Factors Important to the Prioritization and Development of Successful Topical Microbicides for HIV-1

Table 1

Comparison of EC₅₀ and EC₉₉ values determined in the standard transmission inhibition assay to MTSA defined sterilizing concentration.

Compound	EC50 in entry transmission assay	EC99 in entry transmission assay	Sterilizing concentration determined in MTSA
			Experiment 1	Experiment 2
IQP-0528 (μM)	0.017	1.0	0.25	1.25
IQP-0410 (μM)	0.059	1.0	>12.5	>12.5
IQP-1187 (μM)	0.053	1.0	0.02	0.1
AZT (μM)	>0.5	>0.5	>31.25	>31.25
UC781 (μM)	0.009	2.98	0.37	1.9
CV-N (μg/mL)	0.001	0.1	12.5	12.5
Efavirenz (μM)	0.03	0.5	0.05	0.05
Tenofovir (μM)	>10	>10	>97.7	>97.7

The dual acting (entry inhibition and NNRTI) pyrimidinediones IQP-0528, IQP-0410, IQP-1187 [69] nonnucleoside RT inhibitors UC781 and efavirenz, nucleoside RT inhibitor AZT, entry inhibitor cyanovirin-N (CVN), and nucleotide RT inhibitor tenofovir (TFV) were evaluated in the MTSA, and the sterilizing concentration was compared to the EC₅₀ and EC₉₀ determined in a standard virus transmission assay. The concentrations utilized for each compound in the MTSA were derived from their respective EC₅₀ concentrations in a cytopathic effect assay and their TIs (EC₅₀/TC₅₀). The concentrations which were utilized are as follows: IQP-0528, IQP-0410, and IQP-1187: 10 through 31,250 times the EC₅₀ concentration; AZT and UC781: 10 through 31,250 times the EC₅₀ concentration; cyanovirin-N: 10 through 6,250 times the EC₅₀ concentration; efavirenz: 10 through 31,250 times the EC₅₀ concentration; tenofovir: 2.5 through 97.7 times the EC₅₀ concentration. All concentrations evaluated represented 5-fold serial increases in drug concentration with the exception of tenofovir which was in 2.5-fold increments. Passages which were positive for virus production were defined by detection of virus in the cell-free supernatant by RT assay. Cells were passaged for 10 passages in the continuous presence of the fixed compound concentration and for an additional 5 passages in the absence of compound. All tested concentrations were significantly below the defined toxic concentration to CEM-SS cells. Passages which were positive for virus production were defined by detection of virus in the cell-free supernatant by RT assay.
The entry assay results used for comparison to the MTSA results were generated from an assay utilizing HeLa-CD4-LTR-𝛽-Gal Cells with HIV-1_IIIB. Compound is added to the preplated cells approximately 15 minutes prior to the addition of virus. Following a 2-hour incubation at 37°/5% CO₂, residual virus and compound are removed through washing. The culture is incubated for an additional 48 hours at which time compound efficacy is determined by evaluating 𝛽-galactosidase in the lysate using a chemiluminescent endpoint.