1178 Journal of Medicinal Chemistry, 1571, Vol. 14, No. 18
Metabolism of ['*C]Verapamil HUGHAI. R~CILHENNY Pjizer Inc., Groton, Connecticut 06340 Received March 9, 1971 The metabolism of [14C]verapamil was studied in rats and dogs a t dose levels similar to those employed during human therapy. Verapamil is well absorbed, rapidly metabolized, and eliminated largely in feces of both species. Little &sue accumulation occurs in rats. The 7 major drug-related substances in t,he urine and feces of dogs were isolated and identified by mass spectrometry as various 0- and N-dealkaylated metabolites of drug. Major urinary and plasma metabolites represent X-dealkylated products of verapamil which retain the a-isopropylphenylacetonitrile portion of the molecule. A gas chromatographic assay was developed which measures 2 of these metabolites, l-isopropyl-l-N-methylpropylamino-(3,4-dimet~hox~phenyl)acet~~1iitrile and l-isopropyl-lp~opylamino-(3,4-dimethoxyphenyl)acetonitrile, in plasma. Major fecal Metabolites reflect the importance of hepatic 0- and N-demethylat,ion processes. It was concluded from comparative studies that pathways of metabolism of verapamil are similar in rats, although different rates of metabolism are indicakd. The major metabolite in the blood stream of dogs was also found in the plasma of several angina patients receiving verapamil.
Verapamil (Isoptin, Cordilox, Knoll A.-G., Ludwigshafen, Germany), 5- [ N -(3,4-dimethoxyphene t hy1)methylamino ]-2-(3,4-dimethoxyp h e nyl)-2-i s o p r o p y 1valerotlitrile hydrochloride, is a phenethyl3mine coro~iary vasodilator which has shown clinical usefulness in the management of angina pectoris.'-j This paper describes the absorption, biotransformation, distribution, and excretion of verapamil labeled with 14C in the nitrile C upon administration to rats and dogs at dose levels similar (on a mg/kg basis) to those used in human therapy. Limited blood level data were also obtained from angina patients.
Experimental Section Synthesis of 14C-Labeled Verapatt1i1.-1~C-labeled verapamil . HC1 was synthesized from veratryl chloride by an adaptation of established methods6 according to the sequence shown in Scheme I. Labeling was accomplished with [I4C]KCN during nitrile formation.
SCHEME I SYNTHESIS O F ['4C]VERAP.4MIL* HC1
CH ,O
I
(1) G. Knooh, M. Schlepper, and E. Witzleb, M e d . Klin. ( M u n i c h ) , Sa, 1485 (1963). (2) J. H. Atterhog and G . Porje, Su. Laekartidn., 63, 2071 (1966). (3) M. Neumann and 4 . A . Luisada, A m e r . J . M e d . S c i . , 261, 552 (1966). (4) G . Sandler, G. A . Clayton, and S. G. Thornioroft, &it. M e d . J., 3, 224 (1968). ( 5 ) Arrneim.-Forsch., 40, 1277 (1970). (6) Knoll A.-G. Chemische Fahriken, U. S. P a t e n t 2,695,319 (19541, C h e m . Abslr., 49, 15963a (1955) ; Knoll Akt.-Ges. Chemische Fabriken, Belgian P a t e n t 615,861 (1962), C h e m . Abstr., 69, 13892d (1963).
The product and an authentic sample of drug were identical by comparison of mp, uv, ir, and chromatographic data. Iiadiochemical purity n-as estimated to be greater than 99% by tlc in 2 solvent systems. Drug metabolism studies were conducted in mature beagle dogs and male Charles Itiver rats with a preparation of specific activity 3.98 pCi/mg of verapamil base. 1)ose levels and concentrations of drug are expressed in terms ( i f verapamil base equivalent. Radiochemical Analyses. Total radioactivity levels in plasma, tissues, and excreta of animals administered [14C]verapamilwere determined in duplicate by standard liquid scintillation spectrometric methods. Plasma and tissue samples required predigestion with strong base solubilizers (Hyamine or Soluene 100, Packard Instrument Co.) before assay. Fecal specimens and the carcass and G I tract of rats were homogenized with water, lyophilized, and combusted to 14[C]COt which was collected ill ethanolamine-ethyl cellosolve (1:2) for assay. The expired air of a rat administered labeled drug and maintained in a sealed drug metabolism apparatus was monitored for [14C]CO,content by collection in 10% S a O H traps. Verapamil levels in dog plasma and urine were determined by reverse isotope dilution. Unlabeled drug was equilibrated with plasma or urine of medicated animals, extracted with EtOAc a t alkaline pH, and recrystallized as the hydrochloride t,o constant specific activity. The distribution of verapamil metabolites in the excreta of rats and dogs was estimated by radioassay following a tlc separation procedure. EtOAc extracts of urine and feces were applied to precoated silica gel glass plates (Brinkmann Instruments, Inc.) and developed in CHCl3-RfeOH-AcOH (90: 5 : 5 ; system 1). The metabolite zones were visualized by autoradiography (Kodak Blue Brand medical X-ray film), individually scraped from the plate, and eluted from the gel in a capillary tube with 0.1 S HC1 in 90% IlleOH. I t was necessary to repeat this procedure for the separation of metabolites D, E, F, and G , using the solvent system 5% Etl" in PhH (system 2 ) . Final silica gel eluates were directly radioassayed; percentage of dose calculations assume 100% recovery from excreta. Whole-body radioautographic investigations were conducted (Arthur D. Little, Inc., Cambridge, Mass.) in male and feniale albino Fischer rats after drug administration by stomach intubation of a saline soln (5.4 mg/kg, 6.7 pCijmg). Animals were frozen in liq Nf at intervals of 45 min, 2 hr, and 6 hr. Whole-body sections and X-ray film were apposed for development of rachlactivity density, according to the method of Liss, et Isolation and Identification of Verapamil Metabolites.-For the isolation and identification of metabo1it)es A, B, and C, day 1 urine from a dog which received a single oral dose of drug, 5 mg/kg, JTas rendered strongly alk with KOH and shaken with EtOAc. The extract contained largely metabolites A and. R. The recovery of metabolite C required enzymatic hydrolysis with a mixture of @-glucuronidase and sulfatase (Glusulase, Endo Products, Inc. j. Following the removal of ether-sol impiirities the hydrolysate was made basic with KOH and extracted with EtOAc. The extract contained metabolite C. ( 7 ) R. H. Liss, D. \V. 1-esair. 0 . P. Watts. F. .\. Cotton, and C y . .J. Krnhler, Pharmncologist. 10, 154 (1968).
Journal of Medicinal Chemistry, 19Y1, Vol. i4,No. 12 1179
METABOLISM OF [14C]VERAPAMIL
ization potentials of 70 and 12-13.5 eV. Useful solid probe spectra a t programmed inlet temps were obtained from verapamil and metabolites A and E (underivatized) as supportive data, Trimethylsilyl (TMS) ether derivatives of metabolites D-G were prepared using an on-column technique with the simultaneous injection of metabolite and bis(trimethylsily1)acetamide (Pierce Chemical Co.) contained in a single syringe. The analysis of metabolite C was supplemented by the examination of bis(trimethylsily1)acetamide-silylated and CHzNz-methylated (Me) derivatives. Pathways of verapamil metabolism following oral administration were also characterized in 0- to 24-hr excreta of 3 rats administered 3 mg of verapamil/kg, and in 0- to 24-hr bile of a female beagle established with a biliary fistula and administered 5 mg of verapamil/kg, orally in soln. The standard procedures of EtOAc extraction a t alkaline pH, before and after Glusulase hydrolysis, followed by tlc analysis were employed. A substantial portion of radioactivity in rat and day 2 dog feces could not be recovered in this manner. Tlc examination of these samples was therefore performed with exhaustive MeOH extracts (Soxhlet, 16 hr) of lyophilized specimens after removal of ether-sol impurities. Mass spectral evidence (see Table 11) for verapamil and its metabolites are given below: verapamil, m/e 4.54 (calcd M + 454), 303 [ -3,4-(CH30)zCaH3CHz;ions+], 260 (ion a + -CHIN=
Metabolites D, E, F, and G were isolated from a lyophilized day 1 fecal sample from the same dog. A slurry was prepared in 1 N KOH and extracted with EtOAc. The extract was evaporated to dryness, reconstituted with 1N HC1, and freed of ethersoluble impurities. The aq phase was made basic with KOH and extracted with EtOAc to yield a fraction which contained the 4 important fecal metabolites D, E, F, and G
TABLE I TIE DISTRIBUTION OF THE MAJORMETABOLITES OF
VERAPAMIL IN DOGS Relative abundance by --autoradiography, system 1 - 7 0-24 h r feces, 0-24 hr urine, 0-24 hr bile 1-7 hr plasma
-Rverapamila--
Metablite
System 2
System 1
+ ++++ + +++++ +++++ + + ++ ++ ++++
0.28,0.17 0.92 0.43,0.28 0.83 A 0.63 0.37 B 0.47 0.42 C 0.30 0.09 As detd from precoated silica gel tlc plates using the solvent systems: CHC13-MeOH-AcOH (90:5:5; system 1, mean of 3 values) and 5% EtZNH in PhH (system 2). Small amounts of verapamil were detectable in all samples (Rf0.52, system 1). D,E
F, G
TABLE I1 PROPOSED
STRUCTURES O F
THE
7 h1.4JOR METABOLITES O F VERAPAMIL I N DOGSA N D THEIR RELATIVE ABUNDANCE IN DAY1 EXCRETA OF DOGSA N D RATS
'CN
Metabolite
R'
A
CH3
B
H
C
CH3
RZ
H H H
R'
R4
CH3 CH3 CH3
CH3 CHI H
CH3
H
Per cent of day 1 urinary --radioactivityu-Dog 2b RatsC
26 7 19
4 3 40
Per cent of day 1 -fecal radioactivity-Dog 2b Ratsc
5 2 6
A-G, 15
5 5
4
CH3
H
15
CH3
CH3
11
4 G
CH3
CH3
H
16
Dog 2 received 5 mg of a Metabolites C-G were present largely as Glusulase-sensitive conjugates in the urine of dogs and rats. verapamil/kg in aq soln. Day 1 urinary and fecal radioactivity represented 17% and 31 % of the dose, respectively. Results are based on a single analysis and are presumed typical. c Percentages are derived from a single analysis of the collective excreta of 3 rats, each receiving verapamil orally in aq soln, 3 mg/kg. Day 1 urinary and fecal radioactivity represented 8.47, and 59.5% of the dose, respectively. All urine and fecal metabolites were next subjected t o tlc separation (see Table I) and purification by successive development in solvent systems 1, 2, and 1, in that order. Final silica gel eluates were evaporated under a stream of NZand reconstituted with dil aq KOH. The soln was extracted with EtOAc, reduced to dryness under NP,and dissolved in acetone for analysis with a LKB 9000 combined gas chromatograph-mass spectrometer equipped with flame ionization and radioactivity detector systems as described by Hobbs.* A 1.5 mm id X 2 m glass column containing 1% OV-1 on Gas-Chrom Q with a He carrier gas flow of 40 ml/min was employed. The mass spectrometer was operated a t ion( 8 ) D. C . IIobbs, American Society for Mass Spectrometry, Annual Meeting. June 15-19, 1970, Sen Francisco, Calif.
CH2), 165 [3,4-(CH30)zCeH3CHzCHz+; ion b +], 151 [3,4-(CH30)~+ C6H3CHz+;ion c+], 58 [(CH3)zN=CH2]; metabolite A,+ m/e 290 (calcdM+290), 275 (-CH3), 262 ( -C2Hz),9 259 (-"a-, CHI),"' 247 (-CHI, -CO); metabolite B , t m/e 276 (calcd M + 276) 261 (-CHI), 259 (-"a), 233 (-CHs, -CO) 216 (233-NHs); metabolitec, m/e276 (calcdM+276),261 (-CHa), 245 (-CzHz), (9) H. Budzikiewics, C . Djerassi, and D. H. Williams, "Mass Spectromet r y of Organic Compounds," Holden-Day, San Francisco, Calif., 1967, p 409. (10) D. L. Smith and M. F. Grostic. J. M e d . Chem., 10, 375 (1967). Spectra obtained from reference samples of proposed metabolites A a n d B (Knoll A.-G.) under t h e same conditions were identical with those of metabolites A and B, respectively.
t
MCILHENNY
1180 JOuTnUl of Medicinal Chemistry, 1971, Vol. 14, No. 12
single dose of 10 mg (unlabeled) of encapsulated drugjkg, and in 245 (-"&HI), 233 (-CH*, -CO); C-TMS, mje348 (calcdM+ + 16 beagles that had received a daily regimen of verapamil fof. 8 348), 333 ( -CH3), 147 [(CHa)~si=osi(CH,),], 57 (CHINHCH= months (40, 62.5, 70, and 85 mg/kg per day, 2 animalsjsex CHZ+),43 (CH3N=CHz+); C-Me, mje 290 (calcd M + 290), same per dose level). Samples were taken from the chronically treated as metabolite A; metabolite D-(TMS)z, m/e 570 (calcd M+570), animals a t 1, 2, and 4 hr after the first of 3 divided daily doses. 555 (-CHI), 361 [ -3,4-(CH30)(TMSO)CeH3CH2; TMSOion a+] Selected dog plasma samples during single dose I4C studies were 209 (3,4-(CH30)(TMSO)CsH3CH1+; T N S O ion c+] ; metabolite also assayed. Blood levels were detd in 6 patients with angina E, m/e 426 (calcd M + 426), 275 [ - ~ , ~ - ( C H ~ O ) Z C B H ~ion C HaZ+; pectoris, generally 1-2 hr after the last dose, during daily therapy -2CHs], 165 (ion b+), 131 (ion c f ) ; E-TMS, m/e498 (calcd %I+ of 320 mg of verapamil administered in capsules in 4 divided 498), 347 [ -3,4-(CH30)zC6H3CHz; TMSO(NH) ion a+], 165 (ion b+), 151 (ion c + ) ; metabolite F-TMS, m/e 512 (calcd LIS- doses. 512), 497 ( -CHI), 303 (ion a+), 209 [ ~ , ~ - ( C H ~ O ) ( T M S O ) C B H , C H Z + ; TMSO ion c+], (512 510):303: :1:12, 223:209:179: Results : 2: 1:1.5; 3'-demethylverapamil (D628, Knoll A.-G.)-TMS, 510):303: :1:>200, 223:209:m/e 512 (calcd >I+ 312)) (512 Excretion of Metabolites in the Dog.-Three-day 179: :1:2:3; metabolite G-TMS, m/e 512 (calcd 31+ .512), 497 cumulative radioactivity in the urine of 3 dogs! conion a+], 165 (ion (-CH,), 361 [ - ~ , ~ - ( C H ~ O ) Z C & C H ZTMSO ; tained from 21 to 33y0 of the dose after oral as well as b+), 151 (ion c+). Gas Chromatographic Assay for Metabolites A and B in im administration of labeled drug. This radioactivity Plasma.-Five ml of plasma contg 500 mg of NaHCO3 was extd n as composed essentially of metabolites, since negin a 50-ml centrifuge tube with 20 ml of EtOAcj by mechanical ligible quantities of verapamil were found (less than shaking for 15 min. After centrifugation the Et'OAc layer was 0.5% of dose). About 80% of total urinary label u a s withdrawn, dried (NazS04, 2.5 g), and evapd under XZ to 1.0 excreted by 2 dogs within the first 24 hr after oral ml. The concentrate was then filtered through a prewashed (EtOAc) column of silica gel (silica gel, 0.05-0.2 mm, 70-325 administration. Three-day cumulative fecal collecmesh ASTM, for column chromatography, E. Merck, Darmstadt), tions contained the balance of ingested radioactivity 0.5 g, coritd iii a 13.1-cm heavy-wall Pasteur capillary pipette. (58yc,65Oj,), about one-half of which was excreted The filtrate including 3 ml of EtOAc wash soln was rejected. during the first day of treatment. High levels of Metabolites A and B were eluted from the column with 5 ml of EtOAc-MeOH-AV-methylpyrrolidine(88: 10: 2 ) . The eluate was label in feces were measured following im injection (40y0 collected in a 15-ml centrifuge tube and evaporated to 0.1 ml. in 3 days) to reveal the probable significance of biliary T o the sample was added 0.2 ml of 0.1 M Na2B407and 0.5 ml of a excretion. a 1% sohi of 4-fluoro-3-nitrobenzotrifluoride (Pierce Chemical EtOAc extraction of 24-hr dog (dog 2 ) urine at alk Co.) in acetone. The tube contents were mixed and heated pH removed 63-69% of the radioactivity present. in a water bath (70-73") for 30 min, then cooled, and dild to 1.0 ml with distd HnO. The corresponding 2-nitro-4-trifluoroAn additional 27% was extracted following hydrolysis methylaniline derivatives" of metabolites .4 and B were extd with a 0-glucuronidase-sulfatase mixture. At least from the reaction medium with three 1-ml portions of hexane. 13 urinary metabolites were detected by radiochromaN1 The combined hexane ext,s were then evapd to dryness under Three of these substances designated as tography. and reconstituted with 5Op1 of EtOAc itnmediately prior to assay. metabolites A, B, and C were predominant; the most Portions (1 pl) were injected into a 6 mm od X 2 m glass column maintained a t 260' and containing a support of silaiiiaed Corning polar metabolite C largely represented the conjugated glass beads glc 110 (Chemical Research Services, Inc.), 60-80 substances freed by enzyme treatment. The major mesh, coated with 0.0.370 OV-1. A Hewlett-Packard F & 121 metabolites in urine were also the major metabolites in Scientific Model 5750 gas chromatograph equipped with a flame plasma by comparative tlc radiography (Table I). ionization detector (280") was employed. With a He carrier gas flow rate of 100 ml/min, typical retention times of chromatoThe extraction of radioactivity from an alk slurry of graphic peaks corresponding to metabolites A and B were 3.8 24-hr dog (dog 2 ) feces was essentially quant. Only min and 5.1 min, respectively. Untreated dog plasma from small quantities of label were excreted in conjugated control animals in acute and chronic studies, and pooled normal form (7Y0) as determined by hydrolysis. Tlc analysis human serum (NHS) were, essentially free of gas chromatoof fecal extract indicated less than 1% of dose (oral graphic background interferences at these retent'ion times under assay conditions. and im) as unchanged drug, and a mixture of metabThe metabolite peak height was referred to a standard curve olites similar to those in urine in number and mobility derived from NHS spiked with increasing concns of each metabbut markedly different in terms of relative proportion. olite (generally ranging from 0.1 to 1.0 fig/ml of plasma for therThe preponderant zones of radioactivity in feces comapeutic dose levels, and to 3 pg/ml for high multiple dose level studies in dogs) and carried t,hrough the assay procedure. Meprised 4 metabolites labeled D, E, F, and G, each of tabolites A and B could be measured with sensitivities of 5 and which as more polar than verapamil (Table I). Rela10 ng, corresponding to 0.05 and 0.10 pg,'ml of plasma (5-ml tively smaller amounts of radioactivity represented sample), respectively. With each compd, response was linear metabolites -4,B, and C. Metabolites A-G comprised and suitably accurate up to 3.0 pgiml of plasma; efficiencies of about 457, of radioactivity in second-day fecal c01recovery averaged about 70%. Sertlm vols of less than 5 ml were dild to 3 ml with HzO prior to assay, and corrected for by lections; the remaining radioactivity was not extractinsertion of the corresponding diln factor in final calcns. The able at alk pH Rith EtOhc. .4n additional 25-4570 potential for increased assay sensitivity exists in principle with could be extracted folloning acidification of the raffithe use of an electron capture detector. nate ; acid hydrolysis released little additional label. Verapamil in NHS at levels of 0.3 pgjml or greater can he Tlc analysis of purified NeOH extracts of day 2 feces, measured simultaneously in the same assay injection sample, with retention time of about 8.1 niin in an area free of plasma confreed of metabolites A-G, revealed substances of instituents. Spiked XHS standards for the metabolite assays creased polarity (Rverapam11
