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 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.

Treatment of isografted 9L rat brain tumors with beta-5-o-carboranyl-2'-deoxyuridine neutron capture therapy.

beta-5-o-Carboranyl-2'-deoxyuridine (D-CDU) is a nontoxic pyrimidine nucleoside analogue designed for boron neutron capture therapy of brain tumors. In vitro studies indicated that D-CDU accumulates to levels 92- and 117-fold higher than the extracellular concentration in rat 9L and human U-251 glioma cells, respectively, and persists for several hours at levels 5-fold higher than the extracellular concentration. Furthermore, D-CDU was not toxic to rats injected i.p. with up to 150 mg/kg. On the basis of these studies, D-CDU was evaluated as a neutron capture therapy agent using rats bearing stereotactically implanted intracranial 9L tumors at single i.p. doses of 30 mg/kg and 150 mg/kg of D-CDU (20% 10B enriched), given 2 h before irradiation with thermal neutrons. Boron concentrations in tumors 2 h after dosing were 2.3 +/- 1.6 and 7.4 +/- 1.3 micrograms boron/g tissue (mean +/- SD), corresponding to tumor/brain ratios of 11.5 +/- 3.6 and 6.8 +/- 2.0 micrograms boron/g tissue for the low and high doses, respectively. All untreated animals died within 28 days, whereas half survived at days 32, 55, and 38 for groups receiving neutrons only, 30 mg/kg D-CDU, and 150 mg/kg D-CDU, respectively. Odds ratios of all treatment groups differed significantly from the untreated group (P < 0.002; logrank test). The median survival time for the 30 mg/kg-treated group but not for the 150 mg/kg-treated group was significantly longer than for rats treated with neutrons only (P = 0.036), which may correlate with the decreased tumor selectivity for D-CDU observed at the higher dose. Additional pharmacodynamic studies are warranted to determine optimal dosing strategies for D-CDU.

Synthesis and biological evaluation of 2',3'-didehydro-2',3'- dideoxy-5-fluorocytidine (D4FC) analogues: discovery of carbocyclic nucleoside triphosphates with potent inhibitory activity against HIV-1 reverse transcriptase.

The discovery of a novel cytosine nucleoside, beta-D-2', 3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D-D4FC), as a potent antihuman immunodeficiency virus (HIV) agent led us to synthesize a series of analogues and derivatives of beta-D-D4FC that could be more selective and also possess increased glycosidic bond stability. The synthesized D-D4FC analogues were evaluated for anti-HIV-1 activity, anticancer activity, and cytotoxicity in various cells. The biological data demonstrated that the 5-substitution of beta-D-D4FC with bromine (6c) and iodine (6d) resulted in the loss of antiviral activity, and the alpha-D anomer (7a) of D-D4FC was also devoid of activity. The 5-fluorouracil analogues (6b and 7b) of D-D4FC were less potent and more cytotoxic than the parent compound, whereas the beta-L-D4FU (11) showed both potent anti-HIV-1 activity and cytotoxicity. N4- and 5'-O-acyl derivatives (17, 15a-c) of beta-D-D4FC exhibited comparable antiviral activity to beta-D-D4FC. In contrast, the N4-isopropyl derivative (20) of beta-D-D4FC was not active against HIV-1, even at 100 microM. The carbocyclic analogues (26a,b) of D4FC demonstrated weak activity against HIV-1 and no toxicity in various cells. The triphosphates (27a,b) of the carbocyclic nucleosides demonstrated potent inhibitory activity against recombinant HIV-1 reverse transcriptase at submicromolar concentrations. Of the compounds tested as potential anticancer agents, beta-D-, alpha-D-, and beta-L-D4FU (6b, 7b, 11) showed inhibitory activity against rat glioma and modest activity against human lung carcinoma, lymphoblastoid, and skin melanoma cells.

A rapid non-culture-based assay for clinical monitoring of phenotypic resistance of human immunodeficiency virus type 1 to lamivudine (3TC).

Monitoring for lamivudine (3TC) resistance is important both for the clinical management of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with 3TC and for surveillance of transmission of 3TC-resistant HIV-1. We developed a novel non-culture-based assay for the rapid analysis of phenotypic resistance to 3TC of HIV-1 in plasma. The assay measures the susceptibility of HIV-1 reverse transcriptase (RT) activity to 3TC triphosphate (3TC-TP) in plasma. RT detection was done by the Amp-RT assay, an ultrasensitive PCR-based RT assay. Under our assay conditions, we found that 5 microM 3TC-TP inhibited RT activity from wild-type (WT), zidovudine-resistant, or nevirapine-resistant HIV-1 but not from HIV-1 carrying either the M184V mutation or multidrug (MD) resistance mutations (77L/116Y/151M or 62V/75I/77L/116Y/151M). Mixing experiments showed a detection threshold of 10% 3TC-resistant virus (M184V) in a background of WT HIV-1. To validate the assay for the detection of phenotypic resistance of HIV-1 to 3TC in plasma samples, HIV-1 RT in 30 plasma specimens collected from 15 patients before and during therapy with 3TC was tested for evidence of phenotypic resistance by the Amp-RT assay. The results were compared with those of genotypic analysis. The RT in 12 samples was found to be 3TC sensitive, while the RT in 18 samples had evidence of phenotypic resistance. All 12 samples with 3TC-sensitive RT had WT genotypes at codon 184 and were retrieved before treatment with 3TC. In contrast, all 18 specimens with 3TC-resistant RT were posttherapy samples. This assay provides a simple, rapid, and reliable method for the detection of phenotypic resistance of HIV-1 to 3TC in plasma.

In-vitro activities of paromomycin and lasalocid evaluated in combination against Cryptosporidium parvum.

Using a chemiluminescence immunoassay, paromomycin and lasalocid were shown to inhibit Cryptosporidium parvum growth in Madin-Darby canine kidney cells in a concentration-dependent manner. The median effective concentrations (EC50s) for paromomycin and lasalocid were 1184 mg/L and 0.4 mg/L, respectively. Neither drug was cytotoxic to host cells at concentrations up to five times their EC50s. Drug combination studies were conducted and the resulting data were analysed by the median-effect principle and combination index method. Statistically significant synergy was observed when combinations of paromomycin and lasalocid were used at ratios of 5000:1 and 2500:1. A possible mechanism for synergy is discussed.

Carboranyl oligonucleotides. 3. Biochemical properties of oligonucleotides containing 5-(o-carboranyl-1-yl)-2'-deoxyuridine.

Boronated oligonucleotides are potential candidates for boron neutron capture therapy, antisense technology, and as tools in molecular biology. The biological properties of dodecathymidylic acids containing one or more 5-(o-carboran-1-yl)-2'-deoxyuridine residues at different locations within the oligonucleotide chain were studied. 5-(o-Carboran-1-yl)-2'-deoxyuridine containing oligonucleotides manifested marked increased lipophilicity and resistance to 3'- or 5'-phosphodiesterases compared to the corresponding unmodified oligomer. They were substrates for T4 polynucleotide kinase and primers for Escherichia coli polymerase I and human immunodeficiency virus type 1 reverse transcriptase but not for human DNA polymerase alpha and beta. They also formed heteroduplexes that were substrates for E. coli RNase H, an essential property for antisense technology. These studies indicate that the carboranyl-containing oligonucleotides have desirable properties that need to be exploited further in the design of novel biopharmaceuticals.