Discovery of potent selective bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model. Part II: optimization studies and demonstration of in vivo efficacy.

Selective phosphodiesterase 2 (PDE2) inhibitors are shown to have efficacy in a rat model of osteoarthritis (OA) pain. We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of phosphodiesterase 4 (PDE4) inhibitors, while minimizing PDE4 inhibitory activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like binding mode orthogonal to the cAMP-like binding mode found in PDE4. Extensive structure activity relationship studies ultimately led to identification of pyrazolodiazepinone, 22, which was >1000-fold selective for PDE2 over recombinant, full length PDEs 1B, 3A, 3B, 4A, 4B, 4C, 7A, 7B, 8A, 8B, 9, 10 and 11. Compound 22 also retained excellent PDE2 selectivity (241-fold to 419-fold) over the remaining recombinant, full length PDEs, 1A, 4D, 5, and 6. Compound 22 exhibited good pharmacokinetic properties and excellent oral bioavailability (F=78%, rat). In an in vivo rat model of OA pain, compound 22 had significant analgesic activity 1 and 3h after a single, 10 mg/kg, subcutaneous dose.

Desferrithiocin analogue iron chelators: iron clearing efficiency, tissue distribution, and renal toxicity.

The current solution to iron-mediated damage in transfusional iron overload disorders is decorporation of excess unmanaged metal, chelation therapy. The clinical development of the tridentate chelator deferitrin (1, Table 1) was halted due to nephrotoxicity. It was then shown by replacing the 4'-(HO) of 1 with a 3,6,9-trioxadecyloxy group, the nephrotoxicity could be ameliorated. Further structure-activity relationship studies have established that the length and the position of the polyether backbone controlled: (1) the ligand's iron clearing efficiency (ICE), (2) chelator tissue distribution, (3) biliary ferrokinetics, and (4) tissue iron reduction. The current investigation compares the ICE and tissue distribution of a series of (S)-4,5-dihydro-2-[2-hydroxy-4-(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids (Table 1, 3-5) and the (S)-4,5-dihydro-2-[2-hydroxy-3-(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids (Table 1, 8-10). The three most effective polyether analogues, in terms of performance ratio (PR), defined as mean ICE(primate)/ICE(rodent), are 3 (PR 1.1), 8, (PR 1.5), and 9, now in human trials, (PR 2.2). At the onset of the clinical trial on 9, no data were available for ligand 3 or 8. This is unfortunate, as 3 has many advantages over 9, e.g., the ICE of 3 in rats is 2.5-fold greater than that of 9 and analogue 3 achieves very high levels in the liver, pancreas, and heart, the organs most affected by iron overload. Finally, the impact of 3 on the urinary excretion of kidney injury molecule-1 (Kim-1), an early diagnostic biomarker for monitoring acute kidney toxicity, has been carried out in rats; no evidence of nephrotoxicity was found. Overall, the results suggest that 3 would be a far superior clinical candidate to 9.

Characterization of benidipine and its enantiomers' metabolism by human liver cytochrome P450 enzymes.

Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)-alpha and (+)-alpha isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CL(int)) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-alpha isomer was similar to those from the (+)-alpha isomer (1.9 +/- 0.1 versus 2.3 +/- 2.3 microl/min/pmol P450 and 0.5 +/- 0.2 versus 0.6 +/- 0.6 microl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CL(int) values of CYP3A4-mediated metabolite formation from (-)-alpha isomer were similar to those from (+)-alpha isomer (17.7 versus 14.4 microl/min/pmol P450, respectively). The total CL(int) values of CYP3A5-mediated metabolite formation from (-)-alpha isomer were also similar to those from (+)-alpha isomer (8.3 versus 11.0 microl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors.

Pharmacological, pharmacokinetic, and clinical properties of benidipine hydrochloride, a novel, long-acting calcium channel blocker.

Benidipine is a dihydropyridine-derived calcium channel blocker developed in Japan, with several unique mechanisms of action, that is, triple calcium channels (L, N, and T) blocking action with a membrane approach. Benidipine has relatively high vascular selectivity and is expected to show protective effects on vascular endothelial cells. Renal protective effects of benidipine also have been shown in several basic and clinical studies. Moreover, anti-oxidative action and enhancing nitric oxide production have been noted with this drug, following its cardio-protective effects in patients with ischemic heart diseases. In fact, benidipine exerted a better prognostic effect than other calcium channel blockers in the therapy for patients with vasospastic angina. In addition, benidipine showed reliable antihypertensive, renoprotective effects if used in combination with angiotensin II type 1 receptor blockers (ARBs) when adequate anti-hypertensive effects are not achieved by ARBs alone, indicating that benidipine is an useful calcium channel blocker in combination therapy for hypertension. Benidipine was launched on the Japanese market 14 years ago, but few severe side effects have been reported, suggesting that this is a drug with established safety and long-acting pharmacological effects.

Iron chelation promoted by desazadesferrithiocin analogs: An enantioselective barrier.

For patients who require lifelong blood transfusions, there is no efficient means, unless chelation therapy is employed, for elimination of excess iron. Alternatives to desferrioxamine, the currently accepted treatment for transfusional iron overload, are being investigated. The current article focuses on an enantiomeric pair of analogs of desferrithiocin, (+)-(S)- and (-)-(R)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (4'-hydroxydesazadesferrithiocin). The crystal structure corroborated the absolute configuration of the two compounds, (+) and (-) for the (S)- and (R)-enantiomers, respectively. Job's plots established the tridentate nature of both analogs and circular dichroism spectra confirmed the ligands' antipodal relationship. (+)-(S)-4'-Hydroxydesazadesferrithiocin is a more efficient deferration agent than is the (-)-(R)-enantiomer in a Cebus apella model of iron overload. Pharmacokinetic analyses and IC(50) measurements in L1210 murine leukemia cells were undertaken in an effort to account for the contrast in efficacy between the two enantiomers. Some differences exist in the plasma pharmacokinetic parameters between the two analogs. However, a more plausible explanation may be the apparent differences in transport across the cell membrane; the IC(50) value in L1210 cells of the (+)-(S)-enantiomer was at least 5-fold lower than that of the (-)-(R)-compound.

Design and synthesis of orally bioavailable inhibitors of inducible nitric oxide synthase. Part 1: synthesis and biological evaluation of dihydropyridin-2-imines.

Dihydropyridin-2-imines were synthesized and biologically evaluated both in vitro and in vivo using a nitric oxide inhibition assay. Compounds 1, 4, 5 and 7-11 exhibited potent activity in the inducible nitric oxide (iNOS) inhibition assay. Of these 5, 6, 9 and 10 showed 5- to 11-fold increases in isoform selectivity. Compounds 1, 5, 9 and 10 showed potent inhibitory activity in the NOx accumulation assay in mice. Compounds 1 and 5 also showed good bioavailability (BA) when given orally.

Vatanidipine hydrochloride: a new long-lasting antihypertensive agent.

Vatanidipine is a novel dihydropyridine (DHP)-type calcium channel blocker with slow-onset pharmacological actions, which are probably due to both its slow uptake into vascular tissues and resistance in its approach to the calcium channel binding site. Vatanidipine once incorporated into vascular tissues is not easily released, even by repeated washing, thus resulting in a long-lasting action of the agent. A slow-onset and long-lasting hypotensive action was observed in various experimental hypertensive models. Clinical trials using human subjects with essential hypertension indicated that vatanidipine exerts an antihypertensive effect with a slow onset and long duration. In spite of its potent hypotensive effect, the incidence of adverse effects by vatanidipine administration has been reported to be lower than that in cases of nitrendipine. In addition to its vasodilatory effects, vatanidipine efficiently suppressed noradrenaline release from sympathetic nerve endings, thus suggesting this agent exhibits a beneficial effect in the treatment of hypertensive patients, in which the reflex activation of peripheral sympathetic nerves is unfavourable to antihypertensive therapy. In a double-blind study, vatanidipine did not show reflex tachycardia, despite producing a potent and long-lasting hypotensive effect, in contrast to the administration of nitrendipine. In an animal study, vatanidipine exhibited a protective effect against cerebrovascular lesions, through a mechanism independent of its hypotensive effect. In addition, a renoprotective effect was also observed in experimental hypertensive models. In cholesterol-fed rabbits, vatanidipine exerted an anti-atherosclerotic action, which is probably attributable to the inhibitory action of the agent on low-density lipoprotein oxidation. In essential hypertensive patients, the plasma levels of cholesterol and triglyceride decreased after vatanidipine treatment, thus suggesting that this agent may have a therapeutic potential in preventing such vascular diseases as atherosclerosis. Taken together, vatanidipine appears to be a novel and useful antihypertensive agent, which can both prevent target-organ damage and reduce cardiovascular morbidity and mortality.

Cytochrome P-450 isoforms involved in carboxylic acid ester cleavage of Hantzsch pyridine ester of pranidipine.

Cytochrome P-450 (CYP) isoforms responsible for the cleavage of Hantzsch pyridine ester at the 3-position of pranidipine were studied in vitro using cDNA-expressed human CYP enzymes. CYP1A1, 1A2, 2D6, and 3A4 cleaved the ester with a catalytic activity of 5.5, 0. 93, 13.1, and 22.4 nmol/30 min/nmol P-450, respectively. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2E1 were not involved in the de-esterification. The Km and Vmax values for the de-esterification were 11.8 microM and 0.47 nmol/min/nmol P-450 in the CYP2D6-catalyzed reaction and 8. 7 microM and 0.84 nmol/min/nmol P-450 in the CYP3A4-catalyzed reaction. The intrinsic clearance (Vmax/Km) of the de-esterification by CYP3A4 was 2-fold greater than that by CYP2D6. Quinidine almost completely inhibited the CYP2D6-mediated de-esterification at the concentration of 1 x 10(-6) M. Ketoconazole and troleandomycin inhibited the CYP3A4-mediated reaction in a dose-related manner. The results indicate that although the multiple CYP isoforms can catalyze the de-esterification, CYP3A4 and 2D6 are the major isoforms.

Preclinical and phase 1 clinical characterization of CI-979/RU35926, a novel muscarinic agonist for the treatment of Alzheimer's disease.

In vitro and in vivo characterization in rodents and monkeys shows that CI-979/RU35926 is a partial muscarinic agonist with equal affinity for the five subtypes of muscarinic receptors. It activates central cholinergic receptors as shown by its ability to decrease body temperature, enhance local cortical blood flow and increase cortical arousal measured by QEEG. Further, it reverses spatial memory deficits in rats with ibotenic acid-induced lesions of forebrain cholinergic neurons. Signs of peripheral cholinergic stimulation appear at doses higher or equal to those necessary to produce central activity. In a single-dose tolerance study in young, healthy human volunteers, CI-979/RU35926 was well tolerated at doses of 0.002-1.0 mg with cholinergic symptoms such as hypersalivation and sweating, observed at 2-4 mg. It demonstrated linear pharmacokinetic behavior over a dose range of 0.1 to 4 mg and elimination half-life varied from 2-5 hours. Measurement of unchanged drug in urine suggests that the drug was extensively metabolized. Thus, the safety profile supported further clinical evaluation and CI-979/RU35926 is currently in Phase II clinical trials.

Isradipine dynamics and pharmacokinetics in the isolated rabbit heart.

The cardiac effects of increasing concentrations of isradipine (racemic) from 1.64 pM to 232 nM were studied in isolated spontaneously beating rabbit hearts. Inhibitory responses with regard to contraction amplitude, contraction velocity and oxygen consumption exhibited a biphasic progressive course at increasing drug exposure. Computer derived inhibitory Emax-values of the second phase were 104, 103 and 87% (IC50: 7.1, 6.3 and 28.7 nM), respectively, whereas those of the initial phase were 24.7, 25.9 and 19.5% (IC50: 0.012, 0.038 and 0.026 nM). A progressive inhibition of frequency reached a maximum of only 21%. The ECG-derived PQ-interval showed a rapid increase (maximum 46%) at drug concentrations above 1 nM. Complete AV-block and ventricular asystolia occurred in half of the hearts at the second highest (99 nM) and in all except one at the highest concentration. SA-node activity was retained in 9 of 10 hearts at the second highest and in 3 at the highest drug exposure. The QRS-and the frequency-corrected QT-interval did not increase significantly. Coronary flow-rate showed no initial increase, but a decrease to 70% of control at the highest concentration. Supplementary in vitro studies on rabbit coronary artery ring-preparations contracted with 124 mM K+ showed, however, an relaxant Emax-value for isradipine of about 100% and an inhibitory EC50-value of 0.63 nM with a 'Hill' coefficient of 1.1. At toxic concentrations isradipine showed a kinetic monophasic accumulation in the rabbit heart of about 44-fold with a half-time of 10.6 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and dialysability of isradipine in chronic haemodialysis patients.

The pharmacokinetics of isradipine, a calcium-channel blocker, have been studied in eight patients on chronic haemodialysis. A single oral dose of 5 mg was administered on both a non-haemodialysis and a haemodialysis day and the plasma concentrations of isradipine were analyzed. The mean cmax, tmax, AUC, and t1/2 in plasma on the non-haemodialysis day were 5.2 ng.ml-1, 1.4 h, 23.8 ng.h.ml-1, and 3.1 h, respectively. The dialysis clearance of isradipine was negligible (5.0 ml.min-1). The t1/2 values during haemodialysis were not significantly different from those observed during the same period post dose on the non-haemodialysis day. The study demonstrates that supplemental doses of isradipine are not necessary in these patients since isradipine is not significantly removed by haemodialysis.

Different inhibition and induction profiles of hepatic drug metabolism in rats and dogs by two structurally related pyridyl diazinone cardiotonic agents.

ICI 153,110 and ICI 170,777, two pyridyl diazinone cardiotonic agents, produced a different profile of effects on hepatic microsomal mixed function oxidase enzymes following multiple oral dosing to rats and dogs; these differences may be related to the molecular dimensions of the two molecules. ICI 153,110 significantly increased levels of total P450, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase in rat microsomes, indicating an induction profile (P448) similar to that of beta-naphthoflavone. This was supported by gel electrophoresis (SDS-PAGE) of microsomal proteins; a similar type of induction was observed in dog microsomes. In contrast, ICI 170,777 produced no changes indicating enzyme induction in either rat or dog. Instead, ICI 170,777 appeared to inhibit specifically the activity of aldrin epoxidase in the rat. Inhibitory activity was also indicated in the rat by prolongation of pentobarbitone sleeping time following single oral doses of either ICI 153,110 or ICI 170,777. The time-course of this effect appeared to correlate more closely with the profile of circulating metabolites, although both parent compounds were found to produce type II spectral changes on interaction with control rat microsomes. The molecular dimensions (area/depth2) of the compounds supported the finding that only ICI 153,110 should interact with or induce P448 isozymes.