Search results for: liver X receptor

Authors: Osama M. Ahmed, Walaa G. Hozayen, Haidy Tamer Abo Sree

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The effect of doxorubicin (4 mg/kg b.w.week) without or with oral administration of ethanolic purslane (Portulaca oleracea) shoot (leaves and stems) extract (50 mg/kg b.w.day) or ethanolic purslane seeds extract (50 mg/kg b.w.day) co-treatments for 6 weeks was evaluated in adult male rats. There was an increase in serum levels of ALT, AST, ALP, GGT and total bilirubin. In addition, hepatic glutathine, glutathione transferase, peroxidase, SOD, CAT activities were decreased while lipid peroxidation in the liver was increased. Co-administration of ethanolic purslane and seed extracts successfully improved the adverse changes in the liver functions with an increase in antioxidants activities and reduction of lipid peroxidation.

Keywords: antioxidants, doxorubicin, hepatotoxicity, purslane

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Authors: Anjani Kumar Tiwari, Neelam Kumari, Anil Mishra

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The 18-kDa translocator protein (TSPO) previously called as peripheral benzodiazepine receptor, is proven biomarker for variety of neuroinflammation. TSPO is tryptophane rich five transmembranal protein found on outer mitochondrial membrane of steroid synthesising and immunomodulatory cells. In case of neuronal damage or inflammation the expression level of TSPO get upregulated as an immunomodulatory response. By utilizing Benzoxazolone as a basic scaffold, series of TSPO ligands have been designed followed by their screening through in silico studies. Synthesis has been planned by employing convergent methodology in six high yielding steps. For the synthesized ligands the ‘in vitro’ assay was performed to determine the binding affinity in term of Ki. On ischemic rat brain, autoradiography studies were also carried to check the specificity and affinity of the designed radiolabelled ligand for TSPO.Screening was performed on the basis of GScore of CADD based schrodinger software. All the modified and better prospective compound were successfully carried out and characterized by spectroscopic techniques (FTIR, NMR and HRMS). In vitro binding assay showed best binding affinity Ki = 6.1+ 0.3 for TSPO over central benzodiazepine receptor (CBR) Ki > 200. ARG studies indicated higher uptake of two analogues on the lesion side compared with that on the non-lesion side of ischemic rat brains. Displacement experiments with unlabelled ligand had minimized the difference in uptake between the two sides which indicates the specificity of the ligand towards TSPO receptor.

Keywords: TSPO, PET, imaging, Acetamidobenzoxazolone

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Authors: Aya Gamal Khattab

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Chronic infection with the hepatitis C virus (HCV) is now the leading cause of liver-related morbidity and mortality; Currently, alanine aminotransferase ALT measurement is not only widely used in detecting the incidence, development, and prognosis of liver disease with obvious clinical symptoms, but also provides reference on screening the overall health status during health check-ups. Exercise is a low-cost, reliable and sustainable therapy for many chronic diseases. Low-volume high intensity interval training HIT is time efficient while also having wider application to different populations including people at risk for chronic inflammatory diseases. Purpose of this study was to investigate the effect of low volume high intensity interval training on ALT, AST in HCV patients. All practical work was done in outpatient physiotherapy clinic of Suez Canal Authority Hospitals. Forty patients both gender (27 male, 13 female), age ranged (40-60) years old submitted to low volume high intensity interval training on treadmill for two months three sessions per week. Each session consisting of five min warming up, two bouts for 10 min each bout consisting of 30 sec - 1 min of high intensity (75%-85%) HRmax then two to four min active recovery at intensity (40%-60%) HRmax, so the sum of high intensity intervals was one to two min for each session and four to eight min active recovery, and ends with five min cooling down. ALT and AST were measured before starting exercise session and 2 months later after finishing the total exercise sessions through blood samples. Results showed significant decrease in ALT, AST with improvement percentage (18.85%), (23.87%) in the study, so the study concluded that low volume high intensity interval training had a significant effect in lowering the level of circulating liver enzymes (ALT, AST) which means protection of hepatic cells and restoration of its function.

Keywords: alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatitis C (HCV), low volume high intensity interval training

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Authors: Öznur Özge Özcan, Canan Sercan, Hamza Kulaksız, Mesut Karahan, Korkut Ulucan

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Genetic structure is very important to understand the brain dopamine system which is related to athletic performance. Hopefully, there will be enough studies about athletics performance in the terms of addiction-related genetic markers in the future. In the present study, we intended to investigate the Receptor-2 Gene (DRD2) rs1800497, which is related to brain dopaminergic system. 10 sprinter and 10 endurance athletes were enrolled in the study. Real-Time Polymerase Chain Reaction method was used for genotyping. According to results, A1A1, A1A2 and A2A2 genotypes in athletes were 0 (%0), 3 (%15) and 17 (%85). A1A1 genotype was not found and A2 allele was counted as the dominating allele in our cohort. These findings show that dopaminergic mechanism effects on sport genetic may be explained by the polygenic and multifactorial view.

Keywords: addiction, athletic performance, genotype, sport genetics

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Authors: Mahya Naghipoor

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Purpose: Non-small cell lung cancer (NSCLC) is the most common lung cancer type. Epidermal growth factor receptor (EGFR) mutation is the main reason which causes NSCLC. Computed tomography (CT) is used for diagnosis and prognosis of lung cancers because of low price and little invasion. Semantic analyses of qualitative CT features are based on visual evaluation by radiologist. However, the naked eye ability may not assess all image features. On the other hand, radiomics provides the opportunity of quantitative analyses for CT images features. The aim of this review study was comparing accuracy of semantic and radiomics features in prognosis of EGFR mutation in NSCLC. Methods: For this purpose, the keywords including: non-small cell lung cancer, epidermal growth factor receptor mutation, semantic, radiomics, feature, receiver operating characteristics curve (ROC) and area under curve (AUC) were searched in PubMed and Google Scholar. Totally 29 papers were reviewed and the AUC of ROC analyses for semantic and radiomics features were compared. Results: The results showed that the reported AUC amounts for semantic features (ground glass opacity, shape, margins, lesion density and presence or absence of air bronchogram, emphysema and pleural effusion) were %41-%79. For radiomics features (kurtosis, skewness, entropy, texture, standard deviation (SD) and wavelet) the AUC values were found %50-%86. Conclusions: In conclusion, the accuracy of radiomics analysis is a little higher than semantic in prognosis of EGFR mutation in NSCLC.

Keywords: lung cancer, radiomics, computer tomography, mutation

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Authors: A. Newman-Tancredi, R. Depoortère, P. Gruca, E. Litwa, M. Lason, M. Papp

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The N-methyl-D-aspartic acid (NMDA) receptor antagonist, ketamine, can elicit rapid-acting antidepressant (RAAD) effects in treatment-resistant patients, but it requires parenteral co-administration with a classical antidepressant under medical supervision. In addition, ketamine can also produce serious side effects that limit its long-term use, and there is much interest in identifying RAADs based on ketamine’s mechanism of action but with safer profiles. Ketamine elicits GABAergic interneuron inhibition, glutamatergic neuron stimulation, and, notably, activation of serotonin 5-HT1A receptors in the prefrontal cortex (PFC). Direct activation of the latter receptor subpopulation with selective ‘biased agonists’ may therefore be a promising strategy to identify novel RAADs and, consistent with this hypothesis, the prototypical cortical biased agonist, NLX-101, exhibited robust RAAD-like activity in the chronic mild stress model of depression (CMS). The present study compared the effects of a novel, selective 5-HT1A receptor-biased agonist, NLX-204, with those of ketamine and NLX-101. Materials and methods: CMS procedure was conducted on Wistar rats; drugs were administered either intraperitoneally (i.p.) or by bilateral intracortical microinjection. Ketamine: 10 mg/kg i.p. or 10 µg/side in PFC; NLX-204 and NLX-101: 0.08 and 0.16 mg/kg i.p. or 16 µg/side in PFC. In addition, interaction studies were carried out with systemic NLX-204 or NLX-101 (each at 0.16 mg/kg i.p.) in combination with intracortical WAY-100635 (selective 5-HT1A receptor antagonist; 2 µg/side) or muscimol (GABA-A receptor agonist, 12.5 ng/side). Anhedonia was assessed by CMS-induced decrease in sucrose solution consumption; anxiety-like behavior was assessed using the Elevated Plus Maze (EPM), and cognitive impairment was assessed by the Novel Object Recognition (NOR) test. Results: A single administration of NLX-204 was sufficient to reverse the CMS-induced deficit in sucrose consumption, similarly to ketamine and NLX-101. NLX-204 also reduced CMS-induced anxiety in the EPM and abolished CMS-induced NOR deficits. These effects were maintained (EPM and NOR) or enhanced (sucrose consumption) over a subsequent 2-week period of treatment. The anti-anhedonic response of the drugs was also maintained for several weeks Following treatment discontinuation, suggesting that they had sustained effects on neuronal networks. A single PFC administration of NLX-204 reversed deficient sucrose consumption, similarly to ketamine and NLX-101. Moreover, the anti-anhedonic activities of systemic NLX-204 and NLX 101 were abolished by coadministration with intracortical WAY-100635 or muscimol. Conclusions: (i) The antidepressant-like activity of NLX-204 in the rat CMS model was as rapid as that of ketamine or NLX-101, supporting targeting cortical 5-HT1A receptors with selective, biased agonists to achieve RAAD effects. (ii)The anti-anhedonic activity of systemic NLX-204 was mimicked by local administration of the compound in the PFC, confirming the involvement of cortical circuits in its RAAD-like effects. (iii) Notably, the effects of systemic NLX-204 and NLX-101 were abolished by PFC administration of muscimol, indicating that they act by (indirectly) eliciting a reduction in cortical GABAergic neurotransmission. This is consistent with ketamine’s mechanism of action and suggests that there are converging NMDA and 5-HT1A receptor signaling cascades in PFC underlying the RAAD-like activities of ketamine and NLX-204. Acknowledgements: The study was financially supported by NCN grant no. 2019/35/B/NZ7/00787.

Keywords: depression, ketamine, serotonin, 5-HT1A receptor, chronic mild stress

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Authors: Alireza Barari, Maryam Firozi-Niyaki, Maryam Kamarlouei

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Background: Herbs have a strong anti-cancer effect. Also, exercise is one of several lifestyle factors known to lower the risk of developing cancer. The aim of this study was to investigate the effect of endurance training and taxol on cyclooxygenase-2 and prostaglandin E2 in the liver tissue of mice with cervical cancer. Materials and Methods: In this experimental study, 35 female C57 mice were randomly divided into 5 groups (n=7 in each group): control (healthy), control (cancer), complement (cancer), training-supplementary (cancer) and training (cancer). The implantation of cancerous tumors was performed under the skin of the upper pelvis. The training group completed the endurance training protocol, which included 3 sessions per week, 50 minutes per session, at a speed of 14-18 m/s for six weeks. A dose of 60 mg/kg/day of pure taxol was injected intra peritoneally. The dependent variables of this study were measured 24 hours after the last training session by ELISA. Results: The results showed that the use of taxol and endurance training reduced the levels of cyclooxygenase-2 and prostaglandin E2 in the liver tissues of C57 mice with cervical cancer. Conclusion: Induction of the cancerous tissue in mice with cervical cancer increases the levels of cyclooxygenase-2 and prostaglandin E2 and endurance training along with taxol may reduce these levels.

Keywords: cervical cancer, taxol, endurance training, cyclooxygenase-2, prostaglandin E2

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Authors: Y. A. Shettima, M. A. Tijjani, S. Modu, F. I. Abdulrahman, B. M. Abubakar

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The toxicity profile of the methanolic extract of Punica granatum L. fruit rind was studied in normal rats. The rats were administered orally by intubating graded doses of 150, 250, 500 and 750 mg/kg body weight of the extract for 28 days and the effects on biochemical parameters and histology of the liver and kidney were evaluated. There was a significant increase (P<0.05) in the levels of liver enzymes of the rats that received the highest dose of 750 mg/kg body weight. The AST and ALT levels were 41.59±0.18 ALP and 9.25±0.29 IU/L, respectively, while the ALP level was 15.68±10 IU/L.There was a significant difference in the albumin and globulin levels; 3.72±0.05 and 4.05±0.13 g/dl, respectively. Serum urea and creatinine levels remained normal, as well as the electrolyte levels. The increase in sodium concentration observed was not statistically significant (P≥0.05) when the control group (131.50±3.11) was compared with the experimental groups (132.25±3.86, 132.75±3.86, 133.50±3.11 and 134.00±1.83). The increase in potassium concentration was not statistically significant (P≥0.05) when the control group with a value of 95.50±3.51 mmol/L was compared with the experimental groups 98.00±3.16, 99.25±2.22, 99.79±0.36 and 99.99±0.02 mmol/L. The increase observed in bicarbonate concentration was not statistically significant (P≥0.05) when the control group with a value of 20.75±1.71 mmol/L was compared with the experimental groups 21.68±0.62, 24.25±2.99, 24.50±3.42, 25.50±2.65 mmol/L.

Keywords: punical granatum, methanolic, ALT, AST, electrolytes, histology

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Authors: Hatem A. El-Mezayen, Hossam Darwesh

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Background/Aim: Hepatocellular carcinoma (HCC) is often diagnosed at advanced stage where effective therapies are lacking. Identification of new scoring system is needed to discriminate HCC patients from those with chronic liver disease. Based on the link between vascular endothelial growth factor (VEGF) and HCC progression, we aimed to develop a novel score based on combination of VEGF and routine laboratory tests for early prediction of HCC. Methods: VEGF was assayed for HCC group (123), liver cirrhosis group (210) and control group (50) by Enzyme Linked Immunosorbent Assay (ELISA). Data from all groups were retrospectively analyzed including α feto protein (AFP), international normalized ratio (INR), albumin and platelet count, transaminases, and age. Areas under ROC curve were used to develop the score. Results: A novel index named hepatocellular carcinoma-vascular endothelial growth factor score (HCC-VEGF score)=1.26 (numerical constant) + 0.05 ×AFP (U L-1)+0.038 × VEGF(ng ml-1)+0.004× INR –1.02 × Albumin (g l-1)–0.002 × Platelet count × 109 l-1 was developed. HCC-VEGF score produce area under ROC curve of 0.98 for discriminating HCC patients from liver cirrhosis with sensitivity of 91% and specificity of 82% at cut-off 4.4 (ie less than 4.4 considered cirrhosis and greater than 4.4 considered HCC). Conclusion: Hepatocellular carcinoma-VEGF score could replace AFP in HCC screening and follow up of cirrhotic patients.

Keywords: Hepatocellular carcinoma, cirrhosis, HCV, diagnosis, tumor markers

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Authors: Aml M. El-Sharkawy, Hossam M. Darwesh

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Abstract— Background/Aim: Hepatocellular carcinoma (HCC) is often diagnosed at advanced stage where effective therapies are lacking. Identification of new scoring system is needed to discriminate HCC patients from those with chronic liver disease. Based on the link between tumor associated trypsin inhibitor (TATI) and HCC progression, we aimed to develop a novel score based on combination of TATI and routine laboratory tests for early prediction of HCC. Methods: TATI was assayed for HCC group (123), liver cirrhosis group (210) and control group (50) by Enzyme Linked Immunosorbent Assay (ELISA). Data from all groups were retrospectively analyzed including α feto protein (AFP), international normalized ratio (INR), albumin and platelet count, transaminases, and age. Areas under ROC curve were used to develop the score. Results: A novel index named hepatocellular carcinoma-vascular endothelial growth factor score (HCC-TATI score) = 3.1 (numerical constant) + 0.09 ×AFP (U L-1) + 0.067 × TATI (ng ml-1) + 0.16 × INR – 1.17 × Albumin (g l-1) – 0.032 × Platelet count × 109 l-1 was developed. HCC-TATI score produce area under ROC curve of 0.98 for discriminating HCC patients from liver cirrhosis with sensitivity of 91% and specificity of 82% at cut-off 6.5 (ie less than 6.5 considered cirrhosis and greater than 4.4 considered HCC). Conclusion: Hepatocellular carcinoma-TATI score could replace AFP in HCC screening and follow up of cirrhotic patients.

Keywords: Hepatocellular carcinoma, cirrhosis, HCV, diagnosis, TATI

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Authors: Huong M. T. Nguyen, Hoa A. P. Nguyen, Chi V. Phan, Mai P. T. Nguyen, Ngoc D. Ngo, Van T. Ta, Hai T. Le

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Wilson’s disease is an autosomal recessive disorder of copper metabolism, which is caused by mutation in copper- transporting P-type ATPase (ATP7B). The mechanism of this disease is a failure of hepatic excretion of copper to the bile, and it leads to copper deposits in the liver and other organs. Most clinical symptoms of Wilson’s disease can present as liver disease and/or neurologic disease. Objective: The goal of the study is prenatal diagnosis for pregnant women at high risk of Wilson’s disease in Northern Vietnam. Material and method: Three probands with clinically diagnosed liver disease were detected in the mutations of 21 exons and exon-intron boundaries of the ATP7B gene by direct Sanger-sequencing. Prenatal diagnoses were performed by amniotic fluid sampling from pregnant women in the 16th-18th weeks of pregnancy after the genotypes of parents with the probands were identified. Result: A total of three different mutations of the probands, including of S105*, P1052L, P1273G, were detected. Among three fetuses which underwent prenatal genetic testing, one fetus was homozygote; two fetuses were carriers. Conclusion: Genetic testing provided a useful method for prenatal diagnosis, and is a basis for genetic counseling.

Keywords: ATP7B gene, genetic testing, prenatal diagnosis, pedigree, Wilson disease

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Authors: Swetha Priya Darshini Thammadi, Sateesh Kumar Pisini, Sanjay Kumar Shukla

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Source apportionment using Dispersion model depends primarily on the quality of Emission Inventory. In the present study, a CMB receptor model has been used to identify the sources of PM_2.5, while the AERMOD dispersion model has been used to account for missing sources of PM_2.5 in the Emission Inventory. A statistical approach has been developed to quantify the missing sources not considered in the Emission Inventory. The inventory of each grid was improved by adjusting emissions based on road lengths and deficit in measured and modelled concentrations. The results showed that in CMB analyses, fugitive sources - soil and road dust - contribute significantly to ambient PM_2.5 pollution. As a result, AERMOD significantly underestimated the ambient air concentration at most locations. The revised Emission Inventory showed a significant improvement in AERMOD performance which is evident through statistical tests.

Keywords: CMB, GIS, AERMOD, PM₂.₅, fugitive, emission inventory

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Authors: Khaled S. Al Salhen

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Aldehyde oxidase is molybdo-flavoenzyme involved in the oxidation of hundreds of endogenous and exogenous and N-heterocyclic compounds and environmental pollutants. Uncharged N-heterocyclic aromatic compounds such phenanthridine are commonly distributed pollutants in soil, air, sediments, surface water and groundwater, and in animal and plant tissues. Phenanthridine as uncharged N-heterocyclic aromatic compound was incubated with partially purified aldehyde oxidase from rainbow trout fish liver. Reversed-phase HLPC method was used to separate the oxidation products from phenanthridine and the metabolite was identified. The 6(5H)-phenanthridinone was identified the major metabolite by partially purified aldehyde oxidase from fish liver. Kinetic constant for the oxidation reactions were determined spectrophotometrically and showed that this substrate has a good affinity (Km = 78 ± 7.6 µM) for hepatic aldehyde oxidase, coupled with a relatively high oxidation rate (0.77± 0.03 nmol/min/mg protein). In addition, the kinetic parameters of hepatic fish aldehyde oxidase towards the phenanthridine substrate indicate that in vitro biotransformation by hepatic fish aldehyde oxidase will be a significant pathway. This study confirms that partially purified aldehyde oxidase from fish liver is indeed the enzyme responsible for the in vitro production 6(5H)-phenanthridinone metabolite as it is a major metabolite by mammalian aldehyde oxidase.

Keywords: aldehyde oxidase, fish, phenanthridine, specificity

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Authors: Inas Raafat, Azza El Bassiouny, Waldemar L. Olszewsky, Nagui E. Mikhail, Mona Nossier, Nora E. I. El-Bassiouni, Mona Zoheiry, Houda Abou Taleb, Noha Abd El-Aal, Ali Baioumy, Shimaa Attia

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Background: Orthotopic liver transplantation is an established treatment for patients with severe acute and end-stage chronic liver disease. The shortage of donor organs continues to be the rate-limiting factor for liver transplantation throughout the world. Hepatocyte transplantation is a promising treatment for several liver diseases and can, also, be used as a "bridge" to liver transplantation in cases of liver failure. Aim of the work: This study was designed to develop a highly efficient protocol for isolation and transplantation of hepatocytes in experimental Lewis rat model to provide satisfactory guidelines for future application on humans.Materials and Methods: Hepatocytes were isolated from the liver by double perfusion technique and bone marrow cells were isolated by centrifugation of shafts of tibia and femur of donor Lewis rats. Recipient rats were subjected to sub-lethal dose of irradiation 2 days before transplantation. In a laparotomy operation the spleen was injected by freshly isolated hepatocytes and bone marrow cells were injected intravenously. The animals were sacrificed 45 day latter and splenic sections were prepared and stained with H & E, PAS AFP and Prox1. Results: The data obtained from this study showed that the double perfusion technique is successful in separation of hepatocytes regarding cell number and viability. Also the method used for bone marrow cells separation gave excellent results regarding cell number and viability. Intrasplenic engraftment of hepatocytes and live tissue formation within the splenic tissue were found in 70% of cases. Hematoxylin and eosin stained splenic sections from 7 rats showed sheets and clusters of cells among the splenic tissues. Periodic Acid Schiff stained splenic sections from 7 rats showed clusters of hepatocytes with intensely stained pink cytoplasmic granules denoting the presence of glycogen. Splenic sections from 7 rats stained with anti-α-fetoprotein antibody showed brownish cytoplasmic staining of the hepatocytes denoting positive expression of AFP. Splenic sections from 7 rats stained with anti-Prox1 showed brownish nuclear staining of the hepatocytes denoting positive expression of Prox1 gene on these cells. Also, positive expression of Prox1 gene was detected on lymphocytes aggregations in the spleens. Conclusions: Isolation of liver cells by double perfusion technique using collagenase buffer is a reliable method that has a very satisfactory yield regarding cell number and viability. The intrasplenic route of transplantation of the freshly isolated liver cells in an immunocompromised model was found to give good results regarding cell engraftment and tissue formation. Further studies are needed to assess function of engrafted hepatocytes by measuring prothrombin time, serum albumin and bilirubin levels.

Keywords: Lewis rats, hepatocytes, BMCs, transplantation, AFP, Prox1

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Authors: Qing Cao, Fei Wang, Yu-Qiang Wang, Li-Ya Huang, Tian-Tian Sang, Shu-Yan Chen

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Aims: TNF Receptor-Associated Factor 6 (TRAF6) acts as a multifunctional regulator of the Transforming Growth Factor (TGF)-β signaling pathway, and mediates Smad-independent JNK and p38 activation via TGF-β. This study was performed to test the hypothesis that TGF-β/TRAF6 is essential for angiotensin-II (Ang II)-induced differentiation of rat c-kit+ Cardiac Stem Cells (CSCs). Methods and Results: c-kit+ CSCs were isolated from neonatal Sprague Dawley (SD) rats, and their c-kit status was confirmed with immunofluorescence staining. A TGF-β type I receptor inhibitor (SB431542) or the small interfering RNA (siRNA)-mediated knockdown of TRAF6 were used to investigate the role of TRAF6 in TGF-β signaling. Rescue of TRAF6 siRNA transfected cells with a 3'UTR deleted siRNA insensitive construct was conducted to rule out the off target effects of the siRNA. TRAF6 dominant negative (TRAF6DN) vector was constructed and used to infect c-kit+ CSCs, and western blotting was used to assess the expression of TRAF6, JNK, p38, cardiac-specific proteins, and Wnt signaling proteins. Physical interactions between TRAF6 and TGFβ receptors were studied by coimmunoprecipitation. Cardiac differentiation was suppressed in the absence of TRAF6. Forced expression of TRAF6 enhanced the expression of TGF-β-activated kinase1 (TAK1), and inhibited Wnt signaling. Furthermore, TRAF6 increased the expression of cardiac-specific proteins (cTnT and Cx-43) but inhibited the expression of Wnt3a. Conclusions: Our data suggest that TRAF6 plays an important role in Ang II induced differentiation of c-kit+ CSCs via the non-canonical signaling pathway.

Keywords: cardiac stem cells, differentiation, TGF-β, TRAF6, ubiquitination, Wnt

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Authors: Lydia Foucan, Christine Rambhojan, Rachel Billy, Christophe Armand, Carl-Thony Michel, Jean-Marc Lacorte, Laurent Larifla

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Leptin, an adipocyte-derived hormone, modulates insulin secretion and action via the leptin receptor (LEPR) that is expressed in pancreatic beta cells, adipose tissue, and muscle. Several polymorphisms have been described in the human LEPR gene including p.K109R (rs1137100), p.Q223R (rs1137101) and p.K656N (rs1805094) polymorphisms. The role of these polymorphisms is not yet studied in Guadeloupian population. Our aim was to explore the association of LEPR polymorphisms (K109R, Q223R and K656N) with leptin levels and obesity in non-diabetic Afro-Caribbean subjects. Genotypic analysis of the three polymorphisms was performed in 425 subjects using TaqMan and KASPar Assays. Serum leptin was measured with ELISA kits Biovendor® (RD191001100). Logistic regressions were used for assessment of statistical associations. Mean age was 47.6 ± 12.7 years. Among the participants, 238 (56 %) were women, 124 (30%) were obese and 155 (36.5%) had abdominal obesity. Carriers of LEPR K656N rs1805094 rare allele had significant higher frequencies of obesity (P = 0.007), abdominal obesity (P = 0.004) and metabolic syndrome (P = 0.021) but mean leptin level was not significantly different between both groups (P = 0.075). Odds ratios, adjusted for age and sex associated with presence of rs1805094 rare allele were 1.8 (1.1-2.9), P = 0.012 for obesity, 2.0 (1.2-3.3), P = 0.008 for abdominal obesity and 1.8 (1.1-3.0), P = 0.031 for MetS. No significant association was found with K109R, Q223R. These findings suggest that the K656N polymorphism (but not the K109R or Q223R polymorphism) of LEPR is associated with obesity, abdominal obesity and metabolic syndrome in this Afro-Caribbean non-diabetic population.

Keywords: Afro-Caribbean, leptin levels, leptin receptor gene polymorphisms, obesity

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Authors: David Tanujaya Kurniawan

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Background: Liver injury caused by paracetamol is marked by increased serum transaminase levels. Turmeric is a local herb that is available in large quantities and inexpensive in contradiction to its substantial benefits, including its potency to increase glutathione production and regenerate hepatocyte into normal condition. Aim: The aim of this study was to analyze the potencial treatment effect of turmeric rhizome extract to reduce serum transaminase level on paracetamol induced liver toxicity in rats. Methods: This study was a laboratory experimental research with post-test only controlled group design. A group of 24 Wistar white male rats was induced with paracetamol 360 mg/kg body weight for 10 days. The group was then separated into four groups: the first and the second was treated with 100 mg/kg body weight and 150 mg/kg body weight of turmeric rhizome extract, subsequently, the third as positive control was given 27 mg/kg body weight of lesichol, while the fourth as negative control was given CMC-Na 1%. Each of this treatment was given for seven days. At the end of the study, the blood samples were taken to measure SGOT and SGPT levels. The one-way Anova test revealed significant difference in mean of SGPT level (p=0,001). The LSD test showed significant differences of SGPT levels in both treatment groups and negative control group. However, there was no sgnificant difference between positive control and both treatment groups. Conclusion: Curcuma domestica Val. rhizome extract could not reduce SGOT level, but it reduced SGPT level significantly.

Keywords: Curcuma domestica val., SGOT, SGPT, paracetamol, liver toxicity

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Authors: Maha Z. Rizk, Sami A. Fattah, Heba M. Darwish, Sanaa A. Ali, Mai O. Kadry

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Although it is known that nano-TiO2 and other nanoparticles can induce liver toxicity, the mechanisms and the molecular pathogenesis are still unclear. The present study investigated some biochemical indices of nano-sized Titanium dioxide (TiO2 NPS) toxicity in mice liver and the ameliorative efficacy of individual and combined doses of idebenone, carnosine and vitamin E. Nano-anatase TiO2 (21 nm) was administered as a total oral dose of 2.2 gm/Kg daily for 2 weeks followed by the afore-mentioned antioxidants daily either individually or in combination for 1month. TiO2-NPS induced a significant elevation in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic oxidative stress biomarkers [lipid peroxides (LP), and nitric oxide levels (NOX), while it significantly reduced glutathione reductase (GR), reduced glutathione (GSH) and glutathione peroxidase(GPX) levels. Moreover the quantitative RT-PCR analysis showed that nano-anatase TiO2 can significantly alter the mRNA and protein expressions of the fibrotic factors TGF-B1, VEGFand Smad-2. Histopathological examination of hepatic tissue reinforced the previous biochemical results. Our results also implied that inflammatory responses and liver injury may be involved in nano-anatase TiO2-induced liver toxicity Tumor necrosis factor-α (TNF-α) and Interleukin -6 (IL-6) and increased the percent of DNA damage which was assessed by COMET assay in addition to the apoptotic marker Caspase-3. Moreover mRNA gene expression observed by RT-PCR showed a significant overexpression in nuclear factor relation -2 (Nrf2), nuclear factor kappa beta (NF-Kβ) and the apoptotic factor (bax), and a significant down regulation in the antiapoptotic factor (bcl2) level. In conclusion idebenone, carnosine and vitamin E ameliorated the deviated previously mentioned parameters with variable degrees with the most pronounced role in alleviating the hazardous effect of TiO2 NPS toxicity following the combination regimen.

Keywords: Nano-anatase TiO2, TGF-B1, SMAD-2

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Authors: Mark P. Mayala, Henry Mayala, Khuzeima Khanbhai

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Background: Heart failure has been a rising concern in Tanzania. New drugs have been introduced, including the group of drugs called Angiotensin receptor Neprilysin Inhibitor (ARNI), but due to their high cost, angiotensin-converting enzymes inhibitors (ACEIs) and Angiotensin receptor blockers (ARBs) have been mostly used in Tanzania. However, according to our knowledge, the efficacy comparison of the two groups is yet to be studied in Tanzania. The aim of this study was to compare the efficacy of ACEIs and ARBs among patients with heart failure. Methodology: This was a hospital-based prospective cohort study done at Jakaya Kikwete Cardiac Institution (JKCI), Tanzania, from June to December 2020. Consecutive enrollment was done until fulfilling the inclusion criteria. Clinical details were measured at baseline. We assessed the relationship between ARBs and ACEIs users with N-terminal pro-brain natriuretic peptide (NT pro-BNP) levels at admission and at 1-month follow-up using a chi-square test. A Kaplan-Meier curve was used to estimate the survival time of the two groups. Results: 155 HF patients were enrolled, with a mean age of 48 years, whereby 52.3% were male, and their mean left ventricular ejection fraction (LVEF) was 37.3%. 52 (33.5%) heart failure patients were on ACEIs, 57 (36.8%) on ARBs, and 46 (29.7%) were neither using ACEIs nor ARBs. At least half of the patients did not receive a guideline-directed medical therapy (GDMT), with only 82 (52.9%) receiving a GDMT. A drop in NT pro-BNP levels was observed during admission and at 1-month follow-up on both groups, from 6389.2 pg/ml to 4000.1 pg/ml for ARB users and 5877.7 pg/ml to 1328.2 pg/ml for the ACEIs users. There was no statistical difference between the two groups when estimated by the Kaplan-Meier curve, though more deaths were observed in those who were neither on ACEIs nor ARBs, with a calculated P value of 0.01. Conclusion: This study demonstrates that ACEIs have more efficacy and overall better clinical outcome than ARBs, but this should be taken under the patient-based case, considering the side effects of ACEIs and patients’ adherence.

Keywords: angiotensin converting enzymes inhibitors, angiotensin receptor blockers, guideline direct medical therapy, N-terminal pro-brain natriuretic peptide

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Authors: Fakhrosadat Sajjadian

Abstract:

The majority of deaths in cancer patients are caused by distant metastasis. Breast cancer shows a unique spread pattern, often affecting bone, liver, lung, and brain. Breast cancer can be categorized into various subtypes according to gene expression patterns, and these subtypes exhibit specific preferences for organs where metastasis occurs. Breast tumors with luminal characteristics have a preference for spreading to the bone, whereas basal-like breast cancer (BLBC) shows a tendency to metastasize to the lungs. Still, the mechanisms behind this particular pattern of metastasis in organs have yet to be fully understood. In this evaluation, we will outline the latest progress in molecular signaling pathways and treatment methods for breast cancer lung metastasis.

Keywords: lung cancer, liver cancer, diagnosis, BLBC, metastasis

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Authors: Alaa F. A. Bakr, Sherein S. Abdelgayed, Osama. S. EL-Tawil, Adel M. Bakeer

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Objective: This study was carried out to evaluate the cytotoxic and genotoxic effect of tartrazine in both male and female albino rats. Methodology: Forty adult male (20) and female (20) Sprague Dawley albino rats (120 - 150g) were obtained and distributed into four experimental groups; Group I; 10 untreated males, Group II; 10 untreated females, Group III; 10 treated males, and Group IV; 10 treated females. Body weight was recorded weekly, reduced glutathione (RGH), lipid peroxidation (SOD), and superoxide dismutase activity (MDA) in liver tissue were carried out, histopathological studies of brain, liver, and kidneys were performed, COMET assay was performed, all values were statistically analyzed. Results: Decrease in the activity of RGH and SOD in the treated groups were reported, but there was a more significant decrease in the female treated group. MDA was increased in treated groups with tartrazine, moreover, it was more significant in the female treated group. Multiple histological lesions were developed in brain, liver, and kidneys. COMET showed positive results. Conclusion: Our study concluded that Tartrazine has a cytotoxic and genotoxic effect on albino rats and it was more significant in females than males.

Keywords: tartrazine, cytotoxicity, genotoxicity, histopathology, albino rats

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Authors: Yousuf M. Al Suleimani, Robin Hiley

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The endogenous phospholipid lysophosphatidylinositol (LPI) was recently identified as a novel ligand for the G protein-coupled receptor 55 (GPR55) and an inducer of intracellular Ca2+ [Ca2+]i release. This study attempts to characterize Ca2+ signals provoked by LPI in HEK293 cells engineered to stably express human GPR55 and to test cannabinoid ligand activity at GPR55. The study shows that treatment with LPI stimulates a sustained, oscillatory Ca2+ release. The response is characterized by an initial rapid rise, which is mediated by the Gαq-PLC-IP3 pathway, and this is followed by prolonged oscillations that require RhoA activation. Ca2+ oscillations are initiated by intracellular mechanisms and extracellular Ca2+ is only required to replenish Ca2+ lost from the cytoplasm. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoid anandamide, however, rimonabant and the CB1 receptor antagonist AM251 evoked GPR55-mediated [Ca2+]i. Thus, LPI is likely to be a key plasma membrane mediator of signaling events and changes in gene expression through GPR55 activation.

Keywords: lysophosphatidylinositol, calcium, GPR55, cannabinoid

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Authors: Salem Abdalla

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Objective: Dipyrone is a widely used, well tolerated analgesic drug which, however, is compromised by agranulocytosis as an adverse effect. Subsequent to no enzymatic hydrolysis, the primary metabolic step is N-demethylation of 4-methylaminoantipyrine (4-MAA) to 4-aminoantipyrine (4-AA). The aim of the present study was to identify the cytochrome P-450 enzyme (CYP) mediating this reaction. Methods: We identified the relevant CYP using virus expressed isolated rat liver microsomes with chemical inhibition studies. The substrate of 4-methylaminantipyrine was employed at six different concentrations (25, 50, 100, 400, 800, and 1200 µmol/l) with varying concentrations of selective inhibitors of CYP1A2 (furafylline, fluvoxamine), CYP3A4 (ketoconazole), CYP2A6 (coumarin), CYP2D6 (quinidine), CYP2C19 (omeprazole, fluvoxamine, tranylcypromine), CYP2C9 (sulfaphenazole), and CYP1A1 (alpha-naphthoflavone). 4-MAA and 4-AA were analyzed by HPLC, and enzyme kinetic parameters (Km and Vmax) were determined by regression (Sigma plot 9.0). Results: The N-demethylation of 4-MAA by microsomes prepared from baculovirus-expressing human CYP was pronounced with CYP2C19. Intrinsic clearances of the most active enzymes were 0.092, 0.027, and 0.026 for the CYP enzymes 2C19, 2D6, and 1A2, respectively. Metabolism by rat liver microsomes was strongly inhibited by omeprazole (IC50 of 0.05). Conclusion: The enzyme CYP2C19 apparently has an important role in N-demethylation of 4-methylaminoantipyrine which should be further analyzed in clinical studies and which may also be interesting concerning the agranulocytosis.

Keywords: dipyrone, 4-methylaminoantipyrine (4-MAA), 4- aminoantipyrine (4-AA), metabolism, human CYP2C19

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Authors: Dipanshu Sur, Ratnabali Chakravorty

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Background: Polycystic ovary syndrome (PCOS) is the most common metabolic abnormality such as changes in lipid profile, diabetes, hypertension and metabolic syndrome occurring in young women of reproductive age. Low vitamin D levels were found to be associated with the development of obesity and insulin resistance in women with PCOS. Variants on vitamin D receptor (VDR) gene have also been related to metabolic comorbidities in general population. Aim: The aim of this case-control study was to investigate whether the VDR gene polymorphisms are associated with susceptibility to PCOS. Methods: Women with PCOS and a control group, all aged 16-40 years, were enrolled. Genotyping of VDR Fok-I (rs2228570), VDR Apa-I (rs7975232) as well as GC (rs2282679), DHCR7 (rs12785878) SNPs between groups were determined by using direct sequencing. Serum 25-hydroxyvitamin D [25(OH)] levels were measured by ELISA. Results: Mean serum 25(OH)D in the PCOS and control samples were 19.08±7 and 23.27±6.03 (p=0.048) which were significantly lower in PCOS patients compared with controls. CC genotype of the VDR Apa-I SNP was same frequent in PCOS (25.6%) and controls (25.6%) (OR: 0.9995; 95%CI: 0.528 to 1.8921; p= 0.9987). The CC genotype was also significantly associated with both lower E2 (p=0.031) and Androstenedione levels (p=0.062). We observed a significant association of GC polymorphism with 25(OH)D levels. PCOS women carrying the GG genotype (in GC genes) had significantly higher risk for vitamin D deficiency than women carrying the TT genotype. Conclusions: In conclusion, data from this study indicate that vitamin D levels are lower, and vitamin D deficiency more frequent, in PCOS than in controls. The present findings suggest that the Apa-I, Fok-I polymorphism of the VDR gene is associated with PCOS and seems to modulate ovarian steroid secretion. Further studies are needed to better clarify the biological mechanisms by which the polymorphism influences PCOS risk.

Keywords: vitamin D receptor, polymorphism, vitamin D, polycystic ovary syndrome

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Authors: Mohaya Farzin, Shahin Hassanpour, Morteza Zendehdel, Bita Vazir, Ahmad Asghari

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Aim: There are several differences between birds and mammals in terms of food intake regulation. Therefore, this study aimed to investigate the effects of the intracerebroventricular (ICV) injection of spexin and its interaction with nitric oxide, serotonin, and corticotropin receptors on central food intake regulation in broiler chickens. Materials and Methods: In experiment 1, chickens received ICV injection of saline, PCPA (p-chlorophenyl alanine,1.25 µg), spexin, and PCPA+spexin. In experiments 2-7, 8-OH-DPAT (5-HT1A agonist, 15.25 nmol), SB-242084 (5-HT2C receptor antagonist, 1.5µg), L-arginine (Precursor of nitric oxide, 200 nmol), L-NAME (nitric oxide synthetase inhibitor, 100 nmol), Astressin-B (CRF1/CRF2 receptor antagonist, 30 µg) and Astressin2-B (CRF2 receptor antagonist, 30 µg) were injected to chickens instead of the PCPA. Then, food intake was measured until 120 minutes after the injection. Results: Spexin significantly decreased food consumption (P<0.05). Concomitant injection of SB-242084+spexin attenuated spexin-induced hypophagia (P<0.05). Co-injection of L-arginine+spexin enhanced spexin-induced hypophagia, and this effect was reversed by L-NAME (P<0.05). Also, concomitant injection of Astressin-B + spexin or Astressin2-B + spexin enhanced spexin-induced hypophagia (P<0.05). Conclusions: Based on these observations, spexin-induced hypophagia may be mediated by nitric oxide and 5-HT2C, CRF1, and CRF2 receptors in neonatal broiler chickens.

Keywords: spexin, serotonin, corticotropin, nitric oxide, food intake, chicken

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Authors: Jun Yang, Ching-Liang Hsieh, Yi-Wen Lin

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Chronic inflammatory pain results from peripheral tissue injury or local inflammation to increase the release of protons, histamines, adenosine triphosphate, and several proinflammatory cytokines. Transient receptor potential vanilloid 1 (TRPV1) is involved in fibromyalgia, neuropathic, and inflammatory pain; however, its exact mechanisms in chronic inflammatory pain are still unclear. We investigate the analgesic effect of EA by injecting complete Freund’s adjuvant (CFA) in the hind paw of mice to induce chronic inflammatory pain ( > 14 d). Our results showed that EA significantly reduced chronic mechanical and thermal hyperalgesia in the chronic inflammatory pain model. Chronic mechanical and thermal hyperalgesia was also abolished in TRPV1−/− mice. TRPV1 increased in the dorsal root ganglion (DRG) and spinal cord (SC) at 2 weeks after CFA injection. The expression levels of downstream molecules such as pPKA, pPI3K, and pPKC increased, as did those of pERK, pp38, and pJNK. Transcription factors (pCREB and pNFκB) and nociceptive ion channels (Nav1.7 and Nav1.8) were involved in this process. Inflammatory mediators such as GFAP (Glial fibrillary acidic protein), S100B, and RAGE (Receptor for advanced glycation endproducts) were also involved. The expression levels of these molecules were reduced in EA (electroacupuncture) and TRPV1−/−mice but not in the sham EA group. The present study demonstrated that EA or TRPV1 gene deletion reduced chronic inflammatory pain through TRPV1 and related molecules. In addition, our data provided evidence to support the clinical use of EA for treating chronic inflammatory pain.

Keywords: auricular electric-stimulation, epileptic seizures, anti-inflammation, electroacupuncture

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Authors: Jibran Abbasy, Rizwan Sultan, Ammar Humayun, Tabish Chawla

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Background: Hydatid disease caused by Echinococcus Granulosus affects liver in 70-90% of cases. Dogs are the definitive host while humans are the accidental host. Modalities used for its treatment are especially important for our population as the disease is endemic in many Asian countries. The aim of the study was to perform an audit of the various modalities used for treatment of hydatid disease of liver and the response to each modality in tertiary care center of Pakistan. Materials and Methods: Retrospective audit of patients diagnosed and treated for Hydatid disease of the liver at Aga Khan University Hospital from 1st January 2007 to 31st December 2014 was completed. All patients aged 16 and above were included. Patients who had extra hepatic disease and missing records were excluded. Outcome measures were morbidity, mortality and recurrence of the disease. Results: During the study period 56 patients were treated for isolated hepatic hydatid disease and were included. Mean age was 39 years with 48% being females and 52% males. Most common presenting complaint was abdominal pain seen in 53% of patients(n=41). Duration of symptoms was less than 6 months in 74% (n=38). Mostly right lobe was involved in 69% (n=38).Most common treatment modality used was surgery in 34 patients followed by PAIR in 14 patients while 8 patients were treated medically. At a median follow up of 34 months recurrence was seen in 2 patients treated with PAIR while no patient treated with surgery had recurrence with the median follow up of 20 months. While no morbidity and mortality were observed in PAIR, but in surgery 5 patients had morbidity while 1 patient had mortality. Conclusion: Our data is comparative to other studies in terms of morbidity, mortality, and recurrence. We had adequate follow up. In our study PAIR and surgery both are effective and have less complications and recurrence rate. Surgery is still the gold standard in terms of recurrence.

Keywords: echinococcous granulosus, puncture aspiration irrigation reaspiration (PAIR), surgery, hydatid disease

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Authors: Bakytzhan Bimbetov, Abay Zhangabilov, Saule Aitbaeva, Galymzhan Meirambekov

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Mare’s milk (saumal) contains in total about 40 biological components necessary for the human body. The most significant among them are amino acids, fats, carbohydrates, enzymes (lysozyme, amylase), more minerals and vitamins which are well balanced with each other. In Kazakhstan, Company "Eurasia Invest Ltd.” produces a freeze-dried saumal in form of powder by the use of modern German innovative technology by means of evaporating at low temperature (-35°C) with an appropriate pasteurization. Research of freeze-dried biomilk for the qualitative content showed that main ingredients of freshly drown milk are being preserved. We are currently studying medical and dietary properties of freeze-dried mare's milk for diseases of the digestive system, including for nonalcoholic steatohepatitis (NASH) and liver cirrhosis (LC) viral etiology. The studied group consisted of 14 patients with NASH, and 7 patients with LC viral etiology of Class A severity degree as per Child-Pugh. Patients took freeze-dried saumal, preliminary dissolved in boiled warm water (24 g. powder per 200 ml water) 3-4 times a day for a month in conjunction with basic therapy. The results were compared to a control group (11 patients with NASH and LC) who received only basic therapy without mare’s milk. Results of preliminary research showed an improvement of subjective and objective conditions of all patients, but more significant improvement of clinical symptoms and syndromes were observed in the treatment group compared to the control one. Patients with NASH significantly over time compared to the beginning of therapy decreased asthenic and dyspeptic syndromes (p<0,01). Hepatomegaly, identified on the basis of ultrasound prior to treatment was observed in 92,8±2,4% of patients, and after combination therapy hepatomegaly the rate decreased by 14,3%, amounting to 78,5±2,8%. Patients with LC also noted the improvement of asthenic (p<0,01) and dyspeptic (p<0,05) syndromes and hemorrhagic syndrome (nosebleeds and bleeding gums when brushing your teeth, p<0,05), and jaundice. Laboratory study also showed improvement in the research group, but more significant changes were observed in the experimental group. Group of patients with NASH showed a significant improvement of index in cytolysis in conjunction with a combination therapy (p<0,05). In the control group, these indicators were also improved, but they were not statistically reliable (p>0,05). Markers of liver failure were additionally studied during the study of laboratory parameters in patients with liver cirrhosis, in particular, bilirubin, albumin and prothrombin index (PTI). Combined therapy with the use of basic treatment and mare's milk showed a significant improvement in cytolysis and bilirubin (p<0,05). In our opinion, a very important and interesting fact is that, in conjunction with basic therapy, the use of mare's milk revealed an improvement of liver function in the form of normalized PTI and albumin in patients with liver cirrhosis viral etiology. Results of this work have shown therapeutic efficiency of the use of mare's milk in complex treatment of patients with liver disease and require further in-depth study.

Keywords: liver cirrhosis, non-alcohol steatohepatitis, saumal, mare’s milk

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Authors: Asif Bilal, Fouzia Tanvir, Sibtain Ahmad

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Estrogen receptor alpha, also known as estrogen receptor-1, is highly involved in risk of mammary carcinoma. The objectives of this study were to identify non-synonymous SNPs of estrogen receptor and their association with breast cancer and to identify the chemotherapeutic responses of phytochemicals against it via in-silico study design. For this purpose, different online tools. to identify pathogenic SNPs the tools were SIFT, Polyphen, Polyphen-2, fuNTRp, SNAP2, for finding disease associated SNPs the tools SNP&GO, PhD-SNP, PredictSNP, MAPP, SNAP, MetaSNP, PANTHER, and to check protein stability Mu-Pro, I-Mutant, and CONSURF were used. Post-translational modifications (PTMs) were detected by Musitedeep, Protein secondary structure by SOPMA, protein to protein interaction by STRING, molecular docking by PyRx. Seven SNPs having rsIDs (rs760766066, rs779180038, rs956399300, rs773683317, rs397509428, rs755020320, and rs1131692059) showing mutations on I229T, R243C, Y246H, P336R, Q375H, R394S, and R394H, respectively found to be completely deleterious. The PTMs found were 96 times Glycosylation; 30 times Ubiquitination, a single time Acetylation; and no Hydroxylation and Phosphorylation were found. The protein secondary structure consisted of Alpha helix (Hh) is (28%), Extended strand (Ee) is (21%), Beta turn (Tt) is 7.89% and Random coil (Cc) is (44.11%). Protein-protein interaction analysis revealed that it has strong interaction with Myeloperoxidase, Xanthine dehydrogenase, carboxylesterase 1, Glutathione S-transferase Mu 1, and with estrogen receptors. For molecular docking we used Asiaticoside, Ilekudinuside, Robustoflavone, Irinoticane, Withanolides, and 9-amin0-5 as ligands that extract from phytochemicals and docked with this protein. We found that there was great interaction (from -8.6 to -9.7) of these ligands of phytochemicals at ESR1 wild and two mutants (I229T and R394S). It is concluded that these SNPs found in ESR1 are involved in breast cancer and given phytochemicals are highly helpful against breast cancer as chemotherapeutic agents. Further in vitro and in vivo analysis should be performed to conduct these interactions.

Keywords: breast cancer, ESR1, phytochemicals, molecular docking

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Authors: Manohar Salla, Mark S. Butler, Ruby Pelingon, Geraldine Kaeslin, Daniel E. Croker, Janet C. Reid, Jong Min Baek, Paul V. Bernhardt, Elizabeth M. J. Gillam, Matthew A. Cooper, Avril A. B. Robertson

Abstract:

MCC950 is a potent and selective inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that shows early promise for treatment of inflammatory diseases. The identification of major metabolites of lead molecule is an important step during drug development process. It provides an information about the metabolically labile sites in the molecule and thereby helping medicinal chemists to design metabolically stable molecules. To identify major metabolites of MCC950, the compound was incubated with human liver microsomes and subsequent analysis by (+)- and (−)-QTOF-ESI-MS/MS revealed a major metabolite formed due to hydroxylation on 1,2,3,5,6,7-hexahydro-s-indacene moiety of MCC950. This major metabolite can lose two water molecules and three possible regioisomers were synthesized. Co-elution of major metabolite with each of the synthesized compounds using HPLC-ESI-SRM-MS/MS revealed the structure of the metabolite (±) N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide. Subsequent synthesis of individual enantiomers and coelution in HPLC-ESI-SRM-MS/MS using a chiral column revealed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide. To study the possible cytochrome P450 enzyme(s) responsible for the formation of major metabolite, MCC950 was incubated with a panel of cytochrome P450 enzymes. The result indicated that CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C18, CYP2C19, CYP2J2 and CYP3A4 are most likely responsible for the formation of the major metabolite. The biological activity of the major metabolite and the other synthesized regioisomers was also investigated by screening for for NLRP3 inflammasome inhibitory activity and cytotoxicity. The major metabolite had 170-fold less inhibitory activity (IC50-1238 nM) than MCC950 (IC50-7.5 nM). Interestingly, one regioisomer had shown nanomolar inhibitory activity (IC50-232 nM). However, no evidence of cytotoxicity was observed with any of these synthesized compounds when tested in human embryonic kidney 293 cells (HEK293) and human liver hepatocellular carcinoma G2 cells (HepG2). These key findings give an insight into the SAR of the hexahydroindacene moiety of MCC950 and reveal a metabolic soft spot which could be blocked by chemical modification.

Keywords: Cytochrome P450, inflammasome, MCC950, metabolite, microsome, NLRP3

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