Development and in-vitro evaluation of an optimized carvedilol transdermal therapeutic system using experimental design approach

AbstractThe effect of formulation variables on in-vitro release and permeation properties of carvedilol from transdermal patch was studied by varying one factor at a time as preliminary study. Based on these results, design of experiments technique was applied followed by regression analysis and response surface methodology to optimize formulation variables. Central Composite IV model design was used with four formulation variables: drug loading, matrix thickness, adhesive layer thickness, and propylene glycol concentration. Nineteen formulations were prepared according to the design; and the effect of formulation variables was studied on in-vitro release and permeation profiles of these formulations. In all cases, the permeation profiles paralleled in-vitro release profiles. The drug released at 7 h and 24 h was used as release response parameters while permeation flux obtained was employed as permeation response parameter. All four formulation variables were found to be significant for release properties and three of these exhibited significant effect on permeation profile of carvedilol across artificial membrane. Constrained optimization, using 47.9% of cumulative carvedilol released at 7 h and 99.8% at 24 h as well as 25.7 μg/cm2/h of permeation flux, was applied to obtain desired release and permeation profiles. Experimentally, carvedilol was observed to release from the optimized formulation with 51.4% drug release at 7 h and 98.5% at 24 h with an observed flux value of 27.4 μg/cm2/h across artificial membrane, which showed an excellent agreement with the predicted values. The results of this investigation show that the quadratic mathematical model developed could be used to further predict formulations with desirable release and permeation properties

A COMPARATIVE STUDY OF POSTPARTUM BLOOD LOSS BY VISUAL ESTIMATION METHOD AND BY GRAVIMETRIC METHOD

Objectives: The aim of the study was to estimate postpartum blood loss visually and by gravimetric method and compare the both. Methods: This study was conducted in the Department of Obstetrics and Gynaecology, GMC Amritsar on 100 pregnant women satisfying the inclusion criteria over a period of 1.5 years. Patient’s consent was taken and visual estimation was done by the attending obstetrician and obstetric nurse. Total blood loss was calculated using gravimetric method and was compared to value given by visual estimation. Furthermore, comparison was done between the visual estimation values of the attending obstetrician and the obstetric nurse. Results: Obstetrician observed 21.47% less blood loss than the actual (by gravimetric method) blood loss. Obstetric nurse observed 20.01% less blood loss than the obstetrician and 37.19% less than the actual loss. Conclusion: Visual estimation underestimates the actual blood loss and, hence, an objective gravimetric method should be used for early and effective management of PPH

Functionalization of CD36 Cardiovascular Disease and Expression Associated Variants by Interdisciplinary High Throughput Analysis.

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low-density lipoprotein (oxLDL) results in platelet activation. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk by genome-wide association studies. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We screened 81 expression quantitative trait loci (eQTL) single nucleotide polymorphisms (SNPs) associated with platelet CD36 expression by a Massively Parallel Reporter Assay (MPRA) and analyzed the results with a novel Bayesian statistical method. Ten eQTLs located 13kb to 55kb upstream of the CD36 transcriptional start site of transcript ENST00000309881 and 49kb to 92kb upstream of transcript ENST00000447544, demonstrated significant transcription shifts between their minor and major allele in the MPRA assay. Of these, rs2366739 and rs1194196, separated by only 20bp, were confirmed by luciferase assay to alter transcriptional regulation. In addition, electromobility shift assays demonstrated differential DNA:protein complex formation between the two alleles of this locus. Furthermore, deletion of the genomic locus by CRISPR/Cas9 in K562 and Meg-01 cells results in upregulation of CD36 transcription. These data indicate that we have identified a variant that regulates expression of CD36, which in turn affects platelet function. To assess the clinical relevance of our findings we used the PhenoScanner tool, which aggregates large scale GWAS findings; the results reinforce the clinical relevance of our variants and the utility of the MPRA assay. The study demonstrates a generalizable paradigm for functional testing of genetic variants to inform mechanistic studies, support patient management and develop precision therapies

Expression of rat Na-K-ATPase α2 enables ion pumping but not ouabain-induced signaling in α1-deficient porcine renal epithelial cells

Na-K-ATPase is a fundamental component of ion transport. Four α isoforms of the Na-K-ATPase catalytic α subunit are expressed in human cells. The ubiquitous Na-K-ATPase α1 was recently discovered to also mediate signal transduction through Src kinase. In contrast, α2 expression is limited to a few cell types including myocytes, where it is coupled to the Na+/Ca2+ exchanger. To test whether rat Na-K-ATPase α2 is capable of cellular signaling like its α1 counterpart in a recipient mammalian system, we used an α1 knockdown pig renal epithelial cell (PY-17) to create an α2-expressing cell line with no detectable level of α1 expression. These cells exhibited normal ouabain-sensitive ATPase, but failed to effectively regulate Src. In contrast to α1-expressing cells, ouabain did not stimulate Src kinase or downstream effectors such as ERK and Akt in α2 cells, although their signaling apparatus was intact as evidenced by EGF-mediated signal transduction. Additionally, α2 cells were unable to rescue caveolin-1. Unlike the NaKtide sequence derived from Na-K-ATPase α1, which downregulates basal Src activity, the corresponding α2 NaKtide was unable to inhibit Src in vitro. Finally, coimmunoprecipitation of cellular Src was diminished in α2 cells. These findings indicate that Na-K-ATPase α2 does not regulate Src and, therefore, may not serve the same role in signal transduction as α1. This further implies that the signaling mechanism of Na-K-ATPase is isoform specific, thereby supporting a model where α1 and α2 isoforms play distinct roles in mediating contraction and signaling in myocytes

Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase

The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na+ affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na+ was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1

Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-Gingerol in cervical cancer cells

Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer

Serum vascular endothelial growth factor-A (VEGF-A) as a biomarker in squamous cell carcinoma of head and neck patients undergoing chemoradiotherapy

Background: To evaluate serum VEGF-A levels in squamous cell carcinoma of head and neck (SCCHN) patients and relationships with response to therapy. Materials and Methods: Serum VEGF-A levels in patients (n=72) treated with radiotherapy (RT) or radio-chemotherapy (RCT) and controls (n=40) were measured by ELISA. Results: Serum VEGF-A levels of the SCCHN cases were significantly higher (p=0.001) than in healthy controls, and in patients with positive as compared to negative lymph node status (p=0.004). Similarly, patients with advanced stage (Stage III-IV) disease had more greatly elevated levels of serum VEGF-A level than their early stage (Stage I-II) counterparts (p=0.001). In contrast, there was no significant difference (p=0.57) in serum level of VEGF-A in patients with advanced T-stage (T3-4) as compared to early stage (T1-2). Similarly, patients with distant metastasis had no significant (p=0.067) elevation in serum VEGF-A level as compared to non-metastatic disease. However, the non-responder patients had significantly higher serum VEGF-A level as compared to responders (p=0.001). Conclusions: Our results suggest that the serum VEGF-A level may be a useful biomarker for the prediction of response to therapy in SCCHN

Metabolome and microbiome alterations related to short-term feeding of a micronutrient-fortified, high-quality legume protein-based food product to stunted school age children: A randomized controlled pilot trial

Background & aimsStunting in children is a comorbid condition in undernutrition that may be ameliorated by the provision of high-quality foods that provide protein and micronutrients. Addressing this problem in lower social economic environments requires, in part, affordable and scalable food-based solutions with efficacious food products. Towards this end, biochemical/metabolic indicators for fast-throughput screening of foods and their components are desired. A highly acceptable and economical micronutrient-fortified food product with different levels of legume protein was provided to stunted Indian children for one month, to determine change in their linear growth and explore associated biochemical, metabolomic and microbiome indicators.MethodsA randomized controlled pilot trial was conducted with 100 stunted children (6-10 years of age) to elucidate metabolic and microbiome-based biomarkers associated with linear growth. They were randomized into 4 groups receiving 6, 8, 10 or 12 g of legume-based protein for one month. Anthropometry, blood biochemistry, aminoacidomics, acylcarnitomics and fecal microbiome were measured before and after feeding.ResultsNo significant differences were observed between groups in height, height-for-age Z-score (HAZ) or BMI-for-age Z-score (BAZ); however, 38 serum metabolites were altered significantly (Bonferroni adjusted P < 0.1) in response to the interventions. IGF-1 (Insulin like Growth Factor-1) was positively (ρ > 0.2, P = 0.02), while serine and ornithine (ρ < -0.2, P = 0.08) were negatively associated with change in height. Leucine, isoleucine and valine positively correlated (P = 0.011, 0.023 and 0.007 respectively) with change in BAZ. Three Operational Taxonomic Units belonging to Bacteroidetes and Firmicutes (VIP score > 1.5) were significantly correlated with change in height.ConclusionsIn this pilot trial, a number of fasting serum metabolomic and fecal microbiome signatures were associated with linear growth after a short-term dietary intervention. The alterations of these markers should be validated in long-term dietary intervention trials as potential screening indicators towards the development of food products that favor growth. This trial was registered at www.ctri.nic.in as CTRI/2016/12/007564