A competitive ELISA for the quantitative determination of the novel anti-HIV drug candidate CIGB-210 in biological fluids.

The synthetic peptide CIGB-210 is a promising anti-HIV drug candidate shown to inhibit HIV replication in MT4 cells at the nanomolar range by triggering the rearrangement of vimentin intermediate filaments. Sensitive and specific analytical methods are required for pharmacological studies of CIBG-210 in animals. In this study, we describe the development of a competitive ELISA for the quantitative determination of CIGB-210 using an anti-CIGB-210 hyperimmune serum. After optimization of all the steps, the assay exhibited a dynamic range from 11.87 to 0.0095 µg/mL. The intra-assay coefficient of variation (CV) was lower than or close to 5% for all the six concentrations of the calibrator, and the inter-assay CV was below 10% in five out of the six concentrations tested. No interference of either murine or human plasma was observed. The analyte was stable in plasma after five freeze-thaw cycles, while the hyperimmune serum maintained its binding capacity after 10 freeze-thaw cycles. Furthermore, the ELISA was able to detect the two main metabolites of CIGB-210, although with a tenfold decrease in sensitivity. Our results demonstrate the utility and feasibility of this analytical method for pharmacological experiments in animals as humans.

Simultaneous quantification of four antiretroviral drugs in breast milk samples from HIV-positive women by an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method.

The primary strategy to avoid mother-to-child transmission of human immunodeficiency virus (HIV) through breastfeeding is administration of highly active antiretroviral therapy (HAART) to HIV-positive pregnant women. Because significant changes in the pharmacokinetics of antiretroviral (ARV) drugs occur during pregnancy, quantifying HAART and the viral load in breast milk in this population is essential. Here, we developed an analytical assay for the simultaneous quantification of four ARV drugs in breast milk using ultra-performance liquid chromatography coupled to tandem mass spectrometry. We validated this method following Mexican and international guidelines. ARV drugs. We extracted the ARV drugs from 200 µL samples of breast milk and detected these drugs in a triple quadrupole mass spectrometer with positive electrospray ionization. The validated concentration ranges (ng/mL) for zidovudine, lamivudine, lopinavir, and ritonavir were 12.5-750, 50-2500, 100-5000 and 5 to 250, respectively. Additionally, the absolute recovery percentages (and matrix effects) were 91.4 (8.39), 88.78 (28.75), 91.38 (11.77) and 89.78 (12.37), respectively. We determined that ARV drugs are stable for 24 h at 8°C and 24°C for 15 days at -80°C. This methodology had the capacity for simultaneous detection; separation; and accurate, precise quantification of ARV drugs in human breast milk samples according to Mexican standard laws and United States Food and Drug Administration guidelines.

Discovery of N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives as anti-HIV-1 agents

The discovery and development of novel inhibitors with activity against variants of human immunodeficiency virus type 1 (HIV-1) is pivotal for overcoming treatment failure. As our ongoing work on research of anti-HIV-1 inhibitors, 32 N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives were prepared by introduction of the hydrazone fragments on the N-arylsulfonyl-3-acylindolyl skeleton and preliminarily screened in vitro as HIV-1 inhibitors for the first time. Among of all the reported analogues, eight compounds exhibited significant anti-HIV-1 activity, especially N-(3-nitro)phenylsulfonyl-3-acetylindole benzoyl hydrazone (18) and N-(3-nitro)phenylsulfonyl-3-acetyl-6-methylindole benzoyl hydrazone (23) displayed the most potent anti-HIV-1 activity with EC50 values of 0.26 and 0.31 µg/mL, and TI values of >769.23 and >645.16, respectively. It is noteworthy that introduction of R3 as the methyl group and R2 as the hydrogen group could result in more potent compounds. This suggested that introduction of R3 as the methyl group could be taken into account for further preparation of these kinds of compounds as anti-HIV-1 agents

Association of age, baseline kidney function, and medication exposure with declines in creatinine clearance on pre-exposure prophylaxis: an observational cohort study.

BACKGROUND: As pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate and emtricitabine for the prevention of HIV infection is rolled out internationally, strategies to maintain effectiveness and to minimise adverse effects merit consideration. In this study, we aimed to assess reductions in renal function and predictors of renal toxicity in a large open-label study of PrEP. METHODS: As part of the iPrEx open-label extension (OLE) study, men who have sex with men or transgender women aged 18-70 years who were HIV negative and had participated in three previous PrEP trials from Brazil, Ecuador, Peru, South Africa, Thailand, and the USA were enrolled into an open-label PrEP study. There were no restrictions on current renal function for enrolment into iPrEx OLE, in which participants were given combination tablets of tenofovir disoproxil fumarate (300 mg) and emtricitabine (200 mg) and advised to take one tablet per day. At follow-up sessions every 12 weeks, participants' creatinine clearance on PrEP was estimated and in a subset of participants, hair samples were collected to measure tenofovir and emtricitabine concentrations (a measure of adherence and exposure) via liquid-chromatography-tandem-mass-spectrometry. Reductions in creatinine clearance from baseline were calculated and predictors of decline were identified by use of multivariate models. iPrEx is registered with ClinicalTrials.com, number NCT00458393. FINDINGS: Baseline characteristics were similar between all participants in iPrEx-OLE (1224 participants with 7475 person-visits) and those participating in the hair substudy (220 participants with 1114 person-visits). During a median of 72 weeks, the mean decline in creatinine clearance was -2·9% (95% CI -2·4 to -3·4; ptrend<0·0001), but declines were greater for those who started PrEP at older ages: participants aged 40-50 years at baseline had declines of -4·2% (95% CI -2·8 to -5·5) and participants older than 50 years at baseline had declines of -4·9% (-3·1 to -6·8). In multivariate models, age and baseline creatinine clearance less than 90 mL/min predicted declines in renal function. We identified a monotonic association between percentage decrease in creatinine clearance and the number of doses of tenofovir disoproxil fumarate and emtricitabine taken per week, as estimated by hair concentrations of tenofovir and emtricitabine (ptrend=0·008). INTERPRETATION: Our data suggest that the frequency of safety monitoring for PrEP might need to be different between age groups and that pharmacological measures can monitor for toxic effects as well as adherence. FUNDING: National Institutes of Health.

Microbicidas para la prevención de infecciones de transmisión sexual: estado actual y estrategias para la evaluación preclínica de nuevos candidatos / Microbicides for preventing sexually transmitted infections: current status and strategies for preclinical evaluation of new candidates

Los microbicidas constituyen una nueva herramienta, todavía en proceso de investigación, que podrían ayudar en la prevención de la infección por los virus de la inmunodeficiencia humana (Human immunodeficiency virus: HIV) y de otras infecciones de transmisión sexual (ITS). Una serie de evidencias ha demostrado que la complejidad de la transmisión sexual de patógenos virales requiere de la identificación de compuestos capaces de bloquear los eventos tempranos del ciclo de infección viral. En este manuscrito hacemos una revisión exhaustiva de las diferentes estrategias que se han estudiado o se están considerando para prevenir ITS mediante el uso de microbicidas, haciendo particular énfasis en aquellos con el potencial de bloquear la infección por el HIV. También se revisa el proceso complejo de evaluación preclínica que se requiere para llegar a estudios en humanos y se concluye con un breve análisis de las estrategias que podrían formar parte del futuro inmediato en la investigación de microbicidas

Microbicides are a new tool, still under investigation, which could help prevent infection by the human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs). Increasing evidence shows that the complexity of sexual transmission of viral pathogens requires the identification of compounds able to block the early events during the cycle of viral infection. In this manuscript we provide a comprehensive review of the different microbicide strategies that have been studied or are currently being considered for STI prevention, particularly emphasizing those having the potential to block HIV infection. The manuscript also reviews the complex process that is required to conduct future clinical studies in humans and concludes with a brief discussion of the strategies that could be part of the immediate future in microbicide research

Cariogenic and erosive potential of the medication used by HIV-infected children: pH and sugar concentration.

OBJECTIVE: The aim of this work was to analyze pH and sugar concentration in seven antiretroviral and three antibacterial medications frequently prescribed to HIV infected paediatric patients. METHOD: Sugars (sucrose, glucose, lactose and fructose) and pH were measured from every one of ten medications with different serial numbers in two samples. The pH was determined by a previously calibrated digital pHmeter (Beckman). Analysis of free sugars was performed using thin-layer chromatography (TLC). The pH results and the amount of sugar originated from the two samples in each lot were added. The arithmetic mean of these results were computed. RESULTS: Two antiretrovirals (Zidovudin and Abacavir Sulphate) had pH below critical level (3.55 and 3.93, respectively). All three antibacterials analyzed had pH above 5.5, and one of them (Azithromycin) had the highest pH level of the ten medications examined (9.28). Sugar was present in seven out of 10 of the medications analyzed. The antibacterials contained the highest concentration of sucrose, ranging from 40% to 54%. Glucose was found in one of the ten, sucrose was present in seven of them and none showed lactose. Fructose was not observed with the technique used. CONCLUSIONS: A number of medications frequently used by HIV-infected children may cause a significant risk of both caries and dental erosion.

Efavirenz related compounds preparation by hydrolysis procedure: setting reference standards for chromatographic purity analysis.

A simple procedure for obtaining and purifying two degradation products of efavirenz (amino alcohol and quinoline derivatives) from drug substance hydrolysis is described. These impurities are known to exhibit very different UV absorbance properties from those of the parent compound, making determination using a quantitation factor (QF) inaccurate. The obtained hydrolysis products were characterized by physicochemical methods to assure identity, purity and strength. Quinoline derivative was of high purity degree (100%) and amino alcohol was 98.74% pure. Both were set as reference standards in chromatographic related compounds test for efavirenz drug substance and tablets analyses.

Development and validation of a HPLC-UV method for the determination in didanosine tablets.

A simple, rapid, sensitive and specific reversed-phase high performance liquid chromatographic method involving ultraviolet detection (HPLC-UV) was developed for analysis of didanosine in drug substance and formulated products, tablets. Chromatography was carried out on a pre-packed, Lichrospher 100 Rp-8 (5.0 microm, 250 mm x 4.0 mm) column using 0.01 M sodium acetate solution:methanol (85:15, v/v) adjusted to pH 6.5 with acetic acid as mobile phase at a flow rate of 1.5 ml/min and a 248 nm detection. Hypoxantine was confirmed as the main degradation product. The assay was linear over the concentration range of 50-150 microg/ml (R approximately 0.999). The method was validated for accuracy and precision.

Polysaccharide-based chiral phase under polar organic mode of elution in the determination of the enantiomeric purity of emtricitabine an anti-HIV analogue nucleoside.

This work reports the separation of FTC enantiomers using an amylose tris[(S)-1-phenylethylcarbamate] coated onto APS-Nucleosil (7 microm particle size, 500 A pore size, 20% w/w, 15 x 0.46 cm ID) chiral column under polar organic elution mode. Good enantioselectivity (alpha=1.9) with excellent enantioresolution (R(S)=3.3) was achieved by the use of methanol with 0.02% of triethylamine acetate as mobile phase. The method allows the accurate determination of as low as 0.2% of each enantiomer as an impurity. The validated method proved to be reliable and sensitive for the quantification of both enantiomers as impurity in different batches of emtricitabine and beta-D-(+)-FTC.

Análise do teste de ELISA anti-HIV1 em pacientes com leishmaniose visceral / Soropositivity to ELISA's anti - HIV tests in patients with American Visceral Leishmaniase

As reações falso-positivas em testes de ELISA(Enzima Linked Immuno Sorbent Assay) podem acontecer. O presente trabalho demonstra a existência de reações cruzadas no teste de ELISA anti-HIV em 33 pacientes de 0 - 15 anos com diagnóstico de calazar. Foram observados 5 casos positivos para o teste de ELISA anti-HIV na primeira reação e em 4 (12,12%)houve a permanência da positividade na segunda reação sorológica. Estes pacientes positivos apresentaram Western Blot anti-HIV negativos. Devido o estigma da infecção pelo HIV, fica imperativo, em zonas endêmicas de Leishmaniose Visceral Americana, nos pacientes que tenham apresentado ELISA anti-HIV positivo, a realização de outro teste comprobatório do HIV( Western Blot)