Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic ß-cell mass.

A critical unmet need exists for methods to quantitatively measure endogenous pancreatic ß-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet ß-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R>GLP-1R>NPY-2R>mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R>VMAT2∼GLP-1R>mGluR5. Favorable islet selectivity and biodistribution characteristics suggest several GPCRs as potential targets for PET imaging of pancreatic BCM.

Pharmacological effects of nicotine on norepinephrine metabolism in rat brown adipose tissue: relevance to nicotinic therapies for smoking cessation.

In a two-year carcinogenicity study with administration of high doses of the partial nicotinic agonist varenicline (recently approved for smoking cessation), mediastinal hibernomas occurred in three male rats. To investigate potential mechanisms for partial and full nicotinic agonists to contribute to development of hibernomas, the effects of nicotine on rat brown adipose tissue (BAT) were studied. Male and female rats were administered nicotine at doses of 0, 0.3, and 1 mg/kg subcutaneously for fourteen days. Intrathoracic (mediastinal periaortic and mediastinal perithymic) BAT and interscapular BAT were examined microscopically, and determinations of uncoupling protein-1 (UCP-1) expression and norepinephrine (NE) content were made. Additionally, NE turnover was measured in mediastinal periaortic and perithymic BAT. Nicotine (1 mg/kg) administration resulted in decreased vacuolation only in mediastinal periaortic and mediastinal perithymic BAT of males and elevated UCP-1 in mediastinal periaortic BAT of males and females. Increased NE content occurred only in mediastinal periaortic BAT of males given 0.3 and 1 mg/kg doses, whereas NE turnover was decreased in both males and females given 1 mg/kg. Together, these data demonstrate that nicotine primarily affects mediastinal BAT in male rats, consistent with the gender and location of the hibernomas observed in the two-year carcinogenicity study.

Coordinated expression of multidrug resistance-associated proteins (Mrps) in mouse liver during toxicant-induced injury.

Following acute chemical injury, hepatocytes are generally more resistant to toxicant re-exposure. Alterations in expression of hepatobiliary transport systems may contribute to this resistance by preventing accumulation of potentially toxic chemicals. Previous data demonstrate the concomitant reduction of uptake transporter and induction of efflux transporter mRNA during chemical liver injury. The present study further characterizes the expression of multidrug resistance-associated proteins 1-4 (Mrp1-4), breast cancer resistance protein (Bcrp) and sodium-taurocholate co-transporting polypeptide (Ntcp) in mouse liver following administration of the hepatotoxicants acetaminophen (APAP) and carbon tetrachloride (CCl4). Mice received hepatotoxic doses of APAP (400 mg/kg), CCl4 (10 or 25 microl/kg), or vehicle, ip. Livers were collected at 6, 24, and 48 h for Western blot quantification and immunofluorescence analysis. Protein expression of Bcrp was unchanged with treatment. Ntcp levels were preserved in APAP-exposed livers and reduced to 30-50% of control after CCl4. Conversely, Mrp1-4 expression was differentially up-regulated. CCl4 increased Mrp1 (3.5-fold), Mrp2 (1.4-fold), and Mrp4 (26-fold) while reducing Mrp3 levels to 20% of control. Administration of APAP enhanced expression of Mrp2 (1.6-fold), Mrp3 (3.5-fold), and Mrp4 (16-fold). Immunostaining of liver sections obtained 48 h after hepatotoxicant treatment confirmed expression patterns of a subset of transporters (Bcrp, Ntcp, Mrp3, and Mrp4). Double immunofluorescence imaging demonstrated the simultaneous down-regulation of Ntcp and up-regulation of Mrp4 in hepatocytes adjacent to the central vein after CCl4. Altered expression of transporters may reduce the overall chemical burden of an injured liver during recovery and contribute to the resistance of hepatocytes to subsequent toxicant exposure.

PF-03814735, an orally bioavailable small molecule aurora kinase inhibitor for cancer therapy.

The Aurora family of highly related serine/threonine kinases plays a key role in the regulation of mitosis. Aurora1 and Aurora2 play important but distinct roles in the G(2) and M phases of the cell cycle and are essential for proper chromosome segregation and cell division. Overexpression and amplification of Aurora2 have been reported in different tumor types, including breast, colon, pancreatic, ovarian, and gastric cancer. PF-03814735 is a novel, potent, orally bioavailable, reversible inhibitor of both Aurora1 and Aurora2 kinases that is currently in phase I clinical trials for the treatment of advanced solid tumors. In intact cells, the inhibitory activity of PF-03814735 on the Aurora1 and Aurora2 kinases reduces levels of phospho-Aurora1, phosphohistone H3, and phospho-Aurora2. PF-03814735 produces a block in cytokinesis, resulting in inhibition of cell proliferation and the formation of polyploid multinucleated cells. Although PF-03814735 produces significant inhibition of several other protein kinases, the predominant biochemical effects in cellular assays are consistent with inhibition of Aurora kinases. Once-daily oral administration of PF-03814735 to mice bearing human xenograft tumors produces a reduction in phosphohistone H3 in tumors at doses that are tolerable and that result in significant inhibition of tumor growth. The combination of PF-03814735 and docetaxel in xenograft mouse tumor models shows additive tumor growth inhibition. These results support the clinical evaluation of PF-03814735 in cancer patients. Mol Cancer Ther; 9(4); 883-94. (c)2010 AACR.

Induction of hepatobiliary efflux transporters in acetaminophen-induced acute liver failure cases.

Alterations in transporter expression may represent a compensatory mechanism of damaged hepatocytes to reduce accumulation of potentially toxic compounds. The present study was conducted to investigate the expression of hepatobiliary efflux transporters in livers from patients after toxic acetaminophen (APAP) ingestion, with livers from patients with primary biliary cirrhosis (PBC) serving as positive controls. mRNA and protein expression of multidrug resistance-associated protein (MRP) 1-6, multidrug resistance protein (MDR) 1-3/P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) in normal (n = 6), APAP overdose (n = 5), and PBC (n = 6) human liver samples were determined by branched DNA and Western blot analysis, respectively. Double immunohistochemical staining of P-gp and proliferating cell nuclear antigen (PCNA), a marker of proliferation, was performed on paraffin-embedded tissue sections. Compared with normal liver specimens, MRP1 and MRP4 mRNA levels were elevated after APAP overdose and in PBC. Up-regulation of MRP5, MDR1, and BCRP mRNA occurred in PBC livers. Protein levels of MRP4, MRP5, BCRP, and P-gp were increased in both disease states, with MRP1 and MRP3 protein also being induced in PBC. Increased P-gp protein was confirmed immunohistochemically and was found to localize to areas of PCNA-positive hepatocytes, which were detected in APAP overdose and PBC livers. The findings from this study demonstrate that hepatic efflux transporter expression is up-regulated in cases of APAP-induced liver failure and PBC. This adaptation may aid in reducing retention of byproducts of cellular injury and bile constituents within hepatocytes. The close proximity of P-gp and PCNA-positive hepatocytes during liver injury suggests that along with cell regeneration, increased efflux transporter expression is a critical response to hepatic damage to protect the liver from additional insult.