Journal o f Pharmacokinetics and Biopharmaceutics, Vol. 3, No. 4, 1975
Pharmacokinetics in Man of the N-Acetylated Metabolite of Procainamide John M. Strong, 1 John S. Dutcher, 1 Woong-Ku Lee, 2 and Arthur J. Atkinson, Jr.1'3'4 Received Feb. 7, 1975--Fina1 Mar. 19, 1975
The pharmacokinetics of N-acetylprocainamide (NAPA) have been studied in three normal subjects who received 500 mg of this compound by timed intravenous injection. Plasma NAPA concentrations and urine excretion were measured by quadrupole mass fragmentography, and a three-compartment pharmacokinetic model was used for data analysis. NAPA elimination half-life and total distribution volume averaged 6.0 hr and 1.38 liters/kg, respectively. Renal excretion of unchanged NAPA accounted for 81% of its elimination, and the mean renal NAPA clearance was 179 ml/min. Approximately 2 % of the injected NAPA was deacetylated to procainamide. The fate was not determined of 17 % of the NAPA that was estimated to have been eliminated during the 16-hr study period. KEY WORDS: N-acetylprocainamide; procainamide; pharmacokinefics; drug metabolism; clinical pharmacology.
INTRODUCTION
N-Acetylated procainamide (NAPA) has been identified in urine as a major metabolite of procainamide (1,2), and is usually present in #g/ml concentrations in the plasma of patients on long-term procainamide therapy (3,4). NAPA appears to have antiarrhythmic potency similar to that of procainamide not only in in vitro preparations (5,6) and animal models (7,3) but also in man (3). So NAPA plasma levels, as well as those of the parent drug, need to be considered in treating patients with procainamide. 1Clinical Pharmacology Unit, Departments of Pharmacology and Medicine, Northwestern University Medical School, Chicago, Illinois. 2Cardiology Section, Division of Medicine, Veterans Administration Research Hospital and Department of Medicine Northwestern University Medical School Chicago, Illinois. Burroughs Wellcome Scholar m Chmcal Pharmacology. 4Address requests for reprints to Arthur J. Atkinson, Jr., M.D., 303 E. Superior Street, Chicago, Illinois 60611. 3
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223 9 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system,or transmitted, in any form or by any means,electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permissionof the publisher.
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In addition, it has been suggested that NAPA may have important advantages over procainamide that would warrant its further development as a new antiarrhythmic drug (7). Thus it appears that the elucidation of NAPA pharmacokinetics will be important either for the continued use of procainamide or for possible therapy with NAPA itself. In the present investigation, NAPA was injected intravenously in three normal subjects. Observations were made of their clinical response and of the kinetics of NAPA distribfition and elimination. Metabolites of this compound were also sought in urine collected from the subjects. METHODS Reference Compounds
The dipropyl analogue of procainamide, p-amino-N-(2-dipropylaminoethyl)benzamide hydrochloride (m.p. 188-190~ was supplied by Dr. Thomas Q. Spitzer ofE. R. Squibb & Sons, New Brunswick, N. J. Commercially available procainamide (Pronestyl) and this compound were reacted with acetyl bromide and recrystallized from ethyl acetate to synthesize NAPA Em.p. 138-140~ lit. 136.5-138~ (4) and 135-136.5~ (7)] and its dipropyl analogue, p-acetamido-N-(2-dipropylaminoethyl)benzamide (m.p. 155156~ This analogue was the internal standard for NAPA assay by mass fragmentography and was made up in a concentration of 5 gg/ml of water. The identity of these compounds was confirmed by mass spectrometry. Gas chromatography of the synthesized NAPA indicated that this material contained 0.8 % procainamide. p-Aminobenzoic acid (PABA) (m.p. 189-192~ was recrystallized from water. PABA and its glycine conjugate, p-aminohippuric acid (PAH) (m.p. 203-204~ were reacted with acetic anhydride and recrystallized from methanol-water to give p-acetamidobenzoic acid (PACBA) (m.p. 269-270~ and the acetylated analogue of PAH (dec. 232-234~ These were used as reference compounds in the search for possible metabolites of NAPA, and their synthesis was confirmed by mass spectrometry. Conduct of Experiments
The three senior investigators served as subjects for the experiments. The research protocol was reviewed by the institutional human subjects committee and written consent was obtained at the start of each study. The subjects were fasted overnight and hospitalized under continuous observation for the first 4hr of the study. NAPA was administered as a 2.5 % solution in saline over 5-6 min by timed intravenous injection. Pulse, blood pressure, and electrocardiogram were monitored for the first 30 min.
Pharmacokinetics in Man of the N-Acetylated Metabolite of Procainamide
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At intervals after NAPA injection, blood samples were drawn through a heparin lock for the pharmacokinetic investigations. During the 16-hr study period, urine was monitored for pH with Hydrion test paper within 5 min of voiding and collected for assay of NAPA and its metabolites. Plasma and urine were stored frozen at - 15~ until analyzed.
Assay and Identification Procedures Plasma concentrations of NAPA were measured by the technique of quadrupole mass fragmentography (8). Two milliliters of plasma were placed in a 15-ml glass-stoppered centrifuge tube along with 1 ml of the internal standard solution, 0.1 ml of 5 :< NaOH, and 5 ml of ethyl acetate. The mixture was shaken for 15 sec and centrifuged at 800g for 5 rain, then the ethyl acetate layer was removed and evaporated to dryness in a 10-ml pear-shaped flask. The residue was dissolved in 30/A of benzene and a 2-/A aliquot was injected into the injection port of the gas chromatograph-mass spectrometer. Mass fragmentography was done with a Finnigan model 3000 quadrupole gas chromatograph-mass spectrometer equipped with a model 240-01 automatic peak selector (Finnigan Instrument Corp., Sunnyvale, Calif.). The gas chromatograph was fitted with a small-bore glass column, 1.5 m by 1 mm (i.d.), packed with 3 % OV-17 liquid phase on a solid support of Chromosorb W-HP (Chemical Research Services, Addison, Ill.). The temperature of the injection port, column oven, and separator was 255~ and the transfer line was kept at 230~ The flow rate of helium carrier gas was 5 ml/min. The ionizing energy was 70 eV. The base peaks of the mass spectra of NAPA and the internal standard are role 86 and role 114, respectively, and m/e 120 is a fragment ion in the spectra of both compounds~ So the channels of the peak selector were set to monitor intensities of mass spectral ions at role 86, 114, and 120. The mass fragmentographic peaks from these ions were displayed on a four-pen recorder (model KA-40, Rikadenki, Tokyo), and their areas were simultaneously calculated by a laboratory data system interfaced to the peak selector (model 3352 B, Hewlett-Packard, Avondale, Pa.). The ratio of the areas of the NAPA role 86 to the internal standard role 114 peak was used to determine the plasma NAPA concentration from a standard curve.prepared by analyzing blank plasma samples to which known amounts of NAPA had been added. The ratios of the areas of the two ions chosen from each compound were used to exclude cochromatography. Replicate analysis of control plasma containing known concentrations of added NAPA in the range of 0.5-50/~g/ml indicated an analytical precision of 3% (SD). Only 1 ml of plasma was needed for analysis of samples containing more than 2 pg/ml NAPA.
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Urine samples were diluted and assayed for NAPA by the above method and for procainamide by a gas chromatographic procedure described previously (3). Urine samples also were analyzed for PABA and for its acetylated analogue PACBA. The analysis for PACBA was made by acidifying 2 ml of urine with 0.25 ml of 3 N HC1 and extracting this with two 5-ml portions of dichloromethane. The organic phase was evaporated to approximately 0.5 ml, reacted for 5 rain with an excess of diazomethane in ether, and then evaporated to dryness. The residue was dissolved in 25 #1 of benzene, and a 1-#1 aliquot was analyzed by gas chromatography for the methyl ester of PACBA. Urine was analyzed for PABA by acidifying a 2-ml aliquot with an equal amount of 0.3 M NaH2PO 4 buffer (pH 4.5) and extracting with 5 ml of diethyl ether. The organic phase was evaporated, dissolved in 0.25 ml of methyl alcohol, and reacted with diazomethane as above. The residue was dissolved in 50 #1 of ethyl acetate, and a 1-/~1aliquot was analyzed for the methyl ester of PABA by gas chromatography-mass spectrometry. Based on reports that PABA is also excreted in urine as glycine and glucuronide conjugates (9, 10), further attempts were made to identify these compounds and the analogous metabolites of PACBA. Urine samples were incubated with /~-glucuronidase and assayed for PABA and PACBA as above. Studies with reference PAH indicated that this compound could be converted to PABA by an initial acid hydrolysis step and that the acetylated analogue of PAH could be methylated with diazomethane and chromatographed as its methyl ester. Pharmacokinetic Analysis
Plasma NAPA concentrations and urinary NAPA output were analyzed with the SAAM 23 digital computer program for multicompartment analysis developed by Berman and Weiss (11) and implemented on a model 6400 Control Data Corporation computer. Koch-Weser and Klein have previously reported procainamide concentrations following a single intravenous dose of this drug (12), and the data read from their published graph were analyzed for comparison. A two-compartment model was tried initially for the pharmacokinetic analysis, but in each case there were systematic deviations of the least-squares regression lines from the data points. A threecompartment model gave more satisfactory results and a mammillary system was chosen arbitrarily. The comparative sums of squares for the computer fit of the data with the two- and with the three-compartment model were, respectively, 0.180 and 0.102 for subject 1, 0.713 and 0.090 for subject 2, 0.439 and 0.035 for subject 3, and 0.244 and 0.206 for the procainamide data. The parameters estimated from these analyses were used to calculate the results listed in Table I. The total apparent volumes of NAPA and
119.5
Procainamidc Subject 1a 68
1.76
1.27 1.24 1.64 1.38
2.9
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(ml/min)
7.60
2.87 2,89 3.52 3.09
(ml/min/kg)
Plasma clearance
"Results calculated from data of Koch-Weser and Klein (12). bRange of values reported by Koch-Weser and Klein (12).
105.6 67.2 126.3 99.7
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Elimination T~ (liters) (liters/kg) (hr)
83 54 77 71
NAPA Subject 1 Subject 2 Subject 3 Mean
Weight (kg)
(179-309) b
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(ml/min) 2.27 2.50 2.77 2.51
(40-54) ~
79 86 79 81
Percent renal (ml/min/kg) elimination
Renal clearance
Table I. Results of Pharmacokinetic Analyses
17.2
2,52 0.971 1.74 1.74
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4.73
0.924 0.582 1,22 0,909
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Strong, Dutcher, Lee, and Atkinson
procainamide distribution are the sums of the individual compartment volumes and correspond to Ve~s described by other investigators (13). Elimination clearances were determined from the product of the appropriate excretory rate constant and the central compartment volume. Percent renal NAPA elimination was calculated from the quotient of the renal excretory rate constant and the sum of the renal and nonrenal excretory rate constants. The permeability coefficients (14) or intercompartmental clearances (Q) (15) were estimated to facilitate comparison of the kinetics of NAPA and procainamide distribution into the rapid and slow tissue compartments. These clearances are calculated from the product of the intercompartmental transfer rate constant (k) designated by the subscripts and the appropriate compartment distribution volume (V) such that Q = k~jV~ = k j y j
and thus provide a volume-independent estimate of drug distribution analogous to the volume-independent estimate of elimination that is given by the calculation of drug clearance rather than half-life (15). RESULTS The intravenous injection of NAPA was well tolerated by the subjects. Subject 1, a 36-year-old male, noted no adverse effects during or after the injection of 500 mg NAPA, and his pulse and blood pressure were unchanged from the control values of 80/min and 140/80 mm Hg, respectively. Subject 2, a 34-year-old male, developed a sinus tachycardia of 120/min and noted palpitations during the third minute of a 468-mg NAPA injection. Blood pressure remained unchanged and the tachycardia subsided within 1 min of completing NAPA injection. Subject 3, a 37-year-old male, reported some blurring of vision during the fourth minute of a 440-mg NAPA injection but reported that this had cleared 20 rain later. He also developed a sinus tachycardia of 110/rain without change in blood pressure during the injection period. In no subject did PR, QRS, or QT intervals change from their values on the control electrocardiograms, but in subjects 2 and 3 the QT intervals did not shorten as much as expected for the degree of tachycardia. The kinetics of NAPA distribution and elimination in these subjects are shown in Figs. 1 and 2. The mean deviation of the observed plasma and urine data points from the calculated pharmacokinetic curves was 3.5 % for subject 1, 2.2~ for subject 2 and 2.5~o for subject 3. During the 16-hr study period, an estimated 86 % of the administered NAPA was eliminated by subject 1, 87 % by subject 2, and 85 % by subject 3. The results of the pharmacokinetic analyses are summarized in Table I. This table includes for
Pharmacokinetics in Man of the N-Acetylated Metabolite of Precainam~de
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Pharmacokinetics in Man of the N-Acetylated MetaboHte of Proc~inamide
231
comparison results of the analysis of previously reported procainamide concentration data shown in Fig. 3. Renal excretion of unchanged NAPA accounted for 79 % of the estimated 16-hr elimination of this drug in subject 1, 86% in subject 2, and 79% in subject 3. Deacetylation of NAPA to procainamide occurred to a minor extent, accounting for less than 5 % of total NAPA elimination in subject 1, 2.1%1; in subject 2, and 2.4% in subject 3. Urine pH was not controlled in any of these studies but averaged 4.4 (range 4-6) in subject 2 and 5.8 (range 5-7) in subject 3. Metabolism of NAPA to PABA or PACBA could not be demonstrated. The sensitivity of the methods used was checked by identifying reference PABA, PACBA, and the glycine conjugates of these compounds added to the urine samples in amounts equivalent to 1% of the measured NAPA concentration. Incubation of the urine samples with/?-glucuronidase also failed to demonstrate glucuronide conjugates of either PABA or PACBA. Thus of the NAPA that was estimated by pharmacokinetic analysis to have been eliminated in the 16-hr study period, 21~, 11.9~, and 18.6~ in subjects 1, 2, and 3, respectivebi, remained unaccounted for.
Discussion
Although half of the dose of procainamide administered to man is excreted unchanged (2,4,5,12) and only 10-23% is acetylated to NAPA (4,5,16), plasma levels of this metabolite are generally comparable to, and occasionally greater than, procainamide levels in patients treated with this drug (3,4). The present investigation indicates that this primarily results from the fact that the 6-hr elimination half-life of NAPA is almost twice the 3.5-hr average half-life reported in man for procainamide (12). This contrasts with previous observations in monkeys that the elimination half-lives of procainamide and NAPA are similar (2). The 1.38 liters/kg apparent volume of NAPA distribution is smaller than the 1.76 liters/kg volume calculated for procainamide with the same pharmacokinetic model and the 2.01 liters/kg volume reported for procainamide by Koch-Weser and Klein for normal subjects (12), although this latter volume represents Ve....... rather than Vess.The kinetics of distribution of both NAPA and procainamide after rapid intravenous injection appear to be best described by a three-compartment model. However, the intercompartmental clearances for procainamide are much greater than those for NAPA, as is reflected in the fact that the 88 distribution phase for procainamide is shorter than the 188hr required for NAPA after intravenous injection.
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Strong, Dutcher, Lee, and Atkinson
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Fig. 3. Pharmacokinetic analysis of plasma procainamide concentration data reported by Koch-Weser and Klein (12). Presentation of data is the same as in Fig. 2.
A striking finding has been the apparent bimodal distribution of ratios of NAPA to procainamide plasma concentrations measured several hours after a procainamide dose in different patients (3). Particularly high ratios have been reported in patients who were phenotypically rapid acetylators of isoniazid or who had renal disease (4). This latter observation is explained by the fact that impaired kidney function retards NAPA much more than procainamide elimination, since normally 82% of NAPA is excreted unchanged by the kidney, but only half of an administered procainamide dose is eliminated by this route (2,4,5,12). The actual renal clearance of NAPA may be slightly less than renal procainamide clearance, which ranges from 179 to 309 ml/min in subjects with normal cardiac output and renal function
Pharmacokinetics in Man of the N-Acetylated Metabolite of ProcaJnamide
233
(12). But in addition, the greater metabolism of procainamide accounts for its more rapid clearance from the body, as well as the fact that plasma procainamide levels tend to be elevated less than NAPA levels in patients with renal disease. The fate of approximately 17~o of the injected NAPA was not determined. A concerted effort was made to identify PABA, PACBA, and their glycine and glucuronide conjugates since it had been reported that 2-10~o of an administered procainamide dose is metabolized to PABA and conjugates of this compound (17). However, subsequent investigators have not found PABA and PACBA to be metabolites of procainamide in man (2). It is likely that the PABA measured in the initial studies was a metabonate s resulting from hydrolysis of procainamide and NAPA. We determined the efficiency of the extraction method described by these authors to remove interfering procainamide (t9) to be 98~o for procainamide and 50~0 for NAPA ; and the subsequent step of heating 5 ml of urine with 1 ml of 12 N HCI for 1 hr on a water bath is sufficient to convert these compounds in large part to PABA. NAPA has been proposed as a better therapeutic agent than procainamide on the grounds that it may be less toxic (7). If this is indeed so, the present investigation strengthens this proposal by demonstrating that only 2 ~o of an administered NAPA dose is deacetylated to procainamide in man, in accordance with what has been found previously in monkeys (2). In addition, the longer half-life of NAPA should make this drug more convenient to administer than procainamide, for which a 3-hr dosing interval has been recommended (12). In our investigations, we found that NAPA has local anesthetic activity when injected intracutaneously but that plasma concentrations more than 4 times higher than the probable threshold effective against ventricular premature beats (3) caused no hypotension and were well tolerated by the normal subjects who were studied. Nevertheless, this is no guarantee that comparable levels might not be toxic in patients with heart disease. The brief tachycardia that was noted during NAPA injection in subjects 2 and 3 may reflect the fact that neither of these individuals had previously been subjects in investigations of this type; subject 1, who had been a subject before, showed no tachycardia. Alternatively, this may reflect the positive chronotropic action of NAPA that has been noted in a rat atrial preparation exposed to high concentrations of this drug (20). In any event, these investigations should help lay the groundwork for direct investigation of NAPA antiarrhythmic efficacy in patients. 5M e t a b o n a t e has been defined by Beckett et al. (18) as a substance that is a product of metabolism
but not a true metabolite since it is formed by nonenzymatic processes in the biological system or during isolation or analytical procedures.
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ACKNOWLEDGMENTS T h e v a l u a b l e t e c h n i c a l a s s i s t a n c e o f M r . I h s a n E l e r is g r a t e f u l l y a c k n o w ledged. The authors appreciate the help of the nursing staff of the Coronary Intensive Care Unit of the Passavant Pavilion of Northwestern Memorial Hospital in the conduct of these studies.
REFERENCES 1. J. Dreyfuss, J. J. Ross, Jr., and E. C. Schreiber. Absorption, excretion and biotransformation of procainamide-C t4 in the dog and rhesus monkey. Arzneim. Forsch. 21:948-951 (1971). 2. J. Dreyfuss, J. T. Bigger, Jr., A. I. Cohn, and E. C. Schreiber. Metabolism of procainamide in rhesus monkey and man. Clin. Pharmacol. Ther. 13:366-371 (1972). 3. J. Elson, J. M. Strong, W.-K. Lee, and A. J. Atkinson, Jr. Antiarrhythmic potency of Nacetylprocainamide. Clin. Pharmacol. Ther. 17:134-140 (1975). 4. E. Karlsson, L. Molin, B. Norlander, and F. Sj6qvist. Acetylation of procaine amide in man studied with a new gas chromatographic method. Brit. J. Clin. Pharmacol. 1:467475 (1974). 5. E. Karlsson, G. Aberg, P. Collste, L. Molin, B. Norlander, and F. Sj6qvist. Acetylation of procainamide in man. A preliminary communication. Eur. J. Clin. Pharmacol. 8:79-81 (1975). 6. E. E. Bagwell, D. E. Drayer, M. M. Reidenberg, and J. K. Pruett. Effects of the N-acetyl metabolite of procainamide on transmembrane action potentials of canine His Purkinje cells. Clin. Res. 22:676A (1974). 7. D. E. Drayer, M. M. Reidenberg, and R. W. Sew. N-Acetylprocainamide: An active metabolite of procainamide. Proc. Soc. Exp. Biol. Med. 146:358-363 (1974). 8. J. M. Strong and A. J. Atkinson, Jr. Simultaneous measurement of plasma concentrations of lidocaine and its desethylated metabolite by mass fragmentography. Anal. Chem. 44: 2287-2290 (1972). 9. W. P. Deiss and P. P. Cohen. Studies in para-aminohippuric acid synthesis in the human: Its application as a liver function test. J. Clin. Invest. 29:1014-1020 (1950). 10. C. W. Tabor, M. V. Freeman, J. Baily, and P. K. Smith. Studies on the metabolism of para-aminobenzoic acid. 3. Pharmacol. Exp. Ther. 102:98-102 (1951). 11. M. Berman and M. F. Weiss, SAAM Manual, PHS Publication No. 1073, Government Printing Office, Washington, D.C., 1967. 12. J. Koch-Weser and S. W. Klein. Procainamide dosage schedules, plasma concentrations, and clinical effects. 3. Am. Med. Assoc. 215:1454-1460 (1971). 13. S. Riegelman, J. Loo, and M. Rowland. Concept of a volume of distribution and possible errors in the evaluation of this parameter. Y. Pharm. Sci. 57:128-133 (1968). 14. T. Teorell. Kinetics of distribution of substances administered to the body. I. The extravascular modes of administration. Arch. lnt. Pharrnacodyn. 57:205-225 (1937). 15. D. Perrier and M. Gibaldi. Clearance and biologic half-life as indices of intrinsic hepatic metabolism. J. Pharmacol. Exp. Ther. 191:17-24 (1974). 16. R. L. Galeazzi, T. Lockwood, L. B. Sheiner, and L. Z. Benet. Urinary excretion of procainamide. Clin. Res. 23:219A (1975). 17. L. C. Mark, H. J. Kayden, J. M. Steele, J. R. Cooper, I. Berlin, E. A. Rovenstine, and B. B. Brodie. The physiological disposition and cardiac effects of procaine amide. J. Pharmacol. Exp. Ther. 102:5-15 (1951). 18. A. H. Beckett, J. M. Van Dyk, H. H. Chissick, and J. W. Gorrod. Metabolic oxidation on aliphatic basic nitrogen atoms and their ~-carbon atoms--Some unifying principles. J. Pharm. Pharmacol. 23:809-812 (1971). 19. B. B. Brodie, P. A. Lief, and R. Poet. The fate of procaine in man following its intravenous administration and methods for the estimation of procaine and diethylaminoethanol. J. Pharmacol. Exp. Ther. 94: 359-366 (1948).
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20. H. Refsum, K. Frislid, P. K. M. Lunde, and K. H. Landmark. Effects of N-acetylprocainamide as compared with procainamide in isolated rat atria. Eur. J. Pharmacol. (in press, 1975).
Pharmacokinetics in Man of the N-Acetylated Metabolite of Procainamide John M. Strong, 1 John S. Dutcher, 1 Woong-Ku Lee, 2 and Arthur J. Atkinson, Jr.1'3'4 Received Feb. 7, 1975--Fina1 Mar. 19, 1975
The pharmacokinetics of N-acetylprocainamide (NAPA) have been studied in three normal subjects who received 500 mg of this compound by timed intravenous injection. Plasma NAPA concentrations and urine excretion were measured by quadrupole mass fragmentography, and a three-compartment pharmacokinetic model was used for data analysis. NAPA elimination half-life and total distribution volume averaged 6.0 hr and 1.38 liters/kg, respectively. Renal excretion of unchanged NAPA accounted for 81% of its elimination, and the mean renal NAPA clearance was 179 ml/min. Approximately 2 % of the injected NAPA was deacetylated to procainamide. The fate was not determined of 17 % of the NAPA that was estimated to have been eliminated during the 16-hr study period. KEY WORDS: N-acetylprocainamide; procainamide; pharmacokinefics; drug metabolism; clinical pharmacology.
INTRODUCTION
N-Acetylated procainamide (NAPA) has been identified in urine as a major metabolite of procainamide (1,2), and is usually present in #g/ml concentrations in the plasma of patients on long-term procainamide therapy (3,4). NAPA appears to have antiarrhythmic potency similar to that of procainamide not only in in vitro preparations (5,6) and animal models (7,3) but also in man (3). So NAPA plasma levels, as well as those of the parent drug, need to be considered in treating patients with procainamide. 1Clinical Pharmacology Unit, Departments of Pharmacology and Medicine, Northwestern University Medical School, Chicago, Illinois. 2Cardiology Section, Division of Medicine, Veterans Administration Research Hospital and Department of Medicine Northwestern University Medical School Chicago, Illinois. Burroughs Wellcome Scholar m Chmcal Pharmacology. 4Address requests for reprints to Arthur J. Atkinson, Jr., M.D., 303 E. Superior Street, Chicago, Illinois 60611. 3
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223 9 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system,or transmitted, in any form or by any means,electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permissionof the publisher.
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In addition, it has been suggested that NAPA may have important advantages over procainamide that would warrant its further development as a new antiarrhythmic drug (7). Thus it appears that the elucidation of NAPA pharmacokinetics will be important either for the continued use of procainamide or for possible therapy with NAPA itself. In the present investigation, NAPA was injected intravenously in three normal subjects. Observations were made of their clinical response and of the kinetics of NAPA distribfition and elimination. Metabolites of this compound were also sought in urine collected from the subjects. METHODS Reference Compounds
The dipropyl analogue of procainamide, p-amino-N-(2-dipropylaminoethyl)benzamide hydrochloride (m.p. 188-190~ was supplied by Dr. Thomas Q. Spitzer ofE. R. Squibb & Sons, New Brunswick, N. J. Commercially available procainamide (Pronestyl) and this compound were reacted with acetyl bromide and recrystallized from ethyl acetate to synthesize NAPA Em.p. 138-140~ lit. 136.5-138~ (4) and 135-136.5~ (7)] and its dipropyl analogue, p-acetamido-N-(2-dipropylaminoethyl)benzamide (m.p. 155156~ This analogue was the internal standard for NAPA assay by mass fragmentography and was made up in a concentration of 5 gg/ml of water. The identity of these compounds was confirmed by mass spectrometry. Gas chromatography of the synthesized NAPA indicated that this material contained 0.8 % procainamide. p-Aminobenzoic acid (PABA) (m.p. 189-192~ was recrystallized from water. PABA and its glycine conjugate, p-aminohippuric acid (PAH) (m.p. 203-204~ were reacted with acetic anhydride and recrystallized from methanol-water to give p-acetamidobenzoic acid (PACBA) (m.p. 269-270~ and the acetylated analogue of PAH (dec. 232-234~ These were used as reference compounds in the search for possible metabolites of NAPA, and their synthesis was confirmed by mass spectrometry. Conduct of Experiments
The three senior investigators served as subjects for the experiments. The research protocol was reviewed by the institutional human subjects committee and written consent was obtained at the start of each study. The subjects were fasted overnight and hospitalized under continuous observation for the first 4hr of the study. NAPA was administered as a 2.5 % solution in saline over 5-6 min by timed intravenous injection. Pulse, blood pressure, and electrocardiogram were monitored for the first 30 min.
Pharmacokinetics in Man of the N-Acetylated Metabolite of Procainamide
225
At intervals after NAPA injection, blood samples were drawn through a heparin lock for the pharmacokinetic investigations. During the 16-hr study period, urine was monitored for pH with Hydrion test paper within 5 min of voiding and collected for assay of NAPA and its metabolites. Plasma and urine were stored frozen at - 15~ until analyzed.
Assay and Identification Procedures Plasma concentrations of NAPA were measured by the technique of quadrupole mass fragmentography (8). Two milliliters of plasma were placed in a 15-ml glass-stoppered centrifuge tube along with 1 ml of the internal standard solution, 0.1 ml of 5 :< NaOH, and 5 ml of ethyl acetate. The mixture was shaken for 15 sec and centrifuged at 800g for 5 rain, then the ethyl acetate layer was removed and evaporated to dryness in a 10-ml pear-shaped flask. The residue was dissolved in 30/A of benzene and a 2-/A aliquot was injected into the injection port of the gas chromatograph-mass spectrometer. Mass fragmentography was done with a Finnigan model 3000 quadrupole gas chromatograph-mass spectrometer equipped with a model 240-01 automatic peak selector (Finnigan Instrument Corp., Sunnyvale, Calif.). The gas chromatograph was fitted with a small-bore glass column, 1.5 m by 1 mm (i.d.), packed with 3 % OV-17 liquid phase on a solid support of Chromosorb W-HP (Chemical Research Services, Addison, Ill.). The temperature of the injection port, column oven, and separator was 255~ and the transfer line was kept at 230~ The flow rate of helium carrier gas was 5 ml/min. The ionizing energy was 70 eV. The base peaks of the mass spectra of NAPA and the internal standard are role 86 and role 114, respectively, and m/e 120 is a fragment ion in the spectra of both compounds~ So the channels of the peak selector were set to monitor intensities of mass spectral ions at role 86, 114, and 120. The mass fragmentographic peaks from these ions were displayed on a four-pen recorder (model KA-40, Rikadenki, Tokyo), and their areas were simultaneously calculated by a laboratory data system interfaced to the peak selector (model 3352 B, Hewlett-Packard, Avondale, Pa.). The ratio of the areas of the NAPA role 86 to the internal standard role 114 peak was used to determine the plasma NAPA concentration from a standard curve.prepared by analyzing blank plasma samples to which known amounts of NAPA had been added. The ratios of the areas of the two ions chosen from each compound were used to exclude cochromatography. Replicate analysis of control plasma containing known concentrations of added NAPA in the range of 0.5-50/~g/ml indicated an analytical precision of 3% (SD). Only 1 ml of plasma was needed for analysis of samples containing more than 2 pg/ml NAPA.
226
Strong, Dutcher, Lee, and Atkinson
Urine samples were diluted and assayed for NAPA by the above method and for procainamide by a gas chromatographic procedure described previously (3). Urine samples also were analyzed for PABA and for its acetylated analogue PACBA. The analysis for PACBA was made by acidifying 2 ml of urine with 0.25 ml of 3 N HC1 and extracting this with two 5-ml portions of dichloromethane. The organic phase was evaporated to approximately 0.5 ml, reacted for 5 rain with an excess of diazomethane in ether, and then evaporated to dryness. The residue was dissolved in 25 #1 of benzene, and a 1-#1 aliquot was analyzed by gas chromatography for the methyl ester of PACBA. Urine was analyzed for PABA by acidifying a 2-ml aliquot with an equal amount of 0.3 M NaH2PO 4 buffer (pH 4.5) and extracting with 5 ml of diethyl ether. The organic phase was evaporated, dissolved in 0.25 ml of methyl alcohol, and reacted with diazomethane as above. The residue was dissolved in 50 #1 of ethyl acetate, and a 1-/~1aliquot was analyzed for the methyl ester of PABA by gas chromatography-mass spectrometry. Based on reports that PABA is also excreted in urine as glycine and glucuronide conjugates (9, 10), further attempts were made to identify these compounds and the analogous metabolites of PACBA. Urine samples were incubated with /~-glucuronidase and assayed for PABA and PACBA as above. Studies with reference PAH indicated that this compound could be converted to PABA by an initial acid hydrolysis step and that the acetylated analogue of PAH could be methylated with diazomethane and chromatographed as its methyl ester. Pharmacokinetic Analysis
Plasma NAPA concentrations and urinary NAPA output were analyzed with the SAAM 23 digital computer program for multicompartment analysis developed by Berman and Weiss (11) and implemented on a model 6400 Control Data Corporation computer. Koch-Weser and Klein have previously reported procainamide concentrations following a single intravenous dose of this drug (12), and the data read from their published graph were analyzed for comparison. A two-compartment model was tried initially for the pharmacokinetic analysis, but in each case there were systematic deviations of the least-squares regression lines from the data points. A threecompartment model gave more satisfactory results and a mammillary system was chosen arbitrarily. The comparative sums of squares for the computer fit of the data with the two- and with the three-compartment model were, respectively, 0.180 and 0.102 for subject 1, 0.713 and 0.090 for subject 2, 0.439 and 0.035 for subject 3, and 0.244 and 0.206 for the procainamide data. The parameters estimated from these analyses were used to calculate the results listed in Table I. The total apparent volumes of NAPA and
119.5
Procainamidc Subject 1a 68
1.76
1.27 1.24 1.64 1.38
2.9
6.0 5.8 6.2 6.0 517
238 156 271 222
(ml/min)
7.60
2.87 2,89 3.52 3.09
(ml/min/kg)
Plasma clearance
"Results calculated from data of Koch-Weser and Klein (12). bRange of values reported by Koch-Weser and Klein (12).
105.6 67.2 126.3 99.7
Vd~~
Elimination T~ (liters) (liters/kg) (hr)
83 54 77 71
NAPA Subject 1 Subject 2 Subject 3 Mean
Weight (kg)
(179-309) b
188 135 213 179
(ml/min) 2.27 2.50 2.77 2.51
(40-54) ~
79 86 79 81
Percent renal (ml/min/kg) elimination
Renal clearance
Table I. Results of Pharmacokinetic Analyses
17.2
2,52 0.971 1.74 1.74
Qf~, (liters/rain)
4.73
0.924 0.582 1,22 0,909
Q~low (liters/rain)
5"
~
o
,7
5"
5"
228
Strong, Dutcher, Lee, and Atkinson
procainamide distribution are the sums of the individual compartment volumes and correspond to Ve~s described by other investigators (13). Elimination clearances were determined from the product of the appropriate excretory rate constant and the central compartment volume. Percent renal NAPA elimination was calculated from the quotient of the renal excretory rate constant and the sum of the renal and nonrenal excretory rate constants. The permeability coefficients (14) or intercompartmental clearances (Q) (15) were estimated to facilitate comparison of the kinetics of NAPA and procainamide distribution into the rapid and slow tissue compartments. These clearances are calculated from the product of the intercompartmental transfer rate constant (k) designated by the subscripts and the appropriate compartment distribution volume (V) such that Q = k~jV~ = k j y j
and thus provide a volume-independent estimate of drug distribution analogous to the volume-independent estimate of elimination that is given by the calculation of drug clearance rather than half-life (15). RESULTS The intravenous injection of NAPA was well tolerated by the subjects. Subject 1, a 36-year-old male, noted no adverse effects during or after the injection of 500 mg NAPA, and his pulse and blood pressure were unchanged from the control values of 80/min and 140/80 mm Hg, respectively. Subject 2, a 34-year-old male, developed a sinus tachycardia of 120/min and noted palpitations during the third minute of a 468-mg NAPA injection. Blood pressure remained unchanged and the tachycardia subsided within 1 min of completing NAPA injection. Subject 3, a 37-year-old male, reported some blurring of vision during the fourth minute of a 440-mg NAPA injection but reported that this had cleared 20 rain later. He also developed a sinus tachycardia of 110/rain without change in blood pressure during the injection period. In no subject did PR, QRS, or QT intervals change from their values on the control electrocardiograms, but in subjects 2 and 3 the QT intervals did not shorten as much as expected for the degree of tachycardia. The kinetics of NAPA distribution and elimination in these subjects are shown in Figs. 1 and 2. The mean deviation of the observed plasma and urine data points from the calculated pharmacokinetic curves was 3.5 % for subject 1, 2.2~ for subject 2 and 2.5~o for subject 3. During the 16-hr study period, an estimated 86 % of the administered NAPA was eliminated by subject 1, 87 % by subject 2, and 85 % by subject 3. The results of the pharmacokinetic analyses are summarized in Table I. This table includes for
Pharmacokinetics in Man of the N-Acetylated Metabolite of Precainam~de
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Fig, 2, Presentation of NAPA pharmacokinetics in the first 4 hr after injection expanded from Fig. 1 to show the estimated N A P A concentrations in the fast and slow peripheral compartments of the model (dashed lines). The distribution and excretion microconstants (given in reciprocal minutes) and distribution volumes (given in liters and, in parentheses, liters/kg) of the pharmacokinetic model are also included.
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Pharmacokinetics in Man of the N-Acetylated MetaboHte of Proc~inamide
231
comparison results of the analysis of previously reported procainamide concentration data shown in Fig. 3. Renal excretion of unchanged NAPA accounted for 79 % of the estimated 16-hr elimination of this drug in subject 1, 86% in subject 2, and 79% in subject 3. Deacetylation of NAPA to procainamide occurred to a minor extent, accounting for less than 5 % of total NAPA elimination in subject 1, 2.1%1; in subject 2, and 2.4% in subject 3. Urine pH was not controlled in any of these studies but averaged 4.4 (range 4-6) in subject 2 and 5.8 (range 5-7) in subject 3. Metabolism of NAPA to PABA or PACBA could not be demonstrated. The sensitivity of the methods used was checked by identifying reference PABA, PACBA, and the glycine conjugates of these compounds added to the urine samples in amounts equivalent to 1% of the measured NAPA concentration. Incubation of the urine samples with/?-glucuronidase also failed to demonstrate glucuronide conjugates of either PABA or PACBA. Thus of the NAPA that was estimated by pharmacokinetic analysis to have been eliminated in the 16-hr study period, 21~, 11.9~, and 18.6~ in subjects 1, 2, and 3, respectivebi, remained unaccounted for.
Discussion
Although half of the dose of procainamide administered to man is excreted unchanged (2,4,5,12) and only 10-23% is acetylated to NAPA (4,5,16), plasma levels of this metabolite are generally comparable to, and occasionally greater than, procainamide levels in patients treated with this drug (3,4). The present investigation indicates that this primarily results from the fact that the 6-hr elimination half-life of NAPA is almost twice the 3.5-hr average half-life reported in man for procainamide (12). This contrasts with previous observations in monkeys that the elimination half-lives of procainamide and NAPA are similar (2). The 1.38 liters/kg apparent volume of NAPA distribution is smaller than the 1.76 liters/kg volume calculated for procainamide with the same pharmacokinetic model and the 2.01 liters/kg volume reported for procainamide by Koch-Weser and Klein for normal subjects (12), although this latter volume represents Ve....... rather than Vess.The kinetics of distribution of both NAPA and procainamide after rapid intravenous injection appear to be best described by a three-compartment model. However, the intercompartmental clearances for procainamide are much greater than those for NAPA, as is reflected in the fact that the 88 distribution phase for procainamide is shorter than the 188hr required for NAPA after intravenous injection.
232
Strong, Dutcher, Lee, and Atkinson
FAST
400
200 SLOW
IO.0
z~
8.0 60
40
2 . 0 84
1.0 L 0
I
2
5
4
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Fig. 3. Pharmacokinetic analysis of plasma procainamide concentration data reported by Koch-Weser and Klein (12). Presentation of data is the same as in Fig. 2.
A striking finding has been the apparent bimodal distribution of ratios of NAPA to procainamide plasma concentrations measured several hours after a procainamide dose in different patients (3). Particularly high ratios have been reported in patients who were phenotypically rapid acetylators of isoniazid or who had renal disease (4). This latter observation is explained by the fact that impaired kidney function retards NAPA much more than procainamide elimination, since normally 82% of NAPA is excreted unchanged by the kidney, but only half of an administered procainamide dose is eliminated by this route (2,4,5,12). The actual renal clearance of NAPA may be slightly less than renal procainamide clearance, which ranges from 179 to 309 ml/min in subjects with normal cardiac output and renal function
Pharmacokinetics in Man of the N-Acetylated Metabolite of ProcaJnamide
233
(12). But in addition, the greater metabolism of procainamide accounts for its more rapid clearance from the body, as well as the fact that plasma procainamide levels tend to be elevated less than NAPA levels in patients with renal disease. The fate of approximately 17~o of the injected NAPA was not determined. A concerted effort was made to identify PABA, PACBA, and their glycine and glucuronide conjugates since it had been reported that 2-10~o of an administered procainamide dose is metabolized to PABA and conjugates of this compound (17). However, subsequent investigators have not found PABA and PACBA to be metabolites of procainamide in man (2). It is likely that the PABA measured in the initial studies was a metabonate s resulting from hydrolysis of procainamide and NAPA. We determined the efficiency of the extraction method described by these authors to remove interfering procainamide (t9) to be 98~o for procainamide and 50~0 for NAPA ; and the subsequent step of heating 5 ml of urine with 1 ml of 12 N HCI for 1 hr on a water bath is sufficient to convert these compounds in large part to PABA. NAPA has been proposed as a better therapeutic agent than procainamide on the grounds that it may be less toxic (7). If this is indeed so, the present investigation strengthens this proposal by demonstrating that only 2 ~o of an administered NAPA dose is deacetylated to procainamide in man, in accordance with what has been found previously in monkeys (2). In addition, the longer half-life of NAPA should make this drug more convenient to administer than procainamide, for which a 3-hr dosing interval has been recommended (12). In our investigations, we found that NAPA has local anesthetic activity when injected intracutaneously but that plasma concentrations more than 4 times higher than the probable threshold effective against ventricular premature beats (3) caused no hypotension and were well tolerated by the normal subjects who were studied. Nevertheless, this is no guarantee that comparable levels might not be toxic in patients with heart disease. The brief tachycardia that was noted during NAPA injection in subjects 2 and 3 may reflect the fact that neither of these individuals had previously been subjects in investigations of this type; subject 1, who had been a subject before, showed no tachycardia. Alternatively, this may reflect the positive chronotropic action of NAPA that has been noted in a rat atrial preparation exposed to high concentrations of this drug (20). In any event, these investigations should help lay the groundwork for direct investigation of NAPA antiarrhythmic efficacy in patients. 5M e t a b o n a t e has been defined by Beckett et al. (18) as a substance that is a product of metabolism
but not a true metabolite since it is formed by nonenzymatic processes in the biological system or during isolation or analytical procedures.
234
Strong, Dutcher, Lee, and Atkinson
ACKNOWLEDGMENTS T h e v a l u a b l e t e c h n i c a l a s s i s t a n c e o f M r . I h s a n E l e r is g r a t e f u l l y a c k n o w ledged. The authors appreciate the help of the nursing staff of the Coronary Intensive Care Unit of the Passavant Pavilion of Northwestern Memorial Hospital in the conduct of these studies.
REFERENCES 1. J. Dreyfuss, J. J. Ross, Jr., and E. C. Schreiber. Absorption, excretion and biotransformation of procainamide-C t4 in the dog and rhesus monkey. Arzneim. Forsch. 21:948-951 (1971). 2. J. Dreyfuss, J. T. Bigger, Jr., A. I. Cohn, and E. C. Schreiber. Metabolism of procainamide in rhesus monkey and man. Clin. Pharmacol. Ther. 13:366-371 (1972). 3. J. Elson, J. M. Strong, W.-K. Lee, and A. J. Atkinson, Jr. Antiarrhythmic potency of Nacetylprocainamide. Clin. Pharmacol. Ther. 17:134-140 (1975). 4. E. Karlsson, L. Molin, B. Norlander, and F. Sj6qvist. Acetylation of procaine amide in man studied with a new gas chromatographic method. Brit. J. Clin. Pharmacol. 1:467475 (1974). 5. E. Karlsson, G. Aberg, P. Collste, L. Molin, B. Norlander, and F. Sj6qvist. Acetylation of procainamide in man. A preliminary communication. Eur. J. Clin. Pharmacol. 8:79-81 (1975). 6. E. E. Bagwell, D. E. Drayer, M. M. Reidenberg, and J. K. Pruett. Effects of the N-acetyl metabolite of procainamide on transmembrane action potentials of canine His Purkinje cells. Clin. Res. 22:676A (1974). 7. D. E. Drayer, M. M. Reidenberg, and R. W. Sew. N-Acetylprocainamide: An active metabolite of procainamide. Proc. Soc. Exp. Biol. Med. 146:358-363 (1974). 8. J. M. Strong and A. J. Atkinson, Jr. Simultaneous measurement of plasma concentrations of lidocaine and its desethylated metabolite by mass fragmentography. Anal. Chem. 44: 2287-2290 (1972). 9. W. P. Deiss and P. P. Cohen. Studies in para-aminohippuric acid synthesis in the human: Its application as a liver function test. J. Clin. Invest. 29:1014-1020 (1950). 10. C. W. Tabor, M. V. Freeman, J. Baily, and P. K. Smith. Studies on the metabolism of para-aminobenzoic acid. 3. Pharmacol. Exp. Ther. 102:98-102 (1951). 11. M. Berman and M. F. Weiss, SAAM Manual, PHS Publication No. 1073, Government Printing Office, Washington, D.C., 1967. 12. J. Koch-Weser and S. W. Klein. Procainamide dosage schedules, plasma concentrations, and clinical effects. 3. Am. Med. Assoc. 215:1454-1460 (1971). 13. S. Riegelman, J. Loo, and M. Rowland. Concept of a volume of distribution and possible errors in the evaluation of this parameter. Y. Pharm. Sci. 57:128-133 (1968). 14. T. Teorell. Kinetics of distribution of substances administered to the body. I. The extravascular modes of administration. Arch. lnt. Pharrnacodyn. 57:205-225 (1937). 15. D. Perrier and M. Gibaldi. Clearance and biologic half-life as indices of intrinsic hepatic metabolism. J. Pharmacol. Exp. Ther. 191:17-24 (1974). 16. R. L. Galeazzi, T. Lockwood, L. B. Sheiner, and L. Z. Benet. Urinary excretion of procainamide. Clin. Res. 23:219A (1975). 17. L. C. Mark, H. J. Kayden, J. M. Steele, J. R. Cooper, I. Berlin, E. A. Rovenstine, and B. B. Brodie. The physiological disposition and cardiac effects of procaine amide. J. Pharmacol. Exp. Ther. 102:5-15 (1951). 18. A. H. Beckett, J. M. Van Dyk, H. H. Chissick, and J. W. Gorrod. Metabolic oxidation on aliphatic basic nitrogen atoms and their ~-carbon atoms--Some unifying principles. J. Pharm. Pharmacol. 23:809-812 (1971). 19. B. B. Brodie, P. A. Lief, and R. Poet. The fate of procaine in man following its intravenous administration and methods for the estimation of procaine and diethylaminoethanol. J. Pharmacol. Exp. Ther. 94: 359-366 (1948).
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235
20. H. Refsum, K. Frislid, P. K. M. Lunde, and K. H. Landmark. Effects of N-acetylprocainamide as compared with procainamide in isolated rat atria. Eur. J. Pharmacol. (in press, 1975).
