Erika Cretton-Scott

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.

2'-C-Methyl branched pyrimidine ribonucleoside analogues : potent inhibitors of RNA virus replication

RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain ‘natural’ pyrimidine bases, but possess a β-methyl substituent at the 2′-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA), single-stranded negative (ssRNA−) or double-stranded (dsRNA), revealed potent activities for D-2′-C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5′-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2′-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development.

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.

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.

Monoval-LdC: Efficient Prodrug of 2′-Deoxy-β-L-cytidine (L-dC), A Potent and Selective Anti-HBV Agent

Abstract 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.

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).

Pharmacokinetics of β-L-thymidine and β-L-2′-deoxycytidine in woodchucks and monkeys

Publisher Summary Pharmacokinetic studies in woodchucks and monkeys indicated that the disposition of β-L-thymidine (L-dT) is comparable to the pharmacokinetic characteristics of other nucleoside analogs after administration to these animals. Woodchuck is a useful animal model for studying anti-hepatitis B virus (HBV) agents because of many similarities between woodchuck hepatitis virus (WHV) and HBV, hence providing important information on the in vivo efficacy of anti-HBV candidates. The compound has good oral bioavailability and is eliminated and unchanged in urine. 3-L-2’-deoxycytidine (L-dC) exhibits lower oral bioavailability in both species. Several prodrugs that improve the oral bioavailability of L-dC are currently under evaluation. Chronic HBV infection is a major global health problem, affecting approximately 5% of the worlds population. Currently, the only approved treatment options include alpha interferon (INF-α) and lamivudine. Potent in vitro and in vivo efficacy coupled with favorable pharmacokinetic disposition make both L-dT and L-dC promising antiviral candidates for the treatment of 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.

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%.