Karen M. Zimmerman

Losartan, a nonpeptide angiotensin II (Ang II) receptor antagonist, inhibits neointima formation following balloon injury to rat carotid arteries

Angiotensin-converting enzyme inhibitors have been shown to inhibit intimal thickening following balloon catheterization of rat carotid arteries. To assess the role of the renin-angiotensin pathway and the angiotensin type-I (AT1) receptor in this effect, the nonpeptide Ang II antagonist losartan (DuP 753) or vehicle was infused continuously i.v. in rats from two days before to two weeks after balloon injury to the left common carotid artery; drug effects upon intimal thickening were examined histologically. Losartan produced a dose-dependent reduction in cross-sectional area of intimal lesions determined two weeks post balloon injury. At 5 mg/kg/day a nonsignificant 23% reduction of intimal area was observed. At the higher dose of 15 mg/kg/day, losartan produced a 48% reduction in intimal area (P less than 0.05) compared to the vehicle-infused group. The cellular density of the neointima was not affected by losartan, indicating a probable effect of the drug upon migration and/or proliferation of smooth muscle cells. In separate groups of non-ballooned rats, losartan infusions of 5 and 15 mg/kg/day produced significant rightward shifts (averaging 6.4- and 55-fold, respectively) in curves relating increases in blood pressure to intravenous Ang II in pithed rats determined between 2 and 16 days following initiation of losartan infusion. Mean arterial blood pressure (determined under alpha-chloralose anesthesia) was reduced following continuous losartan infusion for 6 days from 128 +/- 8 mm Hg (vehicle) to 105 +/- 8 mm Hg at 5 mg/kg/day (P less than 0.05), and 106 +/- 4 mm Hg at 15 mg/kg/day (P less than 0.05). Thus, losartan attenuated the vascular response to balloon catheter injury, and this effect was associated with functional block of vascular AT1 receptors. The results support a role for Ang II, acting via AT1 receptors, in myointimal thickening subsequent to balloon injury of rat carotid arteries.

Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure

Cholesteryl ester transfer protein (CETP) catalyses the exchange of cholesteryl ester and triglyceride between HDL and apoB containing lipoprotein particles. The role of CETP in modulating plasma HDL cholesterol levels in humans is well established and there have been significant efforts to develop CETP inhibitors to increase HDL cholesterol for the treatment of coronary artery disease. These efforts, however, have been hampered by the fact that most CETP inhibitors either have low potency or have undesirable side effects. In this study, we describe a novel benzazepine compound evacetrapib (LY2484595), which is a potent and selective inhibitor of CETP both in vitro and in vivo. Evacetrapib inhibited human recombinant CETP protein (5.5 nM IC50) and CETP activity in human plasma (36 nM IC50) in vitro. In double transgenic mice expressing human CETP and apoAI, evacetrapib exhibited an ex vivo CETP inhibition ED50 of less than 5 mg/kg at 8 h post oral dose and significantly elevated HDL cholesterol. Importantly, no blood pressure elevation was observed in rats dosed with evacetrapib at high exposure multiples compared with the positive control, torcetrapib. In addition, in a human adrenal cortical carcinoma cell line (H295R cells), evacetrapib did not induce aldosterone or cortisol biosynthesis whereas torcetrapib dramatically induced aldosterone and cortisol biosynthesis. Our data indicate that evacetrapib is a potent and selective CETP inhibitor without torcetrapib-like off-target liabilities. Evacetrapib is currently in phase II clinical development.

The Relation Between Vascular Relaxant and Cardiac Electrophysiological Effects of Pinacidil

Pinacidil may represent an example of a new class of vasodilators that act by increasing membrane permeability to potassium ions. In the present study, the cardiac electrophysiological and venorelaxant effects of a series of pinacidil analogs in canine tissues in vitro were examined. Piacidil (3 x 10(-5) M) markedly reduced action potential duration in Purkinje fibers (82 +/- 3% decrease) and ventricular muscle (54 +/- 2% decrease) without significantly affecting maximal upstroke velocity of the action potential or conduction time. The EC50 for the reduction in Purkinje fiber action potential duration was 2.6 +/- 0.5 microM. Pinacidil also decreased barium-induced automaticity in Purkinje fibers; the concentration that decreased the rate of firing by 50% was identical to the EC50 for decreasing action potential duration. In some preparations, high concentrations of pinacidil (greater than or equal to 3 x 10(-5) M) were associated with the appearance of spontaneous action potentials that were closely coupled to the preceding driven action potential. The EC50 for pinacidil in relaxing phenylephrine-contracted cephalic veins was 0.43 +/- 0.09 microM, and in isolated cat papillary muscle, pinacidil had a direct negative inotropic effect with an EC50 of 4.1 +/- 0.7 microM. Thus, pinacidil was 6 and 10 times more potent in relaxing phenylephrine-contracted veins than in shortening action potential or decreasing cardiac contractility. There was an excellent correlation (r = 0.933, p = 0.002) between decreases in action potential duration and venorelaxation for all pinacidil analogs, as well as for BRL 34915 and nicorandil, two purported potassium channel openers. Significant correlations were also obtained between negative inotropic effects and reductions in action potential duration for the pinacidil series. Pinacidil (10(-5) M) also inhibited the venoconstrictor responses to the selective alpha 2 agonist, B-HT 920, to a greater extent than the alpha 1 agonist, methoxamine. Since a good correlation exists in vitro among all the compounds studied in reducing action potential duration, relaxing vascular tissue, and decreasing cardiac contractility, it is concluded that pinacidil as well as nicorandil and BRL 34915 affect vascular and cardiac tissues by similar mechanisms, possibly by increases in potassium ion permeability, although other mechanisms may also play a role.

Characterization of distinct angiotensin II binding sites in rat adrenal gland and bovine cerebellum using selective nonpeptide antagonists.

We investigated the characteristics of 125I-AII binding to rat adrenal and bovine cerebellar membranes in the presence and absence of new nonpeptide angiotensin II (All) receptor ligands. The imidazole All ligands, DUP753 and WL19, both produced biphasic competition curves to 125I-All binding in rat adrenal glomerulosa and adrenal medulla particles, suggesting the existence of two distinct All binding sites. Antagonist affinity (Ki) and binding capacity (Bmax) for each binding site was determined using nonlinear analysis of competition data fit to a two-site model. The high capacity site (68% of total specific 125I-All bound) in glomerulosa had high affinity for DUP753 (4.6 ± 0.8 nM) and low affinity for WL19 (29 ± 3 μM), and the low capacity site had high affinity for WL 19 (3.3 ± 1.4 nM) and low affinity for DUP753 (51 ± 9 μM). Conversely, in medulla, the high capacity site (77% total binding) had high affinity for WL 19 (19 ± 6 nM) and low affinity for DUP753 (29 ± 8 μM), and the low capacity site had low affinity for WL19 (25 ± 7 μM) but a high affinity for DUP753 (2.8 ± 2.0 nM). In glomerulosa, binding parameters for the nonpeptide ligands at each site derived from monophasic competition curves obtained in the presence of either 0.3 μM DUP753 or WL 19 to selectively block the high or low capacity binding site, respectively, were similar to values determined from the biphasic competition curves. Unlike the nonpeptide inhibitors, unlabeled All yielded monophasic inhibition curves. WL19 and DUP753 also blocked 125I-All binding to bovine cerebellar membranes (Ki = 0.14 ± 0.03 and 170 ± 21 μM, respectively), and both displayed monophasic competition curves. All-induced contraction of rabbit aorta was potently and competitively blocked by DUP753 (K = 3.9 ± 0.4 nM) but not by WL 19. Thus, the Ki values for DUP753 at the DUP753-sensitive binding site in rat adrenal glomerulosa and medulla are similar to the affinity of DUP753 for All receptors in rabbit aorta, suggesting a similarity among these sites. The Ki values for WL19 at the WL19-sensitive site in rat adrenal gland and bovine cerebellum differ from 7–40-fold and represent binding site(s) distinct from the DUP753-sensitive site.

Chiral recognition of the angiotensin II (AT1) receptor by a highly potent phenoxyproline octanoamide

Abstract The synthesis and in vitro biological evaluation of a novel series of diastereomeric phenoxyproline octanoamides ( 3–h ) as angiotensin II (AT 1 ) receptor antagonists are reported.

LY249933: a cardioselective 1,4-dihydropyridine with positive inotropic activity.

Compound LY249933 and its component diastereomers, (RR) and (SR), were studied for their vascular and cardiac effects in vitro and in vivo. In guinea pig cardiac ventricular membranes, LY249933, (RR), and (SR) potently displaced bound [3H]nitrendipine (Kd values = 2–6 nM). In isolated guinea pig right ventricular strips, LY249933 produced a small but significant increase in contraction, whereas (RR) substantially increased (−log EC50 (M) = 4.6 ± 0.8) and (SR) decreased contraction (−log EC50 (M) = 4.1 ± 0.8). In isolated canine cephalic vein, contracted with 80 mM KC1, an increase in contraction was produced by (RR), whereas relaxation was produced by LY249933 (− log EC50 (M) = 5.9 ± 0.9) and (SR) (−log EC50 (M) = 6.0 ± 0.7). At 20 mM KC1, (RR) increased, (SR) decreased, but LY249933 did not alter contraction. In anesthetized dogs, LY249933 (200 μg/kg/min, i.v.) increased dP/dt60, decreased heart rate, but did not change vascular resistance or rate pressure product. At the same dose, (RR) and (SR) both tended to increase dP/dt60 nonsignificantly, whereas (RR) increased and (SR) decreased vascular resistance. Both (RR) and (SR) tended to decrease heart rate nonsignificantly, whereas (RR) did not change and (SR) decreased rate pressure product. Thus, LY249933 produced potentially beneficial cardiovascular changes resulting from the combined actions of its (RR) and (SR) diastereomers that are postulated to be calcium agonist and antagonist, respectively.

Identification, synthesis and pharmacological activity of moxonidine metabolites

The metabolism of moxonidine, 4-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-6-methoxy-2-methyl-5-pyrimidinamine, LY326869, in rats, mice, dogs, and humans has been examined. At least 17 metabolites were identified or tentatively identified in the different species by HPLC, LC/MS and LC/MS/MS. The identities of seven of the major metabolites have been verified by independent synthesis. The metabolites are generally derived from oxidation and conjugation pathways. Oxidation occurred at the imidazolidine ring as well as the methyl at the 2 position of the pyrimidine ring. All seven metabolites were examined in the spontaneously hypertensive rats (3 mg kg(-1), i.v.) for pressure and heart rate. Only one, 2-hydroxymethyl-4-chloro-5-(imidazolidin-2-ylidenimino)-6-methoxypyrimidine, exerted a short-lasting decrease in blood pressure, albeit attenuated in magnitude compared to moxonidine.

Binding and pharmacologic properties of peptides derived from human and rat angiotensin II (AII) mRNA

We investigated the binding and pharmacologic properties of peptides encoded by complementary mRNA derived from the human and rat angiotensinogen gene (human and rat IIA, respectively). Human IIA (identical with AII in 4 amino acids) inhibited binding of [125I]AII to rat adrenal glomerulosa particles (Ki = 0.62 +/- 0.09 microM) and competitively blocked, with similar potency, the ability of three AII receptor agonists to contract rabbit aorta. Rat IIA affected neither [125I]AII binding to glomerulosa particles nor the contractile response of AII. We conclude that rat IIA does not interact with AII or its receptors and that human IIA acts as a competitive inhibitor of AII at the receptor level.

Synthesis and pharmacological evaluation of a novel series of 5-aryl benzimidazole angiotensin II receptor antagonists

Abstract A novel series of benzimidazole angiotensin II (Ang II) receptor antagonists (4, 13, 5, 17) were synthesized and compared pharmacologically to the previously described imidazole analogues (1, 3a-b).

The Application of PK/PD Modeling and Simulations During Lead Optimization

A quick view of recent periodicals clearly point to the challenges of the pharmaceutical industry as we progress through the first decade of the 21st century — the rising costs of new innovative drugs. Much of the current costs and the continued rising costs of drug prices stem from the high price of drug discovery and development. Many sources have cited studies that estimate the cost of pharmaceutical drug development; all of which conclude the same — pharmaceutical RD DimAsi et al., 2003). The increase in R&D costs is due mainly to the increased cost of animal testing and conducting clinical trials (Dickson and Gagnon, 2004). The cost of an individual NCE, which is quickly approaching 1 billion dollars, is not the complete source of the large increase in pharmaceutical R&D over the past decade. The development failures of NCEs contribute substantially to this bottom line. One out of every 10 compounds that enter into human testing successfully completes clinical development and is made available to patients. The costs of these failures must be reconciled by the successful marketing of innovative new drugs. The Food and Drug Administration (FDA) recently published a white paper titled “Innovation or Stagnation? - Challenge and Opportunity on the Critical Path to New Medical Products” (FDA, 2004).