The expression of interleukin-1ß in patients with chronic hepatitis B treated with pegylated-interferon-alpha combined with tenofovir disoproxil fumarate and monotherapy.

BACKGROUND: Anti-hepatitis B virus (HBV) treatment uses tenofovir disoproxil fumarate (TDF) along with Pegylated-interferon-alpha (Peg-IFN-α), which is more effective than TDF/Peg-IFN-α monotherapy. We have previously shown that interleukin-1beta (IL-1ß) is related to the effectiveness of IFN-α treatment in chronic hepatitis B (CHB) patients. The aim was to investigate the expression of IL-1ß in CHB patients treated with Peg-IFN-α combination with TDF and TDF/Peg-IFN-α monotherapy. METHODS: Huh7 cells infected with HBV were stimulated by Peg-IFN-α and/or Tenofovir (TFV) for 24h. A single-center cohort study of prospective recruitment of CHB patients: untreated CHB (Group A), TDF combined with Peg-IFN-α therapy (Group B), Peg-IFN-α monotherapy (Group C), TDF monotherapy (Group D). Normal donors served as controls. The clinical datas and blood of patients were collected at 0, 12, and 24 weeks. According to the early response criteria, Group B and C were divided into two subgroups: the early response group (ERG) and the non-early response group (NERG). Stimulation of HBV-infected hepatoma cells with IL-1ß to validate the antiviral activity of IL-1ß. To test the blood sample, cell culture supernatant, and cell lysates and to assess the expression of IL-1ß and HBV replication levels in various treatment protocols, Enzyme-Linked Immunosorbent Assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used. SPSS 26.0 and GraphPad Prism 8.0.2 software were used for statistical analysis. P values < 0.05 was considered to be statistically significant. RESULTS: In vitro experiments, Peg-IFN-α plus TFV treatment group expressed higher IL-1ß and inhibited HBV more effectively than monotherapy. Finally, 162 cases were enrolled for observation (Group A (n = 45), Group B (n = 46), Group C (n = 39), and Group D (n = 32)), and normal donors (n = 20) were enrolled for control. The early virological response rates of Group B, C, and D were 58.7%, 51.3%, and 31.2%. At 24 weeks, IL-1ß in Group B(P = 0.007) and C(P = 0.034) showed higher than at 0 week. In Group B, the IL-1ß showed an upward trend at 12w and 24w in the ERG. IL-1ß significantly reduced HBV replication levels in hepatoma cells. CONCLUSION: The increased expression of IL-1ß may enhance the efficacy of TDF combined with Peg-IFN-α therapy in achieving an early response for CHB patients.

A New Approach to Developing Long-Acting Injectable Formulations of Anti-HIV Drugs: Poly(Ethylene Phosphoric Acid) Block Copolymers Increase the Efficiency of Tenofovir against HIV-1 in MT-4 Cells.

Despite the world's combined efforts, human immunodeficiency virus (HIV), the causative agent of AIDS, remains one of the world's most serious public health challenges. High genetic variability of HIV complicates the development of anti-HIV vaccine, and there is an actual clinical need for increasing the efficiency of anti-HIV drugs in terms of targeted delivery and controlled release. Tenofovir (TFV), a nucleotide-analog reverse transcriptase inhibitor, has gained wide acceptance as a drug for pre-exposure prophylaxis or treatment of HIV infection. In our study, we explored the potential of tenofovir disoproxil (TFD) adducts with block copolymers of poly(ethylene glycol) monomethyl ether and poly(ethylene phosphoric acid) (mPEG-b-PEPA) as candidates for developing a long-acting/controlled-release formulation of TFV. Two types of mPEG-b-PEPA with numbers of ethylene phosphoric acid (EPA) fragments of 13 and 49 were synthesized by catalytic ring-opening polymerization, and used for preparing four types of adducts with TFD. Antiviral activity of [mPEG-b-PEPA]TFD or tenofovir disoproxil fumarate (TDF) was evaluated using the model of experimental HIV infection in vitro (MT-4/HIV-1IIIB). Judging by the values of the selectivity index (SI), TFD exhibited an up to 14-fold higher anti-HIV activity in the form of mPEG-b-PEPA adducts, thus demonstrating significant promise for further development of long-acting/controlled-release injectable TFV formulations.

Nucleos(t)ide Analogs Do Not Independently Influence Hepatic Fibrosis and Portal Hypertension beyond Viral Suppression in CBDL-Induced Cirrhotic Rat.

Chronic hepatitis is the major cause of liver cirrhosis and portal hypertension. Several factors affect portal pressure, including liver fibrosis, splanchnic vasodilatation, and pathologic angiogenesis. Nucleos(t)ide analogs (NUCs), the oral antiviral agents, effectively attenuate chronic hepatitis B-related liver cirrhosis and portal hypertension via viral suppression and alleviation of hepatitis. On the other hand, NUCs affect tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and nitric oxide, which participate in fibrogenesis, vasodilatation, and angiogenesis. However, whether NUCs independently influence liver fibrosis and portal hypertension beyond viral suppression is unknown. This study thus aimed to evaluate the influences of three frequently used NUCs in rats with nonviral cirrhosis. Male Sprague-Dawley rats received common bile duct ligation (CBDL) to induce cholestatic cirrhosis and portal hypertension. The rats were randomly allocated into four groups, treated by mouth with lamivudine (30 mg/kg per day), entecavir (0.09 mg/kg per day), tenofovir (50 mg/kg per day), or distilled water (vehicle control) from the 15th day after CBDL. On the 29th day, liver cirrhosis- and portal hypertension-related parameters were evaluated. The results showed that chronic NUCs treatment did not affect hemodynamic parameters, plasma TNF-α concentration, and hepatic fibrogenesis protein expressions in rats with nonviral cirrhosis. Though the mesenteric VEGF receptor 2 phosphorylation was downregulated in NUCs-treated groups, the splanchnic angiogenesis was not influenced. In conclusion, lamivudine, entecavir, and tenofovir had no additional effects on liver cirrhosis and portal hypertension in rats with nonviral cirrhosis.

Computational Modeling of Antiviral Drug Diffusion from Poly(lactic- co-glycolic-acid) Fibers and Multicompartment Pharmacokinetics for Application to the Female Reproductive Tract.

The need for more versatile technologies to deliver antiviral agents to the female reproductive tract (FRT) has spurred the development of on-demand and sustained-release platforms. Electrospun fibers (EFs), in particular, have recently been applied to FRT delivery, resulting in an alternative dosage form with the potential to provide protection and therapeutic effect against a variety of infection types. However, a multitude of fabrication parameters, as well as the resulting complexities of solvent-drug, drug-polymer, and solvent-polymer interactions, are known to significantly impact the loading and release of incorporated agents. Numerous processing parameters, in addition to their combined interactions, can hinder the iterative development of fiber formulations to achieve optimal release for particular durations, doses, and polymer-drug types. The experimental effort to design and develop EFs could benefit from mathematical analysis and computational simulation that predictively evaluate combinations of parameters to meet product design needs. Here, existing modeling efforts are leveraged to develop a simulation platform that correlates and predicts the delivery of relevant small molecule antivirals from EFs that have been recently applied to target sexually transmitted infections (STIs). A pair of mathematical models is coupled to simulate the release of two structurally similar small molecule antiretroviral reverse transcriptase inhibitors, Tenofovir (TFV) and Tenofovir disoproxil fumarate (TDF), from poly(lactic- co-glycolic acid) (PLGA) EFs, and to evaluate how changes in the system parameters affect the distribution of encapsulated agent in a three-compartment model of the vaginal epithelium. The results indicate that factors such as fiber diameter, mesh thickness, antiviral diffusivity, and fiber geometry can be simulated to create an accurate model that distinguishes the different release patterns of TFV and TDF from EFs, and that enables detailed evaluation of the associated pharmacokinetics. This simulation platform offers a basis with which to further study EF parameters and their effect on antiviral release and pharmacokinetics in the FRT.

Influence of Chitosan Swelling Behaviour on Controlled Release of Tenofovir from Mucoadhesive Vaginal Systems for Prevention of Sexual Transmission of HIV.

The main challenges facing efforts to prevent the transmission of human immunodeficiency virus (HIV) are the lack of access to sexual education services and sexual violence against young women and girls. Vaginal formulations for the prevention of sexually transmitted infections are currently gaining importance in drug development. Vaginal mucoadhesive tablets can be developed by including natural polymers that have good binding capacity with mucosal tissues, such as chitosan or guar gum, semisynthetic polymers such as hydroxypropylmethyl cellulose, or synthetic polymers such as Eudragit® RS. This paper assesses the potential of chitosan for the development of sustained-release vaginal tablets of Tenofovir and compares it with different polymers. The parameters assessed were the permanence time of the bioadhesion-determined ex vivo using bovine vaginal mucosa as substrate-the drug release profiles from the formulation to the medium (simulated vaginal fluid), and swelling profiles in the same medium. Chitosan can be said to allow the manufacture of tablets that remain adhered to the vaginal mucosa and release the drug in a sustained way, with low toxicity and moderate swelling that ensures the comfort of the patient and may be useful for the prevention of sexual transmission of HIV.

Tunable Release of Multiclass Anti-HIV Drugs that are Water-Soluble and Loaded at High Drug Content in Polyester Blended Electrospun Fibers.

OBJECTIVES: Sustained release of small molecule hydrophilic drugs at high doses remains difficult to achieve from electrospun fibers and limits their use in clinical applications. Here we investigate tunable release of several water-soluble anti-HIV drugs from electrospun fibers fabricated with blends of two biodegradable polyesters. METHODS: Drug-loaded fibers were fabricated by electrospinning ratios of PCL and PLGA. Fiber morphology was imaged by SEM, and DSC was used to measure thermal properties. HPLC was used to measure drug loading and release from fibers. Cytotoxicity and antiviral activity of drug-loaded fibers were measured in an in vitro cell culture assay. RESULTS: We show programmable release of hydrophilic antiretroviral drugs loaded up to 40 wt%. Incremental tuning of highly-loaded drug fibers within 24 h or >30 days was achieved by controlling the ratio of PCL and PLGA. Fiber compositions containing higher PCL content yielded greater burst release whereas fibers with higher PLGA content resulted in greater sustained release kinetics. We also demonstrated that our drug-loaded fibers are safe and can sustain inhibition of HIV in vitro. CONCLUSIONS: These data suggest that we were able to overcome current limitations associated with sustained release of small molecule hydrophilic drugs at clinically relevant doses. We expect that our system represents an effective strategy to sustain delivery of water-soluble molecules that will benefit a variety of biomedical applications.

Antiviral supramolecular polymeric hydrogels by self-assembly of tenofovir-bearing peptide amphiphiles.

The development of long-acting antiviral therapeutic delivery systems is crucial to improve the current treatment and prevention of HIV and chronic HBV. We report here on the conjugation of tenofovir (TFV), an FDA approved nucleotide reverse transcriptase inhibitor (NRTI), to rationally designed peptide amphiphiles (PAs), to construct antiviral prodrug hydrogelators (TFV-PAs). The resultant conjugates can self-assemble into one-dimensional nanostructures in aqueous environments and consequently undergo rapid gelation upon injection into 1× PBS solution to create a drug depot. The TFV-PA designs containing two or three valines could attain instantaneous gelation, with one displaying sustained release for more than 28 days in vitro. Our studies suggest that minor changes in peptide design can result in differences in supramolecular morphology and structural stability, which impacted in vitro gelation and release. We envision the use of this system as an important delivery platform for the sustained, linear release of TFV at rates that can be precisely tuned to attain therapeutically relevant TFV plasma concentrations.

Chitosan-poly(ethylene oxide) nanofibrous mat as a vaginal platform for tenofovir disoproxyl fumarate - The effect of vaginal pH on drug carrier performance.

In the present work, a solution blow spun nanofibrous mat comprised of chitosan (CS) and poly(ethylene oxide) (PEO) was obtained as vaginal platform for tenofovir disoproxil fumarate (TDF) to prevent sexually transmitted infections. Apart from physicochemical and mechanical analysis, the specific steps involved studies on nanofibrous mat mucoadhesive and swelling characteristics upon pH fluctuations over the physiological range. Physicochemical analysis showed uniform drug distribution within the CS/PEO mat volume and pointed toward physical interactions between the drug and polymers. TDF-loaded CS/PEO nanofibrous mat was shown potentially safe when evaluated by the MTT metabolic activity and JC-1 assays in human vaginal epithelial cells VK2-E6/E7. In vitro antiviral studies indicated inhibition efficacy of TDF-CS/PEO nanofibrous mat toward HSV-2 virus and proved the SBS process does not change the microbicidal activity of drug molecule. Fluctuations in the physiological vaginal pH range of 3.8 to 5.0 substantially affected mucoadhesive and swelling behavior of chitosan which in turn impacted drug dissolution rate from polymer carrier. The rate of permeation and accumulation of TDF in vaginal tissue differed in response to vaginal pH. Faster drug permeation assessed at pH 5.0 suggests that an increase in vaginal pH could improve TDF bioavailability at earlier time points.

In vitro Study on Synergistic Interactions Between Free and Encapsulated Q-Griffithsin and Antiretrovirals Against HIV-1 Infection.

INTRODUCTION: Human immunodeficiency virus (HIV) remains a persistent global challenge, impacting 38 million people worldwide. Antiretrovirals (ARVs) including tenofovir (TFV), raltegravir (RAL), and dapivirine (DAP) have been developed to prevent and treat HIV-1 via different mechanisms of action. In parallel, a promising biological candidate, griffithsin (GRFT), has demonstrated outstanding preclinical safety and potency against HIV-1. While ARV co-administration has been shown to enhance virus inhibition, synergistic interactions between ARVs and the oxidation-resistant variant of GRFT (Q-GRFT) have not yet been explored. Here, we co-administered Q-GRFT with TFV, RAL, and DAP, in free and encapsulated forms, to identify unique protein-drug synergies. METHODS: Nanoparticles (NPs) were synthesized using a single or double-emulsion technique and release from each formulation was assessed in simulated vaginal fluid. Next, each ARV, in free and encapsulated forms, was co-administered with Q-GRFT or Q-GRFT NPs to evaluate the impact of co-administration in HIV-1 pseudovirus assays, and the combination indices were calculated to identify synergistic interactions. Using the most synergistic formulations, we investigated the effect of agent incorporation in NP-fiber composites on release properties. Finally, NP safety was assessed in vitro using MTT assay. RESULTS: All active agents were encapsulated in NPs with desirable encapsulation efficiency (15-100%), providing ~20% release over 2 weeks. The co-administration of free Q-GRFT with each free ARV resulted in strong synergistic interactions, relative to each agent alone. Similarly, Q-GRFT NP and ARV NP co-administration resulted in synergy across all formulations, with the most potent interactions between encapsulated Q-GRFT and DAP. Furthermore, the incorporation of Q-GRFT and DAP in NP-fiber composites resulted in burst release of DAP and Q-GRFT with a second phase of Q-GRFT release. Finally, all NP formulations exhibited safety in vitro. CONCLUSIONS: This work suggests that Q-GRFT and ARV co-administration in free or encapsulated forms may improve efficacy in achieving prophylaxis.

Multipurpose tenofovir disoproxil fumarate electrospun fibers for the prevention of HIV-1 and HSV-2 infections in vitro.

Sexually transmitted infections affect hundreds of millions of people worldwide. Both human immunodeficiency virus (HIV-1 and -2) and herpes simplex virus-2 (HSV-2) remain incurable, urging the development of new prevention strategies. While current prophylactic technologies are dependent on strict user adherence to achieve efficacy, there is a dearth of delivery vehicles that provide discreet and convenient administration, combined with prolonged-delivery of active agents. To address these needs, we created electrospun fibers (EFs) comprised of FDA-approved polymers, poly(lactic-co-glycolic acid) (PLGA) and poly(DL-lactide-co-ε-caprolactone) (PLCL), to provide sustained-release and in vitro protection against HIV-1 and HSV-2. PLGA and PLCL EFs, incorporating the antiretroviral, tenofovir disoproxil fumarate (TDF), exhibited sustained-release for up to 4 weeks, and provided complete in vitro protection against HSV-2 and HIV-1 for 24h and 1 wk, respectively, based on the doses tested. In vitro cell culture and EpiVaginal tissue tests confirmed the safety of fibers in vaginal and cervical cells, highlighting the potential of PLGA and PLCL EFs as multipurpose next-generation drug delivery vehicles.

Development and in vivo safety assessment of tenofovir-loaded nanoparticles-in-film as a novel vaginal microbicide delivery system.

UNLABELLED: Topical pre-exposure prophylaxis (PrEP) with antiretroviral drugs holds promise in preventing vaginal transmission of HIV. However, significant biomedical and social issues found in multiple past clinical trials still need to be addressed in order to optimize protection and users' adherence. One approach may be the development of improved microbicide products. A novel delivery platform comprising drug-loaded nanoparticles (NPs) incorporated into a thin polymeric film base (NPs-in-film) was developed in order to allow the vaginal administration of the microbicide drug candidate tenofovir. The system was optimized for relevant physicochemical features and characterized for biological properties, namely cytotoxicity and safety in a mouse model. Tenofovir-loaded poly(lactic-co-glycolic acid) (PLGA)/stearylamine (SA) composite NPs with mean diameter of 127nm were obtained with drug association efficiency above 50%, and further incorporated into an approximately 115µm thick, hydroxypropyl methylcellulose/poly(vinyl alcohol)-based film. The system was shown to possess suitable mechanical properties for vaginal administration and to quickly disintegrate in approximately 9min upon contact with a simulated vaginal fluid (SVF). The original osmolarity and pH of SVF was not affected by the film. Tenofovir was also released in a biphasic fashion (around 30% of the drug in 15min, followed by sustained release up to 24h). The incorporation of NPs further improved the adhesive potential of the film to ex vivo pig vaginal mucosa. Cytotoxicity of NPs and film was significantly increased by the incorporation of SA, but remained at levels considered tolerable for vaginal delivery of tenofovir. Moreover, histological analysis of genital tissues and cytokine/chemokine levels in vaginal lavages upon 14days of daily vaginal administration to mice confirmed that tenofovir-loaded NPs-in-film was safe and did not induce any apparent histological changes or pro-inflammatory response. Overall, obtained data support that the proposed delivery system combining the use of polymeric NPs and a film base may constitute an exciting alternative for the vaginal administration of microbicide drugs in the context of topical PrEP. STATEMENT OF SIGNIFICANCE: The development of nanotechnology-based microbicides is a recent but promising research field seeking for new strategies to circumvent HIV sexual transmission. Different reports detail on the multiple potential advantages of using drug nanocarriers for such purpose. However, one important issue being frequently neglected regards the development of vehicles for the administration of microbicide nanosystems. In this study, we propose and detail on the development of a nanoparticle-in-film system for the vaginal delivery of the microbicide drug candidate tenofovir. This is an innovative approach that, to our best knowledge, had never been tested for tenofovir. Results, including those from in vivo testing, sustain that the proposed system is safe and holds potential for further development as a vaginal microbicide product.

Prevention of vaginal and rectal herpes simplex virus type 2 transmission in mice: mechanism of antiviral action.

Topical microbicides to stop sexually transmitted diseases, such as herpes simplex virus type 2 (HSV-2), are urgently needed. The emerging field of nanotechnology offers novel suitable tools for addressing this challenge. Our objective was to study, in vitro and in vivo, antiherpetic effect and antiviral mechanisms of several polyanionic carbosilane dendrimers with anti-HIV-1 activity to establish new potential microbicide candidates against sexually transmitted diseases. Plaque reduction assay on Vero cells proved that G2-S16, G1-S4, and G3-S16 are the dendrimers with the highest inhibitory response against HSV-2 infection. We also demonstrated that our dendrimers inhibit viral infection at the first steps of HSV-2 lifecycle: binding/entry-mediated events. G1-S4 and G3-S16 bind directly on the HSV-2, inactivating it, whereas G2-S16 adheres to host cell-surface proteins. Molecular modeling showed that G1-S4 binds better at binding sites on gB surface than G2-S16. Significantly better binding properties of G1-S4 than G2-S16 were found in an important position for affecting transition of gB trimer from G1-S4 prefusion to final postfusion state and in several positions where G1-S4 could interfere with gB/gH-gL interaction. We demonstrated that these polyanionic carbosilan dendrimers have a synergistic activity with acyclovir and tenofovir against HSV-2, in vitro. Topical vaginal or rectal administration of G1-S4 or G2-S16 prevents HSV-2 transmission in BALB/c mice in values close to 100%. This research represents the first demonstration that transmission of HSV-2 can be blocked by vaginal/rectal application of G1-S4 or G2-S16, providing a step forward to prevent HSV-2 transmission in humans.

Biophysical characterization of small molecule antiviral-loaded nanolipogels for HIV-1 chemoprophylaxis and topical mucosal application.

UNLABELLED: Nanocarriers are versatile vehicles for drug delivery, and emerging as platforms to formulate and deliver multiple classes of antiretroviral (ARV) drugs in a single system. Here we describe the fabrication of hydrogel-core and lipid-shell nanoparticles (nanolipogels) for the controlled loading and topical, vaginal delivery of maraviroc (MVC) and tenofovir disoproxil fumarate (TDF), two ARV drugs with different mechanisms of action that are used in the treatment of HIV. The nanolipogel platform was used to successfully formulate MVC and TDF, which produced ARV drug-loaded nanolipogels that were characterized for their physical properties and antiviral activity against HIV-1 BaL in cell culture. We also show that administration of these drug carriers topically to the vaginal mucosa in a murine model leads to antiviral activity against HIV-1 BaL in cervicovaginal lavages. Our results suggest that nanolipogel carriers are promising for the encapsulation and delivery of hydrophilic small molecule ARV drugs, and may expand the nanocarrier systems being investigated for HIV prevention or treatment. STATEMENT OF SIGNIFICANCE: Topical, mucosal intervention of HIV is a leading strategy in the efforts to curb the spread of viral infection. A significant research thrust in the field has been to characterize different dosage forms for formulation of physicochemically diverse antiretroviral drugs. Nanocarriers have been used to formulate and deliver small molecule and protein drugs for a range of applications, including ARV drugs for HIV treatment. The broad significance of our work includes evaluation of lipid-shell, hydrogel-core nanoparticles for formulation and topical, vaginal delivery of two water-soluble antiretroviral drugs. We have characterized these nanocarriers for their physical properties and their biological activity against HIV-1 infection in vitro, and demonstrated the ability to deliver drug-loaded nanocarriers in vivo.