Laurent Placidi

Antiviral l-Nucleosides Specific for Hepatitis B Virus Infection

ABSTRACT 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 β-l-2′-deoxyribose of the β-l-2′-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides β-l-2′-deoxycytidine, β-l-thymidine, and β-l-2′-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (α, β, and γ) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 108 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.

Pharmacology of β-l-Thymidine and β-l-2′-Deoxycytidine in HepG2 Cells and Primary Human Hepatocytes: Relevance to Chemotherapeutic Efficacy against Hepatitis B Virus

ABSTRACT β-l-Thymidine (l-dT) and β-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 μM 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/106 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/106 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/106 cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to l-dC, the 5′-monophosphate and 5′-triphosphate derivatives of β-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/106 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/106 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.

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

Abstract 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 3.54μM. 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.

Reduction of 3'-azido-3'-deoxythymidine to 3'-amino-3'-deoxythymidine in human liver microsomes and its relationship to cytochrome P450

The formation of 3′‐amino‐3′‐deoxythymidine (AMT) in patients receiving 3′‐azido‐3′‐deoxythymidine (zidovudine) and the potential role of this metabolite in zidovudine‐induced toxicity was recently demonstrated by our laboratory. This study evaluated the formation of AMT versus cytochrome P450 (P450) content, cytochrome B5 (B5) content and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)‐cytochrome P450 reductase activity in human liver microsomes obtained from 24 different donors. Significant interindividual differences in total P450 content and P450 reductase activity were observed, whereas no variation was observed in B5 content. Of particular importance, metabolism of zidovudine to AMT varied widely and correlated with P450 content but not with B5 content or P450 reductase activity. The apparent values for the Michaelis‐Menten constant and the maximum rate of metabolism of the reaction were 46.1 mmol/L and 3.5 nmol/min/mg microsomal protein. These large variations of AMT levels as a function of P450 suggest that major interindividual differences may be observed in the pharmacokinetics and formation of this metabolite that may affect the pharmacodynamic properties of zidovudine. Potential drug‐drug interactions may occur with therapeutic agents that interact with or induce P450 (zidovudine).

ANTI-HBV SPECIFIC β-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 β-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 β-L-2′,3'Dideoxyadenosine, a Potent Inhibitor of HIV and HBV Replication

The β-L-nucleoside analogue β-L-2′,3′-dideoxy adenosine (β-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 β-L-2′,3′-dideoxyadenosine-5′-monophosphate (β-L-ddAMP) (Placidi et al., 2000). However, the nucleotide β-L-2′,3′-dideoxyadenosine-5′-triphosphate (β-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 μM without inhibiting human DNA polymerases α, β, or γ up to a concentration of 100 μM. These results suggested that prodrugs of β-L-ddAMP may bypass the poor metabolic activation of β-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of β-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, β-L-ddAMP-bis(tButylSATE) was synthesized. β-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 β-L-ddAMP-bis(tButylSATE) demonstrated that β-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of β-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCS, respectively. The intracellular concentrations of β-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.

Intracellular Metabolism of β-l-2′,3′-Dideoxyadenosine: Relevance to Its Limited Antiviral Activity

ABSTRACT The intracellular metabolism of the β-l- enantiomer of 2′,3′-dideoxyadenosine (β-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 μM [2′,3′,8-3H]-β-l-ddA resulted in an increased intracellular concentration of β-l-ddA with time, demonstrating that these cells were able to transport β-l-ddA. However, it did not result in the phosphorylation of β-l-ddA to its pharmacologically active 5′-triphosphate (β-l-ddATP). Five other intracellular metabolites were detected and identified as β-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/106 cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a β-glucuronic derivative of β-l-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with β-l-ddA in the presence of increasing concentrations of 5′-methylthioadenosine resulted in decreased phosphorolysis of β-l-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of β-l-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of β-l-ddA by methylthioadenosine phosphorylase (EC2.4.2.28).

Intravenous Infusion of Cereport Increases Uptake and Efficacy of Acyclovir in Herpes Simplex Virus-Infected Rat Brains

ABSTRACT The outcome of herpes simplex virus (HSV) infections manifesting as encephalitis in healthy or immunocompromised individuals is generally very poor with mortality rates of about 8 to 28% with treatment. The long-term prognosis of survivors is often problematic, posing the need for alternative treatments that may decrease the mortality and morbidity associated with herpes encephalitis. This study addresses one such approach that includes a temporary permeabilization of the blood-brain barrier during treatment with acyclovir (ACV). In these studies we utilized a synthetic bradykinin analog, Cereport (RMP-7), in conjunction with ACV to treat HSV infection of the brain in a rat model. Cereport, infused intravenously via the jugular vein, was shown to increase [14C]ACV uptake in both the HSV-1-infected and -uninfected rat brain by approximately two- to threefold, correlating with enhanced efficacy of ACV in various brain compartments. In another series of experiments to determine efficacy, various doses of unlabeled ACV were administered during infusion with RMP-7. The decrease in viral titers in the temporal regions of the brain after 5 days of treatment suggested that this approach enhanced the efficacy of ACV treatment. These data indicated that Cereport infused with ACV enhances both the penetration and efficacy of this drug in the treatment of an experimental HSV-1 infection of the rat brain.

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

Abstract 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/106 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/106 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%.

Toxicological study of the anti-HBV agent β-L-thymidine

Publisher Summary β-L-thymidine (L-dT) is a potent antiviral nucleoside against hepatitis B virus (HBV) replication in cell culture, and it has a favorable in vitro toxicity profile. L-dT is an unsubstituted, unmodified P-L-deoxynucleoside with potent antiviral activity against hepadnaviruses in vitro and in vivo and a favorable pharmacological profile. As part of its development as a potential oral anti-HBV agent, a series of preclinical studies in vitro and in laboratory animals were conducted to delineate the cytotoxic, genotoxic, toxicologic, and toxicokinetic profiles of L-dT. In vivo efficacy studies conducted on the chronically infected woodchucks indicated that L-dT has a potent antihepadnaviral activity, decreasing plasma viral DNA loads by up to 8 logs. L-dT is nontoxic and nonclastogenic at doses much greater than those most likely to be used clinically. The potent antiviral efficacy and low toxicity in vitro and in vivo make L-dT an attractive candidate for further development as an anti-HBV agent.