Antiviral Activity of Lipophilic Nucleoside Tetraphosphate Compounds.

We report on the synthesis and characterization of three types of nucleoside tetraphosphate derivatives 4-9 acting as potential prodrugs of d4T nucleotides: (i) the δ-phosph(on)ate is modified by two hydrolytically stable alkyl residues 4 and 5; (ii) the δ-phosph(on)ate is esterified covalently by one biodegradable acyloxybenzyl moiety and a nonbioreversible moiety 6 and 7; or (iii) the δ-phosphate of nucleoside tetraphosphate is masked by two biodegradable prodrug groups 8 and 9. We were able to prove the efficient release of d4T triphosphate (d4TTP, (i)), δ-monoalkylated d4T tetraphosphates (20 and 24, (ii)), and d4T tetraphosphate (d4T4P, (iii)), respectively, by chemical or enzymatic processes. Surprisingly, δ-dialkylated d4T tetraphosphates, δ-monoalkylated d4T tetraphosphates, and d4T4P were substrates for HIV-RT. Remarkably, the antiviral activity of TetraPPPPro-prodrug 7 was improved by 7700-fold (SI 5700) as compared to the parent d4T in CEM/TK- cells, denoting a successful cell membrane passage of these lipophilic prodrugs and an intracellular delivery of the nucleotide metabolites.

Incidence and Predictors of Major Adverse Drug Reactions Among Human Immunodeficiency Virus-infected Children on Antiretroviral Treatment in West Amhara Comprehensive Specialized Hospitals, Northwest Ethiopia: A Multicenter Retrospective Follow-up Study.

PURPOSE: Major adverse drug reactions (ADRs) are the leading causes of poor adherence, switching of drugs, morbidity, and mortality. A limited studies was conducted to investigate major ADR in developing countries including Ethiopia, and the purpose of this study was to assess the incidence and predictors of major ADRs among HIV-infected children receiving antiretroviral therapy (ART) in West Amhara Comprehensive Specialized Hospitals, Northwest Ethiopia. METHODS: An institutional-based retrospective follow-up study was conducted among 460 children receiving ART from January 1, 2014 to December 31, 2021. A simple random sampling technique was employed, and data were collected using Kobo Toolbox software and then deployed to STATA 14 for analysis. The Kaplan-Meier survival curve and the log-rank test were used to estimate and compare survival times. Both bivariable and multivariable Weibull regression models were fitted to identify predictors. Finally, an adjusted hazards ratio (AHR) with a 95% CI was computed, and variables with P < 0.05 were considered statistically significant predictors of major ADR. FINDINGS: The overall incidence rate of major ADRs was 5.8 (95% CI, 4.6-7.3) per 1000 child months. Being female (AHR, 2.71; 95% CI, 1.52-4.84), tuberculosis (TB)-HIV co-infection (AHR, 2.49; 95% CI, 1.32-4.68), World Health Organization stage (III and IV) (AHR, 2.52; 95% CI, 1.39-4.56), zidovudine-based (AHR, 2.84; 95% CI, 1.11-7.31), and stavudine-based (AHR, 5.96; 95% CI, 1.63-21.84) regimens were found to be significant predictors of major ADRs. IMPLICATIONS: The major ADR incidence rate was high. Health professionals should employ early screening and close follow-up for children with advanced World Health Organization clinical staging, females, those with TB-HIV co-infection, and those receiving stavudine- and zidovudine-based initial regimens to reduce the incidence of major ADRs.

Nucleoside Analog Reverse-Transcriptase Inhibitors in Membrane Environment: Molecular Dynamics Simulations.

The behavior of four drugs from the family of nucleoside analog reverse-transcriptase inhibitors (zalcitabine, stavudine, didanosine, and apricitabine) in a membrane environment was traced using molecular dynamics simulations. The simulation models included bilayers and monolayers composed of POPC and POPG phospholipids. It was demonstrated that the drugs have a higher affinity towards POPG membranes than POPC membranes due to attractive long-range electrostatic interactions. The results obtained for monolayers were consistent with those obtained for bilayers. The drugs accumulated in the phospholipid polar headgroup region. Two adsorption modes were distinguished. They differed in the degree of penetration of the hydrophilic headgroup region. Hydrogen bonds between drug molecules and phospholipid heads were responsible for adsorption. It was shown that apricitabine penetrated the hydrophilic part of the POPC and POPG membranes more effectively than the other drugs. Van der Waals interactions between S atoms and lipids were responsible for this.

Machine learning aided multiscale modelling of the HIV-1 infection in the presence of NRTI therapy.

Human Immunodeficiency Virus (HIV) is one of the most common chronic infectious diseases in humans. Extending the expected lifetime of patients depends on the use of optimal antiretroviral therapies. Emergence of the drug-resistant strains can reduce the effectiveness of treatments and lead to Acquired Immunodeficiency Syndrome (AIDS), even with antiretroviral therapy. Investigating the genotype-phenotype relationship is a crucial process for optimizing the therapy protocols of the patients. Here, a mathematical modelling framework is proposed to address the impact of existing mutations, timing of initiation, and adherence levels of nucleotide reverse transcriptase inhibitors (NRTIs) on the evolutionary dynamics of the virus strains. For the first time, the existing Stanford HIV drug resistance data have been combined with a multi-strain within-host ordinary differential equation (ODE) model to track the dynamics of the most common NRTI-resistant strains. Overall, the D4T-3TC, D4T-AZT and TDF-D4T drug combinations have been shown to provide higher success rates in preventing treatment failure and further drug resistance. The results are in line with the genotype-phenotype data and pharmacokinetic parameters of the NRTI inhibitors. Moreover, we show that the undetectable mutant strains at the diagnosis have a significant effect on the success/failure rates of the NRTI treatments. Predictions on undetectable strains through our multi-strain within-host model yielded the possible role of viral evolution on the treatment outcomes. It has been recognized that the improvement of multi-scale models can contribute to the understanding of the evolutionary dynamics, and treatment options, and potentially increase the reliability of genotype-phenotype models.

Effect of different antiretroviral therapy on muscle mass, bone mineral density, and trabecular bone score in Chinese HIV-infected males.

This is the first study to report both greater BMD loss and muscle loss in Chinese HIV-infected males with lamivudine (3TC)-tenofovir disoproxil fumarate (TDF)-efavirenz (EFV) regimen, which highlights the importance of closely monitoring muscle mass and bone mineral density in patients treated with 3TC-TDF-EFV regimen and provides a foundation for the clinical intervention of sarcopenia and osteoporosis. PURPOSE: To compare the effect initiating different antiretroviral therapy (ART) regimens have on muscle mass, bone mineral density (BMD), and trabecular bone score (TBS). METHODS: We designed a retrospective study of ART-naive Chinese males with HIV (MWH) undergoing two different regimens at 1-year follow-up. All subjects underwent dual-energy absorptiometry (DXA) for BMD and muscle mass prior to ART initiation, and again 1 year later. TBS iNsight software was used for TBS. We analyzed differences in muscle mass, BMD, and TBS after different treatment arms and associations between ART regimens and changes in them. RESULTS: A total of 76 men were included (mean age 31.83 ± 8.75 years). Mean absolute muscle mass decreased significantly from baseline to follow-up after initiation of lamivudine (3TC)-tenofovir disoproxil fumarate (TDF)-efavirenz (EFV), whereas increased significantly after initiation of 3TC-zidovudine(AZT)/Stavudine(d4T)-Nevirapine(NVP). Assignment to 3TC-TDF-EFV resulted in greater percentage loss in BMD at lumbar spine (LS) and total hip (TH) compared to 3TC-AZT/d4T-NVP, but this difference was not statistically significant at the femoral neck BMD and TBS. In the multivariable logistic regression model adjusted for covariates, the 3TC-TDF-EFV regimen was associated with higher odds of decreased appendicular and total muscle mass, LS and TH BMD. CONCLUSIONS: This is the first study to report not only greater BMD loss but also muscle loss in Chinese MWH with 3TC-TDF-EFV regimen. Our work highlights the importance of closely monitoring muscle mass and BMD in patients treated with 3TC-TDF-EFV regimen and provides a foundation for the clinical intervention of sarcopenia and osteoporosis in them.

Critical Pronociceptive Role of Family 2 Voltage-Gated Calcium Channels in a Novel Mouse Model of HIV-Associated Sensory Neuropathy.

Some people living with HIV present painful sensory neuropathy (HIV-SN) that is pharmacoresistant, sex-associated, and a major source of morbidity. Since the specific mechanisms underlying HIV-SN are not well understood, the aim of our study was to characterize a novel model of painful HIV-SN by combining the HIV-1 gp120 protein and the antiretroviral stavudine (d4T) in mice and to investigate the pronociceptive role of the family 2 voltage-gated calcium channel (VGCC) α1 subunit (Cav2.X channels) in such a model. HIV-SN was induced in male and female C57BL/6 mice by administration of gp120 and/or d4T and detected by a battery of behavior tests and by immunohistochemistry. The role of Cav2.X channels was assessed by the treatment with selective blockers and agonists as well as by mRNA detection. Repeated administration with gp120 and/or d4T produced long-lasting touch-evoked painful-like behaviors (starting at 6 days, reaching a maximum on day 13, and lasting up to 28 days after treatment started), with a greater intensity in female mice treated with the combination of gp120 + d4T. Moreover, gp120 + d4T treatment reduced the intraepidermal nerve fibers and well-being of female mice, without altering other behaviors. Mechanistically, gp120 + d4T treatment induced Cav2.1, 2.2, and 2.3 transcriptional increases in the dorsal root ganglion and the Cav2.X agonist-induced nociception. Accordingly, intrathecal selective Cav2.2 blockade presented longer and better efficacy in reversing the hyperalgesia induced by gp120 + d4T treatment compared with Cav2.1 or Cav2.3, but also presented the worst safety (inducing side effects at effective doses). We conclude that the family 2 calcium channels (Cav2.X) exert a critical pronociceptive role in a novel mouse model of HIV-SN.

Determinants of therapy failure among adults on first-line antiretroviral therapy in Asmara, Eritrea: a multicenter retrospective matched case-control study.

BACKGROUND: Information on treatment failure (TF) in People living with HIV in a data-poor setting is necessary to counter the epidemic of TF with first-line combined antiretroviral therapies (cART) in sub-Saharan Africa (SSA). In this study, we examined the risk factors associated with TF in Asmara, Eritrea from 2001 to 2020. METHODS: A multicenter, retrospective 1:2 matched (by age and gender) case-control study was conducted in four major hospitals in Asmara, Eritrea on adults aged ≥ 18 years who were on treatment for at least 6 months. Cases were patients who fulfills at least one of the WHO therapy failure criterion during the study period. Controls were randomly selected patients on first-line treatment and plasma viral load < 1000 copies/ml in their latest follow-up measurement. Multivariable logistic regression analysis was conducted to identify risk factors for TF. All P-values were 2-sided and the level of significance was set at P < 0.05 for all analyses. RESULTS: Of the 1068 participants (356 cases; 712 controls), 585 (54.7%) were females. The median age at treatment initiation was 46 years [interquartile range (IQR): 39-51]. Median time to combined antiretroviral therapy (cART) failure was 37 months (IQR = 24-47). In the multivariate analysis, factors associated with increased likelihood of TF included initial nucleoside reverse transcriptase inhibitors (NRTI) backbone (Zidovudine + Lamivudine (AZT + 3TC): adjusted odds ratio (aOR) = 2.70, 95% Confidence interval (CI): 1.65-4.41, P-value < 0.001), (Abacavir + lamivudine (ABC + 3TC): aOR = 4.73, 95%CI: 1.18-18.92, P-value = 0.028], and (Stavudine + Lamivudine (D4T + 3TC): aOR = 5.00; 95% CI: 3.03-8.20, P-value < 0.001) in comparison to Emtricitabine and Tenofovir diproxil fumarate (FTC + TDF). Additional associations included prior exposure to cART (aOR = 2.28, 95%CI: 1.35-3.86; P- value = 0.002), record of sub-optimal drug adherence (aOR = 3.08, 95%CI: 2.22-4.28; P < 0.001), ambulatory/bedridden at presentation (aOR = 1.61, 95%CI: 1.12-4.28; P-value = 0.010), presence of comorbidities (aOR = 2.37; 95%CI: 1.36-4.10, P-value = 0.002), duration of cART (< 5 years: aOR: 5.90; 95% CI: 3.95-8.73, P-value < 0.001), and use of SMX-TMP prophylaxis (aOR = 2.00, 95%CI, 1.44-2.78, P-value < 0.001). CONCLUSION: Our findings underscore the importance of optimizing cART adherence, diversification of cART regimens, and interventions directed at enhancing early HIV diagnosis, prompt initiations of treatment, and improved patient-focused monitoring of treatment response.

REVERSIBILITY OF LIPOATROPHY IN HIV-INFECTED PATIENTS TAKING ANTIRETROVIRAL THERAPY: A COHORT STUDY WITH ULTRASOUND ASSESSMENT.

The aim of this study was to characterize and compare changes in subcutaneous fat in the malar, brachial and crural region in a cohort of HIV-infected patients taking antiretroviral therapy. This prospective longitudinal study included 77 patients who were selected from the initial cohort evaluated in 2007 and 2008. We examined reversibility of lipoatrophy measured by ultrasound over at least five-year period and factors related to its reversibility. All 46 patients who used stavudine switched from stavudine to another combination. Of 58 patients on zidovudine, 16 (28%) were on a zidovudine based regimen at the second follow up. There was evidence for subcutaneous fat increase in the malar area (p<0.001) and no increase in the brachial and crural areas. Patients who were smokers and had poor adherence to the Mediterranean diet had a thinner malar area at the follow up measurement (p=0.030) and smaller increase in subcutaneous malar fat compared to others (p=0.040). Our study suggested that modest increase of subcutaneous fat in malar area coincided with stopping stavudine and fewer usage of zidovudine. Lifestyle with non-adherence to the Mediterranean diet and smoking were associated with a smaller increase in subcutaneous malar fat.

The antiretroviral agents azidothymidine, stavudine, and didanosine have the identical mutational fingerprint in the rpoB region of Escherichia coli.

We looked at the mutational fingerprints of three antiretroviral (anti-HIV) agents, azidothymidine (AZT), stavudine (STAV), and didanosine (DIDA) in the rpoB system of Escherichia coli and compared them with each other and with the fingerprints of trimethoprim and of spontaneous mutations in a wild-type and a mutT background. All three agents gave virtually identical fingerprints in the wild-type background, causing only A:T→C:G changes at 3 of the 12 A:T→C:G possible sites among the total of 92 possible base substitution mutations, even though AZT and STAV are thymidine analogs but DIDA is an adenosine analog. As all three agents are reverse transcriptase inhibitors, and act as chain blockers, the common fingerprint may be a property of chain blocking agents.

Liver steatosis and fibrosis in people with human immunodeficiency virus in West Africa and the relationship with hepatitis B virus coinfection.

There is a heavy burden of liver disease in West Africa. While the role of hepatitis B virus (HBV) infection is well recognized, less is known about the contributing role of liver steatosis and how the two interact in the context of human immunodeficiency virus (HIV) infection. Adults with HIV in Ghana underwent FibroScan measurements to determine prevalence of liver steatosis (expressed as controlled attenuation parameter [CAP]) and fibrosis (expressed as liver stiffness [LS]). We explored contributing factors in linear regression models, including demographics, lifestyle characteristics, medical history, HIV and HBV status, and measurements of metabolic syndrome. Among 329 adults (72.3% women; median age, 47 years), 322 (97.9%) were on antiretroviral therapy (median duration, 8.9 years). CD4 counts were preserved (median, 619 cells/mm3 ); plasma HIV RNA was fully suppressed in 162 (50.3%) of the treated participants. Cigarette smoking, excessive alcohol consumption, and use of traditional or herbal remedies were uncommon (6.1%, 1.8%, 3.3%, respectively). Largely undiagnosed metabolic syndrome was detected in 87 (26.4%) participants. We obtained readings indicative of ≥S2 steatosis and ≥F2 fibrosis in 43 (13.1%) and 55 (16.7%) participants, respectively. Higher CAP values were associated with metabolic syndrome and longer prior stavudine exposure. Higher LS values were associated with male sex, higher HIV RNA, and higher CAP values. Relative to people without HBV, those with HBV (n = 90) had a similar prevalence of ≥S2 steatosis but a higher prevalence of ≥F2 fibrosis (36.7% vs. 9.2%, p < 0.0001) and concomitant ≥S2 steatosis and ≥F2 fibrosis (9.1% vs. 1.3%, p < 0.001). Conclusion: Both HBV and liver steatosis pose a threat to long-term liver health among people with HIV in West Africa. Urgently required interventions include improving HIV suppression and diagnosing and managing determinants of the metabolic syndrome.

Risk Factors for Incident Hypertension Within 1 Year of Initiating Antiretroviral Therapy Among People with HIV.

Hypertension (HTN) is a common comorbidity among people with HIV and associated with an increased risk for atherosclerotic cardiovascular disease and chronic kidney disease. The relationship of antiretroviral therapy (ART) initiation to incident HTN remains a clinical question. We determined HTN incidence at 48 weeks of follow-up among ART-naive participants without HTN and not taking antihypertensive medications at ART initiation through randomized clinical trials through the AIDS Clinical Trial Group between 1999 and 2011. We assessed the association of baseline characteristics, including randomized ART agents with HTN incidence at 48 weeks using Poisson regression models. Incident HTN was defined as blood pressure ≥130/80 mmHg, or use of antihypertensive medication. Among 2,614 participants, mean age was 37 ± 10 years, 79% male sex, and 36% African American race. After 48 weeks, 839 participants (32%) developed HTN. Receiving a non-nucleoside reverse transcriptase inhibitor (NNRTI) was associated with an increased relative risk (RR) of incident HTN, while the risk was lower for protease inhibitor use. Stavudine and efavirenz were associated with an increased RR of developing HTN, while tenofovir disoproxil fumarate, darunavir/ritonavir, and atazanavir/ritonavir were associated with a decreased risk of developing HTN. Additionally, older age, higher body mass index (BMI), and having hepatitis C were associated with an increased risk for developing HTN, while women and participants with a higher baseline CD4 count were at a decreased risk of developing HTN at 48 weeks. One third of these ART naive participants developed HTN after ART initiation. NNRTIs, notably efavirenz, and stavudine were associated with an increased risk of HTN. Additional factors associated with HTN included traditional factors like older age and higher BMI, and advanced HIV disease (lower CD4 count). (Clinicaltrials.gov: NCT00001137).

Modulation of MAPK- and PI3/AKT-Dependent Autophagy Signaling by Stavudine (D4T) in PBMC of Alzheimer's Disease Patients.

BACKGROUND: Aß42 deposition plays a pivotal role in AD pathogenesis by inducing the activation of microglial cells and neuroinflammation. This process is antagonized by microglia-mediated clearance of Aß plaques. Activation of the NLRP3 inflammasome is involved in neuroinflammation and in the impairments of Aß-plaque clearance. On the other hand, stavudine (D4T) downregulates the NLRP3 inflammasome and stimulates autophagy-mediated Aß-clearing in a THP-1-derived macrophages. METHODS: We explored the effect of D4T on Aß autophagy in PBMC from AD patients that were primed with LPS and stimulated with Aß oligomers in the absence/presence of D4T. We analyzed the NLRP3 activity by measuring NLRP3-ASC complex formation by AMNIS FlowSight and pro-inflammatory cytokine (IL-1ß, IL-18 and Caspase-1) production by ELISA. The phosphorylation status of p38, ERK, AKT, p70, and the protein expression of CREB, LAMP2A, beclin-1, Caspase-3 and Bcl2 were analyzed by Western blot. RESULTS: Data showed that D4T: (1) downregulates NLRP3 inflammasome activation and the production of down-stream pro-inflammatory cytokines in PBMC; (2) stimulates the phosphorylation of AKT, ERK and p70 as well as LAMP2A, beclin-1 and Bcl2 expression and reduces Caspase-3 expression, suggesting an effect of this compound on autophagy; (3) increases phospho-CREB, which is a downstream target of p-ERK and p-AKT, inducing anti-inflammatory cytokine production and resulting in a possible decrease of Aß-mediated cytotoxicity; and (4) reduces the phosphorylation of p38, a protein involved in the production of pro-inflammatory cytokines and tau hyperphosphorylation. CONCLUSIONS: D4T reduces the activation of the NLRP3 inflammasome, and it might stimulate autophagy as well as the molecular mechanism that modulates Aß cytotoxicity, and D4T might reduce inflammation in the cells of AD patients. It could be very interesting to check the possible beneficial effects of D4T in the clinical scenario.

Sustainable Protocol for the Synthesis of 2',3'-Dideoxynucleoside and 2',3'-Didehydro-2',3'-dideoxynucleoside Derivatives.

An improved protocol for the transformation of ribonucleosides into 2',3'-dideoxynucleoside and 2',3'-didehydro-2',3'-dideoxynucleoside derivatives, including the anti-HIV drugs stavudine (d4T), zalcitabine (ddC) and didanosine (ddI), was established. The process involves radical deoxygenation of xanthate using environmentally friendly and low-cost reagents. Bromoethane or 3-bromopropanenitrile was the alkylating agent of choice to prepare the ribonucleoside 2',3'-bisxanthates. In the subsequent radical deoxygenation reaction, tris(trimethylsilyl)silane and 1,1'-azobis(cyclohexanecarbonitrile) were used to replace hazardous Bu3SnH and AIBN, respectively. In addition, TBAF was substituted for camphorsulfonic acid in the deprotection step of the 5'-O-silyl ether group, and an enzyme (adenosine deaminase) was used to transform 2',3'-dideoxyadenosine into 2',3'-dideoxyinosine (ddI) in excellent yield.

Optimization and Quest of HPMC loaded Stavudine Controlled Release Dosage Development by Central Composite Design utilizing Reduced Factorial Screening Technique

Abstract The current research focused on screening and finding the significant independent variables in stavudine loaded tablet, followed by optimizing the best formulation using central composite design. The objective of the study to develop stavudine loaded controlled release tablet utilizing reduced factorial design, followed by optimization technique as well as characterization of prepared tablets. Preliminary trial batches were prepared using different grades of hydroxypropyl methylcellulose. The resolution-IV reduced factorial design was selected to screen the significant independent variables in the dosage form design. A total number of eight runs were prepared and responses were recorded. The signified factors identified by half-normal and Pareto chart. The prepared tablets are evaluated for various physiochemical characterizations. Three dependent responses such as hardness, dissolution at 6 hour and 12 hours are considered in optimization process. Later on, drug-polymer interaction study was carried out. The principal of the study design based on finding the best formulation with prefixed set parameter values utilizing the concept of screening technique. It observed that HPMC K15M (57.18 %), HPMC K100 (66.32 %) and PVP K30 (7.97 %) as best composition in a formulation batch would fulfill the predetermined parameter with specific values.

Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positively Charged and Hydrophobic Residues as Molecular Transporters.

The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side chain [(RW)5]K(RW)X (X = 1-5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. In vitro cytotoxicity of the peptides showed that the peptides did not exhibit any significant cytotoxicity at the concentration of 10 µM in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) after 3 h incubation. The cellular uptake of a fluorescence-labeled phosphopeptide (F'-GpYEEI) and anti-human immunodeficiency virus (HIV) drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), Stavudine (F'-d4T)), where F' is carboxyfluorescein, was measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW)5K](RW)5 (10 µM) was the most efficient transporter that improved the cellular uptake of F'-GpYEEI (2 µM) by 18- and 11-fold in CCRF-CEM and SK-OV-3, respectively, compared with F'-GpYEEI alone. Fluorescence-activated cell sorting (FACS) analysis results indicated that the cellular uptake of fluorescence-labeled peptide (F'-[(RW)5K](RW)5) was only partially inhibited by chlorpromazine as an endocytosis inhibitor after 3 h incubation in MDA-MB-231 cells. These data suggest the potential of this series of hybrid cyclic-linear peptides as cell-penetrating peptides and molecular transporters.

Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis.

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

Multistep Continuous Flow Synthesis of Stavudine.

Herein, we demonstrate an elegant multistep continuous flow synthesis for stavudine (d4T), a potent nucleoside chemotherapeutic agent for human immunodeficiency virus, acquired immunodeficiency syndrome (AIDS) and AIDS-related conditions. This was accomplished via six chemical transformations in five sequential continuous flow reactors from an affordable starting material, 5-methyluridine. In the first instance, single step continuous flow synthesis was demonstrated with an average of 97% yield, 21.4 g/h throughput per step, and a total of 15.5 min residence time. Finally, multistep continuous flow synthesis of d4T in 87% total yield with a total residence time of 19.9 min and 117 mg/h throughput without intermediate purification was demonstrated.

Characterization of a mouse neuropathic pain model caused by the highly active antiviral therapy (HAART) Stavudine.

BACKGROUND: Although highly active antiviral therapies (HAART) exert control over viral replication in persons with Acquired Immunodeficiency Syndrome (AIDS), neuropathic pain is a side effect. Symptoms include hyperalgesia and allodynia. Stavudine, also known as D4T, is a HAART used to treat Human Immunodeficiency Virus (HIV). This study examined the extent to which D4T produces neuropathic pain and examined pharmacological management with a standard opioid analgesic. METHODS: Male and female C57BL/6 J mice were injected intraperitoneally with one dose of vehicle or D4T (10-56 mg/kg). Mice were tested through day 92 post injection for mechanical allodynia, assessed with von Frey filaments, and thermal hyperalgesia, assessed via the hotplate test. Separate cohorts received vehicle or 56 mg/kg D4T, the presence of allodynia and thermal hyperalgesia confirmed, and mice received intraperitoneal vehicle, morphine, or 0.032 mg/kg naltrexone + morphine. RESULTS: D4T produced dose- and time-dependent mechanical allodynia and thermal hyperalgesia. The smallest effective D4T dose was 17.8 mg/kg. This dose produced mechanical allodynia but not thermal hyperalgesia. Larger D4T doses (32 and 56 mg/kg) produced mechanical allodynia and thermal hyperalgesia lasting 92 days. Morphine dose-dependently alleviated both mechanical allodynia and thermal hyperalgesia in D4T-treated mice with ED50 values of 4.4 and 1.2 mg/kg, respectively. Naltrexone produced a rightward shift of the morphine dose-response function, i.e., increased the ED50 value of morphine by at least 3.8-fold. CONCLUSION: Stavudine produced neuropathic pain as a function of dose and time in mice. Opioid analgesics appear to be effective in alleviating neuropathic pain in a D4T-induced mouse model.

Structural dynamics and susceptibility of anti-HIV drugs against HBV reverse transcriptase.

Hepatitis B virus (HBV), a small enveloped DNA virus, attacks the human liver causing both acute and chronic diseases. Current therapeutic drugs use the nucleos(t)ide analogues (NAs) as a competitive inhibitor against HBV reverse transcriptase (HBV-RT), an essential enzyme pivotally involved in viral replication. Unfortunately, this treatment still causes the development of resistant variants of HBV against NAs. As HBV-RT is homologous to the human immunodeficiency virus reverse transcriptase (HIV-RT), it is reasonable to treat HBV-RT with anti-HIV drugs. In the present study, we aimed to investigate the structural dynamics and susceptibility of the known anti-HIV drugs (stavudine [d4T], didanosine [DDI], and zidovudine [ZDV]) against HBV-RT enzyme in comparison to the anti-HBV drug lamivudine (3TC) and deoxythymidine triphosphate (dTTP) substrate using several computational approaches. The ΔGbindresidue calculations revealed that seven polar residues (K32, R41, D83, S85, D205, N236, and K239) and three hydrophobic residues (A86, A87, and F88) of HBV-RT as well as the adjacent DNA strands play an important role in the ligand binding. In addition, the H-bond pattern of d4T is similar to that of 3TC, especially at the residues A86 and A87. Such interactions promote the favorable conformation of ligand in the HBV-RT binding pocket, while the several different conformations of ligand are found in the unbound state. The predicted binding free energy results based on QM/MM-GBSA and MM/GB(PB)SA methods suggested that the susceptibility towards HBV-RT of d4T and ZDV is higher than that of 3TC and dTTP. Altogether, this work sheds light on the potentiality of d4T and ZDV as a promising drug for HBV-infected patients harboring 3TC resistance.Communicated by Ramaswamy H. Sarma.

Effects of Reverse Transcriptase Inhibitors on Proliferation, Apoptosis, and Migration in Breast Carcinoma Cells.

High telomerase activity in human breast cancer is associated with aggressive tumors resulting in decreased survival. Recent studies have shown that telomerase inhibitors may display anticancer properties in some human cancer cell lines. In the present study, we examined the effects of 4 reverse transcriptase inhibitors (RTIs), used for the treatment of HIV; Abacavir (AC), Lamivudine (LV), Stavudine (SV), and Tenofovir (TF) on proliferation, apoptosis, and migration in the normal human mammary epithelial cell line, hTERT-HME1, and the human breast cancer cell line, MCF-7. Cells were treated with AC, LV, SV, or TF alone or in combination with paclitaxel (PAC), a known drug used to treat breast cancer. Conduct of the thiazolyl blue tetrazolium bromide assay demonstrated that AC, SV, and TF had stronger cytotoxic effects on MCF-7 cells than in hTERT-HME1 cells. The combined treatment of RTIs and PAC caused high rates of cell death in MCF-7 and low rates of cell death in HTERT-HME1 by apoptosis. The percentages of apoptotic cells in the treatment of AC and SV in combination with PAC for 48 and 72 hours were higher than PAC. Significantly increased apoptosis and decreased migration levels were found in MCF-7 cells treated with AC and co-treatment of AC+PAC or SV+PAC than HME1 cells. These treatments can also prevent migration capacity more than PAC. Therefore, a combination strategy based on telomerase inhibitors such as AC or SV and anticancer drugs may be more effective in the treatment of certain breast cancers.