Liquid chromatography-mass spectrometry method for the quantification of tamoxifen and its metabolite 4-hydroxytamoxifen in rat plasma: application to interaction study with biochanin A (an isoflavone)

Tamoxifen is the agent of choice for the treatment of estrogen receptor-positive breast cancer. Tamoxifen is a substrate of P-glycoprotein (P-gp) and microsomal cytochrome P450 (CYP) 3A, and biochanin A (BCA) is an inhibitor of P-gp and CYP3A. Hence, it could be expected that BCA would affect the pharmacokinetics of tamoxifen. In the present study we have developed and validated a simple, sensitive and specific LC-ESI-MS/MS method for the simultaneous quantification of tamoxifen and its metabolite 4-hydroxytamoxifen with 100 μL rat plasma using centchroman as an internal standard (IS). Tamoxifen, 4-hydroxytamoxifen and IS were separated on a Supelco Discovery C18 (4.6 mm × 50 mm, 5.0 μm) column under isocratic condition using 0.0 1M ammonium acetate (pH 4.5):acetonitrile (10:90, v/v) as a mobile phase. The mobile phase was delivered at a flow rate of 0.8 mL/min. The method was proved to be accurate and precise at linearity range of 0.78-200 ng/mL with a correlation coefficient (r) of ≥ 0.996. The intra- and inter-day assay precision ranged from 1.89 to 8.54% and 3.97 to 10.26%, respectively; and intra- and inter-day assay accuracy was between 87.63 and 109.06% and 96 and 103.89%, respectively for both the analytes. The method was successfully applied to study the effect of oral co-administration of BCA (an isoflavone) on the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen in female rats. The coadministration of BCA caused no significant changes in the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen. However, the peak plasma concentration (C(max)) of 4-hydroxytamoxifen in BCA pretreated rats was significantly (P<0.05) lower than those from control group.     

Caring for Caregivers of People Living with HIV in the Family: A Response to the HIV Pandemic from Two Urban Slum Communities in Pune,India

Introduction

In low resource settings, the vast majority of ‘Person/people Living with HIV’ (PLHIV/s) and inadequate healthcare delivery systems to meet their treatment and care needs, caregivers play a vital role. Home based caregivers are often unrecognized with limited AIDS policies and programs focusing on them. We explored the perceptions and norms regarding care being provided by family caregivers of PLHIVs in India.

Methodology

A community based qualitative study to understand the issues pertaining to home based care for PLHIV was conducted in urban settings of Pune city, in Maharashtra, India. Eight Focus Group Discussions (FGDs) among men, women and peer educators were carried out. A total of 44 in-depth Interviews (IDIs) with PLHIVs (20) and their caregivers (24), were conducted using separate guides respectively. Data was analyzed thematically.

Results

Home based care was perceived as economically viable option available for PLHIVs. ‘Care’ comprised of emotional, adherence, nursing and financial support to PLHIV. Home based care was preferred over hospital based care as it ensured confidentiality and patient care without hampering routine work at home. Women emerged as more vital primary caregivers compared to men. Home based care for men was almost unconditional while women had no such support. The natal family of women also abandoned. Their marital families seemed to provide support. Caregivers voiced the need for respite care and training.

Discussion

Gender related stigma and discrimination existed irrespective of women being the primary family caregivers. The support from marital families indicates a need to explore care and support issues at natal and marital homes of the women living with HIV respectively. Home based care training and respite care for the caregivers is recommended. Gender sensitive interventions addressing gender inequity and HIV related stigma should be modeled while designing interventions for PLHIVs and their family caregivers.     

Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection

The ventromedial medulla (VM), subdivided in a rostral (RVM) and a caudal (CVM) part, has a powerful influence on the spinal cord. In this study, we have identified the distribution of glycine and GABA containing neurons in the VM with projections to the cervical spinal cord, the lumbar dorsal horn, and the lumbar ventral horn. For this purpose, we have combined retrograde tracing using fluorescent microspheres with fluorescent in situ hybridization (FISH) for glycine transporter 2 (GlyT2) and GAD67 mRNAs to identify glycinergic and/or GABAergic (Gly/GABA) neurons. Since the results obtained with FISH for GlyT2, GAD67, or GlyT2 + GAD67 mRNAs were not significantly different, we concluded that glycine and GABA coexisted in the various projection neurons. After injections in the cervical cord, we found that 29% ± 1 (SEM) of the retrogradely labeled neurons in the VM were Gly/GABA (RVM: 43%; CVM: 21%). After lumbar dorsal horn injections 31% ± 3 of the VM neurons were Gly/GABA (RVM: 45%; CVM: 12%), and after lumbar ventral horn injections 25% ± 2 were Gly/GABA (RVM: 35%; CVM: 17%). In addition, we have identified a novel ascending Gly/GABA pathway originating from neurons in the area around the central canal (CC) throughout the spinal cord and projecting to the RVM, emphasizing the interaction between the ventromedial medulla and the spinal cord. The present study has now firmly established that GABA and glycine are present in many VM neurons that project to the spinal cord. These neurons strongly influence spinal processing, most notably the inhibition of nociceptive transmission.     

Structure-based discovery of C-2 substituted imidazo-pyrrolopyridine JAK1 inhibitors with improved selectivity over JAK2

Herein we describe our successful efforts in obtaining C-2 substituted imidazo-pyrrolopyridines with improved JAK1 selectivity relative to JAK2 by targeting an amino acid residue that differs between the two isoforms (JAK1: E966; JAK2: D939). Efforts to improve cellular potency by reducing the polarity of the inhibitors are also detailed. The X-ray crystal structure of a representative inhibitor in complex with the JAK1 enzyme is also disclosed.     

Nitric oxide alleviates arsenic toxicity by reducing oxidative damage in the roots of Oryza sativa (rice)

Nitric oxide (NO) is a bioactive gaseous, multifunctional molecule playing a central role and mediating a variety of physiological processes and responses to biotic and abiotic stresses including heavy metals. The present study investigated whether NO applied exogenously as sodium nitroprusside (SNP) has any protective role against arsenic (As) toxicity in Oryza sativa (rice). Treatment with 50 μM SNP (a NO donor) significantly ameliorated the As-induced (25 or 50 μM) decrease in root and coleoptile length of rice. Further, As-induced oxidative stress measured in terms of malondialdehyde (MDA), superoxide ion (), root oxidizability and H₂O₂ content was lesser upon supplementation of NO. It indicated a reactive oxygen species (ROS) scavenging activity of NO. NO addition reversed (only partially) the As-induced increase in activities of antioxidant enzymes – superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and catalase. The study concludes that exogenous NO provides resistance to rice against As-toxicity and has an ameliorating effect against As-induced stress.     

Ethnic‐Specific Differences in Abdominal Subcutaneous Adipose Tissue Compartments

South Asians have a higher prevalence of cardiovascular disease (CVD) than Europeans. Studies have identified distinct subcompartments of subcutaneous adipose tissue (SAT) that provide insight into the relationship between abdominal obesity and metabolic risk factors in different ethnic groups. Our objective was to determine the relationship between SAT compartments and fat‐free mass (FFM) between South Asian and European cohorts, and between men and women. Healthy Europeans and South Asians (n = 408) were assessed for FFM via dual energy X‐ray absorptiometry, and SAT areas by computed tomography (CT). SAT was subdivided into superficial subcutaneous abdominal adipose tissue (SSAT) and deep subcutaneous abdominal adipose tissue (DSAT). Linear regression analyses were performed using DSAT and SSAT as separate dependent variables and FFM and ethnicity as primary independent variables adjusting for age, gender, income, education, and smoking status. Results showed that South Asian men had significantly higher amounts of DSAT (median 187.65 cm² vs. 145.15 cm², P < 0.001), SSAT (median 92.0 cm² vs. 76.1 cm², P = 0.046), and body fat mass (BFM) (25.1 kg vs. 22.6 kg, P = 0.049) than European men. In a fully adjusted model, South Asians showed significantly greater DSAT at any FFM than Europeans. Women had more SSAT at any given FFM than men and less DSAT at any given FFM than men, irrespective of ethnic background. In conclusion, South Asians had more DSAT than Europeans and men had relatively more DSAT than women. These data suggest that specific fat depots are influenced by ethnicity and gender; therefore, could provide insight into the relationship between ethnicity, gender and subsequent risk for CVD.     

An antisuppressor mutation of Schizosaccharomyces pombe affects the post-transcriptional modification of the "wobble" base in the anticodon of tRNAs

The screening of antisuppressor mutants of the yeast Schizosaccharomyces pombe has been successfully accomplished with high resolution liquid chromatographic methods for the analysis of tRNA nucleosides. Antisuppressor mutations reduce or abolish the function of nonsense suppressor-tRNAs or other informational suppressors. Nonradioactive or 35S-labeled unfractionated tRNA from various strains was digested to nucleosides and analyzed by high performance liquid chromatography. The mutant sin3 has lost the nucleoside 5-(methoxycarbonylmethyl)-2-thiouridine from its tRNA in comparison to parental strains. In eukaryotes this nucleoside is found at the first position of the anticodon (wobble position) in several isoacceptor tRNAs that preferentially recognize codons ending with adenosine. The sin3 mutation reduces the efficiency of UGA and UAA suppressor tRNASer and suppressor tRNALeu. The genetic cosegregation of modification loss, antisuppressor phenotype, and a change in cell size is demonstrated. This indicates that a single mutation in the structural gene for a tRNA modification enzyme causes the three different phenotypes.     

Significance of isomerization in hydroxocobalamin

Hydroxocobalamin is present in fairly large proportions both in foods and in the human body and apparently plays an important biological role. Since cyanocobalamin seems to play hardly any significant biochemical role in healthy humans, several physicians prefer to administer hydroxocobalamin to vitamin B12 deficient patients. We find that hydroxocobalamin in solution isomerizes very readily at room and lower temperatures. Our observations raise the question whether "Mother Nature" has gone awry in using an easily convertible substance like hydroxocobalamin or that the new isomeric forms play some significant role. These observations may also have a bearing on the reported occurrence of unidentified corrinoids in animal tissues, human red cells, liver and brain.