The development of new drugs effective against human viral diseases has proven to be both difficult and time-consuming. Indeed, there are but 10 drugs licensed for such applications in the United States today. An attractive solution to this problem may be to optimize the efficacy and selectivity of existing antiviral drugs by combining them with agents that strategically block carefully selected metabolic pathways. This approach was used in the rational design of a three-drug combination to increase the apparent potency of acyclovir against herpes simplex virus. Recent advances in analytical techniques have made the evaluation of this complex drug strategy both possible and practical. A modified version of a previously described analytical method was used to identify optimal drug concentrations and to quantitate statistically significant synergy. Concentrations of 0.25 ,M 5-fluorodeoxyuridine, 3.6 ,uM 2-acetlpyridine thiosemicarbazone, and 0.3 ,uM acyclovir were determined to be optimal in terms of antiviral activity. The volume of synergy produced was nearly 2,000 ,uM3% at a 95% level of confidence (corresponding to a 186-fold decrease in the apparent 50%o inhibitory concentration of acyclovir with the addition of 0.25 ,uM 5-fluorodeoxyuridine and 3.6 ,uM 2-acetylpyridine thiosemicarbazone). We anticipate that this strategic approach and the supporting three-dimensional analytical method will prove valuable in designing and understanding multidrug therapies.Antiviral drugs derive their specificity through the preferential inhibition of virus-encoded enzymes present in infected cells (13). The usefulness of these agents is limited by the degree to which they concomitantly affect uninfected cells, producing untoward toxicity. Additionally, the emergence of drug-resistant mutants is a significant problem in single-drug therapy (3). Treatment with combinations of antiviral agents is generally thought to minimize drug resistance (1, 6, 14) but may or may not offer increased selectivity and efficacy. Traditionally, drug combinations have been chosen on the basis of the potency and/or efficacy of the individual agents. The component drugs often have been active against the same target, and detailed evaluations of drug interactions often have been lacking. Combiwitions of drugs that inhibit different targets, however, often offer surprising potency, even though one or more of the constituent c6mpounds may be weakly active. We now describe the rational design of an antiviral combination of three drugs that strategically inhibits the replication of herpes simplex virus (HSV) by blocking carefully chosen metabolic pathways. Evaluation of this strategy and the determination of optimal drug concentrations were made possible by recent advances in existing three-dimensional analytical procedures (21, 22).The rational design of a combination of drugs relies on a thorough understanding of drug metabolism in both virusinfected and uninfected cells. The antiherpesvirus drug acyclovir (ACV; Zovirax) is a potent inhibitor of HSV re...

The combined cytotoxicity of zidovudine and ganciclovir in three cell lines of human origin was examined. The data were generated by a new rapid cell proliferation assay and a more sensitive plating efficiency assay. A three-dimensional analytical approach was used to evaluate the drug-drug interactions, and the results were compared with those obtained by two conventional methods of analysis. Synergistic cytotoxicity was observed in all cell lines examined and by both assays. Moreover, this synergistic cytotoxicity was statistically significant at physiologically relevant concentrations. It is not known whether these drug-drug interactions manifest themselves in vivo as granulocytopenia or other untoward side effects. These results, however, indicate that further investigation is warranted and that the coadministration of zidovudine and ganciclovir may be contraindicated.Cytomegalovirus retinitis is a common sequela of AIDS and occurs in at least 25% of patients with human immunodeficiency virus disease (3,15). Ganciclovir (DHPG) therapy impedes the progress of this sight-threatening condition but requires lifelong therapy to prevent recurrent infections (9, 13, 18), and neutropenia is seen in 30% of patients (17). Zidovudine (AZT) is useful in treating AIDS (10, 29), but like DHPG, it causes at least some bone marrow suppression in most patients (23). Although the coadministration of AZT with drugs which interfere with erythrocyte and leukocyte number or function is not recommended (3a), the concomitant administration may be inevitable because few alternatives currently exist. Recently, clinical studies have suggested that combination AZT and DHPG therapy causes severe toxicity in patients, confirming initial concerns (14,16,21).We examined the cytotoxicity of combinations of AZT and DHPG in vitro and analyzed the drug-drug interactions as part of a larger effort to evaluate and develop potent combinations of antiviral drugs. The data were obtained in three human cell lines, including one primary cell line. Cytotoxicity was evaluated by two methods, a cellular proliferation assay and a plating efficiency assay. The cellular proliferation assay was developed to measure efficiently the cellular growth rate in the presence of many drug combinations. The data obtained from this assay identified concentrations of AZT and DHPG at which anomalous effects were seen. These concentrations were then specifically targeted by a plating efficiency assay, which is a more sensitive indicator of cytotoxicity. The data obtained from both of these assays were analyzed by a new three-dimensional (3-D) analytical method. The 3-D method was used because it can identify concentrations at which synergistic or antagonistic interactions occur, and then it is able to quantitate and statistically analyze the interactions (22

Symptomatic cytomegalovirus (CMV) disease has been the standard endpoint for clinical trials in organ transplant recipients. Viral load may be a more relevant endpoint due to low frequency of disease. We performed a meta-analysis and systematic review of the literature. We found several lines of evidence to support the validity of viral load as an appropriate surrogate end-point, including the following: (1) viral loads in CMV disease are significantly greater than in asymptomatic viremia (odds ratio, 9.3 95% confidence interval, 4.6-19.3); (2) kinetics of viral replication are strongly associated with progression to disease; (3) pooled incidence of CMV viremia and disease is significantly lower during prophylaxis compared with the full patient follow-up period (viremia incidence: 3.2% vs 34.3%; P < .001) (disease incidence: 1.1% vs 13.0%; P < .001); (4) treatment of viremia prevented disease; and (5) viral load decline correlated with symptom resolution. Based on the analysis, we conclude that CMV load is an appropriate surrogate endpoint for CMV trials in organ transplant recipients.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

In cells infected with herpes simplex virus type 1, intracellular dNTP pools increased markedly. Treatment of these cells with 3 microM acyclovir resulted in an additional expansion in pyrimidine deoxyribonucleoside triphosphate pools with dTTP increasing 32-fold and dCTP 8-fold. Both thymidine and deoxycytidine, however, compete with acyclovir for phosphorylation by the viral pyrimidine deoxyribonucleoside kinase and thus reduce the amount of drug that is anabolized to the active form. Theoretically, agents which inhibit thymidylate synthase or dihydrofolate reductase should reduce intracellular pools of thymidine, resulting in the potentiation of the antiviral effects of acyclovir. We explored this strategy by quantitating the synergy produced by combinations of acyclovir and other drugs using three-dimensional dose-response surface methodology (MacSynergy II). Significant synergy was seen with both 5-FdUrd and methotrexate whereas BrVdUrd, 5-CldUrd, 5-IdUrd, and 5-BrdUrd exhibited little to no synergistic activity. It is suggested that inhibitors of thymidylate synthase and dihydrofolate reductase warrant further exploration as potentiators of acyclovir.

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Current treatments for human immunodeficiency virus (HIV) include both reverse transcriptase and protease inhibitors. Results from in vitro and clinical studies suggest that combination therapy can be more effective than single drugs in reducing viral burden. To evaluate compounds for combination therapy, stavudine (d4T), didanosine (ddI), or BMS-186,318, an HIV protease inhibitor, were combined with other clinically relevant compounds and tested in a T-cell line (CEM-SS) that was infected with HIV-RF or in peripheral blood mononuclear cells infected with a clinical HIV isolate. The combined drug effects were analyzed by the methods described by Chou and Talalay (Adv. Enzyme Regul. 22:27-55, 1984) as well as by Prichard et al. (Antimicrob. Agents Chemother. 37:540-545, 1993). The results showed that combining two nucleoside analogs (d4T-ddI, d4T-zidovudine [AZT], and d4T-zalcitabine [ddC]), two HIV protease inhibitors (BMS-186,318-saquinavir, BMS-186,318-SC-52151, and BMS-186,318-MK-639) or a reverse transcriptase and a protease inhibitor (BMS-186,318-d4T, BMS-186,318-ddI, BMS-186,318-AZT, d4T-saquinavir, d4T-MK-639, and ddI-MK-639) yielded additive to synergistic antiviral effects. In general, analysis of data by either method gave consistent results. In addition, combined antiviral treatments involving nucleoside analogs gave slightly different outcomes in the two cell types, presumably because of a difference in phosphorylation patterns. Importantly, no strong antagonism was observed with the drug combinations studied. These data should provide useful information for the design of clinical trials of combined chemotherapy.

Objective To determine frequency of hospital-acquired viral respiratory infections (HA-VRI) and associated outcomes in a NICU. Study design Prospective cohort study conducted from 4 October 2016 to 21 March 2017. Infants hospitalized from birth in the NICU had a weekly nasal swab collected for testing using a multiplex PCR assay capable of detecting 16 different respiratory viruses. Results Seventy-four infants enrolled, with 5 (6.8%) testing positive for a virus (incidence rate of 1.3/1000 patient days). VRI positive infants had a younger gestational age (median 27 w vs. 32 w, p = 0.048); were hospitalized longer (97 d vs 43 d, p = 0.013); required more antibiotics (8 d vs. 4 d, p = 0.037) and were more likely to be diagnosed with bronchopulmonary dysplasia (p = 0.008) compared to VRI negative infants. Conclusion Respiratory viruses are a frequent cause of HAI in the NICU and are associated with negative outcomes.Results of this study were presented in poster format at St Jude's PIDS