Discovery of a Potent and Orally Efficacious TGR5 Receptor Agonist.

TGR5 is a G protein-coupled receptor (GPCR), activation of which promotes secretion of glucagon-like peptide-1 (GLP-1) and modulates insulin secretion. The 2-thio-imidazole derivative 6g was identified as a novel, potent, and selective TGR5 agonist (hTGR5 EC50 = 57 pM, mTGR5 = 62 pM) with a favorable pharmacokinetic profile. The compound 6g was found to have potent glucose lowering effects in vivo during an oral glucose tolerance test in DIO C57 mice with ED50 of 7.9 mg/kg and ED90 of 29.2 mg/kg.

Differential gene expression analysis of a known hepatotoxin, N-acetyl-p-amino-phenol (APAP) as compared to its non-toxic analog, N-acetyl-m-amino-phenol (AMAP) in mouse liver.

Drug-induced hepatotoxicity is one of the most common adverse events associated with drug withdrawal from the market. Elucidating the molecular mechanism of hepatotoxicity is essential to predict the safety of a new molecule. To examine genes involved in hepatotoxicity, we have used oligonucleotide CodeLink Bioarrays and determined the transcriptional profile of mice liver treated with hepatotoxic drug N-acetyl-p-amino-phenol (APAP) as well as its non-toxic analog N-acetyl-m-amino-phenol (AMAP). Out of 20,000 genes analyzed, 896 showed differential expression of > or = 2-fold (648 upregulated and 248 downregulated) within the liver of APAP treated mice as compared to control. In comparison to AMAP treated mice, 62 genes were upregulated and 70 genes were downregulated in mice liver after APAP treatment. Functional classification of these differentially expressed genes identified genes associated with stress response, cell cycle, growth inhibition, cell death, structural components, cell signaling and inflammation. Gene expression profile was further correlated with biochemical analysis and histopathological lesions. These data show that gene expression profiling would help in better understanding the molecular basis of drug-induced hepatotoxicity that will lead to rational development of safer drugs, particularly in pre-clinical stages.

Combination of dipeptidylpeptidase IV inhibitor and low dose thiazolidinedione: preclinical efficacy and safety in db/db mice.

Thiazolidinediones (TZDs) are currently the most efficacious class of oral antidiabetics. However, they carry the burden of weight gain and haemodilution, which may lead to cardiovascular complications. The present study was designed to ascertain whether a combination of dipeptidyl peptidase IV (DPP IV) inhibitor with low dose of a thiazolidinedione absolves TZD associated weight gain and oedema without compromising its efficacy. In this study, we examined the efficacy and safety of lower dose (1 mg/kg/day) of rosiglitazone, a thiazolidinedione, in combination with 5 mg/kg/day dose of LAF-237 (vildagliptin), a known DPP IV inhibitor, in aged db/db mice after 14 days of treatment and compared the combination with therapeutic dose (10 mg/kg) of rosiglitazone. The combination therapy showed similar efficacy as that of 10 mg/kg/day rosiglitazone in lowering random blood glucose (53.8%, p<0.001 and 54.3%, p<0.001 respectively), AUC ((0-120) min) during oral glucose tolerance test (OGTT) (38.6 %, p<0.01; 38.3%, p<0.01 respectively) and triglyceride levels (63.9% and 61% respectively; p<0.01). Plasma active glucagon like peptide-1 (GLP-1) and insulin levels were found to be elevated significantly (p<0.01 and p<0.05 respectively) in both LAF-237 and combination treated groups following oral glucose load. LAF-237 alone had no effect on random glucose and glucose excursion during OGTT in severely diabetic db/db mice. Interestingly, the combination treatment showed no significant increase in body weight as compared to the robust weight gain by therapeutic dose of rosiglitazone. Rosiglitazone at 10 mg/kg/day showed significant reduction (p<0.05) in haematocrit, RBC count, haemoglobin pointing towards haemodilution associated with increased mRNA expression of Na(+), K(+)-ATPase-alpha and epithelial sodium channel gamma (ENaCgamma) in kidney. The combination therapy escaped these adverse effects. The results suggest that combination of DPP IV inhibitor with low dose of thiazolidinedione can interact synergistically to represent a therapeutic advantage for the clinical treatment of type 2 diabetes without the adverse effects of haemodilution and weight gain associated with thiazolidinediones.

Molecular cloning, stable expression and cellular localization of human alpha1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of alpha1D -adrenergic receptor.

The cDNAs encoding for three subtypes of adrenergic receptors, alpha1A-, alpha1B- and alpha1D-ARs, were cloned and expressed in HEK 293 cells. Expression of alpha1A- and alpha1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of alpha1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of alpha1A- and alpha1B-ARs was primarily localized on the cell membrane whereas expression of alpha1D-AR was predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing alpha1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of alpha1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.

High level stable expression of pharmacologically active human M1-M5 muscarinic receptor subtypes in mammalian cells.

cDNAs encoding for five mAChR subtypes (M1-M5) were cloned under different promoters in various eukaryotic vectors and each subtype was expressed in different mammalian cell lines. CHO-K1 cell line was the best for generating stable cell lines expressing muscarinic receptors. Immunofluorescence and flow cytometry revealed that expression of M1-M5 was primarily localized on the cell membrane. Western blotting and radio-ligand binding studies revealed that expression of each receptor was stable at higher passages.

Use of a synthetic peptide epitope of Asp f 1, a major allergen or antigen of Aspergillus fumigatus, for improved immunodiagnosis of allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is an immunologically complex allergic disorder caused by the fungal pathogen Aspergillus fumigatus. Elevated levels of total immunoglobulin E (IgE), specific IgE, and IgG antibodies in sera are important immunodiagnostic criteria for ABPA. International reference standards or standardized immunodiagnostic assays are not available due to a lack of well-defined diagnostic antigens. The present study was carried out to identify and evaluate the immunodiagnostic relevance of synthetic epitopic peptides of Asp f 1, a major allergen, antigen, or cytotoxin of A. fumigatus. Five overlapping peptides were synthesized from the N terminus of Asp f 1, one of the potential immunodominant regions predicted by algorithmic programs. The 11-amino-acid synthetic peptide (P1) significantly inhibited both IgG binding (89.10% +/- 4.45%) and IgE binding (77.32% +/- 3.38%) of the standardized diagnostic antigen (SDA) (a well-defined pool of diagnostically relevant allergens and antigens of A. fumigatus). With a panel of sera of ABPA patients, allergic patients with skin test negativity to A. fumigatus, and healthy individuals, P1 showed a higher diagnostic efficiency than SDA (specific IgG, 100%; specific IgE, 98.3%). The diagnostic efficiency of P1 could be attributed to the presence of homologous epitopes in various immunodominant allergens or antigens of A. fumigatus. The ability of P1 to induce histamine release from sensitized mast cells and a Th2 type of cytokine profile in peripheral blood mononuclear cells of ABPA patients suggests its potential for use in intradermal testing. P1 could be further explored for development of a standardized, specific, and sensitive immunodiagnostic test for aspergillosis.

Expression and characterization of Asp fI, an immunodominant allergen/antigen of A. fumigatus in insect cell.

Asp fI is a major allergen/antigen/cytotoxin of Aspergillus fumigatus and exhibits ribonuclease activity. This allergen plays a role in allergic and invasive Aspergillosis and reported as a major cytotoxin with ribonuclease activity. To express the protein in large quantity and to characterize the multifunctional nature of Asp fI, we have generated recombinant baculovirus by introducing the gene in pFastBac HTa expression vector and expressed in insect cell. The baculovirus expression vector system has been used as a versatile system for the efficient expression of proteins with most eukaryotic posttranslational modification. Recombinant Asp fI was expressed as approximately 1% of the total cellular protein in infected Sf9 insect cells. The protein was purified using Ni2+ affinity column chromatography and the yield of purified protein was approximately 10 mg/l g of total cellular protein. Immunoreactivity of the protein was determined by immunoblot analysis using both poly His monoclonal antibody, IgG and IgE antibodies present in the sera of ABPA patients. The protein was glycosylated as revealed by the glycoprotein staining and was observed to retain both ribonuclease and cytotoxic activities. These results suggest that Asp fI expressed in insect cell was post translationally modified and biologically active that can be used as a diagnostic marker for biochemical studies.