Evolution of genotypic and phenotypic resistance during chronic treatment with the fusion inhibitor T-1249.

T-1249 is a peptide HIV fusion inhibitor (FI) previously under development for use in FI-naive and experienced patients. Here we present prospectively planned longitudinal analyses of FI resistance during 48 weeks of T-1249 dosing in patients with extensive prior FI exposure. T1249-105 was a single-arm rollover study in patients with prior resistance to enfuvirtide (ENF) and 10 days of T-1249 functional monotherapy exposure. The phenotype and genotype of plasma virus envelopes were analyzed at baseline and at study weeks 8, 16, and 48. At study entry, viruses had a geometric mean decrease in susceptibility to ENF of 51.8-fold but to T-1249 of 1.8-fold; extensive genotypic resistance to ENF was observed. A median viral load response of - 1.5 log(10) copies/ml was observed at week 2 that was partially sustained (- 0.5 log(10) copies/ml) through 48 weeks. Resistance to T-1249 gradually increased to a geometric mean 92.7-fold decrease from FI-naive baseline; this occurred concomitant with further evolution of gp41 amino acids 36-45, most commonly the G36D (n = 6, 16%) or N43K (n = 9, 24%) substitutions. A novel substitution, A50V (n = 12, 32%), was also common, as were the N126K and S138A substitutions in heptad-repeat 2 (HR-2). These data point toward a primary role for the gp41 36-45 locus in modulating FI binding and suggest that residues in HR-2 may contribute in a more limited manner to development of peptide FI resistance. These data also point toward a substantial genetic barrier and fitness cost to development of resistance to next-generation fusion inhibitors.

Characterization of envelope glycoprotein gp41 genotype and phenotypic susceptibility to enfuvirtide at baseline and on treatment in the phase III clinical trials TORO-1 and TORO-2.

Enfuvirtide (T-20) is the first entry inhibitor approved for treatment of HIV infection and acts by inhibiting conformational changes in the viral envelope protein gp41 that are necessary for fusion of the virus and host cell membranes. Here we present genotypic and phenotypic data on viral envelopes obtained at baseline (n = 627) and after 48 weeks of enfuvirtide treatment (n = 302) from patients in the TORO (T-20 versus Optimized Regimen Only)-1 and -2 phase III pivotal studies. The amino acid sequence at residues 36-45 of gp41 was highly conserved at baseline except for polymorphism of approximately 16% at position 42. Substitutions within gp41 residues 36-45 on treatment were observed in virus from 92.7% of patients who met protocol defined virological failure criteria and occurred in nearly all cases (98.8%) when decreases in susceptibility to enfuvirtide from baseline of greater than 4-fold were observed. Consistent with previous observations, a wide range of baseline susceptibilities (spanning 3 logs) was observed; however, lower in vitro baseline susceptibility was not significantly associated with a decreased virological response in vivo. Virological response was also independent of baseline coreceptor tropism and viral subtype.

Phase I/II study of 3TC (lamivudine) in HIV-positive, asymptomatic or mild AIDS-related complex patients: sustained reduction in viral markers. The Lamivudine European HIV Working Group.

OBJECTIVE: To evaluate the efficacy of 3TC (lamivudine), a synthetic nucleoside analogue that inhibits HIV reverse transcriptase in vitro, as treatment for HIV-positive, asymptomatic or mild AIDS-related complex patients. DESIGN: Open-label, multinational and multicentre, non-comparative, escalating dose study. METHODS: Patients who meet the selection criteria (n = 104) were enrolled in three European countries. Ten to 15 patients were included at each of the six dose levels of 3TC (0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 20.0 mg/kg daily in two divided doses every 12 h). Virological parameters--immune-complex dissociation (ICD) assay for HIV p24 antigenaemia, plasma HIV RNA load, whole blood assay and cellular viraemia--were evaluated at weeks 0, 4, 12 and 24. RESULTS: Sustained reductions in HIV RNA load and in ICD p24 antigen levels were observed and maintained over the 12-week assessment period. Greater reductions were noted at higher doses but this trend did not reach statistical significance. In 38 patients, reductions of cell viraemia were significantly greater at 4 weeks for patients treated at higher doses of 3TC. CONCLUSION: These virological data show that 3TC is a potent inhibitor of HIV replication in HIV-positive, asymptomatic or mild ARC patients as assessed by ICD p24 antigenaemia, plasma HIV RNA load and cell viraemia.

Development of HIV-1 resistance to (-)2'-deoxy-3'-thiacytidine in patients with AIDS or advanced AIDS-related complex.

OBJECTIVE: To determine the rate of development of in vitro HIV resistance to (-)2'-deoxy-3'-thiacytidine (3TC) and relate the effect of dose to emergence of resistance. METHODS: HIV-infected men and non-pregnant women, aged > or = 18 years, with a CD4 count < or = 300 x 10(6)/l cells were followed in a Phase I/II study, in which they were evaluated for tolerance to 3TC and effect of this agent with regard to viral susceptibility. Peripheral blood and plasma samples were collected at regular intervals for analysis. HIV was isolated using umbilical cord blood mononuclear cells as targets. These cells were also used in determinations of median inhibitory drug concentration. Specific amplification of the 184 mutation site, associated with HIV resistance to 3TC, was performed by polymerase chain reaction, using specific primer pairs, on DNA harvested from infected peripheral blood mononuclear cells (PBMC) of donors or, alternatively, on DNA that had been reverse transcribed from plasma-associated HIV RNA. RESULTS: Phenotypic resistance was detected in approximately one-third of individuals studied, who were followed between 8 and 56 weeks. Development of 3TC resistance occurred independently of dose, although time of first appearance of resistant HIV-1 variants appeared reduced at high 3TC doses. Amino-acid changes at codon 184 in HIV-1 reverse transcriptase were associated with, and preceded, the development of phenotypic 3TC resistance. Most commonly, a Met to Ile substitution appeared transiently before being superceded by a Val substitution at codon 184. CONCLUSIONS: In vitro resistance to 3TC developed in a high proportion of subjects who received prolonged monotherapy with this drug. The development of resistance to 3TC was associated with appearance of mutated viral forms and the disappearance of wild-type virus, with regard to codon 184, in both patient plasma and PBMC.

HIV protease genotype and viral sensitivity to HIV protease inhibitors following saquinavir therapy.

OBJECTIVE: To examine the relationship between HIV protease genotype and altered protease inhibitor sensitivity of isolates from patients after therapy with saquinavir (SQV) in its hard gelatin formulation. DESIGN: Forty-one post-therapy isolates and corresponding baseline samples were obtained from 37 patients in four different clinical trials after therapy with SQV for 16-147 weeks. Post-therapy isolates were selected on the basis of preliminary sequence or drug sensitivity data. RESULTS: Fifteen out of 17 isolates without detectable Val-48 or Met-90 mutations retained sensitivity to SQV. (The remaining isolates showed only a marginal increase in median inhibitory concentration.) In addition, three out of 15 isolates with Met-90 retained sensitivity to all other protease inhibitors tested (indinavir, ritonavir, amprenavir, nelfinavir). Of the isolates showing reduced sensitivity to SQV, six out of 22 retained sensitivity to all other protease inhibitors, whereas only four out of 22 showed broad cross-resistance to all protease inhibitors tested. The reduction in sensitivity correlated closely with the presence of Val-48 or Met-90. Subsequent accessory substitutions were also linked to reduced sensitivity. However, significant linkage was observed only between mutations at residues 48 and 82 and between those at residues 82 and 74. CONCLUSIONS: Recruitment of Val-48/Met-90 mutations was not found to be synonymous with cross-resistance. Indeed, the majority of isolates with these mutations retained sensitivity to at least one protease inhibitor (Val-48, 86%; Met-90, 77%). The recruitment of accessory mutations may occur only after the selection of key resistance mutations. Furthermore, Met-90 was found to be a poor marker of cross-resistance in SQV-treated patients.

Correlation of response to treatment and HIV genotypic changes during phase III trials with saquinavir and reverse transcriptase inhibitor combination therapy.

OBJECTIVES: Assessment of genotypic change in HIV protease during treatment with saquinavir (SQV) in combination with zidovudine (ZDV) and/or zalcitabine (ddC), to determine the influence of such changes on viral phenotype and response to treatment. DESIGN: Virologic substudies of Phase III clinical trials NV14256 and SV14604. METHODS: Population sequencing of HIV protease genes amplified from pre- and post-treatment plasma. Phenotyping of peripheral blood mononuclear cell (PBMC)-derived virus isolates, and genotyping of proviral DNA clones amplified from PBMC used in the expansion of virus isolates. RESULTS: In both trials the incidence of Met90 remained at < or = 20% in subjects receiving SQV in combination with ddC (with or without ZDV) for 1 year. A Val48 substitution was observed in two out of 81 subjects after 24 weeks and in two out of 75 subjects after 48 weeks. In 12 out of 13 NV14256 subjects with viral load rebound during SQV monotherapy these substitutions were associated with the rebound. In subjects treated with SQV plus ddC, rebound was associated with SQV resistance in six out of 22 cases and ddC resistance in five out of 22 cases. The incidences of non-BRU residues at positions 10, 63 and 71 were increased significantly (P < 0.05, Fisher's exact test) after SQV treatment with or without ZDV. However, comparison of genotypic and phenotypic data showed that these changes were not associated with reduced sensitivity to SQV. CONCLUSIONS: Virological failure during combination therapy can be due to resistance to either treatment drug, emphasising the need to change both the reverse transcriptase inhibitor and the protease inhibitor. Only Val48 and Met90 correlated directly with the development of reduced drug sensitivity during treatment with SQV in vivo.

Measuring the effectiveness of antiretroviral agents.

Considerable progress has been made recently in developing effective antiretroviral combination therapy that can suppress viral replication and delay disease progression in individuals infected with HIV. A range of up to 15 approved antiretroviral agents is now available, which target two different viral enzymes, while several agents are in clinical development. The rapid development and approval of antiretroviral agents, driven by the urgency of clinical need as well as the complexity of possible combinations, has precluded the extensive comparative clinical testing of regimens, which is necessary to establish the relative efficacy of various different agents. The lack of an appropriate animal model for HIV disease also increases reliance on in vitro measures. Several different in vitro and in vivo parameters have been defined in an attempt to quantify the effectiveness of antiretroviral agents, most importantly the 50% inhibitory and effective concentrations (IC50 and EC50). However, the clinical relevance of these measures is uncertain. Additionally, considerable variation exists in the usage of the terms 'IC50' and 'EC50' in recent publications in the literature. These issues pose interpretation problems to clinicians seeking information on the relative clinical efficacy of the agents. In this brief review, we attempt to clarify the different measures available and their potential utility for clinical decision-making, focusing particularly on the example of HIV protease inhibitors. There are many different quantifiable parameters that give information regarding the effectiveness of an antiviral drug. These include: inhibition of the viral target enzyme (inhibition constant, Ki); selectivity for viral versus host enzymes; inhibition of viral replication in cell culture (IC50); ratio of efficacy to cytotoxicity in vitro (therapeutic index); inhibition of viral replication or symptoms in an appropriate animal model of the disease (EC50); and the effect on surrogate markers, such as viral load or CD4 cell count, after administration to humans (in vivo EC50). Each of these different parameters gives valid information about the properties of an antiretroviral agent, which can help to build up a picture of its potential clinical utility relative to other drugs. However, to gain meaningful results, it is important to apply this information intelligently, understanding the limitations of each parameter.

Resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC) in HIV-1 isolated from paediatric patients.

We conducted detailed virological evaluations of 16 HIV-1-infected paediatric patients treated with 3TC (lamivudine) monotherapy. High-level phenotypic resistance against this compound (up to 2,500-fold) was seen in virtually all cases, usually within 8-12 weeks of initiation of therapy. This was concomitant with the appearance of the M184V mutation in viral reverse transcriptase, previously shown to be responsiblefor such resistance. Viral burden fell in virtually all cases after commencement of therapy, and remained below baseline in each instance studied, despite a rebound effect and the appearance of drug resistance. Viral isolates from some patients underwent a switch from a non-syncytium-inducing (NSI) to a syncytium-inducing (SI) phenotype during the course of the study, although no relationship was apparent between dose of drug employed, time to development of drug resistance or time of appearance of SI phenotype.

Synthesis and structure-activity relationships of a series of penicillin-derived HIV proteinase inhibitors: heterocyclic ring systems containing P1' and P2' substituents.

As an extension of our earlier work based upon a single penicillin-derived thiazolidine moiety we have found that the decahydroisoquinoline grouping, also present in Ro 31-8959, is an effective replacement for one of the thiazolidine units in C2 symmetric penicillin-derived dimers. Reaction of racemic epoxide 6 with [3S-[3 alpha, 4a alpha, 8a alpha]]-decahydro-N-(1,1-dimethylethyl)-3- isoquinolinecarboxamide gave diasteroisomers 34a and 34b. The stereochemistry of the hydroxyl grouping of 34a was determined to be (S). Reaction of the amines derived from 34a and 34b with thiazolidine 8a gave 50 and 51, respectively. Compound 50 was a potent inhibitor of HIV proteinase (IC50 = 23 nM) with antiviral activity against HIV-1 in vitro (EC50 C8166 cells = 50 nM). However, a poor pharmacokinetic profile in the dog for compound 50 and its analogues, in keeping with earlier studies on penicillin-derived dimers in three species, precluded their development as potential antivirals.

A series of penicillin derived C2-symmetric inhibitors of HIV-1 proteinase: synthesis, mode of interaction, and structure-activity relationships.

The C2-symmetric diester 1 was identified by random screening as a novel inhibitor of HIV-1 proteinase. This led to the preparation of a series of related more potent amides from readily accessible penicillins. Many of the compounds showed potent antiviral activity in HIV-1-infected MT-4 cells and an ability to inhibit syncytia formation in infected C8166 cells, with no evidence of cytotoxicity. The compounds showed no activity against other aspartyl proteinases (renin, pepsin, and cathepsin D). Structure-activity relationships support a symmetrical interaction with the enzyme. Pharmacokinetic evaluation of the ethylamide 3 revealed it was subject to rapid plasma clearance and had low oral bioavailability.

Synthesis and structure-activity relationships of a series of penicillin-derived HIV proteinase inhibitors containing a stereochemically unique peptide isostere.

A series of HIV-1 proteinase inhibitors was synthesized based upon a single penicillin derived thiazolidine moiety. Reaction of the C-4 carboxyl group with (R)-phenylalaninol gave amide 10 which was a moderately potent inhibitor of HIV-1 proteinase (IC50 = 0.15 microM). Further modifications based on molecular modeling studies led to compound 48 which contained a stereochemically unique statine-based isostere. This was a potent competitive inhibitor (Ki = 0.25 nM) with antiviral activity against HIV-1 in vitro (5 microM). Neither modification to the benzyl group in an attempt to improve interaction with the S2' pocket, nor introduction of a hydrogen bond donating group to interact with residue Gly48' resulted in improved inhibitory or antiviral activity.

Benzophenone derivatives: a novel series of potent and selective inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

A series of benzophenone derivatives has been synthesized and evaluated as inhibitors of HIV-1 reverse transcriptase (RT) and the growth of HIV-1 in MT-4 cells. Through the use of the structure-activity relationships within this series of compounds and computational chemistry techniques, a binding conformation is proposed. The SAR also indicated that the major interactions of 1h with the RT enzyme are through hydrogen bonding of the amide and benzophenone carbonyls and pi-orbital interactions with the benzophenone nucleus and an aromatic function separated from the benzophenone by a suitable spacer group. The crystal structure of compound 1h has been determined. A number of compounds with potent inhibitory activity against HIV-1 RT and HIV in cellular assays at levels comparable with AZT and our efforts to identify a metabolically stable analogue are described.

Synthesis and anti-HIV-1 activity of a series of imidazo[1,5-b]pyridazines.

A series of substituted imidazo[1,5-b]pyridazines have been prepared and tested for inhibitory activity against the reverse transcriptase of HIV-1 (RT) and their ability to inhibit the growth of infected MT-4 cells. Crystal data are reported on two compounds, 15c and 33. From the structure-activity relationships developed within this and other series, it is proposed that key features of the interaction with RT include hydrogen-bond acceptor and aromatic pi-orbital bonding with the imidazopyridazine nucleus and a benzoyl function separated from the heterocycle by a suitable spacer group. Exceptional activity against the reverse transcriptase of HIV-1 (IC50 = 0.65 nM) was obtained with a 2-imidazolyl-substituted derivative, 7-[2-(1H-imidazol-1- yl)-5-methylimidazo-[1,5-b]pyridazin-7-yl]-1-phenyl-1-heptanone (33) which is attributed to additional binding of the imidazole sp2 nitrogen atom. A number of the compounds in this series also inhibit the replication of HIV-1 in vitro in MT-4 and C8166 cells at levels observed with the nucleoside AZT.

Cellular metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine.

The metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine (3TC) was examined in human immunodeficiency virus type 1 (HIV-1)-infected and mock-infected human cells. 3TC 5'-triphosphate levels accumulated comparably in HIV-1-infected and mock-infected phytohaemagglutinin-stimulated peripheral blood lymphocytes (PBL) and reached 40% or more of total intracellular 3TC metabolites after 4 hr. The rate of decay of 3TC triphosphate in HIV-1-infected and mock-infected PBL measured as a half-life (T1/2) ranged from 10.5 to 15.5 hr. 3TC did not significantly affect metabolism of deoxynucleotides in the U937 cell line, and was shown to be resistant to the action of human platelet pyrimidine nucleoside phosphorylase.

Antigenic analysis of yellow fever virus glycoproteins: use of monoclonal antibodies in enzyme-linked immunosorbent assays.

A sensitive enzyme-linked immunosorbent assay with biotin and streptavidin (B/SA ELISA) was developed for the specific detection of yellow fever (YF) viruses. Monoclonal antibodies (MCA) specific for the envelope (E) glycoprotein or the non-structural glycoprotein (NV3) of YF virus-infected cell lysates were tested by antigen and antibody capture B/SA ELISA against YF viruses and a large number of other flaviviruses. In an antigen capture assay, with suckling mouse brain antigens, MCA directed against the envelope glycoprotein clearly differentiated YF viruses from any other flaviviruses, wild-type YF viruses from YF vaccine viruses and YF17D-204 substrains from other YF vaccine viruses. Specific MCA could identify YF viruses in an antibody capture assay with concentrated YF-infected tissue culture supernatant medium as the solid phase. On the basis of the principles established above, MCA binding profiles of purified YF virus strains were compared. No qualitative differences in immunoreactivity could be detected between closely related strains of YF virus. An antibody capture assay on infected tissue culture monolayers was developed to enable in situ detection of YF viruses using MCA specific for both the envelope glycoprotein and the non-structural NV3 glycoprotein.

Use of the biotin-streptavidin interaction to improve flavivirus detection by immunofluorescence and ELISA tests.

The specificity and sensitivity of immunofluorescence microscopy and ELISA tests were compared both with and without the use of biotinylated anti-species antibody and streptavidin, conjugated with either fluorescein isothiocyanate or horse radish peroxidase. In the biotin-streptavidin system monoclonal and polyclonal antibodies against yellow fever virus had titres between ten- and fifty-fold higher and background readings, particularly in ELISA tests, were noticeably reduced. Plaque variants of yellow fever virus, selected for their apparent loss of antigenic determinants against some monoclonal antibodies in conventional fluorescence tests, were found to possess the antigenic determinants when tested with the more sensitive system.

Human immunodeficiency virus type 1 entry and chemokine receptors: a new therapeutic target.

After the identification of CD4 as the primary receptor for human immunodeficiency virus (HIV) type 1 entry into cells of the immune system, it soon became clear that CD4 alone was not sufficient to establish a productive infection. The search for the second receptors or co-receptors started over 10 years ago, and it was not until 1996 that G protein-coupled 7-transmembrane receptors, CXCR4 and CCR5 were finally identified as the co-receptors for HIV-1 entry. The 7-transmembrane receptor family is a familiar therapeutic target for a number of diseases, and therefore these recent findings represent an exciting opportunity for new therapeutic approaches to the treatment of HIV-1 infection.

In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.