Pharmacokinetic and Chemical Synthesis Optimization of a Potent d-Peptide HIV Entry Inhibitor Suitable for Extended-Release Delivery.

Peptides often suffer from short in vivo half-lives due to proteolysis and renal clearance that limit their therapeutic potential in many indications, necessitating pharmacokinetic (PK) enhancement. d-Peptides, composed of mirror-image d-amino acids, overcome proteolytic degradation but are still vulnerable to renal filtration due to their small size. If renal filtration could be slowed, d-peptides would be promising therapeutic agents for infrequent dosing, such as in extended-release depots. Here, we tether a diverse set of PK-enhancing cargoes to our potent, protease-resistant d-peptide HIV entry inhibitor, PIE12-trimer. This inhibitor panel provides an opportunity to evaluate the PK impact of the cargoes independently of proteolysis. While all the PK-enhancing strategies (PEGylation, acylation, alkylation, and cholesterol conjugation) improved in vivo half-life, cholesterol conjugation of PIE12-trimer dramatically improves both antiviral potency and half-life in rats, making it our lead anti-HIV drug candidate. We designed its chemical synthesis for large-scale production (CPT31) and demonstrated that the PK profile in cynomolgous monkeys supports future development of monthly or less frequent depot dosing in humans. CPT31 could address an urgent need in both HIV prevention and treatment.

Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors.

HIV-1 entry and fusion with target cells is an important target for antiviral therapy. However, a few currently approved treatments are not effective as monotherapy due to the emergence of drug resistance. This consideration has fueled efforts to develop new bioavailable inhibitors targeting different steps of the HIV-1 entry process. Here, a high-throughput screen was performed of a large library of 100,000 small molecules for HIV-1 entry/fusion inhibitors, using a direct virus-cell fusion assay in a 384 half-well format. Positive hits were validated using a panel of functional assays, including HIV-1 specificity, cytotoxicity, and single-cycle infectivity assays. One compound-4-(2,5-dimethyl-pyrrol-1-yl)-2-hydroxy-benzoic acid (DPHB)-that selectively inhibited HIV-1 fusion was further characterized. Functional experiments revealed that DPHB caused irreversible inactivation of HIV-1 Env on cell-free virions and that this effect was related to binding to the third variable loop (V3) of the gp120 subunit of HIV-1 Env. Moreover, DPHB selectively inhibited HIV-1 strains that use CXCR4 or both CXCR4 and CCR5 co-receptors for entry, but not strains exclusively using CCR5. This selectivity was mapped to the gp120 V3 loop using chimeric Env glycoproteins. However, it was found that pure DPHB was not active against HIV-1 and that its degradation products (most likely polyanions) were responsible for inhibition of viral fusion. These findings highlight the importance of post-screening validation of positive hits and are in line with previous reports of the broad antiviral activity of polyanions.

Baseline CD4(+) T-cell counts and weighted background susceptibility scores strongly predict response to maraviroc regimens in treatment-experienced patients.

BACKGROUND: Maraviroc-containing regimens are known to achieve virological suppression in many treatment-experienced patients. This study aimed to evaluate a more rigorous methodological approach to resistance-response analysis in large clinical studies and to better establish which subpopulations of patients were most likely to benefit from maraviroc by refining and extending previous subgroup analyses from the MOTIVATE studies. METHODS: Individual weighted optimized background therapy (OBT) susceptibility scores were calculated by combining genotypic or phenotypic resistance testing with prior drug use information. Virological response (HIV-1 RNA<50 copies/ml at week 48) using each of these methods was compared with a commonly used method of counting active drugs. Baseline predictors of virological response, including weighted or unweighted scoring, maraviroc use, baseline CD4(+) T-cell count, HIV-1 plasma viral load and tropism, were assessed by logistic regression modelling. RESULTS: Genotypic or phenotypic weighted methods were similarly predictive of virological response and better than counting active drugs. Weighted scoring and baseline CD4(+) T-cell count were the strongest predictors of virological response (P<0.0001): ≈70% of maraviroc patients with a weighted score ≥2 had a virological response, rising to ≈80% when the baseline CD4(+) T-cell count was ≥50 cells/mm(3). CONCLUSIONS: Approximately 80% of patients with a CD4(+) T-cell count ≥50 cells/mm(3) receiving maraviroc with the equivalent of at least two fully active agents achieved HIV-1 RNA<50 copies/ml at week 48 in the MOTIVATE studies. Genotypic and phenotypic weighted scores were similarly predictive of virological response.

Cost-effectiveness of optimized background therapy plus maraviroc for previously treated patients with R5 HIV-1 infection from the perspective of the Spanish health care system.

OBJECTIVE: The aim of this work was to evaluate the cost-effectiveness, from the perspective of the Spanish health care system, of optimized background therapy (OBT) plus maraviroc 300 mg BID versus OBT plus placebo in previously treated patients with R5 HIV-1 infection. METHODS: A lifetime cohort model was developed, based on 24- and 48-week pooled results from the Maraviroc Versus Optimized Therapy in Viremic Antiretroviral Treatment-Experienced Patients (MOTIVATE) studies 1 and 2, to reflect the Spanish health care system's perspective. Treatment duration was based on clinical trial follow-up from MOTIVATE 1 and 2. Clinical data, cohort characteristics, success probability, CD4 increase rate, CD4 cell status link to disease states, and adverse-event probability were taken from the MOTIVATE trials and other published literature. Other input parameters were taken from published sources. Antiretroviral (ARV) costs were derived from local sources. Non-ARV drug costs were obtained from published literature and a cost database. All costs were calculated as year-2009 euros. The annual discount rate was set at 3.0%. The main outcomes were cost per life-year gained (LYG) and cost per quality-adjusted life-year (QALY) gained. Uncertainty was assessed with one-way and probabilistic sensitivity analyses. RESULTS: In the model analysis, adding maraviroc to OBT was associated with an increase of 0.952 LYG and 0.909 QALY. Total costs were €275,970 for maraviroc plus OBT and €254,655 for placebo plus OBT (difference: €21,315). The incremental cost per LYG was €22,398 and the incremental cost per QALY gained was €23,457. The model appeared to be robust for variations in key parameters. Results from the probabilistic sensitivity analyses indicated that the probability of the cost per QALY being below €30,000 was 99%. CONCLUSION: Despite the limitations of the model, our analysis suggested that OBT plus maraviroc 300 mg BID is a clinically valuable option, and cost-effective from the perspective of the Spanish health care system, for previously treated patients with R5 HIV-1 infection.

Preclinical assessment of the distribution of maraviroc to potential human immunodeficiency virus (HIV) sanctuary sites in the central nervous system (CNS) and gut-associated lymphoid tissue (GALT).

1. Growing knowledge of the pathogenesis of human immunodeficiency virus (HIV)-1 infection has led to the identification of potential virus sanctuary sites within the central nervous system and gut-associated lymphoid tissue. 2. Maraviroc is a novel CCR5 antagonist for the treatment of HIV-1 infection. Disposition studies have been performed within the preclinical testing of maraviroc to determine its distribution to these anatomical sites. 3. Maraviroc, which is a substrate of the efflux transporter P-glycoprotein, shows limited distribution to the central nervous system as evidenced by cerebrospinal fluid concentrations that were 10% of the free plasma concentration following intravenous infusion to rats. Tissue distribution studies also indicated limited distribution of radioactivity into brain tissue of rats. 4. Radioactivity in gut-associated lymphoid tissue lymph nodes exceeded the concentrations in blood and concentrations in the contents of thoracic ducts of the lymphatic system were similar to blood levels following intravenous administration to rats.

Entry inhibitor-based microbicides are active in vitro against HIV-1 isolates from multiple genetic subtypes.

Inhibitors of viral entry are under consideration as topical microbicides to prevent HIV-1 sexual transmission. Small molecules targeting HIV-1 gp120 (BMS-378806) or CCR5 (CMPD167), and a peptide fusion inhibitor (C52L), each blocks vaginal infection of macaques by a SHIV. A microbicide, however, must be active against multiple HIV-1 variants. We therefore tested BMS-C (a BMS-378806 derivative), CMPD167, C52L and the CXCR4 ligand AMD3465, alone and in combination, against 25 primary R5, 12 X4 and 7 R5X4 isolates from subtypes A-G. At high concentrations (0.1-1 microM), the replication of most R5 isolates in human donor lymphocytes was inhibited by >90%. At lower concentrations, double and triple combinations were more effective than individual inhibitors. Similar results were obtained with X4 viruses when AMD3465 was substituted for CMPD167. The R5X4 viruses were inhibited by combining AMD3465 with CMPD167, or by the coreceptor-independent compounds. Thus, combining entry inhibitors may improve microbicide effectiveness.

A randomized pilot study comparing combination therapy plus enfuvirtide versus a treatment interruption followed by combination therapy plus enfuvirtide.

Most individuals with multidrug-resistant HIV who switch to a new therapeutic regimen containing a single fully effective agent experience incomplete viral suppression. We postulated that interruption of antiretroviral therapy prior to the introduction of such a regimen would improve long-term virological outcomes. Thirty, three-class experienced, enfuvirtide-naive individuals with detectable drug-resistant viraemia were randomized to an immediate enfuvirtide/optimized-background treatment regimen or a 16-week treatment interruption followed by enfuvirtide/optimized-background treatment regimen. The median CD4+ T-cell count and viral load at study entry were 39 cells/mm and 4.72 log10 copies RNA/ml, respectively. There was no evidence of any virological or immunological benefit associated with the interruption. In multivariate analysis, only the baseline phenotypic susceptibility score was predictive of treatment response at week 48 (P=0.009). Only 40% of individuals had evidence of a shift in drug-resistance genotype during the interruption. In summary, interrupting therapy prior to initiating salvage therapy in patients with advanced disease did not result in an improved virological response to enfuvirtide. The collective predictive activity of an enfuvirtide-containing regimen was important in predicting treatment response.

HIV entry and fusion inhibitors.

Human immunodeficiency virus (HIV) is a retrovirus that is the causative agent of acquired immunodeficiency syndrome (AIDS). Current HIV therapy is based on targeting two critical enzymes in the viral replication machinery: reverse transcriptase and a virally encoded protease. Although mortality rates due to HIV infection have been dramatically reduced, AIDS remains a major health problem throughout the world. The emergence of HIV variants that are resistant to current therapies and potential toxicity associated with their chronic use has highlighted the need for new approaches to HIV inhibition. Identification of the mechanisms underlying viral entry into the host cell has provided a number of novel therapeutic targets and the first of these HIV fusion inhibitors (enfuvirtide [pentafuside, T-20, Fuzeon; Roche Laboratories and Trimeris]) has recently been approved in the US and Europe. This review will focus on recent progress in the development of therapeutics that target the HIV entry process.