Blair Weig

Hypocholesterolemic activity of a novel inhibitor of cholesterol absorption, SCH 48461

The amount of cholesterol that circulates in the plasma as lipoproteins can be affected by the balance of cholesterol metabolism within and between the intestines and liver. In the present report, we describe a novel hypocholesterolemic agent and document its pharmacological effects in animal models of hypercholesterolemia. The oral administration of (3R,4S)-1,4-bis-(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone (SCH 48461) reduced plasma cholesterol concentrations in cholesterol-fed hamsters, rats and rhesus monkeys with ED50s of 1, 2 and 0.2 mg/kg per day, respectively, SCH 48461 was also highly effective in reducing hepatic cholesteryl ester accumulation in cholesterol-fed hamsters and rats after 7 days of treatment. In one 3 week study, rhesus monkeys were fed a 0.25% cholesterol/22% saturated fat diet with or without SCH 48461. At the end of the 3 week period the control groups VLDL + LDL-cholesterol increased to 180 Mg/dl from a baseline of approximately 65 mg/dl while plasma apolipoprotein B levels had doubled. Animals treated daily with 1 mg/kg SCH 48461 maintained their baseline levels of VLDL + LDL-cholesterol, HDL-cholesterol, and plasma apolipoproteins B and A-I. After 3 weeks the diets of the two groups were switched. Within 1 week SCH 48461 (1 mg/kg per day) rapidly reversed the elevated VLDL + LDL-cholesterol levels of the previous control group to near baseline values. SCH 48461 exerted its hypocholesterolemic effect through the inhibition of cholesterol absorption. A dose of 10 mg/kg per day inhibited cholesterol absorption in cholesterol-fed hamsters by 68% while a similar reduction was achieved in chow-fed monkeys with 3 mg/kg per day. This latter dose inhibited cholesterol absorption in cholesterol-fed monkeys by 95%. Treatment of cholesterol-fed monkeys with 10 mg/kg per day SCH 48461 significantly increased fecal neutral sterol excretion (52 vs. 32 mg/kg) but had no effect on acidic sterol excretion. Using a 2-h absorption model in cholesterol-fed hamsters, SCH 48461 caused a 46% inhibition of unesterified [14C]cholesterol accumulation in the intestinal wall and a 90% inhibition of cholesteryl ester formation at a dose of 10 mg/kg. Similar data were observed when the plasma radioactivity was assessed, indicating inhibition of both free (61%) and esterified (85%) cholesterol appearance. In contrast, CI-976, a potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, did not affect the uptake of free cholesterol into the intestines while inhibiting cholesterol esterification (98% inhibition).(ABSTRACT TRUNCATED AT 400 WORDS)

Nociceptin (ORL‐1) and μ‐Opioid Receptors Mediate Mitogen‐Activated Protein Kinase Activation in CHO Cells Through a Gi‐Coupled Signaling Pathway: Evidence for Distinct Mechanisms of Agonist‐Mediated Desensitization

Abstract: The recently identified 17‐amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor‐like‐1 (ORL‐1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL‐1 receptor (OFQR) may be to modulate opioid‐induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL‐1 (OFQR) modify opioid‐stimulated effects, however, is unclear. Both ORL‐1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)‐sensitive signal transduction, indicating these receptors are capable of coupling to Gi/Go proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen‐activated protein (MAP) kinase in CHO cells expressing ORL‐1 receptor (OFQR). Nociceptin (OFQ)‐stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of β‐adrenergic receptor kinase (βARKct), which specifically blocks Gβγ‐mediated signaling. Expression of the proline‐rich domain of SOS (SOS‐PRO), which inhibits SOS interaction with p21ras, also attenuated nociceptin (OFQ)‐stimulated MAP kinase activation. The phosphatidylinositol 3‐kinase (PI‐3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)‐stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing μ‐opioid receptor, [d‐Ala2,N‐Me‐Phe4,Gly‐ol]‐enkephalin (DAMGO; a μ‐opioid receptor‐selective agonist) stimulated PTX‐sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, βARKct expression, or SOS‐PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL‐1 (OFQR) and μ‐opioid receptors mediate MAP kinase activation via a signaling pathway using the βγ‐subunit of Gi, a PI‐3K, and SOS, independent of PKC activity. In cells expressing both ORL‐1 (OFQR) and μ‐opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)‐ or DAMGO‐stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO‐stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)‐stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL‐1 (OFQR) can modulate μ‐opioid receptor signaling in a cellular system.

Nociceptin activation of the human ORL1 receptor expressed in Chinese hamster ovary cells: functional homology with opioid receptors.

Opioid receptor-like 1 (ORL1) receptor, a member of the superfamily of G-protein-coupled receptors has significant primary sequence homology to the mu-, delta- and kappa-opioid receptors. The ORL1 receptor is selectively activated by the recently discovered peptide nociceptin. To probe the functional homology amongst these receptors, a Chinese hamster ovary (CHO) cell line expressing the human ORL1 receptor has been characterized. Nociceptin inhibited forskolin-stimulated increases in intracellular cAMP with an IC50 of 70 pM. Stimulation by nociceptin caused a 2-fold increase in the rate of [35S]GTPgammaS binding to membranes derived from CHO cells expressing the ORL1 receptor. Following incubation with nociceptin mitogen-activated protein kinase activity was increased by 2-fold in cells expressing the ORL1 receptor. In non-transfected CHO cells, nociceptin had no effect on cAMP accumulation, the rate of [35S]GTPgammaS binding or mitogen-activated protein kinase activity. Human ORL1 receptors expressed in CHO cells selectively bound [125I][Tyr14]nociceptin with a Kd of 2.1 pM and a Bmax of 2.6 pmol/mg protein. Similar to opioid receptors, nociceptin binding to the ORL1 receptor was altered by Na+, GTPgammaS and dithiothreitol. Na+ increased the Kd of nociceptin binding to the ORL1 receptor. GTPgammaS decreased the apparent Bmax of [125I][Tyr14]nociceptin binding but had no effect on the Kd of the remaining sites. Pretreatment with dithiothreitol inhibited nociceptin binding to the ORL1 receptor. Nociceptin binding was insensitive to low nanomolar concentrations of opioid receptor-selective agonists and antagonists. However, high micromolar levels of opioid receptor-selective agents inhibited the binding. Morphine, naloxone, naltrindole, nor-Binaltorphimine and CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) inhibited nociceptin binding to ORL1 receptor with Ki values of 36, 24, 0.4, 8 and 28 microM, respectively. These results imply that ORL1 is a G-protein-coupled receptor with functional as well as structural homology to opioid receptors. In addition, opioid receptor ligands may serve as starting templates for the development of ORL1 specific ligands.