * 0.00  c  o  12.00  o  10.00  20.00  30.00  Total cone. M X 1 0  1.00  40.00  50.00  6  0.50 0.00 0.00  3.00  6.00  9.00  [Bound] X 1 0  12.00  15.00  6  F i g u r e 7. R e l a t i o n s h i p between t h e r a t i o o f bound c o n c e n t r a t i o n / f r e e c o n c e n t r a t i o n and bound c o n c e n t r a t i o n o f 5 - h y d r o x y p r o p a f e n o n e in p u r i f i e d human AAG ( R o s e n t h a l p l o t ) , t h e i n s e r t e d g r a p h shows t h e r e l a t i o n s h i p between bound and t o t a l c o n c e n t r a t i o n of 5-hydroxypropafenone.  60.C  54  protein) versus the ratio of r'/free 5-hydroxypropafenone concentration, is shown in Figure 8.  The concentration of AAG (M.W. = 44,100) was used in  the calculation of r' where a linear relationship was found with a r = 0.935.  One type of binding site was observed over the concentration range  studied.  3.2.3 Free Fraction versus Total Concentration Plot  The free fraction of 5-hydroxypropafenone vs total concentration of 5-hydroxypropafenone is shown in Figure 9. ng/ml),  the  binding was  independent  of  fraction of 0.3066 ± 0.025 ( C V . = 8 %). 20,000 ng/ml), linear  At lower concentrations (50-800 concentration with  a mean free  At higher concentrations (2000 to  binding was found to be dependent on concentration and a  relationship  between free  fraction  and total  concentration  was  observed (r = 0.9644).  3.3 Protein Binding Displacement Effect  3.3.1  Effect  of  Buffer  Strength  on  the  Binding  of  PF  and  5-  Hydroxypropafenone  Table 2 shows the free fraction of PF in a solution of purified human AAG or albumin dissolved  in varying ionic strengths of phosphate buffer,  the pH of which was 7.4.  In purified human AAG solution, the mean free  fraction  in  0.067  M phosphate  buffer  was  0.075  ±  0.01,  which  was  significantly greater than the mean free fraction in 0.1 M phosphate buffer of 0.057 ± 0.004 (two sample t-test with p < 0.05).  In purified human  180.00 r 150.00 120.00 -  Q  90.00 60.00 30.00 0.00 0.00  150.00  300.00 450.00 r X 10  600.00  750.00  6  F i g u r e 8. R e l a t i o n s h i p b e t w e e n [ r ' / b r j u n d c o n c e n t r a t i o n ] and r ' o f 5 - h y d r o x y p r o p a f e n o n e i n p u r i f i e d human AAG ( S c a t c h a r d p l o t ) ; c o n c e n t r a t i o n o f AAG was u s e d i n t h e c a l c u l a t i o n o f [ > ' ] • cn  Figure 9.  Relationship between the ratio of free concentration/total concentration (free fraction) and total concentration of 5 -hydroxypropafenone over the concentration of ng/ml.  20-20000  Table 2.  Free fraction of PF in a solution of AAG or albumin dissolved in 0.067 M or 0.1 M phosphate buffer  sample  buffer cone.  PF free fraction in AAG 0.067M  0.1M  PF free fraction in albumin 0.067M  0.1M  1 2 3 4  0.089 0.079 0.064 0.076  0.055 0.054 0.064  0.427 0.421 0.457 0.421  0.339 0.344 0.344 0.330  mean ± s.d.  0.075* ± 0.01  0.057* ± 0.004  0.431* ± 0.017  0.339* 0.006  C.V.%  14.1  6.5  4.0  1.9  3.8 g/dL  3.9 g/dL  albumin cone. concentration of initial cone, of pH of phosphate: *: statistically at p < 0.05  -  AAG: 90 mg/dL PF: 1 /zg/ml 7.4 significant differences with two sample t-test  58  albumin solution, buffer were 0.431  the mean free fraction in 0.067 M and 0.1 M phosphate ± 0.017  and 0.339 ± 0.006,  respectively.  The free  fraction in 0.067 M phosphate buffer was significantly greater than in 0.1 M phosphate buffer (two sample t-test with p < 0.05).  Table 3 shows the free fraction of 5-hydroxypropafenone in a solution of purified human AAG or albumin dissolved phosphate buffer (pH = 7.4). fraction  in either 0.067 M or 0.1 M  In purified human AAG solution, the mean free  in 0.067 M phosphate buffer was 0.321  ± 0.029, which was not  different from the mean free fraction in 0.1 M phosphate buffer of 0.321 ± 0.028.  In purified human albumin solution, the mean free fraction in 0.067  M phosphate buffer of 0.271 ± 0.017 was significantly lower than the mean free fraction of 0.354 ± 0.013 in 0.1 M phosphate buffer (two sample t-test with p < 0.05).  In summary, buffer strength did affect the binding of PF in purified human AAG and albumin solutions and 5-hydroxypropafenone in purified human albumin, but had no apparent effect on the binding of 5-hydroxypropafenone in purified human AAG solution.  Therefore, comparison of the effect of  different displacing agents on the binding of PF and 5-hydroxypropafenone had to be conducted at the same phosphate buffer concentrations.  3.3.2  Effect  of  Anticoagulant  on the  Protein  Binding of  PF and  5-  Hyd roxypropafenone  Table 4 shows the free fraction of PF in whole blood using heparin and EDTA as anticoagulants.  PF was dissolved in 0.067 M and 0.1 M isotonic  Table 3.  Free fraction of 50HPF in a solution of AAG or albumin dissolved in 0.067 M or 0.1 M phosphate buffer  sample  buffer cone.  50HPF free fraction in AAG 0.067M  1 2 3 4  0.306 0.302 0.354  mean ± s.d. C.V.%  0.1M  50HPF free fraction in albumin 0.067M  0.1M  0.308 0.351 0.338 0.289  0.256 0.302 0.354  0.326 ± 0.029  0.321 ± 0.028  0.271* ± 0.017  0.354* 0.013  9.0  8.9  6.3  3.7  3.8 g/dL  3.9 g/dL  -  albumin cone. concentration of initial cone, of pH of phosphate: *: statistically at p < 0.05  -  0.339 0.360 0.363 -  AAG: 90 mg/dL 50HPF: 0.5 //g/ml 7.4 significant differences with two sample t-test  Table 4.  sample  buffer cone.  Free fraction of PF in whole blood using heparin or EDTA as the anticoagulant PF free fraction in "heparin" blood  PF free fraction in "EDTA" blood  0.067M  0.1M  1 2 3 4  0.084 0.069 0.083  0.069 0.064 0.051 0.043  0.048 0.048 0.049 0.054  0.040 0.048 0.056 0.044  mean ± s.d.  0.079* ± 0.008  0.057 ± 0.012  0.050* ± 0.003  0.047 0.007  C.V.%  10.7  20.9  5.8  14.6  -  0.067M  0.1M  initial cone, of PF: 1 //g/ml pH of phosphate: 7.4 *: statistically significant difference with two sample t-test at p < 0.05  61 phosphate buffer (pH7.4), respectively.  In 0.067 M phosphate buffer, the  mean free fractions were 0.078 ± 0.008 in heparinized whole blood and 0.050 ± 0.003 in whole blood using EDTA as the anticoagulant.  The two values  were significantly different from each other (two sample t-test with p < 0.05).  In 0.1 M phosphate buffer, the mean free fraction in heparinized  whole blood was 0.057 ± 0.012, and was not significantly different from the value of 0.047 ± 0.007, the free fraction in whole blood using EDTA as the anticoagulant  (two sample t-test with p < 0.05).  However, there was a  tendency for the free fraction in heparinized blood to be higher than in whole blood when EDTA was used as the anticoagulant.  Table 5 shows the blood  using  either  free  fraction of  heparin  or  Hydroxypropafenone was dissolved  EDTA  5-hydroxypropafenone as  the  in whole  anticoagulant.  5-  M phosphate  buffer  in 0.067 M and 0.1  before dialysis, respectively.  In 0.067 M phosphate buffer, the mean free  fraction  blood was  in heparinized whole  0.174  ± 0.012  which was  significantly different from a mean free fraction of 0.163 ± 0.01 blood using EDTA as the anticoagulant.  from mean free  fraction  in whole  In 0.1 M phosphate buffer, the mean  free fraction in heparinized whole blood was 0.162 different  not  in  whole  blood  ± 0.011 using  and was not EDTA  as  an  anticoagulant (0.163 ± 0.007, two sample t-test with p < 0.05).  In conclusion, the binding of PF in whole blood was affected by the use of anticoagulant while the binding of 5-hydroxypropafenone was not. Therefore, comparision of the effect of binding displacers in whole blood must be conducted using the same anticoagulant.  Table 5.  sample  Free fraction of 50HPF in whole blood using heparin or EDTA as the anticoagulant 50HPF free fraction in "heparin" blood  50HPF free fraction in "EDTA" blood  buffer cone. 0.067M  0.1M  0.067M  0.1M  1 2 3 4  0.156 0.156 0.161 0.178  0.171 0.155 0.161 0.166  0.184 0.183 0.169 0.159  0.149 0.174 0.170 0.156  mean ± s.d.  0.163 ± 0.01  0.163 ± 0.007  0.174 ± 0.012  0.162 0.011  C.V.%  6.4  4.3  6.9  7.2  initial cone, of 50HPF: 0.5 ^g/ml pH of phosphate: 7.4 *: statistically significant difference with two sample t-test at p < 0.05  63 3.3.3  Protein Binding Displacement Studies  using Purified Human Protein  Solutions (AAG and Albumin) as the Biological Fluids  3.3.3.1 Protein Binding Displacement Effect of Various Drugs on PF Binding to Purified Human AAG  Neither propranolol, verapamil nor ASA displace PF from its binding sites as shown in Figure 10.  Similarly, phenytoin and quinidine did not  displace PF from its binding sites, however, when lidocaine was used as the displacing agent, statistically were observed  (Figure  11,  inequality at p < 0.05).  two  significant sample  increases  t-test  in PF free fraction  combined with Bonferroni  The PF free fraction seemed to increase with the  addition of ASA, although the apparent difference did not reach statistical significance  due to  the  large  variation  in the  free  fraction  in  the  solution of PF spiked with ASA. The maximum displacing effect was observed with lidocaine (124% increase in PF free fraction).  3.3.3.2 Protein Binding Displacement Effect of Various Drugs on the Binding of PF to Purified Human Albumin  Similar  to  the  effects  observed  in  AAG solution,  verapamil and ASA did not cause a statistically significant free  fraction  (Figure  12,  inequality at p < 0.05). significant  increases  two  sample  t-test  fraction  increase in PF  combined with Bonferroni  Phenytoin and lidocaine caused  in PF free  propranolol,  (Figure  13).  statistically Although the  increase in PF free fraction caused by quinidine seemed to be quite large (70%, Figure 13),  it failed to reach statistical  significance due to the  64  PF free fraction in pure AAG solution  < 0.075 ± 0.01 (n-4)  -s CD c+  a  PF + Propranolol  CO Z3  »-  H 0.091 ± 0.005 (n-3)  -i c+  cn o c  XJ  0.088 ± 0.008 (n=3)  P F + ASA  Figure 10.  0.095 ± 0.024 (n=4)  Protein binding displacing effect of propranolol, verapamil and ASA on the free fraction of PF in purified human AAG dissolved in 0.067 M phosphate buffer. The pH in each treatment group is 7.4, the value is the mean ± s.d. and n is the number of replicates.  65  PF free fraction in pure AAG solution  PF  s  CD W r+  0.057 ± 0 . 0 0 4 (n-3)  P F + Phenytoim-  -* 0.083 ± 0.016 (n-4)  3 n> =3  -5 O  c  X3  P F + Quinidine  PF + Lidocaine  Figure 11.  ^ 0.105 ± 0.016 (n-3)  0.129 ± 0.013* (n-4)  Protein binding displacing effect of phenytoin, quinidine and lidocaine on the free fraction of PF in purified human AAG dissolved in 0.1 M phosphate buffer. The pH in each treatment group is 7.4 and the value is the mean ± s.d., * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05, n is the number of replicates.  66  PF free fraction in pure albumin solution  PF  CD 0» .  r+ 3 CD  PF + Propranolol  =3  ^ 0.431 ± 0.017 (n=4)  H 0.452 ± 0.038 (n=4)  r+  CD -5 O  c •a  0.405 ± 0.015 (n=3)  PF + ASA  H0.443 ± 0.047 (n=4)  Figure 12. Protein binding displacing effect of propranolol, verapamil and ASA on the free fraction of PF in purified human albumin dissolved in 0.067 M phosphate buffer. The pH in each treatment group is 7.4, the value is the mean ± s.d. and n is the number of replicates.  67  PF free fraction in pure albumin solution.  PF  V 0.339 ± 0.006 (n-4)  PF + Phenytoin  H 0.649 ± 0.055* (n=3)  PF + Quinidine  PF + Lidocaine  Figure 13.  -«0.577 ± 0.1824 (n=4)  +0.333  ± o.oos*  (n=4)  Protein binding displacing effect of phenytoin, quinidine and lidocaine on the free fraction of PF in purified human albumin dissolved in 0.1 M phosphate buffer. The pH in each treatment group is 7.4 and the value is the mean ± s . d . , * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05, n is the number of replicates.  68 large  variation within the  group.  The maximum displacing  effect  was  observed with phenytoin (91 % increase), followed by lidocaine (13%).  3.3.3.3 Protein Binding Displacement Effect of Various Drugs on the Binding of 5-hydroxypropafenone to Human AAG  5-Hydroxypropafenone was dissolved in a phosphate buffer solution and subsequently spiked with various displacing agents (Figures  14 and 15)  before dialysis against a purified human AAG solution; the free fraction of 5-hydroxypropafenone increased significantly by 47%, in lidocaine treatment group (two sample t-test at p < 0.05,  Figure 15).  Verapamil, ASA and  propranolol did not cause any significant changes in 5-hydroxypropafenone free  fraction  (Figure  14,  two  sample  t-test  combined with Bonferroni  inequality at p < 0.05).  3.3.3.4 Protein Binding Displacement Effect of Various Drugs on the Binding of 5-hydroxypropafenone to Human Albumin  ASA significantly  displaced  5-hydroxypropafenone  from its  sites on purified human albumin by 47% (Figures  16,  combined with Bonferroni inequality at p < 0.05).  Lidocaine (Figure 17),  verapamil significant  and propranolol  (Figure 16)  did not  two sample  binding  cause any  t-test  statistically  changes in 5-hydroxypropafenone free fraction (two sample t-  test combined with Bonferroni inequality at p < 0.05).  69  50HPF free fraction in pure AAG solution  50HPF  -<0.321 ± 0.029 (n-3)  50HPF+VerapaTTTi  -"0.304 ± 0.068 (n=3)  5 0 H P F + ASA  50HPF+Propranolol  Figure 14.  0.476 ± 0.087 (n=3)  -t0.355 ± 0.029 (n=3)  Protein binding displacing effect of verapamil, ASA and propranolol on the free fraction of 5-hydroxypropafenone in purified human AAG dissolved in 0.067 M phosphate buffer. The pH in each treatment group is 7.4, the value is the mean ± s.d. and n is the number of replicates.  70  50HPF free fraction in pure AAG solution  -s CD & r+ 3  50HPF  -*0.321 ± 0.028 (n-4)  CD  c+ CO  ~i o cz  -o  Figure 15.  50HPF + Lidocaine  ^ 0.472 ± 0.03* (n-4)  Protein binding displacing effect of lidocaine on the free fraction of 5-hydroxypropafenone in purified human AAG dissolved in 0.1 M phosphate buffer. The pH in each treatment group is 7.4 and the value is the mean ± s . d . , * indicates statistically significant difference with two sample t-test at p < 0.05, n is the number of replicates.  71  50HPF free fraction in pure albumin soluti on  5-OHPF  H0.271 ± 0.017 (n-3)  CO r+  3  ro  50HPF+Verapamil  0.281 ± 0.045 (n-3)  -s o c -o 50HPF + ASA  50HPF+Propranolol  Figure 16.  H0.398 ± 0.028*  (n-3)  0.304 ± 0.01 (n-3)  Protein binding displacing effect of verapamil, ASA and propranolol on the free fraction of 5-hydroxypropafenone in purified human albumin dissolved in 0.067 M phosphate buffer. The pH in each treatment group is 7.4 and the value is the mean ± s . d . , * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05 and n is the number of replicates.  50HPF free fraction in pure albumin solution  50HPF  0.354 ± 0.013 (n-3)  50HPF + L i d o c a i n e 4 * ° - 3 4 2  17.  ± (n-4)  o.oi  Protein binding displacing effect of lidocaine on the free fraction of 5-hydroxypropafenone in purified human albumin dissolved in 0.1 M phosphate buffer. The pH in each treatment group is 7.4, the value is the mean ± s.d. and n is the number of replicates.  73  3.3.4  Protein  Binding  Displacement  Effect  Using  Human  Serum as  the  Biological Fluid  In human serum,  quinidine,  phenytoin  and lidocaine  significantly  increased the free fraction of PF as shown in Figure 18 (two sample t-test combined with Bonferroni inequality at p < 0.05).  The maximum increase in  the free fraction was observed with lidocaine (580%), followed by quinidine (210%) and phenytoin (150%).  Figure  19 demonstrates  that  neither  phenytoin,  ASA nor quinidine  displaces 5-hydroxypropafenone from its binding sites in human serum (two sample t-test combined with Bonferroni inequality at p < 0.05).  3.3.5 Protein Binding Displacement Effect on PF Free Fraction using Human Whole Blood as the Biological Fluid  Since quinidine and phenytoin caused a significant  increase  in the  free fraction of PF in serum, a further study was performed to determine the displacing ability of these two agents in whole blood.  Statistically  significant  in quinidine  treatment  increase  in  PF free  group and phenytoin  fraction  was observed  did not cause  statistically  significant  increase in PF free fraction (Figure 20, two sample t-test combined with Bonferroni inequality at p < 0.05).  PF free fraction in serum  PF  0.017 ± 0.003 (n=4)  PF+QuinidirVe-  PF+DPH  H0.052 ± 0.009* (n=4)  HO.041 ± 0.007*  (n=4)  PF+Lidocaine  Figure 18.  nO.114 ± 0.006* (n=4)  Protein binding displacing effect of DPH (phenytoin) quinidine and lidocaine on the free fraction of PF in human serum. The pH in each treatment group is 7.4 and the value is the mean ± s . d . , * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05 and n is the number of replicates.  75  50HPF  free f r a c t i o n in serum  50HPF  HO.189 ± 0.015  (n-4)  CD  3  0.182 ± 0.009 (n-4)  CD  3  £75 -s o  50HPF+Quinidine  50HPF+ASA  -10.211 ± o . o i (n-3)  H O . 2 2 4 ± 0.045  (n-4)  Figure 19.  Protein binding displacing effect of phenytoin, ASA and quinidine on the free fraction of 5-hydroxypropafenone in human serum. The pH in each treatment group is 7 . 4 , the value is the mean ± s . d . and n is the number of r e p l i c a t e s .  76  P F Free Fraction in Heparinized Whole blood  PF  H0.057 ±  0.012  (n-4)  CD  a  3 CD 13  r-t-  O  o c  T3  PF+Quinidine  PF+Phenytoin  Figure 20.  -.0.101 ± o.oi* (n-4)  -<0.079 ± 0.01 (n-4)  Protein binding displacing effect of quinidine and phenytoin on the free fraction of PF in human whole blood. The pH in each treatment group is 7.4 and the value is the mean ± s . d . , * indicates statistically significant difference with two sample t-test combined with Bonferroni inequality at p < 0.05 and n is the number of replicates.  77  3.3.6 The Nature of the Displacement Interaction between PF and Quinidine and PF and Lidocaine using Purified Human AAG as the Biological Fluid  3.3.6.1 Rosenthal  Plot of the  Binding of  PF to  Purified  AAG over the  Therapeutic Concentration Range of PF  The Rosenthal plot of the binding of PF to purified human AAG over the therapeutic concentration range of PF is shown in Figure 21.  A linear  relationship exists between the binding ratio and bound concentration of PF (r = -0.953) and indicating that PF binds to one class of binding sites over the therapeutic concentration range with an intrinsic association or affinity constant of K = 3.44 X 10  6  M" . 1  3.3.6.2 The Nature of the Displacement of PF from Human AAG by Quinidine and Lidocaine  A linear relationship between the reciprocal of the concentration of unbound PF and the reciprocal of the moles of PF bound per mole of AAG over the concentration  range was observed  quinidine and lidocaine,  (Figure 22).  In the presence  less PF was bound to AAG (Figure 22).  of  Common  ordinate intercepts indicate that both quinidine and lidocaine compete with PF for the same binding site on AAG. quinidine  and lidocaine  against  the  The inhibitory constant binding  of  (Ki) of  PF by human AAG was  estimated using the equation Ki = i / (Kp/K - 1), where i equals the molar concentration of inhibitor, K and Kp are the slopes of the line absence and presence of the inhibitor.  in the  The Ki value for both quinidine and  lidocaine was approximately 12 nM. Therefore, the nature of the displacing  1.20  ^0.90 cn 3  28.00  § 0.60 • o  ~u c  24.00  ° 0.30  20.00 0.30  £ 16.00 Li_  c 12.00  0.60 0.90 Total cone, ug/ml  1.20  L i n e a r r e g r e s s i o n : Y=-0.344X + 24.909 r=-0.953  O CD  8.00 4.00 0.00 0.00  5.00  10.00  15.00 20.00 25.00 30.00 Bound M X 10  F i g u r e 21.  35.00 40.00  7  R e l a t i o n s h i p between t h e r a t i o o f bound c o n c e n t r a t i o n / f r e e c o n c e n t r a t i o n a n d bound c o n c e n t r a t i o n o f PF i n p u r i f i e d human AAG ( R o s e n t h a l p l o t ) , t h e i n s e r t e d g r a p h i n d i c a t e d t h e r e l a t i o n s h i p b e t w e e n b o u n d and t o t a l c o n c e n t r a t i o n o f P F .  —i  co  160  r  20 -20 1/PF (moles' ) 1  Figure 22.  Relationship between the reciprocal of the moles of PF bound per mole of AAG and the reciprocal of unbound PF without displacing agento-o ; in the presence of quinidine a-a ; in the presence of lidocaine A - A .  80  effect exhibited by quinidine and lidocaine in purified AAG was competitive i n h i b i t i o n of binding of PF to AAG.  3.4 HPLC Analysis of Indocyanine Green (ICG)  3.4.1  HPLC  Analysis  of  Indocyaine  Green  according  to  the  Method of  Rappaport and Thiessen The HPLC separation and measurement method was attempted on a Hewlett Packard Model  1090 HPLC equipped with a Model  1040A Diode-Array Detector.  Unfortunately, the detection l i m i t attained by Rappaport and Thiessen (0.4 /zg/ml) was not obtained,  in large part due to the small volume of sample  which  into  can  be  contamination  injected due to  our  incomplete  microbore  protein  HPLC  system.  precipitation  Also,  in the  the  injection  solution produced a high column pressure which reduced the l i f e time of the column  dramatically.  completely  The protein  eliminated  using  contamination  centrifugation  prior  present to  could  sample  not be  introduction  into the HPLC due to the limited range of rpm available using our bench top centrifuge.  3.4.2 HPLC Analysis of Indocyanine Green with Fluorometric Detection  The  fluorometric method reported by H o l l i n s '  sensitivity  than  Thiessen (1982) nm,  we  other .  obtained  existing  methods  such  as  Although the maximum wavelength an  excitation  spectrum  under  (1987) that  offered  of  better  Rappaport and  in our system was 600 similar  experimental  conditions to that published by Hollins so that the assay was conducted at different  excitation  and emission  wavelengths.  An excitation  spectrum  81  obtained on our HPLC system using the same sample preparation procedure and HPLC conditions as Hoi Tins'  (1987) is shown in Figure 23.  The emission  spectrum at an excitation wavelength of 260 nm was also shown in the same figure.  Excitation wavelengths  response.  Various excitation wavelengths within this range were tried on  our system.  The  between  210-260  did not show better  Array detection.  the  highest  fluorometric detection of ICG was compared with Diode  Array UV detection at 216 nm, simultaneously. detection  nm gave  sensitivity  Unfortunately, fluorometric than the corresponding Diode  A representative chromatogram is  shown in Figure 24.  Furthermore, changing the HPLC conditions such as mobile phase composition, flow rate did not improve separation or sensitivity.  Another excitation  spectrum using the method reported by Dorr and  Pollack (1989) is shown in Figure 25 .  In this method pH 4.0  buffer was used as part of the mobile phase. emission wavelengths  (214  citrate  The reported excitation and  and 370 nm, respectively)  were tried on our  system, and there was no separation between the solvent front peak and ICG.  3.4.3 HPLC Analysis of ICG with Diode Array Detection  A representative chromatogram obtained using the modification method described before (section 2.12.2 ) is shown in Figure 26. standard curve is shown in Figure 27.  A representative  190  240  290  340  390  440  490  540  Excitation wavelength (nm) Figure 23.  Excitation o-o and emission A-A (at excitation wavelength of 260 nm) spectra of ICG using pH 6.0 phosphate buffer as part of the mobile phase.  co ro  LC l 00:1 LOT  R  254,4 FTLD  5 5 0 , i 00 of J W . D  of  90  JH.D  •ICG with UV detection  80 701 60 73  501 Id-  II <J)  40  ICG with fluorometric detection  30 20: 10:  0"  Figure 24.  4 T i me  (min. )  6  A representative chromatogram that compares fluorometric detection with UV detection.  1  0  30-p 282624222018161412108 6 4 2 0 — 0  50  100 150 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 excitation wavelength  Figure 25.  (nm)  Excitation spectrum of ICG using pH 4.0 citrate buffer as part of the mobile phase. co 4=>  85 ins-  31-  CL  cu N  27-  Figure 26.  A representative chromatogram obtained using the modification method (section 2.12.2).  1.000  o  0.800  CL 0J N  L i n e a r r e g r e s s i o n : Y=0.933X r=0.9999  0.017  O  ^  o o D  0.600  0.400  a CD  <  0.200  0.000 0.000  0.200  0.400  0.600  0.800  1.000  K200  A m o u n t of [CG a d d e d ( u g ) Figure 27.  A representative standard curve obtained using the modification method (section 2.12.2). co o-i  87  3.4.4 Comparison of Sample and Blank Chromatograms Using a Modified Method of Christie (1986) and Burns (1989)  When using 0.1 ml serum spiked with ICG and diazepam, there was no interference from blank.  However, when 0.25 ml serum was used, there was a  small peak in the blank which interfered with the diazepam peak as shown in Figure 28.  This interference  peak was minimized by changing the mobile  phase composition from 40% to 39% of acetonitrile and flow rate from 0.5 ml/min to 0.2 ml/min.  Also, instead of using area ratio, peak height ratio  was used for standard curve and calculations.  A standard curve obtained  under these HPLC conditions is shown in Figure 29.  3.4.5 ICG Extraction Efficiency  The conditions  extraction described  efficiency  of  indocyanine  green  in section 2.12.5 is shown in Table 6.  recovery is 73%. Table 6.  Extraction efficiency of ICG in serum  Amount added  0.40 0.60 0.80 1.00  using  Amount recovered  0.27 0.45 0.58 0.76  Recovery  mean  68 75 72 76 73  the HPLC The mean  tC  Ft  2 i 6', 4 16,4  S 5  o f  "550, 1 0 0  b f  SERUM'S  0 D  Top: serum s p i k e d w i t h ICG Bottom: serum b l a n k  D  4  G  Time  Figure 28.  1 0  (min.)  Representative chromatograms of serum (0.25 ml) spiked with ICG and serum blank.  12  1.000  r  o.OOO '  0.000  1  0.200  1  0.400  1  0.600  1  0.800  '  1.000  Amount of ICG added (ug) Figure 29.  Representative standard curve obtained using 0.25 ml serum.  1  1.200  90  3.4.6 Post Extraction Stability  Table 7 shows the peak height preparation.  ratios of  ICG/diazepam 48 h after  The ratios were not significantly different from each other  and indicated that ICG was stable for at least 48 h after preparation when stored at 4°C and protected from light. Table 7.  Peak height ratios of ICG/diazepam at various times after serum extraction  Time after extraction h  Peak height ratio ICG/diazepam  0 1.0 2.5 4.5 5.5 20.5 24.0 26.0 28.0 44.5 48.0  1.51 1.52 1.53 1.48 1.47 1.52 1.50 1.52 1.52 1.48 1.48  3.4.7 Plasma Data of ICG in Rat  A representative chromatogram obtained using the sample preparation procedure in section 2.12.8 is shown in Figure 30.  A standard curve and  the plasma concentration of ICG vs time curve are shown in Figure 31 and 32, respectively.  The calculated ICG plasma clearance was 9.216 ml/min,  and was in agreement with the literature value (10.17 ml/min for a 300 g body weight rat, Burns et al.,  1989).  The on-column detection with our  method was 3.8 ng (1.1 uq/m\ using 50 ul plasma).  The method of Burns et  LC LC  R P  2 16,4 2 16,4  550,100 55 0 , 1 0 0  B  f  RBDI.D RflTBLRNK. D  rt  Q. QJ Nl (O •r—  58'  40-  O f  o  Q  CD  A  <_>  Top: rat plasma sample Bottom: rat plasma blank 30<L  £  20"  10-  0-V 0  Figure 30.  T i me  (min. )  A representative chromatogram of ICG in rat plasma obtained using the extraction procedure shown in Figure 6.  «5  Figure 31.  Amount of ICG added (ug)  A representative standard curve obtained in rat plasma  94 al.,  1989 reported a detection  limit of 30 ng/ml.  Since there was no  volume of injection reported, the on-column detection limit was not known.  3.4.8 The Potential to Detect ICG in Low Concentration Samples with the New Method  When the sample injection volume was increased from 10 to 50 /il in the attempt a7.(1989),  to detect low concentration sample as reported by Burns et the  resolution  of  the  ICG peak  from  diminished and the method could no longer be applied.  plasma  debris  peak  This led to a search  for another method of analysis for ICG.  3.4.9 Solid Phase Extraction  A representative chromatogram is shown in Figure 33. sample  preparation  procedure  allowed  separation s t i l l was not complete.  higher  injection  Although this volume,  the  I 00  LC R 2 1 6 , 4 LC n 2 1 6  550,100 550,100  of of  90:  ICG  80:  JN2 0R13R.D JN20R14R.D  Diazepam  70  Top: serum spiked with ICG Bottom: serum blank  601 D  50  £  401 30 20 l 0 0 0  2  3  T1 me  (min. )  Figure 33. A representative chromatogram obtained using solid phase extraction method  96  4. DISCUSSION  4.1 Protein Binding Technique  Several techniques have been used to study the nature of ligandprotein  interaction.  Commonly employed  methods  are  equilibrium  dialysis, ultrafiltration, ultracentrifugation and gel filtration for in vitro protein binding studies (Kurz et a7.,  1977); and, most recently,  microdialysis has been developed and used as an in vivo method in rats (Scott  et  al.,  1990).  ultrafiltration  are  laboratory setting.  Among these methods, those  most  equilibrium dialysis and  frequently  used  the advantage that the free during  equilibrium  of  the  data.  research  binding  Equilibrium dialysis has  and bound amount of drug are in direct  dialysis  procedure (Kurtz et a7., inexpensive,  the  Equilibrium dialysis is the "classical" method to  determine protein binding (Kurz et a7., 1977).  contact  in  will  process not  1977).  be  and,  therefore,  affected  by  the  the  dynamic  experimental  In addition, equilibrium dialysis  is  has good reproducibility and gives accurate quantitative  However, the total drug concentrations before and after dialysis  are not the same due to the free passing of unbound drug across the membrane and/or the adsorption of drug to the dialysis device.  In ultrafiltration, the protein-free  ultrafiltrate  is  separated  from the protein containing phase by forcing it through a semipermeable membrane  or  filter  Ultrafiltration concentrated  is  protein  either less  time  solutions  by  pressure  consuming or  or  and  tissue  centrifugal applicable  homogenates.  to  force. highly However,  97  appreciable binding of drug to the membrane will interfere with proteinbinding determinations since only the free drug in the ultrafiltrate is measured  and the  corrected.  non-specific  In addition,  binding to  a sieve  effect,  the  membrane can not  viz.,  increasing  be  protein  concentration due to filtration of small molecules and water, and the narrowing of pores in the membrane, due to possible binding of protein molecules to membrane material, make the method less reliable (Kurtz et al.,  1977)  In ultracentrifugation, molecule  from the  accomplished  by  higher  the  separation  molecular  centrifugal  weight  force.  of  the  small  free  drug  drug-protein complex  Ultracentrifugation  offers  is an  alternative choice to ultrafiltration when membrane binding problems are encountered.  However, when the drugs bind to low-density lipoproteins,  it is difficult to separate the free drug from the drug-protein complex by the centrifugal  force as the low density drug-1ipoprotein complex  tends to stay at the surface of solvent media (Rowland, 1980).  Also,  free drug molecules with high molecular weight will tend to move to the bottom of the centrifugation tube due to the influence of centrifugal force.  This will lead to higher binding values as more drug is confined  in the drug-protein complex compartment (Kurz et al., to other methods, ultracentrifugation is expensive,  1977).  Relative  complicated and its  theoretical basis is not well established (Lindup, 1975).  In gel separated  filtration,  provided that  bound and unbound amounts the  protein molecules  complex cannot penetrate the gel pores.  of  and the  a drug  are  protein drug  The method can be applied to  98  drugs of relatively high molecular weight but is not suitable for drugs with low affinity to protein, also relatively large volumes of protein solution are needed to obtain correct binding values.  The microdialysis method gives equivalent in vitro protein binding values as compared to ultrafiltration (Herrera et al.,  1990).  The in  vivo application of this method provides several advantages over the in vitro  method.  Samples can be collected  at high rates from several  sites, without fluid loss, with a single animal (Scott et al., The method is also associated with some disadvantages.  1990).  It is invasive,  for in vivo use, as a small probe has to be implanted in the tissue of interest  (Scott  et  al.,  1989,  1990),  the  available requires much higher sensitivity  small  volume  of  sample  of drug assay method.  So  far, the method is limited in animal study only.  Propafenone  exhibits  extensive  adsorption  (16.2%)  to  the  ultrafiltration device, thus this method is not suitable for PF protein binding  studies  concentration  as  it  or free  leads  to  fraction  an  underestimation  (Chan et  al.,  1989).  of  free  drug  Although the  adsorption of PF to the equilibrium dialysis cell is similar to that of ultrafiltration device, since  samples  from  it does not affect  both  sides  concentration after equilibrium.  of  the  the calculation of free PF, cell  are  assayed  for PF  Therefore, equilibrium dialysis is the  method of choice for PF protein binding study.  There are several complications in the use of equilibrium dialysis in addition to non-specific  binding (binding of drug to the  dialysis  99  cell or membrane). These include volume shift (Miller et al., 1981) and pH change (Lui et al., 1986) after equilibrium; protein leakage into the dialysate due to poor membrane quality, improper pore size and/or poor technique in mounting the dialysis membrane; bacterial growth due to dialysis temperature and long dialysis time (Lui et al., 1986; Ilett et al., 1975).  In the present PF studies, volume and pH before and after  each dialysis were measured, there was no significant change in either volume or pH.  The protein leakage was also tested by adding 3%  trichloroacetic acid to a sample of the buffer side of the dialysis cell; absence of precipitation was used as an indicator of no protein leakage. The possibility of bacterial growth was not studied since the dialysate appeared to be nonturbid and the dialysis time (8 hours) was not sufficiently long enough to encourage bacterial growth. 4.2 Protein Binding of 5-Hydroxypropafenone  In purified isolated human AAG solution, 5-hydroxypropafenone binds to one class of binding sites with affinity constant of 1.6 X 10^ (section 3.2.1).  During displacement experiments which conducted at  similar initial concentrations (0.5 jug/ml), the free fraction of 5hydroxypropafenone is similar in AAG and albumin solution (Table 8), whereas, the free fractions of PF are significantly different in AAG and albumin. This may indicate that at very low concentrations, the binding of 5-hydroxypropafenone is not specific to either AAG or albumin. Also, the free fractions in serum and blood are much lower than that in isolated protein solutions, which further indicates that the binding of 5-hydroxypropafenone to blood protein might be nonspecific.  If the  100  Table 8. Free fraction of 50HPF and PF in AAG, albumin, serum and whole blood 50HPF free fraction AAG Albumin serum blood  0.321 ± 0.028 0.354 ± 0.013 0.189 ± 0.015 0.163 ± 0.007  PF free fraction 0.057 ± 0.339 ± 0.017 ± 0.057 ±  0.004 0.006 0.003 0.012  101  binding is specific to a certain protein, one would expect that the free fraction would be the same in either isolated protein solution or mixture of a number of proteins, as long as the binding sites on that particular protein are not saturated.  But in the serum and whole blood  the free fraction of 5-hydroxypropafenone is approximately half of that seen in the pure isolated protein solution.  The nonspecific binding  characteristic of 5-hydroxypropafenone was also characterized by Tonn (1990).  4.3  Displacing  Effect of Verapamil, Propranolol,  ASA,  Phenytoin,  Lidocaine and Quinidine  4.3.1 Verapamil  Verapamil is a calcium channel blocking drug which has been used extensively as an antiarrhythmic  agent to control superventricular  tachycardia (Krikler and Spurrell, 1974).  Verapamil is approximately  90% bound to plasma proteins in a concentration independent manner over the concentration range from 35 to 1,557 ng/ml suggesting a great excess of binding sites (Keefe et al.,  1981). When verapamil (200 ng/ml) was  used as a displacing agent for the protein binding of PF and 5hydroxypropafenone in an solution of purified isolated AAG (Figures 10 and 14) or albumin (Figures 12 and 16), no displacing effect was observed. One possible explanation could be the concentrations of PF (1 //g/ml) and 5-hydroxypropafenone (0.5 //g/ml) were sufficiently low that even with the addition of verapamil, there were still enough binding sites to accommodate each drug in the presence of each other.  It is  102  also possible that the binding affinity of verapamil  is too low to  displace PF or 5-hydroxypropafenone from their binding sites, or verapamil binds to different sites on AAG and albumin from those of PF and 5-hydroxypropafenone. Therefore, no displacement interaction could be observed. Higher concentrations of PF and 5-hydroxypropafenone could have been used, but the results would be less meaningful clinically since the concentration used were maximal therapeutic concentrations encountered during routine clinical use of PF.  4.3.2 Propranolol  Propranolol  is an beta-adrenergic  receptor  blocking  agent  frequently used as an antiarrhythmic drug. In human serum, propranolol binds mainly to AAG (Sager et al., 1978; Piafsky et al., 1978). In our study, when propranolol was used as a displacer on PF binding to AAG and albumin, no binding interaction was observed {i.e. no change in PF free fraction was seen with the addition of propranolol, Figures 10 and 12). There was also no change in 5-hydroxyprdpafenone free fraction in AAG or albumin before and after the in vitro  solution was spiked with  propranolol (Figures 14 and 16). The lack of displacing effect of propranolol on PF binding was probably due to the very low concentration of propranolol used (1 ug/ml), nonsignificant binding of both PF and propranolol to albumin and lower binding affinity of propranolol to AAG than PF.  Both PF and propranolol are basic compounds.  They both bind to  AAG with similar affinity to one class of binding sites (in a magnitude  of 10° M" ) (Gill is et al.,  1985).  1  However, the binding affinity to a  second class of binding sites is much lower for propranolol (« 10^ M"*) than that for PF (« 10^).  This, together with the low concentration of  propranolol used ( 1 //g/ml) indicates that propranolol can not displace PF from its binding sites on AAG and therefore, no apparent effect on PF free fraction in purified human AAG solution would be observed.  As both  PF and propranolol are not bound specifically to albumin due to their chemical  nature  (basic  compound),  the  lack of  displacing  effect  propranolol in purified human albumin solution is not surprising.  of  Since  protein binding of 5-hydroxypropafenone is nonspecific to either AAG or albumin, the observed no change in 5-hydroxypropafenone free fraction in purified  isolated  propranolol  human  albumin  is anticipated.  solution  upon  the  addition  of  Higher concentration of propranolol might  exhibit displacing interaction, but a plasma concentration of 1 //g/ml is the maximum therapeutic concentration for this drug.  4.3.3 Acetyl salicylic Acid (ASA)  ASA has been widely used as an antipyretic, analgesic inflammatory agent for over 100 years.  and anti-  Salicylate serum concentrations  of up to 100 //g/ml are required for effective analgesia, whereas serum levels  of  300-400 //g/ml  are  rheumatic fever (Evans et al,  expected 1986).  in  the  management  of  acute  The displacing effect of ASA on  the protein binding of phenytoin has been well documented (Ehrnebo and Odar-Cederlof, 1981;  Lunde  1977; et  al.,  Odar-Cederlof 1971;  and Borga,  Paxton,  1980).  1977;  Leonard et  ASA caused  al.,  permanent  acetylation of the lysine residues of human albumin under physiologic  104  conditions in vitro (Pinckard et al.,  1973).  In our displacement study,  ASA did not cause any change in PF free fraction in isolated AAG and albumin (Figures fraction  10 and 12),  no change  in 5-hydroxypropafenone  free  in a solution of AAG (Figure 14) and serum (Figure 19) was  observed, whereas an increased free fraction of 5-hydroxypropafenone in a solution  of albumin was found (Figures 16).  Due to the  specific  binding of PF to AAG, the lack of a displacing effect of ASA on the binding of PF to AAG is expected.  Compared with the concentration of  ASA and PF, the relatively large concentration of albumin is probably the main reason for the observation of a stable PF free fraction.  It is  also possible that PF binding sites in albumin are not around the lysine residue area and therefore, acetylation of albumin molecules exerted by ASA has no effect on PF free fraction in purified isolated human albumin solution. not  Therefore, the displacing interaction between PF and ASA is  important.  Although  ASA caused  significant  increase  in  5-  hydroxypropafenone free fraction, this interaction is not significant in serum, it is, therefore, unlikely to be important c l i n i c a l l y .  4.3.4 Phenytoin  Phenytoin arrhythmia.  is  used  to  treat  seizure  disorders  and  cardiac  When it is used as an antiarrhythmic agent, the therapeutic  concentration range is between 10-18 /xg/ml (Bigger et al.,  1968).  In  plasma, phenytoin is 90% bound and it is bound mainly to albumin (Evans et  al.,  1986).  phenylbutazone, et  al.,  1986).  The binding can be displaced by weak acids, salicylic acid, valproic acid and sulfisoxazole  such as (Evans  When phenytoin was used as displacing agent in our  105  study,  the  free  (Figure 11),  fraction of PF increased 45% in purified human AAG  91% in albumin (Figure 13),  150% in human serum (Figure  18), and 39% in whole blood (Figure 20), although the increases observed in AAG and whole blood did not reach a level of statistical  significance  (two sample t-test combined with Bonferroni inequality at p < 0.05). On the other hand, the free fraction of 5-hydroxypropafenone in serum was not affected by the addition of phenytoin (Figure 19). be that the concentration of 5-hydroxypropafenone  One reason could  is too low (half of  that of PF), and even with the addition of phenytoin, there are s t i l l enough binding sites to adapt both 5-hydroxypropafenone and phenytoin, therefore,  no displacing effect could be observed.  It may also be due  to the nonspecific binding of 5-hydroxypropafenone to albumin that even if  there  is displacement  of 5-hydroxypropafenone from albumin binding  sites by phenytoin, the free 5-hydroxypropafenone binds subsequently to other proteins existing in serum. human albumin is high (1.3 X 10  4  The binding affinity of phenytoin to M" , Levy et al., 1  1982), it  is thus  easy to understand that the extent of increase in free fraction of PF is higher in purified isolated solution.  human albumin solution than that  in AAG  The large increase in PF free fraction in serum (150%, Figure  18) is almost equal to the summation of the individual increase in free fraction indicates  in AAG (45%, that  Figure 11) and albumin (91%,  PF does  not  lipoprotein to a significant  bind  to  degree.  other This  Figure 13), which  serum proteins, is  such  as  in agreement with the  observation that the degree of binding of PF is similar in normal serum to  lipoprotein  deficient  serum  (Tonn,  1990).  The  statistically  nonsignificant increase in PF free fraction in whole blood could be due to the uptake of free PF by the red blood cells as PF exits in these  106  cells (Tonn, 1990) Higher concentration of 5-hydroxypropafenone could be used, but 0.5 jug/ml is close to the clinical situation as the maximum therapeutic concentration for PF is only 1  ug/m].  4.3.5 Quinidine  Quinidine is a well-known antiarrhythmic agent. In human plasma, quinidine is approximately 90% protein bound (Reidenberg and Affrime, 1973), and it binds mainly to albumin and low and high density lipoproteins (Nilsen, 1976; Nilsen and Jacobsen, 1975). When quinidine was used as the displacing agent in our study, the free fraction of PF increased significantly in serum (210%, Figure 18) and whole blood (78%, Figure 20).  no change in 5-hydroxypropafenone free fraction in serum  was observed (Figure 19).  Further study indicates that quinidine  competes with PF for binding sites in purified isolated human AAG solution (Figure 22).  Since quinidine binds mainly to albumin and low  and high density lipoprotein (Nilsen, 1976; Nilsen and Jacobsen, 1975), it is thus expected that the free fraction of PF should also increase in a solution of AAG or albumin.  In fact, the free fraction of PF did  increase in AAG or albumin in our study (84% in AAG, Figure 11; 70% in albumin, Figure 13), but it failed to reach statistical significant due to the large variation within the treatment group and the conservative statistical test used. Again, the uptake of free PF by the red blood cells may contribute to the lower degree of increase in PF free fraction in whole blood displays  (78%) than in serum (210%).  concentration  independent  binding  5-hydroxypropafenone in serum  over the  concentration range from 0.12 to 35.3 Mg/ml (Tonn, 1990), which suggests  107  the large excess of binding sites.  5-hydroxypropafenone also binds to  lipoprotein to a significant degree as increased free fraction of 5hydroxypropafenone in lipoprotein deficient serum compared to normal serum is observed (Tonn, 1990). It is thus not difficult to understand that there is no significant displacement interaction between quinidine and 5-hydroxypropafenone in human serum.  4.3.6 Lidocaine  Lidocaine is considered the parenteral drug of choice for the acute treatment or prevention of ventricular arrhythmias associated with acute myocardial  infarction or cardiovascular surgery and ventricular  tachycardia.  The binding of lidocaine is correlated well with AAG  concentration  (Shand, 1984).  Basic drugs such as amitriptyline,  chlorpromazine, disopyramide, imipramine, meperidine, nortriptyline, propranolol and quinidine did not displace lidocaine from serum AAG binding sites to a great extent. bupivacaine (Goolkasian et al.,  However, lidocaine was displaced by 1983).  When lidocaine is used as a  displacing agent for the binding of PF, significant increases in PF free fraction were observed in AAG (124%, Figure 11), albumin (12%, Figure 13) and serum (580%, Figure 18). The extent of increase was much higher in AAG and serum than in albumin. Due to the specific binding of PF and lidocaine to AAG, greater displacing ability in AAG than in albumin is expected.  Further study indicates that lidocaine competes with PF for  binding sites in purified isolated human AAG solution (Figure 22). The binding affinities of PF and lidocaine to solution of isolated human AAG are similar (1.7 X 10 for PF and 1.4 X 10 for lidocaine, Gill is et 5  5  108  al., 1985), but the molar concentration of lidocaine is much higher than that of PF (approximately 9 times higher) in our displacement study. This might contribute to the observed increase in PF free fraction in AAG as the drugs compete for available binding sites.  The pKa of  lidocaine is 7.9 (Courtney, 1980) and it is less basic than PF (pKa = 9.0, Kohlhardt and Seifert, 1980). It is thus possible that the binding affinity of lidocaine to albumin is higher than that of PF, and displaces PF from its binding sites in albumin causing a subsequent increase in free fraction of PF in albumin.  The large, statistically  significant increase in PF free fraction in serum (5.8 fold) implies that this displacement interaction might be clinically important.  4.4 Clinical Significance of Displacing Effect  Combinations of drugs are often necessary in the treatment of diseases.  For drugs that bind extensively to both plasma and tissue  proteins, such as PF, the binding displacement interaction may not be clinical  important, as the displaced  free  drug can be quickly  distributed in tissues and reach a new equilibrium.  Since the volume of  distribution is large, the free drug concentration in the plasma would be the same or at least approaches the pre-interaction level (D'Arcy and McElnay, 1982; McElnay and D'Arcy, 1983).  Although phenytoin and  quinidine display PF from its binding sites significantly in serum, or whole blood, there might be no significant change in free PF concentration in vivo (when the drugs are given orally) due to the rapid distribution in tissues, and even there is, it will be transient in nature.  However, the binding displacement interaction between PF and  109  lidocaine could be clinically important.  Since lidocaine is given  intravenously and if it is given by bolus injection, displacement of PF from its binding sites will be much faster than distribution and reequalization of displaced free PF, and accumulation of free PF may occur.  This increased free PF plasma concentration may lead to  increased drug reaching receptors and/or entering such a compartment as the CSF and, hence, toxic effects.  4.5 HPLC Analysis of ICG  4.5.1 HPLC Analysis of ICG with Fluorometric Detection The two reported HPLC analysis methods of ICG with fluorometric detection (Hollins et al., our system without success.  1987; Dorr and Pollack, 1989) were tried on The excitation and emission maxima used in  the method of Hollins' were 780 and 810 nm, respectively. With 200 fi\ injection, plasma concentrations as low as 3.2 ng/ml can be detected by this method.  We could not reproduce this method as our fluorometric  detector wavelength range is from 190 to 600 nm. Although we did try to get an excitation spectrum and a corresponding emission spectrum (Figure 23) on our system, the fluorometric response was much lower than the corresponding DAD response (Figure 24). Dorr and Pollack used 214 nm as excitation wavelength and 370 nm as emission wavelength, however, we could not get any detector response at this excitation and emission wavelength pair. Therefore, we concentrated our effort on HPLC analysis of ICG with DAD detection.  110  4.5,2 HPLC Analysis of ICG with Diode Array Detection 4.5.2.1 Sample Preparation  Direct deproteinization of serum sample with acetonitrile before injection into HPLC as reported by Rappaport and Thiessen  is not  feasible with our system, as the microbore system we have is easily contaminated by debris contained in serum and the 5 am, 2.3 i.m.column is easily plugged and produces high column back pressure.  The chemical  nature of ICG (Figure 4) limited the use of solvent-solvent extraction by either acidification or alkalization.  The phase separation method  with ammonium sulphate as reported by Christie et al. (1985) is one way to minimize protein contamination.  The method reported by Burns et al.  (1989) involved phase separation  and was employed  in the sample  preparation procedure described in Experimental section 2.12.2 (Figure 5).  The utilization of syringe sample filter made the sample cleaner  and allowed it to be injected into our HPLC system. This procedure was used to develop a standard curve (using peak area ratio) ranging from 0.1 ug to 1.0 ug (Figure 27).  With the use of 0.1 ml serum in this  procedure, a 0.1 ug detection limit corresponds to a serum concentration of 1 /ig/ml.  This detection limit is not adequate for our future use.  When the serum volume was increased to 0.25 ml, blank interference with internal standard was encountered (Figure 28). the  limited  centrifuge. and Thiessen  centrifugation speed  This is probably due to  (3,000 rpm) of our bench top  The same problem was displayed in the method of Rappaport (1982), where the interference peak from rabbit blank  plasma sample contributes to 5% of the internal standard peak height.  Ill  With  modification  of the chromatographic  conditions,  the blank  interference was minimized and a second standard curve was developed using peak height ratio(Figure 29).  With the increase in serum volume,  it was possible to detect serum ICG concentration of 0.8 /zg/ml. The extraction efficiency of ICG was 73% (section 3.5.5) and the prepared sample was stable for at least 48 hours when stored at 4°C in black vial holder (section 3.5.6), it seems possible to use this method for our future HPLC analysis of ICG in human serum with -automatic sample injection.  4.5.1.2 HPLC Analysis of ICG Plasma Concentration in Rat  Due to the small volume of plasma (100 #1) which can be obtained from rat during  frequent  serial  sampling, micro-centrifuge tubes  normally have to be used for plasma sample preparation.  Since our  centrifuge did not have the necessary rotor to accommodate the small tubes, another high speed centrifuge (17,000 rpm) was used.  With the  use of high speed centrifugation, the supernatant obtained was so clear that direct injection into HPLC was possible. Therefore, another sample preparation procedure described in Experimental section 2.12.8 (Figure 6) was used. fig/m\  The detection limit obtained with this method was 1.1  using 50 fi\ of plasma. This detection limit was much higher than  30 ng/ml, using 35 /zl plasma, as reported by Burns et al. (1989). The difference in detection wavelength (they used 784 nm and we used 216 nm) was the principal reason for the observed high detection limit of this method. It is known that the maximum absorbance wavelength for ICG is between 780 - 806 nm depending on the solvent media (Owen, 1973).  112  Moreover,  the relative difference in absorbance  wavelength 216 and 784 nm (Figure 34).  is 0.16 between  Therefore, the low detection  wavelength might be the main reason for the high detection limit we obtained.  We could not use 784 nm as our detection wavelength as our  DAD detector does not cover the visible wavelength range.  Another  factor contributing to the failure of the method could be the difference in injection volume.  In this study, 10 /zl was injected into the HPLC  system while the volume used by earlier investigation was not reported. An increase in the injection volume from 10 fi\ to 50 #1 in our method leads to loss of separation due probably to the low capacity of our 10 cm, 5 (im particle size, 2.3 i.m. column.  The differences in sample  preparation procedure might also contribute to the high detection limit. Since a clean sample was obtained with the utilization of high speed centrifuge in our method, phase separation using ammonium sulphate in sample preparation employed by Burns et al. was not used.  4.5.2.3 Summary  The HPLC analysis system we have is a microbore system and the maximum detection wavelength is 600 nm.  Since the maximum absorption  wavelength of ICG is around 800 nm (Landsman et al, 1976), better sensitivity would be obtained if this wavelength could be used as the detection wavelength in our HPLC system. Using the DAD, we were forced to use 216 nm as the detection wavelength, which is the maximum absorption wavelength in the range of 190-600 nm. wavelength used is 550 nm.  The reference  This may contribute partly to the observed  lower sensitivity as there might be some absorption at the reference  0.3008?  0.2t070-  i=c  Q.130S2  /  0.12Q3S-  N  0.OGO1?  0.0000-  Annotated  '—i— — — — —i— —'—•— —r— 1  200  300  400  1  1  1  1  500 UfiVELENGTH  1  500  700  Wavelengths:  1  :  Wavelength  = 216  Result  =  0.134811  2  :  Wavelength  = 784  Result  =  8.293243  Figure 34. Ultraviolet/visible spectrum of ICG in distilled water (10 /zg/ml)  soo  114  wavelength.  The low sample capacity of the micropore system limits the  increase in injection volume, detection of low concentration samples by increasing  injection  volume  is  sharply limited  by column capacity.  However, with the use of long column, for example, 25 cm instead of 10 cm as we did, a higher volume injection may be possible. is a weak acid with pKa = 3.27 (Bjornsson et al.,  1982),  Although ICG solvent-solvent  extraction for ICG sample preparation is unlikely due to the physicochemical properties of ICG.  The two sulfonate groups of ICG and its  high water solubility (Figure 4) limits the use of acid or base in the attempt  to  get  free  ICG acid.  Solid  phase  extraction  represents  the best choice for ICG sample preparation.  separation  is  not complete at the HPLC conditions  possible to get better separation with modifications.  used,  probably  Although the it might be  115  5. SUMMARY AND CONCLUSIONS 1. 5-Hydroxypropafenone binds to one class of binding site in purified human AAG solution.  The displacing effect of various agents on the binding of PF and 5-hydroxypropafenone to various protein solutions is summarized in Table 9 and 10, respectively.  The results can be concluded as follows:  2. Verapamil did not displace either PF or 5-hydroxypropafenone from their binding sites in either purified human AAG or albumin solution.  3. Propranolol did not affect the binding of PF or 5-hydroxypropafenone in solutions of AAG and albumin.  4. ASA caused significant increase in 5-hydroxypropafenone free fraction in a solution of albumin but not AAG; ASA did not affect PF binding.  5. Lidocaine caused significant increase in PF free fraction in either purified human AAG, albumin or serum. It is a potent displacing agent for  PF protein  binding.  It also  affected  the binding  of 5-  hydroxypropafenone in a solution of AAG but not in a pure albumin solution.  6. Phenytoin affected the binding of PF in solution of albumin and serum, but not in AAG and whole blood.  Table 9 PF free fraction before and after treatment with differing displacing agents  biological fluid buffer conc.(M)  albumin  AAG  serum  bl ood  0.067  0.1  0.067  0.1  0.1  0.1  control  0.075 ± 0.01  0.057 ± 0.004  0.431 ± 0.017  0.339 ± 0.006  0.017 ± 0.003  0.057 ± 0.012  ASA  0.095 ± 0.024  -  0.443 ± 0.047  -  -  -  propranolol  0.091 ± 0.005  -  0.452 ± 0.038  -  -  -  verapamil  0.088 ± 0.008  -  0.405 ± 0.015  -  -  -  -  0.129 ± 0.013*  -  0.383 ± 0.008*  0.114 ± 0.006*  -  -  0.105 ± 0.016  -  0.577 ± 0.182  0.052 ± 0.009*  0.101 ± 0.01*  -  0.083 ± 0.016  -  0.649 ± 0.055*  0.041 ± 0.007*  0.079 ± 0.01  treatment  1idocaine quinidine phenytoin  *: statistical significance using two sample t-test combined with Bonferroni inequality at p < 0 . 0 5 .  Table 10 50HPF free fraction before and after treatment with differing displacing agents  biological fluid buffer conc.(M)  AAG 0.067  albumin 0.1  0.067  serum  0.1  0.1  treatment control  0.321 ± 0.321 + 0.271 ± 0.354 ± 0.189 ± 0.029 0.028 0.017 0.013 0.015  ASA  0.476 ± 0.087  -  0.398 ± 0.028*  -  propranolol  0.355 ± 0.029  -  0.304 ± 0.01  -  -  verapamil  0.304 ± 0.068  -  0.281 ± 0.045  -  -  0.472 ± 0.03*  1idocaine quinidine phenytoin  -  0.224 ± 0.045  0.342 ± 0.114 ± 0.01 0.006  -  -  -  -  0.211 ± 0.01  -  -  -  -  0.182 ± 0.009  *: statistical significance using two sample t-test combined with Bonferroni inequality at p < 0.05.  118 serum, but not in AAG and whole blood.  7.  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