BRAF inhibitor cessation prior to disease progression in metastatic melanoma: Long-term outcomes.

BACKGROUND: BRAF mutant melanoma treated with BRAF ± MEK inhibitor (targeted therapy) has a high response rate; however, most patients progress (PD). Some patients have durable response, but it is unknown whether treatment can be discontinued in these patients. We describe the recurrence risk, progression patterns, response to subsequent treatment, and survival of patients with advanced melanoma who ceased targeted therapy prior to PD. PATIENTS AND METHODS: Ninety-four patients who ceased targeted therapy without progression were identified retrospectively from 11 centres: 45 were male; 81 V600E; 88 stage IV. Fifty-nine were treated with BRAF + MEK inhibitor, and 35 were treated with BRAF inhibitor alone. Median treatment duration was 29.6 months (range 0.36-77.9). At cessation, 67 were in complete response, 21 in partial response, and 2 stable disease. RESULTS: After median follow-up from cessation of 42.9 months (range 0.0-88.7), 36 (38%) progressed; median time to progression was 4.7 months (range 0.7-56.9); 30 (83%) were asymptomatic and 7 (19%) had new brain metastases. Progression rates did not differ by best response: 34% for complete response and 43% for partial response (P = 0.65). Treatment duration was strongly associated with risk of progression: Median treatment duration was 18.3 (range 0.85-65.7) months for those who progressed and 34.6 (range 0.36-77.9) months for those who did not (P = 0.0004). Twenty-two received further targeted therapy with 15 (68%) responses. CONCLUSION: Risk of progression after cessation of targeted therapy is strongly associated with treatment duration. Response to retreatment with targeted therapy is high.

Development of extended-release metformin core tablet and synergistic coating of sitagliptin for the treatment of type-II diabetes mellitus - A comparative drug release evaluation with reference product.

Diabetes is getting a common disease and is spreading rapidly, affecting 6.6 percent world's population. Metformin HCl is an effective pharmacological treatment for type 2 diabetic patients because of its lowering blood glucose level ability, better weight-neutral effects and reduced risk of hypoglycemia. Nevertheless, gastrointestinal (GI) sensitivities are a concern in many patients using its immediate-release formulations. This study aimed to develop extended-release (ER) formulations to control the release into the body and minimize the dosage-related side effects of metformin and to develop an effective method of coating Sitagliptin immediate-release (IR) formulation over the core tablet. This study evaluated different formulations of Metformin HCl ER tablet using hydrophilic polymers. Different concentrations of Sitagliptin were used to develop immediate release coating. The dissolution profile of the designed formulation was compared with the reference 50/500mg tablet. In vitro dissolution of Metformin HCl (MT5), containing Methocel K4M and Methocel K100 polymer, showed 37.62% release at 1hr, 53.46% at 2hr, 84.75% at 6hr and 94.81% at 10hr. The Sitagliptin (ST8) with 10% excess released 103.64% in 30min. Similarity factor values suggested that developed Metformin ER and Sitagliptin IR formulation were like the reference product.

Activation of the calcium sensing receptor increases claudin-14 expression via a PLC -p38-Sp1 pathway.

Activation of the basolateral calcium sensing receptor (CaSR) in the renal tubular thick ascending limb (TAL) increases claudin-14 expression, which reduces paracellular calcium (Ca2+ ) permeability, thus increasing urinary Ca2+ excretion. However, the upstream signaling pathway contributing to altered CLDN14 gene expression is unknown. To delineate this pathway, we identified and then cloned the CaSR responsive region including the promoter of mouse Cldn14 into a luciferase reporter vector. This 1500 bp sequence upstream of the 5' UTR of Cldn14 variant 1, conferred increased reporter activity in the presence of high extracellular Ca2+ (5 mM) relative to a lower (0.5 mM) concentration. Assessment of Cldn14 reporter activity in response to increased extracellular Ca2+ in the presence or absence of specific inhibitors confirmed signaling through PLC and p38, but not JNK. Overexpression of SP1 attenuated Cldn14 reporter activity in response to CasR signaling. SP1 is expressed in the TAL and phosphorylation was attenuated by CaSR signaling. Finally, activating mutations in the CaSR increased Cldn14 reporter activity while a dominant negative mutation in the CaSR inhibited it. Together, these studies suggest that basolateral activation of the CASR leads to increased Cldn14 expression via a PLC- stimulated p38 pathway that prevents Sp1 mediated repression.

Identification of Preferred Learning Style of Medical and Dental Students Using VARK Questionnaire.

Students have unique preferences when it comes to knowledge acquisition, information processing, retention in memory, and recall. This study is aimed at examining the preferred learning styles of medical and dental undergraduate students of Pakistan. It is also aimed at investigating the influence of gender, preclinical or clinical academic year, and academic record on the preferred learning style. A descriptive cross-sectional study was conducted in Pakistan. The learning styles of undergraduate students were identified using visual, aural, read/write, and kinesthetic (VARK) questionnaire. Students were also asked about their satisfaction towards teaching style of their teachers in institute. Descriptive statistics were done to characterize the learning styles of the students. The Fisher test and chi-square test were used to compare the learning preferences between genders and public/private sector students and among preclinical/clinical years. A p value of less than 0.05 was considered significant. A total of 1473 students participated in the study. Among the students, 39.37% preferred unimodal learning style whereas 60.62% preferred multimodal style. Kinesthetic (K) and visual (V) were the most preferred unimodal styles. The preferred learning styles of female students are aural (A), visual (V), and kinesthetic (K), whereas male students preferred visual (V) and kinesthetic more (K). Students with lower academic record chose unimodal styles in comparison to high achievers that chose multimodal styles. Students of clinical year preferred multimodal and quadmodal styles in comparison to preclinical year students. An alarming 78% of students were dissatisfied with their teacher's instructional style. Majority of students prefer multimodal learning styles over unimodal style. Gender, public/private sector, and academic record have influence on the preference of learning styles. Majority of the students are dissatisfied with their teacher's instructional style and rely on social media platforms for understanding. Academics need to adapt their teaching methods according to student preferences in order to get better graduates.

Antioxidant and lipoxygenase inhibition studies of 4-(4-bromophenyl)-2,2'-bipyridine and its metal complexes Synthesis, characterization and biological screening.

Synthesis of new antioxidants and enzyme inhibitors is an active area of research in pharmaceutical sciences. This can be used for development of new active product ingredients which can prevent body from different diseases. This study comprises of preparation of transition metal complexes using 4-(4-bromophenyl)-2,2'-bipyridine (BPBP) and their screening for antioxidant and lipoxygenase inhibition properties. 4-(4-bromophenyl)-[2,2'-bipyridine]-6-carboxylic acid was used as starting material and its decarboxylation resulted in BPBP. Decarboxylation by conventional heating method was compared with microwave decarboxylation method. Selected metal complexes of the ligand were synthesized with Ruthenium (II), Iron (II) and Cobalt (II) ions. The complexes were characterized using UV, IR, 1H-NMR, ESI-MS and CHNS techniques. It was observed that BPBP acted as a bidentate ligand. The metal to ligand stoichiometry was 1:3 for all the synthesized complexes. The complexes had octahedral structure with C3 symmetry. The antioxidant activity was evaluated using free radical scavenging assay. BPBP showed insignificant antioxidant and lipoxygenase activities while its transition metal complexes showed promising activities. Antioxidant activity of Fe and Co-complexes was found significantly higher than the reference drug used in this study.

Synthesis, lipoxygenase inhibition activity and molecular docking of oxamide derivative.

In this study, a range of oxamide ligands were synthesized by the reaction of amines with oxalyl chloride in basic medium. Spectroscopic and analytical techniques such as IR, 1H-NMR and ESI-MS techniques were used for characterization of the synthesized oxamides. The synthesized oxamides were screened for Lipoxygenase inhibition. Biological screening revealed that the oxamides possessed good lipoxygenase inhibition activities, whereas, the unsubstituted oxamide did not show any distinct lipoxygenase inhibition activity. Molecular docking studies of the oxamides were also carried out for lipoxygenase inhibition. The results obtained from molecular docking were well correlated with the empirical data.

Broad-spectrum receptor tyrosine kinase inhibitors overcome de novo and acquired modes of resistance to EGFR-targeted therapies in colorectal cancer.

It is increasingly appreciated that 3D cultures are more predictive of in vivo therapeutic efficacy than 2D cultures. Using in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR, we previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody cetuximab. The de novo mode of cetuximab resistance in SC cells could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. We now show that crizotinib also overcomes acquired cetuximab resistance in CC-CR cells. Phospho-RTK array analysis showed increased phosphorylation of several RTKs, including MET and RON, in SC and CC-CR cells compared to cetuximab-sensitive CC counterparts. Furthermore, other multi-RTK inhibitors cabozantinib and BMS-777607 helped overcome cetuximab resistance, as measured by 3D colony growth and activation state of key signaling molecules. Conversely, addition of RTK ligands HGF and NRG1 induced cetuximab resistance in CC cells, which could be blocked by addition of crizotinib. We further determined the mechanism of the cooperative effect of cetuximab and crizotinib by FACS analysis and observed increased cell cycle arrest in G1 phase in cetuximab-resistant CRC 3D cultures. Finally, we show that crizotinib overcomes cetuximab resistance in vivo in SC nude mice xenografts. Thus, our work shows that multi-RTK inhibition strategy is a potent, broadly applicable strategy to overcome resistance to EGFR-targeted therapeutics in CRC and highlights the relevance of 3D cultures in these studies. Statement of implication: Using in vitro 3D CRC cultures and in vivo CRC xenografts, we show that parallel inhibition of multiple RTKs with small molecule inhibitors overcomes de novo and acquired resistance to EGFR-directed therapies in CRC.

Endocrine disrupting chemicals and impact on male reproductive health.

Endocrine disrupting chemicals (EDCs) have been known to adversely affect the endocrine system leading to compromised functions of hormones. The presence of these compounds in everyday products such as canned food, water bottles, plastics, cosmetics, fertilizers, kid's toys and many others goods is a greater concern for general population. The persistent and long-term use of EDCs has deleterious effects on human reproductive health by interfering with the synthesis and mechanism of action of sex hormones. Any change during the synthesis or action of the sex hormones may result in abnormal reproductive functions which includes developmental anomalies in the reproductive tract and decline in semen quality. The present paper provides an overview of the EDCs and their possible impact on male reproductive health with major focus on semen quality which leads to male infertility.

Assay Conditions Influence Affinities of Rat Organic Cation Transporter 1: Analysis of Mutagenesis in the Modeled Outward-Facing Cleft by Measuring Effects of Substrates and Inhibitors on Initial Uptake.

The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium+ (MPP+) and tetraethylammonium+ (TEA+) or inhibition of MPP+ uptake by the nontransported inhibitors tetrabutylammonium+ (TBuA+), tetrapentylammonium+ (TPeA+), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant (Km) values, different IC50 values, and varying effects of mutations were determined. In addition, varying IC50 values for the inhibition of MPP+ uptake and varying effects of mutations were obtained when different MPP+ concentrations far below the apparent Km value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 µM MPP+ for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent Km values for TEA+ and/or MPP+ Mutation of Trp218 and Asp475 led to altered IC50 values for TBuA+, TPeA+, and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC50 values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.

Potential of Nigella sativa seed aqueous extract in ameliorating quinine-induced thrombocytopenia in rats.

Dengue infection is rapidly spreading in most of the countries of south Asia. It is of utmost importance to explore the plants with "anti-thrombocytopenic activity" the dreadful response of dengue fever. The present study was conducted to investigate the potential of aqueous extract of Nigella sativa (black cumin) seeds in alleviating the severity of dengue disease by raising the platelet count (PLT). Serum samples of thirty patients with dengue hemorrhagic fever (DHF) were analysed for different biochemical parameters. When compared with control groups, the patients were found with very low PLT count (7.62 fold), reduced antioxidant levels; catalase (1.4 fold), ascorbic acid (1.1 fold), bilirubin (1.06 fold), and severe deficiency of micronutrient concentrations; cobalt (2.27 fold), iron (2.35 fold) and nickel (71.46 fold). Similar parameters were studied in albino rats to observe the changes in serum levels of biochemical markers, after administration of single dose of choloroquine phosphate (IM, 1.5 mL saline). The drug successfully induced thrombocytopenia along with significant decrease in levels of antioxidants and trace metals. Administration of N. sativa aqueous seed extract (15.25 mg/kg/bw) for 12 days resulted in an increase in PLT count (1.59 fold) as compared to control group. N. sativa post-treatment was found effective in elevating the serum levels of catalase, ascorbic acid, and bilirubin (1.06, 1.58 and 0.4 folds respectively). However, the N. sativa pre-treatment was useful in increasing the levels of micronutrients; iron, nickel and cobalt when compared to quinine-induced group. From the above findings it was suggested that N. sativa seed aqueous extract supplementation would be a promising solution for declined PLT count and associated consequences.

New carbazole linked 1,2,3-triazoles as highly potent non-sugar α-glucosidase inhibitors.

In the present study, a series of new carbazole linked 1H-1,2,3-triazoles (2-27) were synthesized via click reaction of N-propargyl-9H-carbazole (1) and azides of appropriate acetophenones and heterocycles. Synthesized carbazole triazoles including 7, 9, 10, 19, 20, and 23-26 (IC50=0.8±0.01-100.8±3.6µM), exhibited several folds more potent α-glucosidase inhibitory in vitro activity as compared to standard drug, acarbose. Compounds 2-5, 7-13, and 17-27 did not show any cytotoxicity against 3T3 cell lines, except triazoles 6, and 14-16. Among the series, carbazole triazoles 23 (IC50=1.0±0.057µM) and 25 (IC50=0.8±0.01µM) were found to be most active, and could serve as an attractive building block in the search of new non-sugar derivatives as anti-diabetic agents.

In vitro α-Glucosidase Inhibition by Non-sugar based Triazoles of Dibenzoazepine, their Structure-Activity Relationship, and Molecular Docking.

BACKGROUND: α-Glucosidase inhibitors (AGIs) have been reported for their clinical potential against postprandial hyperglycemia, which is responsible for the risks associated with diabetes mellitus 2 and cardiovascular diseases (CVDs). Besides, a number of compounds have been reported as potent AGIs, several side effects are associated with them. METHODS: The aim of present work is to explore new and potent molecules as AGIs. Therefore, a library of dibenzoazepine linked triazoles (1-15) was studied for their in vitro α-glucosidase inhibitory activity. The binding modes of potent compounds in the active site of α-glucosidase enzyme were also explored through molecular docking studies. RESULTS AND CONCLUSION: Among the reported triazoles, compounds 3-9, 11, and 13 (IC50 = 6.0 ± 0.03 to 19.8 ± 0.28 µM) were found to be several fold more active than the standard drug acarbose (IC50 = 840 ± 1.73 µM). Compound 5 (IC50 = 6.0 ± 0.03 µM) was the most potent AGIs in the series, about 77- fold more active than acarbose. Therefore, dibenzoazepine linked-triazoles described here can serve as leads for further studies as new non-sugar AGIs.

Impact of Plant Extracts and Antibiotics on Biofilm Formation of Clinical Isolates From Otitis Media.

BACKGROUND: Otitis media can lead to severe health consequences, and is the most common reason for antibiotic prescriptions and biofilm-mediated infections. However, the increased pattern of drug resistance in biofilm forming bacteria complicates the treatment of such infections. OBJECTIVES: This study was aimed to estimate the biofilm formation potential of the clinical isolates of otitis media, and to evaluate the efficacy of antibiotics and plant extracts as alternative therapeutic agents in biofilm eradication. MATERIALS AND METHODS: The ear swab samples collected from the otitis media patients visiting the Mayo Hospital in Lahore were processed to isolate the bacteria, which were characterized using morphological, biochemical, and molecular (16S rRNA ribotyping) techniques. Then, the minimum inhibitory concentrations (MICs) of the antibiotics and crude plant extracts were measured against the isolates. The cell surface hydrophobicity and biofilm formation potential were determined, both qualitatively and quantitatively, with and without antibiotics. Finally, the molecular characterization of the biofilm forming proteins was done by amplifying the ica operon. RESULTS: Pseudomonas aeruginosa (KC417303-05), Staphylococcus hemolyticus (KC417306), and Staphylococcus hominis (KC417307) were isolated from the otitis media specimens. Among the crude plant extracts, Acacia arabica showed significant antibacterial characteristics (MIC up to 13 mg/ml), while these isolates exhibited sensitivity towards ciprofloxacin (MIC 0.2 µg/mL). All of the bacterial strains had hydrophobic cellular surfaces that helped in their adherence to abiotic surfaces, leading to strong biofilm formation potential (up to 7 days). Furthermore, the icaC gene encoding polysaccharide intercellular adhesion protein was amplified from S. hemolyticus. CONCLUSIONS: The bacterial isolates exhibited strong biofilm formation potential, while the extracts of Acacia arabica significantly inhibited biofilm formation among the isolates and, therefore, could be executed in the development of cost-effective biofilm inhibitor medicines.