Safe and sustained vaginal delivery of pyrimidinedione HIV-1 inhibitors from polyurethane intravaginal rings.

The potent antiretroviral pyrimidinediones IQP-0528 (PYD1) and IQP-0532 (PYD2) were formulated in polyurethane intravaginal rings (IVRs) as prophylactic drug delivery systems to prevent the sexual transmission of HIV-1. To aid in the selection of a pyrimidinedione candidate and the optimal loading of the drug in the IVR delivery system, four pyrimidinedione IVR formulations (PYD1 at 0.5 wt% [PYD1(0.5 wt%)], PYD1(1 wt%), PYD2(4 wt%), and PYD2(14 wt%)) were evaluated in pigtail macaques over 28 days for safety and pyrimidinedione vaginal biodistribution. Kinetic analysis of vaginal proinflammatory cytokines, native microflora, and drug levels suggested that all formulations were safe, but only the high-loaded PYD2(14 wt%) IVR demonstrated consistently high pyrimidinedione vaginal fluid and tissue levels over the 28-day study. This formulation delivered drug in excess of 10 µg/ml to vaginal fluid and 1 µg/g to vaginal tissue, a level over 1,000 times the in vitro 50% effective concentration. The in vitro release of PYD1 and PYD2 under nonsink conditions correlated well with in vivo release, both in amount and in kinetic profile, and therefore may serve as a more biologically relevant means of evaluating release in vitro than typically employed sink conditions. Lastly, the pyrimidinediones in the IVR formulation were chemically stable after 90 days of storage at elevated temperature, and the potent nanomolar-level antiviral activity of both molecules was retained after in vitro release. Altogether, these results point to the successful IVR formulation and vaginal biodistribution of the pyrimidinediones and demonstrate the usefulness of the pigtail macaque model in evaluating and screening antiretroviral IVR formulations prior to preclinical and clinical evaluation.

Development of dual-acting pyrimidinediones as novel and highly potent topical anti-HIV microbicides.

In the absence of an effective vaccine against the human immunodeficiency virus (HIV), topical microbicides to prevent the sexual transmission of HIV represent an important strategy to prevent the continued spread of infection. The recent trend in the development of new microbicide candidates includes the utilization of FDA-approved therapeutic drugs that target the early stages of the HIV life cycle, including entry inhibitors and reverse transcriptase inhibitors. We have investigated 12 pyrimidinedione compounds with potent HIV activities and their abilities to inhibit both virus entry and reverse transcription, in an effort to determine a lead microbicide for product development. The candidate compounds were evaluated for efficacy against subtype B, C, and E clinical virus strains in fresh human peripheral blood mononuclear cells and against CCR5-tropic virus strains in both monocyte-macrophages and dendritic cells. Microbicide-specific biological assays and toxicity evaluations were also performed in a variety of established and fresh human cells as well as against Lactobacillus strains common to the vaginal environment. These evaluations resulted in the identification of congeners with cyclopropyl and cyclobutyl substituents at the N-1 of the pyrimidinedione as the most active molecules in the structure-activity relationship series. The pyrimidinediones represent excellent microbicide candidates in light of their significantly high efficacies against HIV-1 (subnanomolar concentration range), potencies (therapeutic index, >1 million), solubility profiles, and dual mechanism of antiviral action that includes two early steps of virus replication prior to the integration of the virus that are considered most important for microbicidal activity.

Selection and characterization of viruses resistant to the dual acting pyrimidinedione entry and non-nucleoside reverse transcriptase inhibitor IQP-0410.

The 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT)-like compounds with homocyclic moieties at the N-1 of the pyrimidinedione, including the highly potent lead compound IQP-0410, inhibit HIV-1 at sub-nanomolar concentrations primarily through a typical non-nucleoside mechanism involving allosteric inhibition at the hydrophobic binding pocket of the HIV-1 RT. Like all NNRTIs, the pyrimidinediones have no activity against HIV-2 RT. The pyrimidinediones, however, also possess a second mode of action involving inhibition of virus entry at nanomolar concentrations which extends their range of action to include HIV-2. Entry inhibition occurs through recognition of a complex conformational binding site formed upon interaction of the virus with target cells, but does not involve direct inhibition of gp120-CD4 binding. In order to further explore the means by which the pyrimidinediones act, resistant strains of HIV-1 and HIV-2 were selected in cell culture and molecularly and biologically characterized. With HIV-1, three phases of resistance selection occurred which involve an initial appearance of single amino changes in the NNRTI binding pocket, followed by changes in the envelope glycoproteins gp120 and gp41, and subsequent multiple additional changes in the RT, resulting in high level resistance to IQP-0410. With HIV-2, resistance to entry inhibition was achieved with no resistance-engendering mutations detected in the HIV-2 RT. Detailed molecular and biological characterization of IQP-0410-resistant viruses was performed to define the resistance-engendering mutations present in the RT and envelope and to quantify cross-resistance to other HIV inhibitors.