Edmund J. Sybertz

Diet-induced obese mice develop peripheral, but not central, resistance to leptin.

Leptin administration reduces obesity in leptin-deficient ob/ob mice; its effects in obese humans, who have high circulating leptin levels, remain to be determined. This longitudinal study was designed to determine whether diet-induced obesity in mice produces resistance to peripheral and/or central leptin treatment. Obesity was induced in two strains of mice by exposure to a 45% fat diet. Serum leptin increased in proportion to body weight (P < 0.00001). Whereas C57BL/6 mice initially responded to peripherally administered leptin with a marked decrease in food intake, leptin resistance developed after 16 d on high fat diet; mice on 10% fat diet retained leptin sensitivity. In AKR mice, peripheral leptin significantly decreased food intake in both 10 and 45% fat-fed mice after 16 d of dietary treatment. However, after 56 d, both groups became resistant to peripherally administered leptin. Central administration of leptin to peripherally leptin-resistant AKR mice on 45% fat diet resulted in a robust response to leptin, with a dose-dependent decrease in food intake (P < 0.00001) and body weight (P < 0.0001) after a single intracerebroventricular infusion. These data demonstrate that, in a diet-induced obesity model, mice exhibit resistance to peripherally administered leptin, while retaining sensitivity to centrally administered leptin.

Comparison of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its glucuronide, SCH60663.

Previous studies described the metabolism‐based discovery of a potent, selective inhibitor of intestinal absorption of cholesterol, SCH58235 (Ezetimibe). Here we demonstrate that the phenolic glucuronide (SCH60663) of SCH58235, was more potent at inhibiting cholesterol absorption in rats than SCH58235, when administered by the intraduodenal route. To understand the increased potency of the glucuronide, the metabolism and distribution of SCH58235 and SCH60663 were studied in bile duct‐cannulated rats. One minute after intraduodenal delivery of SCH58235, significant levels of compound were detected in portal plasma; >95% was glucuronidated, indicating that the intestine was metabolizing SCH58235 to its glucuronide. When intraduodenally delivered as SCH58235, the compound was glucuronidated, moved through the intestinal wall, into portal plasma, through the liver, and into bile. However, when delivered as SCH60663, >95% of the compound remained in the intestinal lumen and wall, which may explain its increased potency. Significant inhibition of cholesterol absorption and glucuronidation of SCH58235 occurred when SCH58235 was intravenously injected into bile duct‐cannulated rats. Autoradiographic analysis demonstrated that drug related material was located throughout the intestinal villi, but concentrated in the villus tip. These data indicate that (a) SCH58235 is rapidly metabolized in the intestine to its glucuronide; (b) once glucuronidated, the dose is excreted in the bile, thereby delivering drug related material back to the site of action and (c) the glucuronide is more potent than the parent possibly because it localizes to the intestine. Taken together, these data may explain the potency of SCH58235 in the rat (ID50=0.0015 mg kg−1) and rhesus monkey (ID50=0.0005 mg kg−1).

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)

Effects of selective inhibitors on cyclic nucleotide phosphodiesterases of rabbit aorta

In this study three forms of cyclic nucleotide phosphodiesterase (PDE) isolated from rabbit aorta were pharmacologically characterized, and the consequence of selective inhibition of calmodulin-stimulated PDE (CaM-PDE) and cGMP specific PDE (cG-PDE) was evaluated using PDE inhibitors. The cG-PDE (F1) was selectively inhibited by M&B 22948 (IC50 = 0.5 microM) and dipyridamole (IC50 = 7 microM). The cAMP-PDE (cA-PDE, F3) was inhibited more effectively by the cA-PDE inhibitor milrinone than by other PDE inhibitors. The cA-PDE preparation appeared to contain both cG-inhibited PDE and cG-insensitive PDE based on an additive inhibition of the activity by milrinone and SQ 65442, respective inhibitors of these enzymes. Vinpocetine, 8-methoxymethyl isobutylmethylxanthine (8-MeOMeMIX) and M&B 22948 effectively inhibited CaM-PDE (F2). Vinpocetine was a more selective inhibitor of CaM-PDE than M&B 22948 or 8-MeOMeMIX. CaM-PDEs isolated from rabbit aorta and bovine brain exhibited a similar sensitivity to these inhibitors. Seventy-two percent of the cGMP-hydrolyzing activity of this rabbit aortic CaM-PDE preparation was immunoadsorbed to monoclonal antibody (ACC-1) against CaM bound to brain CaM-PDE. Vinpocetine, 8-MeOMeMIX and M&B 22948 at concentrations (30 and 100 microM) which inhibit CaM-PDE greater than 60% increased cGMP but not cAMP levels in l-norepinephrine (NE) preincubated rabbit aortic slices. At concentrations selectively inhibiting cG-PDE, dipyridamole and M&B 22948 increased cGMP levels in untreated slices but failed to increase cGMP levels significantly in NE-treated slices. By contrast, vinpocetine failed to increase cGMP significantly in untreated slices, although it increased cGMP levels in NE or KCl preincubated slices. These data indicate that, in activated (precontracted) aorta, CaM-PDE is a major enzyme, whereas in untreated aorta cG-PDE is a predominant enzyme for the hydrolysis of cGMP. This study also shows a usefulness of selective inhibitors in identifying different forms of PDE and similar drug sensitivities and immunoadsorption of aortic and brain CaM-PDEs by a monoclonal antibody.

Evidence for the Presence of a Proteinase-Activated Receptor Distinct From the Thrombin Receptor in Vascular Endothelial Cells

The thrombin receptor was the first cloned G protein-coupled receptor reported to be activated by proteolytic cleavage of its extracellular amino terminus. A second proteinase-activated receptor (PAR-2) was cloned recently and expressed in Xenopus laevis oocytes. PAR-2 was activated by trypsin and by a peptide (SLIGRL) derived from the new amino terminus. Since PAR-2 mRNA was detected in highly vascularized organs, we compared the physiological functions of the thrombin receptor and PAR-2 in vascular endothelium. Thrombin and trypsin both elicited endothelium-dependent relaxations in prostaglandin F2alpha (PGF2alpha)-contracted strips of porcine coronary artery. Whereas high doses of both thrombin or trypsin (10 U/mL) caused homologous desensitization, trypsin caused further relaxation of thrombin-desensitized tissues. Thrombin and PAR-2-derived peptides (SFLLRN and SLIGRL) both induced endothelium-dependent relaxations in PGF2alpha-contracted porcine coronary arteries. SFLLRN or SLIGRL (30 micronmol/L) also showed homologous desensitization but not cross desensitization. In the presence of the NO synthase inhibitor NG-monomethyl-L-arginine (1 mmol/L), both SFLLRN- and SLIGRL-induced relaxations were partially inhibited. SFLLRN elicited weak contraction in coronary arteries without endothelium, whereas SLIGRL had no effect. Intravenous injection of SFLLRN (1 mg/kg, bolus) into anesthetized rats elicited a transient depressor response followed by pronounced pressor response. In contrast, intravenous administration of SLIGRL (1 mg/kg, bolus) produced only a marked depressor response. Consistent with the in vivo data, SFLLRN contracted the endothelium-rubbed rat aortic rings and aggregated human platelets in vitro, whereas SLIGRL had no effect. The finding that both trypsin and SLIGRL induced endothelium-dependent relaxations indicates the presence of PAR-2 on endothelial cells. In addition, both trypsin and SLIGRL elicited relaxations in thrombin- or SFLLRN-desensitized tissue, suggesting that PAR-2 is distinct from thrombin receptor in vascular endothelium. The lack of PAR-2-mediated platelet aggregation or smooth muscle contraction suggested it might not share the pathogenic properties associated with the thrombin receptor in the vasculature.

Inhibition of endothelial derived relaxing factor (EDRF) aggravates ischemic acute renal failure in anesthetized rats

SummaryThe relative importance of endothelial derived relaxing factor (EDRF)/nitric oxide (NO) in maintaining kidney function in normal condition and in acute renal failure (ARF) were evaluated in inactin anesthetized rats. ARF was induced by unilateral occlusion of the left renal artery (40 min) followed by reperfusion, with the contralateral kidney serving as normal control. This protocol resulted in marked reductions in renal plasma flow (RPF), glomerular filtration rate (GFR) and increases in fractional sodium excretion (FENa) and urinary protein excretion in the post-ischemic kidney in comparison to the contralateral normal kidney. Administration of the nitric oxide (NO) synthase inhibitor NG — monomethyl-L-arginine (0.25 mg/kg per min, L-NMMA) exacerbated the ischemia-induced changes in renal functions as reflected by further reductions in urine flow (V), GFR, marked sodium wasting and renal edema. Pretreatment of the animals with NO precursor L-arginine (2.5 mg/kg per min, L-Arg) abolished the detrimental effects of L-NMMA in ARE In contrast, D-Arginine (2.5 mg/kg per min, DArg) failed to reverse the detrimental effects of L-NMMA. Infusion of L-Arg alone also resulted in improvements in RPF and GFR in the ischemic kidney. The results of the present study suggest that the function of the ischemic kidney is sustained by EDRF/NO and is thus more sensitive to NO synthase inhibition.

Ca/CaM-Stimulated and cGMP-Specific Phosphodiesterases in Vascular and Non-Vascular Tissues

A large body of evidence indicates that guanosine 3’,5’-cyclic monophosphate (cGMP) plays an essential role in vasorelaxant actions of various agents such as atrial natriuretic factor (ANF), nitrogen oxide containing compounds (e.g., nitroprusside) and endothelium dependent vasodilators (e.g., acetylcholine) (1). These agents elevate cGMP by stimulating either soluble or particulate guanylate cyclase (1).

Modulation of circulating endothelin levels in hypertension and endotoxemia in rats

We have developed separate radioimmunoassays to measure circulating ET-1 and ET-3 levels in normotensive and different hypertensive rat models so that the role of endothelin in the regulation of vasomotor function can be studied. We also assessed the stimulatory effects of endotoxin on plasma and liver lymph ET-1 and ET-3 levels. The circulating ET-1 levels in normotensive rats, SHRs, and DOCA-salt hypertensive rats were 2.3 ± 0.5, 2.1 ± 0.4, and 2.1 ± 0.9 pg/ml, respectively. Similarly; the plasma ET-3 levels in normotensive and different hypertensive rats were similar, ranging from 19.7 ± 1.5 to 24.7 ± 2.2 pg/ml. The data indicate that steadystate circulating levels of endothelins are a poor correlate of the hypertensive state. Endotoxin (30 mg/kg i.v. over 15 min) reduced blood pressure significantly and augmented plasma ET-1 levels by sevenfold (29.1 ± 3.7 vs. 4.1 ± 0.6 pg/ml in the vehicle group; p < 0.05) and ET-3 levels by twofold (47.7 ± 7.0 vs. 22.7 ± 4.0 pg/ml in the vehicle group; p < 0.05).Human TNF-α (30 ng/kg/min x 30 min), a putative mediator of endotoxin shock, enhanced plasma ET-1 (18.3 ± 1.0 vs: 2.7 ± 0.4 pg/ml in the vehicle group; p < 0.05) by sevenfold and ET-3 levels by twofold (45.7 ± 2.0 vs. 27.1 ± 4.0 pg/ml in the vehicle group; p < 0.05) without affecting blood pressure. In con-trash; PAF (50 ng/kg/min x 30 min); another mediator liberated during endotoxin shock; exerted asimilar hypotensive response to endotoxin but did not alter either plasma ET-1 or ET-3 levels. In a separate study, endotoxin augmented plasma and liver lymph ET-1 levels by eight- and twofold, respectively. In comparison, endotoxin caused a twofold increase in both plasma and lymph ET-3 levels. Pretreatment with indomethacin (10 mg/kg p.o.)significantly attenuated the endotoxin-induced in-creases in plasma ET-1 and ET-3 levels without affecting the depressor responses to endotoxin. The endotoxininduced increase in ET-1 levels in the plasma and liver lymph is most likely derived from the endothelial cells. However, since ET-3 is not produced by endothelial cells, the source of endotoxin-induced increases in plasma and liver lymph ET-3 levels remains to beidentified. The data suggest that the endotoxin-stimulated re-lease of ET-1 and ET-3 can be dissociated from blood pressure changes.Eicosanoids may also be involved in the release of endothelins due to endotoxin.

Atrial natriuretic factor-potentiating and antihypertensive activity of SCH 34826. An orally active neutral metalloendopeptidase inhibitor.

The effects of SCH 34826, an orally active neutral metalloendopeptidase inhibitor, on responses to atrial natriuretic factor-(103–125) or -(99–126) and on blood pressure were evaluated in rats. SCH 34826 (10,30, and 90 mg/kg s.c. and 90 mg/kg p.o.) potentiated the antihypertensive action of atrial natriuretic factor (30 /tg/kg i.v.) in conscious spontaneously hypertensive rats. SCH 34826 (90 mg/kg) also potentiated the diuretic and natriuretic responses to atrial natriuretic factor (30 /tg/kg i.v.) as well as the plasma levels achieved after pcptide injection. SCH 34826 significantly reduced blood pressure in the conscious deoxycorticosterone acetate-salt hypertensive rat, at doses of 90 mg/kg s.c. (−35±12 mm Hg), 10 mg/kg p.o. (−30±7 mm Hg), and 90 mg/kg p.o. (−45±6 mm Hg). SCH 34826 was devoid of acute antihypertensive activity in the spontaneously hypertensive rat but reduced blood pressure by day 3 of a 5-day treatment schedule. SCH 34826 (90 mg/kg s.c.) enhanced urine volume output in the deoxycorticosterone acetate-salt rat (2.78±0.6 vs. l27±03 ml/100 g/3 hr in vehicle-control rats, p < 0.05). SCH 34826 (90 mg/kg s.c) increased plasma levels of atrial natriuretic factor at 1 hour (753±89 vs. 451 ±79 pg/ml in vehicle-treated rats, p< 0.05) but not 3 hours after dosing. The renal excretion of atrial natriuretic factor (3,092 ±1,089 vs. 21 ±6 pg/100 g/3 hr in vehicle-treated rats, p< 0.05) and cyclic guanosine monophosphate (2,131 ±509 vs. 879±168 pg/100 g/3 hr in vehicle-treated rats, p < 0.05) was markedly elevated by SCH 34826 in deoxycorticosterone acetate-salt rats. These studies suggest that neutral endopeptidase inhibition may represent a new approach to treatment of some forms of hypertension.

Antiplatelet and antiproliferative effects of SCH 51866, a novel type 1 and type 5 phosphodiesterase inhibitor.

SCH 51866 is a potent and selective PDE1 and PDE5 inhibitor. The antiplatelet, antiproliferative, and hemodynamic effects of SCH 51866 were compared with those of E4021, a highly selective PDE5 inhibitor. SCH 51866 inhibited PDE1 and PDE5 isozymes with a 50% inhibitory concentration (IC50) of 70 and 60 nM, respectively. SCH 51866 and E4021 inhibited washed human platelet aggregation induced by collagen with an IC50 of 10 and 4 microM, respectively, and attenuated (p < 0.05) the adhesion of 111indium-labeled platelets to the nylon filament-injured rat aorta. The doses of SCH 51866 and E4021 that inhibited platelet adhesion caused significant increases in platelet cyclic guanosine monophosphate (cGMP; p < 0.05). SCH 51866 (1-10 mg/kg, p.o. twice daily) but not E4021 (3-30 mg/kg, p.o twice daily) inhibited neointima formation in the carotid arteries of spontaneously hypertensive rats (SHRs) subjected to balloon angioplasty. Moreover, SCH 51866 (0.3-10 mg/kg, p.o.) elicited dose-dependent reduction in blood pressure in SHRs, whereas E4021 (3-30 mg/kg, p.o.) did not affect blood pressure in SHRs. In conclusion, the data suggest that inhibition of PDE1 and PDE5 isozymes by SCH 51866 exerts antiplatelet and vascular protective effects. In comparison, inhibition of PDE5 alone by E4021 exhibited antiplatelet effects without affecting neointima formation.