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1.
Toxicol Appl Pharmacol ; 223(3): 246-56, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17663016

ABSTRACT

We previously reported prevention of urolithiasis and associated rat urinary bladder tumors by urine acidification (via diet acidification) in male rats treated with the dual peroxisome proliferator-activated receptor (PPAR)alpha/gamma agonist muraglitazar. Because urine acidification could potentially alter PPAR signaling and/or cellular proliferation in urothelium, we evaluated urothelial cell PPARalpha, PPARdelta, PPARgamma, and epidermal growth factor receptor (EGFR) expression, PPAR signaling, and urothelial cell proliferation in rats fed either a normal or an acidified diet for 5, 18, or 33 days. A subset of rats in the 18-day study also received 63 mg/kg of the PPARgamma agonist pioglitazone daily for the final 3 days to directly assess the effects of diet acidification on responsiveness to PPARgamma agonism. Urothelial cell PPARalpha and gamma expression and signaling were evaluated in the 18- and 33-day studies by immunohistochemical assessment of PPAR protein (33-day study only) and quantitative real-time polymerase chain reaction (qRT-PCR) measurement of PPAR-regulated gene expression. In the 5-day study, EGFR expression and phosphorylation status were evaluated by immunohistochemical staining and egfr and akt2 mRNA levels were assessed by qRT-PCR. Diet acidification did not alter PPARalpha, delta, or gamma mRNA or protein expression, PPARalpha- or gamma-regulated gene expression, total or phosphorylated EGFR protein, egfr or akt2 gene expression, or proliferation in urothelium. Moreover, diet acidification had no effect on pioglitazone-induced changes in urothelial PPARgamma-regulated gene expression. These results support the contention that urine acidification does not prevent PPARgamma agonist-induced bladder tumors by altering PPARalpha, gamma, or EGFR expression or PPAR signaling in rat bladder urothelium.


Subject(s)
Acids/urine , Diet , Epidermal Growth Factor/biosynthesis , Peroxisome Proliferator-Activated Receptors/biosynthesis , Urinary Bladder/metabolism , Urothelium/metabolism , Animals , Cell Proliferation , Immunohistochemistry , Male , Peroxisome Proliferator-Activated Receptors/agonists , Phosphorylation , Pioglitazone , Rats , Rats, Sprague-Dawley , Signal Transduction , Thiazolidinediones/pharmacology , Urinary Bladder/cytology , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/prevention & control , Urothelium/cytology
2.
Proc Natl Acad Sci U S A ; 104(16): 6846-51, 2007 Apr 17.
Article in English | MEDLINE | ID: mdl-17428923

ABSTRACT

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.


Subject(s)
Molecular Probes/physiology , Oxazepines/pharmacology , Parathyroid Hormone/physiology , Peptide Fragments/physiology , Receptor, Parathyroid Hormone, Type 1/antagonists & inhibitors , Amino Acid Sequence , Animals , Binding, Competitive , Cell Line , Drug Evaluation, Preclinical , Humans , Male , Molecular Probe Techniques , Molecular Sequence Data , Oxazepines/agonists , Parathyroid Hormone/agonists , Parathyroid Hormone/metabolism , Peptide Fragments/agonists , Peptide Fragments/metabolism , Protein Binding , Rats , Rats, Sprague-Dawley , Receptor, Parathyroid Hormone, Type 1/agonists , Receptor, Parathyroid Hormone, Type 1/metabolism
3.
J Pharmacol Exp Ther ; 321(1): 107-15, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17259449

ABSTRACT

There are two major defects in type 2 diabetes: 1) insulin resistance and 2) insulin deficiency due to loss of beta-cell function. Here we demonstrated that treatment with muraglitazar (a dual peroxisome proliferator-activated receptor alpha/gamma activator), when initiated before or after the onset of diabetes in mice, is effective against both defects. In study 1, prediabetic db/db mice were treated for 12 weeks. The control mice developed diabetes, as evidenced by hyperglycemia, hyperinsulinemia, reduced insulin levels in the pancreas, blunted insulin response to glucose, and impaired glucose tolerance. The muraglitazar-treated mice had normal plasma glucose, and insulin levels, equivalent or higher pancreatic insulin content than normal mice, showed a robust insulin response to glucose and exhibited greater glucose tolerance. In study 2, diabetic db/db mice were treated for 4 weeks. The control mice displayed increased glucose levels, severe loss of islets, and their isolated islets secreted reduced amounts of insulin in response to glucose and exendin-4 compared with baseline. In muraglitazar-treated mice, glucose levels were reduced to normal. These mice showed reduced loss of islets, and their isolated islets secreted insulin at levels comparable to baseline. Thus, muraglitazar treatment decreased both insulin resistance and preserved beta-cell function. As a result, muraglitazar treatment, when initiated before the onset of diabetes, prevented development of diabetes and, when initiated after the onset of diabetes, prevented worsening of diabetes in db/db mice.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Glycine/analogs & derivatives , Hypoglycemic Agents/pharmacology , Oxazoles/pharmacology , PPAR alpha/agonists , PPAR gamma/agonists , Animals , Body Weight/drug effects , C-Peptide/metabolism , Diabetes Mellitus, Experimental/genetics , Disease Progression , Fatty Acids, Nonesterified/blood , Female , Glucose Tolerance Test , Glycated Hemoglobin/metabolism , Glycine/pharmacology , Insulin/metabolism , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Pancreas/drug effects , Pancreas/metabolism , Triglycerides/blood
4.
J Bone Miner Res ; 18(1): 88-96, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12510809

ABSTRACT

Statins, which are inhibitors of 3-hydroxy-3-glutaryl-coenzyme A (HMG-CoA) reductase, decrease the hepatic biosynthesis of cholesterol by blocking the mevalonate pathway. Nitrogen-containing bisphosphonate drugs also inhibit the mevalonate pathway, preventing the production of the isoprenoids, which consequently results in the inhibition of osteoclast formation and osteoclast function. Therefore, we hypothesized that statins could affect bone metabolism in vivo through effects on osteoclastic bone resorption. In vitro, cerivastatin inhibited the parathyroid hormone (PTH)-stimulated bone resorption. Using a panel of 40 statin analogs, which showed variable effects on HMG-CoA reductase activity, we found that the ability of compounds to inhibit bone resorption is directly related to HMG-CoA reductase activity. However, in the thyro-parathyrodectomy (TPTX) model for bone resorption in the rat in vivo, cerivastatin did not prevent experimentally induced increases in bone resorption. The lack of effect of cerivastatin in this model is not related to a limited penetration of the target tissue (bone marrow), because a significant effect on HMG-CoA reductase activity was demonstrated in the total rat bone marrow cell extracts of rats posttreatment in vivo. Furthermore, cerivastatin inhibited protein prenylation in osteoclasts isolated from the rabbit bone marrow of rabbits after treatment in vivo. In contrast to other studies, none of the statins tested showed anabolic effects in parietal bone explant cultures. Taken together, we conclude that statins inhibit bone resorption in vitro, which correlates directly with the potency of the compounds for inhibition of HMG-CoA reductase activity. However, cerivastatin does not affect bone resorption in the rat TPTX model in vivo.


Subject(s)
Bone Resorption/enzymology , Bone Resorption/prevention & control , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Animals , Bone Resorption/metabolism , Calcium/metabolism , Female , Hydroxymethylglutaryl CoA Reductases/metabolism , In Vitro Techniques , Lovastatin/pharmacology , Male , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteogenesis/drug effects , Parathyroidectomy , Pravastatin/pharmacology , Protein Prenylation/drug effects , Pyridines/pharmacology , Rabbits , Rats , Rats, Sprague-Dawley , Simvastatin/pharmacology , Thyroidectomy
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