Ganesh D. Kini

Enhanced oral absorption and antiviral activity of 1-O-octadecyl-sn-glycero-3-phospho-acyclovir and related compounds in hepatitis b virus infection, in vitro☆

Acyclovir (ACV) triphosphate and azidothymidine (AZT) triphosphate inhibit the DNA polymerase of human hepatitis B virus (HBV) by 50% at submicromolar concentrations, but no effects of ACV or AZT treatment have been noted on the clinical manifestations of hepatitis B. We synthesized 1-O-octadecyl-sn-glycero-3-phospho-acyclovir (ODG-P-ACV), 1-O-hexadecylpropanediol-3-phospho-acyclovir (HDP-P-ACV), and 1-O-octadecyl-sn-glycero-3-phospho-azidothymidine (ODG-P-AZT), and evaluated their antiviral activity in human hepatoma cells that constitutively produce HBV (2.2.15 cells). ACV and AZT up to 100 microM caused only slight inhibition of HBV replication in 2.2.15 cells. However, HDP-P-ACV and ODG-P-ACV inhibited viral replication by 50% at 0.5 and 6.8 microM, respectively. ODG-P-AZT also showed increased antiviral activity, with a 50% reduction in HBV replication at 2.1 microM. Based on the EC50, HDP-P-ACV, ODG-P-ACV, and ODG-P-AZT were > 200, > 14.7, and > 48 times more active than their free nucleosides in reducing HBV replication in 2.2.15 cells. To evaluate the biochemical basis for the increased antiviral activity, we studied the uptake and metabolism of 1-O-octadecyl-sn-glycero-3-phospho-[3H]acyclovir (ODG-P-[3H]ACV) in HepG2 cells. Cellular uptake of ODG-P-[3H]ACV was found to be substantially greater than that of [3H]ACV, and cellular levels of ACV-mono-, -di-, and -triphosphate were much higher with ODG-P-ACV. ODG-P-[3H]ACV was well absorbed orally. Based on urinary recovery of tritium after oral or parenteral administration of the radiolabeled compounds, oral absorption of ODG-P-ACV in mice was 100% versus 37% for ACV. ODG-P-ACV plasma area under the curve was more than 7-fold greater than that of ACV. Lipid prodrugs of this type may be useful orally in treating viral diseases.

Alkylglycerol Prodrugs of Phosphonoformate Are Potent In Vitro Inhibitors of Nucleoside-Resistant Human Immunodeficiency Virus Type 1 and Select for Resistance Mutations That Suppress Zidovudine Resistance

ABSTRACT Phosphonoformate (foscarnet; PFA) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), but its use for the treatment of HIV-1 infection is limited by toxicity and the lack of an orally bioavailable formulation. Alkylglycerol-conjugated prodrugs of PFA (1-O-octadecyl-sn-glycero-3-PFA [B-PFA]) having sn-2 substituents of hydrogen (deoxybatyl-PFA [DB-PFA]), methyl (MB-PFA), or ethyl (EB-PFA) are more-potent inhibitors of wild-type HIV-1 in vitro than unmodified PFA and are orally bioavailable in mice. We have evaluated the activities of these compounds against a panel of nucleoside-resistant HIV-1 variants and have characterized the resistant variants that emerge following in vitro selection with the prodrugs. Except for an HIV-1 variant encoding the K65R mutation in RT that exhibited 3.3- to 8.2-fold resistance, the nucleoside-resistant viruses included in the panel were sensitive to the PFA prodrugs (<3-fold increase in 50% inhibitory concentration), including multinucleoside-resistant variants encoding the Q151M complex of mutations or the T69S[SA] insert. Viruses resistant to the PFA prodrugs (>10-fold) were selected in vitro after 15 or more serial passages of HIV-1 in MT-2 cells in escalating prodrug concentrations. Mutations detected in the resistant viruses were S117T, F160Y, and L214F (DB-PFA); M164I and L214F (MB-PFA); and W88G and L214F (EB-PFA). The S117T, F160Y, and M164I mutations have not been previously identified. Generation of recombinant viruses encoding the single and double mutations confirmed their roles in prodrug resistance, including 214F, which generally increased the level of resistance. When introduced into a zidovudine (AZT)-resistant background (67N 70R 215F 219Q), the W88G, S117T, F160Y, and M164I mutations reversed AZT resistance. This suppression of AZT resistance is consistent with the effects of other foscarnet resistance mutations that reduce ATP-dependent removal of AZT monophosphate from terminated template primers. The favorable activity and resistance profiles of these PFA prodrugs warrant their further evaluation as clinical candidates.

Lipid prodrugs of phosphonoacids: greatly enhanced antiviral activity of 1-O-octadecyl-sn-glycero-3-phosphonoformate in HIV-1, HSV-1 and HCMV-infected cells, in vitro.

Phosphonoformate (PFA) effectively inhibits viral polymerases but is relatively ineffective in virus-infected cells in tissue culture. A lipid prodrug of phosphonoformate was synthesized by coupling the phosphonate residue of phosphonoformate to the sn-3 hydroxyl of 1-O-octadecyl-sn-glycerol. This prodrug, 1-O-octadecyl-sn-glycero-3-phosphonoformate (ODG-PFA), was 93-fold more active than phosphonoformate in cells infected with the AD169 strain of cytomegalovirus (CMV), and 111-147-fold more active in cells infected with three human clinical isolates of CMV. The compound was also 44-fold more active in human immunodeficiency virus-1 (HIV-1) infected cells and 43-fold more active in cells infected with herpes simplex virus (HSV). Studies of the mechanisms of increased antiviral activity indicate that 1-O-octadecyl-sn-glycero-3-[14C]phosphonoformate is taken up more extensively than the free drug by the host MRC-5 human lung fibroblasts. Intracellular enzymes convert 1-O-octadecyl-sn-glycero-3-phosphonoformate to phosphonoformate. This conversion does not occur in the tissue culture medium containing fetal bovine serum (FBS) or in MRC-5-conditioned medium. In view of its greatly increased in vitro potency and selectivity, 1-O-octadecyl-sn-glycero-3-phosphonoformate may be useful in treating viral diseases.

Alkoxy propane prodrugs of foscarnet: effect of alkyl chain length on in vitro antiviral activity in cells infected with HIV-1, HSV-1 and HCMV.

The identification of more effective and less toxic foscarnet (PFA) analogs for antiviral therapy would be useful. We recently synthesized 1-O-octadecyl-sn-glycero-3-phosphonoformic acid (ODG-PFA) and noted a 93-fold increase in its anti-HCMV activity relative to PFA. In addition, the antiviral activity of ODG-PFA in herpes simplex virus type-1 (HSV-1) and human immunodeficiency virus type-1 (HIV-1) infected cells was increased 40-fold relative to PFA (Hostetler et al., 1996. Antiviral Res. 31, 59). To evaluate structure-activity relationships further, we synthesized alkoxypropyl esters of foscarnet with varying alkyl chain lengths and degrees of saturation. These compounds were tested in vitro for activity and selectivity in comparison with PFA and ODG-PFA in cells infected with HCMV, HSV-1 or HIV-1. Antiviral activity was strongly dependent on chain length with alkyl ethers 14-18 carbon atoms long exhibiting the greatest antiviral activity against HCMV and HSV-1. In HIV-infected HT4-6C cells, optimal activity was observed at 18-22 carbon chain lengths. The antiviral activities of 1-octadecyloxypropane-3-PFA and 1-docosyloxypropane-3-PFA were 135- and 338-fold greater than that of PFA in HT4-6C cells infected with HIV-1. This also represents a 2.6-6-fold improvement in antiviral activity over ODG-PFA, the previously reported analog.

Synthesis and Antiviral Evaluation of 1-O-Hexadecylpropanediol-3-P-acyclovir: Efficacy Against HSV-1 Infection in Mice

Abstract We synthesized, 1-O-hexadecylpropanediol-3–P-acyclovir, an orally bioavailable lipid prodrug of acyclovir and evaluated it for in vitro and in vivo activity against herpes simplex virus infections. Although 1-O-hexadecylpropanediol-3–P-acyclovir was less active in vitro than acyclovir, on a molar basis it was 2.4 times more active orally in preventing mortality from acute HSV-1 infection in mice. In vitro, 1–O-hexadecylpropanediol-3–P-acyclovir was also more active than acyclovir in a thymidine kinase negative mutant strain of HSV-1 (DM21) and had somewhat higher activity in cytomegalovirus infection in vitro due to its ability to bypass thymidine kinase.

Synthesis and antiviral activity of 1-O-octadecyl-2-O-alkyl-sn-glycero-3-foscarnet conjugates in human cytomegalovirus-infected cells

A series of new lipid prodrugs with the general structure, 1-O-octadecyl-2-X-sn-glycero-3-PFA were synthesized and evaluated for antiviral activity in HCMV-infected human lung fibroblasts (X is -H, -OH or an O-alkyl group of increasing chain length) in order to study structure-activity relationships of PFA lipid prodrugs. The EC50 values for the 2-O-octyl, 2-O-butyl, 2-H, 2-OH, 2-O-methyl and 2-O-ethyl substituted analogs were 1.96, 0.36, 1.0, 0.7, 0.53 and 0.18 microM respectively versus 40 microM for PFA, representing increases in antiviral activity of 20-220 fold. We also synthesized the enantiomer of ODG-PFA, 3-O-octadecyl-sn-glycero-1-PFA, and found that the antiviral activity of both enantiomers as well as the racemate were not significantly different, with EC50 values in the range of 0.67-0.71 microM.

In vitro anti-HIV-1 activity of sn-2-substituted 1-O-octadecyl-sn-glycero-3-phosphonoformate analogues and synergy with zidovudine.

Monoalkyl ether lipid analogues of foscarnet (phosphonoformate, PFA) exhibit substantially greater in vitro antiviral activity than unmodified PFA against human immunodeficiency virus type 1 (HIV-1). Our previous studies indicate that the length of the alkyl chain must be 14–22 carbons for optimal antiviral activity. To further evaluate the structure-activity relationship, we prepared 1-O-octadecyl-sn-glycerol analogues of PFA with various substitutions at the sn-2 position of glycerol and determined the effect of structure on in vitro antiviral activity and selectivity against HIV-1 in MT-2 and CD4-expressing HeLa cells (HT4–6C). We also studied combinations of zidovudine with PFA, 1-O-octadecyl-2-O-methyl-sn-glycero-3-PFA, or 1-O-octadecyl-sn-glycero-3-PFA and calculated their combination index values against HIV-1 in HT4–6C cells. Alkyl substitutions of one to four carbons at the sn-2 position of glycerol showed optimal antiviral activity. Both alkyl ether lipid analogues were strongly synergistic with zidovudine over a wide range of drug ratios and concentrations. 1-O-octadecyl-sn-glycerol analogues of PFA have selective antiviral properties and warrant further evaluation as potential antiretroviral drugs.

Inhibition of pyrimidine metabolism in myeloid leukemia cells by triazole and pyrazole nucleosides.

Two triazole nucleosides, 1 (3-beta-D-ribofuranosyl-1,2,4-triazole-5-carboxamide) and 2 (2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide), and a pyrazole nucleoside, 3 (1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide), were found to inhibit pyrimidine nucleotide biosynthesis in the human myeloid leukemia cell line, K562. Cells treated with these inhibitors released orotate in quantities of 8-35 nmol/10(5) cells/day. Treatment with these compounds caused the K562 cells to accumulate in the S phase of the cell cycle and induced the cells to synthesize hemoglobin.

Nucleoside Peptides. 8. Synthesis of Certain Peptide Derivatives of Ribavirin and Tiazofurin

Abstract The synthesis of certain peptide derivatives of ribavirin and tiazofurin in which the peptide linkage is on the carbamoyl group of the aglycon moiety has been accomplished. Condensation of 1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic acid (3) with glycine ethyl ester, L-aspartic acid dimethyl ester, L-glutamic acid diethyl ester or L-phenylalanine methyl ester in the presence of HOBT/EDC gave the corresponding protected nucleoside peptide esters (4, 6, 8 and 10) respectively, which on ammonolysis furnished the glycineamide (5), aspartic acid diamide (7), glutamic acid diamide (9) and phenylalanineamide (11) derivatives of ribavirin. A similar treatment of 2-β-D-ribofuranosylthiazole-4-carboxylic acid with L-glutamic acid diethyl ester or L-aspartic acid dimethyl ester, followed by ammonolysis gave glutamic acid diamide (17) and aspartic acid diamide (13) derivatives of tiazofurin. None of these compounds exhibited any significant antitumor or antiviral activity in cell cul...

Antiviral and Immunoenhancing Properties of 7-Thia-8-Oxoguanosine and Related Guanosine Analogues

7-thia-8-oxoguanosine (TOGuo) is the first reported structure of a family of modified guanosine analogues exhibiting antiviral activity in rodent models. Its spectrum of action includes interferon-sensitive viruses such as alphaviruses, bunyaviruses, corona viruses, flaviviruses, picornaviruses and, to a lesser extent, herpesviruses. Early treatment before or shortly after virus challenge is necessary to protect animals from mortality. The protective effect of TOGuo against Semliki Forest and Punta Toro viruses can be eliminated by co-treatment with antibody to alpha/ beta-interferon. indicating that interferon induction is of prime importance for antiviral activity against these two viruses. Immunological studies indicate that the nucleoside induces alpha-interferon and activates B cells, natural killer cells, antibody-dependent cytotoxic cells and macrophages. TOGuo also has adjuvant activity, since its use in combination with a killed Ll210 leukemia cell vaccine greatly reduced the mortality of mice inoculated with live Ll210 leukemia cells compared with the vaccine used alone. Several related guanosine analogues show similar antiviral and immunoenhancing properties, including 7-methyl-8-oxoguanosine, 7-methyl-8-thioxoguanosine and 7-deazaguanosine. These studies indicate that certain modifications at the 7 and 8 positions of guanosine may confer antiviral and immunostimulatory properties to nucleoside analogues.