Antiplasmodial activity of extracts of Tridax procumbens and Phyllanthus amarus in in vitro Plasmodium falciparum culture systems.

BACKGROUND: Aqueous extracts of Tridax procumbens (TP) (Compositae) and Phyllanthus amarus (PA) (Euphorbiaceae) are used in traditional medicine in Ghana to treat malaria. Previous studies have demonstrated the anti-trypanosoma, anti-bacterial and anti-HIV effects of TP and PA. OBJECTIVE: To assess the antiplasmodial activity of extracts of TP and PA. METHOD: Aqueous extracts of TP and PA were prepared. A portion of each was freeze-dried and the remaining extracted sequentially with ethyl acetate and chloroform. Ethanolic extracts were also prepared. The antiplasmodial activity of the extracts was assessed with the 3H-hypoxanthine assay using chloroquine-resistant (Dd2) Plasmodium falciparum parasites. Chloroquine was used as the reference drug. The modified tetrazolium-based colorimetric assay was also used to evaluate the red blood cell (RBC)-protective/antiplasmodial activities and cytotoxicities of the extracts. RESULTS: Results showed that TP and PA have antiplasmodial activities. The aqueous and ethanolic extracts of PA were the most active, yielding EC50 values of 34.9 µg/ml and 31.2 µg/ml, respectively in the tetrazolium-based assay. The TP and PA produced and IC50 values of 24.8 µg/ml and 11.7 µg/ml, respectively in the hypoxanthine assay. Protection of human RBCs against P. falciparum damage by the extracts highly correlated with their antiplasmodial activities. None of the extracts, within the concentration range (1.9-500 µg/ml) studied produced any overt toxicity to human RBCs. CONCLUSION: The results indicate that both PA and TP have activities against chloroquine-resistant P. falciparum (Dd2) parasites. The antiplasmodial principles extracted into water and ethanol but not chloroform or ethyl acetate.

Evaluations of unformulated and formulated dendrimer-based microbicide candidates in mouse and guinea pig models of genital herpes.

Prevention of sexually transmitted infections is a priority in developed and developing countries. One approach to prevention is the use of topical microbicides, and one promising approach is the use of dendrimers, highly branched macromolecules synthesized from a polyfunctional core. Three new dendrimer products developed to provide stable and cost-efficient microbicides were initially evaluated in vitro for anti-herpes simplex virus activity and then in vivo by using a mouse model of genital herpes. From these experiments one product, SPL7013, was chosen for further evaluation to define the dose and duration of protection. Unformulated SPL7013 provided significant protection from genital herpes disease and infection at concentrations as low as 1 mg/ml and for at least 1 h following topical (intravaginal) administration of 10 mg/ml. This compound was then formulated into three vehicles and further evaluated in mouse and guinea pig models of genital herpes infection. In the murine evaluations each of the formulations provided significant protection at concentrations of 10 and 50 mg/ml. Formulated compounds provided protection for at least 1 h at a concentration of 10 mg/ml. From these experiments formulation 2V was chosen for dose ranging experiments using the guinea pig model of genital herpes. The guinea pig evaluations suggested that doses of 30 to 50 mg/ml were required for optimal protection. From these studies a lead compound and formulation (2V of SPL7013) was chosen for ongoing evaluations in primate models of simian immunodeficiency virus and Chlamydia trachomatis infection.

Phylogenetic analysis of human immunodeficiency virus 1 in Ghana.

Eleven human immunodeficiency virus 1 (HIV-1) isolates from Ghanaian acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) patients obtained by our serosurvey in 1986-1994 were genomically analyzed and phylogenetically compared with other known strains. A phylogenetic tree constructed by analyzing the env region indicated that heterogeneous HIV-1 strains were circulating in Ghana and the majority of them (9 of 11 isolates) belonged to clade (subtype) A which is now furiously epidemic in Africa. Another isolate (1 of 11) belonged to clade D, and the remaining one (1 of 11) belonged to "clade G". This "clade G" virus grouped by the env analysis belonged to clade A by its pol sequence, suggesting an A/G intersubtype recombinant. The characteristic sequences in the V3 tip which have not yet been reported were observed in these Ghanaian isolates, which should be taken into account for future vaccine programs.

PIP: The molecular epidemiology of HIV-1 in Ghana was investigated through genomic and phylogenetic analysis of isolates from 11 AIDS or AIDS-related complex patients obtained in 1986-94. A phylogenetic tree constructed by analyzing the env region indicated that heterogeneous HIV-1 strains are circulating in Ghana. 9 of the isolates belonged to clade A, 1 to subtype D, and 1 to "clade G"--an A/G intersubtype recombinant. The V3 loops of all isolates were composed of 35 amino acid residues--a characteristic not previously described. These molecular data on the genetic variability of the envelope glycoprotein of HIV-1 should be useful for future vaccine studies in West Africa.

T-lymphocytopaenia, opportunistic infections and pathological findings in Ghanaian AIDS patients and their sexual partners.

Ninety-nine patients at Center for Disease Control (CDC) clinical stage IV were studied. Twelve (12.12%) of these patients turned out to be HIV seronegative. Ten out of the 12 HIV negative patients were immunocompetent whereas the other two had proportional decreases in both CD4+ and CD8+ T-lymphocytes. HIV-1, HIV-2, and dual infection, were detected in 51.5%, 2%, and 22.2% respectively of clinical AIDS patients. The other 12.12% of clinical AIDS patients were indeterminate for HIV antibodies. All HIV positive patients with the exception of two, were immunocompromised with respect to CD4+ and CD8+ T-lymphocyte counts. Two healthy spouses and three children of patients who died from the disease were seronegative for HIV antibodies. Herpes simplex virus type 2 (HSV-2) and cytomegalovirus (CMV) antibody titres were higher in HIV infected than uninfected blood. Patients with chronic diarrhoea, lymphadenopathy, pneumonia, and tuberculosis, either alone or in combination of two or more of such symptoms, were found to be more likely to be confirmed by serology and immunology as definitive AIDS patients in Ghana. In postmortem studies on 20 patients, pneumonia due to tuberculosis constituted the major cause of death. Toxoplasmosis, cytomegaloviral eosophagitis and enteritis, and cryptococcosis were the major opportunistic infections detected. Programmed cell death (apoptosis) was found by the DNA gel electrophoresis method to be an unlikely major mechanism of accelerated culture induced death of PBMCs from CDC stage IV AIDS patients.

PIP: Since HIV infection was first diagnosed in Ghana in 1986, the incidence of HIV infection has increased steadily in the country over the years. Until 1990, most people infected with HIV in Ghana were infected with HIV-2. However, in 1990, most people tested were found to be dually infected with HIV-1 and HIV-2, and recently, most HIV-infected people in Ghana are only HIV-1 positive. Findings are presented from the study of 99 US Centers for Disease Control and Prevention (CDC) clinical stage IV AIDS patients. Polymerase chain reaction assay identified 12 of these patients as HIV-seronegative. HIV-1, HIV-2, and dual infection were identified in 51.5%, 2%, and 22.2% of clinical AIDS patients, respectively, with the remaining patients being indeterminate for HIV antibodies. All but 2 HIV-positive patients were immunocompromised with regard to CD4+ and CD8+ T-lymphocyte counts. 2 healthy spouses and 3 children of patients who died from AIDS were seronegative for HIV antibodies. Herpes simplex virus type 2 and cytomegalovirus antibody titers were higher in HIV-infected than in uninfected blood. Patients with chronic diarrhea, lymphadenopathy, pneumonia, and tuberculosis (TB), either alone or in combination of 2 or more such symptoms, were more likely to be confirmed by serology and immunology as definitive AIDS patients in this study. Pneumonia due to TB was the major cause of death identified through postmortem studies conducted upon 20 patients. Toxoplasmosis, cytomegaloviral esophagitis and enteritis, and cryptococcosis were the major opportunistic infections detected. Programmed cell death was probably not a major mechanism of accelerated culture-induced death of peripheral blood mononuclear cells.

Differential cytopathicity and susceptibility of Ghanaian highly divergent HIV-2 -GH2-, prototype HIV-2 -GH1-, and prototype HIV-1 -GH3- to inhibition by ddCyd and ddIno.

The cytopathicity and susceptibility of prototype HIV-1 [HTLVIIIB] and Ghanaian HIV isolates [GH1, GH2, GH3] to inhibition by ddCyd and ddIno were determined by the tetrazolium-based colorimetric method. HIV-1 [HTLVIIIB] caused the most cytopathic effect followed by HIV-2 [GH2]. At low MOI, HIV-2 [GH1] was more cytopathic than HIV-1 [GH3] but the reverse was true at high MOI. Using EC90 concentrations for comparison at similar cytopathicities, both HIV-1 [HTLVIIIB] and HIV-1 [GH3] which belong to prototype HIV-1 group, were effectively inhibited by one or both drugs. In contrast, HIV-2 [GH1] which belongs to prototype HIV-2 group, and especially, the highly divergent HIV-2 [GH2] which belongs to HIV-2b group were relatively resistant to inhibition by ddCyd and ddIno.

Serological, virological, and polymerase chain reaction studies of HIV type 1 and HIV type 2 infections in Ghanaian patients with AIDS and AIDS-related complex.

We have used a particle agglutination (PA) test, Western blot (WB) test, polymerase chain reaction (PCR) test, and virus isolation to define the human immunodeficiency virus (HIV) status of 17 acquired immunodeficiency syndrome (AIDS), 6 AIDS-related complex (ARC), and 2 asymptomatic Ghanaians. HIV-1 antibodies were more frequently detected. The PCR detected 66.7% HIV-1, 11.1% HIV-2, and 5.6% of both HIV-1 and HIV-2 proviral DNA in peripheral blood mononuclear cell (PBMCs) and PBMC-Molt 4 coculture samples tested. Of the 12 viruses isolated from the 25 Ghanaians, 9 were HIV-1, 2 were HIV-2, and both HIV-1 and HIV-2 were isolated from 1 individual. Two of the HIV-1 isolates were from ARC patients who have been PA negative and either HIV-1 or HIV-2 WB indeterminate for more than 1 year without developing antibodies to HIV envelope proteins. Our results indicate that HIV-1 is now predominant in Ghanaian AIDS and ARC patients and that dual infection can occur.

PIP: While HIV is believed to be the causative agent for AIDS, many clinically diagnosed AIDS and AIDS-related complex (ARC) cases in Ghana have been reported to be negative or indeterminate for HIV antibodies. Dual seropositive reactions have also been common among AIDS and ARC cases in the country. A particle agglutination (PA) test, Western blot (WB), polymerase chain reaction (PCR), and virus isolation were used to define the HIV status of 17 AIDS, 6 ARC, and 2 asymptomatic Ghanaians. The PA test detected HIV-1 antibodies in 72% of the plasma samples, 94.4% of which were also positive according to WB. 1 sample was indeterminate by WB and 2 HIV-1 negative samples were determined to be positive by WB. HIV-2 was detected by PA in 32% of all samples, of which 87.5% were confirmed by WB. PCR detected 66.7% of HIV-1 cases, 11.1% of HIV-2, and 5.6% of both HIV-1 and HIV-2 proviral DNA in peripheral blood mononuclear cells (PBMCs) and PBMC-Molt 4 coculture samples tested. 12 viruses were isolated from the 25 subjects; 9 were identified as HIV-1, 2 as HIV-2, and 1 person was infected with both HIV-1 and HIV-2. 2 of the HIV-1 isolates were from ARC patients who had been PA-negative and either HIV-1 or HIV-2 WB indeterminate for more than 1 year without developing antibodies to HIV envelope proteins.

Serological survey of HIV-1, HIV-2 and human T-cell leukemia virus type 1 for suspected AIDS cases in Ghana.

OBJECTIVE: To determine seroprevalence among suspected AIDS patients in Ghana in relation to clinical manifestations. MATERIALS AND METHODS: Blood samples and medical records were collected from 290 Ghanaian patients with suspected AIDS in 1990 and 1992. Seroprevalence of HIV-1, HIV-2 and human T-cell leukemia virus (HTLV-1) were investigated by the particle agglutination method, indirect immunofluorescence assay, the monoepitope enzyme-linked immunosorbent assay and Western blot. RESULTS: The specimens were classified into five serologic categories: 78 were HIV-1-positive (26.9%), 25 were HIV-2-positive (8.6%), 17 dual-positive (5.9%), 16 indeterminate (5.5%) and 154 seronegative (53.1%). No significant difference was found between the clinical symptoms of patients with HIV-1 and HIV-2 infection. Of the patients, 14 (4.8%) were HTLV-1-seropositive, of whom 11 were also HIV-positive, indicating a significant correlation between the two groups of viral infections (P < 0.01). However, there was no evidence of an increase in severity of symptoms in cases of dual infection with HTLV-1 and HIV. CONCLUSIONS: HIV-1 infection is now dominant in Ghana in contrast to our previous survey in 1986 which showed the dominance of HIV-2. The change in seroprevalence suggests that an HIV-1 epidemic has been developing in recent years in this country, where HIV-2 was originally endemic. A relatively high prevalence of dual-reactive specimens implies the existence of highly cross-reactive strains of HIV or frequent coinfection with HIV-1 and HIV-2 in the region. The large number of seronegative patients with clinically diagnosed AIDS raises the question of the inadequacy of AIDS definitions based on clinical manifestations only.

Differential reactivities of antibodies to HIV and HTLV-I in sera of suspected AIDS and ARC patients.

Sera collection from 255 clinically diagnosed AIDS and ARC patients were analyzed for antibodies to HIV and HTLV-I by Western blot and particle agglutination methods respectively. Antibodies to HIV were detected in 37.3% of the sera collected as compared to 5.5% for HTLV-I. Most (95%) of the HIV positive sera had dual reactivity to both HIV-I and HIV-2. Antibodies to HTLV-1 were more frequently detected in HIV positive sera (11.58%) than in HIV negative sera (1.88%). Conversely, antibodies to HIV were detected twice as frequently in HTLV-1 positive sera (78.6%) than in HTLV-1 negative sera (34.85%).

Comparative analysis of HIV-1 and HIV-2 indeterminate western blot patterns.

A comparison of HIV-1 and HIV-2 indeterminate Western blot patterns of Ghanaian sera collected between 1989 and 1990 was made. Antibodies to group specific antigen (GAG) gene products were most frequently detected both HIV-1 and HIV-2 indeterminate sera. HIV-2 GAG gene product p26 was shown to be a non-specific indicator of infection. Antibody to gp120, and envelope gene product of HIV-1 never occurred in indeterminate sera whereas antibodies to all the envelope gene products of HIV-2 were detected in indeterminate sera.

PIP: A comparison of HIV-1 and HIV-2 indeterminate Western blot patterns of Ghanaian sera collected between 1989 and 1990 was made and interpreted according to new World Health Organization (WHO) criteria. For HIV-1 antibodies detection, Novopath Immunoblot assay kits were used. New LavblotII kits were used for detecting HIV-2 antibodies. In both cases, proteins of the specific virus type were used to detect anti-HIV proteins in sera by the enzyme linked immunoelectrotransfer blot (Western blot) technique. Sera and plasma after screening by enzyme-linked immunosorbent assay (ELISA) were used. Antibodies to group specific antigen (GAG) gene products were most frequently detected both HIV-1 and HIV-2 indeterminate sera. HIV-2 GAG gene product p26 was shown to be a non-specific indicator of infection. Analysis of HIV-1 indeterminate Western blot patterns showed the frequency of protein bands for 231 HIV-1 indeterminate sera. Antibodies for the GAG gene products, i.e., anti-55, anti-p24, and anti-p18 had high frequencies of occurrence with anti-p24 occurring 90.5% of the time. Analysis of HIV-2 indeterminate Western blot patterns showed the frequency of appearance of antibodies to the various viral gene products in 396 HIV-2 indeterminate sera. The GAG gene product p26 reacted with most (91.2%) of the 396 indeterminate sera. Comparison of HIV-1 and HIV-2 indeterminate Western blot patterns indicated that antibodies to p26 appeared 25% of the time in the negative control sera for HIV-2, whereas no antibodies to HIV-1 products were detected in any of the HIV-1 negative control sera. In the Western blot analysis using the WHO criteria, the frequencies of appearance of antibodies to the high molecular weight core proteins of p24 (HIV-1) and p26 (HIV-2) were both quite high (90%). No antibody to the external envelope protein was detected for HIV-1 (anti-gp120), whereas antibody to a similar protein (anti-gp 105) was detected in 4.3% of the HIV-2 indeterminate sera.

Modified tetrazolium-based colorimetric method for determining the activities of anti-HIV compounds.

A modified tetrazolium-based colorimetric assay was used to determine the anti-HIV activities of ddAzThd, ddCyd, ddIno, and PFA. In this assay, poly-1-lysine-coated plates were used to attach the MT-2 cells to the bottom of the plates. A fixed amount of virus (50 TCID50) was used in each well. A modified version of the formula published by Pauwels et al. (1988) was used for calculating the percentage cell protection from virus infection. Using CC10/EC90 to calculate the selective indices, the decreasing order of selectivity against HIV-1 strain A87SF, was: ddAzThd greater than PFA greater than ddCyd greater than ddIno. Against HIV-1 strain A79SK-1 the decreasing order of selectivity was: PFA greater than ddIno greater than AzThd greater than ddCyd. The modified formula showed lack of anti-HIV activity for thymidine at non-toxic concentrations.

Comparative metabolism of E-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in herpes simplex virus-infected cells.

The antiviral activities and metabolic fates of E-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd) and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BrVaraUra) were compared in a dThd kinase-deficient human fibroblast cell line, infected with parental strains of herpes simplex virus, and other strains expressing no viral dThd kinase activity. Metabolic experiments were performed at concentrations well above the ID50 for each compound because radiolabeled agents were not available. BrVaraUra and its nucleotides qualitatively displayed chromatographic and anabolic characteristics which closely paralleled those of BrVdUrd and its nucleotides. Monophosphorylation of both drugs was dependent upon the presence of viral dThd kinase activity except in the case of one dThd kinase-negative type 1 mutant (SC16R5C1) which retained BrVdUrd/BrVaraUra kinase activity. Intracellular uptake of either parent compound was absent during mock-infection and minimal in the cases of infection with mutants unable to phosphorylate the parent compound. Parental type 1 strains were able to induce diphosphorylation and triphosphorylation of both compounds to a similar, dose-dependent degree. Extracts of type 2-infected cells contained greater quantities of BrVdUrd and its monophosphate compared with BrVaraUra and its monophosphate, after identical drug exposure and infection conditions. As previously observed for BrVdUrd, diphosphorylated and triphosphorylated nucleotides of BrVaraUra were not detected after type 2 infection. BrVdUrd and BrVaraUra metabolic breakdown pathways differed, however, as evidenced by the formation of E-5-(2-bromovinyl)uracil (BrVUra). Unlike BrVdUrd, BrVaraUra formed no BrVUra in infected cells, suggesting that replacement of 2'-deoxyribose with arabinose makes the compound biologically more stable, presumably because of resistance to enzymatic breakdown by pyrimidine nucleoside phosphorylases. In this dThd kinase-negative cell line, BrVdUrd and BrVaraUra displayed qualitatively similar susceptibility profiles in that activities were type 1 selective and dThd kinase dependent. Antiviral activities against dThd kinase-positive type 1 strains were similar with both compounds. These data would suggest that BrVdUrd and BrVaraUra have identical type-specific dThd-dTMP kinase-dependent mechanisms of cellular uptake and phosphorylation, but that the latter is not subjected to phosphorolysis and resultant formation of an inactive metabolite. Furthermore, the absence of presence of phosphorolysis of the parent nucleoside does not apparently adversely affect in vitro antiviral activity.

Toxicity of 5-methoxymethyl-2'-deoxyuridine in hamsters and evaluation of its mutagenicity by sister chromatid exchanges and hypoxanthine guanine phosphoribosyl transferase assays.

Toxic effects of 5-methoxymethyl-2'-deoxyuridine (MMdUrd), a drug with antiviral activity against herpes simplex virus, were investigated in Chinese golden hamsters. Pathological, hematological, and clinical chemistry parameters were studied. Systemic toxicity was not observed in hamsters after intraperitoneal (ip) administration of MMdUrd in single doses up to 3000 mg/kg and in repeated dosages of 600 mg/kg daily for 15 days. No gross or microscopic lesions related to MMdUrd treatment were observed in the tissues examined from the animals of subchronic toxicity study. Administration of a single dose of 6000 mg/kg of MMdUrd ip caused cloudy swelling, increased mitotic figures, varied nuclear sizes, and scattered coagulation necrosis of hepatocytes on the second day after drug administration. These hepatic lesions were not observed in animals sacrificed on the fourth and sixth days postadministration of MMdUrd. Microscopic examination of marrow smears from control and MMdUrd-treated animals revealed no differences in morphological features of hematopoiesis, nor were significant differences observed between treated and control animals for hematological parameters. Increased concentrations of alanine amino transferase were observed on the first and second day in animals, given a single dose of 6000 mg/kg of MMdUrd. Increases in sister chromatid exchanges and the number of azaguanine-resistant mutants were observed after exposure to a high concentration of MMdUrd (1024 micrograms/ml).

Efficacy of 5-methoxymethyl-2'-deoxyuridine in combination with arabinosyladenine for the treatment of primary herpes simplex genital infection of mice and guinea pigs.

The relative efficacy of 5-methoxymethyl-2'-deoxyuridine (MMdUrd), arabinosyladenine (ara-A) and the combination of MMdUrd and ara-A in the treatment of experimental genital herpes (GH) was investigated using mouse and guinea pig models. The infection was initiated by intravaginal inoculation using either HSV-2, strain X-265 or HSV-2, strain MS. Treatment was initiated 3 h post virus inoculation. The parameters used to evaluate efficacy were: percent mortality; mean day of death; virus yield from the vaginal secretions; and mean lesion score. The simultaneous application of 5% MMdUrd and 5% ara-A was an effective treatment for controlling primary GH in both animal models. Combination chemotherapy was also effective in preventing recurrence of infection as well as the emergence of drug resistant virus. At 20% concentration, ara-A was effective in providing protection against GH. However, lesions due to recurrent GH appeared after cessation of treatment and the virus isolated from vaginal secretions of ara-A treated animals required higher concentration of drug for inhibition of virus replication in cell culture. 20% MMdUrd was only partially effective in controlling GH. The production of infectious virus particles (virus yield) in cell culture after exposure to either ara-A of MMdUrd alone or in combination was determined. When MMdUrd and ara-A were used together, a substantially lower amount of each drug was needed to inhibit virus production completely and removal of drugs did not result in an increase in virus yield.

Antiviral activity, antimetabolic activity, and cytotoxicity of 3'-substituted deoxypyrimidine nucleosides.

The antiviral activity, effect on cellular DNA and RNA synthesis, and cytotoxicity toward mammalian cells of 5-fluoro-2'-deoxyuridine, 5-methoxymethyl-2'-deoxyuridine, 2'-deoxythymidine, and their corresponding 3'-p-nitrophenylphosphate and 3'-p-aminophenylphosphate derivatives were determined. The 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine derivative was as potent as 5-methoxy-methyl-2'-deoxyuridine in inhibiting herpes simplex viruses; however, 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine was less potent than 5-fluoro-2'-deoxyuridine in inhibiting viral replication. The results suggest that the deoxypyrimidine ribonucleoside kinase has bulk tolerance for substituents at the 3-position of the ribofuranose moiety. The effect on cellular DNA and RNA synthesis and cytotoxicity toward mammalian cells were monitored by studying the incorporation of radioactive precursors. 5-Methoxymethyl-2'-deoxyuridine and 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine failed to inhibit DNA or RNA synthesis. 5-Fluoro-2'-deoxyuridine and 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine decreased incorporation of [3H]deoxyuridine by 50% at 1.0 and 40 microM, respectively. Cytotoxicity (microscopic lesions using monolayer cells) on exposure to 5-methoxymethyl-2'-deoxyuridine, 3'-p-aminophenylphosphate-2'-deoxy-5-methoxymethyluridine, 5-fluoro-2'-deoxyuridine, and 3'-p-aminophenylphosphate-2'-deoxy-5-fluorouridine was observed at 3800, 1600, 1.6, and 110 microM, respectively.

Combination chemotherapy: interaction of 5-methoxymethyldeoxyuridine with trifluorothymidine, phosphonoformate and acycloguanosine against herpes simplex viruses.

Methoxymethyldeoxyuridine (MMUdR) when used in combination with either trifluorothymidine (F3TdR) or phosphonoformate (PFA) showed synergistic activity against herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) in vitro, whereas MMUdR and acycloguanosine (ACG) combination was antagonistic against herpes viruses. HSV-1 mutants resistant to ACG, arabinofuranosyladenine (Ara-A), MMUdR or PFA were isolated. Drug-resistant HSV-1 virus mutants were analyzed for cross sensitivity to ACG, Ara-A, F3TdR, MMUdR, MMUdR-5'-monophosphate (MMUdR-MP) and PFA. The Ara-A-resistant (Ara-AR) virus exhibited 3-fold resistance to MMUdR-MP (ID50 = 105 microM). The ACG-resistant (ACGR) mutant was 160-fold less sensitive to MMUdR (ID50 greater than 1138 microM). The MMUdR-resistant (MMUdRR) mutant remained sensitive to all other antiviral drugs in vitro. Ara-A provided protection against HSV-1 encephalitis in immunosuppressed mice inoculated with a low dose (200 PFU/mouse) of MMUdRR virus or wild-type HSV-1. F3TdR decreased incorporation of tritiated deoxyuridine [( 3H]UdR) in RK-13 cells by 50% at 0.068 microM. Under similar conditions, MMUdR (up to 600 microM) and PFA (up to 208 microM) were without effect on incorporation of [3H]UdR into DNA. In combination chemotherapy experiments, MMUdR (up to 300 microM) used along with F3TdR (up to 1.08 microM) neither decreased nor enhanced cytotoxicity of F3TdR as measured by incorporation of [3H]UdR into cellular DNA. Similarly, MMUdR (up to 300 microM) in combination with PFA (up to 166 microM) was nontoxic to host cells.

Metabolic fate of (E)-5-(2-bromovinyl)-2'-deoxyuridine in herpes simplex virus- and mock-infected cells.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine is a potent antiherpes compound with far better activity against herpes simplex virus type 1 than type 2. To understand the role of drug metabolism in this differential antiviral activity, we examined the metabolic fate of this drug in virus-infected and mock-infected Vero cells by high-pressure liquid chromatography. After 8 h of incubation in which cells were exposed to 10 micrograms of the drug per ml, 63 pmol/10(6) cells of the parent compound was detected in acid-soluble extracts of mock-infected cells. Herpes simplex virus-infected cells, however, incorporated or metabolized, or both, up to 11,310 pmol/10(6) cells. Type 1-infected cells metabolized the drug to the triphosphate where as many as 5,565 pmol/10(6) cells were detected. In contrast, three strains of type 2-infected cells metabolized the drug to the monophosphorylated nucleotide and no further. The amount of drug getting into the cells was virus strain and inoculum dependent. These studies indicate that poor substrate acceptance of (E)-5-(2-bromovinyl)-2'-deoxyuridine monophosphate by herpes simplex virus type 2-specified thymidylate kinase is an important factor in situ in infected cells, preventing anabolism of the parent compound to its active triphosphorylated form. This may account for its type specificity.

Comparison of the antiviral effects of 5-methoxymethyldeoxyuridine-5'-monophosphate with adenine arabinoside-5'-monophosphate.

Methoxymethyldeoxyuridine-5'-monophosphate (MMUdR-MP) and arabinofuranosyladenine-5'-monophosphate (Ara-AMP) had significant antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in RK-13 and Vero cells. MMUdR-MP and Ara-AMP were more potent than methoxymethyldeoxyuridine (MMUdR) and arabinofuranosyladenine (Ara-A) against the MS strain of HSV-2. MMUdR-MP inhibited replication of HSV-1r (mutant resistant to MMUdR). MMUdR in combination with Ara-AMP showed additive activity; whereas the MMUdR-MP and Ara-AMP combination was antagonistic against herpes viruses. MMUdR in combination with Ara-A was synergistic in reducing the log virus yield. Cytotoxicity (microscopic lesions) was observed on exposure to MMUdR-MP and Ara-AMP at 450 and 90 microM, respectively. Rapidly proliferating RK-13 cells exposed to Ara-AMP (64 microM) were killed. In the same system, the cells surviving after incubation with MMUdR-MP (650 microM), multiplied at an almost normal rate.