In vitro and in vivo studies on the metabolism of tirofiban.

Tirofiban hydrochloride [L-tyrosine-N-(butylsulfonyl)-O-[4-(4-piperidinebutyl)] monohydrochloride, is a potent and specific fibrinogen receptor antagonist. Radiolabeled tirofiban was synthesized with either (3)H-label incorporated into the phenyl ring of the tyrosinyl residue or (14)C-label in the butane sulfonyl moiety. Neither human liver microsomes nor liver slices metabolized [(14)C]tirofiban. However, male rat liver microsomes converted a limited amount of the substrate to a more polar metabolite (I) and a relatively less polar metabolite (II). The formation of I was sex dependent and resulted from an O-dealkylation reaction catalyzed by CYP3A2. Metabolite II was identified as a 2-piperidone analog of tirofiban. There was no evidence for Phase II biotransformation of tirofiban by microsomes fortified with uridine-5'-diphospho-alpha-D-glucuronic acid. After a 1 mg/kg i.v. dose of [(14)C]tirofiban, recoveries of radioactivity in rat urine and bile were 23 and 73%, respectively. Metabolite I and unchanged tirofiban represented 70 and 30% of the urinary radioactivity, respectively. Tirofiban represented >90% of the biliary radioactivity. At least three minor biliary metabolites represented the remainder of the radioactivity. One of them was identified as I. Another was identified as II. When dogs received 1 mg/kg i.v. of [(3)H]tirofiban, most of the radioactivity was recovered in the feces as unchanged tirofiban. The plasma half-life of tirofiban was short in both rats and dogs, and tirofiban was not concentrated in tissues other than those of the vasculature and excretory organs.

Species differences in the metabolism of a potent HIV-1 reverse transcriptase inhibitor L-738,372. In vivo and in vitro studies in rats, dogs, monkeys, and human.

In vivo and in vitro metabolism of 6-chloro-4(S)-cyclopropyl-3,4-dihydro-4-((2-pyridyl) ethynyl)quinazolin-2(1H)-one (L-738,372), a potent human immunodeficiency virus-type 1 reverse transcriptase inhibitor, has been investigated in rats, dogs, and monkeys. Following 0.9 mg/kg iv and 9 mg/kg po doses, systemic blood clearance (CLB) and bioavailability (F) of L-738,372 were species-dependent and inversely related (CLB = 48, 15, and 3 ml/min/kg; F = 6, 62 and 94%, in dogs, rats, and monkeys, respectively). Incubation of L-738,372 with rat liver slices and liver microsomes from all species studied led to the formation of two hydroxylated metabolites, M1 and M2. Kinetic studies of the microsomal metabolism of L-738,372 indicated that M1 was formed by a much higher affinity, but lower capacity enzyme(s) than that which catalyzed M2 formation in rats, dogs, and monkeys. The total intrinsic clearance of metabolite formation (CL(int) total = CL(int) M1 + CL(int) M2) was highest in dogs, followed by rats and monkeys. In dogs, CL(int) total was caused almost exclusively by CL(int) M1. Extrapolation of the CL(int) total values to the hepatic clearances (19, 8.4, and 0.9ml/min/kg in dogs, rats, and monkeys, respectively) showed a similar rank order to the CLB observed in vivo. Good agreement between these in vivo and in vitro results suggests that the species differences in hepatic first-pass metabolism, and not the intrinsic absorption, contributed significantly to the observed differences in F.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro metabolism of L-696,229, an HIV-1 reverse transcriptase inhibitor in rats and humans. Hepatic and extrahepatic metabolism and identification of enzymes involved in the hepatic metabolism.

The metabolism of L-696,229, 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-o ne, a potent human immunodeficiency virus-type 1 reverse transcriptase inhibitor, by rat liver, lung, gut, and kidney microsomes has been studied. L-696,229 was metabolized by rat liver microsomes to several products: the 5 alpha-hydroxyethyl (M1); 5,6-dihydrodiol (M2); 6'-hydroxy (M3); 6-hydroxymethyl (M4); and 5-vinyl (M5) metabolites. For these pathways, liver was the most active metabolizing organ, whereas lung was the major extrahepatic organ in the drug metabolism. In all tissues tested, M1 was the major metabolite. With the exception of M3, gender differences in the hepatic formation of all metabolites were observed. Enzymes responsible for the hepatic metabolism of L-696,229 in rats were also investigated using various enzyme inducers and polyclonal antibodies to rat P-450. Treatment of male rats with dexamethasone (DX) or phenobarbital (PB) caused significant increases in the hepatic formation of the gender-dependent metabolites. Methylcholanthrene (3-MC) greatly enhanced the hepatic formation of M1, M3, and M4. Immunoinhibition studies suggested that CYP2B1/2 and 2E1 were not involved in L-696,229 metabolism, whereas CYP1A was partly responsible for the formation of M1 in untreated rats. CYP3A played an important role in the formation of M1, M2, M4, and M5 in untreated and DX-treated rats. In PB-treated rats, CYP2B1/2 was involved in the increased formation of M1 and M4, whereas CYP3A was partly involved in the enhanced M2 and M4 formation, and primarily responsible for the increased M5 formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the antimalarial arteether and its deethylated metabolite dihydroartemisinin in plasma by high-performance liquid chromatography with reductive electrochemical detection.

A rapid, sensitive, and specific reversed-phase HPLC method using reductive electrochemical detection has been developed for the determination of arteether and its O-dealkylated metabolite dihydroartemisinin (DQHS) in plasma. The internal standard assay was validated between 25 and 1000 ng/mL, with a coefficient of variation of greater than 10% for both intra- and interday accuracy and precision. An assay for determination of arteether only, using the n-propyl ether of DQHS as internal standard, was also developed and validated from 5-500 ng/mL. A new automated sample deoxygenation and injection system is used to greatly increase sample throughput. These methods have been used to study the pharmacokinetics of arteether after intravenous and intramuscular administration.

Hepatic metabolism of artemisinin drugs--I. Drug metabolism in rat liver microsomes.

1. In this communication, metabolism of the semisynthetic antimalarial drugs of the artemisinin class (beta-arteether, beta-artelinic acid and dihydroartemisinin) in rat liver microsomes, is reported. 2. Dihydroartemisinin was the major early metabolite of arteether (57%) and artelinic acid (80%); in addition, arteether was hydroxylated in the positions 9 alpha- and 2 alpha- of the molecule. 3. Dihydroartemisinin was further metabolized by extensive hydroxylation of its molecule; we were able to identify four hydroxylated derivatives of DQHS, but not the exact positions of the hydroxyl groups. 4. The rates of NADPH-supported metabolism of arteether, artelinic acid and dihydroartemisinin in rat liver microsomes were: 4.0, 2.5 and 1.3 nmol/min/mg of microsomal protein, respectively. 5. The apparent affinity constants of arteether and artelinic acid for the microsomal metabolizing system, calculated from the rates of product formation, were 0.54 mM and 0.33 mM (for arteether) and 0.11 mM (for artelinic acid), respectively. The appearance of two affinity constants indicated that arteether was metabolized by two different isoenzymes of cytochrome P-450 in rat liver microsomes.

Hepatic metabolism of artemisinin drugs--II. Metabolism of arteether in rat liver cytosol.

1. In this communication, in vitro metabolism of a semisynthetic antimalarial drug arteether in rat liver cytosol is reported. 2. Whenever 14C-labeled arteether was mixed with rat liver cytosol, a crude postmicrosomal fraction of liver cell homogenates, an appearance of three major 14C-labeled metabolites was always attested: deoxy-dihydroartemisinin, AEM-1 (Baker et al., 1988) and metabolite MW286. 3. Transformation of arteether into deoxyDQHS was catalyzed by an enzyme present in the rat liver cytosol, whose activity depended on the presence of NAD+/NADH and a low molecular, dialyzable factor present in the cytosol. The maximal activity of this enzyme was 0.31 nmol of deoxyDQHS formed/min/mg of cytosolic protein. 4. AEM-1 and metabolite mol. wt 286 have been formed directly from arteether by a chemical interaction of the drug with the cytosolic fraction, probably in a non-enzymatic reaction. 5. Taking together the in vitro data of arteether metabolism in rat liver cytosol, presented in this communication, and in vitro data in rat liver microsomes, presented in the preceding communication (Leskovac and Theoharides, 1991), we were able to postulate an integral pathway of Phase I metabolism of arteether in a whole rat liver cell.

The disposition of an antileishmanial 8-aminoquinoline drug in the isolated perfused rat liver: thermospray liquid chromatography-mass spectrometry identification of metabolites.

1. The disposition of the candidate antileishmanial drug 8-(diethylaminohexylamino-6-methoxy-4-methyl quinoline dihydrochloride (I) has been investigated in the isolated perfused rat liver preparation after the administration of 5 mg/kg (25 microCi) of 14C-I. 2. The perfusate concentration of unchanged I declined biexponentially over the 4 h study period, with a distribution t1/2 of 3.3 +/- 0.3 min and a terminal t1/2 of 35.4 +/- 13.6 min. The area under the perfusate plasma concentration/time curve (AUC0-last time point) was 53.3 +/- 15.7 micrograms min/ml, representing 96% of the area under the curve extrapolated to infinity. the perfusate contained predominantly the carboxylic acid metabolite of I, as well as trace quantities of metabolites detected and identified in bile. 3. Biliary excretion of total 14C accounted for 18.2 +/- 5.0% of the dose, only 2.8 +/- 0.7% was identified by h.p.l.c. analysis as unchanged I. The remainder of the bile contained the desethyl metabolite of I as well as a minimum of 12 more polar metabolites. After 4 h, a total of 39.0 +/- 8.3% of dosed 14C was recovered from the liver tissue. Subcellular fractionation of the livers revealed 24.6 +/- 2.2% of 14C to be located in the 10,000 g pellet. 4. Thermospray liquid chromatography-mass spectrometry analysis of untreated bile and bile treated with beta-glucuronidase or aryl sulphatase permitted identification of some of these metabolites, revealing the presence of the parent drug, desethyl metabolite, 6-desmethyl glucuronide, the 6-desmethyl desethyl glucuronide and the side-chain cleaved 8-amino N-glucuronide metabolites of I, as well as the 6-desmethyl sulphate and the 6-desmethyl desethyl sulphate. Two dihydroxylated metabolites were also detected; however, further structure elucidation is required for unambiguous identification.

Metabolism of methoxychlor by hepatic P-450 monooxygenases in rat and human. 1. Characterization of a novel catechol metabolite.

Previous investigations demonstrated that the incubation of the chlorinated hydrocarbon pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] with rat liver microsomes generates phenolic estrogenic metabolites. The current study shows that the incubation of liver microsomes from untreated and phenobarbital-treated rats and human donors, in the presence of NADPH, yields three phenolic metabolites. Identification of the metabolites was achieved by TLC, HPLC, GC/MS, and LC/MS and by hydrodynamic voltammetric analysis. These metabolites were identified as the mon- and didemethylated phenolic derivatives (mono-OH-M and bis-OH-M, respectively) and as a novel trihydroxy derivative (tris-OH-M). The tris-OH-M was demonstrated to be a catechol [1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3,4-dihydroxyphenyl)ethane]. Furthermore, the tris-OH-M becomes radiolabeled by [methyl-3H3]-S- adenosylmethionine (SAM) in a reaction catalyzed by catechol O-methyltransferase (COMT), indicating that tris-OH-M behaves like a catechol. Incubation of the monohydroxy metabolite with liver microsomes from phenobarbital-treated rats (PB microsomes) yields the dihydroxy and the trihydroxy metabolites. Furthermore, the time course of methoxychlor metabolism by PB microsomes demonstrated a rapid appearance and disappearance of the monohydroxy metabolite with the subsequent formation of the dihydroxy and trihydroxy metabolites. On the basis of these findings, it is proposed that the metabolic route of methoxychlor by mono-oxygenases involves sequential demethylations to the dihydroxy derivative and a subsequent ring hydroxylation.

Methemoglobin formation resulting from administration of candidate 8-aminoquinoline antiparasitic drugs in the dog.

In vivo methemoglobin (MHb) formation caused by five 8-aminoquinoline compounds was tested in beagle dogs. Male beagle dogs were dosed orally once per day at 0.0116 mmol/kg for 4 consecutive days with primaquine (8-[4-amino-1-methylbutyl)amino]-6-methoxyquinoline, diphosphate), three candidate 8-aminoquinoline antimalarial drugs (WR 225,448 5-(3-trifluoromethyl)phenoxy-4-methyl primaquine, succinate); WR 238,605 2,6-dimethoxy-5-(3-trifluoromethyl)phenoxy-4-methyl primaquine, succinate; or WR 242,511 5-hexoxy-4-methyl primaquine, diphosphate dihydrate), or a candidate 8-aminoquinoline antileishmanial drug WR 6026 (8-[(6-diethylamino)amino]-6-methoxy-4-methyl quinoline, dihydrochloride). MHb and total hemoglobin levels were determined daily prior to dosing and for 29 days after drug administration. All compounds caused prolonged levels of MHb that peaked at Days 4 to 5 with disappearance half-lives of 5 to 9 days. Peak percentage MHb of primaquine, WR 6026, WR 238,605, WR 225,448, and WR 242,511 was 6.3, 20.7, 16.0, 25.3, and 48.1%, respectively. Total MHb as measured by area under the time-concentration curve was highest for WR 242,511, followed by WR 225,448, WR 238,605, WR 6026, and primaquine, respectively. The results of this study, in conjunction with other toxicity and efficacy studies, have been utilized to select one of these compounds for development as a replacement for the antimalarial drug primaquine, and also to characterize the MHb-forming properties of WR 6026.

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: formation of a novel metabolite by a female-specific P-450.

Previous studies demonstrated that liver microsomes from untreated rats catalyze the omega, omega-1, and omega-2 hydroxylation of prostaglandins [K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489]. The current study examined the regioselectivity of hydroxylation of PGE1 and PGE2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH-P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE1, RLM 5 catalyzed solely the omega-1 hydroxylation and 5a catalyzed primarily the omega-1 and little omega and omega-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE1 and PGE2 at the omega-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is omega-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE1 hydroxylation demonstrated that the affinity of f4 for PGE1 is approximately 100-fold higher than that of RLM 5; the Km values for f4, monitoring 19- and 17-hydroxylation of PGE1, were about 10 microM. Surprisingly, cytochrome b5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE2 by RLM 5 was at the omega, omega-1, and omega-2 sites, demonstrating a lesser regioselectivity than with PGE1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b5 requirement.

Analysis of artesunic acid and dihydroqinghaosu in blood by high-performance liquid chromatography with reductive electrochemical detection.

A new high-performance liquid chromatography (HPLC) method using reductive electrochemical detection has been developed for the analysis of the antimalarial drugs artesunic acid (ARTS) and dihydroqinghaosu (DQHS) in blood. Presently, this method has been validated to 4 micrograms/ml for ARTS and 200 ng/ml for DQHS. Pharmacokinetic studies in the rabbit show that after intravenous administration (100 mg/kg) ARTS is metabolized rapidly to DQHS and has a t1/2 of 1.7 min in blood. DQHS data were fit to non-linear regression models consisting of the sum of two exponential terms. For phases 1 and 2, t1/2 values of 3.0 +/- 0.4 and 29 +/- 2 min were calculated, respectively. In vitro studies in which ARTS was incubated with blood from various species show that rabbit blood hydrolyzes ARTS at a much greater rate than rat or human blood. Incubation of ARTS with rabbit blood in the presence or absence of diisopropylfluorophosphate suggested that this hydrolysis reaction is catalyzed by plasma and red blood cell esterases. These results suggest that future pharmacokinetic studies in both animals and man should focus on the measurement of DQHS rather than ARTS.

omega-1 and omega-2 hydroxylation of prostaglandins by rabbit hepatic microsomal cytochrome P-450 isozyme 6.

Incubation of prostaglandin E1 (PGE1) with liver microsomes from control rabbits and from rabbits treated with ethanol or imidazole yielded 18-, 19-, and 20-hydroxy metabolites, representing hydroxylation at omega-2, omega-1, and omega carbons, respectively. The current investigation demonstrates that rabbit liver P-450 isozyme 6 effectively catalyzes the omega-1 and omega-2 hydroxylation of PGE1 and PGE2. Additionally, a small amount of product with chromatographic characteristics of the corresponding 20-hydroxy metabolite has been detected. The incorporation of cytochrome b5 into the reconstituted system did not enhance the rate of PGE1 hydroxylation and had no effect on the ratio of products formed. The Km value for the omega-1 and omega-2 hydroxylation of PGE1 with P-450 isozyme 6 from imidazole-treated rabbits was approximately 140 microM; the Vmax's (nmol product min-1 nmol P-450-1) were 2.1 and 1.1 for the omega-1 and omega-2 hydroxylations, respectively. These rates represent the highest activities by hepatic P-450 isozymes for hydroxylation of PGs, and suggest that isozyme 6 is responsible for the omega-2 hydroxylation of PGEs observed in rabbit liver microsomes.

Structure-activity relationships of putative primaquine metabolites causing methemoglobin formation in canine hemolysates.

A rapid and reproducible in vitro test system was developed to measure the methemoglobin (MHb)-forming properties of various 8-aminoquinoline derivatives. Initial rates and extents of reaction were measured spectrophotometrically with either canine hemolysates from which ferrihemoglobin reductase was removed, or with purified human oxyhemoglobin (Hb). The results demonstrate that primaquine derivatives that can be oxidized to quinones or iminoquinones (5-hydroxy,6-desmethyl primaquine; 5-hydroxyprimaquine; 5,6-dihydroxy-8-aminoquinoline; and 5-hydroxy, 6-methoxy-8-aminoquinoline) are potent MHb-forming compounds. Studies on the extent of reaction in hemolysates and purified oxyhemoglobin suggest that the extent of MHb formation may be limited by the rate at which the corresponding iminoquinones or quinones arylate nucleophiles. The effects of glutathione, mannitol, ascorbate, and superoxide dismutase on the rate and extent of hemoglobin oxidation by 5,6-dihydroxy-8-aminoquinoline suggest that these compounds oxidize Hb similar to the mechanism known for dimethylaminophenol (DMAP), in which Hb oxidizes the quinoline to semiquinone radical and quinone species which are the oxidizing and arylating agents.

Metabolism of a potential 8-aminoquinoline antileishmanial drug in rat liver microsomes.

The metabolism of the 8-aminoquinoline, 8-(6-diethylaminohexylamino)-6-methoxy-lepidine dihydrochloride (WR 6026 X 2HCl), was studied in a rat hepatic microsomal system. The results show that WR 6026 X 2HCl was metabolized into two more polar compounds. The structures of these metabolites as proven by gas chromatography-mass spectrometry, ultraviolet absorption, and high performance liquid chromatography were: 8-(6-ethylaminohexylamino)-6-methoxy-lepidine (metabolite 1) and 8-(6-diethylaminohexylamino)-6-methoxy-4-hydroxymethyl quinoline (metabolite 2). The formation of both metabolites was NADPH dependent and also linearly dependent on incubation time and microsomal protein concentration at 0.24 mM WR 6026 X 2 HCl. Studies on the effects of pretreatment of animals with either phenobarbital or Aroclor 1254 suggest that cytochrome P-450 isozymes catalyzed both N-deethylation and hydroxylation reactions. N-deethylase activity was induced by either pretreatment: however, hydroxylase activity was unaffected by phenobarbital pretreatment and significantly elevated by Aroclor 1254 pretreatment. These results suggest that these two reactions are catalyzed by different cytochrome P-450 isozymes. The formation of these two metabolites in vivo may play an important role in the antileishmanial activity of WR 6026 X 2HCl.

High-performance liquid chromatographic method for the analysis of a candidate 8-aminoquinoline antileishmanial drug using oxidative electrochemical detection.

An analytical method was developed for the quantitation of a candidate antileishmanial drug, 6-methoxy-8-(6-diethylaminohexylamino)-4-methylquinoline, dihydrochloride, in canine plasma. The assay utilized internal standard technique with a structural similar 8-aminoquinoline, 6-methoxy-8-(7-diethylaminoheptylamino)-4-methylquinoline, dihydrochloride, as the internal standard. The method employs a liquid-solid extraction procedure with prepackaged silica gel columns upon which the drug and internal standard are adsorbed, then selectively washed and eluted. Reversed-phase chromatography was then employed to analyze the extracted sample by means of oxidative electrochemical detection at +0.75 V. Good accuracy and precision were obtained over the range of concentration tested (10-1500 ng/ml plasma). Analyses of plasma samples from human volunteers given the drug demonstrate the method is also suitable for analysis of human plasma samples. The entire procedure is relatively simple and requires only 1 ml of plasma.

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5.

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.