A validated stability indicating LC-MS compatible RP-HPLC assay and dissolution methods for marketed formulation Stribild

Objective: The prime objective of the current work is to develop a simple, rapid, efficient, economical and stability indicating LC-MS (liquid chromatography–mass spectroscopy) compatible RP - HPLC (reverse phase – high performance liquid chromatography) method for the analysis of emtricitabine (EMT), tenofovir disoproxil fumarate (TDF), cobicistat (COB) and elvitegravir (ELV) in bulk, marketed formulation (Stribild) and in In-vitro dissolution method. Method: The chromatography was achieved on Unisol C18 column (250 × 4.0 mm, 3 µ) with a mobile phase combination of acetate buffer (adjusted with dilute glacial acetic acid to pH 4) and acetonitrile in gradient mode at a flow rate of 1mL/min and the detection was performed at 260 nm using PDA (photo diode array) detector. Forced degradation studies were performed and the % degradation under various stress conditions was calculated. The developed RP-HPLC method was applied for Stribild tablets to study the dissolution profile. Results: The retention times for emtricitabine, tenofovir disoproxil fumarate, cobicistat and elvitegravir were 5.7, 12.1, 16.3 and 19.4 min respectively. The % degradation was below 20% which is within the limits. The percent drug release was found to meet USP specification, i.e. not less than 80% of amount of labeled drug EMT, TDF, COB and ELV dissolved in 30min. Conclusion: The method was validated as per ICH guidelines and all the validation parameters were within the compendial requirements. The proposed method can be successfully adopted for the analysis of Stribild tablets in pharmaceutical industries. Keywords: Stribild, emtricitabine, tenofovir disoproxil fumarate, cobicistat, elvitegravi

DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC AND REVERSED PHASE‑HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ‑ PDA METHODS FOR THE ESTIMATION OF ALOGLIPTIN BENZOATE

Objective: To develop and validate simple, rapid, precise, accurate, and economical UV spectrophotometric and reverse phase high performanceliquid chromatographic methods for the estimation of alogliptin benzoate (AGP).Methods: UV spectrophotometric method was performed using UV/Vis double beam spectrophotometer with a spectral bandwidth of 1 nm and1.0 cm matched quartz cells. The maximum absorbance of AGP was observed at 276 nm using methanol as solvent. Reversed phase-high performanceliquid chromatography (RP-HPLC) method was carried out on a Unisol reverse phase C18 column (150 mm × 4.6 mm, 3 μm) with a mobile phasecomposed of methanol and 10 mM ammonium acetate buffer (adjusted to pH 5.0 with glacial acetic acid) in the ratio of 80:20 v/v with a flow rate of0.8 ml/minutes.Results: The linearity of methods was found to be in the range of 5-35 µg/ml (UV) and 20-100 µg/ml (RP-HPLC) and the correlation coefficient was0.999 for both the methods. The regression equations were y = 0.028x + 0.023 (UV) and y = 28,58,942x - 4,33,647 (HPLC). The retention time of AGPwas 2.37 minutes.Conclusion: The proposed methods were validated in terms of linearity, precision, accuracy, specificity, robustness, limit of detection, and limit ofquantitation as per International Conference on Harmonization Q2 R1 guidelines. Thus, the proposed methods are novel, sensitive, and reliable andcan be successfully used for the quantitation of AGP.Keywords: Alogliptin benzoate, UV-visible spectrophotometer, Reversed phase-high performance liquid chromatography, International Conferenceon Harmonization guidelines

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING REVERSE PHASE HIGH‑PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE ESTIMATION OF PIRIBEDIL IN BULK DRUG

ABSTRACTObjective: A simple, precise, fast, economic, accurate, robust, and stability indicating isocratic reverse phase high-performance liquid chromatographicmethod was developed for the analysis of Piribedil.Method: The chromatographic conditions were standardized using Unisol C-18 (4.6 × 150 mm × 3.0 μ) column with UV detection at 244 nm, and themobile phase composed of methanol:acetate buffer-pH 5.0 (85:15, v/v).Results: The retention time of Piribedil was found to be 3.4 minutes. The calibration curve was linear with correlation coefficient of 0.999 over aconcentration range of 20-100 μg/ml with linear regression equationy=74,69,224.37x−39,46,924.90. The limit of detection and limit of quantitationwere found to be 0.04 and 0.4 μg/ml, respectively.Conclusion: The proposed method has been validated according to the ICH guidelines. Piribedil was subjected to stress conditions includingacidic, alkaline, oxidation, photolysis, and thermal degradation. Piribedil is more sensitive to photolytic stress. There are no interfering peaks fromdegradation products at analyte retention time, and thus the method is specific for the estimation of Piribedil in the presence of degradation products.Thus, the proposed method can be successfully applied in the routine quality control and stability samples of Piribedil in bulk drug.Keywords: Piribedil, Validation, Stability indicating, Reverse phase high-performance liquid chromatographic

Simultaneous estimation of telmisartan and atorvastatin calcium in API and tablet dosage form

A new method has been established for the simultaneous estimation of Telmisartan and Atorvastatin calcium by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Telmisartan and Atorvastatin calcium by using boston ODS C18 column, flow rate was 1.0ml/min, mobile Phase consists of methanol:Acetonitrile:buffer in ratio of 35:25:40. Detection wave length was 235nm.The instrument used was SHIMADZU HPLC auto sampler. The retention time of Atorvastatin calcium and Telmisartan was found to be 2.350 and 3.490 minutes respectively. The analytical method was validated according to ICH guidelines (ICH Q2b). The correlation coefficient (r2) was found to be 0.997 and 0.999 for Telmisartan and Atorvastatin calcium respectively. % mean recovery was found to be 100.943% and 100.576% for Telmisartan and Atorvastatin calcium respectively. %RSD for precision on replicate injection was 0.46 and 0.70 for Telmisartan and Atorvastatin calcium respectively. The validation study was found to be precise, robust, and repeatable. Keywords: Telmisartan, Atorvastatin calcium, ICH guidelines, Validation

Hubungan Fungsi Seksual dengan Kecemasan Pasien Pasca-Infark Miokard Akut

Background: Sexual dysfunction and anxiety frequently happens on patients after acute myocardial infarction (AMI) and can affect patients’ quality of life. The purpose of this study was to examine the assosiation of sexual function post-AMI patients with anxiety. Methods: It was a cross-sectional study. Respondents are patients in Integrated Cardiac Clinic of Cipto Mangunkusumo Hospital that meet inclusion and exclusion criteria. They signed informed consent. Sexual function was assessed using International Index of Erectyle Function (IIEF) and anxiety was assessed using Hamilton Anxiety Rating Scale (HAM-A). Results: Post-AMI patients had erectile dysfunction (82.5%), orgasm dysfunction (72.5%) and libido dysfunction (93.8%). Respondents expressed sexual intercourse dissatisfaction (97.5%) and overall dissatisfaction (90%). The proportion of post-AMI anxiety was 52.5%. There was no assosiation between sexual function post-AMI with anxiety. Conclusion: Anxiety and sexual dysfunction post-AMI is a considerable problem. Factors that affect anxiety and sexual dysfunction post-AMI needs to be explored further so that an integrated management guidelines could be proposed.Latar Belakang: Disfungsi seksual dan kecemasan sering dialami oleh pasien pasca-infark miokard akut (acute myocardial infarct, AMI) dan dapat memengaruhi kualitas hidup. Tujuan penelitian ini adalah untuk menguji hubungan fungsi seksual dengan kecemasan pasien pasca-AMI. Metode: Penelitian ini menggunakan desain potong lintang. Responden adalah pasien rawat jalan Poliklinik Jantung Terpadu RS Cipto Mangunkusumo yang memenuhi kriteria inklusi dan eksklusi serta menandatangani informed consent. Fungsi seksual dinilai dengan International Index of Erectyle Function (IIEF) sedangan kecemasan dinilai dengan Hamilton Anxiety Rating Scale (HAM-A). Hasil: Pasien pasca-AMI mengalami disfungsi ereksi (82,5%), disfungsi orgasme (72,5%), dan disfungsi libido (93,8%). Responden pada umumnya menyatakan ketidakpuasan dalam hubungan seksual (97,5%) dan terhadap kehidupan seksual secara keseluruhan (90%). Proporsi kecemasan pasca-AMI adalah 52,5%. Tidak terdapat hubungan antara fungsi seksual dengan kecemasan pasca-AMI. Kesimpulan: Kecemasan dan disfungsi seksual merupakan masalah yang perlu diperhatikan pada pasien pasca-AMI. Faktor-faktor yang memengaruhi kecemasan dan disfungsi seksual pasca-AMI perlu dieksplorasi lebih lanjut sehingga panduan tatalaksana yang terintegrasi dapat disusun dengan baik

Vulnerability to Climate Change: Adaptation Strategies and Layers of Resilience - Agro-climatic analysis of Climatic Trends in Semi-Arid Tropics of India (Andhra Pradesh and Maharashtra). Research Report No. 22

Climate resources inventory using micro-regional weather data is essential to understand and prepare the strategies to cope up with climate change/vulnerability over a region. Semi-arid Tropical (SAT) regions in India are vulnerable to climate extremes and the food grain production of these regions is often affected. Therefore, to prepare viable adaptation strategies to cope with climate risks, we carried out detailed climatic analysis with respect to rainfall and temperature variability in respect of four vulnerable districts, two in Andhra Pradesh (Anantapur and Mahbubnagar) and two in Maharashtra (Akola and Solapur)..

The antibody response to Plasmodium falciparum Merozoite Surface Protein 4: comparative assessment of specificity and growth inhibitory antibody activity to infection-acquired and immunization-induced epitopes

<p>Abstract</p> <p>Background</p> <p>Malaria remains a global public health challenge. It is widely believed that an effective vaccine against malaria will need to incorporate multiple antigens from the various stages of the parasite's complex life cycle. <it>Plasmodium falciparum </it>Merozoite Surface Protein 4 (MSP4) is a vaccine candidate that has been selected for development for inclusion in an asexual stage subunit vaccine against malaria.</p> <p>Methods</p> <p>Nine monoclonal antibodies (Mabs) were produced against <it>Escherichia coli</it>-expressed recombinant MSP4 protein and characterized. These Mabs were used to develop an MSP4-specific competition ELISA to test the binding specificity of antibodies present in sera from naturally <it>P. falciparum</it>-infected individuals from a malaria endemic region of Vietnam. The Mabs were also tested for their capacity to induce <it>P. falciparum </it>growth inhibition <it>in vitro </it>and compared against polyclonal rabbit serum raised against recombinant MSP4</p> <p>Results</p> <p>All Mabs reacted with native parasite protein and collectively recognized at least six epitopes. Four of these Mabs recognize reduction-sensitive epitopes within the epidermal growth factor-like domain found near the C-terminus of MSP4. These sera were shown to contain antibodies capable of inhibiting the binding of the six Mabs indicating infection-acquired responses to the six different epitopes of MSP4. All of the six epitopes were readily recognized by human immune sera. Competition ELISA titres varied from 20 to 640, reflecting heterogeneity in the intensity of the humoral response against the protein among different individuals. The IgG responses during acute and convalescent phases of infection were higher to epitopes in the central region than to other parts of MSP4. Immunization with full length MSP4 in Freund's adjuvant induced rabbit polyclonal antisera able to inhibit parasite growth <it>in vitro </it>in a manner proportionate to the antibody titre. By contrast, polyclonal antisera raised to individual recombinant fragments rMSP4A, rMSP4B, rMSP4C and rMSP4D gave negligible inhibition. Similarly, murine Mabs alone or in combination did not inhibit parasite growth.</p> <p>Conclusions</p> <p>The panel of MSP4-specific Mabs produced were found to recognize six distinct epitopes that are also targeted by human antibodies during natural malaria infection. Antibodies directed to more than three epitope regions spread across MSP4 are likely to be required for <it>P. falciparum </it>growth inhibition <it>in vitro</it>.</p

Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost

Adenovirus-5-Vectored P. falciparum Vaccine Expressing CSP and AMA1. Part B: Safety, Immunogenicity and Protective Efficacy of the CSP Component

Background: A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.\ud \ud Methodology/Principal Findings: NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at 1x1010 particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.\ud \ud Significance: The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection