br TGR Agonists br FXR
FXR/TGR5 Dual Agonists In 2010, a FXR/TGR5 dual agonist, 51 (INT-767), was reported . Using an AlphaScreen coactivator recruitment assay, the potency of 51 at FXR was 30nM. In NCI-H716 cells, 51 stimulated intracellular cAMP secretion with an EC50 of 0.63μM. Its TGR5 potency was comparable to that of the selective TGR5 agonist 46 (EC50=0.8μM). Compound 51 also induced a dose-dependent increase of GLP-1 secretion from NCI-H716 cells. In DBA/2J mice, a streptozotocin-induced type 1 diabetes model, plasma cholesterol levels were significantly higher in mice fed a western diet (WD) compared with standard chow. A 3-week treatment of these mice with 51 admixed at doses of 10 or 30mg/kg/day in the WD resulted in a significant dose-dependent decrease of plasma total cholesterol levels and a significant decrease of TG levels only at the 30mg/kg/d dose. The marked inhibition of total cholesterol induced by compound 51 treatment was correlated with normalization of LDL cholesterol levels; HDL cholesterol levels were not affected. In db/db mice, a model of type 2 diabetes, intraperitoneal administration of 51 for 2 weeks at doses of 10 and 30mg/kg/day significantly and dose-dependently decreased plasma total cholesterol and TG levels.
Clinical Studies and Outlook To date, only limited number of BA receptor agonists have been studied in humans. In 2009, a phase II trial result was reported on a FXR agonist, INT-747, in type 2 diabetes patients with comorbid fatty thapsigargin synthesis disease . From this double-blind placebo-controlled study of 64 patients, INT-747 therapy (25 and 50mg for 6 weeks) significantly improved insulin sensitivity, induced weight loss, and reduced liver damage. In 2010, a phase II study of a TGR5 agonist, SB-756050, for treatment of type 2 diabetes was completed . Further development of this compound was discontinued after the highest dose failed to meet the predetermined efficacy threshold.
TGR5 receptor biology TGR5 was identified through the exploration of GPCRs in the GenBank™ database with human spleen cDNAs and finding a genomic DNA sequence (AC021016) coding for a novel GPCR, designated TGR5 . The TGR5 cDNA is encoded by a single exon that maps to chromosome position 2q35 in humans and to a syntenic region on mouse chromosome 1c3. The initiation codon of human TGR5 was found on the basis of the nucleotide sequence information of AC055884 and AC021016, and the open reading frame (ORF) of human receptor was estimated to consist of 993 base pairs, with a deduced amino acid sequence of a protein consisting of 330 amino acid residues . The motif analysis predicted the protein to be a seven-transmembrane receptor. The subsequent isolation of TGR5 cDNAs in various other species, including identification of homologues of TGR5 in aquatic vertebrates, highlighted the sequence conservation amongst mammals and a role in vertebrate physiology as well. Human TGR5 (GPBAR1 or BG37 or GPCR19 or GPR131 or M-BAR or MGC 40597) shares 82, 83, 86 and 90% amino acid identity with rat (Gpbar1), mouse (Gpbar1 or BG37 or GPR131 or M-BAR), bovine (GPBAR1 or MGC 152072) and rabbit (Gpbar1) receptor respectively . Despite the fairly strong conservation amongst vertebrates, phylogenic analysis highlights that human TGR5 is convergent to monkey and rabbit receptors and is quite divergent from rodent (rat and mice) receptors . Among the known GPCRs, TGR5 shared moderate amino acid sequence similarity with the endothelial cell differentiation gene (EDG) family of receptors being approximately 25, 26, 29 and 30% sequence identity with EDG7, EDG1, EDG8 and EDG6 respectively . Northern hybridization using RNAs from human tissue samples, revealed the ubiquitous expression of TGR5 in various tissues, such as heart, spleen, skeletal muscle, kidney, liver, small intestine, placenta, lung and peripheral blood leukocytes . Similar expression analyses in day-7, day-11, day-15 and day-17 mouse embryos and adult mouse identified the expression of TGR5 gene in heart, spleen, lung, liver, kidney, skeletal muscle, and testis. . Northern hybridization studies were corroborated in human samples through reverse transcription PCR, revealing the highest level of expression in human placenta and spleen, followed by moderate expression in other tissues including lungs, liver, stomach, small intestine and adipose tissue with relatively low level of expression in kidney, skeletal muscles and pancreas. The relative expression level of human TGR5 mRNA in adipose tissues and intestine was nearly threefold to fivefold lower compared with spleen and placenta. Likewise, the relative expression level in the human skeletal muscles was nearly 25–30 fold lower compared with the expression level in the spleen and placenta . TGR5 mRNA was detected in the resting CD14+ monocytes in fractionated human leukocytes and in adherent alveolar macrophage cells, indicating the potential involvement of TGR5 in immune responses mediated by monocytes/macrophages . The expression of TGR5 in different diseased conditions, however, is not yet known.