Multipurpose Prevention Technologies: Biomedical Tools to Prevent HIV-1, HSV-2, and Unintended Pregnancies

Statistics clearly show an unmet need for highly effective contraception, especially in less developed countries. Many of these countries are at the core of the HIV/AIDS epidemic and show very high prevalence rates for other sexually transmitted infections (STIs) such as that caused by HSV-2. A woman at risk of unintended pregnancy due to unprotected intercourse is also at risk for HIV/STI. Owing to their causative interrelationship, combining protection against these conditions will result in enhanced prevention and health benefits. Existing multipurpose prevention modalities such as condoms and physical barriers, albeit efficacious, face cultural hurdles that have so far hindered their widespread use. Success has recently been demonstrated in large clinical trials, demonstrating proof of concept of microbicides in reducing the incidence of HIV-1 and HSV-2 among at-risk populations. The challenge heretofore is to refine these products to make them more potent, convenient, accessible, and acceptable. Potent antiviral drugs released topically in the female reproductive tract by innovative delivered systems and formulations will provide safe, effective, and acceptable multipurpose prevention tools. This paper provides an overview of existing and novel approaches to multipurpose prevention strategies

Synthesis, antiviral and contraceptive activities of nucleoside–sodium cellulose sulfate acetate and succinate conjugates

Chemical conjugates between sodium cellulose sulfate (CS), displaying contraceptive and HIV-entry inhibiting properties, and nucleoside reverse transcriptase inhibitors (NRTIs) (3′-azido-2′,3′-dideoxythymidine (AZT), 3′-fluoro-2′,3′-dideoxythymidine (FLT), or 2′,3′-dideoxy-3′-thiacytidine (3TC)) were designed to simultaneously provide contraceptive and anti-HIV activity. Two linkers, acetate and succinate, were used to conjugate the nucleoside analogs with CS. The conjugates containing cellulose sulfate-acetate (CSA) (e.g., AZT–CSA and FLT–CSA) were found to be more potent than CS and other conjugates (e.g., AZT–succinate–CS, and FLT–succinate–CS). The presence of both sulfate and the acetate groups on cellulose were critical for generating maximum anti-HIV activity. In addition to showing equal potency against wild-type and multidrug resistant HIV-1, the AZT–CSA conjugate displayed significant contraceptive activity in an animal model, providing the initial proof-of-concept for the design and synthesis of dual-activity compounds based on these combinations. [Refer to PDF for graphical abstract

Microbicides 2008 conference: From discovery to advocacy

Recently revised statistics show the number of individuals living with HIV at over 33 million worldwide, with 68% being in sub-Saharan Africa. Current HIV prevention methods, such as condom use, monogamy and abstinence, are not always feasible. The need for improved HIV preventative technologies remains urgent. Of these, microbicides represent a promising female-initiated preventative method. Microbicides are designed to be applied vaginally to prevent HIV and STI acquisition. Research is also being undertaken to assess the safety of the product during rectal application

Preclinical evaluation of lime juice as a topical microbicide candidate

Background: The continued growth of the global HIV epidemic highlights the urgent need to develop novel prevention strategies to reduce HIV transmission. The development of topical microbicides is likely to take a number of years before such a product would be widely available. This has resulted in a call for the rapid introduction of simpler vaginal intervention strategies in the interim period. One suggested practice would be vaginal douching with natural products including lime or lemon juice. Here we present a comprehensive preclinical evaluation of lime juice (LiJ) as a potential intervention strategy against HIV. Results: Pre-treatment of HIV with LiJ demonstrated direct virucidal activity, with 10% juice inactivating the virus within 5 minutes. However, this activity was significantly reduced in the presence of seminal plasma, where inactivation required maintaining a 1:1 mixture of neat LiJ and seminal plasma for more than 5 minutes. Additionally, LiJ demonstrated both time and dosedependent toxicity towards cervicovaginal epithelium, where exposure to 50% juice caused 75–90% toxicity within 5 minutes increasing to 95% by 30 minutes. Cervicovaginal epithelial cell monolayers were more susceptible to the effects of LiJ with 8.8% juice causing 50% toxicity after 5 minutes. Reconstructed stratified cervicovaginal epithelium appeared more resilient to LiJ toxicity with 30 minutes exposure to 50% LiJ having little effect on viability. However viability was reduced by 75% and 90% following 60 and 120 minutes exposure. Furthermore, repeat application (several times daily) of 25% LiJ caused 80–90% reduction in viability. Conclusion: These data demonstrate that the virucidal activity of LiJ is severely compromised in the presence of seminal plasma. Potentially, to be effective against HIV in vivo, women would need to apply a volume of neat LiJ equal to that of an ejaculate, and maintain this ratio vaginally for 5–30 minutes after ejaculation. Data presented here suggest that this would have significant adverse effects on the genital mucosa. These data raise serious questions about the plausibility and safety of such a prevention approach

Synthesis and anti-HIV activities of bis-(cycloSaligenyl) pronucleotides derivatives of 3′-fluoro-3′-deoxythymidine and 3′-azido-3′-deoxythymidine

Anti-HIV nucleoside monophosphates have limited cellular uptake due to the presence of negatively-charged phosphate group. Bis-(cycloSaligenyl) derivatives containing two anti-HIV nucleosides, 3′-fluoro-3′-deoxythymidine (FLT) and 3′-azido-3′-deoxythymidine (AZT) were synthesized to increase intracellular delivery of nucleoside monophosphates. 2,5-Bis(hydroxymethylene)benzene-1,4-diol was selected as a monocyclic bidentate scaffold and synthesized by three different methods from bis(hydroxymethylene)cyclohexan-1,4-diene-1,4-diol, or diethyl 2,5-dihydroxyterephthalate. The reaction of the tetraol with diisopropylphosphoramidous dichloride in the presence of 2,6-lutidine, followed by conjugation reactions with nucleosides (i.e., FLT and AZT) and oxidation afforded symmetrical and unsymmetrical bis-(cycloSaligenyl) diphosphate triester products, AZT–AZT, FLT–FLT, and FLT–AZT conjugates, in 63–74% overall yields and modest anti-HIV activities (IC50 = 2.8–69.6 μM). [Refer to PDF for graphical abstract

Enhancing Interferon Regulatory Factor 7 Mediated Antiviral Responses and Decreasing Nuclear Factor Kappa B Expression Limit HIV-1 Replication in Cervical Tissues

Establishment of a productive HIV-1 infection in the female reproductive tract likely depends on the balance between anti-viral and pro-inflammatory responses leading to activation and proliferation of HIV target cells. Immune modulators that boost anti-viral and depress pro-inflammatory immune responses may decrease HIV-1 infection or replication. Polyinosinic: polycytidylic [Poly (I:C)] has been reported to down-regulate HIV-1 replication in immune cell subsets and lymphoid tissues, yet the scope and mechanisms of poly (I:C) regulation of HIV-1 replication in the cervicovaginal mucosa, the main portal of viral entry in women remain unknown. Using a relevant, underexplored ex vivo cervical tissue model, we demonstrated that poly (I:C) enhanced Interferon Regulatory Factor (IRF)7 mediated antiviral responses and decreased tissue Nuclear Factor Kappa B (NF κ B) RNA expression. This pattern of cellular transcription factor expression correlated with decreased HIV-1 transcription and viral release. Reducing IRF7 expression up-regulated HIV-1 and NFκB transcription, providing proof of concept for the critical involvement of IRF7 in cervical tissues. By combining poly (I:C) with a suboptimal concentration of tenofovir, the leading anti-HIV pro-phylactic microbicide candidate, we demonstrated an earlier and greater decrease in HIV replication in poly (I:C)/tenofovir treated tissues compared with tissues treated with tenofovir alone, indicating overall improved efficacy. Poly (I:C) decreases HIV-1 replication by stimulating IRF7 mediated antiviral responses while reducing NFκB expression. Early during the infection, poly (I:C) improved the anti-HIV-1 activity of suboptimal concentrations of tenofovir likely to be present during periods of poor adherence i.e. inconsistent or inadequate drug use. Understanding interactions between anti-viral and pro-inflammatory immune responses in the genital mucosa will provide crucial insights for the identification of targets that can be harnessed to develop preventative combination strategies to improve the efficacy of topical or systemic antiviral prophylactic agents and protect women from HIV-1 and other sexually transmitted infections

Synthesis and Biological Evaluation of 5′-O-Fatty Acyl Ester Derivatives of 3′-Fluoro-2′,3′-dideoxythymidine as Potential Anti-HIV Microbicides

A number of 5′-O-fatty acyl derivatives of 3′-fluoro-2′,3′-dideoxythymidine (FLT, 1) were synthesized. These conjugates were evaluated for their potential as topical microbicides with anti-HIV activity against cell-free (X4 and R5), cell-associated, and multidrug-resistant viruses. Compared to FLT and 3′-azido-2′,3′-dideoxythymidine (AZT), 5′-O-(12-azidododecanoyl) (5), 5′-O-myristoyl (6), and 5′-O-(12-thioethyldodecanoyl) (8) derivatives of FLT were found to be more active against both cell-free viruses (lymphocytotropic and monocytotropic strains) with EC50 values of 0.4 μM, 1.1 μM, and \u3c0.2 μM, respectively, as well as cell-associated virus with EC50 values of 12.6, 6.4, and 2.3 μM, respectively. Conjugates 5, 6, and 8 exhibited \u3e4 and \u3e30 times better antiviral index than FLT and AZT, respectively. Conjugates 5 and 8 were significantly more potent than FLT against many multidrug-resistant strains. A comparison of the anti-HIV activity with the corresponding non-hydrolyzable ether conjugates suggested that ester hydrolysis to FLT and fatty acids is critical to enable anti-HIV activity. Cellular uptake studies were conducted using fluorescent derivatives of FLT attached with 5(6)-carboxyfluorescein through either β-alanine (23) or 12-aminododecanoic acid (24) spacers. The lipophilic fluorescent analog with a long chain (24) showed more than 12 times higher cellular uptake profile than the fluorescent analog with a short chain (23). These studies further confirmed that the attachment of fatty acids improved the cellular uptake of nucleoside conjugates. In addition, 5, 6, and 8 were the least cytotoxic and did not alter vaginal cell and sperm viability compared to the positive control, a commercial topical spermicide (N-9), which significantly decreased sperm and vaginal cell viability inducing the generation of proinflammatory cytokines

Preferred product attributes of a multipurpose vaginal ring: Findings from a phase 1 trial

IntroductionMost women face multiple and co-occurring risks from unwanted pregnancy, human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs) at some point during their lifetime. While a range of contraceptive methods exist and options for HIV prevention are increasing, to date, only male and female condoms provide multipurpose protection from both pregnancy and disease.MethodsFrom September 2017 to December 2018, 60 women from the United States and the Dominican Republic, randomized 1:1 to continuous or interrupted use and 4:1 to active vs. placebo ring, participated in a Phase I trial to assess the safety and tolerability of a three-month multipurpose intravaginal ring (IVR) containing the antiviral tenofovir and the contraceptive levonorgestrel. This study examines survey responses from all participants and qualitative data from a subset of 17 women to assess acceptability of and preferences for IVR characteristics.ResultsOverall, women liked the concept of a multipurpose IVR and found it easy to insert and remove. Initial concerns about the size or thickness of the ring generally disappeared with use experience. Women weighed trade-offs between the ease of continuous use for a longer duration against concerns about hygiene and discoloration of the ring when left in place during menses. Whether randomized to continuous or interrupted use, most women found ring attributes (size, thickness, flexibility) very acceptable. They provided recommendations via survey and qualitative interviews for ring modifications that would further increase acceptability. Insights into women's use experiences also suggest the need for clear counseling messages and introduction strategies that can facilitate women's choice and use of prevention methods.DiscussionStudy findings suggest that a multipurpose IVR would make a valuable contribution to women's sexual and reproductive health options, and that both continuous and interrupted use strategies may be preferred

Detection and Quantification of Antiviral Drug Tenofovir Using Silver Nanoparticles and Surface Enhanced Raman Spectroscopy (SERS) With Spatially Resolved Hotspot Selection

This study introduces a convenient and ultra-sensitive method of detection and quantification of the antiviral drug, tenofovir (TFV), by surface-enhanced Raman spectroscopy (SERS). Novel spatially resolved instrumentation for spectral acquisition and subsequent statistical analysis for hot spot selection was developed for convenient quantification of TFV in an aqueous matrix. Methods of statistical analysis include the use of partial least squares (PLS) regression vector analysis and spectral ranking by quality indices computed using CHAOS theory. Hydroxylamine-reduced Ag colloidal nanoparticles evaporated to dryness on an aluminum well-plate were used as the SERS substrate. To our knowledge, quantification of TFV down to 25 ng/mL by SERS, comprising clinically relevant concentrations, has not been previously reported. Furthermore, in this work we propose a novel method of quantification of aqueous TFV standards by SERS using statistical treatment of data by PLS and CHAOS theory. Based on these data, we propose future studies to develop a method of TFV detection and quantification in biological samples, beneficial to clinicians for rapid assessment of drug adherence during the treatment and prevention of viral diseases

Synthesis and Evaluation of Anti-HIV Activity of Mono- and Di-Substituted Phosphonamidate Conjugates of Tenofovir

The activity of nucleoside and nucleotide analogs as antiviral agents requires phosphorylation by endogenous enzymes. Phosphate-substituted analogs have low bioavailability due to the presence of ionizable negatively-charged groups. To circumvent these limitations, several prodrug approaches have been proposed. Herein, we hypothesized that the conjugation or combination of the lipophilic amide bond with nucleotide-based tenofovir (TFV) (1) could improve the anti-HIV activity. During the current study, the hydroxyl group of phosphonates in TFV was conjugated with the amino group of L-alanine, L-leucine, L-valine, and glycine amino acids and other long fatty ester hydrocarbon chains to synthesize 43 derivatives. Several classes of derivatives were synthesized. The synthesized compounds were characterized by 1H NMR, IR, UV, and mass spectrometry. In addition, several of the synthesized compounds were evaluated as racemic mixtures for anti-HIV activity in vitro in a single round infection assay using TZM-bl cells at 100 ng/mL. TFV (1) was used as a positive control and inhibited HIV infection by 35%. Among all the evaluated compounds, the disubstituted heptanolyl ester alanine phosphonamidate with naphthol oleate (69), pentanolyl ester alanine phosphonamidate with phenol oleate (62), and butanolyl ester alanine phosphonamidate with naphthol oleate (87) ester conjugates of TFV were more potent than parent drug TFV with 79.0%, 76.5%, 71.5% inhibition, respectively, at 100 ng/mL. Furthermore, two fatty acyl amide conjugates of tenofovir alafenamide (TAF) were synthesized and evaluated for comparative studies with TAF and TFV conjugates. Tetradecanoyl TAF conjugate 95 inhibited HIV infection by 99.6% at 100 ng/mL and showed comparable activity to TAF (97–99% inhibition) at 10–100 ng/mL but was more potent than TAF when compared at molar concentration