Integrative Placental Multi-Omics Analysis Reveals Perturbed Pathways and Potential Prognostic Biomarkers in Gestational Hypertension.

BACKGROUND: Gestational hypertension (GH) is a severe complication that occurs after 20 weeks of pregnancy; however, its molecular mechanisms are not yet fully understood. OBJECTIVE: Through this case-control discovery phase study, we aimed to find disease-specific candidate placental microRNAs (miRNAs) and metabolite markers for differentiating GH by integrating next-generation sequencing and metabolomics multi-omics analysis of placenta. Using small RNA sequencing and metabolomics of placental tissues of healthy pregnant (HP, n = 24) and GH subjects (n = 20), the transcriptome and metabolome were characterized in both groups. RESULTS: The study identified a total of 44 downregulated placental miRNAs which includes three novel, three mature and 38 precursor miRNAs. Six miRNAs including three mature (hsa-miR-181a-5p, hsa-miR-498-5p, and hsa-miR-26b-5p) and three novel (NC_000016.10_1061, NC_000005.10_475, and NC_000001.11_53) were considered for final target prediction and functional annotation. Integrative analysis of differentially expressed miRNAs and metabolites yielded five pathways such as purine, glutathione, glycerophospholipid, inositol phosphate and ß-alanine to be significantly perturbed in GH. We present fourteen genes (LPCAT1, LPCAT2, DGKH, PISD, GPAT2, PTEN, SACM1L, PGM2, AMPD3, AK7, AK3, CNDP1, IDH2, and ODC1) and eight metabolites (xanthosine, xanthine, spermine, glycine, CDP-Choline, glyceraldehyde 3-phosphate, ß-alanine, and histidine) with potential to distinguish GH and HP. CONCLUSION: The differential expression of miRNAs, their target genes, altered metabolites and metabolic pathways in GH patients were identified for the first time in our study. Further, the altered miRNAs and metabolites were integrated to build their inter-connectivity network. The findings obtained from our study may be used as a valuable source to further unravel the molecular pathways associated with GH and also for the evaluation of prognostic markers.

From co-delivery to synergistic anti-inflammatory effect: Studies on chitosan-stabilized Janus emulsions having chloroquine phosphate and flavopiridol in Complete Freund's Adjuvant induced arthritis rat model.

For the first time, the co-delivery of chloroquine phosphate and flavopiridol by intra-articular route was achieved to provide local joint targeting in Complete Freund's Adjuvant-induced arthritis rat model. The presence of paired-bean structure onto the dispersed oil droplets of o/w nanosized emulsions allows efficient entrapment of two drugs (85.86-96.22 %). The dual drug-loaded emulsions displayed a differential in vitro drug release behavior, near normal cell viability in MTT assay, better cell uptake (internalization) and better reducing effect of mean immunofluorescence intensity of inflammatory proteins such as NF-κB and iNOS at in vitro RAW264.7 macrophage cell line. The radiographical study, ELISA test, RT-PCR study and H & E staining also indicated a reduction in joint tissue swelling, IL-6 and TNF-α levels diminution, fold change diminution in the mRNA expressions for NF-κB, IL-1ß, IL-6 and PGE2 and maintenance of near normal histology at bone cartilage interface respectively. The results of metabolomic pathway analysis performed by LC-MS/MS method using the rat blood (plasma) collected from disease control and dual drug-loaded emulsions treatment groups revealed a new follow-up study to understand not only the disease progression but also the formulation therapeutic efficacy assessment.

Preclinical pharmacokinetic and in vitro metabolic stability study of lysosomotropic autophagy inhibitor, IITZ-01 in mice by using UPLC-MS/MS.

The present study evaluates the pharmacokinetics and metabolic stability of a novel lysosomotropic autophagy inhibitor, IITZ-01 using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). It is required as this lead molecule awaits pre-clinical studies for development because of significant therapeutic outcomes in triple-negative breast cancer and renal cancer. A bioanalytical method for the quantitative determination of IITZ-01 in the plasma of mice was developed using the UPLC-MS/MS technique. The UPLC-MS/MS method was validated according to US-FDA bioanalytical guidance and successfully applied to study the pharmacokinetics and metabolic stability. Separation of IITZ- 01 and ZSTK474 (IS) from endogenous components with high selectivity and sensitivity (0.5 ng/mL) was achieved using Waters Acquity BEH C-18 column (50 mm × 2.1 mm, 1.7 µm). A gradient mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile was applied at a flow rate of 0.2 mL/min. Electrospray ionization was employed in positive ion mode for detection, while quantification utilized the multiple reaction monitoring (MRM) mode. This involved using [M+H]+fragment ions at m/z 483.19 â†’ 235.09 for IITZ-01 and m/z 418 â†’ 138 for the internal standard (IS). The method was validated over the calibration range of 0.5-800 ng/mL. The LLOQ of IITZ-01 was 0.5 ng/mL in mice plasma. The method demonstrated good in terms of intra- and inter-day precision and accuracy. The matrix effect was found to be negligible, and the stability data were within acceptable limits. The validated technique supports suitability, reliability, reproducibility, and sensitivity for the pre-clinical investigation of IITZ-01 pharmacokinetics in mice and metabolic stability in human liver microsomes.

Ambient particulate matter (PM2.5) exposure contributes to neurodegeneration through the microbiome-gut-brain axis: Therapeutic role of melatonin.

Exposure to ambient particulate matter (PM2.5) has been shown to disturb the gut microbiome homeostasis and cause initiation of neuroinflammation and neurodegeneration via gut-brain bi-directional axis. Polyaromatic hydrocarbons (PAHs), which are carcinogenic and mutagenic, are important organic constituents of PM2.5 that could be involved in the microbiome-gut-brain axis-mediated neurodegeneration. Melatonin (ML) has been shown to modulate the microbiome and curb inflammation in the gut and brain. However, no studies have been reported for its effect on PM2.5-induced neuroinflammation. In the current study, it was observed that treatment with ML at 100 µM significantly inhibits microglial activation (HMC-3 cells) and colonic inflammation (CCD-841 cells) by the conditioned media from PM2.5 exposed BEAS2B cells. Further, melatonin treatment at a dose of 50 mg/kg to C57BL/6 mice exposed to PM2.5 (at a dose of 60 µg/animal) for 90 days significantly alleviated the neuroinflammation and neurodegeneration caused by PAHs in PM2.5 by modulating olfactory-brain and microbiome-gut-brain axis.

Urinary Metabolomics Identified Metabolic Perturbations Associated with Gutka, a Smokeless Form of Tobacco.

Gutka, a form of smokeless tobacco, is widely used in the Indian subcontinent and in other regions of South Asia. Smokeless tobacco exposure is most likely to increase the incidence of oral cancer in the Indian population, and metabolic changes are a hallmark of cancer. The development of biomarkers for early detection and better prevention measures for smokeless tobacco users at risk of oral cancer can be aided by studying urinary metabolomics and offering insight into altered metabolic profiles. This study aimed to investigate urine metabolic alterations among smokeless tobacco users using targeted LC-ESI-MS/MS metabolomics approaches to better understand the effects of smokeless tobacco on human metabolism. Smokeless tobacco users' specific urinary metabolomics signatures were extracted using univariate, multivariate analysis and machine learning methods. Statistical analysis identified 30 urine metabolites significantly associated with metabolomic alterations in humans who chew smokeless tobacco. Receiver operator characteristic (ROC) curve analysis evidenced the 5 most discriminatory metabolites from each approach that could differentiate between smokeless tobacco users and controls with higher sensitivity and specificity. An analysis of multiple-metabolite machine learning models and single-metabolite ROC curves revealed discriminatory metabolites capable of distinguishing smokeless tobacco users from nonusers more effectively with higher sensitivity and specificity. Furthermore, metabolic pathway analysis depicted several dysregulated pathways in smokeless tobacco users, including arginine biosynthesis, beta-alanine metabolism, TCA cycle, etc. This study devised a novel strategy to identify exposure biomarkers among smokeless tobacco users by combining metabolomics and machine learning algorithms.

Studies on spray dried topical ophthalmic emulsions containing cyclosporin A (0.05% w/w): systematic optimization, in vitro preclinical toxicity and in vivo assessments.

Cyclosporin A (CsA, 0.05% w/w)-loaded positively charged emulsions were prepared based on castor oil, chitosan, poloxamer 188, glycerin and double-distilled water. To augment the shelf/storage-stability of original emulsions, the solid-dry powder for reconstitution was made by spray drying technique. The screening (Taguchi OA) and optimization (face-centered central composite) designs produced the optimized conditions for spray drying: 40 Nm3/h aspirator flow rate, 15 ml/min feed rate, 115 °C inlet temperature, 10% mannitol and 1.25% trehalose. The % drug entrapment efficiency values of original and reconstituted emulsions ranged from 73.20 ± 0.13 to 71.55 ± 1.25%. At 20 min post-dissolution, two times higher CsA release was seen from reconstituted emulsions than the original emulsions (85.78 ± 1.14 vs. 42.25 ± 1.84%) in simulated tear fluid. Using MTT assay, the reconstituted emulsions with or without CsA produced 94.512 ± 2.12 to 99.941 ± 1.89% cell viability values in HCE-2 cells. No appreciable change in capillary integrity was visualized in HET CAM following reconstituted emulsions treatment. At equivalent 15 µg drug, the in vitro protein denaturation assay showed augmented inhibition value (~ 85%) for tested CsA emulsions compared to diclofenac reference (68.30 ± 2.05) indicating enhanced anti-inflammatory activity. The CsA concentrations in multiple ocular matrices of rabbit eyes determined by the UPLC-MS/MS method attained the therapeutic drug level of 50-300 ng/ml even at 90 min post-topical instillation of both emulsions. Overall, the CsA emulsion eyedrops can be supplied as a spray dried storable intermediate product for reconstitution.

Environmental phenol exposure associates with urine metabolome alteration in young Northeast Indian females.

Urinary biomonitoring delivers the most accurate environmental phenols exposure assessment. However, environmental phenol exposure-related biomarkers are required to improve risk assessment to understand the internal processes perturbed, which may link exposure to specific health outcomes. This study aimed to investigate the association between environmental phenols exposure and the metabolome of young adult females from India. Urinary metabolomics was performed using liquid chromatography-mass spectrometry. Environmental phenols-related metabolic biomarkers were investigated by comparing the low and high exposure of environmental phenols. Seven potential biomarkers, namely histidine, cysteine-s-sulfate, 12-KETE, malonic acid, p-hydroxybenzoic acid, PE (36:2), and PS (36:0), were identified, revealing that environmental phenol exposure altered the metabolic pathways such as histidine metabolism, beta-Alanine metabolism, glycerophospholipid metabolism, and other pathways. This study also conceived an innovative strategy for the early prediction of diseases by combining urinary metabolomics with machine learning (ML) algorithms. The differential metabolites predictive accuracy by ML models was >80%. This is the first mass spectrometry-based metabolomics study on young adult females from India with environmental phenols exposure. The study is valuable in demonstrating multiple urine metabolic changes linked to environmental phenol exposure and a better understanding of the mechanisms behind environmental phenol-induced effects in young female adults.

Integrated metabolomics and machine learning approach to predict hypertensive disorders of pregnancy.

BACKGROUND: Hypertensive disorders of pregnancy account for 3% to 10% of maternal-fetal morbidity and mortality worldwide. This condition has been considered one of the leading causes of maternal deaths in developing countries, such as India. OBJECTIVE: This study aimed to discover hypertensive disorders of pregnancy-specific candidate urine metabolites as markers for hypertensive disorders of pregnancy by applying integrated metabolomics and machine learning approaches. STUDY DESIGN: The targeted urinary metabolomics study was conducted in 70 healthy pregnant controls and 133 pregnant patients having hypertension as cases. Hypertensive disorders of pregnancy-specific metabolites for disease prediction were further extracted using univariate and multivariate statistical analyses. For machine learning analysis, 80% of the data were used for training (79 for hypertensive disorders of pregnancy and 42 for healthy pregnancy) and validation (27 for hypertensive disorders of pregnancy and 14 for healthy pregnancy), and 20% of the data were used for test sets (27 for hypertensive disorders of pregnancy and 14 for healthy pregnancy). RESULTS: The statistical analysis using an unpaired t test revealed 44 differential metabolites. Pathway analysis showed mainly that purine and thiamine metabolism were altered in the group with hypertensive disorders of pregnancy compared with the healthy pregnancy group. The area under the receiver operating characteristic curves of the 5 most predominant metabolites were 0.98 (adenosine), 0.92 (adenosine monophosphate), 0.89 (deoxyadenosine), 0.81 (thiamine), and 0.81 (thiamine monophosphate). The best prediction accuracies were obtained using 2 machine learning models (95% for the gradient boost model and 98% for the decision tree) among the 5 used models. The machine learning models showed higher predictive performance for 3 metabolites (ie, thiamine monophosphate, adenosine monophosphate, and thiamine) among 5 metabolites. The combined accuracies of adenosine from all models were 98.6 in the training set and 95.6 in the test set. Moreover, the predictive performance of adenosine was higher than other metabolites. The relative feature importance of adenosine was also observed in the decision tree and the gradient boost model. CONCLUSION: Among other metabolites, adenosine and thiamine metabolites were found to differentiate participants with hypertensive disorders of pregnancy from participants with healthy pregnancies; hence, these metabolites can serve as a promising noninvasive marker for the detection of hypertensive disorders of pregnancy.

Alternanthera brasiliana L. extract alleviates carbon tetrachloride-induced liver injury and fibrotic changes in mice: Role of matrix metalloproteinases and TGF-ß/Smad axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Alternanthera brasiliana L. is a flowering plant belonging to the family Amaranthaceae and is popularly known as "penicillin". It is used in folk medicine to treat infections, coughs, wound healing, and inflammatory diseases. AIM OF THE STUDY: We investigated the effect of Alternanthera brasiliana L. leaves hydroalcoholic extract (AB) against oxidative stress, inflammation, and fibrotic changes in an experimental model of carbon tetrachloride (CCl4)-induced liver injury and fibrosis in mice. MATERIALS AND METHODS: Thirty-six male Balb/C mice were randomized into five groups: normal control, AB control, CCl4 control, CCl4 + AB-200 mg/kg, and CCl4 + AB-400 mg/kg. In mice, liver injury was induced by intraperitoneal injection of CCl4 (20% in corn oil, 5 ml/kg body weight) thrice a week for six consecutive weeks. AB extract at two doses (200 mg/kg and 400 mg/kg body weight) was administered orally for six consecutive weeks. Liver injury-related serum markers (ALT, AST, ALP), antioxidants (GSH, GST, SOD, and vitamin C), pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-18, ultrasonographic and histological alterations, proteins of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1), nuclear factor-κB (p65) (NF-κB), nod-like receptor protein 3 (NLRP3), and TGF-ß/Smad signaling were accessed. LC-Q-TOF-MS/MS analysis of AB was performed. RESULTS: AB treatment significantly decreased the CCl4-induced rise in serum ALT, AST, and ALP activities and improved the histological alterations. Compared with the CCl4-treated group, treatment with AB significantly restored the hepatic antioxidants and reduced the pro-inflammatory cytokines in the liver. The antioxidant activity of AB may be attributed to its terpenoid constituents, which was confirmed by LC-Q-TOF-MS/MS analysis. The CCl4-induced rise in expression of MMP-2 and MMP-9 and decrease in TIMP-1 were markedly restored in the AB-treated groups. Further findings revealed a significant reduction in the protein levels of phospho-NF-κB (p65), NLRP3, TGF-ß, pSmad2/3, collagen I, and α-smooth muscle actin (α-SMA) in the AB treatment groups. CONCLUSIONS: The hepatoprotective effect of AB may be attributed to the high content of terpenoid compounds and alleviates liver injury and associated fibrotic changes through modulating MMPs, NF-κB (p65), and the TGF-ß/Smad axis.

Piperine Attenuates Cigarette Smoke-Induced Oxidative Stress, Lung Inflammation, and Epithelial-Mesenchymal Transition by Modulating the SIRT1/Nrf2 Axis.

Piperine (PIP) is a major phytoconstituent in black pepper which is responsible for various pharmacological actions such as anti-inflammatory, antioxidant, and antitumor activity. To investigate the effects and mechanisms of PIP on cigarette smoke (CS)-induced lung pathology using both in-vitro and in-vivo models. BEAS-2B and A549 cells were exposed to CS extract (CSE) for 48 h; BALB/c mice were exposed to CS (9 cigarettes/day, 4 days) to induce features of airway disease. PIP at doses of (0.25, 1.25, and 6.25 µM, in vitro; 1 and 10 mg/kg, in vivo, i.n) and DEX (1 µM, in vitro; 1 mg/kg, in vivo, i.n) were used to assess cytotoxicity, oxidative stress, epithelial−mesenchymal transition (EMT), Sirtuin1 (SIRT1), inflammation-related cellular signaling, and lung function. PIP treatment protects cells from CSE-induced lung epithelial cell death. PIP treatment restores the epithelial marker (p < 0.05) and decreases the mesenchymal, inflammatory markers (p < 0.05) in both in vitro and in vivo models. The PIP treatment improves the altered lung function (p < 0.05) in mice induced by CS exposure. Mechanistically, PIP treatment modulates SIRT1 thereby reducing the inflammatory markers such as IL-1ß, IL-6 and TNF-α (p < 0.05) and enhancing the epigenetic marker HDAC2 (p < 0.05) and antioxidant marker Nrf2 (p < 0.05) expressions. Thus, PIP alleviates pulmonary inflammation by modulating the SIRT1-mediated inflammatory cascade, inhibits EMT, and activates Nrf2 signaling.

Dual Delivery of Cyclosporin A and Etodolac Using Polymeric Nanocapsules in a Rabbit Eye Model: Ocular Biodistribution and Pharmacokinetic Study.

Purpose: Commercially available eye drops are loaded only with a single drug. By using the polymeric nanocapsules, dual delivery of 0.05% w/w cyclosporin A (CsA) and 0.2% w/w etodolac (Edc) was achieved. An ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for determining simultaneously the biodistribution and pharmacokinetic profile of CsA and Edc in ocular tissues. Methods: After one single drop instillation of nanocapsules into healthy right eyes of rabbits, the eyeballs were enucleated at 5, 15, 30, 60, and 90 min time periods to separate the 5 different ocular tissues. A liquid/liquid extraction method was used for ocular sample extraction using darunavir as internal standard. Using 3 diverse conditions such as bench-top, autosampler, and freeze-thaw, the stability of the analytes at 3 quality control samples in ocular tissues was also checked. Results: Intra- and interday precisions for both CsA and Edc in multiple ocular tissues were <10.32%, and the accuracy was <11.98%. The % bias and % RSD values for CsA and Edc were found within the acceptable limit of ±15%. The highest Cmax values were attained in cornea for both the drugs at 60 min postinstillation time point. Despite molecular size and structural differences, both CsA and Edc after liberation from nanocapsule drops can permeate into the tissues of the anterior as well as posterior segments of the eye. Conclusion: The biodistribution and pharmacokinetic data might help and strengthen our understanding of synergetic anti-inflammatory activity of CsA and Edc from nanocapsules after its ocular topical application for managing keratoconjunctivitis sicca.

Perillyl alcohol attenuates chronic restraint stress aggravated dextran sulfate sodium-induced ulcerative colitis by modulating TLR4/NF-κB and JAK2/STAT3 signaling pathways.

BACKGROUND: Ulcerative colitis (UC) is the most prevalent chronic inflammatory immune bowel disease. The modernization of lifestyle accompanied by the stress to cope with the competition has resulted in a new range of complications where stress became a critical contributing factor for many diseases, including UC. Hence there is an urgent need to develop a dual role in curtailing both systemic and neuroinflammation. Perillyl alcohol (POH) is a natural essential oil found in lavender, peppermint, cherries etc and has been widely studied for its strong anti-inflammatory, antioxidant and anti-stress properties. HYPOTHESIS/PURPOSE: POH regulates the various inflammatory signaling cascades involved in chronic inflammation by inhibiting farnesyltransferase  enzyme. Several studies reported that POH could inhibit the phosphorylation of  NF-κB, STAT3 and promote the endogenous antioxidant enzymes like Nrf2 via farnesyltransferase enzyme inhibition.  Also, the effects of POH against UC is not known yet. Thus, this study aims to explore the anti-ulcerative properties of POH on stress aggravated ulcerative colitis in C57BL/6 mice. METHODS: Ulcerative colitis was induced by duel exposure of chronic restraint stress (day 1 to day 28) and 2.5% dextran sulphate sodium (day8 to day14) in mice. POH treatment 100 and 200 mg/kg was administred from day14 ti day28 following oral route of administration. Disease activity index, colonoscopy, western blot analysis and histological analysis, neurotransmitter analysis and Gene expression studies were perofomerd to asses the anti-colitis effects of POH. RESULTS: The treatment reversed the oxidative stress and inflammatory response by inhibiting TLR4/NF-kB pathway, and IL-6/JAK2/STAT3 pathway in both isolated mice colons and brains. The inhibition of these pathways resulted in a decrease in pro-inflammatory cytokines like IL-6, IL-1ß and TNF-α. The treatment improved the physiological and histological changes with decreased ulcerations as examined by colonic endoscopy and Haematoxylin and Eosin staining. The treatment also improved the behavior response as it increased mobility time which was reduced by chronic restrained stress. This was due to increased satiety neurotransmitters like dopamine and serotonin and decreased cortisol in mice brains. CONCLUSION: These results infer that POH has significant anti-colitis activity on chronic restraint stress aggravated DSS-induced UC in mice.

Implications of endocrine-disrupting chemicals on polycystic ovarian syndrome: A comprehensive review.

Polycystic ovarian syndrome (PCOS) is a complex multifactorial disorder of unknown pathogenesis in which genetic and environmental factors contribute synergistically to its phenotypic expressions. Endocrine-disrupting chemicals (EDCs), a group of widespread pollutants freely available in the environment and consumer products, can interfere with normal endocrine signals. Extensive evidence has shown that EDCs, environmental contributors to PCOS, can frequently induce ovarian and metabolic abnormalities at low doses. The current research on environmental EDCs suggests that there may be link between EDC exposure and PCOS, which calls for more human bio-monitoring of EDCs using highly sophisticated analytical techniques for the identification and quantification and to discover the underlying pathophysiology of the disease. This review briefly elaborated on the general etiology of PCOS and listed various epidemiological and experimental data from human and animal studies correlating EDCs and PCOS. This review also provides insights into various analytical tools and sample preparation techniques for biomonitoring studies for PCOS risk assessment. Furthermore, we highlight the role of metabolomics in disease-specific biomarker discovery and its use in clinical practice. It also suggests the way forward to integrate biomonitoring studies and metabolomics to underpin the role of EDCs in PCOS pathophysiology.

Vitamin D Supplementation Modulates Platelet-Mediated Inflammation in Subjects With Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Recently, our group identified increased platelet-mediated inflammation in type 2 diabetes (T2DM) patients, and it is a well-established risk factor for diabetes complications, particularly for the development of cardiovascular diseases (CVD). Furthermore, vitamin D is reported to play an important role in the modulation of platelet hyperactivity and immune function, although the effect of vitamin D on platelet-mediated inflammation is not well studied. Hence, we aimed to investigate the effect of vitamin D supplementation on platelet-mediated inflammation in T2DM patients. Methods: After screening a total of 201 subjects, our randomized, double-blind, placebo-controlled trial included 59 vitamin-D-deficient T2DM subjects, and the participants were randomly assigned to placebo (n = 29) or vitamin D3 (n = 30) for 6 months. Serum vitamin D metabolite levels, immunome profiling, platelet activation, and platelet-immune cell aggregate formation were measured at baseline and at the end of the study. Similarly, the serum levels of inflammatory cytokines/chemokines were assessed by a multiplex assay. Results: Six months of vitamin D supplementation increases the serum vitamin D3 and total 25(OH)D levels from the baseline (p < 0.05). Vitamin D supplementation does not improve glycemic control, and no significant difference was observed in immune cells. However, platelet activation and platelet immune cell aggregates were altered after the vitamin D intervention (p < 0.05). Moreover, vitamin D reduces the serum levels of IL-18, TNF-α, IFN-γ, CXCL-10, CXCL-12, CCL-2, CCL-5, CCL-11, and PF-4 levels compared to the baseline levels (p < 0.05). Our ex vivo experiment confirms that a sufficient circulating level of vitamin D reduces platelet activation and platelet intracellular reactive oxygen species. Conclusion: Our study results provide evidence that vitamin D supportive therapy may help to reduce or prevent the disease progression and cardiovascular risk in T2DM patients by suppressing oxidative stress and platelet-mediated inflammation. Clinical Trial Registration: Clinical Trial Registry of India: CTRI/2019/01/016921.

In Vitro and In Vivo Evaluations of Berberine-Loaded Microparticles Filled In-House 3D Printed Hollow Capsular Device for Improved Oral Bioavailability.

The low oral bioavailability, short biological half-life, high dose, and frequent dosing of berberine (BBR) contribute to its restricted clinical use despite its extensive pharmacological activity. Thus, the objective of this study was to formulate sustained-release microparticles (MPs) using a pH-independent release polymer and to evaluate their potential to improve the oral bioavailability of BBR. BBR loaded MPs were prepared using the emulsion crosslinking method and evaluated for particle size, circularity, morphology, entrapment efficiency, solid-state analysis, swelling index, and in vitro BBR release study fitted with different models of release kinetics. The MPs exhibited desired particle sizes ranges between 11.09-11.62 µm and were almost spherical in shape, as confirmed by the circularity value and micrographic images. A loss of BBR crystallinity was observed after encapsulation in MPs, as evident from various solid-state analyses. The final optimized batch (F3) showed highest % BBR entrapment efficiency value of 81.63% ± 4.9. The in vitro BBR release performance in both acidic and alkaline media showed the desired sustained release behavior from the crosslinked MPs, where the maximum BBR release was observed at alkaline pH, which is in accordance with the swelling study data. In the in vivo study, the oral absorption profiles of BBR from both pristine and MPs formats were investigated using in-house prototyped 3D printed hollow capsules as a unit dose carrier. In vivo data showed sustained and prolonged absorption behavior of BBR from MPs compared to their pristine counterparts, which resulted in a cumulative increment of relative oral bioavailability to mitigate the aforementioned issues related to BBR. Graphical Abstract.

Determination of bisphenols and parabens in cow urine distillate from India: implication of human exposure and risk assessment.

The exposures of a wide range of endocrine-disrupting chemicals to cows have been linked to diseases and are a major concern. In Indian scripts, cow urine is believed to be a treatment for many diseases. Nonetheless, exposure of bisphenols and parabens to cow urine distillates is unknown. Hence, in this study, we determined the concentration of bisphenols and parabens in commercially available cow urine distillate collected from India. The mean concentration of total bisphenols and parabens ranges from the limit of quantification (LOQ) to 149.3 ng/mL and 1479.88 ng/mL, respectively. Predominant bisphenol-F and bisphenol-A were accounted for 88.6% and 6% of total bisphenol concentration, respectively, in marketed cow urine distillate, whereas methyl and propyl parabens were dominant and accounted for 33% and 65%, respectively. The estimated mean daily intake (EDI) of bisphenol for males and females were 45.94 ng/kg-bw/day and 54.29 ng/kg-bw/day, respectively, while for parabens EDI was 455.35ng/kg-bw/day and 538.14 ng/kg-bw/day for males and females, respectively. Hazard quotient, to evaluate the potential risk of exposure, showed no risk in the studied samples. Even though the EDI results from the Monte-Carlo risk assessment analysis did not exceed the acceptable daily intake, their estrogenic actions cannot be ignored in general populations. The estrogenic activities contributed by parabens and bisphenol A measured by estradiol equivalency quotient (EEQ) ranged from 0.00033-42 pg/mL and 2.3 pg/mL, respectively. Our results revealed higher concentrations of bisphenols and parabens in cow urine distillates; hence, special attention should be given to the quality and safety of cow urine distillates. Moreover, strict guidelines should be enforced for the quality of cow urine distillates.

Levels of parabens and bisphenols in personal care products and urinary concentrations in Indian young adult women: Implications for human exposure and health risk assessment.

Limited information is available about the levels of exposure of paraben and bisphenols emerging from personal care products (PCPs) use in Indian women and the risk associated with it. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the concentrations of six parabens (methyl-, ethyl-, propyl-, butyl, benzyl-, and heptyl-parabens) and 8 bisphenols (Bisphenol A, B, F, P, S, Z, AP, and AF) in PCPs samples (n = 114) obtained from Indian market and in the urine samples of young adult females (n = 52). The concentrations measured in PCPs and urine samples were used to determine the estimated daily intake. The mean concentrations of 6 parabens and 8 bisphenols in PCPs ranged from 38.3 to 2.38 × 105 ng/g and 2.71-148 ng/g, respectively. In urine samples analysed, the mean concentrations of 6 parabens and 8 bisphenols ranged from 0.007 to 293 ng/mL and 0.10-10.8 ng/mL, respectively. There was no significant correlation of EDCs with age, BMI and waist-to-hip ratio (WHR), but significant correlations (p < 0.05) were observed between urinary paraben and bisphenol concentrations. A statistically significant difference (p < 0.05) exists between the BMI and WHR groups by bisphenol concentrations. Estimated daily intake and exposure risks for parabens and bisphenols revealed no possible concerns for Indian young adult females. Hitherto, this is the first study to show that Indian young adult females were exposed to parabens and bisphenols. This study provides evidence on PCPs usage contribute to the urinary concentrations of EDCs.

A sensitive UPLC/ESI/MS/MS method for concomitant quantification of active plant constituent combinations in rat plasma after single oral administration.

Ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC/ESI/MS/MS) for the concomitant quantification of active plant constituents, namely quercetin and piperine, in rat plasma was developed and validated to assess pharmacokinetics after a single oral administration. Liquid-liquid extraction technique with ethyl acetate and n-hexane (1 : 1) was used, and fisetin was added as an internal standard (IS). Effective chromatographic separation of quercetin, piperine and IS was executed on a Waters Acquity BEH C18 column (50.0 mm × 2.1 mm, 1.7 µm) using formic acid both (0.1% w/v) in water (A) and acetonitrile (B) as the mobile phase in gradient mode. For detection purposes, positive electrospray ionization (ESI) mode was used with multiple reaction monitoring (MRM) mode for estimation using [M + H]+ fragment ions m/z 303.04 → 152.9 for quercetin, 286.12 → 201.04 for piperine and 287.01 → 136.93 for IS. The method was linear over the calibration range of 0.1-200 ng mL-1. The lower limit of quantification (LLOQ) of quercetin and piperine was obtained as 0.1 ng mL-1 in rat plasma, along with negligible matrix effect and acceptable stability. Furthermore, the bioanalytical method was successfully implemented to determine the pharmacokinetic profiles of quercetin-and piperine-enriched nanostructured lipid carriers (NLCs) in rat plasma after oral administration. The enhancement in the oral bioavailability of quercetin and piperine was 20.72 and 4.67 fold, respectively, compared to their native pristine dispersions. Future exploration of the concentrations of these active constituents in human plasma and organs is feasible using this sensitive, validated UPLC/ESI/MS/MS method.

Estimation of parabens and bisphenols in maternal products and urinary concentrations in Indian pregnant women: daily intake and health risk assessment.

The presence of parabens and bisphenols in maternal products and usage during pregnancy have raised serious concern about their possible harm to pregnant women. The concentrations of six parabens and eight bisphenols were quantified by high-performance liquid chromatography-tandem mass spectrometry in the samples of commercially available herbal-based ayurvedic maternal products and urine of healthy pregnant women from Assam, India. Methyl paraben (MP) and bisphenol AF (BPAF) were found to be more dominant in the maternal products, whereas MP, bisphenol A (BPA), and BPAF were dominant in urine samples of healthy pregnant women. The sum of the mean concentrations of all forms of parabens and bisphenols in maternal products were 48,308.50 ng/g and 542.42 ng/g, respectively, and urine 101.33 ng/mL and 23.42 ng/mL, respectively. The estimated daily intake (EDI) of total parabens and bisphenols in maternal products were 7378.02 and 19.78 ng/kg body weight/day, respectively. EDI of total parabens and bisphenols from urinary concentrations were 690.12 and 111.33 µg/kg body weight/day, respectively. The concentrations of butyl (BP) and heptyl (HP) parabens have a significant positive correlation with birth weight. The hazard quotient (HQ) value of MP, EP, and BPA was less than 1, and margin of exposure (MOE) identified potential risk associated with propyl paraben. Results from Monte-Carlo risk assessment analysis did not exceed the acceptable daily intake (ADI). Our results showed that higher concentrations of parabens and bisphenols are present in maternal products and the urine of healthy pregnant women. Hence maternal products containing bisphenols and parabens should be used cautiously during pregnancy to avoid maternal and fetal complications.

Transporter-mediated drug-drug interactions: advancement in models, analytical tools, and regulatory perspective.

Drug-drug interactions mediated by transporters are a serious clinical concern hence a tremendous amount of work has been done on the characterization of the transporter-mediated proteins in humans and animals. The underlying mechanism for the transporter-mediated drug-drug interaction is the induction or inhibition of the transporter which is involved in the cellular uptake and efflux of drugs. Transporter of the brain, liver, kidney, and intestine are major determinants that alter the absorption, distribution, metabolism, excretion profile of drugs, and considerably influence the pharmacokinetic profile of drugs. As a consequence, transporter proteins may affect the therapeutic activity and safety of drugs. However, mounting evidence suggests that many drugs change the activity and/or expression of the transporter protein. Accordingly, evaluation of drug interaction during the drug development process is an integral part of risk assessment and regulatory requirements. Therefore, this review will highlight the clinical significance of the transporter, their role in disease, possible cause underlying the drug-drug interactions using analytical tools, and update on the regulatory requirement. The recent in-silico approaches which emphasize the advancement in the discovery of drug-drug interactions are also highlighted in this review. Besides, we discuss several endogenous biomarkers that have shown to act as substrates for many transporters, which could be potent determinants to find the drug-drug interactions mediated by transporters. Transporter-mediated drug-drug interactions are taken into consideration in the drug approval process therefore we also provided the extrapolated decision trees from in-vitro to in-vivo, which may trigger the follow-up to clinical studies.