Monoval-LdC: efficient prodrug of 2'-deoxy-beta-L-cytidine (L-dC), a potent and selective anti-HBV agent.

In order to improve the oral bioavailability of LdC, valinyl esters were prepared as prodrugs. We report here the syntheses of the 3'-mono-, 5'-mono, and 3',5'-di-O-valinyl esters of LdC. The comparison of their ease of synthesis, their physicochemical properties, as well as their pharmacokinetic parameters in cynomologus monkeys has revealed 3'-mono-O-valinyl derivative as the most promising of the studied prodrugs. This compound is being developed as a new anti-HBV agent.

Pharmacology of beta-L-thymidine and beta-L-2'-deoxycytidine in HepG2 cells and primary human hepatocytes: relevance to chemotherapeutic efficacy against hepatitis B virus.

beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 microM in 2.2.15 cells). The intracellular metabolisms of L-dT and L-dC were investigated in HepG2 cells and primary cultured human hepatocytes. L-dT and L-dC were extensively phosphorylated in both cell types, with the 5'-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5'-triphosphate levels were 27.7 +/- 12.1 and 72.4 +/- 1.8 pmol/10(6) cells for L-dT and L-dC, respectively. In primary human hepatocytes, the 5'-triphosphate levels were 16.5 +/- 9.8 and 90.1 +/- 36.4 pmol/10(6) cells for L-dT and L-dC, respectively. Furthermore, a choline derivative of L-dCDP was detected at concentrations of 15.8 +/- 1.8 and 25.6 +/- 0.1 pmol/10(6) cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of beta-L-2'-deoxyuridine (L-dUMP and L-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 +/- 0.4 and 18.2 +/- 1.0 pmol/10(6) cells, respectively. In human hepatocytes, L-dUMP and L-dUTP were detected at concentrations of 5.7 +/- 2.4 and 43.5 +/- 26.8 pmol/10(6) cells, respectively. It is likely that deamination of L-dCMP by deoxycytidylate deaminase leads to the formation of L-dUMP, as the parent compound, L-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of L-dTTP, L-dCTP, and L-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to L-dT in combination with L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of L-dT and L-dC are associated with their extensive phosphorylation.

Antiviral beta-L-nucleosides specific for hepatitis B virus infection.

Three simple, related nucleosides, beta-L-2'-deoxycytidine (LdC), beta-Lthymidine (LdT), and beta-L-2'-deoxyadenosine (LdA), have been discovered to be potent, specific and selective inhibitors of the replication hepatitis B virus (HBV), as well as the closely related duck and woodchuck hepatitis viruses (WHV). Structure-activity relationship analysis indicates that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific anti-hepadnavirus activity. The simple nucleosides had no effect on the replication of 15 other RNA and DNA viruses, and did not inhibit human DNA polymerases (alpha, beta and gamma) or compromise mitochondrial function. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. Once-daily oral administration of these compounds in the woodchuck efficacy model of chronic HBV infection reduced viral load by as much as 10(8) genome equivalents/ml serum and there was no drug-related toxicity. In addition, a decline in WHV surface antigen (WHsAg) paralleled the decrease in viral load. This class of nucleosides displays an excellent overall safety profile. The first compound, LdT, has already entered clinical trials and LdC, currently being developed as a prodrug, is expected to enter the clinic in the near future. These compounds have the potential for use in combination therapy with the goal of achieving superior viral suppression and diminishing the onset of resistance.

Anti-HBV specific beta-L-2'-deoxynucleosides.

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

Antiviral activity and intracellular metabolism of bis(tButylSATE) phosphotriester of beta-L-2',3'dideoxyadenosine, a potent inhibitor of HIV and HBV replication.

The beta-L-nucleoside analogue beta-L-2',3'-dideoxy adenosine (beta-L-ddA) has been shown to exhibit limited antiviral activities. This was attributed to its rapid catabolism through cleavage of the glycosidic bond and poor phosphorylation to the nucleotide beta-L-2',3'-dideoxyadenosine-5'-mono phosphate (beta-L-ddAMP) (Placidi et al., 2000). However, the nucleotide beta-L-2',3'-dideoxyadenosine-5'-triphosphate (beta-L-ddATP) inhibited the activity of both HIV-1 reverse transcriptase (RT) and viral DNA polymerase isolated from woodchuck hepatitis virus-infected serum (a model of hepatitis B) with an inhibitory concentration (IC50) of 2.0 microM without inhibiting human DNA polymerases alpha, beta, or gamma up to a concentration of 100 microM. These results suggested that prodrugs of beta-L-ddAMP may bypass the poor metabolic activation of beta-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of beta-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, beta-L-ddAMP-bis(tButylSATE) was synthesized. Beta-L-ddAMP-bis(tButylSATE) inhibited HIV replication in human peripheral blood mononuclear cells (PBMCs) and HBV replication in 2.2.15 cells with effective concentrations (EC50s) of 2 and 80 nM, respectively. Intracellular metabolism of beta-L-ddAMP-bis(tButylSATE) demonstrated that beta-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of beta-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCs, respectively. The intracellular concentrations of beta-L-ddATP were maintained above the EC50 for the inhibition of HIV RT and hepatitis B virus (HBV) for as long as 24 h after removal of the drug.

Antiviral L-nucleosides specific for hepatitis B virus infection.

A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides beta-L-2'-deoxycytidine, beta-L-thymidine, and beta-L-2'-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (alpha, beta, and gamma) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10(8) genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

In vitro and in vivo metabolism and pharmacokinetics of bis [(t-butyl)-S-acyl-2-thioethyl]-beta-L-2',3'-dideoxy-5-fluorocytidine monophosphate.

Exposure to 10 &M L-FddCMP-bisSATE led to formation of intracellular L-FddCTP levels of 410.1(+/-) +/- 46.2 and 242.1 +/- 13.2 pmol/10(6) cells in unstimulated and PHAstimulated PBM cells, respectively; whereas, exposure of cells to the parent nucleoside, L-FddC, generated 5-10-fold less L-FddCTP. In Hep-G2 cells and EGF/HGF stimulated and unstimulated primary cultured hepatocytes, the active metabolite reached 113 +/- 29, 23.9 +/- 15.6, and 20.6 +/- 10.5 pmol/10(6) cells. Three other metabolites, L-FddCMP-monoSATE, L-FddCMP-SH, and M I, were detected intracellularly and extracellularly in all cell types examined. Intravenous administered dose of 3 mg/kg L-FddCMP-bisSATE to rhesus monkeys resulted in plasma concentration levels of 2.06 +/- 1.00 and 0.39 +/- 0.15 &M of L-FddCMP-monoSATE and L-FddC, respectively, while the prodrug was completely cleared metabolically within 15 min. Following oral administration of an equivalent dose, the absolute oral bioavailability of L-FddC derived from L-FddCMP-bisSATE administration was 65%.

Intracellular metabolism of beta-L-2',3'-dideoxyadenosine: relevance to its limited antiviral activity.

The intracellular metabolism of the beta-L- enantiomer of 2', 3'-dideoxyadenosine (beta-L-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 microM [2',3',8-(3)H]-beta-L-ddA resulted in an increased intracellular concentration of beta-L-ddA with time, demonstrating that these cells were able to transport beta-L-ddA. However, it did not result in the phosphorylation of beta-L-ddA to its pharmacologically active 5'-triphosphate (beta-L-ddATP). Five other intracellular metabolites were detected and identified as beta-L-2', 3'-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 +/- 0.95, 8.15 +/- 2.64, and 15.60 +/- 1.74 pmol/10(6) cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a beta-glucuronic derivative of beta-L-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with beta-L-ddA in the presence of increasing concentrations of 5'-methylthioadenosine resulted in decreased phosphorolysis of beta-L-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of beta-L-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of beta-L-ddA by methylthioadenosine phosphorylase (EC 2.4.2.28).

Intracellular metabolism of beta-L-ddAMP-bis(tbutylSATE), a potent inhibitor of hepatitis B virus replication.

beta-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC50 = 0.1 microM. Following a 0- to 72-hrs exposure of human hepatocytes to a 10 microM [2',3'-3H] beta-L-ddAMP-bis(tbutylSATE), the pharmacologically active beta-L-ddATP was the predominant metabolite attaining a concentration of 268.53 +/- 107.97 pmoles/10(6) cells at 2 hrs. In Hep-G2 cell, beta-L-ddATP accounted for 146.8 +/- 29.8 pmoles/10(6) cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of beta-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.

Pharmacokinetics of beta-L-2',3'-dideoxy-5-fluorocytidine in rhesus monkeys.

beta-L-2',3'-Dideoxy-5-fluorocytidine (beta-L-FddC), a novel cytidine analog with an unnatural beta-L sugar configuration, has been demonstrated by our group and others to exhibit highly selective in vitro activity against human immunodeficiency virus types 1 and 2 and hepatitis B virus. This encouraging in vitro antiviral activity prompted us to assess its pharmacokinetics in rhesus monkeys. Three monkeys were administered an intravenous dose of [3H] beta-L-FddC at 5 mg/kg of body weight. Following a 3-month washout period, an equivalent oral dose was administered. Plasma and urine samples were collected at various times for up to 24 h after dosing, and drug levels were quantitated by high-pressure liquid chromatography. Pharmacokinetic parameters were obtained on the basis of a two-compartment open model with a first-order elimination from the central compartment. After intravenous administration, the mean peak concentration in plasma (Cmax) was 29.8 +/- 10.5 microM. Total clearance, steady-state volume of distribution, terminal-phase plasma half-life (t1/2 beta), and mean residence time were 0.7 +/- 0.1 liters/h/kg, 1.3 +/- 0.1 liters/kg, 1.8 +/- 0.2 h, and 1.9 +/- 0.2 h, respectively. Approximately 47% +/- 16% of the intravenously administered radioactivity was recovered in the urine as the unchanged drug with no apparent metabolites. beta-L-FddC exhibited a Cmax of 3.2 microM after oral administration, with a time to peak drug concentration of approximately 1.5 h and a t1/2 of 2.2 h. One monkey in the oral administration arm of the study had a significant delay in the absorption of the aqueous administered dose. The absolute bioavailability of orally administered beta-L-FddC ranged from 56 to 66%.

Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine.

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.

Comparative metabolism of the antiviral dimer 3'-azido-3'-deoxythymidine-P-2',3'-dideoxyinosine and the monomers zidovudine and didanosine by rat, monkey, and human hepatocytes.

AZT-P-ddI is an antiviral heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (AZT) and one molecule of 2',3'-dideoxyinosine (ddI) linked through their 5' positions by a phosphate bond. The metabolic fate of the dimer was studied with isolated rat, monkey, and human hepatocytes and was compared with that of its component monomers AZT and ddI. Upon incubation of double-labeled [14C]AZT-P-[3H]ddI in freshly isolated rat hepatocytes in suspension at a final concentration of 10 microM, the dimer was taken up intact by cells and then rapidly cleaved to AZT, AZT monophosphate, ddI, and ddI monophosphate. AZT and ddI so formed were then subject to their respective catabolisms. High-performance liquid chromatography analyses of the extracellular medium and cell extracts revealed the presence of unchanged dimer, AZT, 3'-azido-3'-deoxy-5'-beta-D-glucopyranosylthymidine (GAZT), 3'-amino-3'-deoxythymidine (AMT), ddI, and a previously unrecognized derivative of the dideoxyribose moiety of ddI, designated ddI-M. Trace extracellular but substantial intracellular levels of the glucuronide derivative of AMT (3'-amino-3'-deoxy-5'-beta-D-glucopyranosylthymidine [GAMT]) were also detected. Moreover, the extent of the formation of AMT, GAZT, and ddI-M from the dimer was markedly lower than that with AZT and ddI alone by the hepatocytes. With hepatocytes in primary culture obtained from rat, monkey, and human, large interspecies variations in the metabolism of AZT-P-ddI were observed. While GAZT and ddI-M, metabolites of AZT and ddI, respectively, as well as AZT 5'-monophosphate (MP) and ddI-MP were detected in the extracellular media of all species, AMT and GAMT were produced only by rat and monkey hepatocytes. No such metabolites were formed by human hepatocytes. The metabolic fate of the dimer by human hepatocytes was consistent with in vivo data recently obtained from human immunodeficiency virus-infected patients.

Synthesis, biotransformation, and pharmacokinetic studies of 9-(beta-D-arabinofuranosyl)-6-azidopurine: a prodrug for ara-A designed to utilize the azide reduction pathway.

As a part of our efforts to design prodrugs for antiviral nucleosides, 9-(beta-D-arabinofuranosyl)-6-azidopurine (6-AAP) was synthesized as a prodrug for ara-A that utilizes the azide reduction biotransformation pathway. 6-AAP was synthesized from ara-A via its 6-chloro analogue 4. The bioconversion of the prodrug was investigated in vitro and in vivo, and the pharmacokinetic parameters were determined. For in vitro studies, 6-AAP was incubated in mouse serum and liver and brain homogenates. The half-lives of 6-AAP in serum and liver and brain homogenates were 3.73, 4.90, and 7.29 h, respectively. 6-AAP was metabolized primarily in the liver homogenate microsomal fraction by the reduction of the azido moiety to the amine, yielding ara-A. However, 6-AAP was found to be stable to adenosine deaminase in a separate in vitro study. The in vivo metabolism and disposition of ara-A and 6-AAP were conducted in mice. When 6-AAP was administered by either oral or intravenous route,the half-life of ara-A was 7-14 times higher than for ara-A administered intravenously. Ara-A could not be found in the brain after the intravenous administration of ara-A. However, after 6-AAP administration (by either oral or intravenous route), significant levels of ara-A were found in the brain. The results of this study demonstrate that 6-AAP is converted to ara-A, potentially increasing the half-life and the brain delivery of ara-A. Further studies to utilize the azide reduction approach on other clinically useful agents containing an amino group are in progress in our laboratories.

In vitro and in vivo evaluation of 6-azido-2',3'-dideoxy-2'-fluoro-beta-D-arabinofuranosylpurine and N6-methyl-2',3'-dideoxy-2'-fluoro-beta-D-arabinofuranosyladenine as prodrugs of the anti-HIV nucleosides 2'-F-ara-ddA and 2'-F-ara-ddI.

In an effort to improve the pharmacokinetic properties and tissue distribution of 2'-F-ara-ddI, two lipophilic prodrugs, 6-azido-2'-3'-dideoxy-2'-fluoro-beta-D- arabinofuranosylpurine (FAAddP, 4) and N6-methyl-2'-3'-dideoxy-2'-fluoro-beta-D-arabinofuranosyladenine (FMAddA, 5), were synthesized and their biotransformation was investigated in vitro and in vivo, in mice. Compounds 4 and 5 were synthesized via the intermediate 2. For the in vitro studies, FAAddP and FMAddA were incubated in mouse serum, liver homogenate, and brain homogenate. FAAddP was metabolized in liver homogenate by the reduction of the azido to the amino moiety followed by deamination, yielding 2'-F-ara-ddI. The conversion of FAAddP to 2'-F-ara-ddA was mediated by microsomal P-450 NADPH reductase system, as shown by the liver microsomal assay. FAAddP was also converted to 2'-F-ara-ddI at a slower rate in the brain than in the liver. FMAddA, however, was stable in brain homogenate and was slowly metabolized in the liver homogenate. Metabolic conversion of FMAddA in vitro was stimulated by the addition of adenosine deaminase. In the in vivo metabolism study, FAAddP underwent reduction to 2'-F-ara-ddA followed by deamination to 2'-F-ara-ddI. FMAddA did not result in increased brain delivery of 2'-F-ara-ddI in vivo, probably due to the slow conversion as observed in the in vitro studies. However, there was an increase in the half-life of 2'-F-ara-ddI produced from FMAddA. This report is the first example in the design of prodrugs using the azido group for adenine- and hypoxanthine-containing nucleosides. This interesting and novel approach can be extended to other antiviral and anticancer nucleosides.

Pharmacokinetics and metabolism of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470, AGM-1470) in rhesus monkeys.

The metabolic disposition and pharmacokinetics of TNP-470 were investigated in rhesus monkeys following intravenous administration of 5 mg/kg of [3H]-TNP-470. Rapid and extensive metabolism of parent drug to six metabolites occurred as demonstrated by the absence of unchanged drug in plasma and urine at time points as early as 6 min after administration. Substantial, yet variable, plasma levels of M-IV were detected in all three monkeys with a mean Cmax value of 3.54 microM. Five other metabolites, labeled M-I, M-II, M-III, M-V and M-VI, were also detected in biological fluids of monkeys. M-II, M-V and M-VI exhibited similar kinetic profiles with apparent plasma elimination half-life values of 0.91 +/- 0.37, 2.42 +/- 0.13 and 1.19 +/- 0.29 h respectively. In contrast, M-I, M-III and M-IV exhibited much shorter apparent plasma half-life values of 30 min or less. Urinary recovery within 36 h represented only 19.90 +/- 6.09% of the total administered dose. No radioactivity was detected beyond 36 h and during a 15-day sample collection period, suggesting that nonrenal (biliary) elimination of TNP-470 metabolites is a predominant excretion route in nonhuman primates. This study provides the first detailed in vivo analysis of TNP-470 metabolism and disposition using an animal model highly predictive of humans, consistent with the detection of the same TNP-470 metabolites in human tissues. A detailed understanding of TNP-470 metabolism and disposition is critical to fully elucidate the pharmacodynamic properties of this new anticancer drug as clinical investigations proceed.

Disposition and metabolism of the angiogenic moderator O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM-1470) in human hepatocytes and tissue microsomes.

The biotransformation of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM 1470), a potent in vitro inhibitor of angiogenesis, was investigated in primary cultured human hepatocytes and microsomal fractions of various human tissues. Exposure of human hepatocytes to 5 microM [3H]TNP-470 led to a rapid metabolism of unchanged drug to six metabolic derivatives within 30 min. The predominant extracellular metabolites were M-II and M-IV, attaining a maximum level of 3.23 +/- 0.34 and 0.88 +/- 0.10 microM, respectively. M-II leveled off, while M-IV rapidly declined to 0.06 +/- 0.05 microM by 3 h. TNP-470 was undetectable after 60 min. M-V and M-VI slowly reached maximal concentrations of 0.26 +/- 0.12 and 0.32 +/- 0.16 microM, respectively. M-I only reached a concentration of 0.18 +/- 0.07 microM at 60 min and leveled at 0.13 +/- 0.06 microM for the remaining time of the experiment. The intracellular profile was different, with M-III and M-V representing the major metabolites detected. Studies using human liver microsomes demonstrated that M-IV formation was associated with an esterase-like enzymatic cleavage of TNP-470 and that this metabolite was then further metabolized by microsomal epoxide hydrolase to M-II, as evidenced by inhibition of this metabolic step by cyclohexene oxide, a microsomal epoxide hydrolase inhibitor. Extrahepatic metabolism of TNP-470 was also demonstrated using different sites of human intestinal, stomach, and kidney microsomes, with metabolite M-IV as the principal derivative detected in these tissues. Hepatic microsomal samples from seven different donors demonstrated large interindividual variations in the formation of both M-II and M-IV. In summary, this study demonstrates a rapid and extensive metabolism of TNP-470 in human tissues. The data emphasize the need to evaluate the in vivo formation and extent of TNP-470 metabolites to adequately assess the pharmacodynamic effects of this novel anticancer drug with a novel mechanism of action.