5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

Synthesis and antiviral effect of phosphamide modified vidarabine for treating HSV 1 infections.

Vidarabine (ARA) was one of the earliest marine-related compounds to be used clinically for antiviral therapy, however, its fast metabolism is the main defect of this drug. To overcome this, we designed and synthesized a group of phosphamide-modified ARA compounds using ProTide technology. With a phosphamide modification, these compounds could become the substrate of specific phospholipase enzymes expressed in the liver. Among all 16 synthesized compounds, most showed stronger activity against herpes simplex virus type 1 (HSV-1) than ARA (EC50 of approximately 10 µM). The top three compounds were compound 2 (EC50 = 0.52 ± 0.04 µM), compound 6 (EC50 = 1.05 ± 0.09 µM) and compound 15 (EC50 = 1.18 ± 0.08 µM) (about 2 times higher than Sp type compound 2). This study provides evidence for use of the phosphamide modification, which could give ARA higher activity and liver cell targeting.

Downregulated SOCS1 expression activates the JAK1/STAT1 pathway and promotes polarization of macrophages into M1 type.

Macrophage polarization is flexible, and involves in different signaling pathways and various transcription factors. Suppressor of cytokine signaling (SOCS) is an important inhibitor of cytokine signaling pathways and also a key physiological regulator for natural and acquired immunity systems. Following transfection of SOCS1 short hairpin (sh)RNA into mouse macrophage cells, reverse transcription­quantitative polymerase chain reaction demonstrated that the mRNA levels of Janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)1 increased significantly. In addition, western blotting indicated that JAK1, STAT1 and p­STAT1 expression was significantly enhanced. Fludarabine can inhibit phosphorylation of STAT1 and SOCS1 expression. When fludarabine was added and SOCS1 shRNA was transfected, the inhibition of fludarabine was weakened, and p­STAT1 expression was upregulated. Flow cytometry detection indicated that, following the downregulation of SOCS1 expression, M1­type cells significantly increased, but the proportion of M2­type cells did not change significantly. Fludarabine can reduce the effect of SOCS1 shRNA on promoting M1­type cell polarization, and macrophages can polarize into both M1 and M2 phenotypes. Further ELISA results presented that, when downregulating SOCS1 expression, interleukin (IL)­4 and IL­10 expression was both downregulated, and tumor necrosis factor (TNF)­α and interferon (IFN)­Î³ expression was significantly upregulated. When adding fludarabine or injecting with the traditional Chinese medicine Xuebijing, IL­4 and IL­10 expression was both significantly upregulated, and TNF­α and IFN­Î³ expression was significantly downregulated. When adding fludarabine and downregulating SOCS1, IL­4, IL­10, TNF­α and IFN­Î³ expression presented no significant changes. The above results indicated that, when SOCS1 expression is downregulated, it will activate the JAK1/STAT1 pathway, and thereby promote the polarization of macrophages into M1 type. The findings are of great importance for understanding occurrence, development and treatment of various immune­related diseases.

Telomerase contributes to fludarabine resistance in primary human leukemic lymphocytes.

We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

Omega 3 fatty acids increase the chemo-sensitivity of B-CLL-derived cell lines EHEB and MEC-2 and of B-PLL-derived cell line JVM-2 to anti-cancer drugs doxorubicin, vincristine and fludarabine.

BACKGROUND: B-Cell chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the United States. Clinical treatment of CLL is often limited due to drug resistance and severe therapy-induced toxicities. We hypothesized that the omega 3 (n-3) fatty acids, eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), would increase the sensitivity of malignant B-lymphocytes to anti-cancer drugs doxorubicin, vincristine and/or fludarabine in vitro and that increased sensitivity is achieved by alterations in cell-cycle progression leading to growth inhibition and/or enhanced cell death. We further postulate that enhanced sensitivity is dependent on the formation of lipid peroxides and to the generation of reactive oxygen species (ROS). METHODS: In the present study, B-CLL-derived leukemic cell lines EHEB and MEC-2 and the B-Prolymphocytic leukemic-derived (PLL) cell line JVM-2 were tested for in vitro sensitivity against doxorubicin, vincristine or fludarabine in the presence or absence of vehicle, arachidonic acid (omega 6), EPA or DHA. Cell cycle analysis and Annexin-V assays were performed to determine cell cycle progression and % apoptotic cells, respectively. Assays for malondialdehyde, a measure of lipid peroxidation, and DCF fluorescence assays, a measure of intracellular ROS, were performed to determine if enhanced sensitivity of cells to the drugs by n-3 was dependent on the formation of ROS. RESULTS: Our results indicated that: 1) EPA and DHA differentially sensitized B-leukemic cell lines EHEB, JVM-2 and MEC-2 to doxorubicin, vincristine and fludarabine in vitro; 2) n-3 alone and with drug treatment increased cell death and induced G2/M arrest in a cell-type specific manner; 3) lipid peroxidation increased in the presence of n-3; 4) there was higher lipid peroxidation in MEC-2 cells in presence of DHA and doxorubicin than with either alone; 5) n-3 increased generation of ROS in MEC-2, and 6) the addition of vitamin-E abrogated the increase in ROS generation and chemo-sensitivity of MEC-2 to doxorubicin by DHA. CONCLUSION: N-3's are promising chemo-sensitizing agents for the treatment of CLL. Selective enhancement of chemo-sensitivity of EHEB, JVM-2 and MEC-2 to drugs by n-3 that is not dependent on increased lipid peroxidation and ROS generation indicates alternative mechanisms by which n-3 enhances chemo-sensitivity.

N-(4-hydroxyphenyl)retinamide promotes apoptosis of resting and proliferating B-cell chronic lymphocytic leukemia cells and potentiates fludarabine and ABT-737 cytotoxicity.

The in vitro effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR, fenretinide) on primary B-cell chronic lymphocytic leukemia (CLL) cells from previously untreated CLL patients were investigated. 4HPR promoted the intrinsic apoptotic pathway by reactive oxygen species (ROS) generation and was accompanied by drop of Mcl-1 protein expression. The latter was not attributable to transcriptional downregulation but to protein degradation mediated by jun N-terminal kinase activation, and likely by NF-kB downregulation and Noxa upregulation. CLL cells stimulated in vitro with CD40L did not increase 4HPR chemoresistance if activation was accompanied by proliferation. Intra-patient analysis confirmed that the proliferating pool of CLL cells was more sensitive to the cytotoxic action of 4HPR than the activated but resting CLL subpopulation. The different 4HPR susceptibility of the two subpopulations was associated with higher Noxa expression in proliferating CLLs. Combination experiments revealed that 4HPR strongly potentiated ABT-737 cytotoxicity, especially in proliferating CLL cells that displayed amplified chemoresistance to ABT-737 alone. Synergic cytotoxicity was also demonstrated in combination with fludarabine, in both resting and stimulated CLL samples. This study entitles 4HPR to be assayed as a chemotherapeutic adjuvant for the treatment of CLL.

Preclinical assessment of curcumin as a potential therapy for B-CLL.

Curcumin, the principle component of the spice turmeric, has been used as an anti-inflammatory medication in India and China for centuries. Recent studies, predominantly using actively dividing cell lines, have suggested that this compound could be used as a chemopreventative or therapeutic agent for epithelial tumors. As curcumin has been reported to inhibit the NIK/IKK complex, an activity that would be expected to induce apoptosis in B cell malignancies, we sought to determine whether curcumin induces apoptosis in vitro in primary chronic lymphocytic leukemia (B-CLL) cells. Primary leukemic cells were incubated with varying dosages of curcumin, followed by assessment for apoptosis. The role of PPARgamma or NF-kappaB signaling in curcumin-induced apoptosis was examined by cotreatment with a PPARgamma antagonist or EMSA of nuclear NFkappaB complexes. We also examined whether a clinically achievable concentration of curcumin (1 microM) would augment the apoptotic effects of fludarabine, dexamethasone, vincristine or the PDE4 inhibitor rolipram. In B-CLL cells from 14 patients, curcumin-induced apoptosis with a mean EC(50) of 5.5 microM. In contrast, the EC(50) for whole mononuclear cells from a healthy donor was 21.8 microM. In a 48 hr wash-out time course, curcumin-induced apoptosis was time-dependent, with a substantial reduction in apoptosis observed when curcumin was removed after 5 hr. Curcumin treatment reduced basal nuclear NF-kappaB levels and 1 microM curcumin augmented both vinca alkaloid and PDE4 inhibitor-induced apoptosis in B-CLL cells. Our studies suggest that curcumin may augment the efficacy of established or experimental therapies for B-CLL.

Preclinical evaluation of gemcitabine combination regimens for application in acute myeloid leukemia.

The DNA antimetabolite gemcitabine is an anticancer agent with shown preclinical and clinical utility and a low toxicity profile. In this study, we sought to identify and optimize drug partners for binary and tertiary combinations with gemcitabine for use in the treatment of acute myelogenous leukemia (AML). Drug interaction was assessed by growth inhibition assay with metabolic end points. The combination index method was used to evaluate combinations of gemcitabine with fludarabine, paclitaxel, chlorambucil, doxorubicin, mitoxantrone, and SN-38 in U937 human AML cells. A three-dimensional method was used to determine the effect of dose ratio and schedule on drug interaction. Mechanisms underlying interactions related to cell cycle effects and apoptosis were assessed by flow cytometric and caspase-3 and -7 assays, respectively. The most synergistic binary combination was gemcitabine + fludarabine. The most synergistic tertiary combination was gemcitabine + fludarabine + paclitaxel, where the interaction was sequence dependent with paclitaxel given before gemcitabine + fludarabine, producing a 2-fold increase in synergy. Cell cycle analysis did not reveal a significant G(2)-M arrest, suggesting that the synergistic effect of paclitaxel in this combination, which produced the greatest caspase activation, might be independent of microtubule stabilization. In contrast, the gemcitabine + fludarabine + mitoxantrone combination was synergistic and schedule independent. Moreover, few ratios of gemcitabine + fludarabine to mitoxantrone were antagonistic, which could be important for clinical translation. In conclusion, synergistic interactions with gemcitabine occurred with several drugs, the most promising being gemcitabine + fludarabine, gemcitabine + fludarabine + paclitaxel, and gemcitabine + fludarabine + mitoxantrone. These findings provided a rationale for clinical trials of gemcitabine + fludarabine and gemcitabine + mitoxantrone where responses were observed in heavily pretreated AML patients.

Osteosarcoma cells, resistant to methotrexate due to nucleoside and nucleobase salvage, are sensitive to nucleoside analogs.

PURPOSE: To test a novel strategy for overcoming intrinsic resistance to methotrexate (MTX) in osteosarcoma (OS) due to nucleoside and nucleobase salvage (NS). METHODS: Four OS cell lines, found to be highly resistant to MTX, were tested to determine the dominant mechanism of resistance. Sensitivity to MTX was tested in the presence of dialyzed serum or the transport inhibitor dipyridamole (DP) to confirm the contribution of NS to MTX resistance. We then investigated whether increased NS activity could be exploited using cytotoxic nucleoside analogs. RESULTS: Like other cell types, OS cells are capable of circumventing inhibition of de novo nucleotide synthesis by relying on NS. MTX, at concentrations as high as 1 m M did not inhibit cell growth in culture medium supplemented with undialyzed serum. In contrast, when NS was inhibited by DP or in medium depleted of nucleosides and nucleobases, sensitivity to MTX was seen at nanomolar concentrations. In medium with dialyzed serum, thymidine and hypoxanthine provided dose-dependent protection from MTX toxicity at concentrations similar to those seen in human plasma. No evidence of other significant mechanisms of resistance were found. All four cell lines were sensitive to 3-day exposures to cytarabine (IC50 0.22 to 2.88 micro M) and vidarabine (IC50 0.09 to 0.95 micro M). CONCLUSIONS: Salvage of de novo nucleotide synthesis inhibition by extracellular thymidine and hypoxanthine, at physiologically relevant concentrations, contributes to resistance to MTX in OS. However, this same process may impart a collateral sensitivity to nucleoside analogs. These findings support clinical trials for patients with OS using nucleoside analogs, either alone or in combination.

Antiherpetic activities of acidic protein bound polysacchride isolated from Ganoderma lucidum alone and in combinations with acyclovir and vidarabine.

To investigate antiherpetic activity, an acidic protein bound polysaccharide (APBP) was isolated from carpophores of Ganoderma lucidum. This brownish APBP was isolated from water soluble substances of the carpophores by activity-guided isolation method. APBP was tested for its antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by plaque reduction assay in tissue culture. APBP showed potent antiviral activity against HSV-1 and HSV-2 in Vero cells at its 50% effective concentration (EC(50)) of 300 and 440 microg/ml, respectively. APBP had no cytotoxicity on Vero cells at a concentration of 1 x 10(4) microg/ml. APBP exhibited a potent antiviral activity with selectivity index (SI) of more than 22.73. The combined antiherpetic effects of APBP with nucleoside antiherpetic agents, acyclovir (ACV) and vidarabine (ara-A), were examined on the multiplication of these two strains of herpesviruses in Vero cells by the combination assay. The results of combination assay were evaluated by the combination index (CI) that was calculated by the multiple drug effect analysis. CI values were in the range 0.47-0.51 for a combination of APBP with ACV, and in the range of 1.02-1.18 for a combination of APBP with ara-A. The combinations of APBP with ACV on HSV-1 and HSV-2 showed potent synergistic effects, and these results suggest that the possibility of developing APBP as a new antiherpetic agent.

Successful modulation of high-dose cytosine arabinoside metabolism in acute myeloid leukaemia by haematopoietic growth factors: no effect of ribonucleotide reductase inhibitors fludarabine and gemcitabine.

High-dose cytosine arabinoside (AraC)-containing regimens have shown the highest antileukaemic efficacy of all currently used regimens in the treatment of acute myeloid leukaemia (AML). This study aimed at increasing the antileukaemic potential of high-dose AraC by raising intracellular levels of AraC triphosphate (AraCTP), which is the mediator of cytotoxicity, via biochemical modulation by inhibitors of ribonucleotide reductase (RR) or haematopoietic growth factors (HGFs). Blasts from patients with de novo AML were analysed for their formation of AraCTP under high-dose AraC conditions (20 microM over 3 h) without prior modulation (n = 47) after a 2-h pre-exposure with fludarabine (50 microg/ml) (n = 40) or gemcitabine (30 ng/ml) (n = 40) and after a 48-h pre-exposure to granulocyte colony-stimulating-factor (G-CSF; 100 ng/ml) (n = 27) or granulocyte-macrophage colony-stimulating-factor (GM-CSF; 100 U/ml) (n = 28). Unmodulated formation of AraCTP (median 239.8 ng/107 cells) could not be increased via modulation by gemcitabine (232.4 ng/107 cells) or fludarabine (247.8 ng/107 cells). The lack of effect of RR inhibitors was also observed for all other known metabolites of AraC [Ara-cytosine monophosphate (CMP), Ara-cytosine diphosphate (CDP), AraCDP-choline, Ara-uridine monophosphate (UMP), Ara-uridine diphosphate (UDP) and Ara-uridine triphosphate (UTP)]. In contrast, pre-exposure to HGFs led to significant increases in AraCTP formation (G-CSF 556.0 ng/107 cells, 2.31-fold increase, P < 0.001; GM-CSF 447.9 ng/107 cells, 1.87-fold increase, P < 0.0001). To establish the mechanism responsible for these effects, the activity of the rate-limiting enzyme of AraC metabolism, deoxycytidine kinase (dCK), was investigated (n = 33). In vivo exposure to GM-CSF led to increases in dCK activity from unmodulated values at 0 h (29.8 pmol/min/mg protein) to 34.3 pmol/min/mg protein at 24 h (1.15-fold increase) and 54.5 pmol/min/mg protein at 48 h (1. 83-fold increase). The raise in dCK activity over 48 h was significant (P < 0.013).

Foscarnet-resistant herpes simplex virus infection in patients with AIDS.

Six human immunodeficiency virus-infected patients had clinical lesions of herpes simplex virus (HSV) type 2 that showed in vitro resistance to foscarnet. In each patient, lesions were unresponsive to foscarnet therapy or developed during daily suppressive foscarnet. Five patients had a history of intermittent or chronic foscarnet use for the management of acyclovir-resistant HSV infection, and 1 was receiving daily foscarnet for suppression of cytomegalovirus retinitis. Seven of 10 foscarnet-resistant isolates from 6 patients were susceptible to acyclovir in vitro, and 1 was of borderline susceptibility. In 3 patients, the administration of acyclovir, either alone or in combination with foscarnet, resulted in healing. Clinically significant resistance to foscarnet may occur in immunosuppressed patients with prior foscarnet exposure. Addition or substitution of acyclovir to foscarnet therapy may be a useful strategy for patients in whom foscarnet resistance is suspected, pending the results of in vitro susceptibility testing.

[Effects of extracts from Phyllanthus urinaria L. on HBsAg production in PLC/PRF/5 cell line].

Experiment on the effect of extracts from Phyllanthus urinaria on HBsAg production in PLC/PRF/5 human hepatoma cell line showed that the production was decreased markedly 48 hours after the treatment of cells with 2-4 mg/ml Phyllanthus urinaria, especially in combination with 10(-5) mol/L of Ara-A. The decrease of extracellular HB-sAg excretion seems to stem from the inhibition of intracellular HBsAg formation.

Inhibition of in vitro HBsAg production by amphotericin B and ketoconazole.

The effects of amphotericin B, ketoconazole, and adenine arabinoside on production of hepatitis B surface antigen (HBsAg) particles by the human hepatoma cell line PLC/PRF/5 were examined. In addition, the effects of these drugs on cellular protein synthesis were determined. These drugs caused a dose-dependent decrease in HBsAg production that was paralleled by a decrease in cellular protein synthesis. Ketoconazole was the most active of these drugs and the most specific, causing a 72% reduction in HBsAg production with only a 38% reduction in protein synthesis. These data suggest that further studies evaluating ketoconazole for the treatment of chronic hepatitis B virus infection in animals are warranted.

Pharmacological regulation in vitro of meiotic resumption in oocytes of Xenopus laevis.

The pharmacological control in vitro of meiotic resumption in Xenopus laevis oocytes was undertaken with a variety of drugs or agents which either mimic a steroidal hormone, progesterone, the physiological inducer of meiosis in vivo and in vitro, potentiate progesterone-induced meiosis (e.g. insulin) or inhibit the action of steroidal and non-steroidal inducers. Most steroidal and non-steroidal inducers trigger a cascade of cellular events which seem to be initiated at the oocyte membrane. One membrane target seems to be the enzyme adenylate cyclase which is inhibited (with concomitant dephosphorylation of yet unknown cellular substrates) by progesterone as well by several other inducers, or activated by inhibitors of meiosis such as cholera toxin or forskolin. However, additional, complementary or subsequent interaction(s) of inducers with other membrane and/or cellular constituents, such as calcium, phospholipids and protein kinase C have been also suggested by experiments with cycloheximide (a protein synthesis inhibitor) or with a serine protease inhibitor, which both inhibit progesterone-induced meiosis. Furthermore, dideoxyadenosine was shown to decrease particulate adenylate cyclase, although it did not induce meiosis in vitro. It is hoped that the great variety of pharmacological agents which can be used at the present time to study in vitro the meiotic resumption of Xenopus laevis oocytes may help to clarify the complex sequence of meiotic events which start at the cell membrane level and progress to the second meiotic metaphase.

Evaluation of the anti-herpesvirus drug combinations: virazole plus arabinofuranosylhypoxanthine and virazole plus arabinofuranosyladenine.

Combinations of Virazole plus arabinofuranosylhypoxanthine (ara-Hx) and Virazole plus arabinofuranosyladenine (ara-A) were investigated in KB or BHK cells infected with types 1 or 2 herpes viruses. Combinations of Virazole and ara-Hx exhibited significant synergy as evaluated graphically (isobolograms) or by fractional inhibitory concentration (FIC) indices. Optimal ratios for the combination were 1:1 to 1:10 for Virazole to ara-Hx. At these ratios, FIC indices in the range of 0.5-0.2 were commonly observed. Combinations of Virazole and ara-A were antagonistic when observed in the presence of pentostatin, an adenosine deaminase inhibitor. In the absence of pentostatin, the minimum inhibitory concentration (MIC) of ara-A and degree of synergy with Virazole were variable.

Different in vitro effects of dual combinations of anti-herpes simplex virus compounds.

Five promising antivirals have been tested individually and in pairs on herpes simplex virus (HSV) types 1 (Strain F) and 2 (Strain G) in Vero cells. These are: 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV), 9-beta-D-arabinofuranosyladenine (ara-A), 5-trifluorothymidine (TFT), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and phosphonoformate (PFA). Various types of interactions depending on virus type were observed: synergistic [ara-A/TFT; ara-A/BVDU (G); BVDU/PFA (F)]; additive [ACV/ara-A; ACV/TFT; ACV/BVDU; ACV/PFA (G); BVDU/TFT; PFA/ara-A; PFA/TFT]; and sub-additive [ACV/PFA (F); ara-A/BVDU (F) and BVDU/PFA (G)]. Neither antagonism nor interference was noted for any combinations.

Anti-herpesvirus activity of adenine arabinoside analogues in tissue culture and a genital infection of mice and guinea pigs.

Four analogues of adenine arabinoside (ara-A) were compared for activity against herpes simplex virus (HSV) in tissue culture and in a genital infection of mice and guinea pigs. These analogues, 5'-monophosphate (ara-AMP), 5'-valerate ester (ara-AV), 2'3'-diacetate ester (ara-ADA), and 2',3',5'- triacetate ester (ara-ATA) have greater water and lipid solubility and resistance to deamination than ara-A. In mouse embryo fibroblast cells, similar viral inhibitory levels were noted with ara-A, AMP, and ara-Av, while ara-ADA and ara-ATA were 6-10 time less active. In mice infected intravaginally with HSV type 2 (HSV-2), intravaginal treatment with 10% concentrations of each of the compounds beginning 3 h after viral challenge, had no effect on infection rates, titers of virus in vaginal secretions, mortality rates or the mean day of death as compared with placebo-treated controls. In the HSV-2 genital infection of guinea pigs, treatment with 10% vaginal creams or placebo vehicle was initiated 6 or 24 h after viral inoculation. In animals treated at 6 h with ara-A, ara-AMP and ara-AV, there was complete inhibition of viral replication in the vaginal tract and development of external genital lesions. When treatment with these three drugs was delayed 24 h after infection, there was no effect on vaginal virus titers, but lesions severity was reduced by ara-A or ara-AMP therapy. Ara-ATA was ineffective whether begun at 6 or 24 h. The greater solubility in water and lipid as well as the resistance to deamination of ara-AMP and ara-AV did not appear to enhance their antiviral activity over that of ara-A. Additionally, ara-ADA and ara-ATA exhibited less activity both in tissue culture and in the experimental genital infections.