Glucuronidation in the chimpanzee (Pan troglodytes): studies with acetaminophen, oestradiol and morphine.

The chimpanzee has recently been characterized as a surrogate for oxidative drug metabolism in humans and as a pharmacokinetic model for the selection of drug candidates. In the current study, the glucuronidation of acetaminophen, morphine and oestradiol was evaluated in the chimpanzee to extend the characterization of this important animal model. Following oral administration of acetaminophen (600 mg) to chimpanzees (n=2), pharmacokinetics were comparable with previously reported human values, namely mean oral clearance 0.91 vs. 0.62+/-0.05 l h-1 kg-1, apparent volume of distribution 2.29 vs. 1.65+/-0.25 l kg-1, and half-life 1.86 vs. 1.89+/-7h, for chimpanzee vs. human, respectively. Urinary excretions (percentage of dose) of acetaminophen, acetaminophen glucuronide and acetaminophen sulfate were also similar between chimpanzees and humans, namely 2.3 vs. 5.0, 63.1 vs. 54.7, and 25.0 vs. 32.3%, respectively. Acetaminophen, oestradiol and morphine glucuronide formation kinetics were investigated using chimpanzee (n=2) and pooled human liver microsomes (n=10). V(max) (app) and K(m)(app) (or S(50)(app)) for acetaminophen glucuronide, morphine 3- and 6-glucuronide, and oestradiol 3- and 17-glucuronide formation were comparable in both species. Eadie-Hofstee plots of oestradiol 3-glucuronide formation in chimpanzee microsomes were characteristic of autoactivation kinetics. Western immunoblot analysis of chimpanzee liver microsomes revealed a single immunoreactive band when probed with anti-human UGT1A1, anti-human UGT1A6, and anti-human UGT2B7. Taken collectively, these data demonstrate similar glucuronidation characteristics in chimpanzees and humans.

Design, synthesis, and structure-activity relationships of macrocyclic hydroxamic acids that inhibit tumor necrosis factor alpha release in vitro and in vivo.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of

Synthesis and biological activities of potential metabolites of the non-nucleoside reverse transcriptase inhibitor efavirenz.

Studies on the biotransformation of the clinically important non-nucleoside reverse transcriptase inhibitor efavirenz have shown that oxidation and secondary conjugation are important components of the processing of this molecule in vivo. We have synthesized metabolites of efavirenz to confirm their structure and to evaluate their activity as antivirals.

The species-dependent metabolism of efavirenz produces a nephrotoxic glutathione conjugate in rats.

Efavirenz, a potent nonnucleoside reverse transcriptase inhibitor widely prescribed for the treatment of HIV infection, produces renal tubular epithelial cell necrosis in rats but not in cynomolgus monkeys or humans. This species selectivity in nephrotoxicity could result from differences in the production or processing of reactive metabolites, or both. A detailed comparison of the metabolites produced by rats, monkeys, and humans revealed that rats produce a unique glutathione adduct. The mechanism of formation and role of this glutathione adduct in the renal toxicity were investigated using both chemical and biochemical probes. Efavirenz was labeled at the methine position on the cyclopropyl ring with the stable isotope deuterium, effectively reducing the formation of the cyclopropanol metabolite, an obligate precursor to the glutathione adduct. This substitution markedly reduced both the incidence and severity of nephrotoxicity as measured histologically. Further processing of this glutathione adduct was also important in producing the lesion and was demonstrated by inhibiting gamma-glutamyltranspeptidase with acivicin pretreatment (10 mg/kg, IV) prior to dosing with efavirenz. Again, both the incidence and severity of the nephrotoxicity were reduced, such that four of nine rats given acivicin were without detectable lesions. These studies provide compelling evidence that a species-specific formation of glutathione conjugate(s) from efavirenz is involved in producing nephrotoxicity in rats. Mechanisms are proposed for the formation of reactive metabolites that could be responsible for the renal toxicity observed in rats.

Mass spectrometric and NMR characterization of metabolites of roxifiban, a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor.

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.

Identification and characterization of efavirenz metabolites by liquid chromatography/mass spectrometry and high field NMR: species differences in the metabolism of efavirenz.

Efavirenz (Sustiva, Fig. 1) is a potent and specific inhibitor of HIV-1 reverse transcriptase approved for the treatment of HIV infection. To examine the potential differences in the metabolism among species, liquid chromatography/mass spectrometry profiles of efavirenz metabolites in urine of rats, guinea pigs, hamsters, cynomolgus monkeys, and humans were obtained and compared. The metabolites of efavirenz were isolated, and structures were determined unequivocally by mass spectral and NMR analyses. Efavirenz was metabolized extensively by all the species as evidenced by the excretion of none or trace quantities of parent compound in urine. Significant species differences in the metabolism of efavirenz were observed. The major metabolite excreted in the urine of all species was the O-glucuronide conjugate (M1) of the 8-hydroxylated metabolite. Efavirenz was also metabolized by direct conjugation with glucuronic acid, forming the N-glucuronide (M2) in all five species. The sulfate conjugate of 8-OH efavirenz (M3) was found in the urine of rats and cynomolgus monkeys but not in humans. In addition to the aromatic ring-hydroxylated products, metabolites with a hydroxylated cyclopropane ring (at C14) were also isolated. GSH-related products of efavirenz were identified in rats and guinea pigs. The cysteinylglycine adduct (M10), formed from the GSH adduct (M9), was found in significant quantities in only rat and guinea pig urine and was not detected in other species. In vitro metabolism studies were conducted to show that the GSH adduct was produced from the cyclopropanol intermediate (M11) in the presence of only rat liver and kidney subcellular fractions and was not formed by similar preparations from humans or cynomolgus monkeys. These studies indicated the existence of a specific glutathione-S-transferase in rats capable of metabolizing the cyclopropanol metabolite (M11) to the GSH adduct, M9. The biotransformation pathways of efavirenz in different species were proposed based on some of the in vitro results.

Liquid chromatography/mass spectrometry and high-field nuclear magnetic resonance characterization of novel mixed diconjugates of the non-nucleoside human immunodeficiency virus-1 reverse transcriptase inhibitor, efavirenz.

Efavirenz (Sustiva) is a potent and specific inhibitor of the HIV-1 reverse transcriptase and is approved for the treatment of HIV infection. The metabolism of efavirenz in different species has been described previously. Efavirenz is primarily metabolized in rats to the glucuronide conjugate of 8-OH efavirenz. Electrospray ionization-liquid chromatography/mass spectrometry analyses of bile samples from rats dosed with either efavirenz or with 8-OH efavirenz revealed three polar metabolites, M9, M12, and M13, with pseudomolecular ions [M-H](-) at m/z 733, 602, and 749, respectively. The characteristic mass spectral fragmentation patterns obtained for metabolites M9 and M13 suggested that these were glutathione-sulfate diconjugates, and the presence of a glutathione moiety in metabolite M9 was confirmed by liquid chromatograpy/nuclear magnetic resonance (NMR) analysis of bile extracts. Metabolite M12 was characterized by liquid chromatography/mass spectrometry as a glucuronide-sulfate diconjugate. Unambiguous structures of M9, M12, and M13 were obtained from one-dimensional proton and carbon NMR as well as proton-proton (correlated spectroscopy, two-dimensional shift correlation), proton-carbon heteronuclear multiple quantum correlation, and long-range proton-carbon (heteronuclear multiple bond correlation) correlated two-dimensional NMR analyses of metabolites isolated from rat bile. The mass spectral and NMR analyses of M10, which was isolated from rat urine, suggested a cysteinylglycine-sulfate diconjugate. The isolation of these polar metabolites for further characterization by NMR was aided by mass spectral analyses of HPLC fractions and solid phase extraction extracts during the isolation steps. The complete characterization of these novel diconjugates demonstrates that further phase II metabolism of polar conjugates such as sulfates could take place in vivo.

Pharmacokinetic-pharmacodynamic relations of losartan and EXP3174 in a porcine animal model.

The pharmacokinetics of losartan and EXP3174, an active metabolite of losartan, were evaluated in the anesthetized pig after both a single intravenous dose (3 mg/kg) and during constant intravenous infusion. The pharmacodynamic activities of losartan and EXP3174 were determined during constant intravenous infusion as the degree of inhibition of angiotensin II-induced increase in the diastolic pressure. The systemic plasma clearance of losartan was 22.1 +/- 4.4 ml/min/kg (mean +/- SEM) and had an apparent volume of distribution at steady state of 0.56 +/- 0.16 L/kg after a 3-mg/kg intravenous dose. The elimination half-life of losartan was 40 +/- 6 min. Less than 2% of the intravenous losartan doses was estimated to be present as unconjugated EXP3174. The plasma clearance of EXP3174 was approximately 50% that of losartan, 11.8 +/- 1.5 ml/min/kg, and had a smaller steady-state apparent volume of distribution, 0.18 +/- 0.04 L/kg. The elimination half-life for EXP3174 was slightly longer than that of losartan (52 min). The time course of the pharmacodynamic effects of losartan and EXP3174 closely followed their respective plasma concentrations. The apparent dissociation constant of EXP3174 to the angiotensin II receptor was estimated, based on the total plasma concentrations, to be approximately 5 times lower than that for losartan.

DuP 532, an angiotensin II receptor antagonist: first administration and comparison with losartan.

We investigated the tolerability and angiotensin II antagonist activity of oral DuP 532 in healthy male subjects. DuP 532 (1 to 200 mg) was well tolerated, with no effect on blood pressure or heart rate. Compared with losartan (100 mg), DuP 532 (200 mg) was a weak antagonist of pressor responses to intravenous angiotensin II. Maximum inhibition of diastolic pressor response was 86% (95% confidence interval [CI], 84%, 88%) approximately 4.6 hours after losartan and 48% (95% CI, 38%, 56%) 8.7 hours after DuP 532. Twenty-four hours after dosing, inhibition by losartan and DuP 532 was similar (40% to 45%). DUP 532 is extensively bound in human plasma, with an in vitro free fraction of 0.06. Although DuP 532 and EXP3174 (losartan's active metabolite) have similar AT1-receptor potency, and plasma concentrations of DuP 532 were much greater than losartan/EXP3174, the level of antagonism was much less for DuP 532. These results indicate that multiple factors determine the in vivo potency of angiotensin II antagonists, including affinity for and distribution to the receptor as modulated by plasma binding.

In vitro and in vivo effects of the arylamine human immunodeficiency virus protease inhibitor 4R-(4alpha,5alpha,6beta, 7beta)-1-[(3-(1-imidazoylcarbamoyl)phenyl)methyl]-3-[(3-aminophenyl)m ethyl]hexahydro-5,6-dihydroxy-4,7-bis(phenylmethyl)-2H-1, 3-diazepin-2-one (SD894) on rat hepatic cytochrome P450 2B and 3A.

The human immunodeficiency virus-1 protease inhibitor SD894 was evaluated as an inhibitor and inducer of cytochromes P450 (CYPs) in rats. After addition of 10 microM SD894 and 2 mM NADPH to liver microsomes from dexamethasone-treated rats, a type II spectrum appeared. Within 2 min, it was replaced by a type III spectrum, with absorbance maxima at 426 and 456 nm, similar to those observed with alkylamines (SKF-525A) and arylamines (p-chloroaniline). Preincubation of microsomes from dexamethasone-treated rats with SD894 and NADPH resulted in a time-dependent inhibition of testosterone 6beta-hydroxylation (CYP 3A1/2 activity), which was decreased to 25% of controls after 30 min. Testosterone 16beta-hydroxylation (CYP 2B1/2 activity) was unaffected under these conditions. Testosterone 6beta-hydroxylation rates in liver microsomes from pregnenolone 16alpha-carbonitrile-treated rats incubated with 10 microM SD894 and NADPH, washed, and reisolated by ultracentrifugation were reduced by 71%, whereas 16beta-hydroxylation was unaffected by SD894. Immunoblots of liver microsomes from rats dosed iv with SD894 or ip with TAO displayed increased CYP 2B1 and CYP 3A1 levels, respectively. Testosterone 6beta-hydroxylase activity in microsomes from TAO-treated rats was greater than controls. Preincubation of these microsomes with potassium ferricyanide produced an additional 50% increase, consistent with disruption of a metabolite-CYP complex. Microsomes from SD894-treated rats displayed a 3-fold increase in testosterone 16beta-hydroxylation. Potassium ferricyanide preincubation did not increase activity. Thus, although SD894 appears to inhibit CYP in vitro in a manner typical of other amine-containing, mechanism-based inhibitors, in vivo induction by 10 mg/kg daily doses of SD894 affects a different isozyme than does inhibition. The mechanism of induction is unknown.

Nonpeptide angiotensin II receptor antagonist: pharmacokinetics and pharmacodynamics in rats of EXP3174, an active metabolite of losartan.

The pharmacokinetics and pharmacodynamics of EXP3174 (2-n-butyl-4-chloro-1-[(2'-(1H-tetrazole-5-yl)biphenyl-4- yl-)methyl]imidazole-5-carboxylic acid), an angiotensin II receptor antagonist, were studied in conscious rats. Elimination half-life, systemic clearance, and apparent volume of distribution of EXP3174 at a dose of 10 mg/kg i.v. were 2.9 h, 1.8 ml/min/kg, and 0.25 l/kg, respectively. Inhibition of the angiotensin II pressor response correlated with the log of the steady state plasma EXP3174 concentration in a sigmoidal fashion with an IC50 of about 200 ng/ml. When corrected for plasma protein binding, the IC50 (free) for EXP3174 was 0.4 ng/ml (0.9 nmol/l). This study indicates a predictable plasma concentration-effect relationship of EXP3174 in rats which would be helpful in designing more rational dosing schemes for pharmacodynamic studies.

Human plasma protein binding of the angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) and its pharmacologically active metabolite EXP3174.

The in vitro protein binding characteristics of the prototypical angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) and its pharmacologically active metabolite EXP3174 were determined by ultrafiltration with plasma from naive donors, volunteers dosed with losartan, and purified human plasma proteins. The binding of losartan was high, with a percent unbound (free) of 1.4 +/- 0.2% to 1.2 +/- 0.1% at concentrations ranging from 0.5 to 5.0 micrograms/mL; that is, approximately 98.6 to 98.8% bound. EXP3174 was more highly bound than losartan (P < .05) with 0.2 +/- 0.0% free at concentrations ranging from 0.1 to 10.0 micrograms/mL; or, greater than 99.7% bound. The binding in the plasma from volunteers given oral losartan was similar to that determined with plasma from naive donors, with 1.5 +/- 0.3 versus 1.4 +/- 0.1% free for losartan, and 0.5 +/- 0.1 versus 0.4 +/- 0.0% for EXP3174, respectively. This extensive plasma binding of both acidic compounds occurs primarily to albumin, with negligible binding to the alpha 1-acid glycoprotein. Although highly bound, neither losartan nor EXP3174 were displaced in vitro by pharmacologically relevant concentrations of non-steroidal antiinflammatory drugs (NSAIDs), warfarin, or diazepam; however, suprapharmacologic concentrations of the NSAIDs increased the free fraction of both compounds. These data show that the angiotensin II receptor antagonists losartan and EXP3174 are highly bound to plasma albumin in humans, although clinically significant drug interactions due to displacement from binding sites are unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the thrombin inhibitor DuP 714 by an enzyme-linked immunosorbent assay.

A competition enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative detection in plasma of DuP 714, a boroarginine tripeptide (Ac-(D)-Phe-Pro-boroArg) with potent antithrombin activity. The assay has been used to calculate the half-life after i.v. administration of DuP 714, as well as the percent bioavailability after oral administration of the agent. Following i.v. administration, in dogs, the clearance of compound from the circulation could best be fit to a biexponential decay with an initial half-life of approximately 9 min, and a slower elimination phase with a half-life of 40 min. There was a significant correlation between pharmacokinetic and pharmacodynamic characteristics (r = 0.9570, P < 0.01) as measured with the ELISA and the clotting assay, aPTT, following i.v. infusion in conscious dogs. A plasma concentration of 311 ng/ml doubled the aPTT. After oral administration of 1 mg/kg DuP 714, peak concentration ranged from 384 to 584 ng/ml. Oral bioavailability, determined by comparing the areas under concentration vs time curves after oral and i.v. administration, was 53 +/- 8% (n = 4). In summary, this assay offers a rapid, sensitive and specific method of examining the peptide's pharmacokinetic characteristics.

The pharmacokinetics and pharmacodynamics of the angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) in the dog.

The pharmacokinetics and plasma concentration-effect relationship for the nonpeptide angiotensin II (Ang II) receptor antagonist losartan potassium (losartan) have been determined with conscious and anesthetized dogs. The p.o. bioavailability of single doses of 5 to 20 mg/kg was low, 23 to 33%, and independent of the dose. Absorption was rapid, with peak plasma levels observed within 1 hr, and the Cmax and area under the concentration vs. time curve to infinity were proportional to the dose, P < .05. The elimination half-life, 108 to 153 min, was longer than that observed after a single i.v. dose, 41 min, and may reflect both continuous absorption and enterohepatic recirculation because the major route of excretion was via the bile. Single i.v. doses were eliminated rapidly, with a systemic plasma clearance of 22.2 ml/min/kg. When corrected for the blood:plasma distribution ratio, 0.66 to 0.72, the systemic clearance approximates hepatic blood flow, suggesting that clearance is primarily via hepatic metabolism and biliary excretion. Losartan was not distributed extensively to tissues; apparent volume of distribution at steady-state of 0.30 liters/kg and was highly but not extensively bound to plasma proteins; 2.7 to 2.9% unbound (free). The plasma concentration vs. blockade of exogenous Ang II-induced vasopressor response was also determined after a single 3-mg/kg i.v. dose of losartan with a sigmoidal Emax model. Blockade of the pressor response was rapid, 89% at 5 min, and declined to 11% at 240 min postdose. The relationship between concentration and effect was highly significant (r = 0.922, P < .01), with an IC50 (total) of 96 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of cytochromes P-450 2B and 3A in mice following the dietary administration of the novel cognitive enhancer linopirdine.

The effects of the novel cognitive enhancer linopirdine [3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one] on hepatic cytochromes P-450 (CYP) and linopirdine metabolism were determined in female mice fed 0, 10, 50, and 250 mg/kg/day of linopirdine in the diet for 4, 7, 14, and 28 days. Linopirdine induced CYP maximally by day 4 only at the highest dose, as demonstrated by significant (p < 0.05) increases in total spectral CYP and liver weight. SDS-PAGE revealed induced 52 kDa microsomal protein(s), identified as CYP2B and 3A by immunoblotting. Linopirdine also increased the rates of reactions selectively catalyzed by CYP2B and 3A (pentoxyresorufin O-dealkylation, benzphetamine N-demethylation, erythromycin N-demethylation, and testosterone 2 beta-, 6 beta-, 16 beta-hydroxylation), 1.7- to 3.0-fold vs. control, similar to increases produced by the prototypical CYP2B and 3A inducers phenobarbital and dexamethasone. No increase in microsomal CYP1A or 2E levels was demonstrated by immunoblotting or selective substrate assays. CYP induction increased the metabolism of linopirdine. The area under the plasma concentration-time curve of linopirdine after a 250 mg/kg/day dose decreased 11-fold from day 1-28, and microsomes from a parallel 250 mg/kg/day group metabolized linopirdine 1.9-fold faster than control (p < 0.05). This autoinduction was due primarily to the induced CYP3A, because antibodies recognizing CYP3A inhibited the microsomal metabolism of linopirdine by 85%, whereas antibodies to CYP2B were not inhibitory. In summary, the dietary consumption of 250 mg/kg/day of linopirdine by female mice coinduced CYP2B and 3A maximally by day 4, and resulted in an increased rate of metabolism of linopirdine, predominantly due to CYP3A.

Enhancement of losartan (DuP 753)-induced angiotensin II receptor antagonism by PD123177 in rats.

Losartan (DuP 753) and PD123177 are angiotensin II type 1 (AT1) and type 2 (AT2) receptor selective ligands, respectively. In rats, PD123177 did not exhibit angiotensin II antagonism or hypotensive activity but enhanced these activities of a submaximal dose of losartan. As PD123177 displaced losartan from its rat plasma protein binding sites and thus increased the free concentration of losartan, this may account for its enhancement of the in vivo activities of losartan in rats.

Monoclonal antibodies to the nonpeptide angiotensin II receptor antagonist, losartan.

Two murine monoclonal antibodies were produced to losartan (DuP 753), a nonpeptide angiotensin II receptor antagonist. Using a solid phase competitive enzyme-linked immunosorbent assay (ELISA), each antibody was examined for its ability to bind to a set of losartan analogs that differ structurally in varying degrees. Both antibodies distinguished fine structural changes in the analogs, particularly at the R5 position of the imidazole ring. No cross-reactivity towards either antibody was observed with the natural ligand angiotensin II, the peptide antagonist saralasin, or the AT2 selective nonpeptide antagonist PD123177.