Efficacy of ganciclovir in combination with other antimicrobial agents against cytomegalovirus in vitro and in vivo.

In MRC-5 cell cultures, the efficacy of the acyclic nucleoside ganciclovir (GCV) against human cytomegalovirus (CMV) was unaffected when combined with either amphotericin B (AMP B), ketoconazole (KCZ), dapsone (DAP), or trimethoprim/sulfamethoxazole (TMP/SMX). When differences in 3-dimensional plots for antiviral activity and cytotoxicity of GCV alone and in combination were compared, the anti-CMV activity of GCV (IC50 8 microM, 5-9 microM range) was not affected by concentrations of up to 10 microM AMP B, 1000 microM KCZ, 100 microM DAP or 320 microM TMP/SMX (higher concentrations could not be tested due to cytotoxicity). In Swiss Webster mice, the anti-CMV activity of GCV was also unaffected when administered in combination with any of the four other antimicrobial agents against murine CMV. GCV (s.c.) alone had an ED50 of 7 mg/kg (2-12 mg/kg range) which was unaffected by daily doses of 1 mg/kg AMP B (i.p.), 60 mg/kg KCZ (i.p.), 32 mg/kg DAP (p.o.) or 80/400 mg/kg TMP/SMX (p.o.). These results suggest that GCV can be administered in combination with these other drugs for treatment of various opportunistic infections in AIDS patients without compromising the efficacy of GCV against CMV.

Prevalence of resistance in patients receiving ganciclovir for serious cytomegalovirus infection.

Seventy-two AIDS patients treated with ganciclovir for cytomegalovirus (CMV) disease were prospectively monitored for the development of drug-resistant virus. No resistant strains were found in 31 patients before therapy or among seven culture-positive patients treated for less than or equal to 3 months. Of 13 culture-positive patients treated for greater than or equal to 3 months, 5 excreted virus resistant (ED50, greater than 12 microM, or ED90, greater than 30 microM) to ganciclovir. Thus, 38% of patients and receiving ganciclovir for greater than 3 months and excreting virus or, overall, 7.6% of the patients were excreting CMV resistant to the drug.

Lack of antiviral activity of ketoconazole alone or in combination with the acyclic nucleoside ganciclovir against a herpes virus type 2 infection in mice.

Ketoconazole and ganciclovir were tested for antiviral activity, each alone and in combination, against a herpes simplex virus type 2 (HSV-2) systemic infection in Swiss-Webster mice. When given once daily for 5 days starting 24 hr after infection, the ED50 for ketoconazole either alone or in combination was greater than 60 mg/kg; for ganciclovir, the ED50 was 7.1 mg/kg alone and 10.8 mg/kg in combination. Thus, ketoconazole did not potentiate or antagonize the antiviral activity of an acyclic nucleoside. Consequently, AIDS patients could perhaps receive ketoconazole and ganciclovir simultaneously for fungal and viral opportunistic infections without interference with their respective efficacies.

Increased efficacy of ganciclovir in combination with foscarnet against cytomegalovirus and herpes simplex virus type 2 in vitro and in vivo.

In tissue culture, efficacy against either murine CMV or HSV-2 was increased 27-fold for the acyclic nucleoside ganciclovir and 3-fold for foscarnet (trisodium phosphonoformate) when the 2 drugs were combined; whereas against human CMV, efficacy was increased 3-fold for both drugs. In mice, efficacy was increased 2-fold for ganciclovir and 4- to 5-fold for foscarnet when used in combination against either murine CMV or HSV-2. These results suggest an additive interaction between the two drugs in vivo.

Efficacy of interleukin-1 beta against systemic Candida albicans infections in normal and immunosuppressed mice.

Prophylactic treatments with either recombinant human interleukin-1 beta (rHuIL-1 beta) or a muramyl dipeptide analog ([Abu1]MDP) enhanced the resistance of mice to systemic infection with Candida albicans. The optimum treatment regimen in both normal and cyclophosphamide-treated mice was intraperitoneal administration of 100 ng of rHuIL-1 beta or 1.6 mg of [Abu1]MDP per mouse once daily for 3 consecutive days before infection. Neither rHuIL-1 beta nor [Abu1]MDP was efficacious when started after the infection or when given before cyclophosphamide to mice infected subsequently. Continuing to treat after the infection with either drug neither enhanced nor antagonized the efficacy of prophylactic treatments.

Susceptibility testing of herpes viruses.

The plaque reduction or CPE assay has been the mainstay of virus sensitivity testing for both research and clinical work. These procedures have undergone a significant amount of methologic change in recent years. Almost completely lacking are results that clearly define the best parameters for correlating virus sensitivity to clinical outcome. Those few results that are available do not appear to show a direct correlation between virus sensitivity and clinical response.

Kinetics and inhibition of reverse transcriptase from human and simian immunodeficiency viruses.

Reverse transcriptase from the simian immunodeficiency virus (SIV) was found to have kinetic behavior similar to that of enzyme from the human immunodeficiency virus (HIV). Michaelis constants for the substrates TTP and dGTP and inhibition constants for the inhibitors 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2'-3'-dideoxyguanosine 5'-triphosphate were obtained for SIV reverse transcriptase and were found to be similar to the corresponding values for HIV reverse transcriptase. Thus, the interaction of SIV reverse transcriptase with nucleotide analogs appears to be indistinguishable from that of the HIV enzyme, suggesting that SIV/simian acquired immunodeficiency syndrome (SAIDS) is a potentially good model of AIDS.

Effect of ketorolac on phagocytosis of Candida albicans by peritoneal macrophages.

The effect of ketorolac tromethamine, a nonsteroidal anti-inflammatory drug, on normal phagocytosis has been studied. When peritoneal macrophages, taken from mice treated intraperitoneally 4 times daily for 7 days with 2 mg ketorolac per day, were cultured ex vivo or when untreated macrophages were cultured with 1-10 microM of ketorolac in vitro, the number of engulfed Candida albicans was no different from saline-treated or untreated controls. In contrast, macrophages from mice treated intraperitoneally 4 times daily for 7 days with 0.4 mg dexamethasone per day or cultured with 1-10 microM dexamethasone had a greater than or equal to 54% reduction in phagocytosis (p less than 0.001). Thus, ketorolac is a nonsteroidal anti-inflammatory drug which does not inhibit the phagocytic activity of murine mononuclear phagocytes.

New class of antifungal agents: jasplakinolide, a cyclodepsipeptide from the marine sponge, Jaspis species.

Jasplakinolide is a cyclodepsipeptide which represents a new class of antifungal agents with potent activity against Candida albicans. Jasplakinolide is fungicidal against C. albicans with both a MIC and a minimum lethal concentration of 25 micrograms/ml in a broth dilution assay. This activity compares to that of the imidazole miconazole nitrate, which had a MIC of 6.2 micrograms/ml and a minimum lethal concentration of 50 micrograms/ml in the same assay. Topical administration of 2% jasplakinolide cream against a murine vaginal C. albicans infection was equivalent in efficacy to administration of miconazole nitrate at 2%. Subcutaneous administration of jasplakinolide was not effective against a systemic murine C. albicans infection.

Effect of ketorolac on Pseudomonas aeruginosa ocular infection in rabbits.

Corticosteroids can exacerbate bacterial ocular infections, even in the presence of antibiotics. Ketorolac tromethamine is a new non-steroidal compound being considered as an anti-inflammatory ophthalmic drug. In this study, rabbits ocularly infected with Pseudomonas aeruginosa were treated topically with 0.4 percent tobramycin sulfate 4 times daily for 7 days to control infection. At the same times, either 0.5 percent ketorolac, 0.1 percent dexamethasone or vehicle was also given topically. Animals were scored for severity of both conjunctivitis (maximum severity rates score of 10) and corneal opacity (maximum of 4) using the Draize scale. Severity of the infection was determined by counting the number of punctate lesions which developed on the cornea. Nine days after treatment ended, the number of these lesions was the same for ketorolac as for the vehicle (respectively, 16.7 +/- 3 and 13.8 +/- 3, mean +/- SE, n = 24), indicating no exacerbation of the infection, whereas with dexamethasone these parameters increased (30.2 +/- 4, n = 24). During treatment, ketorolac reduced conjunctivitis (1.8 +/- 0.2, n = 120) when compared with the vehicle (2.9 +/- 0.2, n = 120), whereas dexamethasone did not (3.8 +/- 0.2, n V 120); neither ketorolac nor dexamethasone reduced corneal opacity (respectively, 2.3 +/- 0.05 and 2.6 +/- 0.1, n = 24) compared with vehicle (2.2 +/- 0.05, n = 24). After treatment, both conjunctivitis and corneal opacity became more severe only in dexamethasone treated eyes (respectively, 4.4 +/- 0.2, n = 120 and 3.0 +/- 0.02, n = 24). Thus, ketorolac appears to be an anti-inflammatory agent that does not worsen bacterial ocular infection.

Effect of ketorolac on herpes simplex virus type one ocular infection in rabbits.

Corticosteroids can exacerbate viral ocular infections. Ketorolac tromethamine is an effective nonsteroidal anti-inflammatory agent that may be a useful substitute for corticosteroids following ocular surgery. In this study, rabbits ocularly infected with herpes simplex virus type 1 (HSV-1) were treated topically four times daily with 0.5 percent ketorolac or 0.1 percent dexamethasone for 7 days after infection. Severity of the infection was determined by scoring corneal opacity and HSV-1 corneal ulcerations with the Draize scale as well as iritis and conjunctivitis. Ten days after treatment ended both the corneal opacity scores (1.5 out of 4) and HSV-1 corneal ulcerations (0.3 to 0.7 out of 4) were similar for ketorolac and the vehicle, indicating no exacerbation of the infection, whereas with dexamethasone these scores were increased (3.6/4 and 3.4/4, respectively). Furthermore, both iritis scores (0.5/2) and conjunctivitis scores (1.3 to 1.4/10) were also similar for ketorolac and the vehicle, while dexamethasone increased both iritis (1.8/2) and conjunctivitis (4.3/10) compared to vehicle. Thus, ketorolac appears to be an anti-inflammatory agent that does not worsen viral ocular infection.

The efficacy of an N-substituted imidazole, RS-49676, against a Trypanosoma cruzi infection in mice.

In vitro studies against epimastigotes and intracellular amastigotes and in vivo studies in inbred C3H/He mice infected with Trypanosoma cruzi Brazil strain were performed to determine the activities of two N-substituted imidazole compounds, RS-49676 and ketoconazole. Both compounds are extremely active in vitro against the amastigotes (ED50 less than 0.0001 micrograms/ml) yet inactive against the epimastigotes. In vivo, RS-49676 increased the mean survival time over 11 weeks beyond the untreated control when given subcutaneously twice daily at 100 mg/kg/day. Ketoconazole increased the mean survival time 11-18 days beyond the untreated control (mean survival time 22 days) when given subcutaneously twice daily at 100 mg/kg or orally once daily at 100 mg/kg. Approximately 20%-25% of the RS-49676 treated mice were cured as determined by culturing the blood of infected mice with fibroblast lung cells. None of the ketoconazole mice were cured.

Effect of ketorolac on Candida albicans ocular infection in rabbits.

The tendency of corticosteroids to exacerbate ocular infections limits their usefulness. Ketorolac tromethamine, a nonsteroidal anti-inflammatory agent, is under development as an anti-inflammatory ophthalmic drug. In this study, rabbits ocularly infected with Candida albicans were treated topically four times daily with 0.5% ketorolac tromethamine or 0.1% dexamethasone for seven days after infection. Severity of infection was scored using both the Draize scale and Candida colony counts. Seven days after treatment ended, both the overall ocular scores (6/20) and colony count scores (0.5/3) were the same for ketorolac as for the vehicle, indicating no exacerbation of the infection, whereas with dexamethasone these values increased (13/20 and 2.3/3, respectively). During treatment, both ketorolac and dexamethasone reduced conjunctivitis equally (1.5/10) when compared with the vehicle (3.4/10). However, after treatment, conjunctivitis became more severe only in dexamethasone-treated eyes (6/10). Thus, unlike dexamethasone, ketorolac seems to be a drug that can suppress inflammation without exacerbating fungal ocular infection.

Synthesis and anti-herpes-virus activity of acyclic 2'-deoxyguanosine analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.

Several "sugar" modified acyclic nucleoside analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 2) were synthesized and evaluated for antiviral activity. The preparation generally involved the condensation of the acetoxymethyl ether of alcohols 6c-g and 10-12a with diacetylguanine to give adducts 7c-g and 14-16, which were then deprotected to afford analogues 9c-g and 17-19. Alternatively, alcohols 12a and 13a were converted to iodides via their tosylates 12b and 13b and then reacted with the sodium salt of guanine to afford, after deprotection, analogues 22 and 23. A crossed aldol-Cannizzaro reaction on aldehyde 27 readily afforded 28, which was deprotected to give analogue 29. An in vitro assay against HSV-1 showed that all compounds tested were less active than DHPG, though several were good substrates for the viral thymidine kinase. The more promising acyclic nucleosides 9c, 19, and 29 were evaluated in a mouse encephalitis model and proved ineffective at preventing death at a dose of 20 mg/kg.

In vitro and in vivo activities of phosphate derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)-guanine against cytomegaloviruses.

The anti-cytomegalovirus activities of four phosphate derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) were evaluated against human, monkey and murine viruses. The 5'-mono-, 3'5'-bis(mono-), and 3',5'-cyclic monophosphate and 5'-homophosphonate forms of DHPG inhibited virus plaque formation at 1-15 microM. The cyclic phosphate and homophosphonate were more active than the other compounds against murine cytomegalovirus (MCMV) in vitro. In an in vivo MCMV infection model, DHPG homophosphonate and DHPG were equally effective at reducing mortality at greater than or equal to 10 mg/kg. The cyclic phosphate was active at 10-20 mg/kg but toxic at greater than or equal to 40 mg/kg. The phosphorylation of DHPG phosphate and DHPG phosphonate, as well as the inhibition of human cytomegalovirus DNA polymerase by their respective triphosphates, were also examined.

Metabolism of ribavirin in respiratory syncytial virus-infected and uninfected cells.

The metabolism of ribavirin to its mono-, di-, and triphosphate derivatives was examined in uninfected and respiratory syncytial virus-infected cells. The degree of phosphorylation was dose dependent upon extracellular ribavirin concentration. The major species formed was the triphosphate, with mono- and diphosphates being approximately 12 and 4% of the triphosphate, respectively. Amounts of triphosphate formed in infected cells were up to 2.6-fold greater than those in uninfected cells. Upon drug removal, ribavirin triphosphate degradation was very rapid, with decay half-lives of 70 to 100 min. Actinomycin D inhibited triphosphate production and also neutralized the antiviral effect of ribavirin.

Synthesis and antiherpes virus activity of phosphate and phosphonate derivatives of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.

A series of phosphate esters of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) were synthesized and evaluated for antiherpes virus activity. The cyclic phosphate esters were made by a new, efficient method utilizing stannic chloride as a solubilizing agent. Monophosphate 2 and bisphosphate 4 showed comparable activity to DHPG and probably acted as prodrugs of DHPG. On the other hand, the cyclic phosphate of DHPG 3 was taken up by cells and bypassed the virus-specified thymidine kinase. As a result, 3 was active against DHPG-resistant HSV mutants that lacked the viral-specified thymidine kinase and was more toxic than DHPG to uninfected cells. The phosphonate 5, the least toxic of the derivatives tested, was only marginally active against HSV but showed substantial activity against human cytomegalovirus in vitro.

Antitrichomonal activity of mesoionic thiazolo[3,2-a]pyridines.

Screening of mesoionic compounds as potential electron acceptors by analogy with metronidazole led to the finding of in vitro antitrichomonal activity for anhydro-2-phenyl-3-hydroxythiazolo [3,2-a]pyridinium hydroxide (1). In a series of analogues, potent in vitro activity was found to be associated with amino substitution; however, such activity was dependent on specific structural features and not on the reduction potential. The most active compounds showed only poor in vivo activity.

Comparative anti-herpesvirus activities of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, acyclovir, and two 2'-fluoropyrimidine nucleosides.

9-(1,3-Dihydroxy-2-propoxymethyl)guanine (DHPG), was evaluated in cell culture and in animals for its inhibitory effect on herpes simplex viruses. Compounds run for comparison included acyclovir, 2'-fluoro-2'-deoxy-5-iodo-arabinofuranosylcytosine (FIAC), and 2'-fluoro-2'-deoxy-5-methyl-arabinofuranosyluracil (FMAU). In plaque reduction assays DHPG, acyclovir, FIAC, and FMAU were inhibitory to six herpes types 1 and 2 virus strains at concentrations of 0.2-2.4 microM. These concentrations were much lower than those required to inhibit Vero cell proliferation. In guinea pig vaginal infections, DHPG provided significantly greater inhibition of herpetic lesions than did acyclovir. In a herpes type 2 infection model in mice, DHPG, and FMAU were active at 5 mg/kg, whereas acyclovir and FIAC showed no statistically significant effect at 80 mg/kg. In a herpes type 1 encephalitis model, DHPG and FMAU were active at doses less than 10 mg/kg, with FMAU being about 4 times more potent than DHPG in that model.

Synthesis and antiherpes simplex virus activity of 9-[(1,3-dihydroxy-2-propylthio)methyl]guanine.

The synthesis of the thio analogue (thio-DHPG, 2) of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) is described. The synthesis of 2 proceeded via the condensation of acetoxymethyl sulfide 9 with diacetylguanine 10 to give the protected nucleoside analogue 11. Although catalytic hydrogenolysis failed, the benzyl ether functionalities of 11 were successfully cleaved by an acetolysis reaction to furnish 14. Ammonolysis of 14 gave 2, which was also transformed to sulfoxide 15 and sulfone 16. Preliminary in vitro screening indicated that 2 exhibited comparable activity to DHPG against herpes simplex virus type 1 (HSV-1) but was less active against the type 2 virus (HSV-2) and human cytomegalovirus (HCMV). In a mouse encephalitis model (HSV-2), subcutaneous treatment with 2 led to a 53% reduction in mortality at a dose of 100 mg/kg per day.