Synthesis and biological characterization of L-N(6)-(1-iminoethyl)lysine 5-tetrazole-amide, a prodrug of a selective iNOS inhibitor

The 5-tetrazole amide of L-N(6)-(1-iminoethyl)lysine (L-NIL), L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide (1), has been prepared and evaluated. In contrast to L-NIL, 1 is a stable, nonhygroscopic, crystalline solid. Unlike L-NIL, 1 has minimal inhibitory activity in vitro on human inducible nitric oxide synthase (iNOS). However, it is rapidly converted in vivo to L-NIL and produces dose-dependent inhibition of iNOS in acute and chronic models of inflammation in the rodent with efficacy comparable to L-NIL. In addition, both 1 and L-NIL exhibit significant and comparable in vivo selectivity for the inhibition of iNOS vs endothelial NOS. Doses approximately 80-fold greater than those that inhibited inflammation do not elevate systemic blood pressure. In summary, both the physical properties and the pharmacological profile of 1 make it an ideal molecule for preclinical and clinical studies on the role of selective iNOS inhibitors in mediating inflammatory disease processes.     

L-N6-(1-iminoethyl)-lysine (L-NIL) but not S-methylisothiourea sulphate (SMT) displays selectivity towards NOS-2

Our aim was to verify potency and selectiveness of two most widely used drugs regarded as NOS-2 inhibitors: L-N6-(1-iminoethyl)-lysine (L-NIL) and S-methylisothiourea sulphate (SMT). Thioglycolate-elicited rat peritoneal macrophages and coronary endothelium of isolated guinea pig heart were used as assay systems for NOS-2 and NOS-3, respectively. A non-selective NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) was used as a reference compound. We found that L-NIL but not SMT was a selective NOS-2 inhibitor. Interestingly, L-NAME displayed selectivity towards NOS-3.     

Disposition of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5h)-furanone (mx) in b6c3f1 mice and f344 rats

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a mutagenic by-product of chlorination of drinking water, particularly where the water contains humic matter. MX has been estimated to account for 50% of the mutagenic activity in some drinking water. A bioassay in rats demonstrated an increased tumor incidence, primarily in liver and thyroid glands. This study was designed to provide disposition/metabolism information in mice to evaluate the necessity of a National Toxicology Program chronic bioassay and to provide data for female rats. Radioactivity was rapidly absorbed and excreted near equally in urine (42-54%) and feces (40-51%) 72 h following oral administration of (14)C-labeled MX at single doses from 0.2 to 20 mg/kg to male and female mice and female rats. A larger percentage (71-73%) of MX-derived radioactivity was excreted in urine after an iv dose (0.2 mg/kg) in both female rats and male mice. Most MX-derived radioactivity was excreted within the first 24 h postdosing. MX was transformed to urinary and biliary metabolites. A major extremely polar urinary metabolite was tentatively identified as 1-hydroxy-1,2,2-ethanetricarboxylic acid. This metabolite is likely transformed from the MX degradation product 2-hydroxy-3-formyl-4-oxo-2-butenoic acid. Oral administration produced highest tissue/blood ratios in the following order: forestomach (>100), glandular stomach, intestine, and kidney. Intravenous administration resulted in high, prolonged levels of radioactivity in blood compared to oral dosing. Therefore, MX disposition appears to be dominated by its chemical reactivity with highest concentrations of radioactivity being found at the site of administration.     

Pharmacological characterization of 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), a potent and selective hypoxia-inducible factor prolyl hydroxylase inhibitor

The hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) enzymes represent novel targets for the treatment of anemia, ulcerative colitis, and ischemic and metabolic disease inter alia. We have identified a novel small-molecule inhibitor of PHD, 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), through structure-based drug design methods. The pharmacology of JNJ-42041935 was investigated in enzyme, cellular, and whole-animal systems and was compared with other compounds described in the literature as PHD inhibitors. JNJ-42041935, was a potent (pK(I) = 7.3-7.9), 2-oxoglutarate competitive, reversible, and selective inhibitor of PHD enzymes. In addition, JNJ-42041935 was used to compare the effect of selective inhibition of PHD to intermittent, high doses (50 μg/kg i.p.) of an exogenous erythropoietin receptor agonist in an inflammation-induced anemia model in rats. JNJ-42041935 (100 μmol/kg, once a day for 14 days) was effective in reversing inflammation-induced anemia, whereas erythropoietin had no effect. The results demonstrate that JNJ-42041935 is a new pharmacological tool, which can be used to investigate PHD inhibition and demonstrate that PHD inhibitors offer great promise for the treatment of inflammation-induced anemia.     

Population pharmacokinetics of zileution,a selective 5-lipoxygenase inhibitor,in patients with rheumatoid arthritis

The pharmacokinetics of zileuton, a novel selective 5-lipoxygenase inhibitor, were studied in 37 patients with rheumatoid arthritis after administration of 200 mg, 400 mg, and 600 mg zileuton for 4 weeks. Patients had 6-h pharmacokinetic evaluation of zileuton on day 14. Plasma zileuton concentrations were quantitated using HPLC. Zileuton pharmacokinetic parameters were estimated using standard noncompartmental methods. A population analysis of zileuton pharmacokinetics was also performed with the NONMEM computer program. The pharmacokinetics of zileuton in patients with rheumatoid arthritis were similar to those previously estimated in normal healthy humans. The peak concentrations and the areas under the curves during the dosing interval were dose proportional. The noncompartmental means of the CL/f, terminal-phase half-life, and V/f of zileuton were approximately 545 ml min^–1, 1.4 h, and 64.3 1, respectively. The estimate of population typical values of the CL/f for a 70-kg person (540 ml min^–1) and V/f for a 70-kg person (64.8 1) from the NONMEM analysis were in agreement with the noncompartmental estimates. Differences in body weight, but not age or gender, helped explain some of the variability in the pharmacokinetics of zileuton in patients. Therefore, there is no pharmacokinetic basis for alteration of the zileuton dose size or the dosing schedule in patients with rheumatoid arthritis.     

Nonlinear pharmacokinetics of L-N(G)-methyl-arginine in rats: characterization by an improved HPLC assay

L-N(G)-methyl-arginine (L-NMMA) is an inhibitor of nitric oxide synthase (NOS) enzymes. We have characterized the pharmacokinetics of L-NMMA in rats using HPLC. The HPLC assay requires pre-column derivatization, gradient elution and ultraviolet detection. The limit of sensitivity in plasma was 3.0 microM (0.75 microg mL(-1)). Using this assay, the pharmacokinetics of L-NMMA were characterized following iv bolus doses of 25, 50 and 100 mg kg(-1). Compartmental and noncompartmental data analysis suggest that L-NMMA pharmacokinetics are nonlinear at these doses. From the nonlinear compartmental analysis, we estimated the K(m) and V(max) parameters of L-NMMA elimination to be 70.2 microM and 4.59 microM min(-1), respectively. This estimated K(m) value of L-NMMA elimination is consistent with its nonlinear elimination characteristics in humans and its saturable metabolism by the N(G), N(G)-dimethylarginine dimethylamino-hydrolase enzyme in isolated rat tissue.     

Disposition and effect of the new thromboxane synthetase inhibitor 6-(1-imidazolylmethyl)-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid in man

The disposition of a new thromboxane synthetase inhibitor, 6-(1-imidazolylmethyl)-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid (DP-1904) upon administration of a single 200-mg oral dose to normal Japanese volunteers was studied. DP-1904 proved to be rapidly absorbed from the gastrointestinal tract and converted to its ester glucuronide, which appeared in plasma within 30 min after dosing. The AUCs of DP-1904 and its ester glucuronide were 7.23 +/- 0.54 and 7.93 +/- 0.86 micrograms.h/ml (mean +/- S.E., n = 5), respectively. Both compounds were also eliminated very rapidly from the body (half-lives greater than 60 min). The primary route of elimination was renal, with 52.1 +/- 2.2 and 37.6 +/- 1.6% of the dose being excreted in the urine as the unchanged form and the glucuronide conjugate within 48 h, respectively. The cumulative fecal excretion rates of DP-1904 up to 48 h after dosing were approximately 0.5%. The main metabolite of DP-1904 in humans was DP-1904 glucuronide. Serum thromboxane (TX) B2 levels were reduced more than 98% within 1 h after dosing. There was still more than 75% suppression of serum TXB2 levels at 12 h after dosing. At 72 h TXB2 concentrations returned to control levels. These data indicate that DP-1904 is a potent and long-acting thromboxane synthetase inhibitor.     

Disposition of the cholesterol absorption inhibitor ezetimibe in mdr1a/b (-/-) mice

The lipid lowering agent ezetimibe (EZ) and its intestinally formed glucuronide (GLUC) were shown to be substrates of the efflux transporters P-glycoprotein (P-gp) and the multidrug resistance associated protein 2 (MRP2) which markedly influences the disposition and efficacy of EZ in man. This study aims to elucidate the unique meaning of P-gp in the pharmacokinetics of EZ in mice. In brief, serum concentrations, organ distribution and elimination of EZ were determined in 10 male wild-type and mdr1a/b (-/-) mice after oral treatment with EZ (10 mg/kg, 10 days). EZ and GLUC were quantified in serum, urine, feces and various tissues using a validated LC-MS/MS method. Compared to wild-type mice, mdr1a/b knockout was associated with significantly increased serum concentrations of GLUC (5.58 +/- 2.07 versus 2.09 +/- 0.83 ng/ml, p < 0.001) but not of EZ (0.92 +/- 0.73 versus 0.55 +/- 0.40 ng/ml, n.s.). Consequently, urinary excretion of GLUC was about three-fold increased (9.96 +/- 0.27 versus 3.10 +/- 1.37 microg/day, p = 0.049) whereas renal clearance and the amount excreted via feces remained unchanged. Both EZ and GLUC were not over-proportionally distributed into investigated organs. P-glycoprotein primary influences the oral absorption of ezetimibe in mice. Distribution, renal and fecal excretion of the drug seems not to be markedly affected by P-glycoprotein.     

Disposition of 4,4'-thiobis(6-t-butyl-m-cresol) in rats

The absorption, distribution, metabolism, and excretion of 14C-labeled 4,4'-thiobis(6-t-butyl-m-cresol) (TBBC) was studied in male rats. Oral treatment showed a dose-related decrease in the rate of absorption due to a dose-related increase in retention time in the stomach. TBBC was incompletely absorbed after oral treatment, although the rate of absorption was proportional to the dose once the compound reached the small intestine. TBBC was rapidly distributed throughout the body with the liver being the major tissue depot. Significant amounts of the compound were also present in blood, muscle, skin, and adipose tissue. TBBC was initially rapidly cleared from all tissues except adipose, although a small percentage of the total dose tended to persist in liver and skin. Over half of the compound was excreted the first day, primarily via the bile into the feces. Little TBBC-derived radioactivity appeared in the urine. Metabolites of TBBC were present in the tissues at early times after administration, but were rapidly excreted. The major metabolite(s) appeared to be glucuronide conjugates of the parent compound. Thus, TBBC, an important antioxidant in the rubber and plastic industries, would tend to accumulate in liver and lipid-rich tissues upon chronic exposure, which if by the oral route, could also result in direct damage to the gastrointestinal tract.     

The pharmacokinetics and metabolism of (1R, cis)- and (1R, trans)-tetramethrin in rats

1. The pharmacokinetics and metabolism of (1R, cis)- and (1R, trans)-isomers of tetramethrin (i.v. 0.25 mg/kg) were studied in rats. 2. The experimental data for the time course of the concentration of tetramethrin isomers in plasma fit a pharmacokinetic two-compartmental open model. Plasma levels of both isomers were similar. The terminal half-life of the trans-isomer in plasma was greater (125 min) than the cis-isomer (72 min). 3. The concentrations of the two metabolites, 3,4,5,6-tetrahydrophthalimide (TPI) and N-(hydroxymethyl)-3,4,5,6-tetrahydrophthalimide (MTI), were consistently higher in the plasma of rats treated with the trans-isomer than in those treated with the cis-isomer. 4. In rats treated with the trans-isomer, the majority of radioactivity excreted after 96 h was found in urine. The faeces was the major excretory route for rats treated with the cis-isomer (26% urine, 69% faeces with cis-isomer; 64% urine, 29% faeces with trans-isomer). 5. Metabolism of each isomer was rapid and complete. Parent chemical was not detected in urine and only small quantities of the intact cis-isomer were found in the faeces. MTI, TPI, and cyclohexane-1,2-dicarboximide (HPI) were detected in both urine and faeces. 6. The amount of radioactivity excreted into the bile was similar for both isomers. However, levels of the intact parent compound and TPI were higher in the bile isolated from rats treated with the trans-isomer. The trans-isomer was found to undergo enterohepatic circulation.     

Excretion, metabolism, and pharmacokinetics of 1-(8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl)-4-(ethylamino)piperidine-4-carboxamide, a selective cannabinoid receptor antagonist, in healthy male volunteers

The disposition of 1-(8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H- purin-6-yl)-4-(ethylamino)-piperidine-4-carboxamide (CP-945,598), an orally active antagonist of the cannabinoid CB1 receptor, was studied after a single 25-mg oral dose of [(14)C]CP-945,598 to healthy human subjects. Serial blood samples and complete urine and feces were collected up to 672 h after dose. The mean total recovery of radioactivity was 60.1 ± 12.8 from the urine and feces, with the majority of the dose excreted in the feces. The absorption of CP-945,598 in humans was slow with T(max) at 6 h. Less than 2% of the dose was recovered as unchanged drug in the combined excreta, suggesting that CP-945,598 is extensively metabolized. The primary metabolic pathway of CP-945,598 involved N-de-ethylation to form an N-desethyl metabolite (M1), which was then subsequently metabolized by amide hydrolysis (M2), N-hydroxylation (M3), piperidine ring hydroxylation (M6), and ribose conjugation (M9). M3 was further metabolized to oxime (M4) and keto (M5) metabolites. M1, M4, and M5 were the major circulating metabolites, with AUC((0-48)) values 4.7-, 1.5-, and 1.1-fold greater than that of CP-945,598. M1, M2, and M9 accounted for 5.6, 33.6, and 6.30% of the dose, respectively, in excreta. The results from in vitro experiments with recombinant isoforms suggested that the oxidative metabolism of CP-945,598 to M1 is catalyzed primarily by CYP3A4/3A5. The molecular docking study showed that the N-ethyl moiety of CP-945,598 can access to the heme iron-oxo of CYP3A4 in an energetically favored orientation. Together, these data suggest that CP-945,598 is well absorbed and eliminated largely by CYP3A4/3A5-catalyzed metabolism.     

Cardiocirculatory effects of Ro 11-2464, a selective 5-HT (serotonin) uptake inhibitor,in healthy volunteers

Summary The imipramine derivative Ro 11-2465, a potent, selective 5-HT (serotonin) uptake inhibitor, is being developed as an antidepressant agent. The effects of Ro 11-2465 on heart rate, blood pressure, electrocardiogram and systolic time intervals were assessed in nine normotensive volunteers. Ro 11-2465 1 and 2 mg and a placebo were given in a single blind, cross over design study. The placebo did not induce any significant changes. With Ro 11-2465, the ECG-intervals (P,PQ,QRS,QTc) did not change, the blood pressure increased 3–6 h after administration of either dose, and the heart rate was increased 4–6 h after the 2 mg dose. There was also evidence of increased ventricular automaticity in one subject. The total electromechanical systole (QS₂-index) was significantly shortened 4–8 h after administration of 2 mg, whereas neither 1 mg the dose nor the placebo had any such effect. This finding suggests the presence of a positive inotropic effect, which is probably due to a stimulatory effect of serotonin, and is not mediated by an adrenergic mechanism. The findings suggest that Ro 11-2465, as a potential new tricyclic antidepressant, might have favourable cardiocirculatory side effects, particularly in patients with pre-existing heart disease.     

Discovery of ((S)-5-(methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone as a potent and selective I(Kur) inhibitor

Previously disclosed dihydropyrazolopyrimidines are potent and selective blockers of I(Kur) current. A potential liability with this chemotype is the formation of a reactive metabolite which demonstrated covalent binding to protein in vitro. When substituted at the 2 or 3 position, this template yielded potent I(Kur) inhibitors, with selectivity over hERG which did not form reactive metabolites. Subsequent optimization for potency and PK properties lead to the discovery of ((S)-5-(methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone (13j), with an acceptable PK profile in preclinical species and potent efficacy in the preclinical rabbit atrial effective refractory period (AERP) model.     

Disposition and metabolism of 2-(2'(1',3'-dioxolan-2-yl)-2- methyl-4-(2'-oxopyrrolidin-1-Yl)-6-nitro-2h-1-benzopyran (SKP-450) in rats.

The disposition and metabolism of the new antihypertensive agent 2-(2"(1", 3"-dioxolan-2-yl)-2-methyl-4-(2'-oxopyrrolidin-1-yl)-6-nitro -2H-1-benzopyran (SKP-450) were investigated in male rats after single oral and i.v. doses of 14C-labeled compound. After an oral 2.0 mg/kg dose, mean radiocarbon recovery was 98.2 +/- 2.3% with 31.1 +/- 7.3% in the feces and 67.1 +/- 14.3% in the urine. Biliary excretion of radioactivity for the first 24-h period was approximately 40%, suggesting that SKP-450 is cleared either by hepatobiliary excretion or by renal excretion. SKP-450 was well absorbed; bioavailability calculated on the basis of radioactivity was 68 to 97%. Tissue distribution of the radioactivity was widespread with high concentrations in the liver and kidney but low central nervous system penetration. Radio-HPLC analysis of bile and urine from rats indicated the extensive metabolism of SKP-450 into oxidative metabolites. Oxidative metabolism of the dioxolanyl ring resulted in an aldehyde intermediate, subsequently confirmed in vitro, which was further oxidized to the corresponding carboxylic acid (M1) or reduced to the corresponding alcohol (M3). No parent drug was detected in the urine or bile. Glucuronide conjugate of M3 was also detected in urine and bile, accounting for 5.8 +/- 2.1 and 8.9 +/- 3. 7% of the excreted radioactivity, respectively. Quantitative data obtained from plasma samples suggest that the majority of circulating radioactivity was associated with metabolites. Our results suggest that the long duration of pharmacological activity of SKP-450 (>10 h) is largely attributable to its metabolites.     

The pharmacokinetics and pharmacodynamics of a new thromboxane synthetase inhibitor, 6-(1-imidazolylmethyl)-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid (DP-1904) in man after repeated oral doses

The pharmacokinetics of DP-1904, a new potent and selective thromboxane synthetase inhibitor, and its effects on ex-vivo prostanoid formation have been studied in groups of Japanese normal male volunteers, who received repeated oral doses of 200 mg every 12 h for 4 doses, or 400 mg every 24 h for 2 doses, or 200 mg every 12 h for 14 doses. The drug was well tolerated by all subjects without evidence of adverse reactions. Repeated administration showed no significant changes in half-lives, tmax values, cmax values and AUC values. DP-1904 did not exhibit time-dependent kinetics. Its plasma levels were lower than the quantifiable level (50 ng mL-1) at 12 h after each dose. These data suggest no significant accumulation of DP-1904 in normal volunteers. DP-1904 reduced the serum thromboxane B2 by about 80% during the medication, the serum concentrations returning to about 44, 75 and 20% of the predrug control values at 36 h after the last 200 mg doses and 48 h after the last 400 mg dose.     

Mechanism of action of 5-(2-chloroethyl)-2'-deoxyuridine, a selective inhibitor of herpes simplex virus replication

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1). CEDU is preferentially phosphorylated by HSV-infected (Vero) cells, as compared with mock-infected cells or cells infected with a thymidine kinase-deficient strain of HSV-1. The end product of this phosphorylation process, CEDU 5'-triphosphate, is a competitive inhibitor of HSV-1 DNA polymerase activity and, to a lesser extent, of cellular DNA polymerase alpha activity. However, in the absence of the natural substrate dTTP, CEDU 5'-triphosphate also serves as an alternative substrate for viral and cellular DNA polymerase. When exposed to HSV-1-infected cells, [2-14C]CEDU was incorporated into both viral and cellular DNA. The extent to which [2-14C]CEDU was incorporated remained approximately constant over a concentration range of 0.5 to 50 microM. Within this concentration range, CEDU effected a concentration-dependent inhibition of viral DNA synthesis that closely paralleled the inhibition of viral progeny formation. It is postulated that CEDU owes (i) its selectivity as an antiviral agent to its preferential phosphorylation by the virus-infected cell and (ii) its antiviral potency to an inhibition of viral DNA synthesis at the level of the viral DNA polymerization reaction.