icon- folder.gif   Conference Reports for NATAP  
  16th CROI
Conference on Retroviruses and Opportunistic Infections Montreal, Canada
February 8-11, 2009
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New HIV Drug Candidates in Pre-Clinical Development
  Reported by Jules Levin
CROI Feb 8-12 2009 Montreal
There is yet little exciting news in this front but there were several pre-clinical HIV drug candidates reported at CROI. CMX157 is being developed by former GSK virologist Randall Lanier down the road in Durham, NC where he is trying to develop a more potent and safer tenofovir, he presented this first last Summer at the Resistance Workshop in Spain. At this time the most promising and timely help for patients with HIV drug resistance who need new therapies may come from PRO140 and perhaps Bevirimat, as well the Tanox monoclonal antibody drug Ibalizumab appears to have survived and has a new study on clinical trials.gov. PRO140 is an entry inhibitor and data from subcutaneous injection in patients was presented at CROI, and this drug appears potent and is moving ahead in studies in patients. Bevirimat , the maturation inhibitor, was recently sold to Myriad for $7 million from Panacos. Myriad told me they are planning a new bevirimat study to start soon, as well they have their own maturation inhibitors, and Panacos also has further bak in evelopment stages a second generation maturation inhibitor program. As well, both Myriad and Panacos have additional HIV drugs, I recall Panacos has an ora fusion inhibitor, all these are several years back in develoment.
TNX-355 survives Tanox: new Phase 2 Dose-Finding StudyStudy
TaiMed Biologics Inc. will conduct clinical trials of TNX-355 -- also known as Ibalizumab -- from a location on the West Loop near the former Tanox ... www.natap.org/2009/HIV/012809_01.htm
Hexadecyloxypropyl Tenofovir Associates Directly with HIV and Subsequently Inhibits Viral Replication in Untreated Cells
_Randall Lanier*, B Lampert, A Robertson, M Almond, and G Painter Chimerix Inc, Durham, NC, US
Background: CMX157 is a lipid conjugate of tenofovir (TFV). Compared to TFV, CMX157 is >300 times more potent against wild-type and NRTI-resistant HIV in vitro; it effectively penetrates isolated human peripheral blood mononuclear cells (PBMC), producing >30-fold higher intracellular levels of the active anabolite, TFV-diphosphate (TFV-PP). Since CMX157 has a lipid side chain and HIV has a lipid bilayer, it was postulated that CMX157 associates directly with virions. To test this hypothesis, purified HIV was incubated with CMX157 or TFV followed by quantification of virus-associated drug and determination of the tissue culture infectious dose (TCID50).
Methods: Concentrated HIV-1IIIB was incubated with 500 nM CMX157 or TFV for 2 hours, pelleted to remove unbound compound and lysed with 70% methanol. Supernatants were analyzed in triplicate using LC/MS/MS. Analytes were separated by gradient, reverse phase, ion-paring chromatography and detected by positive ion electrospray; separate viral aliquots were used to determine TCID50 by XTT, RT, and p24 assays. To assess the effect of exposure time, concentrated HIV-1IIIB was incubated with 500 nM CMX157 for 1, 15, 30, 60, and 120 minutes prior to TCID50. To determine the effect of drug dose, TCID50 was determined following a 15 minute incubation of virus with eight concentrations of CMX157 ranging from 0.039 to 125 nM. HDP-acyclovir was evaluated in parallel as a control.
Results: Analysis of purified HIV pellets following incubation with 500 nM drug showed ≈30,000 molecules of CMX157 were associated with each virion versus ≈100 molecules/virion for TFV. Incubation of HIV for 1 to 15 minutes with concentrations of CMX157down to 3.9 nM resulted in 3-4 fold decreases in TCID50. No inhibition of HIV replication was observed following incubation with TFV or the lipid control bearing the same alkyl modification as CMX157, HDP-acyclovir.
Conclusions: Overall, these results indicate that CMX157 associates directly with HIV and that this association enhances its antiviral activity in vitro. The most likely mechanism is direct targeting of CMX157 to the cell being infected; this has implications for treatment and prevention of HIV infection. Once inside the cell, CMX157 is presumably converted to TFV-PP which inhibits HIV replication via chain termination. CMX157 could have advantages over TFV via this mechanism of cell targeting as any HIV virion exposed to drug would carry CMX157 into any compartment or cell type it subsequently enters.

β-D-3'-Azido-2,6-diamino-2',3'-dideoxypurine Is a Potent Inhibitor of HIV-1 and Is Bioconverted Intracellularly to both AZD-TP and Its Guanosine-5'-triphosphate Form
Raymond Schinazi*1, S Coats2, H-W Zhang1, M Detorio1, A Obikhod1, G Asif1, B Herman3, J Nettles1, N Sluis-Cremer3, and J Mellors3 1Emory Univ, VAMC, Atlanta, GA, US; 2RFS Pharma LLC, Tucker, GA, US; and 3Univ of Pittsburgh, VAMC, PA, US
Background: New antiviral nucleosides that are potent and safe are urgently needed. Recently, we showed that b-D-3'-azido-2',3'-dideoxyguanosine (AZG) is a potent and selective inhibitor of HIV-1 with a superior resistance profile compared to AZT. This discovery led to the synthesis and evaluation of 3'-azido-2,6-diamino-2',3'-dideoxypurine (AZD) and a 5'-monophosphate (MP) pro-drug of AZD.
Methods: We used a multi-disciplinary approach including medicinal chemistry, molecular virology, rational drug design, biochemistry, cellular pharmacology, HPLC-MS/MS, and enzyme kinetics to uncover the molecular mechanisms of anti-HIV-1 activity for AZD and its MP pro-drug.
Results: AZD was considerably more potent against HIV-1 in human peripheral blood mononuclear cells (PBMC) (EC50 = 0.07 mM) than previously reported in MT-4 cells (EC50 = 2 μM). Incubation of AZD in PBMC for 4 hours and subsequent HPLC-MS/MS analysis revealed low levels of AZD-DP and AZD-TP, but surprisingly high levels of AZG-DP and AZG-TP (ratio of AZD-TP to AZG-TP 1:167). The AZD-MP pro-drug showed greater potency (EC50 = 0.0031 mM) against HIV-1 in PBMC than AZD. This pro-drug increased the intracellular levels of AZD-TP in PBMC approximately 250-fold compared to the levels achieved with AZD and resulted in a reversal of the ratio of AZD-TP to AZG-TP (73:1). Computational evaluation of AZD paired with either T or C in a DNA duplex bound to HIV-1 reverse transcriptase (RT) indicated that AZD behaves like an A-analog rather than a G-analog.
Conclusions: AZD and its pro-drug can deliver both AZD-TP and AZG-TP intracellularly, each of which can inhibit HIV-1 RT. The combined delivery of chain-terminating 3'-azidopurine nucleotide analogs with different incorporation profiles (as A- vs G-analog) may improve the resistance profile of AZD. The potency and intracellular delivery of AZD-TP were substantially increased by phosphate pro-drugs of AZD.

Investigational New Drug-directed Development of IQP-0410, a Safe and Highly Potent Dual-acting Nonnucleoside Pyrimidinedione for the Therapy of HIV-1 Infection
Nick Kaludov*, K Watson, and R Buckheit Jr
ImQuest Pharmaceuticals Inc, Frederick, MD, US
Background: Drug cocktails combining NRTI and NNRTI and PI are the first-line treatment for HIV infection. The primary problems associated with anti-HIV therapy continue to be drug toxicity, drug-drug interactions, patient compliance with prescribed treatment regimens, and the appearance of drug-resistant viruses.
Methods: ImQuest Pharmaceuticals is developing IQP-0410, a highly potent nonnucleoside pyrimidinedione inhibitor of both HIV-1 and HIV-2. A comprehensive program of standard investigational new drug (IND)-enabling good laboratory practices (GLP) studies has been performed in order to establish the acute and multiple dose toxicity, toxico-kinetics, genotoxicity, and safety pharmacology profile of IQP-0410.
Results: Oral dosage of IQP-0410 administered via gavage, up to a maximum feasible dose level of 1000 mg/kg, was well tolerated in beagle dogs. There were no test article-related findings noted during the evaluation of in-life data, clinical pathology or necropsy data. Histopathology evaluation of selected tissues revealed no IQP-0410-related microscopic findings. Preliminary pharmacokinetics studies of IQP-0410 in dogs following oral administration demonstrated significant plasma concentrations of IQP-0410 at 24 hours on day 6, with an average of 37 ng/mL remaining. Cmax values for Day 1 ranged from 52 to 113 ng/mL and from 71 to 165 ng/mL on day 7 and the calculated EC95 value for IQP-0410 was 1 ng/mL. Thus the effective concentration of IQP-0410 was exceeded by 30- to 50-fold at 24 hours, indicating once per day dosage. Metabolic stability assays showed a high degree of liver microsome metabolism reaction phenotyping in human liver microsomes indicated that CYP3A4 is the major enzyme responsible for the metabolism of IQP-0410. Safety pharmacology studies (in vitro effects of IQP-0410 on the hERG channel current, neuropharmacological profile determination and pulmonary assessment in mice) showed no signs of pharmacological or toxicological activity. All genotoxicology testing of IQP-0410-including the Ames, Mouse Lymphoma, Bone Marrow Micronucleus, and CHO Chromosome Abnormality tests-were negative.
Conclusions: This favorable pre-clinical profile suggests that IQP-0410 will be an important addition to the currently available primary therapeutic regimens used to treat HIV. In addition, the activity of IQP-0410 against multi-drug resistant virus strains indicates a significant potential for use in salvage therapy.

Antiviral Active against Different Subtypes in vitro and Resistance Profile of 2 New HIV-1 Protease Inhibitors: CRS-074 and CRS-075
Alexandre Calazans*1, E Xavier1, H Pereira1, R Debom2, O Pacheco2, A Tanuri1, and R Brindeiro1 1Federal Univ of Rio de Janeiro, Brazil and 2Cristalia, Sao Paulo, Brazil
Background: CRS-074 and CRS-075 are 2 new PI with EC50 values of 0.5 and 50.0 nM, respectively. The aims of this study are to characterize the phenotypic and enzymatic antiretroviral activity against wild-type viruses from different HIV-1 subtypes, to investigate the resistance profile against protease inhibitor resistant (PIr) HIV-1 strains from different subtypes, and to describe in vitro selection of HIV-1 variants having increased resistance do CRS-074 and CRS-075.
Methods: Phenotyping assays were performed using a MT-4 cell-based MTT viability assay. A panel of 19 HIV-1 recombinant viruses was generated: 12 viruses from subtypes B and F carrying PIr mutations, and 7 others PI-susceptible, from subtypes B, C, and F. Recombinant HIV-1 protease from subtypes B, C, and F were expressed and its activity determined using a fluorogenic substrate. Selection experiments were performed in vitro by passaging HIV-1 strain NL4-3 into MT-4 cells, in the presence of increasing concentrations of CRS-074 or CRS-075. Viral RNA was extracted from selected viruses and the cDNA subjected to sequencing and viral load determination by quantitative polymerase chain reaction (q-PCR).
Results: Both drugs presented the same EC50 for different subtypes wild-type viruses (B, C, and F). The EC50 values of CRS-074 against resistant viruses varied from 1.0- to 510.0-fold higher than the EC50 for reference virus. For the CRS-075 these EC50 values varied from 0.2 to 64.1; while ritonavir (used as a reference drug) exhibited values from 1.2- to 666.7-fold. The Ki values obtained for CRS-074 (0.02 to 0.07 nM) and for CRS-075 (0.9 to 2.2 nM) were subtype independent. After 43 days, a viral variant carrying the mutation E34G was selected for the CRS-074. No mutations were found from the viruses under selective pressure of CRS-075, after 53 days in culture.
Conclusions: CRS-074 is one of most potent PI ever described (EC50 = 0.5 nM). Although CRS-074 presented high relative values of EC50 against resistant viruses, the absolute value of EC50 reached is still very low when compared to other PI against susceptible viruses. CRS-075 has an EC50 value comparable to the Food and Drug Administration-approved PI, moreover, CRS-075 highly effectiveness against resistant viruses demonstrated its potential as a future drug for second line therapy. Both drugs were highly active against the different HIV-1 subtypes circulating in Brazil.