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We evaluated the in vivo pharmacokinetics and used a complementary ex vivo coculture assay to determine the pharmacodynamics of IQB3002 gel containing 1% IQP-0528, a nonnucleoside reverse transcriptase inhibitor (NNRTI), in rhesus macaques (RM). The gel (1.5 ml) was applied vaginally to 6 simian-human immunodeficiency (SHIV)-positive female RM. Blood, vaginal fluids, and rectal fluids were collected at 0, 1, 2, and 4 h. RM were euthanized at 4 h, and vaginal, cervical, rectal, and regional lymph node tissues were harvested. Anti-human immunodeficiency virus (HIV) activity was evaluated ex vivo by coculturing fresh or frozen vaginal tissues with activated human peripheral blood mononuclear cells (PBMCs) and measuring the p24 levels for 10 days after an HIV-1Ba-L challenge. The median levels of IQP-0528, determined using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) methods, were between 10(4) and 10(5) ng/g in vaginal and cervical tissue, between 10(3) and 10(4) ng/g in rectal tissues, and between 10(5) and 10(7) ng/ml in vaginal fluids over the 4-h period. The vaginal tissues protected the cocultured PBMCs from HIV-1 infection ex vivo, with a viral inhibition range of 81 to 100% in fresh and frozen tissues that were proximal, medial, and distal relative to the cervix. No viral inhibition was detected in untreated baseline tissues. Collectively, the median drug levels observed were 5 to 7 logs higher than the in vitro 50% effective concentration (EC50) range (0.21 ng/ml to 1.29 ng/ml), suggesting that 1.5 ml of the gel delivers IQP-0528 throughout the RM vaginal compartment at levels that are highly inhibitory to HIV-1. Importantly, antiviral activity was observed in both fresh and frozen vaginal tissues, broadening the scope of the ex vivo coculture model for future NNRTI efficacy studies., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Nucleocapsid 7 (NCp7) inhibitors have been investigated extensively for their role in impeding the function of HIV-1 replication machinery and their ability to directly inactivate the virus. A class of NCp7 zinc finger inhibitors, S-acyl-2-mercaptobenzamide thioesters (SAMTs), was investigated for topical drug delivery. SAMTs are inherently unstable because of their hydrolytically labile thioester bond, thus requiring formulation approaches that can lend stability. We describe the delivery of N-[2-(3,4,5-trimethoxybenzoylthio)benzoyl]-β-alaninamide (SAMT-10), as a single agent antiretroviral (ARV) therapeutic and in combination with the HIV-1 reverse-transcriptase inhibitor pyrimidinedione IQP-0528, from a hydrophobic polyether urethane (PEU) intravaginal ring (IVR) for a month. The physicochemical stability of the ARV-loaded IVRs was confirmed after 3 months at 40°C/75% relative humidity. In vitro, 25 ± 3 mg/IVR of SAMT-10 and 86 ± 13 mg/IVR of IQP-0528 were released. No degradation of the hydrolytically labile SAMT-10 was observed within the matrix. The combination of ARVs had synergistic antiviral activity when tested in in vitro cell-based assays. Toxicological evaluations performed on an organotypic EpiVaginal(™) tissue model demonstrated a lack of formulation toxicity. Overall, SAMT-10 and IQP-0528 were formulated in a stable PEU IVR for sustained release. Our findings support the need for further preclinical evaluation. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3426-3439, 2015., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Three nucleoside analogues, 3'-fluoro-2',3'-dideoxythymidine (FLT), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-dideoxy-3'-thiacytidine (3TC) were conjugated with three different dicarboxylic acids to afford the long chain dicarboxylate esters of nucleosides. In general, dinucleoside ester conjugates of FLT and 3TC with long chain dicarboxylic acids exhibited higher anti-HIV activity than their parent nucleosides. Dodecanoate and tetradecanoate dinucleoside ester derivatives of FLT were found to be the most potent compounds with EC(50) values of 0.8-1.0 nM and 3-4 nM against HIV-1(US/92/727) and HIV-1(IIIB) cells, respectively. The anti-HIV activity of the 3TC conjugates containing long chain dicarboxylate diester (EC(50)=3-60 nM) was improved by 1.5-66 fold when compared to 3TC (EC(50)=90-200 nM). This study reveals that the symmetrical ester conjugation of dicarboxylic acids with a number of nucleosides results in conjugates with improved anti-HIV profile., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In light of the increasing worldwide AIDS pandemic, there is a continuing need to develop new prevention strategies to inhibit the transmission of HIV-1. In the absence of a successful vaccine, topical microbicides represent the best strategies to reduce the epidemic. Following the success of HIV therapeutic cocktail strategies, combinations of microbicides including nucleotide reverse transcriptase inhibitors (NtRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) may offer significant protection from infection over single agents. We have developed a combination microbicide gel formulation for the delivery of IQP-0528, a novel NNRTI, and tenofovir (TFV), a NtRTI. Gel formulations were evaluated based on quantitative viscoelastic and physiochemical evaluations defined by a target product profile (TPP). For the majority of the evaluations, the gel formulations behaved similarly; all showed shear thinning behavior, were stable, nontoxic, and active against HIV-1 infection. Gel formulation F2759 displayed increased drug release of 289 ± 100 µg/(cm(2) h(1/2) ) and a tissue permeability of 60 times the half maximal effective concentration (EC(50) ) of TFV and 800 times the EC(50) of IQP-0528. In addition, F2759 showed osmolality within TPP and the highest performance in gel spreading. We have identified a gel formulation to deliver a combination microbicide of IQP-0528 and TFV that has significant potential to prevent infection of HIV-1., (Copyright © 2012 Wiley Periodicals, Inc.)

Two crystal structures have been solved for separate complexes of alkenyldiarylmethane (ADAM) nonnucleoside reverse transcriptase inhibitors (NNRTI) 3 and 4 with HIV-1 reverse transcriptase (RT). The structures reveal inhibitor binding is exclusively hydrophobic in nature and the shape of the inhibitor-bound NNRTI binding pocket is unique among other reported inhibitor-RT crystal structures. Primarily, ADAMs 3 and 4 protrude from a large gap in the back side of the binding pocket, placing portions of the inhibitors unusually close to the polymerase active site and allowing 3 to form a weak hydrogen bond with Lys223. The lack of additional stabilizing interactions, beyond the observed hydrophobic surface contacts, between 4 and RT is quite perplexing given the extreme potency of the compound (IC(50)

As a continuation of efforts to replace the metabolically labile methyl esters of lead alkenyldiarylmethanes (ADAMs) with stable bioisosteres, compounds bearing benzo[d]isoxazole and oxazolidine-2-one rings were designed and evaluated as a new series of potent HIV-1 non-nucleoside reverse transcriptase inhibitors with anti-HIV activity. All of the resulting ADAMs were found to inhibit HIV-1 RT with poly(rC) x oligo(dG) as the template primer. The most promising compound in this series was ADAM 3, with EC(50) values of 40 nM (vs HIV-1(RF)) and 20 nM (vs HIV-1(IIIB)). Compound 3 also inhibited HIV-1 reverse transcriptase with an IC(50) of 0.91 microM. ADAM 4 has an antiviral EC(50) of 0.6 microM in CEM-SS cells and a plasma half-life of 51.4 min.

The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number of ADAM analogues have been identified that protect HIV-infected cells from the cytopathic effects of the virus by an unknown, HIV-1 RT-independent mechanism. Since the phosphodiesterase 4 family is required for HIV infection, the effect of various ADAMs on the activity of PDE4B2 was investigated in an effort to determine if the ADAMs could possibly be targeting phosphodiesterases. Six compounds representative of the ADAM class were tested for inhibition of cAMP hydrolysis by PDE4B2 enzymatic activity. Four ADAMs were found to be weak inhibitors of PDE4B2 and two of them were inactive. The experimental results are consistent with an antiviral mechanism that does not include inhibition of PDE4 isoforms.

Non-nucleoside inhibitors of HIV reverse transcriptase (NNRTIs), albeit not the mainstays of HIV/AIDS treatment, have become increasingly important in highly active antiretroviral therapy (HAART) due to their unique mechanism of action. Several years ago our group identified the alkenyldiarylmethanes (ADAMs) as a potent and novel class of NNRTIs; however, the most active compounds were found to be metabolically unstable. Subsequent work has led to the synthesis of 33 analogues, with improved metabolic profiles, through the replacement of labile esters with various heterocycles, nitriles, and thioesters. As a result, a number of hydrolytically stable NNRTIs were identified with anti-HIV activity in the nanomolar concentration range. Furthermore, an improved pharmacophore model has been developed based on the new ADAM series, in which a salicylic acid-derived aryl ring is oriented cis to the side chain and the aryl ring that is trans to the side chain contains a hydrogen bond acceptor site within the plane of the ring.

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 microM, EC50 = 0.44 microM, and TC50 >/= 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants.

The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are capable of inhibiting HIV-1 reverse transcriptase (RT) through an allosteric mechanism. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties that are hydrolyzed by nonspecific esterases present in blood plasma, resulting in the formation of the inactive carboxylic acid metabolites. Therefore, to discover metabolically stable ADAMs, the design and synthesis of a new class of ADAMs with N-methoxy imidoyl halide and 1,2,4-oxadiazole systems were attempted. The resulting new ADAM 6 displayed enhanced metabolic stability in rat plasma (t1/2 = 61 h) along with the ability to inhibit HIV-1 reverse transcriptase and the cytopathic effect of HIV-1RF and HIV-1IIIB at submicromolar concentrations.

Objective: The objective of this article is to study the effect of PC-815, a novel combination microbicide containing carrageenan and the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150, in blocking HIV-1 and HIV-2 infections in vitro as compared with Carraguard alone., Goal: The goal of this study was to develop a combination microbicide that is more efficacious than Carraguard against HIV-1 and HIV-2., Study Design: The microtiter syncytial assay was used to evaluate: 1) the antiviral and virucidal activity of MIV-150 against HIV-1MN; 2) the additive effect of MIV-150 when combined with carrageenan; and 3) a possible interference of seminal fluid in the antiviral activity of these compounds., Results: MIV-150 effectively inactivated free virus. Combination of MIV-150 and Carraguard demonstrated an additive antiviral effect. Seminal fluid had no effect on the antiviral activity of MIV-150 or Carraguard. The average concentration that blocks 50% of infection (EC50) for PC-815 was approximately 10 times stronger than Carraguard for the different clinical isolates used in the study., Conclusion: Theoretically, PC-815 is likely to be a more efficacious microbicide than Carraguard.

The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors that have potential value in the treatment of HIV/AIDS. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties. A series of novel ADAMs were therefore designed and synthesized in order to replace the metabolically labile methyl ester moieties of the existing ADAM lead compounds with hydrolytically stable, fused isoxazolone, isoxazole, oxazolone, or cyano substituents on the aromatic rings. The methyl ester and methoxy substituents on both of the aromatic rings in the parent compound 1 were successfully replaced with metabolically stable moieties with retention of anti-HIV activity and a general decrease in cytotoxicity.

The HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) constitute a large and structurally diverse set of compounds, several of which are currently used in the treatment of AIDS. A series of novel alkenyldiarylmethanes (ADAMs) were designed and synthesized as part of an ongoing investigation to replace the metabolically labile methyl ester moieties found in the ADAM pharmacophore with stable modifications that retain the potent anti-HIV activity of the parent compounds. Unsurprisingly, the rat plasma half-lives of the new ADAMs were not improved when compared to the parent compounds, but all of the synthesized ADAMs inhibited the cytopathic effect of HIV-1 in cell culture. The most potent compound identified was (E)-5-[1-(3,7-dimethyl-2-oxo-2,3-dihydro-benzoxazol-5-yl)-5-methoxycarbonyl-pent-1-enyl]-2-methoxy-3-methylbenzoic acid methyl ester (7), which inhibited the cytopathic effects of both HIV-1(RF) and HIV-1(IIIB) strains in cell cultures with EC50 values of 30 and 90 nM, respectively, and inhibited HIV-1 reverse transcriptase with an IC50 of 20 nM.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) are part of the combination therapy currently used to treat HIV infection. Based on analogy with known HIV-1 NNRT inhibitors, 18 novel alkenyldiarylmethanes (ADAMs) containing 5-chloro-2-methoxyphenyl, 3-cyanophenyl, or 3-fluoro-5-trifluoromethylphenyl groups were synthesized and evaluated as HIV inhibitors. Their stabilities in rat plasma have also been investigated. Although introducing 5-chloro-2-methoxyphenyl or 3-fluoro-5-trifluoromethylphenyl groups into alkenyldiarylmethanes does not maintain the antiviral potency, the structural modification of alkenyldiarylmethanes with a 3-cyanophenyl substituent can be made without a large decrease in activity. The oxazolidinonyl group was introduced into the alkenyldiarylmethane framework and found to confer enhanced metabolic stability in rat plasma.

We have previously reported the potent in vitro HIV-1 anti-reverse transcriptase activity of a 35-mer of 4-thio-deoxyuridylate [(s(4)dU)(35)]. In efforts to define its activity in a more physiological system, studies were carried out to determine the stage of viral infection that this compound mediates its anti-viral effect. Results of the studies reported herein show that (s(4)dU)(35) is nontoxic and is capable of inhibiting both single and multi-drug resistant HIV strains (IC(50): 0.8-25.4 microg/ml) in vitro. Besides its previously reported anti-RT activity, (s(4)dU)(35) mediated its antiviral action by preventing virus attachment (IC(50): 0.002-0.003 microg/ml), and was stable in vitro and slowly degraded by DNAses. Competition studies and fluorescence resonance energy transfer (FRET) experiments indicated that (s(4)dU)(35) preferentially binds to CD4 receptors, but not to CD48. Confocal laser scanning microscopy (CLSM) studies showed that (s(4)dU)(35) did not penetrate into the cells and colocalized with cell surface thioredoxin. Our studies identify (s(4)dU)(35) as a potential novel HIV entry inhibitor that may have utility as either a systemic antiretroviral or as a preventing agent for HIV transmission.

The increasingly prevalent emergence of drug-resistant virus strains in patients being treated with highly active antiretroviral regimens and the increasing rates of transmission of drug-resistant virus strains have focused attention on the critical need for additional antiretroviral agents with novel mechanisms of action and enhanced potency. Furthermore, novel means of employing highly active antiretroviral therapy are needed to reduce or eliminate the virological treatment failures that currently occur. Over the past several years, evidence has mounted supporting the fact that the emergence of resistant strains is associated with reductions in viral fitness, yielding decreases in plasma virus load in treated patients harbouring resistant populations of the virus. Additional mutations that serve to modify fitness (compensatory mutations) and mutations that impact the viral replication capacity also emerge under the selective pressure of drug treatment, and have both negative and positive effects on virus growth. Fitness is generally accepted to refer to the ability of HIV to replicate in a defined environment and thus is used to describe the viral replication potential in the absence of the drug. Although viral fitness and replication capacity are related in some ways, it is important to recognise that viral fitness is not the same as viral replication capacity. This review will assess the recent literature on antiviral drug resistance, viral fitness and viral replication capacity, and discuss means by which the adaptability of HIV to respond rapidly to antiviral treatment through mutation may be used against it. This would be done by treating patients with an aim to lock the deleterious mutations into the resistant virus genome, resulting in a positive therapeutic outcome despite the presence of resistance to the selecting agents. The review will specifically discuss the literature on nucleoside and non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, fusion inhibitors, as well as other biological factors involved in viral fitness.

The existing methods for the synthesis of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors proceed from symmetrical benzophenones and therefore result in products with identical aromatic rings. New methods have therefore been devised for the preparation of stereochemically defined ADAMs with non-identical aromatic rings. The new routes rely on palladium-catalyzed reactions, including Sonogashira, Suzuki, Stille, and hydroarylation methodology. Several of the new ADAMs inhibited the cytopathic effect of HIV-1 in cell culture and HIV-1 reverse transcriptase at submicromolar concentrations.

Abstract: Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor and IQP-0528 is a non-nucleoside reverse transcriptase inhibitor that also blocks virus entry. TFV and IQP-0528 alone have shown antiviral activity as microbicide gels. Because combination therapy will likely be more potent than mono-therapy, these drugs have been chosen to make a combination microbicide gel containing 2.5% TFV/1% IQP-0528. Safety and efficacy testing was done to evaluate five prototype combination gels. The gels retained TZM-bl cell and ectocervical and colorectal tissue viability. Further, the epithelium of the ectocervical and colorectal tissue remained intact after a 24h exposure. The ED50 calculated from the formulations for IQP-0528 was ∼32nM and for TFV was ∼59nM and their inhibitory activity was not affected by semen. The ED50 of TFV in the combination gels was ∼100-fold lower than when calculated for the drug substance alone reflecting the activity of the more potent IQP-0528. When ectocervical and colorectal tissue were treated with the combination gels, HIV-1 p24 release was reduced by ⩾1log10 and ⩾2log10, respectively. Immunohistochemistry for the ectocervical tissues treated with combination gels showed no HIV-1 infected cells at study end. With the increased realization of receptive anal intercourse among heterosexual couples often in conjunction with vaginal intercourse, having a safe and effective microbicide for both mucosal sites is critical. The safety and efficacy profiles of the gels were similar for ectocervical and colorectal tissues suggesting these gels have the potential for dual compartment use. [Copyright &y& Elsevier]