ABSTRACT
This report describes the characterization of INCB3344, a novel, potent and selective small molecule antagonist of the mouse CCR2 receptor. The lack of rodent cross-reactivity inherent in the small molecule CCR2 antagonists discovered to date has precluded pharmacological studies of antagonists of this receptor and its therapeutic relevance. In vitro, INCB3344 inhibits the binding of CCL2 to mouse monocytes with nanomolar potency (IC(50) = 10 nM) and displays dose-dependent inhibition of CCL2-mediated functional responses such as ERK phosphorylation and chemotaxis with similar potency. Against a panel of G protein-coupled receptors that includes other CC chemokine receptors, INCB3344 is at least 100-fold selective for CCR2. INCB3344 possesses good oral bioavailability and systemic exposure in rodents that allows in vivo pharmacological studies. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological analysis of tissues from the delayed-type hypersensitivity model demonstrates that inhibition of CCR2 leads to a substantial reduction in tissue inflammation, suggesting that macrophages play an orchestrating role in immune-based inflammatory reactions. These results led to the investigation of INCB3344 in inflammatory disease models. We demonstrate that therapeutic dosing of INCB3344 significantly reduces disease in mice subjected to experimental autoimmune encephalomyelitis, a model of multiple sclerosis, as well as a rat model of inflammatory arthritis. In summary, we present the first report on the pharmacological characterization of a selective, potent and rodent-active small molecule CCR2 antagonist. These data support targeting this receptor for the treatment of chronic inflammatory diseases.
Subject(s)
Pyrrolidines/pharmacology , Receptors, Chemokine/antagonists & inhibitors , Animals , Arthritis, Experimental/drug therapy , Cell Line , Chemotaxis, Leukocyte/drug effects , Chemotaxis, Leukocyte/immunology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Female , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Monocytes/drug effects , Protein Binding/drug effects , Protein Binding/immunology , Pyrrolidines/pharmacokinetics , Rats , Rats, Inbred Lew , Receptors, CCR2 , Receptors, Chemokine/deficiency , Receptors, Chemokine/genetics , Structure-Activity RelationshipABSTRACT
The peroxisome proliferator activated receptor alpha (PPARalpha) plays a key role in regulating fatty acid metabolism by regulating expression of genes involved in fatty acid oxidation. To identify endogenous transcripts that could be used as surrogate markers for on-target activity of PPARalpha agonists, we employed a global profiling approach using DNA microarrays. The HK-2 cell line derived from proximal tubules of the human kidney, showed induction of several genes, including pyruvate dehydrogenase kinase 4 (PDK-4) and adipocyte differentiation related protein (ADRP) by PPARalpha ligands. HK-2 cells express detectable levels of PPARalpha and its dimerization partner the retinoid X receptor (RXRalpha) proteins. Induction of PDK-4 in these cells correlates with induction of PDK-4 in the liver of fat-fed hamsters. The magnitude of fibrate induction of PDK-4 in the liver also mirrors the decrease in serum triglyceride levels. Thus, induction of PDK-4 by PPARalpha agonists in the HK-2 cell model closely correlates with its induction in vivo and may represent an early marker for PPARalpha agonist action.
Subject(s)
Fatty Acids/metabolism , Isoenzymes/biosynthesis , Kidney Tubules, Proximal/physiology , Membrane Proteins/biosynthesis , Protein Kinases/biosynthesis , Receptors, Cytoplasmic and Nuclear/agonists , Transcription Factors/agonists , Animals , Butyrates/pharmacology , Cells, Cultured , Cricetinae , Enzyme Activation , Fenofibrate/pharmacology , Gene Expression Regulation/physiology , Humans , Hypolipidemic Agents/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Kidney Tubules, Proximal/enzymology , Kidney Tubules, Proximal/metabolism , Ligands , Liver/enzymology , Male , Membrane Proteins/genetics , Mesocricetus , Oligonucleotide Array Sequence Analysis , Perilipin-2 , Phenylurea Compounds/pharmacology , Protein Kinases/genetics , Protein Kinases/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors/physiology , Triglycerides/bloodABSTRACT
Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.
