Evaluation of three alum-precipitated Aeromonas hydrophila vaccines administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella: immunokinetics, immersion challenge and histopathology

Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was observed in different organs of vaccinated fish whereas minor deteriorative changes were found in fish vaccinated with higher concentrations of the vaccine.

Simultaneous Estimation of Sacubitril and Valsartan Combination of Drug in Tablet Dosage Form Using Hydrotropy by UV Spectrophotometry

Sacubitril/valsartan, traded under the brand name Entresto between others, is a fixed-dose combination medication for use heart failure. Sacubitril is a neprilysin inhibitor (A prodrug) and is used in combination with valsartan to reduce the risk of cardiovascular events in patients with chronic heart failure. It is anti - hypertensive drug. Valsartan is an Angiotensin Receptor Blocker (ARB) that may be used to treat a variety of cardiac conditions including hypertension, diabetic nephropathy and heart failure. Two UV-spectrophotometric methods have been developed and validated for simultaneous estimation of Sacubitril and Valsartan in a tablet dosage form. The first method employed solving of simultaneous equations based on the measurement of absorbance at two wavelengths, 226.0 nm and 254.0 nm, 𝜆max for Sacubitril and Valsartan, respectively. The second method was absorbance ratio method, which involves formation of Q-absorbance equation at 240 nm (isoabsorptive point) and also at 254 nm (𝜆max of Valsartan). The methods were found to be linear between the range of 4-12 𝜇g/mL for Sacubitril and 2-10 𝜇g/mL for Valsartan using Methanol as solvent. The mean percentage recovery was found to be 96.68%and 101.89% for the simultaneous equation method and 100.2% and 104.53% for the absorbance ratio method, for sacubitril and valsartan respectively. It could be concluded from the results obtained in the present investigation that the two methods for simultaneous estimation of sacubitril and valsartan in tablet dosage form are simple, rapid, accurate, precise and economical and can be used, successfully, in the quality control of pharmaceutical formulations and other routine laboratory analysis. The reviewed highlights various analytical techniques such as high-performance liquid chromatography (HPLC), ultra- performance liquid chromatography (UPLC), UV Spectroscopy, high per-formance thin layer chromatography (HPTLC), liquid chromatography coupled to tandem mass spectrometry (LC- MS), RP-HPLC and other chromatographic method used. The combination of these drugs with different method was examine and the commonly use of the drugs in hypertensive.

FORMULATION AND DEVELOPMENT OF FIXED-DOSE COMBINATION OF BI-LAYER TABLETS OF EFAVIRENZ, LAMIVUDINE AND TENOFOVIR DISOPROXIL FUMARATE TABLETS 600 MG/300 MG/300 MG

Objective: This study is to formulate bi-layer tablet as a multidrug regimen against each reference listed drugs of Brand SUSTIVA® (efavirenz tablets 600 mg), EPIVER®(lamivudine tablets 300 mg), and VIREAD®(tenofovir disoproxil tablets 300 mg) to treat human immunodeficiency virus (HIV) infections. Which provides highly active antiretroviral therapy to provide effective treatment. Methods: Bilayer formulation was developed with each blend of layer-I (efavirenz) and layer-II (lamivudine and tenofovir disoproxil fumarate) through wet granulation process and roller compaction process, respectively. Further, both layers were compressed by using bi-layer compression followed by film coating. Layer-I and II formulations were developed by using various concentrations of diluents, surfactants, and disintegrants to improve the solubility of efavirenz and improve the flowability and uniformity of layer-II. Finally, the optimum formulation was developed to compare the in vitro dissolution with each branded formulation. Results: Drug-excipients interaction results revealed that the mixtures of three drug substances in 50 °C/75 % relative humidity (RH) resulted in an increase in tenofovir IMP-E and the highest unknown impurity was significantly increased and additionally decreased tenofovir assay in the presence of efavirenz. Sodium lauryl sulfate is very critical and it acts as a wetting agent and increases the solubility of efavirenz, and directly influences the dissolution of a drug product. Microcrystalline and croscarmellose sodium have a chance to affect the dissolution and friability of tenofovir. Powdered cellulose was acting as a diluent and flow property of the lamivudine part and it also affects the uniformity and dissolution. So, these ranges were optimized. X-ray diffraction (XRD) indicates there are no polymorphic changes for the optimized formulation and there is no interaction between the three active substances, and finally, in vitro dissolution results for the optimized formulation against the reference drugs. Conclusion: Optimum formulation yielded consistent drug release against each branded drug to treat human immunodeficiency virus (HIV1) infections. This formulation is robust and easily scale up for the next stage.

Pembrolizumab plus azacitidine in patients with chemotherapy refractory metastatic colorectal cancer: a single-arm phase 2 trial and correlative biomarker analysis

Background DNA mismatch repair proficient (pMMR) metastatic colorectal cancer (mCRC) is not responsive to pembrolizumab monotherapy. DNA methyltransferase inhibitors can promote antitumor immune responses. This clinical trial investigated whether concurrent treatment with azacitidine enhances the antitumor activity of pembrolizumab in mCRC. Methods We conducted a phase 2 single-arm trial evaluating activity and tolerability of pembrolizumab plus azacitidine in patients with chemotherapy-refractory mCRC (NCT02260440). Patients received pembrolizumab 200 mg IV on day 1 and azacitidine 100 mg SQ on days 1–5, every 3 weeks. A low fixed dose of azacitidine was chosen in order to reduce the possibility of a direct cytotoxic effect of the drug, since the main focus of this study was to investigate its potential immunomodulatory effect. The primary endpoint of this study was overall response rate (ORR) using RECIST v1.1., and secondary endpoints were progression-free survival (PFS) and overall survival (OS). Tumor tissue was collected pre- and on-treatment for correlative studies. Results Thirty chemotherapy-refractory patients received a median of three cycles of therapy. One patient achieved partial response (PR), and one patient had stable disease (SD) as best confirmed response. The ORR was 3%, median PFS was 1.9 months, and median OS was 6.3 months. The combination regimen was well-tolerated, and 96% of treatment-related adverse events (TRAEs) were grade 1/2. This trial was terminated prior to the accrual target of 40 patients due to lack of clinical efficacy. DNA methylation on-treatment as compared to pre-treatment decreased genome wide in 10 of 15 patients with paired biopsies and was significantly lower in gene promoter regions after treatment. These promoter demethylated genes represented a higher proportion of upregulated genes, including several immune gene sets, endogenous retroviral elements, and cancer-testis antigens. CD8+ TIL density trended higher on-treatment compared to pre-treatment. Higher CD8+ TIL density at baseline was associated with greater likelihood of benefit from treatment. On-treatment tumor demethylation correlated with the increases in tumor CD8+ TIL density. Conclusions The combination of pembrolizumab and azacitidine is safe and tolerable with modest clinical activity in the treatment for chemotherapy-refractory mCRC. Correlative studies suggest that tumor DNA demethylation and immunomodulation occurs. An association between tumor DNA demethylation and tumor-immune modulation suggests immune modulation and may result from treatment with azacitidine. Trial registration ClinicalTrials.gov, NCT02260440. Registered 9 October 2014, https://clinicaltrials.gov/ct2/show/NCT02260440.

Safety, antitumor activity and biomarkers of sugemalimab in Chinese patients with advanced solid tumors or lymphomas: results from the first-in-human phase 1 trial

Background This first-in-human phase 1 trial is to evaluate the safety, pharmacokinetics, preliminary efficacy, and biomarkers of sugemalimab, a full-length, fully human anti-PD-L1 monoclonal antibody, in Chinese patients with advanced malignancies. Methods Eligible patients with unresectable advanced or metastatic solid tumors or lymphomas were enrolled in phase 1a to receive sugemalimab following a modified 3 + 3 design. The primary endpoints included safety, tolerability, and the recommended Phase 2 dose (RP2D). In phase 1b, patients with 7 selected types of tumor received sugemalimab at the RP2D alone (monotherapy cohorts) or in combination with standard-of-care (SOC) chemotherapy (combination cohorts). The primary endpoint of phase 1b was investigator-assessed objective response rate (ORR). Results As of 19 February 2020, 29 and 178 patients were treated in phase 1a and 1b, respectively. No dose-limiting toxicities were observed in phase 1a, and the RP2D of sugemalimab was determined as 1200 mg fixed dose once every 3 weeks. Sugemalimab-related adverse events (AEs) were mostly (75.9%) grade 1–2 in phase 1a. Antitumor activity was observed across dose levels with an ORR of 24.1%. In phase 1b, 15.9% and 40.4% of patients in the monotherapy and combination cohorts, respectively, reported grade 3–5 sugemalimab-related AEs. Promising efficacy was observed in all combination cohorts, with ORRs ranging from 47.6 to 75.0%. Exploratory biomarker analysis did not indicate significant differences in responses at different PD-L1 expression/tumor mutation burden levels. Conclusions Sugemalimab was well-tolerated and showed promising antitumor activity as monotherapy or in combination with SOC chemotherapy in advanced malignancies. This trial was registered with ClinicalTrials.gov on Oct 18, 2017, number NCT03312842.