Safety assessment, in vitro and in vivo, and pharmacokinetics of emivirine, a potent and selective nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1.

Emivirine (EMV), formerly known as MKC-442, is 6-benzyl-1-(ethoxymethyl)-5-isopropyl-uracil, a novel nonnucleoside reverse transcriptase inhibitor that displays potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity in vivo. EMV showed little or no toxicity towards human mitochondria or human bone marrow progenitor cells. Pharmacokinetics were linear for both rats and monkeys, and oral absorption was 68% in rats. Whole-body autoradiography showed widespread distribution in tissue 30 min after rats were given an oral dose of [(14)C]EMV at 10 mg/kg of body weight. In rats given an oral dose of 250 mg/kg, there were equal levels of EMV in the plasma and the brain. In vitro experiments using liver microsomes demonstrated that the metabolism of EMV by human microsomes is approximately a third of that encountered with rat and monkey microsomes. In 1-month, 3-month, and chronic toxicology experiments (6 months with rats and 1 year with cynomolgus monkeys), toxicity was limited to readily reversible effects on the kidney consisting of vacuolation of kidney tubular epithelial cells and mild increases in blood urea nitrogen. Liver weights increased at the higher doses in rats and monkeys and were attributed to the induction of drug-metabolizing enzymes. EMV tested negative for genotoxic activity, and except for decreased feed consumption at the high dose (160 mg/kg/day), with resultant decreases in maternal and fetal body weights, EMV produced no adverse effects in a complete range of reproductive toxicology experiments performed on rats and rabbits. These results support the clinical development of EMV as a treatment for HIV-1 infection in adult and pediatric patient populations.

In vitro comparison of selected triple-drug combinations for suppression of HIV-1 replication: the Inter-Company Collaboration Protocol.

Ten different three-drug combinations have been analyzed for their ability to prevent HIV-induced cytopathic effects (CPEs) in a continuous human T-lymphoblastoid cell line. Agents acting at the same as well as at different sites in the HIV-1 replicative cycle were used. Each compound was analyzed at peak and trough plasma levels achieved in monotherapy and in the presence of HIV-1 strains 3B and MN at a viral inoculum varying from 1 x TCID50 (50% tissue culture inhibitory dose) to 1,000 x TCID50. Using a viral inoculum of 10 x TCID50 HIV-1 3B, it was determined that triple-drug combinations had greater antiviral activities than the corresponding double-drug combinations, which had greater antiviral activities than zidovudine (AZT) monotherapy. The most consistent triple-drug combination, demonstrating superior activity at all concentrations of virus, was AZT + dideoxyinosine + lamivudine which reduced the AZT IC95 (95% inhibitory concentration) by 208-, 57-, 133-, and 25-fold at of 1,000, 100, 10, and 1 x TCID50 HIV-1 3B, respectively, as compared with the IC95 for AZT monotherapy. For all antiviral regimens tested, higher viral inoculum resulted in less inhibition of viral replication and a higher IC95 for AZT. This observation argues for therapeutic intervention at an earlier stage in HIV infection, when viral burden is lower.

Inter-Company Collaboration Combination Trials. Clinical Trial Subcommittee of the Inter-Company Collaboration for AIDS Drug Development.

The Inter-Company Collaboration for AIDS Drug Development (ICC) represents a collaborative effort among member companies to facilitate the conduct of clinical trials on AIDS drugs. One of the goals of the ICC is to expedite the development of combination antiretroviral therapy through data and compound sharing. Recently, the ICC formed a consensus master protocol to evaluate rapidly the safety and efficacy of triple-drug combinations of antiretroviral therapy for treatment of HIV-infected patients. This concept builds upon historical work with combination chemotherapy that resulted in treatments to successfully control chronic immunosuppressive, infectious or malignant diseases, such as tuberculosis, leprosy, childhood acute lymphoblastic leukemia, and Hodgkin's lymphoma. Because of limitations on potency and the continuing emergence of drug resistance seen with use of currently available antiretroviral agents in monotherapy and two-drug combination regimens, triple-combination regimens should represent a more promising approach to maximize antiviral activity, maintain long-term efficacy, and reduce the incidence of drug resistance. The ICC master protocol is a randomized, controlled, double-blind study with a treatment duration of 52 weeks. Patients eligible to enroll in this study must have documented HIV infection, with CD4 counts between 200 and 500 cells/mm3, and no history of antiretroviral therapy. The first four triple-drug combinations will be evaluated in two trials. These regimens have been selected based on encouraging data from laboratory and clinical studies. Each ICC trial will consist of three arms, with 75 patients per arm. Protocol ICC 001 will include AZT + zalcitabine (ddC) + saquinavir, AZT + ddC + nevirapine, and AZT + ddC as the control arm.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid screening of antiretroviral combinations.

Increasing evidence supports the view that therapy with combinations of antiretroviral drugs provides greater and more sustained benefits in the treatment of HIV infection than either monotherapy or sequential therapy. As the number of licensed and developmental antiretroviral agents grows, in vitro analysis is increasingly being used to aid in the selection of effective combinations. An assay has been designed to ascertain the inhibitory action of drug combinations on HIV-infected MT4 cells, allowing rapid evaluation of those that may be of use in the clinic. Manipulation of this system also provides data on the efficacy of drugs under conditions of high viral load and against resistant strains, providing valuable information for the treatment of antiretroviral-experienced patients with advanced disease.

Orofacial infection of athymic mice with defined mixtures of acyclovir-susceptible and acyclovir-resistant herpes simplex virus type 1.

Infection of athymic mice with defined populations of acyclovir-susceptible (thymidine kinase [TK]-positive) and acyclovir-resistant (TK-deficient or TK-altered) herpes simplex virus type 1 strains was used to simulate herpetic skin disease of the immunocompromised host. In vitro characterization of the defined virus mixtures revealed that the dye uptake method was quite sensitive in the detection of small amounts (3 to 9%) of acylovir-resistant virus. Mice infected with homogeneous virus populations exhibited a good correlation between clinical response and the in vitro drug susceptibility of the infecting virus. Animals infected with defined mixtures of viruses exhibited varied patterns of infection and responses to acyclovir treatment. However, disease severity was useful in predicting the TK phenotype of virus recovered from lesions. Pathogenic, TK-altered virus was responsible for progressive disease in animals receiving low-dose (0.25-mg/ml) prophylactic acyclovir or high-dose (1.25-mg/ml) delayed therapy. Although this mutant was recovered infrequently, it was responsible for clinically significant disease in the animals from which it was isolated.

Survival experience among patients with AIDS receiving zidovudine. Follow-up of patients in a compassionate plea program.

Through a compassionate plea program (Treatment Investigational New Drug), 4805 patients with acquired immunodeficiency syndrome who previously had experienced Pneumocystis carinii pneumonia (PCP) received zidovudine (Retrovir, formerly azidothymidine). Overall survival at 44 weeks after initiation of therapy was 73% (+/- 2.1%). A positive association was found between survival and pretherapy clinical status as defined by hemoglobin level, functional ability, and stage of disease as measured by time since diagnosis of PCP. For patients with baseline hemoglobin levels of 120 g/L or greater, Karnofsky scores of 90 or greater, and PCP diagnosis within 90 days prior to initiation of therapy, 44-week survival was 88%. Adverse clinical experiences associated with zidovudine therapy were consistent with those from a double-blind, placebo-controlled trial. Survival experience of this large and diverse cohort is consistent with, and extends data from, this clinical trial. Comparison with available natural history data suggests that zidovudine therapy is associated with increased 44-week survival of post-PCP patients with acquired immunodeficiency syndrome.

Spectrum of antiviral activity and mechanism of action of zidovudine. An overview.

Zidovudine is a potent in vitro inhibitor of human immunodeficiency virus (HIV) with varying efficacy against other retroviruses. With the exception of Epstein-Barr virus, all non-retroviruses tested so far have been insensitive to inhibition by zidovudine. In vivo, efficacy of zidovudine was demonstrated against Rauscher murine leukemia virus and feline leukemia virus. In both experimental models, infections completely resolved in animals when the drug was administered soon after infection. These results suggest that prompt initiation of zidovudine therapy, following a known exposure to HIV, should be considered. Mechanism studies show that zidovudine is phosphorylated to the monophosphate and diphosphate derivatives by the host cell cytosolic thymidine kinase and thymidylate kinase, respectively. The identity of the enzyme that phosphorylates zidovudine diphosphate is not known, but is believed to be the cellular nucleoside diphosphate kinase. The triphosphate of zidovudine appears to be the active form of the drug. Zidovudine triphosphate competes well with thymidine 5'-triphosphate for binding to the HIV reverse transcriptase and also functions as an alternative substrate. Incorporation of zidovudine monophosphate results in chain termination. However, it is not clear which mechanism, chain termination or competition with thymidine 5'-triphosphate, or a combination of both, is responsible for the inhibition of HIV replication.

HIV-1 inhibition by azidothymidine in a concurrently randomized placebo-controlled trail.

Two independent measures of human immunodeficiency virus type 1 (HIV-1) infection, virus isolation, and serum levels of p24 antigen were evaluated in a double-blind randomized clinical trial of the safety and efficacy of a nucleoside analogue, 3'-azido-3'-deoxythymidine (AZT) versus placebo in a single center. Pretreatment studies from 38 AIDS and AIDS-related complex (ARC) patients were comparably positive for virus isolation from their lymphocytes; all patients were qualitatively virus positive. Before AZT treatment, there was significantly decreased virus recovery in patients with higher numbers of CD4-positive lymphocytes. Within 1 month of AZT therapy, the time in culture required to register virus positivity was increased markedly in the AZT-treated group, and over the following several months progressive diminution in virus recovery was noted. Similar changes were not seen in patients concurrently receiving placebo treatment. Before treatment, 16 of 20 and 12 of 16 patients in the AZT and placebo groups, respectively, were p24 antigen positive. Marked reduction in serum p24 levels were noted in 11 of 16 (69%) of the p24 antigen-positive AZT-treated patients compared to 3 of 12 (25%) of the p24 antigen-positive placebo-treated patients (p = 0.02). There was a marked virologic response in 14 of 20 (70%) of the AZT-treated patients compared to 4 of 18 (22%) placebo-treated patients (p = 0.004). A higher frequency of positive clinical and immunological effects also were noted in the AZT-treated patients relative to placebo-treated patients (p = 0.02 and p = 0.06, respectively).

Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity.

In vitro and in vivo studies were done on a herpes simplex virus type 2 strain recovered from a patient on acyclovir (ACV) which was ACV resistant but expressed thymidine (dThd) kinase (EC 2.7.1.21) activity. Plaque-purified clones derived from the original clinical sample were heterogeneous with respect to plaque size and drug susceptibility. The heterogeneity of this viral mixture was also evident from varied 125I-labeled 5-iodo-2'-deoxycytidine autoradiographic patterns and from varied expression of dThd kinase-associated phosphorylating activities. Four clones from this mixture were 1-beta-D-arabinofuranosylthymine (ara-T) susceptible and ACV resistant. Extracts of cells infected with these clones catalyzed the phosphorylation of ara-T but little of ACV. The virus-coded dThd kinase was purified from one of these clones to determine whether its substrate specificity was altered. The amount of virus-coded dThd phosphorylating activity with the cell extracts was estimated to be sevenfold lower with the resistant clone than with the MS strain of herpes simplex virus type 2. The dThd kinase eluted from a dThd-agarose affinity column under the same conditions with extracts from both sources and substrate saturations of both enzymes by acyclic nucleoside analog phosphate acceptors were classical hyperbolic functions. However, there were significant differences in the kinetic parameters of substrates between the two enzymes. Apparent Km (Km') values for dThd, deoxycytidine, ara-T, ACV, and the acyclic guanosine analog 9-[[2-hydroxyl-1-(hydroxymethyl)ethoxy]methyl]guaine (BW B759U) were 2- to 60-fold higher with the variant enzyme than with the enzyme from laboratory strain MS. Comparing these two enzymes, relative maximal phosphorylation rates (Vm) were eightfold lower for ACV but unchanged for BW B759U. In contrast, the relative rates for deoxycytidine and ara-T were eight- and twofold higher, respectively. The surprisingly good substrate activity with BW B759U compared with that of ACV (Vm/Km' = 0.39 versus 0.01) coincided with susceptibility of the ACV-resistant virus to BW B759U. This clinical variant retained its pathogenicity for mice and was only moderately less neurovirulent than wild-type virus. Although such mutants have the potential to induce illness less responsive to therapy, the recurrence from which the isolate was obtained was typical for this patient in severity and duration. Since this episode, the patient has been treated successfully with ACV.

Clinical and laboratory experience with acyclovir-resistant herpes viruses.

The emerging field of antiviral sensitivity testing must depend upon some of the lessons learned from prior experience with anti-bacterial sensitivity testing, but also take into account additional factors including intracellular drug distribution and metabolism as well as the unique features of viral replication. There is an urgent need to standardise in-vitro sensitivity testing, and an international collaborative study is essential so that different laboratories may compare and define their sensitivity results in the context of the outcome of therapy in infected patients. It is only by such a study that the influence of patient and therapeutic factors such as immune function, anatomical location of the infection and dosage regimens used can be understood.

Induction of acyclovir-resistant mutants of herpes simplex virus type I in athymic nude mice.

Induction of acyclovir resistance was studied in the immune compromised host by multiple passage of plaque-purified herpes simplex type I strains in athymic nude mice receiving suboptimal antiviral therapy. Mice infected with a highly pathogenic clinical isolate rapidly developed infections that were resistant to therapy. Viruses isolated from these mice had decreased in-vitro sensitivities to acyclovir, as well as altered characteristics when assayed by [125I]plaque autoradiography. In contrast, less virulent laboratory strains, or a genetically stable clinical isolate, showed no indication of mutation to resistance after extended passage in this mouse model. Highly pathogenic viruses may increase the probability of mutation to resistance because of the large amount of infectious virus they produce, while viruses of equivalent virulence may produce different amounts of drug-resistant progeny because of alterations in the replication fidelity of the viral DNA polymerase.

Extended acyclovir therapy for herpes genitalis: changes in virus sensitivity and strain variation.

The in-vitro sensitivity of herpes simplex virus isolated from patients enrolled in clinical trials of orally administered chronic suppressive acyclovir therapy was determined. Approximately 500 isolates from 250 patients were examined. There was no increased incidence of recovery of virus less sensitive to acyclovir in vitro following chronic administration of acyclovir or intermittent therapy for recurrences during treatment periods of four months to four years. Changes in in-vitro sensitivity of virus isolated from two patients who shed virus less sensitive to acyclovir were studied to determine the possible role of continued antiviral therapy in selecting for progressively more resistant phenotypes. Increasing resistance was not documented. Both patients who shed less sensitive virus derived clinical benefit from chronic orally administered acyclovir treatment.

Recurrent genital herpes and suppressive oral acyclovir therapy. Relation between clinical outcome and in-vitro drug sensitivity.

To evaluate the association between in-vitro resistance of herpes simplex virus type 2 to acyclovir and breakthrough recurrences of herpes despite chronic suppressive therapy, we determined the in-vitro sensitivity of herpes simplex virus isolated before, during, and after therapy. One hundred eighty-three virus isolates from 107 patients were tested. Before therapy, the median amount of drug required to inhibit 50% of the virus in tissue culture (ID50) was 0.91 microgram/mL. The median ID50 after therapy was 0.99. Six isolates from patients with culture-positive breakthrough recurrences were evaluated. The median ID50 was 0.90 microgram/mL (range, 0.39 to 1.55). The development of breakthrough recurrences could not be correlated with infection with strains of herpes simplex virus type 2 that were resistant to acyclovir in vitro. Acyclovir-resistant strains are not commonly recovered from patients during acyclovir therapy, nor does there seem to be a high frequency of resistance after 4 months of chronic suppressive therapy.

Administration of 3'-azido-3'-deoxythymidine, an inhibitor of HTLV-III/LAV replication, to patients with AIDS or AIDS-related complex.

In a 6-week clinical trial 4 dose regimens of 3'-azido-3'-deoxythymidine (AZT), a thymidine analogue with potent anti-viral activity against HTLV-III in vitro, were examined in 19 patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). AZT was given intravenously for 2 weeks, then orally for 4 weeks at twice the intravenous dose. AZT was well absorbed from the gut and crossed the blood-brain barrier. Therapeutic levels were maintained with 5 mg given intravenously or 10 mg given orally every 4 h. Treatment was not limited by side-effects, the commonest of which were headaches and depression of white-cell counts. 15 of the 19 patients had increases in their numbers of circulating helper-inducer T lymphocytes (p less than 0.001) during therapy, 6 who were anergic at entry showed positive delayed type hypersensitivity skin test reactions during treatment, 2 had clearance of chronic fungal nailbed infections without specific anti-fungal therapy, 6 had other evidence of clinical improvement, and the group as a whole had a weight gain of 2.2 kg. Also, with the highest dose regimen cultures of peripheral blood mononuclear cells for HTLV III became negative.

3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro.

The acquired immune deficiency syndrome (AIDS) is thought to result from infection of T cells by a pathogenic human retrovirus, human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV). In this report, we describe the antiviral effects of a thymidine analogue,3'-azido-3'-deoxythymidine (BW A509U), which, as a triphosphate, inhibits the reverse transcriptase of HTLV-III/LAV. This agent blocks the expression of the p24 gag protein of HTLV-III/LAV in H9 cells following exposure to virus. The drug also inhibits the cytopathic effect of HTLV-IIIB (a virus derived from a pool of American patients) and HTLV-III/RF-II (an isolate obtained from a Haitian patient that differs by about 20% in the amino acid sequence of the envelope gene from several isolates of HTLV-III/LAV, including HTLV-IIIB, analyzed so far). 3'-Azido-3'-deoxythymidine also completely blocks viral replication as assessed by reverse transcriptase production in normal human peripheral blood mononuclear cells exposed to HTLV-IIIB. Finally, at concentrations of 3'-azido-3'-deoxythymidine that block the in vitro infectivity and cytopathic effect of HTLV-IIIB, the in vitro immune functions of normal T cells remain basically intact.

Plaque autoradiography assay for the detection and quantitation of thymidine kinase-deficient and thymidine kinase-altered mutants of herpes simplex virus in clinical isolates.

A plaque autoradiography assay to detect and quantitate thymidine kinase (TK) mutants of herpes simplex virus type 1 (HSV-1) and HSV-2 in clinical samples is described. This method utilizes the selective incorporation of [125I]iododeoxycytidine, a pyrimidine analog selectively phosphorylated by the HSV TK. Only cells infected with TK-competent virus will efficiently incorporate iododeoxycytidine and are the only cells detected by autoradiography. Furthermore, this assay discriminates between TK+ virus (TK competent) and TKA virus (TK altered or reduced). This ability to differentiate TK+ from TKA virus is enhanced when infected cells are labeled with [14C]thymidine in tandem with iododeoxycytidine labeling. Reconstruction experiments with mixtures of TK+ (HSV-1 Patton) virus and TK-deficient (TK-) (B2006) or TKA (IUDRr) mutants were performed to determine the limits of detection of this technique. Ten percent TK- or TKA virus was the lower limit for the detection of TK mutants in a mixed population, whereas 1 in 1,000 TK+ virus revertants could be detected in a TK- virus population. In reconstructed populations and 45 clinical samples, a good correlation existed between the increase in 50% inhibitory dose for acyclovir and the percent TK mutant virus present. Similarly, the results of this technique correlated well with the acyclovir phosphorylating activity of extracts from cells infected with isolates or reconstructed mixtures. Plaque autoradiography with [125I]iododeoxycytidine was able to distinguish mixed populations of TK+ and TK- virus and homogeneous populations of TKA virus. The tandem use of [125I]iododeoxycytidine and [14C]thymidine readily identified TKA virus, which appeared as TK+ virus when labeled with [14C]thymidine alone. This technique provides a sensitive screen for antiviral resistance due to alterations in the viral TK and can be used to analyze clinical samples.

Oral acyclovir therapy of genital herpes simplex virus type 2 infections in guinea pigs.

Oral acyclovir was evaluated for its effectiveness in treating guinea pigs with primary herpes simplex virus type 2 infections. Guinea pigs inoculated intravaginally with acyclovir-susceptible strains (for which 50% inhibitory concentrations of acyclovir in cell culture were found to be in the range of 0.15 to 1.2 micrograms/ml) and treated with 5.0 mg of acyclovir per ml in the drinking water beginning 48 h postinfection showed significant reductions in lesion severity. This dosage produced serum acyclovir levels of 1.3 micrograms/ml. Lower concentrations of oral acyclovir (less than or equal to 2.5 mg/ml in the drinking water), which produced serum acyclovir levels of less than 1.0 microgram/ml, were less consistently effective against these same virus strains. When an acyclovir-resistant isolate (for which the 50% inhibitory concentration of acyclovir in cell culture was found to be 8.5 micrograms/ml) was used to initiate infection, treatment with 5 or 10 mg/ml (yielding serum levels of 1.3 and 3.5 micrograms/ml) in the drinking water had only minimal clinical benefit. However, the degree of response was difficult to determine because of the attenuated disease produced by the acyclovir-resistant virus. In vitro virus sensitivity may be predictive of the serum drug levels that need to be obtained to produce a successful response to therapy.

Viral resistance, clinical experience.

Over 1500 herpes simplex virus isolates from over 600 patients have been examined in the Wellcome Research Laboratories during the past 5 years using the dye uptake method in Vero cells to determine acyclovir sensitivity. No significant change in sensitivity of those isolates to acyclovir has been noted during that period, and the few isolates whose sensitivity significantly diminished during therapy were generally not associated with a clinical lack of response to therapy. In patients who were severely immunocompromised and who had received prolonged or repeated courses of therapy, however, less sensitive viruses were occasionally associated with poorly healing ulcers. The significance of these findings are discussed, as are problems in the interpretation of results of in vitro antiviral sensitivity testing and their extrapolation to clinical practice.