Metabolic studies of cinnamyl anthranilate in relation to its safety evaluation

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Interaction of sperm with endometrium can regulate genes involved in endometrial receptivity pathway in mice: An experimental study

Background: Many researchers consider implantation and endometrial receptivity as pertinent issues in reproductive science. Although, several experiments have been performed and their results evaluated, yet there is no confirmed evidence about the related factors and the role of sperm in endometrial receptivity. Objective: To investigate the effect of the sperm-endometrium interaction in regulating genes involved in the endometrial receptivity pathway. Materials and Methods: In this experimental study, 10 male and 30 female NMRI mice were included, and half of the male cases were vasectomized. The subjects were divided into two groups as follows; group 1 (case) comprised of 15 females mated with 5 non-vasectomized male mice, while group 2 (control) consisted of 15 females mated with 5 vasectomized males. Cases were sacrificed and assessed after 36 hr and the endometrial tissue was extracted and kept at -80°C until the next use. The expression of the endometrial receptivity pathway genes, including VEGF, HBEGF, FGF2, EGF, LIF, LIFR, HOXA10, MUC1, PGR, and CSF, was examined in both groups. For statistical analysis, an independent samples test (Mean ± SD) was used. Results: The mRNA levels of LIF (p = 0.045), LIFR (p = 0.040), MUC1 (p = 0.032), VEGF (p = 0.022), EFG (p = 0.035), and FGF2 (p = 0.040) were significantly upregulated in the case group compared with the control group. Conclusion: Finally, seminal plasma was observed to be effective in expressing the involved genes in the successful implantation pathway, including LIF, LIFR, MUC1, VEGF, EGF, and FGF2. Key words: Endometrial receptivity, Sperm, Gene expression, Mice

Identification of B and T cell epitope peptide vaccines from IGF-1 Receptor in breast cancer

Introduction: The insulin-like growth factor-1 receptor (IGF-1R) plays a key role in proliferation, growth, differentiation, and development of several human malignancies including breast and pancreatic adenocarcinoma. IGF-1R targeted immunotherapeutic approaches are particularly attractive, as they may potentially elicit even stronger antitumor responses than traditional targeted approaches. Cancer peptide vaccines can produce immunologic responses against cancer cells by triggering helper T cell (Th) or cytotoxic T cells (CTL) in association with Major Histocompatibility Complex (MHC) class I or II molecules on the cell surface of antigen presenting cells.  Methods and Results: In our previous study, we set a technique based on molecular docking in order to find the best MHC class I and II binder peptides using GOLD. In the present work, molecular docking analyses on a library consisting of 30 peptides mimicking discontinuous epitopes from IGF-1R extracellular domain identified peptides 249 and 86, as the best MHC binder peptides to both MHC class I and II molecules. The receptors most often targeted by peptide 249 are HLA-DR4, HLA-DR3 and HLA-DR2 and those most often targeted by peptide 86 are HLA-DR4, HLA-DP2 , and HLA-DR3. Conclusions: These findings, based on bioinformatics analyses, can be conducted in further experimental analyses in cancer therapy and vaccine design

Investigation of the binding specificity of IGF-1R using monoclonal antibodies.

The insulin-like growth factor type one receptor (IGF-IR) plays a critical role in cancer. The receptor is over expressed in many tumours and mediates growth, motility, and survival from apoptosis. Several studies have shown that the inhibition of IGF-1R's expression or its function blocks tumour growth or metastasis, and also enhances sensitivity to cytotoxic drugs and irradiation. To date, IGF-1R is widely considered as a very promising target for cancer treatment. Among different techniques for targeting IGF-1R, blocking the receptor with specific monoclonal antibodies (MAbs) is an attractive way to inhibit the receptor signalling. In addition, antibodies specific for the IGF-1R are potential diagnostic reagents as the IGF-1R is overexpressed in several cancers. A similar approach has been successfully utilized in the treatment of breast cancer using Herceptin, which is a humanised MAb against human epidermal growth factor receptor-2. In this thesis, two high affinity murine MAbs against IGF-1R namely 7C2 and 9E11 were isolated and cloned. To obtain these MAbs, s-IGF-1R (the soluble extracellular part of the IGF-1R, amino acids 1-906) was used as an immunogen. These MAbs were IgG1k isotype and did not cross react with either insulin receptor isoform IR-A or IR-B. MAbs 7C2 and 9E11 successfully detected IGF-1R in a number of immunoassays such as in enzyme linked immunosorbent assays (ELISAs), flow cytometry and immunohistochemistry. The MAbs also immunoprecipitated IGF-1R from lysates of cells overexpressing recombinant IGF-1R. Both MAbs 7C2 and 9E11 showed high affinity to the s-IGF-1R. The binding affinities of the MAbs 7C2 and 9E11 to s-IGF-1R were 0.5±1.6 nM and 2.1±0.4 nM, respectively. The sequences of the variable regions of these MAbs' heavy and light chains were also described in this study. Comparison of the CDR sequences of the MAbs with those of other MAbs against the IGF-1R showed that MAbs 7C2 and 9E11 each had a unique sequence. During this research it was found that MAbs 7C2 and 9E11 blocked the binding of IGF-I to the IGF-1R, but did not show an inhibitory effect on the binding of IGF-II to this receptor. Moreover, epitope mapping of MAbs 7C2 and 9E11 revealed that they both bound to the cysteine-rich domain of the receptor. These findings support a previous study, which demonstrated that the cysteine-rich domain of IGF-1R is critical for specific binding of IGF-I but not IGF-II to the receptor. In this thesis, further epitope mapping studies on MAbs 7C2 and 9E11 using alanine mutants of the IGF-1R in the cysteine-rich domain are reported. The results showed that amino acids F241, F251 and F266 were involved in binding to both MAbs 7C2 and 9E11. On the other hand, another study showed that amino acids F241 and F251 were crucial for IGF-I binding to the receptor. Therefore, binding to these two amino acids by MAbs 7C2 and 9E11 is most likely responsible for the selective blocking of IGF-I but not IGF-II to the receptor. Competition experiments performed during this research revealed that the chimeric IGF analogues IGF-ICII (IGF-I with IGF-II C domain) and IGF-IICI (IGF-II with IGF-I C domain) behaved like IGF-II and IGF-I, respectively in their ability to inhibit the binding of MAbs 7C2 and 9E11 to s-IGF-1R. These findings imply that out of all four domains of IGF-I, it is the C domain, which binds to residues in IGF-1R that form the epitope for MAbs 7C2 and 9E11. Because in the C domain of IGF-I, amino acids R36 and R37 and also Y31 have been shown to be important for binding to IGF-1R, it could be proposed that the binding of the IGF-I C domain to the IGF-1R cysteine-rich domain is through binding amino acids R36, R37 and Y31 of IGF-I to residues F241 and F251, or to the nearby residues, which are sterically affected by the presence of the MAbs binding to the receptor. In this research, in vitro studies, using MAbs as anti-cancer reagents revealed that both MAbs 7C2 and 9E11 inhibited the proliferation of colon cancer cell (HT -29) induced by IGF-I and IGF-II. However, the neutralizing effect for IGF-II was less potent than for IGF-I. Since the MAbs did not inhibit binding of IGF-II to the receptor, their effect on IGF-II induced cell proliferation could be claimed to be due to an indirect effect. This is consistent with the in vitro result for the receptor down-regulation experiment, which showed that 24 h incubation of breast cancer cells (MCF-7) with MAbs 7C2 and 9E11, significantly reduced the IGF-1R expression. In this study it was also shown that MAbs 7C2 and 9E11 inhibited MCF-7 cells migration induced by IGF-I. This could indicate a potential anti-metastatic property of MAbs 7C2 and 9E11. Taken together, the in vitro effects of MAbs 7C2 and 9E11 reported in this thesis revealed that they had inhibiting effects on cancer cell proliferation and migration and they also induced IGF-1R down-regulation. Therefore, they could be potentially valuable prospects for cancer treatment if humanised. Finally, MAbs 7C2 and 9E11 could be used as diagnostic reagents to detect IGF-1R in cells or tissue samples in a range of immunoassays as mentioned earlier. Due to the IGF-1R overexpression in several cancer tumours, MAbs 7C2 and 9E11 could be utilized for malignant tumour recognition. Furthermore, as another application, these MAbs could also be used to identify tumours overexpressing the IGF-1R, allowing tailored anti-cancer treatment including an IGF-1R blocking agent.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

Determination of mebudipine in human plasma by liquid chromatography�tandem mass spectrometry

In previous studies, mebudipine, a dihydropyridine calcium channel blocker, showed a considerable potential to be used in cardiovascular diseases. The aim of the current study was to develop a valid method using reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry to assay mebudipine in the human plasma. Separation was achieved on a Zorbax Eclipse® C18 analytical column using a mobile phase consisted of methanol/water (90:10, v/v). The flow rate was 0.6 mL/min and carbamazepine was used as an internal standard (IS). This method involved the use of M +Na+ ions of mebudipine and IS at m/z 411 and 259, respectively with the selected ion monitoring (SIM) mode. There were no interfering peaks from endogenous components in blank plasma chromatograms. Standard curves were linear (r2>0.99) between 5 to 100 ng/mL. The mean extraction efficiency was about 84% and the limit of quantification for mebudipine was 5 ng/mL in plasma. The coefficient of variation and error at all of the intra-day and inter-day assessments were less than 11%. The results indicated that this method is a fast, accurate, sensitive, selective and reliable method for the determination of mebudipine in the human plasma. The assay method has been successfully used to estimate plasma concentration of mebudipine after the oral administration of 2.5 mg tablet in healthy adults. © 2015 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services

Design and evaluation of oral nanoemulsion drug delivery system of mebudipine

A nanoemulsion drug delivery system was developed to increase the oral bioavailability of mebudipine as a calcium channel blocker with very low bioavailability profile. The impact of nano-formulation on the pharmacokinetic parameters of mebudipine in rats was investigated. Nanoemulsion formulations containing ethyl oleate, Tween 80, Span 80, polyethylene glycol 400, ethanol and deionized water were prepared using probe sonicator. The optimum formulation was evaluated for physicochemical properties, such as particle size, morphology and stability. The particle size of optimum formulation was 22.8 ± 4.0 nm. Based on the results of this study, the relative bioavailability of mebudipine nanoemulsion was enhanced by about 2.6-, 2.0- and 1.9-fold, respectively, compared with suspension, ethyl oleate solution and micellar solution. In conclusion, nanoemulsion is an interesting option for the delivery of poorly water soluble molecules, such as mebudipine. © 2015 Informa UK Limited, trading as Taylor & Francis Group

The role of Olea Europaea L. Fruit on A2780, A172 and HFFF2 proliferation

Olea europaea L. commonly known as olive has been traditionally used for the prevention and treatment of many diseases since ancient times. Olive has been reported to possess a broad spectrum of pharmacological properties. In the present study, we investigated the activity of aqueous extract of Olea europaea L. fruit at various concentrations on A2780, A172, and HFFF2 cell lines proliferation by MTT assay. Aqueous extract of olive significantly increased cell proliferation in a dose dependent manner in the cell lines. It has been previously reported that olive has chemoproventive and anti-tumor effects. These disagreements can be explained by differences in cell line properties, type of olive and different solvents in the extracts. However, further investigation is needed to clarify the exact role of olive in cell proliferation and cancer. In this study fruit extract of Olea europaea L. showed more activatory effects on A2780 cell line in comparison with A172 and HFFF2. These differences in the activatory effects may be related to the activation of different signaling pathways in different cell lines. © 2016, Iranian Association of Pharmaceutical Scientists. All rights reserved

Improved HPLC method for determination of four PPis, omeprazole, pantoprazole, lansoprazole and rabeprazole in human plasma

PURPOSE: To develop a simple and rapid HPLC method for measuring of four proton-pump inhibitors (PPIs), omeprazole (OPZ), pantoprazole (PPZ), lansoprazole (LPZ) and rabeprazole (RPZ) concentrations in human plasma. METHODS: Following a single step liquid-liquid extraction analytes along with an internal standard (IS) were separated using an isocratic mobile phase of phosphate buffer (10 mM)/acetonitrile (53/47, v/v adjusted pH to 7.3 with triethylamine) at flow rate of 1 mL/min on reverse phase TRACER EXCEL 120 ODS-A column at room temperature. RESULTS: Total analytical run time for selected PPIs was 10 min. The assays exhibited good linearity (r2>0.99) over the studied range of 20 to 2500 ng/mL for OPZ, 20 to 4000 ng/mL for PPZ, 20 to 3000 ng/mL for LPZ and 20 to 1500 ng/mL for RPZ. The recovery of method was equal or greater than 80 and lower limit of quantification (LLOQ) was 20 ng/mL for four PPIs. Coefficient of variation and error at all of the intra-day and inter-day assessment were less than 9.2 for all compounds. CONCLUSIONS: The results indicated that this method is a simple, rapid, precise and accurate assay for determination of four PPIs concentrations in human plasma. This validated method is sensitive and reproducible enough to be used in pharmacokinetic studies and also is time- and cost-benefit when selected PPIs are desired to be analyzed

Cancer chemoprevention by oleaster (Elaeagnus angustifoli L.) fruit extract in a model of hepatocellular carcinoma induced by diethylnitrosamine in rats

Hepatocellular carcinoma (HCC) is a frequent and fatal human cancer with poor diagnosis that accounts for over half a million deaths each year worldwide. Elaeagnus angustifolia L. known as oleaster has a wide range of pharmacological activities. This study aimed to investigate the chemopreventive effect of aqueous extract of E. angustifolia fruit (AEA) against diethylnitrosamine (DEN)-induced HCC in rats. HCC was induced in rats by a single injection of DEN (200 mg/kg) as an initiator. After two weeks, rats were orally administered 2-acetylaminofluorene or 2-AAF (30 mg/kg) as a promoter for two weeks. Oleaster-treated rats were orally pretreated with the increasing doses of AEA two weeks prior to DEN injection that continued until the end of the experiment. In the current study, a significant decrease in serum biomarkers of liver damage and cancer, including alfa-fetoprotein (AFP), gamma glutamyl transpeptidase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) was observed in AEA-treated rats when compared to HCC rats. Furthermore, the oleaster extract exhibited in vivo antioxidant activity by elevating reduced glutathione (GSH) contents as well as preventing lipid peroxidation in the liver tissues of DEN-treated rats. The relative weight of liver, a prognostic marker of HCC, was also reduced in oleaster-treated rats. To conclude, our results clearly demonstrated that oleaster fruit possesses a significant chemopreventive effect against primary liver cancer induced by DEN in rats. It can be suggested that the preventive activity of oleaster against hepatocarcinogenesis may be mediated through the antioxidant, anti-inflammation, and antimutagenic effects of the fruit