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Disopyramide pharmacokinetics in non-smoking and smoking volunteers : analytical development and effect… Kapil, Ram Prakash 1985

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"DISOPYRAMIDE PHARMACOKINETICS IN NON-SMOKING AND SMOKING VOLUNTEERS: ANALYTICAL DEVELOPMENT AND EFFECT OF PHENOBARBITAL ON ELIMINATION AND PROTEIN-BINDING" by RAM PRAKASH KAPIL M.Pharm., I n s t i t u t e of Technology, Banaras Hindu University, 1979 M.Sc, The University of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences, D i v i s i o n of Pharmaceutics) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1985 © Ram Prakash Kapil, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of T h OL/-L,'YY\CCC-&JJL\~LCJL The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) II ABSTRACT Disopyramide i s a c l a s s IA antiarrhythmic agent which shares a number of pharmacological properties with quinidine and pro-cainamide. C l i n i c a l l y , i t i s e f f e c t i v e in the treatment of supra-ventricular tachycardia, a t r i a l f i b r i l l a t i o n , v entricular tachy-cardia and premature ve n t r i c u l a r contractions. Disopyramide, a basic drug, has been shown in the past to be bound to acute phase r e a c t a n t , a l p h a s - a c i d g l y c o p r o t e i n (AAG) i n a concentration-dependent manner. Disopyramide has been known to demonstrate a steep serum concentration-pharmacological e f f e c t r e l a t i o n s h i p . The primary metabolic pathway of disopyramide i n humans i s N - d e a l k y l a t i o n which i s s u s c e p t i b l e to enzyme induc-t i o n , as indicated by a few preliminary studies. The pharmacolo-g i c a l role of the mono-N-dealkylated metabolite (MND) of disopyr-amide in humans i s not f u l l y established. This thesis describes the development of an improved gas-l i q u i d c h r o m a t o g r a p h i c (GLC) a s s a y f o r the s i m u l t a n e o u s q u a n t i t a t i o n of t r a c e l e v e l s of disopyramide and MND i n human b i o l o g i c a l f l u i d s and the a p p l i c a t i o n of the method i n i n v i t r o plasma protein-binding of disopyramide. This thesis also describes and discusses the e f f e c t of phenobarbital treatment on the serum protein-binding and single dose pharmacokinetics of disopyramide i n non-smokers and smokers. I l l A GLC-nitrogen/phosphorus s p e c i f i c detection (NPD) assay has been developed which provides improved s e l e c t i v i t y and s e n s i t i v i -t y f o r disopyramide and MND, employing a f u s e d - s i l i c a c a p i l l a r y column f o r chromatographic separation from b i o l o g i c a l specimen components. The q u a n t i t a t i o n of disopyramide and MND i n human serum, s a l i v a and urine was accomplished by i n j e c t i n g t r i f l u o r o -a c e t i c anhydride (TFAA)-treated samples containing the i n t e r n a l standard, p-chlorodisopyramide (PC-Dis), into a gas chromatograph equipped with a nitrogen/phosphorus detector (NPD) and an automa-t i c l i q u i d sampler (ALS). A 25 m ( i . d . = 0.31 mm) f u s e d - s i l i c a column, coated with c r o s s l i n k e d 5 % phenyl methyl s i l i c o n e f l u i d was u t i l i z e d and samples were injected using the s p l i t l e s s sample i n j e c t i o n mode. L i n e a r i t y was observed i n the serum concentra-t i o n range from 0.05 to 5.0 mcg/ml f o r disopyramide and from 0.02 to 3.0 mcg/ml for MND. The average c o e f f i c i e n t of v a r i a t i o n was 5 and 8 % for disopyramide and MND, respectively, over the concentration range studied. The c a p i l l a r y GLC-NPD assay was u t i l i z e d to measure disopyr-amide concentration, unbound to plasma protein, following e q u i l i -brium d i a l y s i s of plasma samples col l e c t e d from healthy volun-teers. Various concentrations of disopyramide ranging from 0.5 to 10 mcg/ml i n 0.4 ml of isoton i c phosphate buffer (pH 7.4) were dialysed for 6 hours at 37°C, against 0.4 ml blank plasma from f i v e healthy volunteers. The concentration-dependent binding of disopyramide was v e r i f i e d . The average unbound fractions for disopyramide, at concentrations 0.5, 1, 2, 3, 4, 5 and 10 mcg/ml, were 0.14, 0.15, 0.20, 0.28, 0.30, 0.35 and 0.55, respectively. IV The e f f e c t of phenobarbital treatment on the pharmacokine-t i c s and b i n d i n g o f disopyramide was s t u d i e d i n non-smoking v o l u n t e e r s : (1) Because of the p o t e n t i a l pharmacological and t o x i c o l o g i c a l significance of the major metabolite of disopyr-amide, (2) due to the need to develop a b e t t e r understanding of the e f f e c t of phenobarbital treatment on the serum concentration of alphas-acid glycoprotein (AAG), the p r i n c i p a l binding protein of disopyramide and other basic drugs and (3) to provide insight into the s e l e c t i v i t y of phenobarbital induction as compared to l i t e r a t u r e data on other inducers (rifampin, phenytoin, carbamazepine and spironolactone). Eight healthy male subjects received a 200 mg o r a l dose of disopyramide on 2 occasions separated by a 3 week period during which phenobarbital was taken (100 mg p.o.) at bedtime y i e l d i n g a mean steady-state concentration of 14 + 2 mcg/ml. Serum, s a l i v a and urine c o l l e c t i o n s were obtained following each disopyr-amide dose. The serum c o n c e n t r a t i o n v e r s u s time d a t a were a n a l y s e d by AU TOAN and NONLIN c o m p u t e r p r o g r a m s and were found t o f i t a one-compartment open p h a r m a c o k i n e t i c model. The a p p a r e n t t e r m i n a l phase e l i m i n a t i o n h a l f - l i f e ( t ^ ^ 2 ) o f d i s o p y r a m i d e was s h o r t e r (p < 0.05) i n s u b j e c t s d u r i n g p h e n o b a r b i t a l treatment (4.6 +^  0.7 hr) (mean + S.D.) than d u r i n g the c o n t r o l experiment (6.5 +_ 1.5). Furthermore, an i n d e x o f t h e amount o f d r u g i n t h e b o d y , t h e a r e a under the disopyramide serum c o n c e n t r a t i o n versus time curve (AUC), was markedly reduced from c o n t r o l (27 +_ 6 hr.mcg/ml) after phenobarbital (17 + 6 hr.mcg/ml, p < 0.05). V The A U C o _ 2 4 h r f o r t * i e P r i n c * P a l m e t a b o l i t e o f disopyramide (mono-N-dealkyl d i s o p y r a m i d e or MND) i n c r e a s e d (3.8 +_ 1.6 hr.mcg/ml f o r c o n t r o l vs 4.1 +_ 2.3 hr.mcg/ml i n pheno-b a r b i t a l - t r e a t e d s u b j e c t s ) but t h i s change was not s t a t i s -t i c a l l y s i g n i f i c a n t . Phenobarbital treatment caused no obser-vable trend towards a change i n disopyramide unbound f r a c t i o n (0.23 + 0.02 vs 0.21 + 0.02) or in serum AAG concentration (50 +_ 15 vs 54 +_ 18 mg/100 m l ) , as has been o b s e r v e d i n dogs i n the past. The r a t i o of MND to disopyramide i n s a l i v a was c o n s i s t e n t l y h i g h e r i n p h e n o b a r b i t a l - t r e a t e d s u b j e c t s . The average p e r c e n t a g e of dose r e c o v e r e d i n u r i n e as unchanged disopyramide i n 48 hrs was 43 + 6 % and 25 + 5 % (p < 0.05) i n c o n t r o l and p h e n o b a r b i t a l - t r e a t e d s u b j e c t s , r e s p e c t i v e l y . The average percentage of dose recovered as MND i n 48 hours was 25 + 6 % i n controls and 3 3 + 7 % i n phenobarbital-treated subjects. It can be concluded from our study that the enzyme inducer phenobarbital enhances disopyramide metabolism but does not a l t e r the serum AAG le v e l s or the binding of disopyramide to t h i s protein i n normal subjects. The e f f e c t o f c h r o n i c c i g a r e t t e smoking and the combined e f f e c t of smoking and phenobarbital treatment on the disopyramide pharmacokinetics and serum AAG l e v e l s were a l s o i n v e s t i g a t e d . The general procedures such as dosing and p h y s i o l o g i c a l monitor-ing of smokers, sampling p r o t o c o l s of smokers before and during phenobarbital-treatment, sample c o l l e c t i o n and a n a l y t i c a l methods were i d e n t i c a l to the non-smoking study. There was no di f f e r e n c e i n the pharmacokinetic parameters or serum AAG co n c e n t r a t i o n s VI between the smoking and the non-smoking volunteers when studied and compared i n the absence and presence of phenobarbital treatment. Therefore, i t appears that the metabolic b i o - t r a n s f o r m a t i o n o f d i s o p y r a m i d e remains u n a f f e c t e d by c h r o n i c c i g a r e t t e s m o k i n g . These f i n d i n g s s u p p o r t t h e general view that the in d u c t i o n of hepatic microsomal o x i d a t i o n o f a p a r t i c u l a r drug i s h i g h l y s e l e c t i v e i n humans and i s a function of substrate or drug s p e c i f i c i t y for a p a r t i c u l a r form of cytochrome P-450. James E. Axelson VTI TABLE OF CONTENTS CHAPTER PAGE ABSTRACT II LIST OF TABLES XII LIST OF FIGURES XIII LIST OF SCHEMES XVIII SYMBOLS AND ABBREVIATIONS XIX ACKNOWLEDGEMENTS • XXIII 1. INTRODUCTION 1.1 General Background 1 1.2 Selected Pharmacodynamic Studies. 1.2.1 Electrophysiological E f f e c t s on the Heart 2 1.2.2 Hemodynamic Eff e c t s 2 1.2.3 Anticholinergic Side E f f e c t s 3 1.2.4 Miscellaneous Adverse Ef f e c t s of Disopyramide 3 1.3 C l i n i c a l Indications 3 1.4 Comparison of Disopyramide to Other Antiarrhythmics i n the Treatment of Ventricular Arrhythmias 3 1.5 Serum Concentration-Antiarrhythmic E f f e c t Relationship 4 1.6 Disopyramide Pharmacokinetics 6 1.6.1 Single Dose Studies 6 1.6.2 Multiple Dose Studies..... 7 1.6.3 Disopyramide Pharmacokinetics i n Diseased States 8 1.6.4 E f f e c t of Disopyramide on the Pharmacokinetics of Other Drugs 10 1.6.5 Controlled or Sustained Release Dosage Form of Disopyramide 11 1.7 Disopyramide Serum Protein-Binding 12 1.7.1 General Concepts of Unbound Drug Concentration versus Pharmacological Response 12 1.7.2 Concentration-Dependent Disopyramide Protein-Binding 13 VIII CHAPTER P A G E 1.7.3 Serum Alpha 1-Acid Glycoprotein and Disopyramide Binding 14 1.7.4 C l i n i c a l Importance of Unbound Disopyramide Concentration 15 1.7.4.1 Pharmacokinetic Studies 15 1.7.4.2 Pharmacodynamic Studies 17 1.7.4.3 C l i n i c a l Relevance of the Measurement of Unbound Disopyramide Concentration i n Serum 17 1.7.5 Disopyramide Binding Experiments.... 19 1.8 Metabolism of Disopyramide 22 1.9 A n a l y t i c a l Methods 25 1.10 Metabolic Induction of Disopyramide and Altered Binding 29 1.10.1 E f f e c t of Enzyme Inducers on Disopyramide Metabolism 29 1.10.2 E f f e c t of Enzyme Inducers on Serum AAG 30 1.10.3 E f f e c t of Cigarette Smoking on Drug Elimination... 31 1.10.4 Cigarette Smoking and Serum AAG Levels 31 1.11 Primary Objectives 33 2. EXPERIMENTAL 2.1 Supplies 34 2.1.1 Drugs Used 34 2.1.2 Chemicals and Reagents 34 2.1.3 Solvents 3 5 2.2 Gas Chromatography Stationary Phases, So l i d Supports and Columns 3 5 2.3 Equipment 35 2.4 Preparation of Stock Solutions 36 IX CHAPTER p A G E 2.4.1 Disopyramide Phosphate Stock Solution 36 2.4.2 > MND and PC-Dis Stock Solutions 36 2.4.3 Preparation of Isotonic, 0.067M, pH 7.4 Phosphate Buffer (Documenta Geigy, 1970) 36 2.5 Preliminary Studies of Packed Column (3 % OV-17) GLC-FID and GLC-NPD Methods 37 for Disopyramide 2.5.1 GLC-FID 37 2.5.2 GLC-FID versus GLC-NPD 38 2.6 Preliminary Results of Fused-Silica C a p i l l a r y GLC Studies of Disopyramide 42 2.6.1 C a p i l l a r y GLC-FID 42 2.6.2 C a p i l l a r y GLC-NPD 45 2.6.3 S p l i t versus S p l i t l e s s Sample Injection Mode 49 2.6.4 Miscellaneous Optimizing Conditions for C a p i l l a r y GLC-NPD 52 2.7 C a p i l l a r y GLC-NPD Chromatography of MND 53 2.8 Optimization of TFAA-Treatment 57 2.9 General Scheme for Simultaneous Analysis of Disopyramide and MND by C a p i l l a r y GLC-NPD 57 2.9.1 Extraction and TFAA-Treatment 57 2.9.2 Quantitation Analysis of Disopyramide and MND 59 2.9.3 Optimum C a p i l l a r y GLC-NPD Conditions 60 2.10 Extraction E f f i c i e n c y of Toluene 60 2.11 General Procedure for Recoating of NPD Col l e c t o r 61 2.12 Measurement of Disopyramide Plasma Protein-Binding Using Equilibrium D i a l y s i s 62 X 2.13 Phenobarbital Treatment i n Non-Smokers for Disopyramide Pharmacokinetics and Binding Studies 64 2.13.1 Volunteers 64 2.13.2 Dosing and Physiological Monitoring of Study Subjects 66 2.13.3 Phenobarbital Treatment Protocol 66 2.13.4 Sample Co l l e c t i o n Techniques 68 2.13.5 A n a l y t i c a l Procedures 69 2.13.6 Data Analysis 72 2.13.7 Assurance of Phenobarbital Compliance 73 2.14 Phenobarbital Treatment i n Smokers for Disopyramide Pharmacokinetics and Binding Studies 73 3 RESULTS 3.1 A n a l y t i c a l Development 75 3.1.1 C a p i l l a r y GLC-NPD 75 3.1.2 L i n e a r i t y and Reproducibility of GLC-NPD Assay methods 78 3.1.3 Kinetics of TFAA-Treatment 78 3.1.4 Extraction E f f i c i e n c y of Toluene 82 3.1.5 Structural Confirmation of Compounds 82 3.2 Measurement of Disopyramide Plasma Protein-Binding Using Equilibrium D i a l y s i s 84 3.3 Phenobarbital Treatment i n Non-Smokers for Disopyramide and MND Pharmacokinetics and Binding Studies 92 3.4 Phenobarbital Treatment i n Smokers: Pharmacokinetics and Binding Studies of Disopyramide and MND 108 XI CHAPTER PAGE 4. DISCUSSION 4.1 Development of A n a l y t i c a l Methods 117 4.1.1 C a p i l l a r y GLC-NPD 117 4.1.2 Optimization of TFAA-Treatment 118 4.1.3 Extraction E f f i c i e n c y of Toluene 119 4.1.4 I d e n t i f i c a t i o n of Untreated and TFAA-Treated Compounds by MS 119 4.1.5 A p p l i c a b i l i t y of the C a p i l l a r y GLC-NPD Assay method 125 4.2 In V i t r o Disopyramide Plasma Protein-Binding 126 4.3 Phenobarbital Treatment in Non-Smokers: Pharmacokinetics and Binding Studies of Disopyramide and MND 127 4.3.1 Pharmacokinetics of Disopyramide and MND: Serum Data 128 4.3.2 Saliva Data 129 4.3.3 Urinary Excretion Data 130 4.3.4 Binding Results 131 4.3.5 C l i n i c a l Implications of Phenobarbital-Induced Disopyramide Metabolism 132 4.4 Phenobarbital Treatment in Smokers: Pharmacokinetics and Binding Studies of Disopyramide and MND 133 4.4.1 Pharmacokinetics of Disopyramide and MND: Serum Data 134 4.4.2 Urinary Excretion Data 135 4.4.3 Serum Protein-Binding of Disopyramide and Phenobarbital Treatment i n Chronic Cigarette Smokers 136 4.4.4 Phenobarbital Induced Disopyramide Pharmacokinetics: Non-Smokers versus Smokers 13 7 5. SUMMARY AND CONCLUSIONS 138 6. REFERENCES 141 7. APPENDIX •• 161 7.1. GLC-EI-MS Conditions 161 7.2 GLC-CI-MS Conditions 161 XII LIST OF TABLES TABLE PAGE 1. Subject Characteristics for Non-Smokers 65 2. Phenobarbital Induction Study Protocol 67 3. Subject Characteristics for Smokers 74 4. C a l i b r a t i o n Data Points for Disopyramide and MND (Higher Concentration Range) 79 5. C a l i b r a t i o n Data points for Disopyramide and MND (Lower Concentration Range) 80 6. Toluene E x t r a c t a b i l i t y of Disopyramide and MND 82 7. Unbound Fraction (fu) of Disopyramide as a Function of Concentration 91 8. Pharmacokinetic Parameters of Disopyramide In Non-Smokers 9 5 9. Mean Disopyramide Serum Concentration (mcg/ml) r and Unbound (fu) as a Function of Time and Phenobarbital Treatment 97 10. Serum AAG Concentrations (mg/100 ml): Eff e c t of Phenobarbital Treatment i n Non-Smokers 98 11. Forty-Eight Hour Cumulative Urinary Excretion Recovery of Disopyramide and MND i n Non-Smokers . 106 12. Pharmacokinetic Parameters of Disopyramide i n Smokers HI 13. Serum AAG Concentration (mg/100/ml): E f f e c t of Phenobarbital Treatment i n Smokers 113 14. Forty-Eight Hour Cumulative Urinary Excretion Recovery of Disopyramide and MND i n Smokers.... 115 15. Comparison of Pharmacokinetic Parameters of Total Serum Disopyramide Concentrations Between Non-Smokers and Smokers 116 XIII LIST OF FIGURES FIGURE PAGE 1. Representative packed column chromatograms of disopyramide (retention time, R.T. = 6.8 min) and PC-Dis (R.T. = 11.4 min) obtained from FID (A) and NPD (B) detection modes (An attenuation of 2 was used i n both cases). A 2 meter ( i . d . = 2mm) glass column packed with 3 % OV-17 stationary phase was used 40 2. Relationship between disopyramide amount and NPD detector response. A 2 meter long ( i . d . = 2 mm) glass column packed with 3 % OV-17 stationary phase was used for the chromatography of disopyramide (retention time, R.T. = 2.64 min) and PC-Dis (R.T. = 4.77 min) 41 3. Representative c a p i l l a r y GLC-FID chromato-grams obtained a f t e r i n j e c t i n g methylene chloride extracts of blank plasma (A) and aqueous solution of disopyramide phosphate (B), (R.T. = 2.61 min.). A 12 meter ( i . d . = 0.27 mm) f u s e d - s i l i c a c a p i l l a r y column coated with crosslinked methyl s i l i c o n e f l u i d was used 43 4. E f f e c t of various s p l i t r a t i o s on the area r a t i o of disopyramide MND and PC-Dis; Jennings insert-1, ( • ); Jennings insert-2 (similar type to 1), ( O ); f u s e d - s i l i c a insert with glass wool, ( X ); and ins e r t without glass wool ( > ) . A l l of the above area r a t i o readings were obtained from the sample measured i n t r i p l i c a t e 44 5. Representative c a p i l l a r y GLC-NPD chromato-grams of various antiarrhythmics.......... 47 6. Baseline c h a r a c t e r i s t i c s of various solvents with c a p i l l a r y GLC employing NPD detection 48 XXV FIGURE 7. Representative c a p i l l a r y GLC-NPD chromato-grams obtained from s p l i t (A) and s p l i t l e s s (B) sample i n j e c t i o n modes. A 2 m i c r o l i t e r aliquot of a TFAA-treated toluene extract of serum sample, containing disopyramide (retention time, R.T. = 10.56 min), MND (R.T. = 10.75 min) and PC-Dis (R.T. = 12.56 min) was injected under each mode. A s p l i t of 10 to 1 was used i n the s p l i t mode * 8. E f f e c t of purge a c t i v a t i o n time versus area counts for TFAA-treated toluene extracts of disopyramide, MND and PC-Dis.... 9. Representative c a p i l l a r y GLC-NPD chromato-grams obtained a f t e r i n j e c t i n g untreated MND (A) and acetic anhydride (100 micro-l i t e r s ) treated MND (B) solutions i n toluene. The untreated MND resulted i n at leas t 3 peaks (A) and acetic anhydride treated MND yielded p a r t i a l acetylation of MND (B); The peak with R.T. = 4.01 min was speculated to be that of acetylated MND Representative c a p i l l a r y GLC-NPD chromato-gram of t r i f l u o r o a c e t i c anhydride (TFAA) derivatized MND Acetylation of the secondary amine group of MND and dehydration of the amide group of disopyramide and MND as a r e s u l t of t r i f l u o r o a c e t i c anhydride (TFAA) treatment Representative c a p i l l a r y GLC-NPD chromato-grams obtained from blank (A) and spiked plasma extracts (B); the spiked sample before extraction contained disopyramide (retention time, R.T. = 10.10 min), 1.0 mcg/ml; MND (R.T. = 10.59 min), 0.7 mcg/ml and PC-Dis (R.T. = 12.91 min), 2.4 mcg/ml. Attenuation, 2 ; voltage, 16 V; NPD detector was i n use for ca. 200 hours. A 12 meter ( i . d . = 0.27 mm) f u s e d - s i l i c a c a p i l l a r y column coated with crosslinked methyl s i l i c o n e f l u i d was used Representative c a p i l l a r y GLC-NPD peaks of TFAA-treated antiarrhythmic agents 10. 11. 12. XV FIGURE PAGE 14. Kinetics of t r i f l u o r o a c e t i c anhydride (TFAA)-treatment of disopyramide/para-chlorodisopyramide (PC-Dis) and mono-N-dealXylated metabolite (MND) of disopyr-amide (n = 2-3) 81 15. The po s i t i v e ion electron impact (EI) data ( t o t a l ion current chromatogram, mass spectrum (MS) and prominent fragments) of untreated MND 85 16. The p o s i t i v e chemical ionization (CI) t o t a l ion current mass chromatogram obtained from untreated MND (A): CI mass spectrum of in t a c t MND (B) and CI mass spectrum of degradation product of untreated MND (C) 86 17. The p o s i t i v e ion EI data ( t o t a l ion current chromatogram, mass spectrum and prominent fragments) of TFAA-treated MND 87 18A. The p o s i t i v e CI t o t a l ion current mass chromatogram (A) and mass spectrum (B) of TFAA-treated MND (incomplete rea c t i o n ) . . . . . . . 88 18B. The p o s i t i v e CI t o t a l ion current mass chromatogram (A) and mass spectrum (B) of excess of TFAA-treated MND (complete acetylation and dehydration) 89 19. Representative chromatograms of TFAA-treated t o l -uene extracts of buffer (A) and plasma (B) of diso-pyramide (R.T. « 10.4 min) and PC-Dis (R.T. = 13.07 min) 90 20. Disopyramide serum concentration versus time curves before (O ) and during (A ) phenobarbital treatment i n non-smokers (n « 8). The disopyramide dose was 200 mg of the base. The data are presented__as the mean + one standard deviation X + S.D.)T 7 93 21. MND serum concentration versus time curves before ( o ) and during ( • ) phenobarbital treatment i n non-smoker s_(n » 8). The data are presented as the X + S.D 22. Disopyramide s a l i v a concentration versus time curves obtained i n non-smoking volunteers before (O ) and (A ) during phenobarbital treatment • XVI FIGURE PAGE 23. MND s a l i v a concentration versus time curves of i n d i v i d u a l non-smoking volunteers before ( • ) and during ( • ) phenobarbital treatment 100 24. Comparison of area under the s a l i v a concen-t r a t i o n versus time curves for disopyramide (A) and MND ("§") i n non-smoking volunteers before and during phenobarbital treatment 101 25. Saliva MND/disopyramide ra t i o s before (O ,n = 6) and af t e r (• ,n = 6) phenobarbital-treatment 103 26. The rel a t i o n s h i p of disopyramide concen-t r a t i o n i n s a l i v a and unbound disopyramide concentration i n serum of non-smoking volunteers (O : untreated, n = 8 and A : phenobarbital treatment, n = 8) 104 27. The percent of dose recovered as diso-pyramide and MND i n 48 hour urine before ( O • , n = 8) and during phenobarbital treatment (A • , n = 8 ) i n non-smoking volunteers 105 28. Renal clearance of t o t a l disopyramide (upper), urine flow-rate (middle) and urinary pH (lower) versus time before ( l e f t panel, n = 8) and during phenobarbital treatment (right panel, n = 8) i n non-smoking volunteers 107 29. Disopyramide serum concentration versus time curves before (O , n = 8) and during ( A , n = 6) phenobarbital treatment i n smokers. The disopyramide was 200 mg_of the base. The data are presented as X + S.D 7 109 30. MND serum concentration versus time curves before ( O , n = 8^  and during ( • , n = 6) phenobarbital treatment i n _ smokers. The data are presented as X + S.D 7 110 31. The percent of dose recovered as disopyramide and MND i n 48 hour urine before ( O , • , n = 8) and during phenobarbital treatment (A , • , n = 6) i n smoking volunteers 114 XVII FIGURES PAGE 32. Postulated degradation reaction of mono-N-dealkylated metabolite of disopyramide (MND) 122 33. Proposed prominent fragment ions based on the EI-MS of TFAA-treated MND (A) dehydrated ( n i t r i l e and t r i f l u o r o a c e t y l a t e d MND and (B) t r i f l u o r o a c e t y l a t e d MND (amide form) 124 34. Total ion current mass chromatogram (A) and EI-MS (B) of disopyramide ... 163 35. Total ion current mass chromatogram (A) and EI-MS (B) of TFAA-treated disopyramide 164 36. Total ion current mass chromatogram (A) and EI-MS (B) of PC-Dis 165 37. Total ion current mass chromatogram (A) and EI-MS (B) of TFAA-treated PC-Dis 166 38. Total ion current mass chromatogram (A) and CI-MS (B) of disopyramide 167 39. Total ion current mass chromatogram (A) and CI-MS (B) of TFAA-treated disopyramide 168 40. Total ion current mass chromatogram (A) and CI-MS (B) of PC-Dis 169 41. Total ion current mass chromatogram (A) and CI-MS (B) of TFAA-treated PC-Dis 170 42. Prominent fragment ions based on the EI-MS of disopyramide 171 43. Prominent fragment ions based on the EI-MS of TFAA-treated disopyramide 17 2 44. Prominent fragment ions based on the EI-MS of PC-Dis 173 45. Prominent fragment ions based on the EI-MS of TFAA-treated PC-Dis 174 46. Prominent fragment ions based on the EI-MS of untreated MND 17 5 XVIII LIST OF SCHEMES SCHEME PAGE I Scheme for Simultaneous Analysis of Disopy-ramide and i t s Mono-N-dealkylated Metabolite by C a p i l l a r y GLC-NPD 58 II Equilibrium D i a l y s i s Scheme for Plasma Protein-Binding of Disopyramide i n Healthy Volunteers 63 XIX ABBREVIATIONS AAG ALS AMI ARE A U C t l - t 2 AUTOAN 1 4 C ca. CI '"peak CR TB unbound CI CI CI CR ^ss,unbound Cunbound C.V. EI EI-MS EMIT® F alphas-acid glycoprotein automatic l i q u i d sampler acute myocardial i n f a r c t i o n amount remaining to be excreted area under serum concentration versus time curve a decision-making pharmacokinetic computer program radi o l a b e l l e d carbon-14 approximately chemical i o n i z a t i o n maximum or peak serum or plasma concentration achieved following o r a l administration of a drug creatinine clearance renal clearance of t o t a l drug (bound and unbound) t o t a l body clearance of t o t a l drug (bound and unbound) clearance of the unbound drug controlled release unbound concentration of a drug at steady state unbound concentration of a drug c o e f f i c i e n t of v a r i a t i o n electron impact electron impact mass spectrum (spectra) enzyme mul t i p l i e d immunoassay technique b i o a v a i l a b i l i t y XX FID fu g GIT GLC H2 HC1 H.P. HPLC hr h. s. i . d. k K e l kg KH 2P0 4 1 log M m mm meg min ml MND MS flame i o n i s a t i o n detector unbound f r a c t i o n gram ga s t r o i n t e s t i n a l t r a c t gas-liquid chromatography hydrogen gas hydrochloric acid high-performance high-performance l i q u i d chromatography hour hora somni (at bedtime) inter n a l diameter pseudo f i r s t - o r d e r absorption rate constant pseudo f i r s t - o r d e r o v e r a l l elimination rate constant kilogram monopotassium phosphate l i t e r logarithm to the base 10 molar (moles/liter) meter millimeter microgram minute m i l l i l i t e r mono-N-dealkylated metabolite mass spectrometer, spectrum or spectra XXI N NaOH NaoHP0. 2 4 ng NONLIN NPD OV-17 PAHs PC-Dis pH PTFE p. o. QRS QT QTc r r 2 RID R.T. +SD h/2 TFAA TLC normal sodium hydroxide dibasic sodium phosphate (dihydrate) nanogram computer program for non-linear l e a s t squares regression analysis of pharmacokinetic systems nitrogen/phosphorus s e l e c t i v e detector 50 % phenylmethyl s i l i c o n e stationary phase polynuclear aromatic hydrocarbons para-chlorodisopyramide negative logarithm of hydrogen ion concentration polytetrafluoroethylene per os (oral route) duration of the electrocardiogram representing v e n t r i c u l a r depolarization of the cardiac action p o t e n t i a l i n t e r v a l of the electrocardiogram representing v e n t r i c u l a r depolarization and r e p o l a r i z a t i o n -1/2 corrected QT i n t e r v a l = QT* (R-R) ' co r r e l a t i o n c o e f f i c i e n t c o e f f i c i e n t of determination radialimmuno d i f f u s i o n retention time + one standard deviation apparent terminal phase elimination h a l f - l i f e t r i f l u o r o a c e t i c anhydride thin-layer chromatography XXII time taken to reach maximum or peak serum or plasma concentration following o r a l administration apparent volume of d i s t r i b u t i o n calculated from AUC versus XXIII ACKNOWLEDGEMENTS I would l i k e to take this opportunity to sincerely thank my supervisor, Professor James E. Axelson for his valuable guidance, constant encouragement and provision of the excellent f a c i l i t i e s during the course of this p r o j e c t . I am grateful to Dr. Frank S. Abbott for his advice and help during the analytical development. I am also grateful to Dr. Charles R. Kerr for his super-visi o n of human experimentation and Dr. David Lalka who was involved in the protein binding studies. I would l i k e to thank Mrs. Barbara McErlane, Ms. Inger Mansfield, Ms. Marcia Mason, Ms. Joyce Chan, Mr. Rainold Lee and Mr. Douglas Tisdale for their technical assistance during the course of this p r o j e c t . I would l i k e to acknowledge Dr. William Godolphin, Dr. Keith M. McErlane and Dr. James M. Orr for their help and encouragement. I am deeply indebted to Ms. Grace L.Y. Chan for her valuable time, kindness and constant support during the completion of the study and preparation of the t h e s i s . This work was funded by the B r i t i s h Columbia Heart Foundati on . XXTV This thesis is dedicated to my dearest wife, J y o t i , whose unfai l i n g support, constant encouragement and boundless patience have carried me through the years of my graduate program and the completion of this t h e s i s . 1 1 INTRODUCTION 1.1 GENERAL BACKGROUND Disopyramide, 4-(diisopropylamino)-2-(2-pyridyl)-2-phenyl-butyramide (pKa = 8.5 - 9.7), i s an established class 1A a n t i -arrhythmic agent. Its antiarrhythmic a c t i v i t y was f i r s t repor-4 ( D i i s o p r o p y l a m i n o ) - 2 - ( 2 - p y r i d y l ) - 2 - p h e n y l b u t y r a m i d e D I S O P Y R A M I D E ( D I S ) ted i n 1962 (Mokler and Van Arman, 1962) and i t has been marketed i n Europe since 1969 (Jequier et a l . , 1970). More than 300 m i l l i o n doses of disopyramide were prescribed world -wide, several hundred publications described i t s actions and 15 years passed before t h i s drug gained approval for o r a l c l i n i c a l use i n the United States i n 1977 (Koch-Weser, 1979). Several excellent review a r t i c l e s on disopyramide have appeared recently, describing various pharmacologic and pharmacokinetic c h a r a c t e r i s -t i c s of t h i s antiarrhythmic agent (Ankier, 1977; Heel et a l . , 1978; Koch-Weser, 1979; Befeler and Lazzara, 1980; Lawrie, 1980; Moses and Paul, 1980; Keefe et a l . , 1981; Schwartz et a l . , 1981; Karim et a l . , 1982; Morady et a l . , 1982; Milne et a l . , 1984 and Roden and Woosley, 1984). 2 1.2 Selected Pharmacodynamic Studies 1.2.1 Electrophysiological Effects on the Heart Disopyramide shares a number of e l e c t r o -p h y s i o l o g i c a l actions on the heart with other chemically un-related Class IA antiarrhythmics or membrane s t a b i l i z e r s such as quinidine and procainamide. For example, i t suppresses automati-c i t y , slows the maximal rate of depolarization and prolongs refractoriness of the cardiac action potential (Roden and Woosley, 1984). The electrophysiological e f f e c t s of disopyra-mide include s l i g h t widening of the QRS complex and prolonged QTc i n t e r v a l of the electrocardiogram (Heel et a l . , 1978). 1.2.2 Haemodynamic Effects Enthusiasm for the use of disopyramide as an antiarrhythmic agent i s s l i g h t l y diminished because of i t s undesirable s i g n i f i c a n t negative inotropic e f f e c t on the heart (Podrid et a l . , 1980). On the other hand, other antiarrhyth-mics including encainide., mexiletine, tocainide (Jewitt, 1980) and propafenone (Trimarco et a l . , 1983 and Angermann and Fahrmarker, 1983) are only minor negative inotropes. The myo-ca r d i a l depressant e f f e c t of disopyramide i s transient (Jensen et a l . , 1976) which indicates that the drug can be safely used with proper precaution. The e f f e c t s on heart rate and blood pre-ssure are usually minimal (Heel et a l . , 1978). 3 1.2.3 Anticholinergic Side Ef f e c t s Disopyramide exerts dose-related reversible a n t i c h o l i n e r g i c side e f f e c t s such as urinary hesitancy and reten-t i o n , b l u r r i n g of v i s i o n , dryness of mouth and constipation (Morady et a l . , 1982). 1.2.4 Miscellaneous Adverse Effects of  Disopyramide A few i n d i v i d u a l case studies suggest that disopyramide may cause fasting hypoglycemia (Semel et a l . , 1982) and hepatic t o x i c i t y (Antonelli et a l . , Doody, 1982). More c l i n i c a l t r i a l s are needed to test these observations. 1. 3 C l i n i c a l Indications Disopyramide has been shown to be e f f e c t i v e i n the suppression of various disorders of e l e c t r i c a l impulse genera-ti o n and conduction i n man. For instance, i t i s used in the treatment of a t r i a l f l u t t e r and f i b r i l l a t i o n (Tonkin et a l . , 1982), supraventricular and v e n t r i c u l a r extrasystoles, supra-v e n t r i c u l a r t a c h y c a r d i a (Tonkin et a l . , 1982, Stewart et a l • , 1984), and the Wolff-Parkinson-White Syndrome (Kerr et a l . , 1982 and Kou et a l . , 1982). 1.4 Comparison of Disopyramide to Other Antiarrhythmics In the Treatment of Ventricular Arrhythmias Disopyramide i s generally better tolerated than quinidine (Oshrain et a l . , 1976) and does not cause a l u p u s - l i k e syndrome as does procainamide (Scwartz et a l . , 1980 and Trimarco 4 et a l . , 1983). Comparative studies have reported that disopyramide i s more e f f e c t i v e than lidocaine (Deano et a l . , 1978 and Sbarbaro et a l . , 1979), mexiletine (Breithardt et  a l . , 1982) and s l i g h t l y less e f f e c t i v e than propafenone (Naccarella et a l . , 1982) i n the treatment of ventricular premature contractions related to various chronic ventricular arrhythmias. Mexiletine and propafenone have less negative inotropic e f f e c t s than disopyramide (Trimarco et a l . , 1983 and Angermann and Fahrmarker, 1983). A recent report has demon-strated that disopyramide i s less potent than f l e c a i n i d e in the treatment of ventricular premature complexes (Kjekshus et a l . , 1984). Tocainide, a st r u c t u r a l analogue of lidocaine, has been found to be equally e f f e c t i v e as disopyramide against ventricular arrhythmias (Allen-Narker et a l . , 1984 and McLaran et a l . , 1984). Although adverse e f f e c t s such as a n t i -cholinergic actions and negative inotropic e f f e c t s occur, diso-pyramide appears to be an important pharmacological agent for use i n the treatment of cardiac arrhythmias. 1.5 Serum Concentration - Antiarrhythmic E f f e c t Relationship I t i s well known that the i n t e r - i n d i v i d u a l v a r i a t i o n i n drug elimination accounts f o r much of the v a r i a b i l i t y i n observed pharmacologic response when a standard fixed dose of a drug i s given to a s e r i e s of p a t i e n t s . A l t e r n a t i v e l y , serum drug concen-t r a t i o n i s often found to correlate with c l i n i c a l response for some drugs. The strategy of c l i n i c a l pharmacokinetics i s to t a i l o r drug dosage such that one may achieve serum concentrations 5 with a high p r o b a b i l i t y of producing the desired response while minimizing or avoiding t o x i c i t y . Experimental data concerning the rela t i o n s h i p between disopyramide serum concentrations and the suppression of arrhyth-mias have been reported in the l i t e r a t u r e . Niarchos (1976) reported that in patients whose arrhythmias were successfully treated, the disopyramide serum l e v e l was s i g n i f i c a n t l y higher (3.7 + 1.4 mcg/ml) (mean + S.D.) than in uncontrolled patients (2.4 + 0.4 mcg/ml). Robert et a l . , (1978) have shown that there are small differences in t o t a l disopyramide serum concentration between controlled (3-4 mcg/ml) and uncon-t r o l l e d (<2 mcg/ml) patients with respect to premature v e n t r i -cular contractions. A i t i o (1981) found the steady-state serum lev e l s of disopyramide immediately before dosing were s i g n i f i -cantly higher i n patients with controlled v e n t r i c u l a r arrhyth-mias (3.1 + 0.2 mcg/ml) as compared to the uncontrolled p a t i e n t s (2.3 + 0.3 mcg/ml). In p a t i e n t s with s u p r a v e n t r i c u l a r arrhythmias, however, no s i g n i f i c a n t differences in serum concentrations were noted between c o n t r o l l e d (3.2 + 0.4 mcg/ml) and uncontrolled (2.8 + 0.4 mcg/ml) subjects. These studies have demonstrated a serum concentration-antiarrhythmic response re l a t i o n s h i p for disopyramide with r e l a t i v e small differences in serum concentrations between controlled and uncontrolled p a t i e n t s . A l l of the aforementioned i n v e s t i g a t i o n s have examined the t o t a l serum concentrations of disopyramide rather than the unbound c o n c e n t r a t i o n . The v a r i a b i l i t y and apparent overlap i n 6 serum concentration data i n controlled and uncontrolled patients may be due to high i n t e r - and i n t r a - i n d i v i d u a l v a r i a b i l i t y i n protein-binding of disopyramide (discussed i n subsequent sections). Nevertheless, any perturbation which may a l t e r disopyramide serum concentration, such as a l t e r a t i o n of i t s elimination by enzyme induction or i n h i b i t i o n , would be expected to a f f e c t i t s pharmacologic response and hence would be c l i n i c a l l y important. 1.6 Disopyramide Pharmacokinetics 1.6.1 Single Dose Studies Disopyramide i s read i l y absorbed from the g a s t r o i n t e s t i n a l t r a c t (GIT) reaching peak plasma concentrations within 3 hours of o r a l dosing and i s ^  85 % bioavailable (Dubetz et a l . , 1978; Karim et a l . , 1982 and Lima et a l . , 1984). The apparent b i o l o g i c a l e l i m i n a t i o n h a l f - l i f e ( t j y 2 ^ o f t o t a l (bound and unbound) disopyramide appears to be 6-8 hours i n healthy i n d i v i d u a l s and i s independent of route of administration (Ashford et a l . , 1979; Karim et a l . , 1982 and Lima et a l . , 1984). However, such v a l u e s m a Y n o t p a r t i c u l a r l y meaningful, since the plasma protein-binding of disopyramide changes dramatically with concentration, and even within the therapeutic range. The approximate values for apparent t o t a l body clearance and apparent volume of d i s t r i b u t i o n of t o t a l disopyramide (bound and unbound) are 1.5 ml/min/kg and 1.0 l/kg respectively (Karim et a l . , 1982). About 50 % of a disopyramide dose i s excreted unchanged i n the urine (Karim et a l . , ]982. Renal clearance of disopyramide has been shown to 7 decline with time (Cunningham et a l . , 1977 and 1978, Lawrence et a l . , 1979 and Giacomini et a l . , 1982) which may, be i n part, due to the declining unbound f r a c t i o n i n serum (unbound f r a c t i o n , fu = unbound concentration/total (bound plus unbound) concentration of disopyramide) with time. It has been shown by Giacomini et a l . (1982) that the renal clearance of unbound disopyramide i s li n e a r and can be reported without q u a l i f i c a t i o n as to the drug concentration. 1.6.2 Multiple Dose Studies It has been reported that disopyramide single dose pharmacokinetics based on t o t a l serum concentration are non-line a r (Hinderling and Garrett, 1976 and Meffin et a l . , 1979), and hence t h i s information cannot be used to predict steady-state serum concentration. For example, i n order to obtain a two-fold increase i n steady-state serum concentration of t o t a l disopyramide, a four - f o l d increase in dosing rate i s required. Moreover, the same increase i n the dosing rate r e s u l t s i n a proportionate increase (four-fold) i n the steady-state serum concen t r a t i o n of unbound disopyramide. The unbound f r a c t i o n of disopyramide follows concentration-independent or l i n e a r k i n e t i c s and hence, should be used for accurate steady-state predictions. The non-linear, concentration-dependent behavior of t o t a l diso-pyramide pharmacokinetics has been attributed to concentration-dependent serum protein-binding within the therapeutic range (Hinderling and Garrett, 1976; Meffin et a l . , 1979; Giacomini et a l . , 1982; Bryson et a l . , 1982 and, Haughey and Lima, 8 1984). Ueda et a l . (1984) have recently attempted to predict steady-state serum concentration of t o t a l disopyramide based on single dose t o t a l disopyramide serum concentration data. The observed steady-state serum values for t o t a l disopyramide were generally lower than the predicted values, an observation repor-ted by Meffin et a l . (1979). It appears that the measurement of serum concentration of unbound disopyramide might be more appropriate for subsequent prediction of unbound disopyramide le v e l s at steady-state which, i n turn, might be a better i n d i c a -tor of drug concentration at the s i t e of action. 1.6.3 Disopyramide Pharmacokinetics i n Diseased  States Renal impairment, as expected, has been shown to diminish disopyramide elimination and r e s u l t s i n a prolonged t j ^ f ° r disopyramide dependent upon the severity of the renal f a i l u r e (Shen et a l . , 1980; Johnston et a l . , 1980; Sevka et a l . , 1981 and Francois et a l . , 1983). A l i n e a r r e l a t i o n s h i p between creatinine clearance (C1 C R) and t j y j ** a s t > e e n shown to exist only when C 1 C R was less than 40 ml/minute (Shen et a l . , 1980 and Francois et a l . , 1983). Therefore, the main-tenance dose should be reduced i n such patients according to t h e i r respective creatinine clearance values. Also, appropriate curtailment of disopyramide dose based on l i v e r function has been recommended by Karim et a l . , (1982) for patients with hepatic i n s u f f i c i e n c y . This suggestion was based on the observation of prolonged °* disopyramide i n 2 of the 3 patients studied with hepatic impairment. 9 The e f f e c t of acute myocardial i n f a r c t i o n (AMI) on disopyr-amide pharmacokinetics has been evaluated i n the past. Most of the investigations have indicated that there i s no apparent change i n the elimination h a l f - l i f e of disopyramide af t e r AMI (Ward et a l . , 1976? Rangno et a l . , 1976 and Jounela et a l . , 1982). However, I l l e t t et a l . , (1979) have concluded that the ^1/2 °^ disopyramide was markedly increased in patients with AMI. The r e s u l t s of I l l e t t et a l . , (1979) are questionable because the v a l u e s ^ n t h e i r study were not derived from the usual regression analysis of serum concentration versus time curve, but from the computer-simulated curves which gave the best f i t to the limited steady-state concentrations obtained during t h e i r study. There seems to be a general agreement that the extent of disopyramide absorption from the g a s t r o i n t e s t i n a l t r a c t i s diminished in AMI (Ward et a l . , 1976; Jounela et a l . , 1982; Weissberg et a l . , 1982 and Bryson et a l . , 1982). The rate of disopyramide absorption has been reported to be unchanged (Ward et a l . , 1976 and Jounela et a l . , 1982), delayed (Kumana et a l . , 1982) or variable (Weissberg et a l . , 1982). The v a r i a b i l i t y i n the rate of disopyramide absorption may, i n part, be related to the concurrent administration of narcotic analgesics and antiemetics (Weissberg et a l . , 1982 and Bryson et a l . , 1982) or, i n part, be the r e s u l t of the a n t i c h o l i n e r g i c side e f f e c t s expressed by the drug and i t s p r i n c i p a l metabolite. The pharmacokinetic r e s u l t s of disopyramide during recovery from AMI are comparable to the r e s u l t s from healthy volunteers 10 (Bryson et a l . , 1982). Dose adjustment according to serum concentrations of disopyramide has been suggested i n order to allow optimal treatment of patients with lower cardiac output (Landmark et a l . , 1981). 1.6.4 E f f e c t of Disopyramide on the Pharmacokinetics of Other Drugs Co-administration of disopyramide during multiple drug therapy did not r e s u l t i n apparent a l t e r a t i o n i n the pharmacokinetics of propranolol, diazepam (Karim et a l . , 1982) or warfarin (Sylven and Anderson, 1983). The area under the serum concentration versus time curve (AUC_ ) of 0—ohr quinidine was s l i g h t l y decreased (16 %) i n the presence of diso-pyramide (Karim et a l • , 1982). The mechanism of t h i s apparent decline i n AUC i s unclear. R i s l e r et a l . (1983) have recently reported on the possible i n t e r a c t i o n between digoxin and disopy-ramide. They found that the t o t a l and renal clearance of digoxin was not changed. However, the volume of d i s t r i b u t i o n (from 672 + 176 1 to 407 +153 1) and terminal h a l f - l i f e (from 40 + 12 hr to 22 + 7 hr) were reduced s i g n i f i c a n t l y i n f i v e healthy subjects a f t e r 600 mg oral disopyramide d a i l y . The c l i n i c a l s i g n i f i c a n c e of t h i s i n t e r a c t i o n i s not c l e a r . It appears from these l i m i t e d studies that disopyramide neither influences hepatic drug metabolic enzyme a c t i v i t y nor the renal clearance of other drugs i n humans. 11 1.6.5 Controlled or Sustained Release Dosage  Form of Disopyramide In practice, owing to i t s r e l a t i v e short ^1/2 (^ -^  ^ r)» disopyramide requires frequent administration d a i l y (3-4 times) to maintain an e f f e c t i v e steady-state concen-t r a t i o n for optimal antiarrhythmic e f f e c t . This circumstance leads to patient inconvenience and noncompliance. In the past five years, a number of studies have successfully demonstrated the effectiveness of controlled or sustained release (CR) disopyramide tablet s . The use of 250-300 mg of CR disopyramide tablets twice d a i l y resulted in comparable r e l a t i v e extent of absorption as opposed to the standard use of 150-200 mg disopyr-amide capsules 3-4 times d a i l y ( F o r s e l l et a l . , 1980; Graffner et a l . , 1981; Torstensson and Hofvendahl, 1982; Arnman et  a l . , 1983 and Fechter et a l . , 1983). The CR formulation resulted i n a slower rate of absorption; nevertheless, the maximum and minimum concentrations as well as the antiarrhythmic e f f e c t achieved from CR disopyramide tablets were comparable to the standard capsules (Torstensson and Hovendahl, 1982; Arnman et a l . , 1983; Fechter et a l . , 1983 and Zema, 1984). A quick onset of antiarrhythmic action of disopyramide can be achieved by intravenous bolus i n j e c t i o n or inf u s i o n ; f o r maintenance therapy, a safe and e f f i c i e n t switch over to CR tablets has been demon-strated (Lien and Bakke, 1983 and Liem and Hollander, 1984). 1.7 Disopyramide Serum Protein-Binding 12 1.7.1 General Concepts of Unbound Drug Concentration Versus Pharmacological  Response Pharmacological response i s generally corre-lated with the degree or rate of drug-receptor in t e r a c t i o n (Paton, 1961 and Ariens and Simonis, 1962) which i n turn i s dependent on the concentration of active species of a given drug at a receptor s i t e . For most drugs, only that f r a c t i o n of the t o t a l amount in serum which remains unbound (free) i s thought to be available for d i f f u s i o n out of the vascular system to the receptor s i t e s to produce a pharmacologic response (Goldstein, 1949; Goldstein et a l . , 1969 and G i l l e t e , 1973 ). A number of investigations have confirmed that the unbound concentration of a drug i n serum often correlates better with the drug e f f e c t (McDevitt et a l . , 1976; Lima et a l . , 1981; Huang and Oie, 1982, and Holt et a l . , 1983). In some instances, the use of t o t a l rather than unbound drug concentration at steady-state to adjust drug dosage may not lead to s i g n i f i c a n t c l i n i c a l e rrors. This i s possible i f the clearance of t o t a l drug i s independent of dose (Meffin et a l . , 1979) and i f the unbound f r a c t i o n of the drug i n serum (fu = unbound/total) does not vary appreciably within and between i n d i -viduals (Wilkinson et a l . , 1975; Rowland, 1980 and Greenblatt et a l . , 1982). 13 1.7.2 Concentration-Dependent Disopyramide Protein-Binding Based on i n v i t r o protein-binding experiments, Hinderling and Garrett i n 1974 demonstrated that the bound f r a c t i o n of disopyramide in serum appears to be concentration-dependent at therapeutic serum concentrations (2-6 mcg/ml). This finding has been confirmed by several more recent investigations (Hinderling and Garrett, 1976; Cunningham et a l . , 1978, Meffin et a l . , 1979; David et a l . , 1980; Lima et a l . , 1982, Bredesen, 1982 and Giacomini et a l . , 1982). However, the extent of binding at a given concentration has varied consider-ably among these studies. This v a r i a t i o n may be related to wide i n t e r - and intra-subject v a r i a b i l i t y i n the serum AAG concentra-t i o n (Piafsky et a l . , 1978) and displacement of disopyramide from the binding s i t e s of serum AAG by a p l a s t i c i z e r ( t r i s - 2 --butoxyethyl phosphate) which i s present i n Vacutainer® stoppers (Haughey and Lima, 1982). Therefore, d i f f e r e n c e s i n the source of serum (blood bank, pooled serum or fresh serum) and method of sample c o l l e c t i o n (Vacutainers^ or glass syringes) might be responsible i n part to the v a r i a b i l i t y i n disopyramide binding. The inter-subject v a r i a b i l i t y of disopyramide binding i s s t i l l quite s i g n i f i c a n t i n normal subjects i n spite of the use of unpooled serum and glass syringes for blood c o l l e c t i o n (Giacomini et a l . , 1982). Meffin et a l . , (1979) have also observed wide inter-patient v a r i a b i l i t y i n unbound f r a c t i o n for given t o t a l concentrations of disopyramide i n patients with cardiac disease. The p r e d i c t i o n of unbound f r a c t i o n of disopyr-amide i n the serum of an i n d i v i d u a l patient may be extremely 14 d i f f i c u l t because of wide i n t e r - i n d i v i d u a l v a r i a b i l i t y and concentration-dependent protein-binding. 1.7.3 Serum Alpha -Acid Glycoprotein and  Disopyramide Binding The v a r i a b i l i t y i n disopyramide protein -binding may also be related to the v a r i a t i o n i n serum concen-t r a t i o n of the binding protein i t s e l f . Disopyramide i s a basic compound which binds to alphas-acid g l y c o p r o t e i n (AAG) (Piafsky and Woolner, 1980, De Leve and Piafsky, 1981, Haughey and Lima, 1982 and Norris et a l . , 1984). AAG, an acute phase reactant, has been shown to be present i n higher than usual serum leve l s (normal serum concentration: 50-100 mg/100 ml, Piafsky et a l . , 1978) i n various pathological states such as myocardial i n f a r c -t i o n (Johannson et a l . , 1972, Johnston et a l . , 1983 and David et a l . , 1983), surgery (Aronsen et a l . , 1972), body stress (Schmid, 1975), cancer (Chio and Oon, 1979), trauma (Edwards et a l . , 1982), a r t h r i t i s , Crohn's disease, renal f a i -lure and c i r r h o s i s (Piafsky et a l . , 1978). Therefore, i t i s apparent that variable serum le v e l s of AAG could lead to a l t e r a -tions i n the disopyramide binding pattern, which i s already complicated by concentration dependency. Hayler and Holt (1983) have reported that neonates and infants have lower AAG l e v e l s . An additional factor contributing to the v a r i a b i l i t y of disopyr-amide binding has been reported by Haughey and Lima (1982). These workers have shown that a p l a s t i c i z e r present i n Vacutainers 4* (tris-2-butoxyethyl phosphate) competitively displaces disopyr-amide from the binding s i t e s of AAG to a s i g n i f i c a n t extent. 15 This observation has been previously noted for other basic drugs, such as propranolol (Cotham and Shand, 1975), lidocaine (Stargel et a l . , 1979) and quinidine (Kessler et a l . , 1979). This r e s u l t s in erroneous estimation of unbound f r a c t i o n and the un-bound concentration ( c u n D O U n < j ) °f disopyramide i n the serum. Also the presence of the primary disopyramide metabolite, mono -N-dealkylated disopyramide (MND), other basic antiarrhythmic drugs and endogenous displacers may also compete for the same s i t e and decrease disopyramide binding. 1.7.4 C l i n i c a l Importance of Unbound Disopyramide  Concentration Generally, for high clearance drugs, the non-linear serum protein-binding does not lead to dose-dependent d i s p o s i t i o n (Tucker et a l . , 1970; Goldstein, 1974 and Wilkinson and Shand, 1975). This i s so because the hepatic clearance of such drugs i s so e f f i c i e n t that not only the unbound drug but also the drug bound to red blood c e l l s are r e a d i l y available for hepatic metabolism (Tucker et a l . , 1970; G i l l e t t e , 1974; Lancet E d i t o r i a l , 1979 and De Leve and Piafsky 1981). In contrast, disopyramide i s a low clearance drug (Meffin et a l . , 1979; Lima et a l . , 1981 and Haughey and Lima, 1983), and under t h i s category, drug elimination i s often r e s t r i c t e d to the unbound fr a c t i o n of drug i n serum (Wilkinson and Shand, 1975 and Levy, 1976). 1.7.4.1 Pharmacokinetic Studies Meffin et a l . (1979) have analysed diso-pyramide pharmacokinetics a f t e r both single and multiple i n t r a -16 venous doses. Two models were used based on the hypothesis that either clearance i s independent of the t o t a l disopyramide serum concentration ( t o t a l concentration model) or that clearance i s independent of the concentration of disopyramide unbound to the serum protein (free clearance model). A two-fold increase i n steady-state serum concentration of t o t a l disopyramide required an approximate f o u r - f o l d increase i n dosing rate ( t o t a l clearance model), whereas the same dosing rate resulted i n a proportionate increase (four-fold) i n the steady-state serum concentration of unbound disopyramide (free drug clearance model). These obser-vations indicate that the free drug clearance model i s l i n e a r . Based on the concentration-dependent serum protein-binding of disopyramide i n the therapeutic range., investigators (Hinderling and Garrett, 1976 and Giacomini et a l . , 1982) have forecasted that the t o t a l body clearance of unbound disopyramide (CI , , ,) and renal clearance of unbound disopyramide (CI _ , unbound " renal, unbound^ a r e t* i e o n * v pharmacokinetic parameters which could be reported without q u a l i f i c a t i o n as to the drug concentrations. In other words, C l ^ . , , _ and CI , . . have been total,unbound renal,unbound speculated to be l i n e a r and dose-independent. Lima and co-workers (1981) have recently confirmed the free drug clearance model for disopyramide pharmacokinetics. They demonstrated that the AUC unbound, as opposed to the AUC t o t a l , increased propor-t i o n a t e l y and l i n e a r l y with an increasing disopyramide dose. Therefore, i t would appear that measurement of unbound rather than t o t a l concentration of disopyramide i n serum i s more appropriate i n designing proper dosage regimens. 17 1.7.4.2 Pharmacodynamic Studies Greenblatt et a l . , (1982) have recently commented that the steady-state concentration of unbound drug ( C g s u n Dound^ d o e s n o t depend on the extent of binding ( i . e . unbound f r a c t i o n , fu) and therefore C . , i s the most ss,unbound important determinant of the in t e n s i t y of a drug's c l i n i c a l action. Huang and Oie (1982) have shown i n rabbits that increased serum protein concentration does not a l t e r the rel a t i o n s h i p between the pharmacologic response and unbound steady-state concentration of disopyramide. The unbound concen-t r a t i o n in serum may, therefore, be considered as the "active" concentration of drug in the body (Huang and Oie, 1982). There are a few examples reported in the l i t e r a t u r e recently regarding the c o r r e l a t i o n of pharmacological response and unbound concentration of disopyramide i n humans (Holt et a l . , 1983 and Lima et a l . , 1981). Lima et a l . , (1981) have c l e a r l y demonstrated that the mean s y s t o l i c time in t e r v a l s correlated far better with the unbound than bound con-centration of disopyramide i n human serum. However, Holt et  a l . (1983) could not d i s t i n g u i s h the hemodynamic e f f e c t of t o t a l from unbound disopyramide serum concentration. This might be due to the narrow range of serum concentrations observed i n t h e i r study. 1.7.4.3 C l i n i c a l Relevance of the Measurement of Unbound Disopyramide Concentration i n Serum Based on the aforementioned studies, i t appears that the measurement of unbound disopyramide serum con-centration i s e s s e n t i a l for the proper characterization of the 18 l i n e a r pharmacokinetics and pharmacologic e f f e c t s of disopyra-mide. The i n t e r - and intra-subject v a r i a t i o n i n disopyramide serum protein-binding i s such that mean data on the disopyramide unbound f r a c t i o n (fu) cannot be used to r e l i a b l y estimate unbound concentration from t o t a l concentration in a given patient at any p a r t i c u l a r time. I t would appear that i n d i v i d u a l monitoring of unbound serum le v e l s i s desirable to provide the best possible therapy. Though t o t a l body and renal clearance of unbound diso-pyramide have been established to be dose-independent, the highly variable and concentration-dependent protein-binding can a l t e r the apparent volume of d i s t r i b u t i o n (Vd ) and the t 1 y 2 of the drug ( G i l l e t t e , 1973; Wilkinson et a l . , 1975; Gibaldi and McNamara, 1978 and, Faed, 1981). The net r e s u l t of altered Vd and m a Y n o t D e o n the t o t a l body clearance and renal clearance, but may a f f e c t the unbound concentration of disopyramide at a p a r t i c u l a r time during the steady-state dosing schedule. Therefore, knowledge of the steady-state pharmaco-k i n e t i c s of unbound disopyramide w i l l aid in the choice of a p a r t i c u l a r dose and i n t e r v a l combination, or a dose of c o n t r o l l e d release tablet, to minimize fluctuations i n maximum and minimum steady-state concentrations. Therefore, i t seems that the thera-peutic concentration of unbound disopyramide i n serum has to be properly established i n i n d i v i d u a l patients to aid the physician i n s e l e c t i n g the appropriate dosage regimen. The technical complexity and the time and expense i n measuring unbound diso-pyramide concentrations i n i n d i v i d u a l patients would seem worthwhile and j u s t i f i e d i f one can achieve better antiarrhythmic 19 therapy. An alternative to binding measurement would be to characterize a measurable indicator of the pharmacological resp-onse which i s highly correlated with unbound drug l e v e l s i n serum (Meffin et a l . , 1979). Monitoring the s a l i v a concentra-ti o n of disopyramide may be another approach, since the drug concentration i n s a l i v a approximates unbound concentration in serum for many drugs (Danhof and Breimer, 1978). The p r e l i m i -nary inves t i g a t i o n i n t h i s d i r e c t i o n by A i t i o et a l . , (1982) has indicated wide i n t e r - i n d i v i d u a l v a r i a t i o n along with a concentration-dependent i n h i b i t o r y e f f e c t of disopyramide on the s a l i v a r y flow rate. Therapeutic drug monitoring of patients on antiarrhythmic therapy with drugs such as quinidine and procaina-mide has increased the su r v i v a l rate s i g n i f i c a n t l y (Myerburg et a l . , 1979) and si m i l a r r e s u l t s may be expected for diso-pyramide following a more appropriate therapeutic management. 1.7.5 Disopyramide Binding Experiments Concentration-dependent disopyramide binding in human plasma or serum has been extensively studied using conventional equilibrium d i a l y s i s (Hinderling et a l . , 1974; Meffin et a l . , 1979; Giacomini et a l . , 1982; Haughey and Lima, 1982 and 1983, and, Norris et a l . , 1984) and u l t r a -f i l t r a t i o n methods (Bryson et a l . , 1982 Norris et a l . , 1983 and David et a l . , 1983). In almost a l l cases, the determination of unbound f r a c t i o n (fu) of disopyramide i s based 14 on the i n d i r e c t measurement of spiked C r a d i o - l a b e l l e d 20 disopyramide on the buffer and serum side of the d i a l y s i s chambers a f t e r the equilibrium d i a l y s i s . The unbound concen-t r a t i o n ( c u nbound^ """s t h e n calculated as the product of fu and the t o t a l i n i t i a l concentration of unlabelled disopyramide ( C t o t a ^ ) . Most of the aforementioned studies malce use of blank serum or plasma from each subject to construct i n d i v i d u a l in  v i t r o binding curves of unbound f r a c t i o n versus c t o t a ^ * Cunbound ^ s t n e n estimated from the binding curve. The i n d i r e c t i n v i t r o b i n d i n g r e s u l t s based on ra d i o - l a b e l l e d disopyramide measurement may not adequately represent disopyramide serum protein-binding, e s p e c i a l l y i f the mono-N-dealkylated metabolite of disopyramide and other displacers (selected antiarrhythmics and/or variable endogenous substances) are present i n the serum. Ideally, the d i r e c t quan-t i t a t i o n of the unbound drug from each serum sample using either measurement of l a b e l l e d disopyramide or a s p e c i f i c and highly s e n s i t i v e gas-liquid chromatographic (GLC) a n a l y t i c a l technique should provide more meaningful r e s u l t s . 14 The binding studies involving C-labelled drug have two p o t e n t i a l l y s i g n i f i c a n t l i m i t a t i o n s . F i r s t , v i r t u a -14 . . l l y a l l r a d i o - l a b e l l e d compounds have traces of C-containing contaminants which e q u i l i b r i a t e i n d i a l y s i s c e l l s and may y i e l d a r t i f a c t u a l estimates of fu. The magnitude of the error gener-ated i s h i g h l y dependent on the r e l a t i v e unbound fr a c t i o n s of the drug and contaminant. The combination of a very t i g h t l y bound drug with an e s s e n t i a l l y unbound contaminant can cause large 21 errors i n the estimation of fu even i f the contaminant comprises only 1-2 % of the t o t a l radio-labelled material. Secondly, the addition of l a b e l l e d compound by d e f i n i t i o n increases the fu for drugs which exhi b i t saturable binding. The magnitude of the error obviously depends on the s p e c i f i c a c t i v i t y of the compound, the e f f i c i e n c y of counting and the rate of change of c u n D O u n d and c t o t a ^ * T ^ e recognition of these and other p o t e n t i a l d i f f i c u l t i e s have led to e f f o r t s to estimate C , , without unbound resorting to the use of l a b e l l e d drug. For example, d i r e c t measurement of unlab e l l e d C . , has been attempted by packed unbound glass column GLC (Bredesen, 1980 and Brien et a l . , 1983) methods. However, these techniques have s e n s i t i v i t y and s p e c i f i c i t y l i m i t a t i o n s . Therefore, further enhancement i n the s e n s i t i v i t y and s e l e c t i v i t y of disopyramide analysis i s required for the d i r e c t measurement of unbound disopyramide in the presence of pote n t i a l displacers. 22 1.8 Metabolism of Disopyramide Like many t e r t i a r y amine drugs, the primary metabolic pathway of disopyramide i n man i s N-dealkylation y i e l d i n g mono-N-dealkylated metabolite (MND) (Karim et a l . , 1972 and Hinderling and Garrett, 1976). In healthy humans about 50 % of disopyramide i s excreted unchanged i n the urine and about 25 % of the administered dose i s recovered as MND (Karim, 1975; Hinderling and Garrett, 1976 and, Cunningham et a l . , 1977), with no apparent differences between oral and intravenous routes of administration or pH of urine (Cunningham et a l . , 1977). The r a t i o of MND to disopyramide has been reported to be 0.23 i n plasma and 0.46 i n urine following a single oral dose of diso-pyramide and the t^^2 °* M N D ** a s D e e n reported to be 12.9 + 6.4 hours ( A i t i o et a l . , 1982). The urinary excretion of MND has been found to be proportional to the disopyramide dose (Haughey and Lima, 1983). M O N O - N - D E A L K Y L A T E D M E T A B O L I T E ( M N D ) 23 The steady-state concentration of MND i n post-myocardial i n f a r c t i o n patients with normal renal function has been reported to be one t h i r d of that of disopyramide ( A i t i o , 1981). Another report has been published stating that the t o t a l serum le v e l s of MND i n patients on maintenance therapy with disopyramide were comparable to the parent drug lev e l s with wide inter-subject v a r i a t i o n s being observed ( A i t i o and Vuorenmaa, 1980 and Bredesen et a l . , 1982). This may be the r e s u l t of increased production of MND due to drug-induced metabolic induction or reduced elimination due to diminished renal function in e l d e r l y patients ( A i t i o and Vuorenmaa, 1980). The serum levels of MND are expected to increase to an appreciable extent in the patients with severe renal impairment (Shen et a l . , 1980 and Francois et a l . , 1983) or in patients who are taking disopyramide concomitantly with enzyme-inducing agents ( A i t i o , 1981 and A i t i o et a l . , 1981). Hinderling and Garrett (1974) have shown that the presence of high serum concentrations of MND lowers the binding of the parent drug disopyramide s i g n i f i c a n t l y . Similar findings of diminished disopyramide binding i n the presence of MND and vice versa were reported recently by A i t i o (1981) and Bredesen et a l . (1982). Although present i n plasma in much smaller concentrations than disopyramide, MND has been found to be excreted i n human breast milk i n concentrations similar to those of the parent compound (Barnett et a l . , 1982). There-fore, the pharmacologic and t o x i c o l o g i c properties of MND need to be considered i n breast-fed infants whose mothers are on disopyramide therapy. 24 Evidence has been presented in the past suggesting a major role for MND i n the antiarrhythmic and anticholinergic e f f e c t s observed during disopyramide treatment. Preliminary data comparing antiarrhythmic action of disopyramide and i t s metabo-l i t e , MND i n dogs with experimentally induced a t r i a l and v e n t r i -cular arrhythmias have shown that the metabolite appears to be only s l i g h t l y l e s s active than the parent compound against a t r i a l arrhythmia but inactive against ventricular arrhythmia (Baines et a l . , 1976). In the same study the investigators, using the i s o l a t e d guinea-pig ileum, noted that the a n t i c h o l i n e r g i c e f f e c t s of MND are approximately 24-fold greater than the in t a c t drug. Use of i s o l a t e d guinea-pig a t r i a (Grant et a l . , 1978), on the other hand suggest that MND i s only 25 % as potent e l e c t r o -p h y s i o l o g i c a l ^ as the parent drug disopyramide. The pharmacological and t o x i c o l o g i c a l role of MND i n humans i s not f u l l y established. Whiting et a l . (1980), have concluded that the serum concentration of MND occurring aft e r a single o r a l dose of disopyramide made no c l i n i c a l l y s i g n i f i c a n t contribution to the pharmacological e f f e c t which was measured as the prolongation of the QT i n t e r v a l of the electrocardiogram. I t has been reported recently that the MND serum concentrations were i d e n t i c a l i n those patients with and without a n t i c h o l i n e r g i c side e f f e c t s ( A i t i o , 1981). However, another group demonstrated that MND caused r e l a t i v e l y more pronounced side e f f e c t s (eg., reduced s a l i v a r y secretion) (Chiang et a l . , 1982). I t has also been shown that no c o r r e l a t i o n between MND concentrations and a n t i -cholinergic a c t i v i t y existed, arguing against the r o l e of MND 25 causing antimuscarinic side e f f e c t s ( I i s a l o and Aaltonen, 1983 and 1984). Clearly, additional studies i n t h i s area are warranted. 1.9 A n a l y t i c a l Methods A great number of diverse a n a l y t i c a l techniques for diso-pyramide measurement i n b i o l o g i c a l f l u i d s have evolved in the past decade including spectrofluorometric, spectrophotometry, thin-layer densitometric, enzyme immunoassay (EMIT®), high -performance l i q u i d chromatographic (HPLC) and GLC methods. The spectrofluorometric (Ranney et a l . , 1971), spectrophotometrie (Martin et a l . , 1978) and thin-layer densitometric (Gupta et  a l . , 1979) techniques are usually nonspecific, less sensitive and are subject to interferences from co-extracted endogenous substances, the c l o s e l y r e l a t e d disopyramide metabolite, MND, and selected antiarrhythmics prescribed in combination. The comparison of the EMIT® disopyramide assays to the currently used HPLC (Colin et a l . , 1980 and Bryson et a l . , 1982) and GLC (Pape, 1981 and Johnston and Hamer, 1981) methods, yielded high c o r r e l a t i o n c o e f f i c i e n t (r > 0.97), a slope of unity (p >0.05) and zero y-intercept (p > 0.05). However, these methods do not allow simultaneous quantitation of disopyramide and MND. Simultaneous measurement of parent drug i n human serum along with metabolite(s) has become a more common pra c t i c e i n the c l i n i c a l s e t t i n g . This i s due to s i g n i f i c a n t technical advances in the f i e l d of drug analysis. The simultaneous quantitation of 26 disopyramide and MND i n various human b i o l o g i c a l f l u i d s i s es s e n t i a l during the course of enzyme-induction or i n h i b i t i o n studies to provide a better insight into metabolic a l t e r a t i o n s . Most of the GLC (Duchateau et a l . , 1975? Hayler and Flanagan, 1978; Hayler et a l . , 1978? Deodens and Forney, 1978? V a s i l i a d e s et a l . , 1979a and 1979b? Johnston and McHaffie, 1978? Foster and Reid, 1979; Gal et a l . , 1980 and Haskins et a l . , 1980) and HPLC ( I l l e t t e t a l . , 1978; Draper et a l . , 1979; Broussard et  a l . , 1979; Ahokas et a l . , 1980; Flood et a l . , 1980; Bridges and Jennison, 1983 and Swezey and Ponzo, 1984) methods prev-iously reported either do not possess the e s s e n t i a l s p e c i f i c i t y or the combined c a p a b i l i t y of quantifying MND i n the presence of disopyramide. The simultaneous quantitation of disopyramide and MND i n human urine, plasma or serum has been successfully achieved by various GLC methods (Hutsell and Stachelski, 1975, Hinderling and Garrett, 1976; A i t i o , 1979; Cunningham et a l . , 1977; Bredesen, 1980; Brien et a l . , 1983 and Kapil et a l . , 1984) and HPLC (Meffin et a l . , 1977; Lagerstrom and Persson, 1978; Nygard et  a l . , 1979; Beltz et a l . , 1979; Lima, 1980, Wesley and Lasky, 1981; Kabra et a l . , 1981; Norris et a l . , 1982; Charette et  a l . , 1983 and Kubo et a l . , 1984). Some of the HPLC methods (Meffin et a l . , 1977; Nygard et a l . , 1979 and Lima, 1980) require cumbersome extraction procedures for sample preparation which s u b s t a n t i a l l y increase analysis time while others describe r e l a t i v e l y simple single extraction methods (Lagerstrom and Persson, 1978; Beltz et a l . , 1979, Kabra et a l . , 1981; 27 Wesley and Lasky, 1981; Norris et a l . , 1982 and Charette et  a l . , 1983). Kubo et a l . (1984) have recently reported an HPLC technique involving a sample preparation step without evaporation; however, t h e i r method lacks the required s e n s i t i -v i t y . The HPLC techniques are s p e c i f i c and convenient but s e n s i t i v i t y l i m i t a t i o n s necessitate i n j e c t i o n of the entire extracted sample volume into the chromatograph, l i m i t i n g duplicate determinations. Therefore, the HPLC methods with the present s e n s i t i v i t y l i m i t a t i o n s r e l a t i v e to the GLC assays cannot u t i l i z e the multiple as well as automatic l i q u i d sample i n j e c t i o n c a p a b i l i t i e s which may be required to achieve economy of labor and time. Nevertheless, any one of the HPLC techniques for disopyramide and MND measurement i s a good al t e r n a t i v e to the e x i s t i n g GLC techniques. The GLC a n a l y t i c a l methodologies (Hinderling and Garrett, 1976, Cunningham et a l . , 1977; A i t i o , 1979 and Bredesen, 1980) are i d e n t i c a l to the method reported by Hutsell and Stachelski (1975), where i n t a c t disopyramide i s measured simultaneously with acetylated MND. D e r i v a t i z a t i o n of the secondary amino group of MND i s required to y i e l d a single chro-matographic peak because i n t a c t MND, when subjected to high GLC temperatures, r e s u l t s i n three unresolved peaks (Hutsell and Stachelski, 1975). Recent findings i n our laboratory have i n d i -cated that the acetylation of the secondary amino group of MND with acetic anhydride i s not complete under usual conditions; extensive peak t a i l i n g i s also associated with the chromatogra-phic peak for acetylated MND on columns conventionally used i n 28 disopyramide analysis, v i z . , 3 % OV-17 (50% phenyl methyl s i l i c o n e ) and 5 % phenyl methyl s i l i c o n e . These two l i m i t a t i o n s may explain the non-linear behavior i n chromatograph response for MND as a function of MND concentration in a few recent publications (Brien et a l . , 1983 and Charette et a l . , 1983). Gal et a l . (1980) have shown that the dehydration of the amide functional group of disopyramide with t r i f l u o r o a c e t i c anhydride improves i t s chromatography through increased symmetry of peak shape and shortened retention time. No mention of MND was made. A l l GLC methods reported so far make use of conventional packed glass columns which demonstrated po t e n t i a l for chromato-graphic interference from other drugs and from endogenous substances i n the serum or plasma. Therefore, therapeutic monitoring of serum disopyramide and MND may be more d i f f i c u l t with conventional packed glass column GLC analysis i f disopyr-amide i s used i n combination with other selected antiarrhythmics. State-of-the-art f u s e d - s i l i c a c a p i l l a r y columns i n general provide superior s e l e c t i v i t y or s p e c i f i c i t y and greater s e n s i t i -v i t y (Jennings, 1980 and Freeman, 1981). The development of a simple, reproducible, more sensi t i v e and s p e c i f i c c a p i l l a r y column ga s - l i q u i d chromatographic-nitrogen/phosphorus s e l e c t i v e (GLC-NPD) technique would be highly desirable i n order to simul-taneously quantitate trace l e v e l s of disopyramide and MND i n human b i o l o g i c a l f l u i d s . Improved s e n s i t i v i t y i s p a r t i c u l a r l y necessary i f measurement of unbound drug serum concentration i s to be performed during the course of protein-binding experiments. 29 1.10 Metabolic Induction of Disopyramide Elimination and  Altered Binding of Disopyramide 1.10.1 E f f e c t of Enzyme Inducers on Disopyramide  Metabolism Preliminary studies ( A i t i o and Vuorenmaa, 1980; A i t i o et a l . , 1981 and A i t i o , 1981) have demonstrated that the disopyramide clearance i s susceptible to the e f f e c t of metabolic induction. Co-administration of known enzyme inducers such as rifampin ( A i t i o et a l . , 1981), phenytoin ( A i t i o and Vuorenmaa, 1980; A i t i o et a l . , 1981 and A i t i o ) , carbamazepine and spironolactone ( A i t i o , 1981) in a small number of volunteers and patients decreased the mean serum leve l s of disopyramide and increased those of MND. Rifampin and phenytoin treatment ( A i t i o et a l . , 1981) r e s u l t e d i n a s i g n i f i c a n t decrease i n e l i m i n a t i o n h a l f - l i f e ^2./2^ a n c^ a r e a under the serum concentration versus time curves (AUC) for disopyramide and caused a two-fold increase in the AUC for MND. The potential e f f e c t of the c l a s s i c a l inducer, phenobarbital, on disopyramide metabolism i n man cannot be predicted on the basis of aforementioned studies because there i s a c e r t a i n degree of s e l e c t i v i t y i n enzyme induction, as has been seen with the e f f e c t of spironolactone (Huffman et a l . , 1973) and rifampin (Toverud et a l . , 1981). A i t i o and A i t i o (1979) have reported that phenobarbital causes induction of N-dealkylation of disopyramide i n the r a t l i v e r . These investigators also reported that disopyramide auto-induces i t s N-dealkylation to a r e l a t i v e l y minor extent i n the r a t l i v e r . These r e s u l t s cannot be extrapolated to man because there i s evidence i n the l i t e r a t u r e regarding the species-dependent 30 -dependent differences i n hepatic metabolic induction. For example, i t has been reported that the metabolism of the antiarrhythmic agent, tocainide, could be r e a d i l y induced i n the ra t (Venkatararaanan and Axelson, 1980), while s i m i l a r experiments with tocainide i n human volunteers f a i l e d to show s i g n i f i c a n t e f f e cts ( E l v i n et a l . , 1980). Thus, the e f f e c t of phenobar-b i t a l on disopyramide metabolism i n man may or may not mimic the e f f e c t of phenobarbital induction of the metabolism of t h i s drug i n rat l i v e r . 1.10.2 E f f e c t of Enzyme Inducers on Serum AAG Chronic phenobarbital-treatment (Bai and Abramson, 1982) has been reported to cause a substantial increase (5- fold) i n s i a l i c a c i d l e v e l s , presumably due to an increase i n serum AAG l e v e l s , accompanied by the anticipated f a l l i n propra-nolol fu ( i . e . fu f a l l s tov*20 % of control) i n the dog. Fur-thermore, human e p i l e p t i c s treated with multiple drugs including phenytoin (Routledge et a l . , 1981) reportedly exhibit a s i g n i -f i c a n t l y increased serum AAG concentration (approximately twice that seen i n control subjects). Interestingly, i t has been observed that the combination of phenobarbital and carbamazepine causes a decrease i n serum AAG l e v e l s i n man (Bruguerolle and Jadot, 1984). Thus, i t would appear to be necessary to assess the e f f e c t of phenobarbital treatment on disopyramide fu and on the serum concentration of i t s p r i n c i p a l binding protein, AAG. 31 1.10.3 Ef f e c t of Cigarette Smoking on Drug  Elimination Chronic cigarette smoking has been suggested to be one of the most potent environmental factors which account for variable rates of oxidative metabolism of drugs i n man (Jusko , 1978 and 1979; Dawson and Vestal, 1982; and Luczynska and Wilson, 1984). This v a r i a t i o n i n bio-transformation may, i n part, contribute to the altered pharmacologic response of drugs in smokers (Jusko, 1979 and Park and Breckenridge, 1981). Cigarette smoking, which i s associated with the intake of various polynuclear aromatic hydrocarbons (PAHs), i s thought to be involved in r e l a t i v e l y s p e c i f i c stimulation of those hepatic microsomal mixed function oxidases that are mediated by cytochrome P-448 heme-protein (Jusko, 1978 and Dawson and Ves t a l , 1982). In general, the microsomal pathways of hepatic drug metabolism are eith e r induced or unaffected by c i g a r e t t e smoke i n a s e l e c t i v e but unpredictable fashion (Jusko, 1978 and 1979). It i s d i f f i c u l t to predict whether the metabolism of any compound w i l l be induced based upon structure or by extrapolation from data obtained i n laboratory animals. The e f f e c t of chronic cigarette smoking on the hepatic clearance of disopyramide has not been investigated i n the past. 1.10.4 Cigarette Smoking and Serum AAG Levels There are c o n f l i c t i n g reports i n jthe l i t e r a -ture regarding the independent e f f e c t s of phenobarbital and cigarette smoking on the l e v e l of serum AAG (McNamara, et a l . , 32 1979; Wolf et a l . , 1982; and Benedek et a l . , 1984). There i s no report i n the l i t e r a t u r e on the combined e f f e c t of pheno-b a r b i t a l and cigarette smoke on the serum AAG l e v e l or on the fu of disopyramide i n serum in smokers. 33 1.11 Primary Objectives The primary objectives of the present project were: (a) to develop a c a p i l l a r y column gas-liquid chromatographic-nitrogen/phosphorus detection technique (GLC-NPD) for the simultaneous quanti-t a t i o n of disopyramide and i t s mono-N-dealkylated metabolite (MND) i n human b i o l o g i c a l f l u i d s ; (b) to apply the c a p i l l a r y GLC-NPD assay to study i n v i t r o plasma protein-binding of disopyramide; (c) to apply the a n a l y t i c a l technique to study disopyramide pharmacokinetics and i t s metabolic induction by the prototype enzyme inducer, phenobar-b i t a l , i n healthy volunteers; (d) to study the e f f e c t of cigarette smoking on diso-pyramide pharmacokinetics in the presence and absence of phenobarbital-treatment i n healthy smokers; (e) to determine serum alphas-acid glycoprotein concentration i n untreated and phenobarbital-treated non-smokers and smokers; and (f) to determine unbound f r a c t i o n (fu) of selected serum disopyramide samples of control and phenobarbital -treated non-smokers and smokers. 34 2. EXPERIMENTAL 2.1 Supplies 2.1.1 Drugs used 4-(Diisopropylamino)-2-(2 pyridyl)-2-phenylbutyramide (disopyramide) ( l o t No. SC-7031) 1 and i t s phosphate s a l t ( l o t No. SC-13957) , disopyramide phosphate (Norpace^, l o t No. 72079) 3, disopyramide (Rhythmodan®, l o t No.0583 FJR), 4-(isopropylamino)-2-(2-pyridyl)-phenylbutyramide (MND) 5 (l o t No. ADAMEKCD3-1854) , para-chlorodisopyramide l o t No. SC-13068) 6 and phenobarbital tablets ( l o t No. A39BBL) 7. 2.1.2 Chemicals and Reagents g Acetic anhydride , t r i f l u o r o a c e t i c anhydride 9 10 (TFAA) , sodium hydroxide (NaOH) , hydrochloric acid (HC1) 1 1, dibasic sodium phosphate (Na 2HP0 4.2H 20) 1 2 and 13 monopotassium phosphate (KHjPO^) 1-3,5,6 G.D. Searle of Canada., Oakville, Ontario., Canada. 4 Rousell Canada Incorporation., Montreal, Quebec, Canada. 7 Stanley Drug Products Ltd., North Vancouver, B.C., Canada. 8,12,13 Fisher S c i e n t i f i c Company., F a i r Lawn, New Jersey., 07410, U.S.A. 9 Pierce Chemical Co., Rockford, I l l i n o i s . , 61105, U.S.A. 10,11 Reagent A.C.S., A l l i e d Chemical Canada, Ltd., Pointe C l a i r e , Quebec, Canada. 35 2.1.3 Solvents 14 15 Methylene chloride and toluene 2.2 Gas Chromatography Stationary Phases, S o l i d Supports  and Columns 16 (a) 3 % OV-17 (50 % phenyl methyl s i l i c o n e ) on Chromosorb- (High P e r f o r m a n c e ) 1 7 , 100/120 mesh s i z e , packed i n a 1.8 meter x 2 mm (i.d.) glass column, (b) A 12 meter (i.d.= 0.27 mm) siloxane deactivated 18 f u s e d - s i l i c a u l t r a performance c a p i l l a r y column coated with crosslinked methyl s i l i c o n e f l u i d , and (c) A 25 meter (i.d.= 0.31 mm) ( f i l m thickness = 0.17 19 micron) f u s e d - s i l i c a u l t r a performance c a p i l l a r y column coated with crosslinked 5 % phenyl methyl s i l i c o n e . 2.3 Equipment 20 Hewlett-Packard (H-P) model 5830A gas chromatograph equipped with (a) dual flame-ionization and nitrogen/phosphorus sel e c t i v e detectors, (b) a model 18835B c a p i l l a r y i n l e t system, and (c) a model 18850A H-P integrator system for peak area i n t e -21 gration and quantitation; an H-P model 5970A mass spectrome-ter was used for c a p i l l a r y GLC electron-impact mass spectrometry 22 (EI-MS); an H-P model 5987A mass spectrometer was used for c a p i l l a r y GLC chemical-ionization mass spectrometry (CI-MS); 14,15 D i s t i l l e d i n glass, Caledon Laboratories Ltd., Georgetown, Ontario., Canada. 16,17 Applied Science, Pennsylvania, U.S.A. 18-22 Hewlett Packard, Avondale, Pennsylvania, 19311, U.S.A. 36 23 24 Vortex-genie mixer , pH meter, model 600 and a Labquake tube shaker, Model 415-110 2 5. 2.4 Preparation of Stock Solutions 2.4.1 Disopyramide Phosphate Stock Solution Disopyramide phosphate (approximately 10 mg) was weighed accurately and dissolved i n deionized d i s t i l l e d water to y i e l d 50 ml solution. An aliquot (2.5 ml) of t h i s solution was dil u t e d to volume with deionized d i s t i l l e d water i n a 100 ml volumetric f l a s k . Different volumes of t h i s solution ranging from 0.05 to 1.0 ml were used for toluene extraction in the preparation of standard curves. 2.4.2 MND and PC-Dis Stock Solutions MND (6 mg) and the int e r n a l standard, para-chlorodisopyramide (PC-Dis) (10 mg) were separately weighed and dissolved using 0.1N HC1 i n 50 ml volumetric f l a s k s . Further d i l u t i o n s were also made i n 0.IN HC1. These solu t i o n s along with disopyramide phosphate were stored at 4°C, following preparation, for up to two months. 2.4.3 Preparation of Isotonic, 0.067 M, pH 7.4  Phosphate Buffer (Documenta Geigy, 1970). Monopotassium phosphate (KI^PO^) (9.07g, stock solution A) and disodium phosphate (NajHPO^2H 20) 23,24 Fisher S c i e n t i f i c Company, S p r i n g f i e l d , Massachusetts., U.S.A.. 25 Labindustries, Berkley, C a l i f o r n i a , 94710, U.S.A.. 37 (11.87 g, stock s o l u t i o n B) were weighed accurately and dissolved separately i n a s u f f i c i e n t volume of deionized d i s t i l l e d water to y i e l d 1 l i t r e solutions ( M/15 ) of each substance. An accurate volume of stock solution A (197 ml) was transferred into a clean 1 l i t r e volumetric f l a s k . To t h i s , sodium chloride (3.9 g) was added for the i s o t o n i c i t y adjustment. The volume was then made up to 1 l i t r e with stock solution B. The f i n a l solution f u r n i -shed a pH of 7.4 which was v e r i f i e d by a pH meter and, i f necessary, adjustment of pH was made by adding appropriate aliquots of stock solutions A or B. 2.5 Preliminary Studies of Packed Column (3 % OV-17) GLC-Flame Ionization Detection (FID) and GLC-NPD  Methods for Disopyramide. Our i n i t i a l approach towards the improvement of the disopyrarfTide assay was to f i r s t repeat the previously published technique and then, i n a stepwise manner to enhance s p e c i f i c i t y , r e p r o d u c i b i l i t y and s e n s i t i v i t y of the disopyramide measurement. 2.5.1 GLC-FID The e f f e c t of temperatures (column or oven, i n j e c t i o n and detector) and c a r r i e r gas (helium) flow rate on the chromatographic c h a r a c t e r i s t i c s of disopyramide were observed with s p e c i a l reference to peak area count, peak shape, solvent front and peak retention time. The preliminary findings of the GLC-FID method are summarized as follows: (a) The optimum column (oven) temperature range for disopyramide and PC-Dis was 245-255°C. Temperatures 38 below 245°C resulted i n r e l a t i v e l y longer retention times for both compounds and temperatures higher than 255°C resulted i n chromatographic peaks too close to the solvent front. (b) The v a r i a t i o n i n FID (250-325°C) and i n j e c t i o n temperature (220-260°C) did not a f f e c t the chromatography of either compound to any appreciable extent. (c) Under the experimental conditions, procainamide did not i n t e r f e r e with the disopyramide peak as has been reported e a r l i e r (Hutsell and Stachelski, 1975). (d) Helium was used as a c a r r i e r gas and a flow rate of 40 ml/minute was found to be optimum with respect to peak shape, number of t h e o r e t i c a l plates and retention times for disopyramide and PC-Dis. (e) FID s e n s i t i v i t y with respect to disopy-ramide and PC-Dis was found to be optimal when helium/hydrogen/ a i r : 40/25/150 flow rate (ml/min) r a t i o combination was used. 2.5.2 GLC-FID vs FID-NPD The performance of FID and NPD modes was compared by i n j e c t i n g i d e n t i c a l volumes of toluene solution cont-aining equal concentrations of disopyramide and PC-Dis into a GLC equipped with a 2 meter (i.d.= 2 mm) glass column packed with 3 % OV-17 stationary phase and measuring the r e s u l t i n g peak areas. 39 The following chromatographic conditions were used for the two detection modes: Parameter FID NPD  Column temperature 250°C 250°C Injection temperature 255°C 255°C Detector temperature 300°C 300°C Carr i e r gas (helium) flow rate. 40 ml/minute 40 ml/minute H 2 : a i r flow rate r a t i o . . . . 25:150 3:50 Voltage to the ceramic bead of NPD 15 vo l t s Relative peak area count for disopyramide 1 14 It i s clear that the NPD mode ( Figure 1), under i d e n t i c a l conditions, i n addition to s p e c i f i c i t y , provided an approximate 14-fold increase i n s e n s i t i v i t y of disopyramide measurement. The results obtained i n the NPD mode are shown i n Figure 2. MtER RRE« * 1517U 53.533 1314B 46.413 FIGURE 1. Representative packed column chromatograms of disopyramide (retention time, R.T. = 6.8 min) and PC-Dis (R.T. = 11.4 min) obtained from FID (A) and NPD (B) detection modes (An attenuation of 24 was used in both cases). A 2 meter long (i.d. = 2 mm) glass column packed with 3Q% OV-17 stationary phase was used. 41 C/3 5 u w o < 06 > C-O 5 o 06 < 06 < . 9 -. 8 -. 7 -. 6 -. 5 -. 4 -. 3 -. 2 -. 1 -0 I 1 1 1 1 1 1 1 1 1 1 0 . 5 1 1 . 5 2 2 . 5 3 3 . 5 4 4 . 5 5 A M O U N T OF D I S O P Y R A M I D E BASE(ng/injection) FIGURE 2. Relationship between disopyramide amount and N/P detector response. A 2 meter long (i.d. = 2 mm) glass column packed with 3% OV-17 stationary phase was used for the chromatography of disopyramide (retention time, R.T. = 2.64 min) and PC-Dis (R.T. = 4.77 min). 42 2.6 Preliminary Results of Fused-Silica C a p i l l a r y GLC Studies of Disopyramide 2.6.1 C a p i l l a r y GLC-FID The use of f u s e d - s i l i c a c a p i l l a r y columns for disopyramide analysis was attempted to enhance the assay s e n s i t i -v i t y and s p e c i f i c i t y . The FID detection mode was used during i n i t i a l a n a l y t i c a l development because of the u n a v a i l a b i l i t y of NPD detection coupled to c a p i l l a r y column technology at the outset of the present study. A 12 meter f u s e d - s i l i c a c a p i l l a r y column coated with phenyl s i l i c o n e f l u i d successfully yielded a sharp, needle-shaped and symmetrical disopyramide peak as shown i n Figure 3. The GLC-FID conditions used were: oven temperature, 240°C; i n j e c t i o n temperature, 255°C; FID temperature, 300°C temperature programming r a t e , 10°C/minute, c a r r i e r gas (helium) flow rate, 1.5 ml/minute; septum purge flow-rate, 2 ml/minute; make-up gas flow rate, 60 ml/minute, H 2 : a i r flow rate r a t i o , 25:150 and i n j e c t i o n volume s p l i t r a t i o , 20:1. Figure 4 presents the influence of various s p l i t r a t i o s on the peak area r a t i o of disopyramide to PC-Dis. These re s u l t s showed that the r a t i o of disopyramide and PC-Dis was v i r t u a l l y constant over a wide range of s p l i t r a t i o s for d i f f e r e n t i n s e r t s . This observation suggested that there was no discrimination between the s p l i t t i n g of disopyramide and PC-Dis vapors i n the i n l e t port and that the l a t t e r would be an appro-priate i n t e r n a l standard for subsequent quantitative analysis of disopyramide. 43 C a p i l l a r y G a s C h r o m a t o g r a p h i c P e a k TEMPI 24B 24B TIME1 15. B INJ TEMP 26B 26B FID TEMP 3BB 3BB TCD TEMP 35B 35B RUX TEMP 26B 26B OVEN MAX 25B CHT SPD B.5B RTTH 2t 4 FID SGML fi SLP SENS B.5B RPER REJ -FLOW ft 3B FLOW B 23 OPTH B b l a n k p l a s m a B d i s o p y r a m i d e *T 2.61 1383 AREA % 1BB.BBB FIGURE 3. Representative capillary GLC-FID chromatograms obtained after injecting methylene chloride extracts of blank plasma (A) and aqueous solution of disopyramide phosphate (B) with retention time (R.T.) of disopyramide being 2.61 min. A 12 meter long (i.d. = 0.27 mm) fused-silica capillary column coated with crosslinked methyl silicone fluid was used. 44 l.S-i (0 o I o Q. • v. 0 TO E co a o CO 1 -I-< < UJ cc < . 5 -0 •-•••err I I I 1 1 1 1 1 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 SPLIT RATIO FIGURE 4. Effect of various split ratios on the area ratio of disopyramide, MND and PC-Dis; (•): Jennings insert-1, (O): Jennings insert-2 (similar type to 1), (*): fused-silica with glass wool, and (>): fused-silica without glass wool. All the above area ratio readings were obtained from the same sample in triplicates. 45 The areas of the chromatographic peaks of disopyramide and PC-Dis were not affected by the a l t e r a t i o n i n the c a r r i e r gas flow rates (0.7 - 2.5 ml/minute) and make-up gas flow rates (20 - 100 ml/minute). Figure 5 represents a t y p i c a l c a p i l l a r y gas-l i q u i d chromatogram obtained by i n j e c t i n g a sample of toluene mixture containing various spiked antiarrhythmics. I t i s evident that a 12 meter f u s e d - s i l i c a c a p i l l a r y column coated with 5 % phenylmethyl stationary phase can be suc c e s s f u l l y employed i n the separation of disopyramide i n the presence of other antiarrhy-thmic agents v i z . , tocainide, lidocaine, mono-ethyl glycine xylidide, procainamide, N-acetyl procainamide, propranolol and quinidine without any interference. 2.6.2 C a p i l l a r y GLC-NPD Additional s e n s i t i v i t y was obtained by u t i l i z i n g the NPD mode. The use of the NPD detection mode resulted i n a consistent loss of baseline at the beginning of each chromatographic run but the baseline was quickly restored without i n t e r f e r i n g with the peaks of i n t e r e s t . The s e l e c t i o n of solvent was based on two c r i t e r i a : (a) baseline c h a r a c t e r i s t i c s i n the NPD mode, and (b) e x t r a c t a b i l i t y . Solvents such as toluene and ethyl acetate, resulted i n stable baseline as opposed to methylene chloride or chloroform which resulted i n wavy and delayed recovery of the baseline r e s p e c t i v e l y (Figure 6). Chlorinated solvents have been 46 reported to cause a reversible s e n s i t i v i t y loss i n the NPD detection mode (Bente, 1976). Toluene was selected as a solvent because i n addition to a stable baseline, i t also demonstrated better e x t r a c t a b i l i t y for disopyramide and PC-Dis. 47 C a p i l l a r y G a s C h r o m a t o g r a p h i c P e a k s REPRESENTRTIVE CHROMRTOGRflM OF URRIOUS fiNTI-flRRHVTHMlCS: Q Q _ l > LU H 2 a to i LU o z < a O a. a. a o o a. a o1 o f LU z a o o o 10 • 10 00 a-LU a H I £ a! i 8 I I C' 5 u 1 5 C5 IT1 H.P. CflPILLflRV CHROMflTOGRPPH, MODEL 5830fl EQUIPPED WITH N/P DETECTOR* FIGURE 5. Representative capillary GLC-NPD chromatograms of various antiarrhythmics. 48 M e t h y l e n e c h l o r i d e CJ C h l o r o f o r m Li • T o l u e n e FIGURE 6. Baseline characteristics of various solvents with capillary GLC employing N/P detection. 49 2.6.3 S p l i t versus S p l i t l e s s Sample Injection Modes The use of commercially available crosslinked f u s e d - s i l i c a c a p i l l a r y column assisted i n improving the s e n s i t i -v i t y of disopyramide detection as i t was possible to i n j e c t a large sample volume into the column without the fear of stationa-ry phase s t r i p p i n g . This was p r a c t i c a l because the stationary phases, methyl s i l i c o n e f l u i d and 5 % phenyl methyl s i l i c o n e (used in subsequent analyses), are covalently bonded ( i . e . cross -linked) to the inner walls of the f u s e d - s i l i c a c a p i l l a r y column. A switchover from the s p l i t to s p l i t l e s s sample i n j e c t i o n mode resulted i n increased s e n s i t i v i t y without any s i g n i f i c a n t band broadening or peak t a i l i n g (Figure 7). The multi-ramp temperature programming techniques which are associ-26 ated with the s p l i t l e s s mode , to a f f e c t solvent e f f e c t or cold trapping, did not provide further improvement i n the chromatography of disopyramide. Therefore, a r e l a t i v e l y simple temperature programming rate was found to be s u f f i c i e n t for disopyramide analysis. The r e l a t i o n s h i p of purge a c t i v a t i o n time during the s p l i t l e s s mode and the respective area count i s shown in Figure 8. 26" C a p i l l a r y I n l e t System: Accessory Manual for Gas Chromatographs, Hewlett-Packard, Avondale, Pennsylvannia, U.S.A., 2.1 - 2.10 (1978). 50 B FIGURE 7. Representative c a p i l l a r y GLC-NPD chromatograms obtained from s p l i t (A) and s p l i t l e s s (B) sample i n j e c t i o n modes. A 2 m i c r o l i t e r a l i q u o t o f a TFAA-treated toluene extract o f serum sample, containing disopyramide (retention time, R.T. = 10.56 min), MND (R.T. - 10.75 min) and PC-Dis (R.T. • 12.56 min) was injected under each mode. A s p l i t o f 10 to 1 was used i n the s p l i t mode. 51 (+) D i s o p y r a m i d e (x) M N D (•) P C - D i s 203330 - i P U R G E A C T I V A T I O N T I M E (MIN) FIGURE 8. E f f e c t o f purge a c t i v a t i o n time versus area counts for TFAA-treated toluene extracts o f disopyramide, MND and PC-Dis. 52 2.6.4 Miscellaneous Optimizing Conditions for  Ca p i l l a r y GLC-NPD Variation i n the column temperature (220-260°C) did not s i g n i f i c a n t l y a l t e r the area counts of disopyr-amide. Higher i n j e c t i o n temperatures (255-260°C) resulted i n sharper peaks. Disopyramide s e n s i t i v i t y increased as a function of NPD temperature i n the range studied (275-350°C). The detector a i r flow rate i n the range studied did not a l t e r disopyramide s e n s i t i v i t y . A l t e r a t i o n in H2 flow rate had a marked e f f e c t on disopyramide area counts. Flow rates > 3 ml/minute for enhanced disopyramide s e n s i t i v i t y as well as the baseline noise. Make-up gas (helium) flow rate (15-60 ml/minute) i n the NPD mode suggested that the disopyramide s e n s i t i v i t y was maximum at the lowest (15 ml/minute) flow rate. In fact, the peak shape of disopyramide was not affected i n the absence of make-up gas flow. However, 15-20 ml/minute of helium was s t i l l u t i l i z e d as a means of a continuous purge of the NPD jet and column ex i t area. Additional s e n s i t i v i t y was achieved^as required, by increasing the voltage ( i . e . o f f s e t ) to the ceramic bead (coated with rubidium chloride) inside the NPD c o l l e c t o r . The positioning of both c a p i l l a r y column ends, the i n l e t (9 mm) and detector end (1 mm below the NPD je t opening), was optimized for the best performance (maximum s e n s i t i v i t y ) . 53 2.7 C a p i l l a r y GLC-NPD Chromatography of MND The acetylation of the secondary amino group of MND with various volumes of acetic anhydride at various incubation times was attempted to prevent thermal degradation of MND under high temperature GLC conditions. Two major drawbacks were found: (a) Derivatization was variable and incomplete. Preliminary findings suggested that t h i s was apparently neither a function of the amount of the acetic anhydride present i n the reaction mixture, nor a function of the incubation period; and (b) There was extensive peak t a i l i n g of the acetylated MND. Figure 9 shows the chromatograms of underivatized as well as acetic anhydride-treated MND samples. Following an unsuccessful and unsatisfactory attempt at complete acetylation of the secondary amine group of MND with acetic anhydride, i t was decided to use a more v o l a t i l e deriva-t i z i n g agent, t r i f l u o r o a c e t i c anhydride (TFAA). Figure 10 represents the c a p i l l a r y gas chromatographic peak of TFAA derivatized MND. Gal et a l . (1980) have reported that TFAA dehydrates the amide functional group of disopyramide. Therefore, i t was speculated that TFAA, i n addition to the acetylation of the secondary amine group of MND also dehydrates the amide functional groups of disopyramide, MND and PC-Dis (Figure 11). The GC-MS-EI and GC-MS-CI data for untreated and TFAA-treated disopyramide, MND and PC-Dis confirmed these findings (for complete d e t a i l s , r e f e r to APPENDIX). 54 9. Representative capillary GLC-NPD chromatograms obtained after injecting untreated MND (A) and acetic anhydride (100 microliters) treated MND (B) solutions in toluene. The untreated MND resulted in at least 3 peaks (A) and acetic anhydride treated MND yielded partial acetylation of MND (B) (The peak with R.T. = 4.01 min was speculated to be that of acetylated MND). 55 C a p i l l a r y G a s C h r o m a t o g r a p h i c P e a k o f T F A A d e r i v a t i z e d m o n o - N - d e a l k y l a t e d m e t a b o l i t e RT 6. 77 7. 88 8. B7 RREft 594 5587B 1 176 flREft 5j 1 . B31 96.929 2, 040 XF: 1.B000 E + B il o (-CO FIGURE 10. Representative capillary GLC-NPD chromatogram of trifluoroacetic anhydride (TFAA) derivatized MND T R I F L U O R O A C E T I C A N H Y D R I D E T R E A T M E N T D e h y d r a t i o n CONH, yCH(CHj)j 2CH,N( 4 ( D i i s o p r o p y l a m i n o ) - 2 - ( 2 - p y r i d y l ) - 2 -p h e n y l b u t y r a m i d e D i s o p y r a m i d e ( D i s ) T F A A C=N * /CH(CH,)2 \CH(CH,) d e h y d r a t e d D i s FIGURE 11. D e h y d r a t i o n a n d A c y l a t i o n H |N N 0 H X H m o n o - N - d e a l k y l a t e d d i s o p y r a m i d e ( m n d ) T F A A CH(CH,} 8 ^ ^ ~ C - - C H l C H 1 N ^ C = N C O C F d e r i v a t i z e d m n d Acetylation of the secondary amine group of MND and dehydration of amide group of disopyramide and MND as a result of trlfluoroacetic anhydride (TFAA)-treatment. 57 2.8 Optimization of TFAA-Treatment A stock solution of toluene containing disopyramide (2.2 mcg/ml), MND (1.8 mcg/ml) and PC-Dis (1.7 mcg/ml) was prepared. Aliquots of 1 ml of the stock solution were trans-ferred to polytetrafluoroethylene (PTFE)-lined screw-capped culture tubes. The samples were incubated with 0.1 ml of TFAA for 0, 15, 30, 60 and 120 minutes at 55°C. The residue was reconstituted in 0.1 ml of toluene and 2 m i c r o l i t e r of t h i s solution was injected into the chromatograph. A p l o t of i n d i v i d u a l peak area counts of disopyramide, MND and PC-Dis against the TFAA incubation time revealed the optimum duration of acylation and dehydration. 2.9 General Scheme for C a p i l l a r y GLC-NPD Analysis 2.9.1 Extraction and TFAA-Treatment The various steps involved i n the simultaneous c a p i l l a r y GLC-NPD analysis of disopyramide and MND i n human b i o l o g i c a l f l u i d s i s shown in Scheme I. An aliquot of blank human b i o l o g i c a l sample ( v i z . , serum, urine and saliva) were spiked with a volume (0.1, 0.2, 0.4, 0.6, 0.8 or 1.0 ml) of the prepared stock solutions of disopyramide phosphate (equivalent to 5 mcg/ml of base) i n deionized d i s t i l l e d water and MND (equiva-lent to 3 mcg/ml of base) i n 0.1N HC1. To t h i s mixture, 0.5 ml of 0.1N HC1 s o l u t i o n of PC-Dis (5.5 mcg/ml) and 0.5 ml of IN NaOH (pH 12) were added. 58 Simultaneous Analysis of Disopyramide and its Mono-N-Dealkylated Metabolite by Capillary GLC-NPD Plasma, saliva or urine 0.025 - 0.5 ml IN NaOH 0.5 ml Deionised distilled H2O q.s. 3.5ml 10 ml Glass tube p-chlorodisopyramide in 0.1 N HC1 0.5 ml of 0.55 mcg/ml Toluene 6 ml Shaken for 20 min Centrifuge to separate layers (discard aqueous layer) J 5 ml toluene layer T Evaporate on a water bath at 40*C and under a gentle stream of nitrogen J Dissolve residue in 0.5 ml toluene Trifluoro acetic anhydride 100 m1 Vortex for 10 seconds reaction allowed to proceea for 45 min at 55'C J — Dissolve residue In 0.2 ml toluene; vortex for 10 sees Evaporate on a water bath at 40*C and under a stream of nitrogen Cool to room temperature 1-2 ul Injected Into the chromatograph S c h e m e I. 59 The aqueous phase was adjusted to a t o t a l volume of 3.5 ml with deionized d i s t i l l e d water. Toluene (6 ml) was added and then shaken. Following centrifugation for 10 minutes, 5 ml of the organic phase was removed and dried under a gentle stream of nitrogen i n a 40°C water bath. The residue was reconstituted to a volume of 0.5 ml with toluene and 0.1 ml of TFAA was added. Each sample was vortexed f o r 10 seconds and the samples incubated i n an oven at 55°C f o r 45 minutes. The excess TFAA was removed by evaporating the sample under a gentle stream of nitrogen i n a 40°C water bath. The residue was reconstituted with 0.2 ml of toluene and 1 to 2 m i c r o l i t e r aliquots were used for c a p i l l a r y GLC-NPD anal y s i s . A s i m i l a r extraction and TFAA-treatment was carried out for d i l u t e stock solutions of disopyramide (0.54 mcg/ml), MND (0.23 mcg/ml) and PC-Dis (0.55 mcg/ml). 2.9.2 Quantitative Analysis of Disopyramide and MND A 2 m i c r o l i t e r aliquot of toluene solution containing TFAA-treated disopyramide, MND and PC-Dis was injected into a gas chromatograph equipped with an automatic l i q u i d sampler. C a l i b r a t i o n curves for disopyramide and MND were constructed by p l o t t i n g area r a t i o s (disopyramide or MND/PC-Dis) against the known concentrations of disopyramide and MND. The c a l i b r a t i o n curves thus obtained were used for the estimation of the unknown concentrations of disopyramide and MND i n b i o l o g i c a l samples. A l l samples were prepared i n t r i p l i c a t e and an aliquot of each sample was injected twice into the chromatograph. 60 2.9.3 Optimum C a p i l l a r y GLC-NPD Conditions The optimum parameters for the simultaneous c a p i l l a r y GLC-NPD analysis of disopyramide and MND were: Oven temperature 1 160°C Oven temperature programming rate 5°C/minute Oven temperature 2 260°C Nitrogen/phosphorus detector temperature .... 300°C Carrier gas (helium) flow rate 1.2 ml/minute Make-up gas (helium) flow rate 20 ml/minute H 2 / a i r flow rate r a t i o 3 / 50 NPD c o l l e c t o r voltage 16 vol t s Sample i n j e c t i o n modes S p l i t l e s s and auto-matic l i q u i d sampling Volume of sample 1-2 m i c r o l i t e r C a p i l l a r y column in use 25 m ( i . d . = 31 mm) crosslinked, 5 % phenyl methyl s i l i c o n e fused-s i l i c a c a p i l l a r y column 2.10 Extraction E f f i c i e n c y of Toluene The extraction e f f i c i e n c y of toluene was evaluated over a concentration range for disopyramide (0.5 - 5 mcg/ml) and MND (0.3 - 3 mcg/ml). Various volumes (0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ml) of the stock solutions of disopyramide phosphate i n deionized d i s t i l l e d water (equivalent to 5 mcg/ml of base) and MND i n 0.1N HC1 (equivalent to 3 mcg/ml of base) were transferred 61 into separate clean 10 ml glass tubes and the f i n a l volume of the aqueous medium was adjusted to 3 ml with deionized d i s t i l l e d water. The extraction procedure was similar to the general analysis Scheme I except that PC-Dis (0.5 ml of 3.3 mcg/ml) was added afte r the extraction step. T r i p l i c a t e samples were made for each concentration and each sample was injected twice into the chromatograph. The r e s u l t i n g area r a t i o s for each concen-t r a t i o n were m u l t i p l i e d by a cor r e c t i o n factor (6/5) because only 5 out of 6 ml of toluene mixture was used for evaporation and subsequent analysis. The amounts of disopyramide and MND extra-cted from these aqueous s a l t solutions by toluene were calculated from the standard curve of disopyramide and MND bases, obtained by using i d e n t i c a l volume of PC-Dis (0.5 ml of 3.3 mcg/ml) i n each tube. 2.11 General Procedure for Recoating of NPD Collector The s e n s i t i v i t y of the NPD c o l l e c t o r (part No. 27 19304-90200) was regenerated by replenishing the rubidium chloride which was l o s t due to v o l a t i l i z a t i o n over time from the inside of the NPD c o l l e c t o r . Rubidium chloride was manually coated on the ceramic bead inside the NPD c o l l e c t o r . Details of the procedure were described within an information sheet supplied 2 8 by Hewlett-Packard . Rubidium chloride rods (part No. 5020-8280) and aluminium oxide (part No. 5080-8875) were obtained from Hewlett-Packard 2 8. "27 Hewlett-Packard, Avondale, Pennsylvania, U.S.A. 28 Gas Chromatography: Nitrogen Phosphorus Element Recoating Information Sheet (1983), Hewlett-Packard, P.O. Box 10301, Palo Alto, C a l i f o r n i a , 94304-0890, U.S.A. 62 The recoating method b a s i c a l l y involved melting of rubidium ch l o r i d e (10 mg) onto the ceramic cylinder present inside the NPD c o l l e c t o r and then coating of the melted rubidium chloride with aluminium oxide (3 mg). The voltage normally applied to the NPD c o l l e c t o r during routine operation was decreased by 2 v o l t s during the coating process. 2.12 Measurement of Disopyramide Plasma Protein-Binding  Using Equilibrium D i a l y s i s The various stages of the technique used to determine the i n v i t r o plasma pr o t e i n - b i n d i n g of disopyramide i n man by conv-entional equilibrium d i a l y s i s are shown in Scheme I I . A 40 ml blood sample was c o l l e c t e d from each of f i v e healthy volunteers and heparinised plasma was immediately separated a f t e r c e n t r i f u -gation. Disopyramide phosphate was dissolved i n isotonic phos-phate buffer (0.067M, pH = 7.4) to y i e l d a series of concentra-tions ranging from 0.5 to 10 mcg/ml (equivalent of disopyramide base). This drug-containing b u f f e r (0.4 ml) was dialysed against blank plasma (0.4 ml) from each i n d i v i d u a l . D i a l y s i s was c a r r i e d out at 37°C i n p l e x i g l a s s d i a l y s i s c e l l s i n which buffer and 29 plasma were separated by a cellophane membrane . This membrane was b o i l e d i n d i s t i l l e d water for one hour and presoaked i n phosphate buff e r for one hour before mounting within the d i a l y s i s c e l l . Precaution was taken to ensure that the portion of 29 Sigma Chemical Company, Stock No. 250-9U, cut-off l i m i t 12,000 daltons, P.O. Box 14508, St Louis, MO, 63178, U.S.A. 63 EQUILIBRIUM DIALYSIS Disopyramide phosphate ( * 10 pg/ml base) In Isotonic phosphate buffer (0.067 M, pH Dilution with isotonic phosphate buffer Series of solutions containing 0.5 to 10.0 ug/ml of disopyramide 40 ml blood (from each 7.4) volunteer) Centrifugation Plasma 0.4 ml of each solution 0.4 ml of plasma Transfer Into 0.4 ml x 2 Capacity Plexi-Glas$) Dialysis cell separated by Cellophane (Sigma Chemical Co.) I Equilibrium dialysis at 37*C for 6 hours Aliquots of buffer and plasma for capillary GLC analysis Notes: 1. Dialysis was performed 1n triplicate at each concentration In all subjects. 2. Free fraction was calculated by comparing the area ratios (disopyramide/para-chlorodisopyramide) of buffer to plasma. Measurement of alpha -add glycoprotein (AAG) was carried out using nephelometry. 1 S c h e m e II. 64 the semipermeable membrane involved i n the d i a l y s i s was not touched by the investigator's fingers during the mounting. At the end of the d i a l y s i s , aliquots of buffer and plasma were transferred into clean glass tubes with the help of clean glass syringes mounted with unbeveled 2 inch long 22 gauge needles. The p o s s i b i l i t y of leakage of protein across the membrane was tested by adding a drop of the dialysed buffer to 0.1 ml of 3 % t r i c h l o r o a c e t i c acid. Absence of t u r b i d i t y was used as an indicator of membrane i n t e g r i t y . D i a l y s i s was performed i n t r i p l i c a t e at each concentration i n a l l subjects. 2.13 Phenobarbital-Treatment i n Non-Smokers for Disopyramide  Pharmacokinetics and Binding Studies 2.13.1 Volunteers Eight healthy male volunteers, without an admitted h i s t o r y of smoking, ranging i n age from 19 to 34 years participated in our study (Table 1). The recruitment of these subjects was made through campus b u l l e t i n s and advertisement i n l o c a l newspaper. Each volunteer received Ethics Committee approved compensation ($80/study) for inconvenience incurred during p a r t i c i p a t i o n i n the research project. None of the volunteers had a h i s t o r y of l i v e r , renal or cardiac disease and a l l had a normal physical examination, biochemical and hemato-l o g i c a l screen, and electrocardiogram (ECG). None was taking other medication and alcohol consumption was disallowed for 48 hours p r i o r to and during the study. TABLE 1. SUBJECT CHARACTERISTICS (NON-SMOKERS) Estimated . Subject Age Weight Cre a t i n i n e 9 SGOT Serum Phenobarbital yrs kg Clearance , IU/L l e v e l (mcg/ml) ml/min/1.73m 10th day 23rd day RH 34 73 95 23 9.9 14.4 MC 25 70 137 30 8.9 10.5 MK 25 75 76 35 9.2 12.7 RS 24 95 162 23 10.4 11.9 RK 27 75 78 42 11.5 17.3 PR 19 75 85 28 8.6 14.1 RM 19 72 85 49 13.7 13.7 ML 25 75 82 23 11.8 13.9 Key a : corrected for body surface, 2 (normal range = 70 to 157 ml/minute/1.73 m ). Key k : serum glumatic oxaloacetic transaminase or aspartate transferase (AST) (normal range = 22 to 47 international u n i t s / l i t e r ) . 66 After informed consent was obtained, each subject received a 200 mg o r a l dose of disopyramide (Rhythmodan®) with approximately 150 ml water on two occasions separated by a three week period during which phenobarbital (100 mg at bedtime) was taken o r a l l y . Phenobarbital concentrations were determined on day 10 and day 23 (Table I) to ensure that serum phenobarbital concentrations were greater than 10 mcg/ml during the test dose experiment with disopyramide. 2.13.2 Dosing and Physiologic Monitoring of Study  Subjects A l l volunteers were studied under the super-v i s i o n of a Cardiologist in the study Unit of the D i v i s i o n of Cardiology, Vancouver General Hospital. Heart rate and blood pressure were measured and an ECG was recorded p r i o r to and 1, 2 and 3 hours following disopyramide administration. No food was ingested u n t i l at l e a s t 3 hours following disopyramide. Water intake was approximately 250 ml hourly during the f i r s t 3 hours of the study to ensure adequate hourly urine flow. 2.13.3 Phenobarbital-Treatment Protocol The dosage schedule of disopyramide and phenobarbital i s depicted in Table 2. Our study involved an o r a l control dose of 200 mg disopyramide on day 1 followed by phenobarbital s t a r t i n g on day 3 of the study. Phenobarbital (100 67 TABLE 2. PHENOBARBITAL INDUCTION STUDY HEALTHY MALE VOLUNTEERS (19 - 34 yrs) WERE CHOSEN STUDY DAY EXPERIMENTS 0 Physical examination, lab tests (biochemical and hematological), blood pressure and ele c t r o -cardiogram recordings. 1 200 mg disopyramide p.o. to fasted subjects. 10 ml of blood samples collected at 0 (blank), 15 minutes, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 16, 24.5, 29, 32 and 48 hours. Saliva and urine samples were also c o l l e c t e d . 3 - 24 100 mg of phenobarbital h.s. 23 Second dose of disopyramide (sample c o l l e c t i o n exactly as on day 1). Sera obtained from blood samples along with s a l i v a and urine samples were frozen (-20°C) u n t i l analysis. 68 mg at bedtime) was continued through day 25, with a second o r a l dose (200 mg) of disopyramide on day 24. The sampling protocols of control (untreated) and phenobarbital-treatment were iden-t i c a l . Blood (10 ml) and s a l i v a (4 ml) samples were c o l l e c t e d at 0 (blank), 15 minute, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 16, 24, 28, 32, and 48 hours. Timed t o t a l urine c o l l e c t i o n was conducted at hourly i n t e r v a l s u n t i l 8 hours and then at 10, 12, 16 hours a f t e r drug administration. In addition, a l l other urine excreted up to 48 hours was c o l l e c t e d . Single blood samples were co l l e c t e d on day 10 and 23 for serum phenobarbital determination i n a l l volun-teers. Single blood samples on days 10, 15, 19, 23 and 28 were co l l e c t e d for serum AAG measurement in selected subjects while serum AAG concentration was measured in control and on day 21 i n a l l subjects. 2.13.4 Sample C o l l e c t i o n Techniques Blood samples (10 ml) were withdrawn using a l l glass syringes and 1.5 inch 22 gauge needles, from an i n -dwelling b u t t e r f l y cannula inserted i n the b r a c h i a l vein. Unstim-ulated mixed s a l i v a samples (4 ml) were c o l l e c t e d into 20 ml clean glass v i a l s at the same time as blood c o l l e c t i o n . Total urine was c o l l e c t e d at appropriate time i n t e r v a l s into p l a s t i c Whirl Pak® urine sample bags. The pH of s a l i v a and urine was measured soon aft e r c o l l e c t i o n with a pH meter. Saliva samples were centrifuged at 2,500 rpm for 10 minutes to remove any 69 insoluble matter and the c l e a r supernatant was used f o r analysis. Serum was obtained from cl o t t e d blood samples i n a similar manner. A l l b i o l o g i c a l samples were kept frozen at -20°C u n t i l analysed. Each serum concentration represented mean of two separate determinations at each time point except 48 hour values which required more than one ml of serum for quantitation. In order to increase the confidence i n the estimates of the apparent elimination rate constant ( K e^)# supplementary blood samples were taken at 28 and 3 2 hour in some subjects. 2.13.5 A n a l y t i c a l Procedures Disopyramide and MND were simultaneously quantitated i n various b i o l o g i c a l f l u i d s using the improved c a p i l l a r y GLC-NPD technique (discussed e a r l i e r ) . Quantitation was carried out using d a i l y standards and selected samples were also measured using a commercially available homogeneous EMIT® technique (Johnston and Hamer, 1981). The serum phenobarbital concentration was determined by a Dupont Automatic C l i n i c a l Analyser® which i s an adaptation of Syva's homogeneous EMIT® assay. The free f r a c t i o n of disopyramide was estimated i n the 2, 3 and 8 hour serum samples from each study using a commercially a v a i l a b l e disposable u l t r a - f i l t r a t i o n device (Johnston and Hamer, 1981). The t o t a l and unbound disopyramide serum concentration i n t h i s series of studies were estimated using a commercially available homogeneous EMIT® k i t (Hammad 70 et a l . , 1982). Free drug concentrations of less than 0.25 mcg/ml were considered below the s e n s i t i v i t y l i m i t of t h i s assay and were excluded from evaluation ( r e g r e t f u l l y t h i s included a l l 8 hour samples obtained during the phenobarbital-treatment phase of the study). The assays were performed on a spectrophoto-3 0 31 meter l i n k e d to a c l i n i c a l p r o cessor . Before f i l t r a t i o n , serum was adjusted to pH = 7.4 with a small volume of 0.5 M H 3P0 4 and centrifugation was carried out i n a refrigerated centrifuge. AAG concentration i n serum samples of volunteers was estimated immunochemically by nephelometry. The method was based on the p r i n c i p l e that the amount of l i g h t s c a t t -ered by a d i l u t e mixture of sali n e , antibody and antigen, i s a li n e a r function of the amount of antigen-antibody complex present in the mixture. Serum AAG (antigen) formed an insoluble complex 3 2 with a s p e c i f i c anti-AAG human serum (antibody) . The AAG-antisera complex thus formed was measured using a Beckman 33 Immunochemistry System . The re s u l t s of t h i s method were 30 G i l f o r d Instruments Laboratories, Inc., Oberlin, Ohio, U.S.A. 31 Syva Company, Palo Alto, C a l i f o r n i a , U.S.A. 32 LN Antisera to human AAG, catalogue No. 449440, Calbiochem Behring Calbiochem-Behring, San Diego, C a l i f o r n i a , U.S.A. 33 Beckman Instruments, Incorporation, F u l l e r t o n , C a l i f o r n i a , U.S.A. 71 compared with those obtained using a r a d i a l immunodiffusion procedure on selected samples. This was achieved using commercially available NOR-Partigen® agarose gel p l a t e s ^ . The square of the diameter of the p r e c i p i t i n ring formed due to AAG-antisera complex was i n l i n e a r relationship to the AAG (antigen) concentration. Standard AAG solutions (NOR-Partigen Standard Set III , Catalogue No. 9410011) were also obtained from 34 the same source 34 Behring Diagnostics, D i v i s i o n of American Hoechst Corporation, 10933 North Torrey Pines Road, La J o l l a , C a l i f o r n i a , 92037, U.S.A. 72 2.13.6 Data Analysis T o t a l serum disopyramide concentration versus time data were subjected to non-linear least squares regression analyses with AUTOAN (Sedman and Wagner, 1976) and i n i t i a l phar-macokinetic parameters for the best f i t open-model of one comp-artment were obtained using equal weighting of a l l data points. These i n i t i a l parameters were further analysed by NONLIN (Metzler, 1974) u t i l i z i n g i t e r a t i v e techniques, to obtain appa-rent f i r s t - o r d e r absorption (k ) and apparent elimination cl (K . ) rate constants. The i n i t i a l estimates of the terminal e l e limination rate constant (Kel) was obtained from l o g - l i n e a r reg-ression analysis of blood concentration data after the 6 hour data points for a l l subjects, using a Hewlett-Packard Model-65 ca l c u l a t o r . Area under the serum concentration vs time curve (AUC„ „ , ) was estimated using the l i n e a r - t r a p e z o i d a l r u l e . x 0-32 hour 3 c Renal clearance of t o t a l disopyramide was estimated by d i v i d i n g the urinary excretion rate by t o t a l serum concentration at the mid-point of the urine c o l l e c t i o n . Other pharmacokinetic para-meters such as peak serum concentration ( Cp e a^)» time to peak serum c o n c e n t r a t i o n ( t v ) , a p p a r e n t o r a l c l earance and C^-VF and apparent volume of d i s t r i b u t i o n (Vd =/F) were estimated as described by G i b a l d i and Per r i e r (1975). S t a t i s t i c a l evalua-tions were performed on the various pharmacokinetic parameters using paired t - t e s t (p <_ 0.05, two-tailed) comparing the con-t r o l values with those obtained from the phenobarbital treatment. 73 2.13.7 Assurance of Phenobarbital Compliance Compliance with the dosage regimen was ensured by c a r e f u l selection of educated individuals as subjects and by careful observation of the volunteers. Phenobarbital concentrations were measured on day 10 and day 23 to ensure compliance and to ensure adequate phenobarbital concentrations (>^ 10 mcg/ml) to induce hepatic enzymes. 2.14 Phenobarbital-Treatment i n Smokers for Disopyramide  Pharmacokinetic and Binding Studies Eight healthy male volunteers, ranging i n age from 25 to 39 years, participated i n our experiment (Table 3). A l l individuals were habitual cigarette smokers who, by history, smoked at l e a s t 20 cigarettes a day for the past 5 years (see Table 3 for smoking and alcohol h i s t o r y ) . The general experimental procedures such as dosing and physiological monitoring of smokers, sampling protocols of control and phenobarbital-treated smokers, sample c o l l e c t i o n techniques, a n a l y t i c a l methodologies, methods for data analysis and assurance of compliance etc, were similar to that employed during the phenobarbital treatment study i n non-smokers (discussed i n a preceding section). S t a t i s t i c a l evaluation were also performed on various pharmacokinetic parameters between non-smokers and smokers using unpaired two-tailed t - t e s t (p _< 0.05). 74 TABLE 3. SUBJECT CHARACTERISTICS (SMOKERS3) Estimated^ Subject Age Weight Creatinine SGOT Serum Phenobarbital yrs kg Clearance _ IU/L l e v e l (mcg/ml) History of ml/min/l.73m 10th 23rd Ethanol day day Consumption AB 26 87 128 21 9.3 11.6 moderate JO 32 73 97 24 10.1 14.8 l i g h t 6 IT 25 86 155 44 6.1 13.2 moderate SD 30 54 125 18 12.8 17.4 occasional' JK 39 62 152 18 10.2 14.7 g moderate SM 27 79 107 18 9.6 12.8 l i g h t DF 23 78 113 22 d l i g h t MC 27 65 80 16 l i g h t f Key a 2 6 + 7 cigarettes a day (mean + S.D.) : corrected for body surface, 2 (normal range = 70 to 157 ml/minute/l.73 m ). ° : serum glutamic oxaloacetic transaminase, or aspartate transferase (normal range = 22 to 47 international u n i t s / l i t e r ) . : volunteers were not available for t h i s part of the study. e : < 1 li t e r of 5% v/v of ethanol per week f : < 0.25 liters of 5% v/v of ethanol per week g : > 2 liters of 5% v/v of ethanol per week 75 3 RESULTS 3.1 A n a l y t i c a l Development 3.1.1 C a p i l l a r y GLC-NPD Representative GLC-NPD chromatograms from the extracts of blank plasma and spiked plasma are shown i n Figure 12. No extraneous (interfering) peaks from endogenous plasma constituents are apparent i n the plasma extracts (Figure 12A). Peaks with retention times (R.T.) of 10.10 and 12.91 minutes are the dehydrated ( i . e . n i t r i l e ) forms of disopyramide and PC-Dis, respectively, and the peak at 10.59 minutes represents the TFAA d e r i v a t i v e of the dehydrated form of MND (Figure 12B). The peaks were sharp and symmetrical. Complete baseline resolution i s achieved between the three peaks. Total analysis time was 14 minutes. Figure 13 represents the chromatogram of a TFAA-treated mixture containing various antiarrhythmic drugs. A l l of the chromatographic peaks were resolved under the following s p l i t l e s s sample i n j e c t i o n mode, GLC-NPD conditions: oven temperatures 1, 160°C, oven temperature programming rate 1, 5 °C/minute; oven temperature 2, 270°C, time 2, 25 minutes; i n j e c t i o n temperature, 260°C; N/P detector temperature, 300 °C; c a r r i e r gas (helium) flow rate, 1.2 ml/minute; make-up gas (helium) flow rate, 27 ml/minute; hydrogen-air flow rate r a t i o , 3:50. Total analysis run time was 15 minutes. 76 C A P I L L A R Y C O L U M N C H R O M A T O G R A P H Y B •H-to FIGURE 12. Representative c a p i l l a r y GLC-NPD chromatograms obtained from blank (A) and spiked plasma extracts (B); the spiked sample before extraction contained disopyramide (retention time, R.T. = 10.10 min), 1.0 mcg/ml; MND (R.T. = 10.59 min), 0.7 mcg/ml and PC-Dis (R.T. = 12.91 min), 2.4 mcg/ml. Attenuation, 2^; voltage, 16 v o l t s ; NPD detector was i n use for ca. 200 hours. A 12 meter ( i . d . = 0.27 mm) f u s e d - s i l i c a c a p i l l a r y column coated with crosslinked methyl s i l i c o n e f l u i d was used. 77 C a p i l l a r y G a s C h r o m a t o g r a p h i c P e a k s REPRESENTATIVE CHROMATOGRAM OF TFPP DERIVATIVES OF VARIOUS ANTIARRHYTHMIC AGENTS : H.P. CAPILLARY CHROMATOGRAPH, MODEL 5S30A EQUIPPED WITH N/'P DETECTOR* FIGURE 13. Representative c a p i l l a r y GLC-NPD peaks o f various TFAA-treated antiarrhythmic agents. 78 3.1.2 Li n e a r i t y and Reproducibility of GLC-NPD  Assay Method GLC-NPD detector response for disopyramide and MND was l i n e a r i n the concentration range studied (0.05 -5.0 mcg/ml for disopyramide and 0.02 - 3.0 mcg/ml for MND). The c a l i b r a t i o n curves were obtained by analyzing blank plasma sam-ples spiked with varying amounts of disopyramide and MND (Tables 4 and 5). The best f i t through the data points was obtained from lin e a r regression analysis (Tables 4 and 5). The c o e f f i c i e n t of determination, r , was >0.98 for a l l regression l i n e s . The average c o e f f i c i e n t of v a r i a t i o n (C.V.), which i s a measure of r e p r o d u c i b i l i t y i n sample an a l y s i s , was found to be 5 and 8 % for disopyramide and MND, respectively, over the concentration range studied. The dehydrated forms of disopyramide and PC-Dis, and the TFAA d e r i v a t i v e of the dehydrated MND were found to be stable for at least 48 hours at room temperature and at least 10 days when stored at -4°C as v e r i f i e d by repeat i n j e c t i o n s showing no s i g n i f i c a n t decline i n peak areas over the period of storage. 3.1.3 Kinet i c s of TFAA-Treatment Figure 14 shows the k i n e t i c s of dehydration of disopyramide, MND and PC-Dis as well as acet y l a t i o n of MND by TFAA-treatment. The optimum re a c t i o n time was evaluated by i n c u -bating samples containing equivalent amounts of disopyramide t MND, PC-Dis and TFAA for various times at 55°C. The peak areas of the dehydrated forms of disopyramide, PC-Dis and the TFAA de r i v a t i v e of the dehydrated form of MND was found to not change TABLE 4. CALIBRATION DATA POINTS FOR DISOPYRAMIDE AND MND (HIGHER CONCENTRATION RANGE) Concentration Concentration Area Ratio Area Ratio of Disopyramide of MND Disopyramide/PC-Dis MND/PC-Dis (mcg/ml) (mcg/ml) Range* Range* 0.50 0.31 0.21 - 0.22 (1.1)** 0.085- 0. 091 (5.0)** 1.00 0.61 0.41 - 0.42 0.17 - 0. 18 1.99 1.23 0.88 - 0.89 0.36 - 0. 37 2.99 1. 94 1.24 - 1.39 0.49 - 0. 55 3.98 2.45 1.75 - 1.88 0.67 - 0. 75 4.98 3.07 2.12 - 2.38 (2.5)** 0.90 - 0. 98 (6.9)** S t a t i s t i c s : linear regression l i n e s for disopyramide: y = (0.47) x + 0.046; r 2 = 0.999; and for MND : i y = (0.31) x - 0.016; r 2 = 0 .999 . * Number of samples, n = 2 (two injections for each sample). ** Coefficient of var i a t i o n (%). TABLE 5. CALIBRATION DATA POINTS FOR DISOPYRAMIDE AND MND (LOWER CONCENTRATION RANGE) Concentration Concentration Area Ratio* Area Ratio of Disopyramide of MND Disopyramide/PC-Dis MND/PC-Dis (mcg/ml) (mcg/ml) Mean+S.D.** C.V.*** Mean+S.D. C.V. 0.054 0.023 0.23+0.01 5.6 0.07+0.01 10.2 0.109 0.046 0.45+0.03 5.7 0.13+0.01 8.1 0.217 0.092 0.88+0.03 3.5 0.27+0.03 9.5 0.326 0.139 1.16+0.09 8.0 0.40+0.03 8.2 0.435 0.185 1.81+0.17 9.4 0.62+0.05 8.7 S t a t i s t i c s : linear regression l i n e s for disopyramide: y = (3.96) x + 0.001; r 2 = 0.98; and for MND : y = (3.32) x - 0.02 5; r 2 = 0.99 . * Number of samples, n = 4 (two injections for each sample). ** Mean + one standard deviation. *** Co e f f i c i e n t of variation (%). 81 1 2 0 0 0 0 n 1 0 0 0 0 0 -4 0 0 0 0 -2 0 0 0 0 -0 d i s o p y r a m i d e •J- B p - c h l o r o d i s o p y r a m i d e m o n o - N - d e a l k y l a t e d m e t a b o l i t e 2 0 "T" 40 60 6 0 100 l 120 T I M E ( M I N U T E S ) FIGURE 14. Kine t i c s o f t r i f l u o r o a c e t i c anhydride (TFAA)-treatment o f disopyramide, parachlorodisopyramide (PC-Dis) and mono-N-dealkylated metabolite (MND) o f disopyramide (n 2-3). 82 s i g n i f i c a n t l y over an incubation period of 2 hours. Excess TFAA was removed according to the method of Walle and Ehrsson (1970), whereby the reaction mixture was evaporated on a water bath at 40°C under a gentle stream of nitrogen. 3.1.4 Extraction E f f i c i e n c y of Toluene Ca l i b r a t i o n curves for a s e r i a l d i l u t i o n of the free base forms of disopyramide and MND i n toluene were pre-pared. Following extraction of known quantities (Table 6) of disopyramide and MND from spiked human plasma, the recovery i n the organic phase (toluene) was determined by using the free base c a l i b r a t i o n curves. The average percentages of disopyramide and MND extracted by toluene were 95 % and 87 %, respectively (Table 6). 3.1.5 Structural Confirmation of Compounds Preliminary i d e n t i f i c a t i o n of the chromato-graphic peaks was made by i n d i v i d u a l l y i n j e c t i n g the standard compounds of i n t e r e s t into the gas chromatograph. Characteri-zation of the structures of untreated and TFAA-treated compounds 'Disopyramide, MND and PC-Dis) was obtained using both electron impact (EI) and chemical i o n i z a t i o n (CI) mass spectra (MS), (for d e t a i l s , r e f e r to APPENDIX). The t o t a l ion current mass chromato-grams of disopyramide and PC-Dis under both i o n i z a t i o n modes yielded single peaks. The GC/MS of a l l regions of each of these TABLE 6. TOLUENE EXTRACTABILITY OF DISOPYRAMIDE AND MND Disopyramide Added (meg) Disopyramide Measured (meg) (Mean+S.D.)a % Recovery (Mean+S.D.) MND Added MND (meg) Measured (Mean+S.D. % Recovery (Mean+S.D.) ) 0.5 0.5+0.03 100+6 0.3 0.3+.02 100+2 1.0 0.9+.02 94+2 0.6 0.6+.01 94+1 2.0 1.9+.02 95+1 1.2 1.1+.02 85+2 3.0 2.8+.2 92+8 1.8 1.4+.1 78+6 4.0 3.8+.2 94+5 2.4 2.0+.1 80+7 5.0 4.8+.2 95+5 3.1 2.6+.1 84+6 Note a : Number of determinations, n = 4 84 single peaks yielded the same spectra. The EI-MS and CI-MS data of untreated MND and i t s degradation products are shown i n Figures 15 and 16. Figures 17 and 18 i l l u s t r a t e EI-MS and CI-MS data of TFAA-treated MND ( n i t r i l e and amide forms). 3.2 Measurement of Disopyramide Plasma Protein-Binding Using Equilibrium D i a l y s i s Figure 19 shows the representative chromatograms of TFAA-treated disopyramide and PC-Dis i n toluene extracts of phos-phate buffer (19A) and plasma (19B) aliquots. No extraneous peaks from endogenous plasma constituents are apparent in the toluene extract of plasma (20B). The r e s u l t s of equilibrium d i a l y s i s experiments are shown in Table 7. There was no appre-cia b l e change i n the volumes of buffer and plasma within the d i a l y s i s c e l l s . Appreciable v a r i a b i l i t y was observed i n the free f r a c t i o n (fu) results between d i f f e r e n t c e l l s at the i d e n t i c a l concentration. There was also s i g n i f i c a n t inter-subject v a r i a -b i l i t y i n the protein-binding of disopyramide i n the entire con-centration range (0.5 - 10 mcg/ml). Plasma protein-binding of disopyramide was found to be concentration-dependent (Table 7). Plasma AAG l e v e l s i n a l l 5 healthy volunteers are also shown i n Table 7. 85 TOTAL ION CURRENT MASS CHROMRTOGRPM OF i MONO-N-DEPLKVLRTED METfiB. TOTAL ABUNDANCE FROM 30 TO 500 ajnu F u l l Scale- 11543 ii ii N a II i . . ri WM,u.iir.ii "^ 1"— IT.- u . I ' I I ELECTRON IMPACT MASS SPECTRUM OF MONO-N-DERLKVLPTED DISOPVRRMIDE <PERK:1> Base Peak «= 194.10 Base Peak Abundance ?A7._JL9i*L£Lb^2SL a n c e " 2285 I I I. 50 100 150 200 250 300 350 400 ELECTRON IMPACT MPSS SPECTRUM OF MONO-N-OEPLKVLPTED DISOPVRRMIDE (PERK:2) Base Peak 195.10 Base Peak Abundance 1^  605 _ _ Jo t a_l_ _ Abu n da nee -7902 J U L a 50 100 150 200 250 PROMINENT FRRSMENT IONS : MASS <<Ve> : 195 212 194 167 196 18* RBUNDPNCE( '4>: 108 57 54 49 18 16 300 350 400 213 239 43 44 288 10 6 5 5 3 FIGURE 15. The pos i t i v e ion electron impact (EI) data ( t o t a l ion current chromatogram, mass spectrum (MS) and prominent fragments) o f untreated MND. CHEHICAL IGNISPTION (HC) H.S. OF HOHO-H-OEflLKVLfiTED METOBOLITE FIGURE 16. The positive chemical i o n i z a t i o n (CI) t o t a l ion current chromatogram obtained from untreated MND (A) ; CI mass spectrum o f intact MND (B) and CI mass spectrum o f degra-dation product of untreated MND (C) . 87 TOTAL ION CURRENT MASS CHROMATOGRAM OF MONO-N-DEALKVLATED METAB.-TFAA TOTAL ABUNDANCE FROM 30 TO 500 amu Fu l l Scale- 11543 • -. - JiL- . . ft- - . ••• • . • .fcl. . . i t ..tl. 4-. .. • r v ^ » j ^ J E ^ T L J H J w y i w W ^ wftj~wujiftnitHiaftm»f,^m fthW^r-1 i<"~>* .^nfyft*-' • m ~w ii«ifmrv | l^S> l f—' 111 S f • • • •** • 1 . . . • »• • I I I E.I.-M.S. OF MONO-N-DEALKYLATED DISOPYRAMIDE-TFAA <PEAK:1> Base Peal, = 194.10 Base Peak Abundance 582 Total Abundance 1844 50 100 150 PROMINENT FRAGMENT IONS : 200 250 300 350 400 MASS (ive> : 194 207 ABUNDANCES): 100 45 193 221 192 167 43 41 37S 21 13 13 5 5 4 h>2 E.I.-M.S. OF MONO-N-DEALKVLATEO DISOPVRAMIDE-TFAA <PEAK:2) Base Peak 182.10 Base Peak Abundance -• l l i t i . l a II i 1 l i i • 50 100 150 PROMINENT FRAOMENT IONS : I - -|—«• 200 250 172 Total Abundance 300 350 MASS <«'e> : 182 195 167 194 212 43 225 193 44 69 ABUNDANCES): 100 55 51 42 26 20 16 16 14 6 1148 400 FIGURE 17. The po s i t i v e ion EI data ( t o t a l ion current chromato-gram, mass spectrum and prominent fragments) o f TFAA-treated MND. r i 1 • >AME 2 6 0 . 0 - 4 9 9 . 0 u u . <• s» i?e i c e ;e« 2 4 0 2 9 9 3 2 0 . I . . . I . . . . . . . I . . . I . . . I . . . I . . . I . . . I I I |1 I I I i T n l H . l l l l 120000-101 9*01 10000-1.0 2.0 3.0 4.0 6.0 6.0 7.8 8.0 9.0 10.0 r u « >ni0C •P* m «i03 60-40-20> H . H.I 3 9 3 B 194 57 / lUy > ' -' -'-99 129 1*5 195 239 <297*96+1) \ 201 308 364 100 150 ZB8 258 V 266 / / , |Mi|iU/i|>/ii'|i'm|iiiif>iii^ Scan 217 -.02 aln. 30 380 3S8 480 CH+*9> 422 j' «3S 4*7 J U T 20 45* rii« >n>0e Ipk Ob 9 384 100 37* 80-60-40-20-M.M.i 375 4" <297-18*96+1) — CM,CM,l)l C O C f . «7 99 122 15* 194 224 IB* 15ft ?8S 351 rcan 104 f .97 am. 30 <H*29> 404 11 488 450 -20 10 r i i . >At.es Bpk Ob 1897 180-1 80-60-48-20-57 99 138 126 1*9 «»« 223 266 291 p^»iyipW|in, i8)r.M|i 158 2«8 158 309 380 Scan 170 5.53 aln. M6 1-12 379 3S0 I 43] 480 431447 *T7 460 •1 r i l . >RB05 tpk Pb J ? 93 1 0 0 - 1 60-281 40- 57 / *- JL*>^4v 26 S 361 309 99 127 1*9 196;|9 188 150 209 250 Scan 171 5.6* a i n . 1-24 h*0 16 12 379 4 2 9 448 4" >«MI>HI.W 450 CO CO FIGURE 18A. The p o s i t i v e CI t o t a l ion current mass chromatogram (A) and mass spectrum (B) of TFAA-treated MND (incomplete reaction). r . i » >ai»r £ 8 . 8 - 6 8 0 . 0 aau. J" 99 i j j j |?« 16B 240 r ' ' t i l i - > ' ^ i i * - - i 1 i i f ' ' ' ' 1 ' » l l l » * 2080* 1 i t m t j 128888-1 •Ml (.0088-48088-/ L — _ * ^ i • • " i m i i n ••• i n i n ••• I' "' l " " l " "I • _ 3.0 3.0 4.0 5.0 6.9 7.8 9.0 9.9 •P» 100-98-68-46-28-S7 B 376 H . H.I 3 7 S 4 < 297-18*9641 >^  298 314 " 108 I SB 200 2St» 36* 359 400 <H*29) Scan 162 6 . 9 3 0ln. t *8 •48 -38 28 -10 « r 1 1 « >ai67 •pk Ob )??B8 10^ 88-60-40-20- 67 / 9? 129 1*9 I9« *• 39 1*0 150 ?00 *S8 26 Si an 148 E.St t i n . r j-38 -28 309 361 ?t-8 379 429 441 468 3C.0 460 r i l » ,0B87 •pk nt> 0 3 9 7 1 0 0 -88-60-48-67 28-1 V 0-137 158 196 I * mi'*, 208 2S8 26B ; 28i JUL 331 / | i l i i | l i n . 388 3S8 Scan 134 8.36 0ln. 38 r i 1 • >nt87 •pk MB 1973 67 379 , 438 473 . \ *v / mftomfwu^ 499 28 18 3SI / 88-68-48-28-8->&»*fj 99 129 169 196 221 *?B 293 \ 334 Scan 136 6.43 a l n . 481 X 447 D n i i X PIGURE 18B. The positive CI t o t a l ion current mass chromato-gram (A) and mass spectrum (B) o f excess o f TFAA-treated MND (complete acetylation and dehydration). 9Q CD 03 B 03 r Representative chromatograms o f TFAA-treated toluene extracts o f buffer (A) and plasma (B) o f disopyramide (R.T. = 10.4) and para-chlorodisopyramide (R.T. =13.07 min.). 91 TABLE 7. UNBOUND FRACTION ( f u) OF DISOPYRAMIDE AS A FUNCTION OF CONCENTRATION Concentration Free fraction in volunteers* Statistical probability of equivalent to disopyramide base (mcg/ml) Subject JA Subject JO Subject RK Subject KM Subject MB replication between adjacent concentration pai rsb 0.5 0.094a 0.162 0.127 0.187 0.121 — 1.0 0.118 0.175 0.106 0.217 0.146 0.50cp 2.0 0.172 0.237 0.169 0.283 0.150 0.10<p<0.50 3.0 0.246 0.326 0.259 0.243 0.294 0.02<p<0.05* 4.0 0.260 0.361 0.242 0.327 0.321 0.10<p<0.50 5.0 0.290 0.376 0.317 0.327 0.402 0.10<p<0.50 10.0 not done 0.584 0.552 0.380 0.614 0.001<p<0.01** AAG level (mq/100 ml) 82 30 44 62 40 . . . Key, a Each number represents mean of 2-3 dialysis experiments; range of coefficient of variation: 5 to 27% (mean C.V. * 12%). b The mean unbound fraction (fu) o f each volunteer at each concentration was compared to the next higher concentration. * Significant ** Highly significant # Assessment of in vitro binding using spiked plasma samples. 92 3.3 Phenobarbital Treatment i n Non-Smokers for Disopyramide and MND Pharmacokinetics and Binding Studies Figures 20 and 21 represent the mean serum disopyr-amide and MND concentration versus time p r o f i l e s for subjects during the untreated and phenobarbital-treated phases. Table 8 summarizes the pharmacokinetic parameters which best f i t these data i n a l l eight non-smoking volunteers. There was a s t a t i s -t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n the time taken to reach maximum serum concentration ( t p e a l c ) of disopyramide between untreated and phenobarbital-treated groups. However, the observed mean peak serum concentrations (Cp e a^) between the two phases were not s i g n i f i c a n t l y d i f f e r e n t (Table 8). The i n t e r - i n d i v i d u a l v a r i a b i l i t y i n the apparent absorption rate constant (k ) i s a indicated by large r e l a t i v e standard deviations about the popula-t i o n mean for t h i s parameter. A lag period during the gastro - i n t e s t i n a l absorption of disopyramide from the capsules was not apparent (evident) from the l i m i t e d data points i n the absorption phase in most of the volunteers and hence was not incorporated in the c a l c u l a t i o n s of ka. This factor may contribute to the v a r i a -b i l i t y i n the estimates of ka. There was a s t a t i s t i c a l l y s i g n i -f i c a n t difference i n the calculated values for the apparent f i r s t - o r d e r elimination rate constant (Kel), apparent elimina-t i o n h a l f - l i f e ^1/2^' a r e a u n <3er serum concentration-time curve (AUC0_2 2^r) a n d apparent o r a l t o t a l body clearance ( C l m o F), between the two groups. Phenobarbital treatment caused a reduction i n A U C q _ j 2 n r a n < * t i / 2 ' b ^ ^ * a n ^ ^ *' respectively. A 74 % increase in C1 T B/F was observed in the 93 O DIS - C o n t r o l • DIS - P h e n o b a r b i t a l FIGURE 20. Disopyramide serum concentration versus time curves before ( O ) and during ( A ) phenobarbital-treatment i n non-smokers (n = 8). The disopyramide dose was 200 mg o f the base. The data are_presented as the mean + one standard deviation (X + S.D.). The Insert represents the semi logarithmic plot of the total serum disopyramide concentration vs time data. 94 O M N D - C o n t r o l • M N D - P h e n o b a r b i t a l T i m e (hours) FIGURE 21. MND serum concentration versus time curves before (O) and during (• ) phenobarbital-treatment_in non-smokers (n = 8). The data are presented as the X + S.D. The insert represents the semi logarithmic plot of the total serum MND concentration vs time data. TABLE 8. PHARMACOKINETIC PARAMETERS OF DISOPYRAMIOE IN NON-SMOKERS Subject tpeak cpeak K e 1 M/2 AUC C1 T B/F v d an (hr ) (mcg/ml) b ( h r " 1 ) 0 ( h r - 1 ) * 1 ( h r ) e (hr.mcg/ml (1/hr/kg)9 (1/kg) 1 Control a a l 1 1.5 1.4 2.6 2.4 0.11 6.6 26.1 0.105 1.00 2 1.5 2.5 2.0 0.9 0.14 5.1 19.9 0.144 1.06 3 3.0 1.8 2.5 1.6 0.11 6.1 26.0 0.103 0.90 4 3.0 1.8 2.5 1.4 0.14 4.9 21.9 0.096 0.68 5 2.5 1.7 2.8 1.9 0.08 8.3 31.5 0.085 1.01 6 4.0 3.1 2.9 0.9 0.08 9.1 38.2 0.070 0.92 7 3.0 2.3 3.8 1.1 0.12 5.9 31.3 0.089 0.75 8 1.0 1.2 3.0 2.9 0.12 5.8 24.4 0.109 0.92 Average 2.4 i S.D. 2.0 2.8 1.6 0.11 6.5 27.4 0.100 0.91 (n-8) * 1.0 t 0.6 t 0.5 t 0.7 * 0.02 t 1.5 ± 6.0 * 0.020 ± 0.13 Phenobarbital -treated a «1 1 1.0 1.3 2.1 2.3 0.14 5.0 15.6 0.175 1.26 2 2.0 2.0 1.5 1.1 0.19 3.7 10.3 0.279 1.50 3 1.5 1.1 2.5 2.7 0.16 4.4 17.4 0.158 0.96 4 1.0 1.5 2.3 1.7 0.18 3.9 15.1 0.140 0.78 5 3.0 1.3 2.9 3.5 0.12 6.0 27.1 0.099 0.85 6 2.5 2.7 2.8 0.8 0.14 4.9 26.1 0.103 0.73 7 2.5 1.3 1.7 2.2 0.16 4.4 13.3 0.210 1.32 8 1.0 1.4 1.4 2.1 0.16 4.4 12.8 0.209 1.32 Average 0.17* 1.09 t S.D. 1.8 1.6* 2.1 2.0 0.16* 4.5* 17.2* (n»8) ± 0.8 ± 0.5 t 0.6 4 0.9 t 0.02 ± 0.7 * 6.2 t 0.06 t 0.30 •ea/F Dose - 200 mg oral l y a estimated from serum cone, vs time curve •1 tpeak ' (2.303/k a-k ei) 1ogTk~a/kei) b estimated from serum cone vs time curve jj from NONLIN computer program e t i / 2 - 0.693/K ei AUCrj-32hr from l i n e a r trapezoidal rule C1tb/f = Dose/AUCo-32 Vd/F = Cl TB/K el S t a t i s t i c a l l y s i g n i f i c a n t from the respectln control value (p < 0.05) 96 phenobarbital-treated subjects. The apparent volume of d i s t r i -bution (Vd /F) between the two groups was not s t a t i s t i c a l l y d i f f e r e n t (Table 8). The AUC for MND determined during phenobarbital treatment (4.1+ 2.3 hr. mcg/ml) was higher than i n the untrea-ated group (3.8+ 1.6 hr. mcg/ml), but t h i s difference did not reach s t a t i s t i c a l s i g n i f i c a n c e . The average apparent elimination t_l/2 of MND during the untreated phase of the study (6.7 hours) was not d i f f e r e n t from that obtained during phenobarbital t r e a t -ment (6.4 hour). The serum leve l s and fu of disopyramide as a function of time and phenobarbital treatment (both measured by EMIT® technique) are shown in Table 9. Table 10 summarizes the serum AAG concentration in healthy volunteers before and a f t e r 21 days of phenobarbital treatment. The s a l i v a concentration-time p r o f i l e s for disopyr-amide and MND for the untreated and phenobarbital-treated groups are shown i n Figures 22 and 23, r e s p e c t i v e l y . The s a l i v a r y l e v e l s of disopyramide were generally higher i n untreated subjects ( F i g -ure 22) while MND l e v e l s were higher in the phenobarbital-treat-ed group (Figure 23). A s e l e c t i o n of the area under the s a l i v a concentration-time curves f o r disopyramide and MND i n both phases are shown i n Figure 24. The area for disopyramide was consistent-l y higher in the untreated group as compared to that obtained afte r phenobarbital treatment. Conversely, the area for MND was consistently higher i n the phenobarbital-treated group as 97 TABLE 9. MEAN DISOPYRAMIDE SERUM CONCENTRATIONS (mcg/ml) AND UNBOUND FRACTION (fu) AS A FUNCTION OF TIME AND PHENOBARBITAL-TREATMENT Control Phenobarbital treated c2hr 2.22 ± 0.19b 1.86 ± 0.25 (fu2 h r) (0.23 ± 0.02) (0.21 ± 0.02) c3hr 2.26 ± 0.30 1.64 ± 0.28 (0.22 ± 0.03) (0.20 ± 0.02) c8hr 1.43 ± 0.11 0.89 ± 0.33 (0.16 ± 0.01) ( — )c a n = 7 b mean ± S.D. c the unbound levels were below the assay limit Note: A l l unbound levels were determined directly from dosed volunteer blood samples. • 9*8 TABLE 10. SERUM AAG CONCENTRATIONS (mg/100 ml): EFFECT OF PHENOBARBITAL—TREATMENT IN NON-SMOKERS Subject RS MK RH ML MC RK PR RM Phenobar-bital -Treatment day 0 41/603 30/44 — 48/70 — 58b 74 48 3 — 17/21 — — — — — 7 — 36/52 61/89 — 26/36 — — — 9 31/47 — — — — — — — 10 — 32/45 — 42/64 — — — — 13 — 36/55 — 45/70 — — — — 14 — — 35/51 — 31/45 — — — 15 30/46 — — — — — — — 19 35/51 — — — — — — — 21 — — 47/68 — 35/51 56 84 49 28 — — 23/27 — 28/40 — — — a ratio of serum AAG concentration measured by nephelometry to that of r a d i a l immunodiffusion technique. Serum AAG concentration as measured by nephelometry. 0 3 6 9 12 15 18 0 4 8 12 16 0 3 6 9 12 15 18 Time (hours) FIGURE 22. Disopyramide sa l i v a concentration versus time curves obtained i n non-smoking volunteers before ( O ) and ( A ) during phenobarbital-treatment. • C o n t r o l T ime (hours) FIGURE 23. MND s a l i v a concentration versuB time curves o f individual non-smoking volunteers before (•) and during (•) phenobarbital-treatment. 101 C O M P A R I S O N O F S A L I V A D I S A U C S CONTROL D PHENOBARB CN-1> CN-1> X • _J z \ LD D < < > »—i _J < 00 6 0 0 0 -i 5 4 0 0 -4 8 0 0 -4 2 0 0 -3 6 0 0 -3 0 0 0 -2 4 0 0 -1 8 0 0 -1 2 0 0 -6 0 D -• - W 1 m i i PR MC RM RK RH C O M P A R I S O N O F S A L I V A M N D A U C S CONTROL 0 PHENOBARB CN-1> CN-1> 2 7 0 0 -i 2 2 4 0 0 -• 2 1 0 0 -z N 1 8 0 0 -CNG 16 hr 1 5 0 0 -CNG 16 hr U o 1 2 0 0 -D < 9 0 0 -< > 6 0 0 --J < 3 0 0 -01 0 -1 i 1 i B 1 1 PR MC RM RK RH FIGURE 24. Comparison o f area under the s a l i v a concentration versus time curve for disopyramide (A) and MND (B) i n non-smoking volunteers before and during phenobarbital-treatment. 102 compared to untreated (control) group (Figure 24). The r a t i o of metabolite (MND) to parent drug was consistently higher i n the phenobarbital-treated subjects (Figure 25). It should be noted that a reasonable c o r r e l a t i o n exists between s a l i v a r y l e v e l s and unbound disopyramide serum concentration at selected time i n t e r v a l s (eg. 2,3 & 8 hr) (Figure 26). These data may be useful since i t may r e f l e c t drug concen-t r a t i o n at an e f f e c t o r s i t e . The c o r r e l a t i o n between t o t a l serum and s a l i v a disopyramide concentration was weak. Figure 27 shows the 48 hour cumulative urinary excre-t i o n of disopyramide and MND i n untreated and phenobarbital trea-ted subjects. The percentage of administered dose recovered as disopyramide was s i g n i f i c a n t l y decreased by phenobarbital t r e a t -ment (43 + 6 % versus 2 5 + 5 % ) . Conversely, the percen-tage of dose recovered as MND was s t a t i s t i c a l l y higher i n the phenobarbital-treated group (31 +5 %) as compared to the controls (25+6 %) (Table I I ) . Figure 28 summarizes the re l a t i o n s h i p of renal clearance (C1 R) of t o t a l disopyramide, urine flow rate and the urinary pH as a function of time in both untreated and phenobarbital-treated groups. There was no apparent difference i n any of these relationships between the two groups. The C1 R of t o t a l disopyramide declined as a function of time i n both groups. This pattern of decline was also supported by the presence of non-linear terminal phases of the logarithm of excretion rate (dXu/dt) and logarithm of amount remaining to be excreted (ARE) versus time curves. The decline 103 FIGURE 25. Saliva MND/disopyramide r a t i o s before (O ,n = 6) and a f t e r (• ,n = 6) phenobarbital-treatment. Y = 0.4 X + 200 100 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 700 8 0 0 9 0 0 1000 1100 1200 Disopyramide concentration in saliva (ng/mO FIGURE 26. The r e l a t i o n s h i p o f disopyramide concentration i n s a l i v a and unbound disopyramide concentration i n serum o f non-smoking volunteers (O : untreated, n = 8 and A: pheno-barbital-treatment, n = 8). The means + standard errors for Y intercept and slope are 200 + 28 and 0.4 + 0.05, respectively. 105 O DIS - C o n t r o l A DIS • P h e n o b a r b i t a l • M N D - C o n t r o l • M N D - P h e n o b a r b i t a l 12 24 T i m e (hours) 3 6 J 48 FIGURE 27. The percent o f dose recovered as disopyramide and MND i n 48 hour urine before ( O • , n - 8) and during phenobarbi-tal-treatment ( • • , n « 8) i n non-smoking volunteers. 106 T A B L E 11. F O R T Y - E I G H T H O U R C U M U L A T I V E U R I N A R Y E X C R E T I O N R E C O V E R Y O F D I S O P Y R A M I D E A N D M N D I N N O N - S M O K E R S DISOPYRAMIDE MND Subject Control Phenobarbital Control Phenobarbital (Untreated) -Treated (Untreated) -Treated •• RH 52 26 33 37 MC 36 21 24 34 MK 40 26 18 25 RS 46 30 27 31 RK 47 22 18 29 PR 39 32 28 25 RM 36 19 28 33 ML 48 32 22 46 Mean + S.D. 43 + 6 26 + 5* 25 + 5 33 + 7* Note : The values are t o t a l percentage of dose administered of disopyramide. Key * : S i g n i f i c a n t (p < 0.05) when compared to the respective control values. 107 R E N A L C L E A R A N C E V S TIME CB 0.1 -. c mJ .08 -• .06 -c 0 o _•> .04 -"5 #»• . 02 -w • o -C o n t r o l (n=8) P h e n o b a r b i t a l (n=8) 3 6 9 12 15 18 T ime (hours) 3 6 9 12 15 Time (hours) URINE F L O W R A T E V S TIME — 200 -3 6 9 12 15 18 T i m e (hours) 3 5 6 9 T i m e (hours) z a ^ o U R I N A R Y pH V S TIME 4 -2 " 4 -2 -3 6 9 12 15 T i m e (hours) 18 3 6 9 12 15 T i m e (hours) FIGURE 28. Renal clearance o f t o t a l disopyramide (upper), urine flow rate (middle) and urinary pH (lower) versus time before ( l e f t panel, n = 8) and during phenobarbital-treatment (right panel, n « 8) i n non-smoking volunteers. 108 of C I of t o t a l disopyramide was not associated with changes in urine flow rate or urinary pH, both which remained r e l a t i v e l y constant as a function of time in both phases (Figure 2 8 ) . 3.4 Phenobarbital Treatment i n Smokers; Pharmacokinetics and Binding Studies of Disopyramide and MND Figures 29 and 30 present the mean serum concentration of disopyramide and MND versus time curves for smokers during the untreated (control) and phenobarbital treatment phases. Table II summarizes the pharmacokinetic parameters which best f i t these data in a l l subjects. There was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e (p > 0.05) i n the time taken to reach t v of d i s o -pyramide between untreated and phenobarbital-treated phase. The Cp e a^ of disopyramide i n phenobarbital-treated smokers was found to be lower than that observed during the untreated phase. However, t h i s did not reach s t a t i s t i c a l s i g n i f i c a n c e . There was a s t a t i s t i c a l s i g n i f i c a n t difference (p < 0.05) i n the values of Kel, apparent elimination t ^ ^ ' A U C 0 - 3 2 hr a n < ^ C 1 T B ^ F between the two treatment phases. Phenobarbital treatment caused a reduction of AUCq_.j2 a n ^ t l / 2 ^ * a n c ^ ^ * respect-i v e l y , i n smokers. A 74 % increase in C 1 T B / F was observed i n the phenobarbital-treated smokers. There were no. s t a t i s t i c a l l y s i g n i f i c a n t differences i n the values of Vd /F between the 3 area two groups (Table 1 2 ) . The area under the serum MND concentration-time curve following phenobarbital treatment (3.9 + 2.0 hr. mcg/ml) was higher than i n the untreated group (3.5 + 1.4 hr. mcg/ml) 109 O DIS - C o n t r o l A DIS • P h e n o b a r b i t a l T i m e (hours) FIGURE 29. Disopyramide serum concentration versus time curves before ( O , n = 8) and during (• , n = 6) phenobarbital-treatment i n smokers. The disopyramide_was 200 mg of the base. The data are presented as X + S.D. The insert represents the semilogarithmic plot of the total serum disopyramide concentration vs time data. 110 O M N D - C o n t r o l • M N D - P h e n o b a r b i t a l 1.0 r 1 c o §0.5H c o E D l_ 0) z 4.0 (-3.0 -2.0 -1.0 -0.5 -0.2 0.1 0.05 8 8 0 011 1111 I I I I I I I I I L_ 0 2 4 6 8 1012 16 20 24 28 32 Time (hours) 16 24 T i m e (hours) FIGURE 3 0 . MND serum c o n c e n t r a t i o n versus time curves b e f o r e (O, n = 8) and during ( • , n = 6) phenobarbital- _ treatment i n smokers. The data are presented as X + S.D. The insert represents the semi logarithmic plot of the total serum MND concentration vs_ time data I l l TABLE 12. PHARMACOKINETIC PARAMETERS OF DISOPYRAMIDE IN SMOKERS Subject tpeak (hr) Cpeak (mcg/ml )"> (hr-l)c K e 1 i A tl/2 (hr)e AUC (hr. mcg/ml )f (l/hr/kg)9 vd area (l/kg)n Control a «1 1 1.0 1.3 2.6 2.6 0.11 6.3 24.4 0.094 0.86 2 1.0 1.2 3.1 2.7 0.12 5.9 23.8 0.115 0.98 3 1.0 1.2 3.5 2.9 0.10 6.9 33.4 0.069 0.69 4 1.0 1.5 2.2 1.9 0.13 5.4 19.6 0.189 1.48 5 1.5 2.1 2.0 1.5 0.08 8.3 21.4 0.118 1.43 6 3.0 2.0 2.8 1.5 0.09 7.9 28.6 0.113 1.28 7 1.5 1.7 1.9 1.8 0.10 6.7 25.4 0.121 1.18 8 2.0 1.7 2.0 1.7 0.12 5.8 21.0 0.120 1.00 Average -* S.D. 1.5 1.6 2.5 2.1 0.11 6.7 24.7 0.117 1.11 (n-B) ± 0.7 t 0.4 t 0.6 t 0.6 i 0.02 t 1.0 ± 4.5 ± 0.034 ± 0.27 -treated a •1 1 1.0 1.2 1.9 2.5 0.14 5.0 16.9 0.135 0.97 2 1.0 1.2 2.0 2.4 0.19 3.7 14.2 0.194 1.04 3 1.5 1.9 2.3 1.6 0.11 6.6 26.6 0.087 0.83 4 1.0 1.3 1.9 2.2 0.17 4.2 13.0 0.286 1.73 5 1.5 1.1 1.7 3.0 0.14 5.1 13.8 0.183 1.34 6 2.0 1.9 2.2 1.2 0.18 4.0 13.7 0.235 1.35 Average ± S.D. 1-3 1.4 2.0 2.1 0.15» 4.7* 16.4* 0.187* 1.20 (n-6) t 0.4 t 0.4 ± 0.2 * 0.7 t 0.03 ± 1.0 * 5.2 ± 0.071 1 0.33 Dose « 200 mg orally a estimated from serum cone, vs time curve •1 tpeak " (Z-303/ka-kel) logTTa/kel) b estimated from serum cone vs time curve c from NONLIN computer program d e ti/2 » 0.693/Kel f AUCo-32hr from linear trapezoidal rule g C1TB/F " Dose/AUCo-32 hr f> VD/F - ClTB/Kel * Statistically significant from the respective control value (p < 0.05) but t h i s difference did not reach s t a t i s t i c a l s i g n i f i c a n c e . The mean *-±l2 °f M N D during the untreated phase of the study (6.4 hours) was not d i f f e r e n t from that following phenobarbital t r e a t -ment (6.5 hours). The serum concentrations of AAG in healthy smokers before and after 21 days of phenobarbital treatment are shown in Table 13. Figure 31 shows the 48 hour cumulative urinary excretion of disopyramide and MND before and aft e r phenobarbital treatment i n smokers. The urinary excretion data are consistent with the serum data. The percentage of administered dose recovered as disopyramide was s i g n i f i c a n t l y decreased during phenobarbital treatment (50+8 % versus 30+ 5 %) (Table 14). Conversely, the percentage of dose recovered i n smokers as MND was s i g n i f i c a n t l y increased during phenobarbital treatment (34+_ 14 %) as compared to the period before treatment in smokers (22+6 % ) . Table 15 permits comparison between the pharmacokinetic parameters for disopyramide in smokers and non-smokers, before and during phenobarbital treatment s i t u a t i o n . There appears to be no difference (p > 0.05) i n the disopyramide pharmacokinetics between the smokers and non-smokers, while phenobarbital treatment uniformly caused an increase i n disopyramide clearance in both study groups (Table 15). 113 T A B L E 13. A A G C O N C E N T R A T I O N S (mg/100 ml): E F F E C T O F P H E N O B A R B I T A L — T R E A T M E N T I N S M O K E R S Control Phenobarbital Subject (Untreated) -Treatment (22 days) AB 65 64 JO 78 86 IT 81 77 SD 43 46 JK 56 67 SM 58 60 MC 54 a DF 65 — Mean .+ S.D. : 63 .+ 13 67 +_ 14* Note : Serum AAG concentration as measured by r a d i a l immunodiffusion technique. Key a : Volunteers were not available for t h i s part of the study. * : Not d i f f e r e n t (p > 0.05) from the control (untreated) value. > 114 7 0 6 0 5 0 4 0 3 0 2 0 10 O DIS - C o n t r o l • DIS - P h e n o b a r b i t a l • M N D - C o n t r o l • M N D - P h e n o b a r b i t a l 8 16 2 4 3 2 T i m e (hours) 4 0 4 8 FIGURE 31. The percent o f dose recovered as disopyramide and MND it\ 48 hour urine before (O , • , n « 8) and during phenobarb-ital-treatment (• , • , n • 6) i n smoking volunteers. 115 T A B L E 1 4 . F O R T Y - E I G H T H O U R C U M U L A T I V E U R I N A R Y E X C R E T I O N R E C O V E R Y O F D I S O P Y R A M I D E A N D M N D I N S M O K E R S DISOPYRAMIDE MND Subject Control Phenobarbital Control Phenobarbital (Untreated) -Treated (Untreated) -Treated • AB 54 40 25 29 JO 44 29 23 35 IT 66 48 10 17 SD 43 19 27 59 JK 45 19 15 31 SM 46 22 24 34 DF 50 a 26 MC 48 23 Mean + S.D. 50 + 8 30 + 12* 22 + 6 34 + 14* Note : The values are t o t a l percentage of dose administered of disopyramide. Key a : Volunteers were not ava i l a b l e for t h i s part of the study. * : S i g n i f i c a n t (p < 0.05) when compared to the respective control values. TABLE 15. COMPARISON OF PHARMACOKINETIC PARAMETERS OF TOTAL SERUM CONCENTRATION OF DISOPYRAMIDE BETWEEN NON-SMOKERS AND SMOKERS IN THE ABSENCE AND PRESENCE OF PHENOBARBITAL NON SMOKERS SMOKERS PHARMACOKINETIC CONTROL PHENOBARBITAL CONTROL PHENOBARBITAL PARAMETER (mean (n=8) + SD) (mean (n-8) + SD) (mean (n-8) SD) (mean (n=6) + SD) tpeak (hr) 2.0 + 0.6 1.6 + 0.5* 1.6 + 0.4 1.4 + 0.4 Cpeak (mcg/ml) 2.8 + 0.5 2.1 + 0.6 2.5 + 0.6 2.0 + 0.2 ka (hr'1) 1.6 + 0.7 2.0 + 0.9 2.1 + 0.6 2.1 + 0.7 kel (hr"1) 0.11 + 0.02 0.16 + 0.02* 0.11 + 0.02 0.15 + 0.03* ti/2 (hr) 6.5 + 1.5 ' 4.6 + 0.7* 6.7 + 1.0 4.7 + 1.0* AUC0-32hr % (hr.mcg/ml) 27.4 + 6.0 17.2 + 6.2* 24.7 4.5 16.4 + 5.2* (1/Sr/kg) 0.10 + 0.02 0.17 + 0.06* 0.12 + 0.03 0.18 + 0.07* Vdarea/F (lAg) 0.91 + 0.13 1.09 + 0.3 1.11 + 0.27 1.20 + 0.33 * Statistically significant from the respective control value (p < 0.05). 117 4 DISCUSSION The a p p l i c a t i o n of c a p i l l a r y column GLC i n the analysis of drugs and metabolites i n b i o l o g i c a l samples has p r o l i f e r a t e d i n the recent l i t e r a t u r e . This technique has been applied i n metabolic p r o f i l i n g of normal and diseased conditions (Jennings, 1980, Freeman, 1981, and Novotny, 1981), the measurement of drug concentration in human urine and plasma (Guerret, 1980, Novotny, 1980, and Jochemsen and Breimer, 1981), and in pharmacokinetic drug studies i n man (Van den heuvel and Zweig, 1980). With the advent of i n e r t f u s e d - s i l i c a c a p i l l a r y columns i n 1979, more r e l i a b l e and reproducible analysis of individual drugs ( a c i d i c , basic, neutral, polar) in complex mixtures became possible (Plotczyk, 1982). Before a pharmacokinetic and protein-binding study of disopyramide in untreated and phenobarbital-treated non-smokers and smokers could be undertaken, an a n a l y t i c a l procedure with high s e l e c t i v i t y and s e n s i t i v i t y was required to permit measure-ment of drug and metabolite i n buffer (dialysate), plasma and serum. 4.1 Development of A n a l y t i c a l Method 4.1.1 C a p i l l a r y GLC-NPD A 25 m (i.d.= 0.31 mm) polysiloxane-deactivated, open-tubular f u s e d - s i l i c a column with a crosslinked 5 % phenyl methyl s i l i c o n e phase was used for the development of a s e l e c t i v e and sensitive assay for disopyramide and MND. 118 Complete baseline resolution between disopyramide and MND was obtained as i l l u s t r a t e d i n Figure 12B and no interference from endogenous plasma components was observed (Figure 12A) following a simple single extraction step. The chromatographic peaks fo r disopyramide and MND were well separated (resolved) under i d e n t i c a l GLC conditions from several antiarrhythmic drugs, v i z . , tocainide, mono-ethyl glycine x y l i d i d e , lidocaine, propranolol and quinidine (Figure 13). The analysis time of the present assay i s r e l a t i v e l y short and r e p l i c a t e injections of plasma extracts every 14 min are possible without interference from plasma components. 4.1.2 Optimization of TFAA-Treatment The present work has demonstrated that the k i n e t i c s of dehydration of the amide functional group of disopyr-amide, PC-Dis and MND, and acetylation of the amine functional group of MND by TFAA i s r a p i d . This i s based on the f a c t that no apparent differences were observed i n the peak areas of TFAA -treated toluene extracts of these substances over the e n t i r e incubation time range of 0 to 2 hours (Figure 14). A 30 min reaction time was subsequently chosen to ensure completeness of the reactions. The hydrolysis of excess d e r i v a t i z i n g reagent (heptafluorobutyric anhydride) and subsequent n e u t r a l i z a t i o n with an excess of ammonia has been reported to be a better technique (Tam and Axelson, 1978). However, the use of a small volume of toluene (0.1 ml) for r e c o n s t i t u t i o n of dried sample extracts d i d not permit an e f f i c i e n t separation of the organic layer from the 119 aqueous layer. Therefore, excess TFAA reagent was removed by evaporation rather than by an hydrolysis technique (Walle and Ehrsson, 1970). Evaporation of the incubation mixture was carr i e d out under a gentle steam of nitrogen, using a water bath at 40°C to prevent evaporation of the v o l a t i l e TFAA deriva-t i v e s . The TFAA-treated samples were found to be stable for at least 48 hours at room temperature and at least 10 days when stored at -4°C with repeat injections showing no s i g n i f i c a n t decline i n peak areas over the stored time period. 4.1.3 Extraction E f f i c i e n c y of Toluene Toluene was found to be an e f f i c i e n t extrac-t i o n solvent (Table 6) with a recovery of 95 % and 87 % for diso-pyramide and MND respectively, over the ent i r e concentration range of disopyramide (0.50-5 mcg/ml) and MND (0.30-3 mcg/ml). A single extraction step was s u f f i c i e n t for the quantitation of the compounds of i n t e r e s t without any interference from plasma constituents. 4.1.4 I d e n t i f i c a t i o n of Untreated and TFAA-Treated  Compounds by MS H u t s e l l and Stachelski i n 1975 and subsequent-l y other workers (Duchateau and Hollander, 1979 and Bredesen, 1980) have reported that the mono-N-dealkylated metabolite of disopyramide, MND, r e s u l t s i n three unresolved chromatographic peaks when injected i n t o a g a s - l i q u i d chromatograph equipped with a 3 % OV-17 packed glass column. Unfortunately, no report has appeared i n the l i t e r a t u r e regarding the i d e n t i t y of these peaks. 120 A similar pattern of at least three peaks was observed i n our laboratory when MND was injected into a GC-MS equipped with a 12 m (i.d.= 0.27 mm) f u s e d - s i l i c a column coated with methyl s i l i c o n e (Figure 16). Previous studies have shown that acetylation of the secondary amino group of MND with acetic anhydride results i n a single chromatographic peak (Hutsell and Stachelski, 1975, Duchateau and Hollander, 1979 and Bredesen, 1980). However, recent findings i n our laboratory have shown two l i m i t a t i o n s to t h i s approach: (1) a c e t y l a t i o n of the secondary amino group with acetic anhydride i s not complete under usual conditions (Figure 9) and (2) acetylated MND i s associated with extensive peak t a i l -ing (Figure 9) on columns conventionally used i n disopyramide analysis, v i z . , 3 % OV-17 and 5 % phenyl methyl s i l i c o n e . T r i f l u o r o a c e t i c anhydride (TFAA) i s a commonly used acylating agent which has been reported to dehydrate a l i p h a t i c amide groups to form n i t r i l e groups (Gerardin, Abadie and Laffont, 1975). Gal et a l . (1980) used TFAA as a dehydrating agent to accomplish such a conversion with disopyramide to improve i t s chromatographic properties. The EI-MS fragmentation of both disopyramide and PC-Dis i n the present study followed a pattern s i m i l a r to that reported by previous workers (Gal et  a l . , 1980, Haskins et a l . , 1980). The dehydrated derivatives ( n i t r i l e s ) obtained upon TFAA-treatment of disopyramide and PC-Dis exhibited shorter retention times than the corresponding untreated amides. The prominent fragment ions for the n i t r i l e 121 derivative of disopyramide were at m/e 194, 221, 128, 114, 193, 32, 44, 72, 70, 195 i n order of decreasing i n t e n s i t y (for d e t a i l s , r e f e r to APPENDIX). The fragment ions at m/e 128, 114, 72, and 42 were also present in the EI-MS p r o f i l e of the n i t r i l e d e r i v a t i v e of PC-Dis. In addition, the l a t t e r contained ion pairs m/e 255-257, 228-230 and 229-231 i n 3:1 r a t i o , thus i n d i r e c t l y supporting the postulated structures reported for fragment ions of the n i t r i l e derivative of disopyramide by Gal et a l . , 1980. Further evidence for dehydration of disopyramide and PC-Dis was obtained from the presence of pseudo-molecular ions (M+l) + i n the CI-MS mode (Figures 39 and 41, respectively, APPENDIX section). In addition to the molecular ions, character-i s t i c ions of (M+29)+ and (M+41)+ were also found when meth-ane was used as an ionizing gas. Following the i n j e c t i o n of untreated MND into the GC-MS system, the t o t a l ion current chromatogram (Figure 9) showed three unresolved MND degradation products from 5.5 to 5.8 min, i n addition to the peak for authentic MND at 6.5 min. It i s proposed that untreated MND i s unstable when exposed to high temperatures encountered i n the i n j e c t i o n port of the gas chromatograph. A comparison of the EI and CI spectra (Figures 15 and 16) of the three peaks from 5.5 to 5.8 min, a l l show i d e n t i c a l spectra. It appears that at le a s t one of the degradation products may be a c y c l i z e d compound (Figure 32) which could a r i s e from MND through a loss of ammonia. 122 m w 297 (\ /Y XCH2CH2K U CH(CH3)2 M O N O - N - D E A L K Y L A T E D D I S O P Y R A M I D E heat — NH3 mw280 i / • CH,-CH2 cyclized product FIGURE 32. Postulated degradation reaction o f mono-N-dealXylated metabolite of disopyramide (MND). 123 This proposed structure i s based largely on the presence of a strong molecular ion at m/e 280 i n the EI spectrum and a corres-ponding pseudomolecular ion of m/e 281 in the CI spectrum. Confirmation of the c y c l i c structure requires further i n v e s t i -gation. The fragment ions of MND i n the EI mode (Figure 15) were similar to those reported by Haskins et a l . , 1980. The CI-MS p r o f i l e (Figure 16B) showing a pseudomolecular ion at m/e 298 confirms the i d e n t i t y of the MND chromatographic peak i n Figure 16A. Preliminary studies i n our laboratory showed the presence of two peaks for TFAA (0.1 ml)-treated MND. The EI and CI-MS data (Figures 17 and 18A) indicated that the peak having the shortest retention time was the dehydrated form of the MND TFAA der i v a t i v e ; whereas, the l a t e r peak was that of the TFAA derivative of MND with the i n t a c t amide group. The EI and CI-MS data of the amide are shown in Figures 17 and 18, respectively . Excess TFAA (0.15 ml), as used i n the present assay, completely dehydrated the amide functional group of MND along with the complete acylation of the secondary amine group of MND. Structures for the c h a r a c t e r i s t i c fragment ions i n the EI mass spectra of the two peaks are shown in Figure 33A and 33B, res-p e c t i v e l y . Some of the ions present i n the d e r i v a t i z e d form of MND such as m/e 195, 194, 212 and 167 are also common to underi-v a t i z e d disopyramide (Gal et a l . , 1980, Haskins et a l . , 1980) or underivatized MND (Haskins et a l . , 1980). 124 C = N m/e 375 COCF, O 7 A Peakl Y / " ( C H O , ^ J ) - C - C M 1 C H I N ( o < / = \ 1^  C M » C=N m/e 221 <^J>-C—CM, m/e 185 9. T 0 - ] -c h' C=N m/e 207 CH m/0 194 base ion CH, COCF, m/e 1B2 * CH, CH< CH, m/e 43 _ V N - C H Q - C = C H m/e 193 B Peak 2 o - T O T ,N' N O H \ O C F , I -C II c / \ H,N OH m/e 393 m/e 212 9. c II c II H,N* m/e 1B5 9 T _,. O f - " - --c II c II NH m/e 104 ( X . m/e 195 ' C H , : C H CONH, m/e 44 CH, COCF, m/e 182 base ion • /CH(CH>), CH, = N<^ COCF, m/e 166 • CH, CH< CH, m/e 43 C H I C H m/e 69 m/e 103 m/e 1M H / X H m/e 167 FIGURE 33. Proposed prominent fragment ions based on the EI-MS o f TFAA-treated MND (A) dehydrated ( n i t r i l e and t r i f l u o r o -acetylated MND and (B) t r i f l u o r o a c e t y l a t e d MND (amide form). 125 4.1.5 A p p l i c a b i l i t y of the C a p i l l a r y GLC-NPD  Assay Method The present a n a l y t i c a l technique has been found to be sens i t i v e , s p e c i f i c and reproducible. I t has been shown to be applicable i n the simultaneous quantitation of diso-pyramide and MND i n human serum, s a l i v a and urine. L i n e a r i t y was observed i n the range of 0.05 to 5.0 mcg/ml for disopyramide and 0.02 to 3.0 mcg/ml for MND. These samples have been charact-erized by r e p l i c a t e c a l i b r a t i o n curves having a c o e f f i c i e n t of determination (r ) of at lea s t 0.98. The method i s highly r e l i a b l e with an average c o e f f i c i e n t of v a r i a t i o n of 5 and 8 % for disopyramide and MND, respectively, over the enti r e concen-t r a t i o n range studied. The use of the s p l i t l e s s mode of sample i n j e c t i o n , c a p i l l a r y columns and nitrogen/phosphorus s p e c i f i c detection provided enhanced s e n s i t i v i t y and s p e c i f i c i t y for the simultaneous quantitation of disopyramide and MND i n human b i o l o g i c a l f l u i d s . The present method was used for subsequent protein-binding, pharmacokinetic and enzyme induction studies involving quantitation of disopyramide and MND. 126 4'2 In V i t r o Disopyramide Plasma Protein-Binding The sensitive c a p i l l a r y GLC-NPD assay permitted the d i r e c t measurement of unbound f r a c t i o n (fu) at plasma concentrations as low as 0.5 mcg/ml, u t i l i z i n g only 0.4 ml of sample. This presents a s i g n i f i c a n t improvement over the previously reported HPLC and packed column GLC methods. Multiple l i q u i d sample i n j e c t i o n s have been made possible and furthermore, we have established that the present method can be automated; thus, economy of labor can also be achieved. The v a r i a b i l i t y of the unbound f r a c t i o n r e s u l t s between d i f f e r e n t c e l l s at i d e n t i c a l disopyramide concentrations (Table 7) necessitated d i a l y s i s to be carried out i n t r i p l i c a t e for each sample. This i s possible with the present method as the size of plasma sample required per d i a l y s i s i s small (0.4 ml). As l i t e r a t u r e data would suggest, the unbound f r a c t i o n (fu) increased with increasing disopyramide plasma concentration (Table 7). The s t a t i s t i c a l analysis of the protein-binding data further substantiates the concentration-dependent plasma protein-binding of disopyramide. For example, there i s a s i g n i f i -cant difference (p < 0.05) i n fu between 2 and 3 mcg/ml and a highly s i g n i f i c a n t difference (p < 0.01) i n fu between 5 and 10 mcg/ml (Table 7). The plasma AAG levels were variable among the healthy volunteers (Table 7) and t h i s may, i n part, contribute to the i n t e r - i n d i v i d u a l binding differences at i d e n t i c a l disopyr-amide plasma concentrations. The method reported i n t h i s text can provide s i g n i f i c a n t advantages i n s e n s i t i v i t y and s p e c i f i c i t y 127 over the previously reported methods for the estimation of unbound disopyramide concentrations in plasma. 4.3 Phenobarbital-Treatment i n Non-Smokerst Pharmacokinetics and Binding Studies of Disopyramide  and MND Antiarrhythmic drugs such as quinidine (Data et a l . , 1976), lidocaine (Harrison and Alderman, 1971), and tocainide (Meffin et a l . , 1977) are known to exhibit steep plasma concentration-antiarrhythmic response curves. A few studies (Niarchos, 1976, Robert et a l . , 1978 and A i t i o , 1981) have demonstrated that small changes in serum concentration of disopyramide may be accompanied by large changes i n antiarrhyth-mic e f f e c t . Therefore, a drug-drug interaction (for example, enzyme induction) leading to an a l t e r a t i o n of i n t e n s i t y and duration of drug action may be c l i n i c a l l y important for disopyr-amide. Induction of disopyramide metabolism by phenobarbital i n the rat ( A i t i o et a l . , 1979) and i n man by rifampin ( A i t i o , 1981 and A i t i o , 1981), phenytoin ( A i t i o and Vuorenmaa 1980 and A i t i o , 1981), carbamazepine ( A i t i o , 1981) and spironolactone ( A i t i o , 1981), has been reported previously. As indicated i n the INTRODUCTION, the e f f e c t of phenobarbital treatment on disopyr-amide elimination i n man could not be adequately predicted on the basis of the r e s u l t s of studies involving other inducers. This may be due to differences i n the s e l e c t i v i t y i n enzyme induction by these substances as well as, possible differences in the capacity of the inducers to a l t e r serum protein-binding. Even phenytoin which most c l o s e l y approximates the e f f e c t of 128 phenobarbital, reportedly causes a decrease i n the AUC for disopyramide and a s i g n i f i c a n t increase i n AUC of MND. We observed only a minimal a l t e r a t i o n i n AUC of MND i n our present study. Phenobarbital has been known to induce metabolism of a number of drugs i n man (Conney, 1967). Our study design incor-porated a 100 mg d a i l y o r a l dose of phenobarbital for 21 days, a regimen previously reported to s a t i s f a c t o r i l y induce hepatic enzymes (Corn, 1966; C u c i n e l l et a l . , 1965 and E l v i n et  a l . , 1980). The serum phenobarbital lev e l s before the administration of the second dose of disopyramide were >10 mcg/ml in a l l volunteers. The aforementioned phenobarbital treatment regimen did not apparently a l t e r the blood pressure or the EKG in the smoking and non-smoking volunteers. 4.3.1 Pharmacokinetics of Disopyramide and MND;  Serum Data The pharmacokinetics of disopyramide in both control and phenobarbital-treated subjects were adequately described by a one-compartment open model with apparent (pseudo) f i r s t - o r d e r absorption and elimination. The res u l t s of t h i s modelling are summarized i n Table 8. The C . of disopyramide observed during the control phase of these studies was larger than that during phenobarbital-treatment. This may be due i n part to a modest (\i 10%) increase i n f i r s t - p a s s metabolism suggested by the observed increase i n or a l clearance (Table 8). The decrease i n the AUC for disopyramide observed i n the phenobarbital-treated subjects indicates (Table 8) that i n order 129 frequency would be required. The f r a c t i o n of the disopyramide dose recovered as disopyramide plus MND (corrected for mass balance) i n urine was not s i g n i f i c a n t l y altered by phenobarbital treatment. Furthermore, the pattern of urinary excretion of drug and metabolite i s as would be expected with metabolic induction. Therefore, i t appears most l i k e l y that increased hepatic c l e a r -ance i s responsible for the s u b s t a n ^ a l decrease i n AUC of the parent drug. The serum MND l e v e l s were highly variable (Figure 21) and there was no s i g n i f i c a n t difference i n the AUC_ ~ A , of'MND between phenobarbital treatment (4.1 + 2.3 0-24 hr — hr.mcg/ml) and the control period (3.8 + 1.6 hr.mcg/ml). The apparent h a l f - l i f e of MND did not s i g n i f i c a n t l y d i f f e r between the two groups (6.7 hr before treatment vs 6.4 hr a f t e r phenobarbital treatment) suggesting that phenobarbital induction had only a modest e f f e c t on the elimination of MND. 4.3.2 Saliva Data Lima (1979) as well as A i t i o et a l . (1980) reported a wide inter-subject v a r i a t i o n i n s a l i v a r y l e v e l s of disopyramide and MND, whereas Lima's study (Lima, 1979) involved only one subject with disopyramide measurement alone. It i s apparent from our data (Figures 22 and 23) that the s a l i v a concentration-time p r o f i l e s of disopyramide and MND are v a r i a b l e . The s a l i v a r y pH was found to be r e l a t i v e l y constant and the s a l i v a r y l e v e l s of disopyramide and MND were approximately 20-30 % of those observed i n serum. Although no pharmacokinetic 130 constants could be calculated r e l i a b l y from the s a l i v a , an in d i c a t i o n of enzyme induction was obtained from s a l i v a data by comparing MND/Disopyramide r a t i o s (Figure 25) and A U Co-24hr* The average AUC for s a l i v a r y disopyramide concentration during the control phase (3.8 + 1.5 hr.mcg/ml) of the study was larger than that observed during phenobarbital treatment (2.1 + 0.5 hr.mcg/ml, p < 0.05) (Figure 24). Conversely, the average s a l i v a r y AUC of MND was smaller during untreated experiments (1.5 + 0.4 hr.mcg/ml) than during phenobarbital treatment (1. 8 +_ 0.8 hr.mcg/ml, p > 0.05) (Figure 24). Ther fore, based on the two aforementioned parameters, the data suggest that s a l i v a c o l l e c t i o n (a non-invasive technique) can be u t i l i z e d i n the assessment of c e r t a i n drug-drug metabolic i n t e r -action studies. 4.3.3 Urinary Excretion Data The urinary excretion data were consistent with the r e s u l t s from serum and s a l i v a . During the control phase, the percent recovery of disopyramide was s t a t i s t i c a l l y higher (43 + 6 %) than during phenobarbital treatment (26 + 5 %, Table 11). Conversely, the percentage of dose recovered as MND was greater ( 3 3 + 7 %) i n the phenobarbital-treeted group than i n the control group (25 + 5 % ) . These r e s u l t s combined with serum and s a l i v a data strongly suggest that disopyramide metabolism to MND i s inducible by phenobarbital i n man. Phenobarbital did not change the renal clearance pattern disopyramide (Cl_) (Figure 28). The decline i n C1 R of 131 disopyramide, as a function of time, i n both groups was s i m i l a r to that observed i n healthy volunteers by previous workers (Cunningham et a l . , 1977; A i t i o , 1982 and Giacomini et a l . , 1982), and there was no i n d i c a t i o n that eit h e r urine flow rate or urinary pH was involved in the decline of C1 R of disopyramide. The decline may be explained in part by the concentration-dependent change i n the f r a c t i o n of unbound disopyramide i n the serum (Cunningham et a l . , 1977; Giacomini et a l . , 1982). 4.3.4 Binding Results The e f f e c t of phenobarbital induction on serum disopyramide unbound f r a c t i o n (Table 7) and AAG concentration (Table 10) was n e g l i g i b l e when contrasted with the previously published e f f e c t s of phenobarbital on serum AAG i n dogs (Bai and Abramson, 1982) and phenytoin i n humans (Routledge et a l . , 1981 and Broguerolle, Jadot 1984). This i s the f i r s t study i n humans to s o l e l y evaluate the e f f e c t s of phenobarbital on AAG plasma concentrations, since previous studies involved combination drug therapy (Routledge et a l . , 1981 and Bruguerolle and Jadot, 1984) i n patients with seizure disorders. In the 7 subjects who had both the 2 and 3 hr blood samples on each study day, the mean unbound f r a c t i o n was 0.22 + 0.02 on the control day and 0.21 + 0.02 during phenobarbital induction (p > 0.05). Even t h i s modest decrease i n unbound f r a c t i o n may not be secondary to changes related to the binding capacity of serum AAG i n induced subjects, since the unbound f r a c t i o n of disopyramide i s highly concentration-dependent (Giacomini et a l . , 1982), and the 132 induced subjects generally exhibited plasma concentrations at 2-3 hr post-dose which were 10-20 % less than those observed i n control subjects (Figure 20). Similarly, the serum AAG on the control study day and during chronic phenobarbital-treatment (21 days) were almost i d e n t i c a l (Table 10). Clearly, at the pheno-b a r b i t a l dose used in t h i s study, no recognizable trend or i d e n t i f i a b l e e f f e c t could be observed i n the binding. 4.3.5 C l i n i c a l Implications of Phenobarbital  Induced Disopyramide Metabolism Our study provides compelling evidence that disopyramide metabolism i n human i s susceptible to induction of hepatic enzymes by phenobarbital. Since there i s a r e l a t i v e l y narrow range between therapeutic, subtherapeutic and toxic concentrations of disopyramide (Niarchos, 1976; Robert et a l . , 1978, and A i t i o , 1981), the pharmacokinetic changes are of a magnitude that may be important c l i n i c a l l y . Therefore, a new regimen may be required in the induced state. Moreover, the increased l e v e l s of MND have to be taken into consideration. The status of MND as an active and/or toxic metabolite i s yet to be f u l l y e s t a b l i s h e d i n man. Therefore, caution should be exercised in the concurrent use of disopyramide and enzyme inducers i n patients undergoing treatment for arrhythmias. 133 4.4 Phenobarbital-Treatment i n Smokers: Pharmacokinetics and Binding Studies of Disopyramide and MND Phenobarbital and cigarette smoking are both known to induce drug metabolism (Jusko, 1978 and 1979; Dawson and Vestal, 1982 and Luczynska and Wilson, 1983). The enhanced metabolism of disopyramide by the c l a s s i c a l enzyme inducer, phenobarbital, i n non-smokers has been discussed i n a previous communication. In humans, there appears to be a ce r t a i n degree of s e l e c t i v i t y i n the enhancement of hepatic drug oxidation rates influenced by cigarette smoking. Many drugs such as phenacetin, antipyrine theophylline, imipramine, propranolol and lidocaine are known to exhibit higher hepatic clearances i n smokers, although other drugs with s i m i l a r metabolic pathways such as diazepam, phenytoin, pethidine and n o r t r i p t y l i n e are unaffected (Jusko, 1978 and 1979). Extensive evidence documenting the existence of multiple forms of human cytochrome P-450 have appeared in the l i t e r a t u r e . For example, long term cigarette smoking causes s i g n i f i c a n t elevations i n the high a f f i n i t y component of 7-ethoxycoumarin O-deethylation i n man (Boobis and Davies, 1983); on the other hand, treatment with anticonvulsant drugs increases the microsomal cytochrome P-450 content and the low a f f i n i t y component of 7-ethoxycoumarin O-deethylation. Theophylline seems to be a common substrate for cytochrome P-450 (Landay et a l . , 1978), and cytochrome P-448 (Grygiel and B i r k e t t , 1981), with higher a f f i n i t y for the l a t t e r . Based on the available reports, there i s no r e l i a b l e method for predicting the metabolic induction of a p a r t i c u l a r drug that undergoes hepatic microsomal 134 oxidation in smokers. 4.4.1 Pharmacokinetics of Disopyramide and MND In Smokers: Serum Data The pharmacokinetics of a single dose of disopyramide i n chronic smokers, before and during phenobarbital treatment, were adequately described by a simple one-compartment open model with apparent f i r s t - o r d e r absorption and elimination. The r e s u l t s obtained from t h i s modelling are summarized i n Table 12. The C p e a k °f disopyramide observed during the untreated phase of these studies was larger than that following phenobar-treatment. This may be, i n part, due to a modest (\x!0 %) increase in the f i r s t - p a s s metabolism suggested by the observed increase i n apparent o r a l clearance (Table 12). The decrease i n the AUCQ_22^ r» *~\/2 a n < ^ concomitant inc r e a s e i n apparent o r a l clearance for disopyramide a f t e r phenobarbital treatment i n smokers indicates (Table 12) that to maintain a constant disopyr-amide concentration i n any i n d i v i d u a l , i t i s l i k e l y that a 35 % increment i n either dose or dosing frequency would be required. The serum MND l e v e l s were highly variable, the average AUC^ of MND was higher during phenobarbital treatment (3.9 0-24hr +_ 2.0 hr.mcg/ml) than during the c o n t r o l l e d p e r i o d (3.5 + 1.4 hr.mcg/ml). The apparent mean elimination h a l f - l i f e ( t ^ ^ ^ °^ MND was not a l t e r e d by treatment with the inducer suggesting that phenobarbital induction had only a modest e f f e c t on the elimina-t i o n of MND i n the smoking population, l i k e that observed i n non-smokers. 135 4.4.2 Urinary Excretion Data i n Smokers The urinary excretion of disopyramide and MND excretion between the two experiments was consistent with the serum data. The percentage of dose recovered as disopyramide was s i g n i f i c a n t l y lower a f t e r phenobarbital treatment (30 + 5 %) as compared to the untreated phase (50 + 8 % ) . Conversely, the percentage of dose recovered as MND was greater (34 + 14 %) during phenobarbital treatment than during pretreatment (22 + 6 % ) . Phenobarbital did not change the concentration-dependent renal clearance of disopyramide. These observations are consistent with the hypothesis that disopyramide metabolism to MND i s inducible by phenobarbital i n smoking volunteers. 136 4.4.3 Serum Protein-Binding of Disopyramide and  Phenobarbital Treatment i n Smokers The serum AAG level s i n untreated smokers (63 + 13 mg/100 ml) were not d i f f e r e n t (Table 13, p > 0.05) from phenobarbital-treated smokers (67 + 14 mg/100 ml). These results are consistent with those of non-smokers (Table 10). Also, the li m i t e d r e s u l t s of the r a d i a l immunodiffusion technique (RID) showed that the serum AAG concentrations i n untreated smo-kers (63 + 13 mg/100 ml) (Table 13, n = 8) were not d i f f e r e n t (p > 0.05) from the AAG level s i n age-matched untreated non-smokers ( 5 8 + 1 3 mg/100 ml) (Table 10, n = 3). These findings are i n agreement with those of Wolf et a l . (n = 132, 1982). In contrast, Benedek et a l . (1984) found that the serum AAG l e v e l s were higher (p < 0.05) i n smokers (84 + 13 mg/100 ml, n = 10) as compared to the non-smokers (63 + 13 mg/100 ml, n = 10). Further investigations are required to evaluate the al t e r a t i o n s i n serum AAG concentrations of chronic cigarette smoking volunteers and the c l i n i c a l implications of such fl u c t u a t i o n s . 137 4.4.4 Phenobarbital-Induced Disopyramide Pharmacokinetics; Non-Smokers Versus Smokers I f one compares the pharmacokinetic r e s u l t s of the phenobarbital induction of disopyramide i n smokers to non -smokers (Table 15), i t i s apparent that the pharmacokinetics of disopyramide i s unaffected by chronic cigarette smoking. The extent of phenobarbital-induced enhancement of disopyramide metabolism i s of the same order of magnitude i n chronic cigarette smokers as i n non-smokers. In conclusion, t h i s study provides evidence that the disopyramide metabolism i s susceptible to phenobarbital—induced induction and the hepatic microsomal metabolism of disopyramide i n man i s mediated primarily by cytochrome P-450. The metabolic bio-transformation of disopyr-amide remains unaffected by chronic cigarette smoking. Our study also supports the general view (Jusko, 1978 and 1979; Dawson and Vestal, 1982, and Luczynska and Wilson, 1983) that the induction of hepatic microsomal oxidation of a p a r t i c u l a r drug i s highly s e l e c t i v e in humans and i s a function of substrate or drug s p e c i f i c i t y for a p a r t i c u l a r form of cytochrome P-450. 138 5 SUMMARY AND CONCLUSIONS A method for the simultaneous quantitation of low concen-trations of disopyramide and i t s mono-N-dealkylated metabolite MND) in human b i o l o g i c a l f l u i d s has been obtained, employing a c a p i l l a r y g a s - l i q u i d chromatographic nitrogen/phosphorus selec-t i v e detection (GLC-NPD) technique. The u t i l i z a t i o n of high re s o l u t i o n c a p i l l a r y column GLC technique combined with s e l e c t i v e and sensitive NPD detection, permits measurement of disopyramide and MND i n the presence of various antiarrhythmic agents ( v i z . , tocainide, lidocaine, mono-ethyl glycine x y l i d i d e , propranolol and quinidine) without interference. L i n e a r i t y ranged from 0.05 to 5.0 mcg/ml and from 0.02 to 3.0 mcg/ml for disopyramide and MND, respectively. The average c o e f f i c i e n t of v a r i a t i o n was 5 and 8 % for disopyramide and MND, respectively, over the concen-t r a t i o n range studied. The s e n s i t i v e c a p i l l a r y GLC-NPD assay permits measurement of the unbound fractions of unlabelled disopyramide concentrations as low as 0.5 mcg/ml, u t i l i z i n g only 0.4 ml volumes of plasma, along with multiple and automatic sample i n j e c t i o n c a p a b i l i t y . The r e s u l t s of plasma protein-binding of disopyramide i n healthy volunteers, following equilibrium d i a l y s i s , indicates that the binding i s both, highly variable as well as concentration-depen-dent. The average i n v i t r o unbound fractions (fu) for t o t a l disopyramide concentrations of 0.5, 1, 2, 3, 4, 5 and 10 mcg/ml were 0.14, 0.15, 0.20, 0.28, 0.30, 0.35 and 0.55, respectively. These r e s u l t s are in agreement with e a r l i e r reports using 14 C-disopyramide. 139 The c a p i l l a r y GLC-NPD method was also employed i n the pharmacokinetic studies of disopyramide and MND, i n the absence and presence of phenobarbital i n healthy non-smoking and smoking volunteers. The e f f e c t of phenobarbital treatment on disopyramide d i s p o s i t i o n and protein-binding was studied i n eight male non-smokers because of the potential pharmacological and t o x i c o l o g i c a l significance of the major metabolite of disopyr-amide (MND) and the possible changes i n serum alphas-acid glycoprotein concentration a f t e r phenobarbital treatment. The apparent elimination h a l f - l i f e ( t ] ^ ^ o f d i s o P y r a m i d e w a s s i g n i f i c a n t l y shorter following phenobarbital treatment (4.6 + 0.7 vs 6.5 + 1.5 hr) and the area under the serum concentra-t i o n vs time curves (AUC) was also reduced (27 + 6 vs 17 + 6 hr.mcg/ml, p < 0.05). Phenobarbital treatment did not s i g n i f i c a n t l y a l t e r the AUC of MND (3.8 + 1.6 vs 4.1 + 2.3 hr.mcg/ml) and also did not change either serum AAG concentra-t i o n (50 + 15 vs 54 + 18 mg/100 ml) or disopyramide unbound f r a c t i o n (0.23 + 0.02 vs 0.21 + 0.02). The 48 hour urinary recoveries of disopyramide and MND were 4 3 + 6 and 26 + 5 %, respectively during the control period, while 2 5 + 5 and 33 +_ 7 % were recovered following phenobarbital treatment. Our results show that phenobarbital a l t e r s disopyramide pharmacokinetics without changing AAG. The e f f e c t of chronic cigarette smoking and the combined e f f e c t of smoking and phenobarbital treatment on the disopyramide pharmacokinetics and serum AAG concentrations were also i n v e s t i -gated. There was no difference, i n either the pharmacokinetic 140 parameters or the serum AAG concentrations between the smoking and non-smoking volunteers, when studied and compared i n the absence and presence of phenobarbital treatment. Therefore, i t appears that the metabolic bio-transformation of disopyramide remains unaffected by chronic cigarette smoking. 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Therapeutic Drug Monitoring 6: 192-198 (1984). 161 7 APPENDIX A thorough gas-liquid chromatographic (GLC) electron impact (EI) as well as chemical i o n i z a t i o n (CI) mass spectrometric (MS) study of untreated and t r i f l u o r o a c e t i c anhydride-(TFAA) treated disopyramide, mono-N-dealkylated metabolite of disopyramide (MND) and p-chlorodisopyramide (PC-Dis) was carried out for the struc-t u r a l confirmation of these compounds and t h e i r derivatives. 7.1 GLC-EI-MS Conditions A computerized gas chromatographic-electron impact mass spectrometer H-P Model 5970A, equipped with a 12 m ( i . d . = 0.27 mm) f u s e d - s i l i c a column coated with crosslinked methylsilicone f l u i d and a mass—selective detector was used to study the frag-mentation pattern of untreated and TFAA-treated toluene sample solutions of disopyramide, MND and PC-Dis. The following s p l i t -l e s s , c a p i l l a r y GLC conditions were used: oven temperature 1, 225°C; time 1, 0.5 min; programming rate, 15°C/min; oven temperature 2, 250°C; time 2, 2.0 min; i n j e c t i o n port tempera-ture 250°C; helium ( c a r r i e r gas) flow rate, 1 ml/min. For the mass spectrometer, the i o n i z i n g beam energy was 70 electron v o l t s , the electron m u l t i p l i e r voltage, 1400 v o l t s and the i n t e r -face temperature was 280°C. 7.2 GLC-CI-MS Conditions An H-P Model 5987A gas chromatograph-mass spectrometer was used to determine the molecular ion of the parent untreated comp-ounds, as well as, TFAA-treated compounds. Samples were in j e c t e d c 162 using the s p l i t l e s s mode and a li n e a r scanning method was used. The instrument was equipped with a 12 m ( i . d . = 0.27 mm) cap-i l l a r y f u s e d - s i l i c a column coated with crosslinked methylsilicone f l u i d . Methane was used as an ionizing gas and the following mass spectrometric conditions were used: i n j e c t i o n port temp-erat u r e , 250°C; i n t e r f a c e oven temperature, 275°C, GLC i n t e r -face probe temperature, 275°C; ion source temperature, 100°C; oven temperature 1, 100°C; programming rate 30°C/min; oven temperature 2, 260°C; m u l t i p l i e r voltage ?000 v o l t s and emiss-ion current, 300 micro ampere. The following pages represent the GLC-EI-MS and GLC-CI-MS results of untreated and TFAA-treated toluene samples of disopy-ramide, MND and PC-Dis. The prominent fragment ions on the basis of GLC-EI-MS of these compounds are also shown in the subsequent pages. 163 TOTAL I OH CURRENT MASS CHROMATOGRAM OF DISOPYRAMIDE TOTAL ABUNDANCE FROM 30 TO 500 amu Fu l l Scale- 11543 i i i . i i i j a .nn " I U I f / U I . Ii' UIII I • <i i i i i • i i i ELECTRON IMPACT MASS SPECTRUM OF DISOPYRAMIDE Base Peak - 195.10 Base Peak Abundance • I. Am I i j l 1 It I ..•,!» I B J 200 250 264 Total Abundance 176 300 50 100 150 PROMINENT FRAGMENT IONS : MASS <«^e> : 195 212 194 114 167 72 196 128 43 239 ABUNDANCES): 106 69 48 36 31 17 16 16 16 12 350 400 FIGURE 34. Total ion current mass chromatogram (A) and EI-MS (B) of disopyramide. 164 TOTAL ION CURRENT MASS CHROMATOGRAM OF DISOPVRAMIDE-TFAA TOTAL ABUNDANCE FROM 30 TO 500 amu. F u l l Scale- 11543 i i i i i i i i i ELECTRON IMPACT MASS SPECTRUM OF DISOPVRAMIDE-TFAA Base Peak - 194.10 Base Peak Abundance - 254 Total Abundance i J.AUI.'j. •.•!.]. 1907 50 100 150 200 250 300 350 400 PROMINENT FRAGMENT IONS : MASS<:«/e>: 194 221 128 114 193 43 44 72 78 195 ABUNDANCES): 108 83 80 41 30 26 24 19 19 15 F I G U R E 3 5 . Total ion current mass chromatogram (A) and EI-MS (B) of TFAA-treated disopyramide. 165 p-CHLORODISOPVRAMIDE t TOTAL ION CURRENT MASS CHROMATOGRAM OF ; TOTAL ABUNDANCE FROM 30 TO 500 ar.u j F u l l Scale- 11543 J i i i i i I * ii ii II II II » ii II II II n II II II ii <i n n n II II n u J I I I u ir in i « Ki. U .... a..* • - ... ..». J* «««l»». I" I I ELECTRON IMPACT MASS SPECTRUM OF P-CHLORODISOPVRAMIDE Base Peak - 229.10 Base Peak Abundance - 1480 Total Abundance -B 11 180 J A A I 50 100 150 200 250 300 350 400 PROMINENT FRAGMENT IONS : MASSU./e): 229 114 246 128 72 228 43 273 56 41 (231) (24S> (238) <275) ABUNDANCE< V.): 108 56 49 26 26 26 25 14 18 11 ( 3 5 ) (19) (8) <4.6) FIGURE 36. Total ion current mass chromatogram (A) and EI-MS (B) of PC-Dis. 166 TOTAL ION CURRENT MASS CHROMATOGRAM OF P-CHLORODISOPVRAMIDE-TFAA TOTAL ABUNDANCE FROM 30 TO 500 ana F u l l Scale= 11543 ELECTRON IMPACT MASS SPECTRUM OF P-CHLORODISOPVRAMIDE-TFAA ? 5^JL?_ealc - 255.10 Base Peak Abundance - 255 T o t a l Abundance -2222 B I it t i I 50 100 150 200 250 300 350 400 PROMINENT FRAGMENT IONS • • MASS(«/e>: 255 123 228 192 114 43 193 72 56 229 (257> (236) (231) ABUNDANCES): 100 94 91 42 38 23 23 21 16 13 (35> (30) <4> FIGURE 37. Total ion current mass chromatogram (A) and EI-MS (B) of TFAA-treated PC-Dis. 2 0 4 0 6 0 . „ * • ' 1 * • • * i.. .. f . 0 0 1 0 0 1 2 0 24eeeH 1 J 4 . t . i •. 11 • i . 1 1 . 1 1 1 . . . . i . i . . i . . i . i r . . . i . *., ! 4 0 160 111 11 111 11 4*«((it?-j i ' ' ' i ' . 1 i ' ; 1 i • : • i ' i — i 1 i 1 i 1 i * i * i * i — i • i ' i ! i 1 i 1 . 1 i — r i . : i .t 2 . 8 z . 4 z.& i.z s.t. 4.e « . 4 4 . & = . . 2 s.t 6 . 0 I F 1 i «• ' ai:ts9 fit 13927 |1 *«i 1 •I 4*4 I 2&H B M.H.: 339.5 ;i2 / 1 1 : 126 J 9 3 3 4 6 324 / 26? 297 <339+1) 4 .46 :«<6 k i n . • 1 1 1 260 256 i <M+29> CM+41) 380 4 * 9 4 3 9 4 9 E . / / \ 3ae S E E 4 » * i 4 5 0 he FIGURE 38. Total ion current mass chromatogram (A) and CI-MS (B) of disopyramide. 168 •»•? s» ire ite zee ?4e .1 i i I—> i t i .—• ; I i * i — ' 1 • • 1 ' * • 1 • ' • t t . , ; . . i i i i i i . . . I ^  • • i . • « I i . 3.00000-288*084 • 1 j! ./\_ 1 .H I r ; .8 — r ~ r - 7.8 • i ' <: .8 c - . e w£*4 32'-i .6-320 .8 i » u . •»* ctii i r e 2ee ;.'888-6-^ B 2. d 3.»S 4.8 5 . * 6.6 248 I i i i l • I : -.».- fly i iesz 1 r J i ! M.H.: 321 -4_ C M , C N , N ( J j C B N V * ?97 322, _L__l (339-18+1> <M+29> / 3 S 8 4 378 411 / ?6 l e * "lie 266 zse 388 3sa 4ee 4se . C a n i i . 6.44 > i n . - 3 0 •2E. [-28 I S 18 •e 446 478 FIGURE 3 9 . Total ion current mass chromatogram (A) and CI-MS (B) o f TFAA-treated disopyramide. 169 F l i t - ."-PB2& £ 0 . 0 - 5 0 0 . 0 a n u . 2 0 >»0 ee ee 1 0 0 1 2 0 1 4 0 1 6 0 ise -> - J — 1 — I — 1 — I — 1 — i — 1 — I — 1 — I — 1 — I — 1 I 1 I 1 1 1 I 1 I • ' • ' * l i t 1 ! 1 ' • 1 5 0 0 0 0 ^ 40000-^ 3 0 0 0 0 -i'0000-1 06 0 6 -1 — 1 — • • . • • • 1 • • • • • 1 • 1 • : ' 1 • . • 1 • 1 • 1 • 1 • 1 • 1 • 1 • 1 ' 1 • 1 ' 1 • 1 ' 1 ' 1 • 1 • 1 • 1 • T . 1 '. . 6 3 . 0 Z. 4 2 . t- 3.2 3 . 6 4 . 0 4 . 4 4 . 8 5 . 2 f. . 6 6 . 0 6 . 4 6 . 6 7 . 2 T i l e >fiK10 Bpi- Hb 1 5 8 2 2 1 0 0 n B 60-J M.W.: 373.5 V - c — c 6 c-OrH , C H , NW«,x 1 2 8 1 5 6 2 4 6 ! ! 1 2 1 2 y 3 7 4 S c a n 1 5 6 6 . 0 6 • i n . (373+1). ! 0 0 1 5 0 3 5 P y 381 3™ I <M+23) 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 FIGURE 4 0 . Total ion current mass chromatogram (A) and CI-MS (B) of PC-Dis. 170 F . i e nhve 5 0 . 0 - 5 0 0 . 0 * » u . 4 0 t i p 1 2 0 • ' • • ' ' ' • i 1 • • • 1 • • 1 • • * 1 1 1 1 * i i i i I i » i i i t * 2 2 4 e zee 3 2 B ' • • ' ' • • • ' • ' * 1 • • ' 1 • • • 1 *' • 1 3 0 0 0 0 - j 1 0 0 0 0 0 - i •i 0 8 0 8 6 -6 6 6 0 0 -4 0 0 0 0 -U I i I i • * — - • — i ' i 11 i » i; i • i 11 • 11 11 11 111 i • . i ) • • • i 111 • • 11 • • • i • i (_ 3.0 3.0 * . 0 5.8 6 . 0 7.8 S . 0 ? . 0 1 8 . 8 1 1 . 0 f; : t wB0t 3 5 0 . 8 - 3 6 0 . 0 a » u . 4 0 6 0 1 3 0 160 2 8 0 2 4 0 2 6 0 3 2 * —.—; . . . i . . . i • • . i i ... i ... i . . . i . . . i . . . i . . . i • • i . . . i . . . i - • i B i • i • • • 1 • • • ' I • • • • i » • • • : • • • • | • ' 5 . 0 6 . 0 7 . 0 8 . 0 9 . 0 1 0 . 0 1 1 . 0 . 0 3.8 4 . 0 rr -e -41:06 T-Z'V. 3D 46649 4 8-! J M.W.: 355 3 6 b 7(373-18+1> Sc a n 1 7 8 + 6 . 7 5 t i n . ( C M , ) , («4 (M+29> (M+41) 14 1 f 8 , 5 6 1 9 5 2 2 8 2 E & S * f , 2 0 I ( / l 2 4 4 4 4 7 4 9 ! -38 -20 -10 1 8 0 1 5 0 2 0 8 25 0 3 8 0 3 5 0 4 9 0 4 5 0 FIGURE 41. Total ion current mass chromatogram (A) and CI-MS (B) of TFAA-treated PC-Dis 171 F r a g m e n t s f o u n d in t h e e l e c t r o n i m p a c t m a s s s p e c t r u m o f D I S O P Y R A M I D E 7 Orc\ C CH— H2N O H < CH^ CH,), CH(CH,), m / e 339 0< CH CH, I -CH, HjN O m / e 2 3 9 C=N m / e 2 2 1 CH, CH, c H,N' SOH m / e 2 1 2 H,N 0 m / e 2 3 7 9> < Q - c — A H , CH II •CH m / e 195 b a s e i o n Q OH c II H,N + m / e 195 b a s e i o n CH, CHCCH,), CH, CH(CH,)2 m / e 128 CH, C^HtCH,), CH, H CH, I CH m / e 194 o-r c II c II NH m / e 194 m / e 86 CH CH^ CH,), m / e 114 CH, = N CHCCH,)2 H m / e 72 ' CH^ CH, m / e 167 CH, m / e 43 CONHH m / e 44 FIGURE 42. Prominent fragment ions based on the EI-MS o f disopyramide. 172 F r a g m e n t s f o u n d in t h e e l e c t r o n i m p a c t m a s s s p e c t r u m o f TFAA-treated DISOPYRAMIDE 0 7 V ^ ~ i ~ C H ' C H , N \ , , Us C H ( C H J . m / e 3 2 1 ^ C M i CH,x CHtCH,), 0<... m / e 1 9 5 m / e 1 9 4 b a s e i o n < G > - { —iH, /'' XCH(CH,) 1  C E N m / e 1 2 8 m / e 2 2 1 CH, + CHtCH,), CH, H m / e 8 6 CH, x CH(CH,), . i . . CH, = N. C = CH \ H m / e 7 2 ( • CH, ^ V N CH< , / \ I CH, C = C H m / e 1 6 7 m / e 4 3 m / e 1 9 3 FIGURE 43. Prominent fragment ions based on the EI-MS o f TFAA -treated disopyramide. 173 F r a g m e n t s f o u n d i n t h e e l e c t r o n i m p a c t m a s s s p e c t r u m o f p - C H L O R O D I S O P Y R A M I D E CI CI < * < C H , C C H , A . H,N O m/e 2 7 3 (& 2 7 5 ) 3 : 1 C H , "-OH* «-Q^  C=N m/e 2 5 5 (& 2 5 7 ) 3 :1 CI r c c H.N O H m/e 2 4 6 [> 2 4 8 ) 3 : 1 T I Y / C H( C H ,)« C C H — N ( H,N O H N C H ( C H , ) , m / e 3 7 3 . 5 C H , CI—(v A-C CH, m / e 2 2 9 b a s e i o n C i m / e 2 2 8 C H , I C H CI f c C H , ^ ^ C H l C H , ) , C H , C H ( C H , J 2 m / e 1 2 B C H , C H f C H . ) C H , H m / e 8 6 C H C H I C H , ) , C H ( C H , ) 2 m / e 1 1 4 CH(CH,) • / CH,= N^ H m / e 7 2 CI o ,7 H,N r c ,7 H / N H N m / e 2 0 1 (& 2 0 3 ) 3 1 m / e 2 2 9 ( & 2 3 1 ) NH 3 1 b a s e i o n m / e 2 2 8 (& 2 3 0 ) 3 :1 C O N H m / e 4 4 • C H , CH<^ C H , m / e 4 3 FIGURE 4 4 . Prominent fragment ions based on the EI-MS o f PC-Dis. 174 F r a g m e n t s f o u n d i n t h e e l e c t r o n i m p a c t m a s s s p e c t r u m o f TFAA-treated P-CHLORODISOPYRAMIDE 7 C I — ^ /f— C — C H , C H , N ^ C H ( C H , ) F C = N m / e 3 5 5 XH(CH,)F CH(CH,1 CH, " Y 1 CH,' XCH(CH , l CI (v />—C CH, V '/ | m / e 1 2 8 C=N m / e 2 5 5 (& 2 5 7 ) 3 :1 b a s e i o n CH, CH(CH,) C H , H ^ N ^ C H 86 CI—^\ A - C C H , m / e 2 2 9 ( & 2 3 1 ) 3 1 + / C H C C H , ) , C H . X H ( C H , ) T m / e 1 1 4 C . - ^ y - C = = C H \ C H J C H , = N ^ m / e 2 2 8 ( & 2 3 0 ) 3 : 1 m / e 4 3 m / e 7 2 FIGURE 45. Prominent fragment ions based on the EI-MS o f TFAA -treated PC-Dis. 175 F r a g m e n t s f o u n d i n t h e e l e c t r o n i m p a c t m a s s s p e c t r u m o f M O N O - N - D E A L K Y L A T E D M E T A B O L I T E m / e 1 9 5 b a s e i o n C H , C H f C H . ) I 'J» C H , H m / e 8 6 CHCCH,), C H , = N i \ H m / e 7 2 • C H , C H < ^ C H , m / e 4 3 C O N H , m / e 44 m / e 1 6 7 FIGURE 46. Prominent fragment ions based on the EI-MS of untreated MND. PUBLICATIONS Kapil, R.P. and Axelson, J.E. Plasma Protein Binding of Phenytoin in disease states, Pharmaceutical Society, Banaras Hindu University Publication (A Solicited Review Article). The Pharm. Student, 20: 8-12 (1980). Tarn, Y.K., Axelson, J.E., McErlane, B., Kapil, R.P., Riggs, W.K., Ongley, R. and Price, J.D.E. The Pharmacokinetics of Metoclopramide in Rats with Experimental Renal and Hepatic Dysfunction, J. Pharmacol. Exp. Therap., 219: 141-6 (1981). Kapil, R.P., Axelson, J.E-, Ongley, R. and Price, J.D.E., "Nonlinear Bioavailability of Metoclopramide in The Rat: Evidence for Saturable First-Pass Metabolism", J. Pharm. Sci., 73: 215-218 (1984). Kapil, R.P., Abbott, F.S., Kerr, C.R., Edwards, D.J., Lalka, D. and Axelson, J.E. "Simultaneous Quantitation of Disopyramide and it's Mono-N-dealkylated Metabolite in Human Plasma by Fused Silica Capillary Gas Chromatography using Nitrogen/Phosphorous Specific Detection", J. Chromatogr., 307: 305-321 (1984). 

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