RESUMO
A stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of dolutegravir sodium and lamivudine encapsulated in the nanoliposomal formulation was developed. The chromatographic parameters namely, organic phase ratio, flow rate, and sample injection volume were selected as independent factors and were optimized by multivariate Box-Behnken design. Responses analyzed were retention time, peak area, and resolution. The optimized chromatographic method with Hypersil BDS C8 CN column as stationary phase and methanol and acetonitrile mixture and acidified Milli-Q water (pH 2.8, adjusted with 0.02% v/v orthophosphoric acid) as the mobile phase in an isocratic elution mode was validated according to parameters of International Conference on Harmonization Q1(R2) guidelines. The validated reversed-phase high-performance liquid chromatography method exhibited specificity for both dolutegravir sodium and lamivudine in the presence of degradation products as well as the liposomal matrix. This method was effectively utilized to determine the amount of drug entrapped and drug loading efficiency of dolutegravir sodium and lamivudine in a nano-liposomal formulation.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Portadores de Fármacos , Inibidores de Integrase de HIV/análise , Compostos Heterocíclicos com 3 Anéis/análise , Lamivudina/análise , Lipossomos , Nanopartículas , Oxazinas/análise , Piperazinas/análise , Piridonas/análise , Inibidores da Transcriptase Reversa/análise , Composição de Medicamentos , Limite de DetecçãoRESUMO
RATIONALE: Drug levels in hair provide a longer window of detection, compared to plasma drug levels, and therefore hair analysis has the advantage of assessing adherence over a longer period of time. No methods for the analysis of antiretroviral drugs in hair currently exist in South Africa, and worldwide there is only one validated method for the determination of efavirenz in hair that has been published. METHODS: Efavirenz was extracted from 0.2 mg of hair through a simultaneous pulverization and extraction step. Separation was achieved on an Agilent Poroshell C18 column using an isocratic elution with a total run time of 3 min. A triple quadrupole mass spectrometer set to electrospray ionization in positive multiple reaction monitoring mode was used for detection. The method was validated over the concentration range 0.625-40 ng/mg. RESULTS: Using ten times less hair than in a previously published method, the lower limit of quantitation was validated at 0.625 ng/mg. The interday and intraday assay precision, expressed as the percentage coefficient of variation (CV), for spiked calibration standards and quality control samples was lower than 7% and accuracy ranged from 97 to 110%. For quality controls prepared from authentic hair the CV was less than 12%. The extraction efficiency for authentic quality control samples was determined to be 83% after repeated extractions of the same samples. CONCLUSIONS: This paper reports the first quantitative method for the determination of efavirenz in hair to be developed in South Africa. The validated method allowed for the successful monitoring of efavirenz in hair collected from HIV-infected patients as part of a clinical study.
Assuntos
Benzoxazinas/análise , Monitoramento de Medicamentos/métodos , Cabelo/química , Inibidores da Transcriptase Reversa/análise , Espectrometria de Massas em Tandem/métodos , Alcinos , Cromatografia Líquida/métodos , Ciclopropanos , Feminino , HIV/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Humanos , Limite de Detecção , MasculinoRESUMO
The development of new and safe anti-human immunodeficiency virus (anti-HIV) drugs has been an urgent task for medical research recently. Herein, based on the norm-index descriptors proposed in this work and previous works, a couple of models were developed for investigating the quantitative structure-activity/toxicity relationship (QSAR/QSTR) of dual-target anti-HIV integrase (IN) and reverse transcriptase (RT) inhibitors. The validation results proved that the developed models were stable and reliable, both in statistical quality and predictive capacity. Moreover, potential dual-target inhibitors with high activity and low toxicity were deduced from the developed models; molecular docking results indicated that these inhibitors could interact with some important residues of HIV IN and RT through H-bonding. Accordingly, the norm indexes descriptors proposed by this work might be helpful for the research and development of dual-target anti-HIV drugs.
Assuntos
Inibidores de Integrase de HIV/química , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Inibidores da Transcriptase Reversa/química , Inibidores de Integrase de HIV/análise , Humanos , Inibidores da Transcriptase Reversa/análiseRESUMO
AIM: The non-nucleoside reverse transcriptase inhibitor efavirenz is one of the most prescribed antiretroviral therapeutics. Efavirenz-containing therapy has become associated with the occurrence of CNS side effects, including sleep disturbances, depression and even psychosis. RESULTS: The investigation of efavirenz distribution required the development of a versatile and sensitive method. In addition to plasma, quantification was required in brain tissue and phosphate-buffered saline. The assay presented here was linear from 1.9 to 500 ng/ml. Accuracy and precision ranged between 93.7 and 99.5%, and 1.5 and 5.6%, respectively. DISCUSSION: The method developed here represents a versatile, sensitive and easy-to-use assay. The assay has been applied to in vitro and in vivo samples demonstrating reliable efavirenz quantification in multiple matrices.
Assuntos
Benzoxazinas/análise , Cromatografia Líquida de Alta Pressão , Testes de Química Clínica/métodos , Inibidores da Transcriptase Reversa/análise , Espectrometria de Massas em Tandem , Alcinos , Animais , Benzoxazinas/sangue , Benzoxazinas/normas , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/normas , Ciclopropanos , Masculino , Controle de Qualidade , Ratos , Ratos Wistar , Inibidores da Transcriptase Reversa/sangue , Inibidores da Transcriptase Reversa/normas , Espectrometria de Massas em Tandem/normasRESUMO
World Health Organization estimates that 34 million individuals globally are living with Human Immunodeficiency Virus (HIV). Doravirine is a non-nucleoside reverse transcriptase inhibitors (NNRTI) being evaluated by Merck for the treatment of HIV-1 infection. Drug regulation authorities require the purity of a pharmaceutical to be fully defined. This is important to ensure that the pharmacological and toxicological effects are truly those of the drug substances and not because of the impurities. Thus, understanding the drug impurity profiles is critical to the safety and potency assessment of the drug candidate for clinical trials. The impurity characterization can also provide useful information for critical assessment of pharmaceutical processes. Advances in mass spectrometry instrumentation and methods allow the identification of impurities in pharmaceuticals with a minimum of sample material and increased sensitivity. In this study, a rapid and sensitive method was developed for the structural determination of the major impurities of doravirine. The study utilizes ultra performance liquid chromatography-high-resolution-tandem mass spectrometry (UHPLC-HRMS/MS) techniques to perform structure elucidation of the unknown structures. This approach has significant impact on impurity structural elucidation, and a total of five trace-level impurities of doravirine were characterized using the developed method. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Fármacos Anti-HIV/análise , Piridonas/análise , Inibidores da Transcriptase Reversa/análise , Triazóis/análise , Cromatografia Líquida de Alta Pressão , Contaminação de Medicamentos , Infecções por HIV/tratamento farmacológico , Humanos , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, is a drug widely used in the treatment of Acquired Immunodeficiency Syndrome (AIDS). The evaluation of NVP stability is of fundamental importance in order to guarantee drug product efficacy, safety and quality. In this study, NVP active pharmaceutical ingredient (API) and tablets were subjected to a detailed study of forced degradation, employing several degrading agents (acid, alkaline, water, metal ions, humidity, heat, light and oxidation agents). In order to determine NVP and the degradation products formed, a stability-indicating UHPLC method using fused core column was developed and validated. The separation was carried out using a Poroshell 120C18 column (100×2.1mm i.d.; 2.7µm particle size) and the mobile phase was composed of acetonitrile and water in a gradient elution, at a flow rate of 0.2ml/min. Chemical structures and mechanisms for the formation of three degradation products were proposed by means of LC/MS-MS. Also, NVP degradation kinetic was studied and its order of degradation evaluated. NVP was degraded in acidic and oxidative conditions and the degradation profile for NVP tablets and API were similar. The stability-indicating method proved to be selective for NVP and its degradation products. Calibration curve was linear in the range of 8-48µg/ml and the method showed to be precise, accurate and robust for both NVP API and tablets, with detection and quantification limits of 0.092µg/ml and 0.174µg/ml, respectively.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Nevirapina/análise , Inibidores da Transcriptase Reversa/análise , Espectrometria de Massas em Tandem/métodos , Calibragem , Contaminação de Medicamentos , Estabilidade de Medicamentos , Temperatura Alta , Umidade , Cinética , Nevirapina/química , Nevirapina/normas , Oxirredução , Reprodutibilidade dos Testes , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/normas , ComprimidosRESUMO
A novel virtual screening approach is implemented herein, which is a further improvement of our previously published "target-bound pharmacophore modeling approach". The generated pharmacophore library is based only on highly contributing amino acid residues, instead of arbitrary pharmacophores, which are most commonly used in the conventional approaches in literature. Highly contributing amino acid residues were distinguished based on free binding energy contributions obtained from calculation from molecular dynamic (MD) simulations. To the best of our knowledge; this is the first attempt in the literature using such an approach; previous approaches have relied on the docking score to generate energy-based pharmacophore models. However, docking scores are reportedly unreliable. Thus, we present a model for a per-residue energy decomposition, constructed from MD simulation ensembles generating a more trustworthy pharmacophore model, which can be applied in drug discovery workflow. This work is aimed at introducing a more rational approach to the field of drug design, rather than comparing the validity of this approach against those previously reported. We recommend additional computational and experimental work to further validate this approach. This approach was used to screen for potential reverse transcriptase inhibitors using the pharmacophoric features of compound GSK952. The complex was subjected to docking, thereafter, MD simulation confirmed the stability of the system. Experimentally determined inhibitors with known HIV-reverse transcriptase inhibitory activity were used to validate the protocol. Two potential hits (ZINC46849657 and ZINC54359621) showed a significant potential with regard to free binding energy. Reported results obtained from this work confirm that this new approach is favorable in the future of the drug design industry.
Assuntos
Fármacos Anti-HIV/análise , Fármacos Anti-HIV/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV/efeitos dos fármacos , HIV/enzimologia , Inibidores da Transcriptase Reversa/análise , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/química , Transcriptase Reversa do HIV/metabolismo , Ligantes , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Inibidores da Transcriptase Reversa/químicaRESUMO
Long-term exposure to efavirenz (EFV) measured in hair samples may predict response to antiretroviral treatment (ART). Polymorphisms in CYP2B6 are known to alter EFV levels. The aim of this study was to assess the relationship between CYP2B6 genotype, EFV levels measured in hair, and virological outcomes on ART in a real-world setting. We measured EFV levels in hair from HIV-positive South African females who had been receiving EFV-based treatment for at least 3 months from the South African Black (SAB) (n = 81) and Cape Mixed Ancestry (CMA) (n = 53) populations. Common genetic variation in CYP2B6 was determined in 15 individuals from each population using bidirectional Sanger sequencing. Prioritized variants (n = 16) were subsequently genotyped in the entire patient cohort (n = 134). The predictive value of EFV levels in hair and selected variants in CYP2B6 on virological treatment outcomes was assessed. Previously described alleles (CYP2B6*2, CYP2B6*5, CYP2B6*6, CYP2B6*17, and CYP2B6*18), as well as two novel alleles (CYP2B6*31 and CYP2B6*32), were detected in this study. Compared to noncarriers, individuals homozygous for CYP2B6*6 had â¼109% increased EFV levels in hair (p = .016) and CYP2B6*18 heterozygotes demonstrated 82% higher EFV hair levels (p = .0006). This study confirmed that alleles affecting CYP2B6 metabolism and subsequent EFV exposure are present at significant frequencies in both the SAB and CMA populations. Furthermore, this study demonstrated that the use of hair samples for testing EFV concentrations may be a useful tool in determining long-term drug exposure in resource-limited countries.
Assuntos
Benzoxazinas/análise , Benzoxazinas/farmacocinética , Citocromo P-450 CYP2B6/genética , Infecções por HIV/tratamento farmacológico , Cabelo/química , Inibidores da Transcriptase Reversa/análise , Inibidores da Transcriptase Reversa/farmacocinética , Adulto , Idoso , Alcinos , Benzoxazinas/administração & dosagem , Ciclopropanos , Etnicidade , Feminino , Humanos , Pessoa de Meia-Idade , Inibidores da Transcriptase Reversa/administração & dosagem , Análise de Sequência de DNA , África do Sul , Adulto JovemRESUMO
A detailed impurity study was conducted on tenofovir, (R)-({[1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid (1), which is the key starting material of manufacturing the active pharmaceutical ingredient (API) tenofovir disoproxil fumarate (2) based on a recently reported procedure. The major impurities generated in the production of tenofovir (1) have been synthesized, characterized and confirmed. The possible formation mechanisms of these impurities were elucidated herein, which would help to understand the process. In addition, this work will also improve the quality control during manufacturing tenofovir and tenofovir disoproxil fumarate (2).
Assuntos
Adenina/análogos & derivados , Organofosfonatos/análise , Inibidores da Transcriptase Reversa/análise , Adenina/análise , Adenina/síntese química , Contaminação de Medicamentos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Organofosfonatos/síntese química , Compostos Organofosforados/análise , Controle de Qualidade , Inibidores da Transcriptase Reversa/síntese química , TenofovirRESUMO
Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.
Assuntos
Descoberta de Drogas/métodos , HIV-1/enzimologia , Pró-Fármacos/isolamento & purificação , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Espectroscopia de Ressonância Magnética , Pró-Fármacos/análise , Inibidores da Transcriptase Reversa/análise , Ribonuclease H/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas , Replicação Viral/efeitos dos fármacosRESUMO
A sensitive and selective high-performance liquid chromatographic (HPLC) method for determination of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor in rat plasma, was developed and validated. Chromatographic separation of W-1 and megestrol acetate (internal standard) was achieved on a reversed-phase C18 column at 25°C. The mobile phase was consisted of acetonitrile-water (60:40, v/v) and pumped at a flow rate of 1.0 mL/min. The ultraviolet (UV) detector was set at the absorption wavelength of 284 nm. The calibration curve for W-1 was linear over the concentration range of 0.01-8 µg/mL and the lower limit of quantification was 10 ng/mL. The intra- and inter-day precision and accuracy were <8.9 and 5.3%, respectively. The extraction recoveries ranged from 97.9 to 101.6%. The validated HPLC method was successfully applied to a pharmacokinetic study of W-1 in rats.
Assuntos
Inibidores da Transcriptase Reversa/farmacocinética , Uracila/análogos & derivados , Animais , Fármacos Anti-HIV/análise , Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/farmacocinética , Calibragem , Cromatografia Líquida de Alta Pressão/métodos , Limite de Detecção , Masculino , Acetato de Megestrol/análise , Ratos Sprague-Dawley , Inibidores da Transcriptase Reversa/análise , Inibidores da Transcriptase Reversa/sangue , Sensibilidade e Especificidade , Raios Ultravioleta , Uracila/análise , Uracila/farmacocinéticaRESUMO
The measurement of antiretroviral concentrations in hair is emerging as an important technology to objectively quantify adherence to combination antiretroviral therapy. Hair levels of antiretrovirals are the strongest independent predictor of virologic success in large prospective cohorts of HIV-infected patients and surpass self-report in predicting outcomes. Hair is easy to collect and store, but validated methods to analyze antiretroviral levels in hair using liquid chromatography tandem mass spectrometry (LC-MS/MS) are expensive. We report here on the development of a thin-layer chromatography (TLC) assay for the semiquantitative analysis of nevirapine in hair. TLC assay results from 11 samples were consistent with results using LC-MS/MS [Spearman correlation coefficient 0.99 (95% CI 0.95-0.996)]. This simple, low-cost method of analyzing nevirapine concentrations in hair may provide a novel monitoring tool for antiretroviral adherence in resource-limited settings and merits further study in clinical settings.
Assuntos
Fármacos Anti-HIV/análise , Cromatografia em Camada Fina/métodos , Adesão à Medicação/estatística & dados numéricos , Nevirapina/análise , Infecções por HIV/tratamento farmacológico , Cabelo/química , Humanos , Estudos Prospectivos , Inibidores da Transcriptase Reversa/análise , Resultado do TratamentoRESUMO
OBJECTIVE: To determine the prevalence rates of nucleotide reverse-transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI)TDRs among HIV-1 ART-naÑve patients in Hunan province using the ultra deep sequencing (UDS) technique. METHODS: ART-naÑve subjects diagnosed in Hunan between 2010 and 2011 were evaluated by both UDS technique and Sanger sequencing techniques, to the 1% variant level. Mutations were scored using the Stanford HIVdb algorithm to infer the status on drug resistance. RESULTS: UDS method was performed on 90 ART-naÑve subjects that seeking service of care, in Hunan. In total, 42.2% (38/90) of the subjects showed major NRTI or nonnucleoside reverse transcriptase inhibitor NNRTI TDRs by UDS technique, at a HIV variant frequency level of ≥1%, 15.6% (14/90) showed NRTI TDR, 16.7% (15/90) showed a major NNRTI TDR and 10% (9/90) were both resistant to NRTI and NNRTI when variants were analyzed by Stanford HIVdb. CONCLUSION: ART-naÑve subjects from Hunan province, which had been predominately infected by subtype AE, would frequently possess HIV variants with NRTI/NNRTI TDRs that would affect the use of first line ART in the region, identified by the UDS technique. Further studies were needed to describe the prevalence of TDRs and to gather related information.
Assuntos
Farmacorresistência Viral/genética , Infecções por HIV/virologia , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Inibidores da Transcriptase Reversa/análise , Fármacos Anti-HIV/uso terapêutico , China , Infecções por HIV/tratamento farmacológico , Humanos , Mutação , PrevalênciaRESUMO
A simple micellar liquid chromatography (MLC) method has been developed and validated for use in stability indicating studies of lamivudine and its carbonate derivatives with proved activity against human immunodeficiency and hepatitis B viruses (HIV and HBV, respectively), in simulated gastric (SGF) and intestinal (SIF) fluids samples. The optimized method involves a C18 column thermostated at 30°C, UV detection at 272 nm, a flow rate of 1.0 mL min(-1) and a micellar mobile phase composed by 0.15M sodium dodecyl sulphate (SDS) - 4% (v/v) 1-butanol - 0.01 M KH2PO4-Na2HPO4 (pH 7), using zidovudine (AZT) as internal standard. Validation under Food and Drug Administration (FDA) guideline of the analytical parameters include: linearity (r(2)>0.9996), LODs (1.6 × 10(-7)-6.9 × 10(-6)M) and LOQ (1 × 10(-5)M), intra (0.02-1.48%) and inter-day precision (0.04-1.66%) expressed as relative standard deviation (R.S.D.), and robustness parameters (less than 1.98%). Using this method, recoveries ranging from 92.9 to 119% were obtained for the eight substances. Thus, this method provides a simple, sensitive, accurate and precise assay for the determination of all compounds that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, we evaluated the stability of carbonates of lamivudine in buffer pH 1.2 and 6.8; SGF (pH 1.2) and SIF one (pH 6.8), all as indicated in United States Pharmacopeia (USP) 32. Finally, this chromatographic method was applied to stability studies which resulted in all the compounds following a pseudo-first-order kinetics, and in the determination of its kinetic constant and half-life time.
Assuntos
Antivirais/análise , HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Lamivudina/análise , Inibidores da Transcriptase Reversa/análise , Antivirais/farmacologia , Cromatografia Líquida de Alta Pressão , Humanos , Lamivudina/farmacologia , Limite de Detecção , Reprodutibilidade dos Testes , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
Dual and triple combinations of antiretroviral drugs are a cornerstone of human immunodeficiency virus type 1 (HIV-1) treatment. Supercritical fluid chromatography (SFC) and reverse phase liquid chromatography (RPLC) methods have been developed for the impurity profiling of a prototype combination tablet containing three such drugs: lamivudine, BMS-986001 and efavirenz. Separation by SFC was achieved using a Princeton 2-ethyl pyridine stationary phase and a mobile phase B consisting of methanol with 10 mM ammonium acetate and 0.1% isopropyl amine. This combination of mobile phase additives was required for both the separation of minor components and to minimize peak tailing of the active pharmaceutical ingredients (APIs). Separation by RPLC was achieved using a Discovery HSF5 stationary phase and a mobile phase consisting of 10 mM ammonium acetate, pH 5.5 and methanol. Mobile phase gradient elution was employed in each case to elute components with a wide range of polarities. Both these methods were found to have advantages and disadvantages. Out of the three APIs and 13 possible impurity/degradation products selected, all were resolved by RPLC. By SFC, 15 peaks were resolved with one co-eluting pair and a high degree of orthogonality was achieved relative to RPLC. A more even distribution of peaks across the separation space, a non-sloping baseline and fewer system peaks were significant advantages associated with the SFC method. Particular attention had to be paid to optimizing the reverse phase diluent strength/initial mobile phase composition to avoid distortion of the peak shapes for early eluting components. This was not an issue with SFC, as the diluent of choice (methanol) was also the solvent of choice (in combination with ≤20% water) for the dissolution of the triple combination tablet. As with RPLC, SFC was found to exhibit the required sensitivity for successful quantitation of potential impurities/degradation products at the 0.05-0.1 area% level.
Assuntos
Fármacos Anti-HIV/análise , Benzoxazinas/análise , Cromatografia Líquida/métodos , Cromatografia com Fluido Supercrítico/métodos , Lamivudina/análise , Inibidores da Transcriptase Reversa/análise , Timidina/análogos & derivados , Alcinos , Fármacos Anti-HIV/administração & dosagem , Benzoxazinas/administração & dosagem , Ciclopropanos , Combinação de Medicamentos , Concentração de Íons de Hidrogênio , Lamivudina/administração & dosagem , Comprimidos , Temperatura , Timidina/administração & dosagem , Timidina/análiseRESUMO
Vaginal rings are currently being developed for the long-term (at least 30 days) continuous delivery of microbicides against human immunodeficiency virus (HIV). Research to date has mostly focused on devices containing a single antiretroviral compound, exemplified by the 25mg dapivirine ring currently being evaluated in a Phase III clinical study. However, there is a strong clinical rationale for combining antiretrovirals with different mechanisms of action in a bid to increase breadth of protection and limit the emergence of resistant strains. Here we report the development of a combination antiretroviral silicone elastomer matrix-type vaginal ring for simultaneous controlled release of dapivirine, a non-nucleoside reverse transcriptase inhibitor, and maraviroc, a CCR5-targeted HIV-1 entry inhibitor. Vaginal rings loaded with 25mg dapivirine and various quantities of maraviroc (50-400mg) were manufactured and in vitro release assessed. The 25mg dapivirine and 100mg maraviroc formulation was selected for further study. A 24-month pharmaceutical stability evaluation was conducted, indicating good product stability in terms of in vitro release, content assay, mechanical properties and related substances. This combination ring product has now progressed to Phase I clinical testing.
Assuntos
Antagonistas dos Receptores CCR5/química , Dispositivos Anticoncepcionais Femininos , Cicloexanos/química , Sistemas de Liberação de Medicamentos , Pirimidinas/química , Inibidores da Transcriptase Reversa/química , Elastômeros de Silicone/química , Triazóis/química , Antagonistas dos Receptores CCR5/administração & dosagem , Antagonistas dos Receptores CCR5/análise , Varredura Diferencial de Calorimetria , Cicloexanos/administração & dosagem , Cicloexanos/análise , Preparações de Ação Retardada/análise , Preparações de Ação Retardada/química , Combinação de Medicamentos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Infecções por HIV/prevenção & controle , Transcriptase Reversa do HIV/antagonistas & inibidores , Temperatura Alta/efeitos adversos , Maraviroc , Fenômenos Mecânicos , Pirimidinas/administração & dosagem , Pirimidinas/análise , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/análise , Solubilidade , Triazóis/administração & dosagem , Triazóis/análiseRESUMO
In quite a few diseases, drug resistance due to target variability poses a serious problem in pharmacotherapy. This is certainly true for HIV, and hence, it is often unknown which drug is best to use or to develop against an individual HIV strain. In this work we applied 'proteochemometric' modeling of HIV Non-Nucleoside Reverse Transcriptase (NNRTI) inhibitors to support preclinical development by predicting compound performance on multiple mutants in the lead selection stage. Proteochemometric models are based on both small molecule and target properties and can thus capture multi-target activity relationships simultaneously, the targets in this case being a set of 14 HIV Reverse Transcriptase (RT) mutants. We validated our model by experimentally confirming model predictions for 317 untested compound-mutant pairs, with a prediction error comparable with assay variability (RMSE 0.62). Furthermore, dependent on the similarity of a new mutant to the training set, we could predict with high accuracy which compound will be most effective on a sequence with a previously unknown genotype. Hence, our models allow the evaluation of compound performance on untested sequences and the selection of the most promising leads for further preclinical research. The modeling concept is likely to be applicable also to other target families with genetic variability like other viruses or bacteria, or with similar orthologs like GPCRs.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Moleculares , Proteômica/métodos , Inibidores da Transcriptase Reversa/análise , Inibidores da Transcriptase Reversa/química , Sequência de Aminoácidos , Sítios de Ligação , Bases de Dados como Assunto , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , Humanos , Ligantes , Dados de Sequência Molecular , Mutação/genética , Reprodutibilidade dos Testes , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
Calophyllum species are sources of calanolides, which inhibit human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The hexane extract of the leaves from C. brasiliense collected in Soconusco, State of Chiapas, Mexico, analyzed by HPLC showed to contain apetalic acid, calanolides B, and C. It showed potent anti-HIV-1 RT inhibition (IC(50)=20.2 µg/ml), but was not toxic in mice (LD(50)=1.99 g/kg). The histological study of the mice treated at the highest dose revealed no alteration on hepatocytes, and an increase in the number of spleen megakaryocytes. These results suggest this extract is suitable to continue studies for developing a phytodrug against HIV-1.
Assuntos
Calophyllum/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Isoflavonas/farmacologia , Fitoterapia , Extratos Vegetais/farmacologia , Piranocumarinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Animais , Calophyllum/efeitos adversos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/enzimologia , Hepatócitos/efeitos dos fármacos , Humanos , Isoflavonas/efeitos adversos , Isoflavonas/análise , Masculino , Megacariócitos/efeitos dos fármacos , México , Camundongos , Extratos Vegetais/efeitos adversos , Extratos Vegetais/química , Folhas de Planta , Piranocumarinas/efeitos adversos , Piranocumarinas/análise , Inibidores da Transcriptase Reversa/efeitos adversos , Inibidores da Transcriptase Reversa/análise , Baço/efeitos dos fármacosRESUMO
This paper describes a stripping method for the determination of nevirapine at the submicromolar concentration levels. The method is based on controlled adsorptive accumulation of nevirapine at thin-film mercury electrode, followed by a linear cyclic scan voltammetry measurement of the surface species. Optimal experimental conditions include a 2.0 x 10(-3) mol L(-1) NaOH solution (supporting electrolyte), an accumulation potential of -0.20 V, and a scan rate of 100 mV s(-1). The response of nevirapine is linear over the concentration range 0.01-0.14 ppm. For an accumulation time of 6 minutes, the detection limit was found to be 0.87 ppb (3.0 x 10(-9) mol L(-1)). More convenient methods to measure the nevirapine in presence of the efavirenz, acyclovir, didanosine, indinavir, nelfinavir, saquinavir, lamivudine, zidovudine and metals ions were also investigated. The utility of this method is demonstrated by the presence of nevirapine together with ATP or DNA.
Assuntos
Álcalis/química , Eletrodos , Eletrólitos/química , Mercúrio/química , Nevirapina/análise , Inibidores da Transcriptase Reversa/análise , AdsorçãoRESUMO
A novel and robust screening method for the determination of the non-nucleoside reverse transcriptase inhibitor, efavirenz (EFV), in human saliva has been developed and validated based on high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). Sample preparation of the saliva involved solid-phase extraction (SPE) on C18 cartridges. The analytes were separated by high performance liquid chromatography (Phenomenex Kinetex C18, 150mm×3mm internal diameter, 2.6µm particle size) and detected with tandem mass spectrometry in electrospray positive ionization mode with multiple reaction monitoring. Gradient elution with increasing methanol (MeOH) concentration was used to elute the analytes, at a flow-rate of 0.4mL/min. The total run time was 8.4min and the retention times for the internal standard (reserpine) was 5.4min and for EFV was 6.5min. The calibration curves showed linearity (r(2), 0.989-0.992) over the concentration range of 3.125-100µg/L. Mean intra- and inter-assay relative standard deviation, accuracy, mean extraction recovery, limit of detection (LOD) and limit of quantification (LOQ) were 0.46-9.43%, 80-120%, 60% (±7.95), 1.84 and 6.11µg/L respectively. The working range was defined as 6.25-100µg/L. This novel LC-MS/MS assay is suitable for reliable detection of low EFV concentrations in saliva and can be used as a screening tool for monitoring EFV compliance.