High DGAT1 expression levels in the small intestine highlight the critical role this enzyme plays in nutrient absorption. Identification of inhibitors which predominantly inhibit DGAT1 in the gut is an attractive drug discovery strategy with anticipated benefits of reduced systemic toxicity. In this report we describe our discovery and optimization of DGAT1 inhibitors whose plasma exposure is minimized by the action of transporters, including the P-glycoprotein transporter. The impact of this unique absorption profile on efficacy in rat and dog efficacy models is presented. KEYWORDS: DGAT1, triglyceride synthesis, efflux O rally ingested triglycerides (TG) undergo hydrolysis and then are reassembled within enterocytes into TG-rich chylomicrons destined for systemic circulation. The final committed step in triglyceride biosynthesis is known to be mediated by at least two distinct intracellular acyl-coA diacylglycerol acyltransferases (DGATs), namely DGAT1 1 and DGAT2. 2 Since the development of whole-body knockout models of these enzymes, there has been intense evaluation of pharmacological approaches to modulate their activity. 3−8 For DGAT1, this interest is inspired by the favorable metabolic phenotype of DGAT1 −/− mice, 9 which are resistant to dietinduced body weight gain, 10 are more insulin-sensitive relative to wild-type littermates, 11 and exhibit a reduced rate of chylomicrons formation when challenged with lipid nutrients. 12 Interestingly, all aspects of this phenotype are lost when DGAT1 is reintroduced via a tissue-specific promoter into the intestines of female DGAT1 −/− mice, implying that intestinal DGAT1 plays a crucial role in the effects observed in the whole-body knockout model. 13 Indeed, DGAT1 mRNA expression levels have been shown to be high in regions of the small intestine in mice and humans. 14,15 Selective inhibition of intestinal DGAT1 therefore becomes an intriguing drug discovery approach to recapitulate aspects of the DGAT1 −/− mouse, especially if this gut-specific inhibition reduces the potential risk of on-and off-target activity for candidate molecules. Particularly relevant for DGAT1 pharmacological inhibition is the observation of functional and morphological abnormalities in the fur and sebaceous glands of DGAT1 −/− mice. 16 In this report we describe a novel approach to specifically inhibit intestinal DGAT1, and demonstrate the potential viability of this strategy with regard to efficacy and safety in multiple preclinical models.High-throughput screening efforts using recombinant human DGAT1 enzyme identified the benzimidazole 1 (DGAT1 IC 50 = 1.3 μM; DGAT2 IC 50 > 20 μM; Figure 1) as a potential starting point for optimization. Initial structural modifications demonstrated that both the ethyl carbamate and the 2,6-dichlorophenyl substituents on the benzimidazole core could be replaced without substantial loss of activity (i.e., 2, DGAT1 IC 50 = 1.4 μM), and in fact introducing an additional substituent at the 4-position of the 2,6-dimethylphenyl ring led to an im...
ABSTRACT:The respectively. Metabolic profiling revealed that CP-544439 was primarily metabolized via glucuronidation, reduction, and hydrolysis. Glucuronidation was the primary route of metabolism in dogs, whereas reduction of the hydroxamate moiety was the major pathway in rats. Human plasma and urine obtained from a dose escalation study in healthy human volunteers were also analyzed in this study to assess the metabolism of CP-544439 in humans and ensure that selected animal species were exposed to all major metabolites formed in humans. Analysis suggested that CP-544439 was metabolized via all three pathways in humans consistent with rat and dog; however, the glucuronide conjugate M1 was the major circulating and excretory metabolite in humans. Preliminary in vitro phenotyping studies indicated that glucuronide formation is primarily catalyzed by UGT1A1, 1A3, and 1A9.The matrix metalloproteinases (MMP) are a family of zincdependent enzymes responsible for breaking down extracellular matrix proteins. Overexpression and activation of MMP have been linked to a range of diseases in which the destruction of connective tissue is an important pathological event, such as osteo arthritis (OA) and rheumatoid arthritis, tumor metastasis and angiogenesis, and corneal ulceration (Beckett et al., 1996;Michaelides and Curtin 1999;Rao, 2005). Collagenase 3 (MMP-13) is one type of MMP that is overexpressed in cartilage tissues of OA patients and is very efficient in the degradation of type II collagen. Thus, MMP-13 has been implicated in the pathology of OA. A selective MMP-13 inhibitor should therefore slow down or prevent cartilage breakdown and improve the quality of life of OA patients by retarding loss of function due to joint deterioration.4-[4-(4-Fluorophenoxy)-benzenesulfonylamino]tetrahydropyran-4-carboxylic acid hydroxyamide (CP-544439) (Scheme 1) was designed and synthesized as a selective inhibitor of MMP-13 (IC 50 ϭ 0.8 nM) (Reiter et al., 2004). In vivo studies using a hamster model, in which the cartilage collagen degradation was induced by intra-articular injection of recombinant human MMP-13 (Otterness et al., 2000), demonstrated that oral administration of CP-544439 inhibits degradation of the cartilage collagen with an ED 50 of 14 mg/kg and efficacious plasma concentrations ranging from 0.5 to 1.0 g/ml.Preclinical pharmacokinetic studies of CP-544439 in rats and dogs demonstrated that the clearance of CP-544439 was high in rats (53 ml/min/kg) and moderate in dogs (10 ml/min/kg). The volume of distribution ranged from 1.6 to 2.0 l/kg in the two species, and the terminal elimination half-life was 0.9 and 6.5 h in rats and dogs, Article, publication date, and citation information can be found at
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