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1. We studied the functional interaction between transport and metabolism by comparing the transport of losartan and its active metabolite EXP 3174 (EXP) across cell monolayers. 2. Epithelial layers of Caco-2 cells as well as MDR1, MRP-1 and MRP-2 overexpressing cells, in comparison to the respective wildtypes, were used to characterize the transcellular transport of losartan and EXP. 3. Losartan transport in MDCK-MDR1 and Caco-2 cells was saturable and energy-dependent with a significantly greater basolateral-to-apical (B/A) than apical-to-basolateral (A/B) flux (ratio=31+/-1 in MDCK-MDR1 and ratio 4+/-1 in Caco-2 cells). The B/A flux of losartan was inhibited by cyclosporine and vinblastine, inhibitors of P-glycoprotein and MRP. In contrast, no active losartan transport was observed in MRP-1 or MRP-2 overexpressing cells. 4. The metabolite was only transported in Caco-2 cells with a B/A-to-A/B ratio of 5+/-1, while lacking active transport in the MDR1, MRP-1 or MRP-2 overexpressing cells. The B/A flux of EXP was significantly inhibited by cyclosporine and vinblastine. 5. In conclusion, losartan is transported by P-glycoprotein and other intestinal transporters, that do not include MRP-1 and MRP-2. In contrast, the carboxylic acid metabolite is not a P-glycoprotein substrate, but displays considerably higher affinity for other transporters than losartan, that again most probably do not include MRP-1 and MRP-2.

1. Cilostazol (OPC-13013) undergoes extensive hepatic metabolism. The hydroxylation of the quinone moiety of cilostazol to OPC-13326 was the predominant route in all the liver preparations studies. The hydroxylation of the hexane moiety to OPC-13217 was the second most predominant route in vitro. 2. Ketoconazole (1 microM) was the most potent inhibitor of both quinone and hexane hydroxylation. Both the CYP2D6 inhibitor quinidine (0.1 microM) and the CYP2C19 inhibitor omeprazole (10 microM) failed to consistently inhibit metabolism of cilostazol via either of these two predominant routes. 3. Data obtained from a bank of pre-characterized human liver microsomes demonstrated a stronger correlation (r2=0.68, P < 0.01) between metabolism of cilostazol to OPC-13326 and metabolism of felodipine, a CYP3A probe, that with probes for any other isoform. Cimetidine demonstrated concentration-dependent competitive inhibition of the metabolism of cilostazol by both routes. 4. Kinetic data demonstrated a Km value of 101 microM for cilostazol, suggesting a relatively low affinity of cilostazol for CYP3A. While recombinant CYP1A2, CYP2D6 and CYP2C19 were also able to catalyze formation of specific cilostazol metabolites, they did not appear to contribute significantly to cilostazol metabolism in whole human liver microsomes.

Two high affinity and selective NK1-receptor antagonists, GR203040 and GR205171, were labelled with 11C and used in a series of experiments in rhesus monkeys. The purpose of these studies was to evaluate the brain uptake pattern and to explore the potential use of these compounds as PET ligands to characterise NK1-receptor binding. Seventeen studies were carried out with [11C]GR205171 and five experiments with [11C]GR203040, including baseline studies and studies performed after a 5 min infusion of cold compound at doses between 0.05 and 1 mg/kg. Both compounds demonstrated a significant and rapid uptake in the brain, but the uptake of [11C]GR205171 was more than double the uptake of [11C]GR203040. At tracer doses of [11C]GR205171 and all doses of [11C]GR203040 the uptake reached a plateau with no washout during the examination time, whereas [11C]GR205171 after pre-treatment with cold GR205171 showed a significant washout. Using a model with the cerebellum as reference, a method for quantitation was applied to the studies with [11C]GR205171 and the results indicated that the highest specific binding was in the striatum. The pre-treatment dose of cold GR205171 needed for 50% inhibition of binding was less than 0.04 mg/kg. The studies indicated that [11C]GR205171 could be used for the in vivo characterisation of NK1-receptor binding.

The angiotensin II type 1 (AT(1)) receptor plays a pivotal role in the regulation of blood pressure and electrolyte balance, and is involved in the control of specific ingestive behaviours. Irbesartan (SR 47436/BMS 186295) is a recently developed angiotensin AT(1) receptor antagonist, chemically described as 2-butyl-3-([2'-¿1H-tetrazol-5-yl¿biphenyl-4-yl]methyl)-1, 3-diazaspiro (4,4)non-1-en-4-one. Irbesartan displays higher affinity for its target receptor than other similar antagonists. In radioligand binding assays performed on membranes from WB-Fischer 344 (WB) rat liver epithelial cells, irbesartan was able to displace [125I]angiotensin II with a K(i) of 4.05 nM as compared to losartan (DuP 753) and tasosartan (WAY 126756), which had K(i) values of 25.2 nM and 46.6 nM, respectively. Similarly, in functional assays, irbesartan exhibited the highest functional potency to block angiotensin II-induced inositol trisphosphate (IP(3)) turnover. The improved affinity of irbesartan for the angiotensin AT(1) receptor does not coincide with a concomitant increase in affinity for the angiotensin AT(2) receptor, as irbesartan and losartan exhibited the same low potency to displace [125I]angiotensin II in radioligand binding assays performed on membranes from PC-12w cells. In binding assays performed on peripheral tissues in rat, irbesartan bound to the angiotensin AT(1) receptor expressed in liver, adrenal, kidney and pituitary with an overall affinity closely approaching that of the high affinity peptidic antagonist [Sar(1), Ile(8)]angiotensin II. Due to the higher affinity of irbesartan over other similar antagonists for the angiotensin AT(1) receptor in many tissues and its greater potency to block receptor activation, irbesartan may be quite useful in the study of the angiotensin AT(1) receptor and its role in controlling ingestive behaviours and, furthermore, shows great potential to improve the treatment of hypertension and other cardiovascular disease states.

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-pro penone, to 2.1-A resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

Cilostazol is a potent cyclic nucleotide phosphodiesterase (PDE) type 3 (PDE3) inhibitor that was recently approved by the Food and Drug Administration (FDA) for the treatment of intermittent claudication. Its efficacy is presumed to be due to its vasodilatory and platelet activation inhibitory activities. Compared with those treated with placebo, patients treated with cilostazol showed a minimal increase in cardiac adverse events. Because of its PDE3 inhibitory activity, however, the possibility that cilostazol exerts positive cardiac inotropic effects is a safety concern. Therefore we compared the effects of cilostazol with those of milrinone, a selective PDE3 inhibitor, on intracellular cyclic adenosine monophosphate (cAMP) levels in platelets, cardiac ventricular myocytes, and coronary smooth muscle cells. We also compared the corresponding functional changes in these cells. Cilostazol and milrinone both caused a concentration-dependent increase in the cAMP level in rabbit and human platelets with similar potency. Furthermore, cilostazol and milrinone were equally effective in inhibiting human platelet aggregation with a median inhibitory concentration (IC50) of 0.9 and 2 microM, respectively. In rabbit ventricular myocytes, however, cilostazol elevated cAMP levels to a significantly lesser extent (p < 0.05 vs. milrinone). By using isolated rabbit hearts with a Langendorff preparation, we showed that milrinone is a very potent cardiotonic agent; it concentration-dependently increased left ventricular developed pressure (LVDP) and contractility. Cilostazol was less effective in increasing LVDP and contractility (p < 0.05 vs. milrinone), which is consistent with the cardiac cAMP levels. The cardiac effect of OPC-13015, a metabolite of cilostazol with about sevenfold higher PDE3 inhibition, was similar to cilostazol. Whereas milrinone concentration-dependently increased cAMP in rabbit coronary smooth muscle cells, cilostazol did not have such an effect. However, both compounds increased coronary flow equally in rabbit hearts. Our results show that although cilostazol and milrinone both inhibit PDE3, cilostazol preferentially acts on vascular elements (platelets and flow). This unique profile of cilostazol is consistent with its beneficial and safe clinical outcomes in patients with intermittent claudication.

The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) for evaluating the metabolic activity of aerobic bacteria has gained wide application in recent years. In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria. In addition, the factors contributing to abiotic reduction of CTC were also examined. CTC was used in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), that targets bacterial cell wall proteins, to quantitate the active fraction of total bacterial numbers. Facultative anaerobic bacteria, including Escherichia coli grown fermentatively, and Pseudomonas chlorophis, P. fluorescens, P. stutzeri, and P. pseudoalcalegenes subsp. pseudoalcalegenes grown under nitrate-reducing conditions, actively reduced CTC during all phases of growth. Greater than 95% of these cells accumulated intracellular CTC-formazan crystals during the exponential phase. Obligate anaerobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desulfovibrio desulfuricans subsp. desulfuricans and D. halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H2 and CO2 all reduced CTC to intracellular CTC-formazan crystals. The optimal CTC concentration for all organisms examined was 5 mM. Anaerobic CTC incubations were not required for quantification of anaerobically grown cells. CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients. CTC was reduced by culture fluids containing ferric citrate as electron acceptor following growth of either G. metallireducens or G. sulfurreducens. Abiotic reduction of CTC was observed in the presence of ascorbic acid, cysteine hydrochloride, dithiothreitol, NADH, NADPH, Fe(II)Cl2, sodium thioglycolic acid and sodium sulfide. These results suggest that while CTC can be used to capture the metabolic activity of anaerobic bacteria, care must be taken to avoid abiotic reduction of CTC.

We have examined the pharmacology of kainate receptors in cultured hippocampal neurons (6-8 days in vitro (DIV)) from embryonic rats (E17). Cultured neurons were pre-treated with concanavalin A to remove kainate receptor desensitization and whole-cell voltage clamp electrophysiology employed to record inward currents in response to glutamatergic agonists and antagonists. N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor responses were blocked using MK801 (3 microM) and the 2,3-benzodiazepine, LY300168 (GYKI53655, 50 microM), respectively. Inward currents were recorded in hippocampal neurons upon application of kainate and the 2S,4R isomer of 4-methyl glutamic acid (SYM2081) with EC50 values of 3.4 +/- 0.4 microM and 1.6 +/- 0.5 microM, respectively (n = 6 cells). The GluR5 selective agonists, LY339434 (100 microM) and (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl) propionic acid (ATPA) (100 microM), did not evoke detectable inward currents in any cell responding to kainate. LY293558 and the selective GluR5 antagonist, LY382884, had weak antagonist effects on responses evoked by either kainate or (2S,4R)-4-methyl glutamate (IC50 > 300 microM). The quinoxalinedione, 2,3-dihyro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), blocked both kainate and (2S,4R)-4-methyl glutamate-activated currents at much lower concentrations (IC50 approximately 10 microM). These results provide pharmacological evidence that ion channels comprised of GluR6 kainate receptor subunits mediate kainate receptor responses in hippocampal neurons cultured 6-8 DIV.

A new epifluorescent microscopy based method using 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) and 5-(4,6-dichlorotriazinyl) aminofluoroscein (DTAF) was developed for quantifying total microbial biomass and evaluating levels of microbial activity. CTC is a tetrazolium dye that forms fluorescent intracellular formazan when biologically reduced by components of the electron transport system and/or dehydrogenases of metabolically active bacteria. DTAF is a fluorescein-based fluorochrome that selectively stains bacterial cell walls thereby enabling quantification of total bacterial biomass. CTC can be used in conjunction with DTAF to provide the optical resolution necessary to differentiate metabolically active cells from inactive cells in microbial populations associated with subsurface soils. The CTC/DTAF staining method has been shown to be effective for quantifying the metabolic activity of not only aerobic bacteria, but also diverse groups of anaerobic bacteria. This method allows for the rapid quantification of total and active bacterial numbers in complex soil samples without enrichment or cell elution. In this study, CTC/DTAF staining was applied to evaluate in-situ microbial activity in petroleum hydrocarbon contaminated subsurface soils from Sites 3 and 13 at Alameda Point, CA. At each site, subsurface microbial activity at two locations within contaminated plumes were examined and compared to activity at two geologically similar but uncontaminated background locations. Significant bacterial populations were detected in all soils examined, and the biomass estimates were several orders of magnitude higher than those obtained by conventional culture-based techniques. Both the total bacterial concentrations and the numbers of active bacteria in soils from contaminated areas were substantially higher than those observed in soils from background locations. Additionally, the percentages of metabolically active bacteria in the contaminated areas were consistently higher than those detected in background areas, suggesting that the enhanced microbial activity was due to microbial contaminant degradation. Although conventional heterotrophic plate counts failed to show significant microbial activity at either of the sites, soil gas carbon dioxide and methane measurements confirmed that hydrocarbon contaminant degradation was occurring in both areas. The CTC/DTAF staining protocol proved to be a rapid, reliable, and inexpensive method to evaluate the progress of in-situ bioremediation., (Copyright 1999 Elsevier Science B.V.)

Candesartan (Atacand) is a novel antagonist of angiotensin II AT1 receptors. It is a specific "insurmountable" antagonist which is characterized by its extremely high specificity and its very slow dissociation from the receptor. In the treatment of essential arterial hypertension it appears very efficacious and shows a dose dependent activity within the dose-range of 4 mg to 32 mg/day. The efficacy is maintained up to 36 hours after drug intake. Candesartan is excreted, unchanged, both in the feces (2/3) and urine (1/3). Side-effects are comparable to placebo.

Binding of the non-peptide angiotensin II AT1 antagonist [3H](2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]- H-benzimidazoline-7-carboxylic acid ([3H]candesartan) to human angiotensin II AT1 receptor-transfected Chinese hamster ovary (CHO-AT1) cells was inhibited to the same extent by angiotensin II and non-peptide angiotensin II AT1 antagonists. No binding was observed in control CHO-K1 cells. Dissociation was slow (k(-1) = 0.0010+/-0.0001 min(-1)) after removal of the free [3H]candesartan but increased 5-fold upon addition of supramaximal concentrations of angiotensin II AT1 antagonists. Angiotensin II responses recovered equally slow from candesartan-pretreatment. When washed and further incubated, these angiotensin II responses also recovered more rapidly in the presence of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphen yl-4-yl)methyl]imidazole (losartan), indicating that unlabelled ligands prevented reassociation. [3 H]candesartan saturation binding experiments required a long time to reach equilibrium. Therefore, the equilibrium dissociation constant (Kd = 51+/-8 pM) was calculated from the association and dissociation rate constants. Our findings indicate that the insurmountable nature of candesartan in functional studies is related to its slow dissociation from the receptor.

The oxidative metabolism of irbesartan, a new nonpeptide angiotensin II receptor antagonist, was investigated on 12 human fully characterized hepatic microsomes and purified cytochrome P-450 (CYP) isoforms. After incubation of microsomes with irbesartan and NADPH, four main hydroxy metabolites were formed, as confirmed by liquid chromatography-mass spectrometry analysis. Irbesartan oxidation follows Michaelis-Menten kinetics, consistent with the involvement of a single CYP isoform in these hydroxylation processes. Only a low interindividual variability (2-fold difference) was observed in drug oxidation, even in preparations lacking CYP2D6. Km and Vmax for irbesartan oxidation were 54 +/- 6.5 microM and 0.62 +/- 0.18 nmol/min/mg, respectively. Irbesartan oxidation correlated (r2 = 0. 769) with tolbutamide (CYP2C9 substrate) 4-methyl-hydroxylation. Oxidation of irbesartan was markedly inhibited by sulfaphenazole (CYP2C9 inhibitor), but not by any of several other CYP inhibitors. In the same manner, both tolbutamide and warfarin (CYP2C9 substrates), were competitive-type inhibitors of irbesartan oxidation with Ki values of 500 and 30 microM, respectively. Moreover, irbesartan was a competitive-type inhibitor of tolbutamide 4-methylhydroxylation (Ki = 317 microM). Nifedipine also potentially decreased irbesartan oxidation, whereas neither ketoconazole and triacetyloleandomycin (CYP3A inhibitors), nor diltiazem and verapamil, (CYP3A4 substrates), exhibited an inhibitory effect. Additional studies demonstrated that nifedipine was an inhibitor of irbesartan (Ki = 20 microM) and tolbutamide oxidation processes, whereas irbesartan had no effect at all on nifedipine dehydrogenation. Enzyme kinetics suggest that nifedipine is a noncompetitive-type inhibitor of CYP2C9-mediated catalytic activities. Finally, only microsomes containing recombinant human liver CYP2C9 were capable of oxidizing irbesartan. These results provide evidence that CYP2C9 plays a major role in irbesartan oxidation.

Inhibition of angiotensin (Ang) II type 1 (AT1) receptors in various target tissues of adult Sprague-Dawley rats was studied after single oral administration of TCV-116. The effects of TCV-116 on Ang II-receptor binding were assessed by quantitative in vitro autoradiography using 125I-[Sar1,Ile8]Ang II as a ligand. Four hours after the administration of TCV-116 (1 mg/kg), Ang II-receptor binding was markedly inhibited in the kidney (20% of control), adrenal cortex (27%), thoracic aorta (57%), heart (55%) and testis (76%) where AT1 receptors predominate. In the brain, orally administered TCV-116 produced a significant inhibition of binding both to the circumventricular organs (38%), which are devoid of the blood-brain barrier (BBB), and to the discrete regions within the BBB such as the paraventricular hypothalamic nucleus (48%), nucleus of the solitary tract (60%). Twenty-four hours after the administration, Ang II-receptor binding had partly recovered to approximately 50-85% of control levels. In contrast, throughout the experimental period, Ang II-receptor binding was little affected in sites where Ang II type 2 (AT2) receptors predominate such as the adrenal medulla and the nucleus of the inferior olive. These data indicate that orally administered TCV-116 specifically binds to AT1 receptors both in peripheral tissues and the central nervous system.

Objective: In vitro results suggest that cilostazol is metabolised by cytochrome P450 (CYP) isoforms 1A2, 2D6, 3A and 2C19. This study investigated the role of CYP3A inhibition on the metabolism of cilostazol., Design: The study was conducted as a single-centre, open-label, nonrandomised, 2-period, crossover pharmacokinetic trial. A single dose of cilostazol 100 mg was administered orally on days 1 and 15. Erythromycin (150 mg orally 3 times daily) was administered on days 8 to 20. 14C-erythromycin (3 microns Ci) was administered intravenously on days 1 and 15 one hour before cilostazol administration to determine baseline and the inhibitory effect of erythromycin treatment on CYP3A activity., Study Participants: 16 healthy nonsmoking male volunteers., Main Outcome Measures: Serial blood and pooled urine samples were collected before and after cilostazol administration to quantitate cilostazol and its metabolites. Serial exhalation samples were collected after intravenous 14C-erythromycin administration and radioactivity was quantitated by scintillation counting. Pharmacokinetics were determined by noncompartmental methods and compared before and after erythromycin administration. Tolerability assessments included adverse events, laboratory tests, vital signs and electrocardiographs., Results: Following erythromycin coadministration, cilostazol maximum plasma concentration (Cmax), area under the plasma concentration-time curve at time t (AUCt), and area under the curve from zero to infinity (AUC infinity) increased significantly by 47, 87, and 73%, respectively, and an approximately 50% reduction in unbound clearance was observed for the major circulating metabolite of cilostazol, OPC-13015. Cmax decreased significantly (p < 0.001) by 24%, while AUCt increased by 8%; this increase was not significant. For the second major metabolite, OPC-13213, the Cmax and AUCt increased by 29 and 141%, respectively (p < 0.001)., Conclusions: In vivo results are in agreement with previous in vitro human microsome studies, indicating that cilostazol is metabolised to OPC-13015 via CYP3A. In addition, OPC-13213 concentrations increased after inhibition of CYP3A because of inhibition of sequential metabolism of OPC-13213 via CYP3A. A starting dose for cilostazol of 50 mg twice daily should be considered during coadministration of inhibitors of CYP3A.

Biotransformation of the M1-muscarinic agonist Lu 25-109 (5-(2-ethyl-2H-tetrazol-5-yl)-1-methyl-1,2,3,6-tetrahydropyridine) , in development for the treatment of Alzheimer's disease, was investigated to obtain information on the identity of human hepatic cytochrome P-450 enzymes involved in its metabolism. The identification of these P-450s was accomplished through studies using 1) simple regression analysis with 14 phenotyped human liver samples, 2) selective chemical inhibitors, and 3) microsomes containing cDNA-expressed enzymes. The production of some metabolites is enhanced in vitro when the pH of the incubation media is shifted from pH 7.4 to 8.5. The metabolite production in human liver microsomes was NADPH-dependent, suggesting that the metabolism of Lu 25-109 in human liver microsomes is primarily P-450-dependent. Lu 25-109 was metabolized by human liver microsomes to Lu 31-126 (de-ethyl Lu 25-109) mainly by CYP2D6; to Lu 29-297 [3-(2-ethyltetrazol-5-yl)-1-methyl-pyridinium] and Lu 25-077 (demethyl Lu 25-109) mainly by CYP1A2, CYP2A6, CYP2C19, and CYP3A4; and to Lu 32-181 (Lu 25-109 N-oxide) by CYP1A2 and possibly by CYP2C19. One metabolite, Lu 32-181 (N-oxide), could be reduced back to Lu 25-109, a reaction not inhibited by the applied cytochrome P-450 inhibitors. This study did not indicate any involvement of FMO3 or MAO in the in vitro metabolism of Lu 25-109 in human liver microsomes.

Objectives: The objectives of this research were to (1) assess the relative bioavailability following administration of a 100 mg cilostazol suspension versus 100 mg tablet; (2) assess dosage form equivalency (2 x 50 mg compared with 1 x 100 mg); (3) compare the relative bioavailability following a single 50 mg dose of cilostazol administered as an ethanolic solution versus a 50 mg tablet; and (4) determine the effects of high fat diet on the pharmacokinetics of cilostazol following a single dose of 100 mg cilostazol in the fed or fasted state. Results were compiled from 3 separate studies to address these objectives., Design: All studies involved healthy adult males receiving single oral doses of cilostazol in the fed or fasted state. The fed state consisted of administering cilostazol after ingestion of a high fat meal. One study compared the relative bioavailability of 100 mg suspension and 2 x 50 mg tablet versus 100 mg tablet in a randomised crossover design. The study involving administration of a 50 mg cilostazol ethanolic solution was a single treatment study. The effects of food on the pharmacokinetics of cilostazol after administration of 100 mg cilostazol in the fed or fasted state as well as the pharmacokinetic profile following administration of a single 50 mg oral dose of cilostazol were assessed in a randomised crossover design., Study Participants: All participants were healthy nonsmoking males aged between 19 and 48 years whose bodyweight was within 15% of ideal bodyweight., Main Outcome Measures: Noncompartmental pharmacokinetic parameters were determined for each study participant., Results: The area under the plasma concentration-time curve (AUC) parameters were within the 80 to 125% criterion for bioequivalence for the cilostazol and its primary metabolite, OPC-13015. The maximum observed plasma concentrations (Cmax) for these formulations were not equivalent and indicated that the absorption of cilostazol from a suspension is more rapid than from a tablet. The apparent terminal half-lives (t1/2z) of cilostazol and OPC-13015 were shorter after administration of the suspension compared with the tablet. Cmax and AUC following administration of a single 50 mg cilostazol tablet were approximately 80% of that from the same dose administered as an ethanolic solution. The t1/2z of cilostazol decreased from 15.5 hours after a tablet to 2.5 hours after an ethanolic solution. Upon coadministration with a high fat meal, the Cmax of cilostazol increased 90% and AUC infinity increased 25% (p < 0.05). The t1/2z decreased from 15.1 +/- 14.5 hours (mean +/- SD) in the fasted state to 5.4 +/- 2.0 hours in the fed state. Single oral doses of 50 and 100 mg cilostazol were well tolerated., Conclusions: The relative bioavailability of the 100 mg cilostazol tablet versus an oral 100 mg cilostazol suspension is 100%. The 2 x 50 mg and 1 x 100 mg tablets are considered to be bioequivalent. The absorption following administration of 50 mg cilostazol ethanolic solution is faster and appears to be greater than that after administration of the 50 mg tablet. Coadministration of food increases the rate and extent of cilostazol absorption. The oral pharmacokinetics of cilostazol and metabolites are absorption-rate limited. The significant differences in the t1/2z observed when comparing cilostazol tablet, suspension, and solution as well as the effects of food suggest 'flip-flop' pharmacokinetics.

Objective: In vitro results suggest that cilostazol is metabolised by cytochrome P450 (CYP) isoforms 1A2, 2D6, 3A4 and 2C19. This study was designed to evaluate the effect of concomitant administration of omeprazole (a CYP2C19 inhibitor) on the pharmacokinetics of a single 100 mg oral dose of cilostazol., Design: This study was conducted as a single-centre, open-label, nonrandomised, 2-period, crossover pharmacokinetic trial. A single 100 mg dose of cilostazol was administered orally on days 0 and 14. Oral omeprazole (40 mg every day) was administered on days 7 to 18., Study Participants: 20 healthy nonsmoking male and female volunteers., Main Outcome Measures: Serial blood samples were collected before and after cilostazol administration to characterise the pharmacokinetics of cilostazol and its metabolites., Results: Following omeprazole coadministration, the increases in cilostazol maximum plasma concentration (Cmax) and area under the plasma concentration-time curve at time t (AUCt) were 18% (p = 0.062) and 26% (p < 0.001), respectively. For the 2 major circulating metabolites, OPC-13015 and OPC-13213, the OPC-13015 Cmax and AUCt increased by 29 and 69%, respectively (p < 0.001). However, for OPC-13213, the Cmax and AUCt decreased by 22 and 31%, respectively (p < 0.001). The plasma protein binding of cilostazol was unaffected by coadministration of omeprazole., Conclusions: Coadministration of cilostazol with omeprazole resulted in an increase in the systemic exposure of cilostazol and its active metabolite, OPC-13015, by 26 and 69%, respectively. For the other active metabolite, OPC-13213, systemic exposure decreased by 31% because of inhibition of cilostazol metabolism to this metabolite. These changes in systemic exposure were well tolerated. A dose of 50 mg cilostazol twice a day should be considered during coadministration of inhibitors of CYP2C19, such as omeprazole.

Objective: The pharmacokinetic profiles of cilostazol and its metabolites following a single oral dose of cilostazol 100 mg were compared between individuals with impaired and normal liver function., Design: The study was conducted as a single-centre, open-label, single dose pharmacokinetic and tolerability trial., Study Participants: 12 patients with impaired and compensated liver function were compared with 12 volunteers with normal liver function. Participants in each group were matched for gender, age and weight. Of the 12 patients with hepatic impairment examined in this study, 10 had mild impairment (Child-Pugh class A) and 2 had moderate impairment (Child-Pugh class B)., Main Outcome Measures: Blood and urine were collected up to 144 hours after drug administration. Pharmacokinetics were determined by noncompartmental methods., Results: Protein binding did not differ between the groups (95.2% healthy volunteers, 94.6% hepatically impaired patients). Mean +/- SD unbound oral clearance of cilostazol decreased by 8.6% because of hepatic impairment (3380 +/- 1400 ml/min in healthy volunteers, 3260 +/- 2030 ml/min in hepatically impaired patients). Total urinary excretion of metabolites was significantly higher in healthy volunteers (26 vs 17% of dose). Overall, the pharmacokinetics of cilostazol and its metabolites, OPC-13213 and OPC-13015, were not substantially different in those with mild and moderate hepatic disease compared with values in healthy volunteers. Except for terminal-phase disposition half-life and apparent terminal-phase volume of distribution for cilostazol, the ratios of geometric means of pharmacokinetic parameters for plasma cilostazol, OPC-13213 and OPC-13015 in those with hepatic impairment versus healthy volunteers were close to 100%., Conclusions: Based on the results of the pharmacokinetic analysis, dose adjustment in patients with mild hepatic impairment is not necessary. However, caution should be exercised when cilostazol is administered to patients with moderate or severe hepatic impairment.

Objective: In vitro results are inconclusive as to whether cilostazol is metabolised by cytochrome P450 isoenzyme 2D6 (CYP2D6). The goals of this study were (1) to assure the dose of quinidine and timing relative to cilostazol used in this study were adequate to cause inhibition of CYP2D6, (2) to evaluate carryover effects of quinidine administration, and (3) to evaluate the effect of CYP2D6 deficiency and administration of quinidine (a CYP2D6 inhibitor) on the pharmacokinetics of a single 100 mg oral dose of cilostazol., Design: This study was conducted as a single-centre, open-label, randomised sequence, 2-period, crossover pharmacokinetic trial. Water alone (treatment without quinidine) or two 200 mg oral doses of quinidine sulfate with water were administered 25 hours and 1 hour prior to a single 100 mg dose of cilostazol in period 1. Study participants were crossed over to opposite treatment in period 2. Metoprolol 25 mg, used as a positive control, was administered 1 hour after quinidine sulfate with water or using water alone to assess the magnitude of CYP2D6 inhibition by quinidine., Study Participants: 22 healthy nonsmoking Caucasian (14 male and 8 female) volunteers participated in the study., Main Outcome Measures: Serial blood and urine samples were collected at predose and after cilostazol administration to characterise cilostazol and its metabolite pharmacokinetics. Additional plasma samples were taken to assess the pharmacokinetics of quinidine. Urine samples were collected to measure metoprolol and hydroxymetoprolol., Results: Administration of metoprolol with quinidine caused a significant (p < 0.001) decrease in the urinary 4-hydroxymetoprolol/metoprolol ratio compared with administration of metoprolol alone (42-fold decrease, 0.065 vs 2.707). Hence, quinidine effectively converted extensive metabolisers of CYP2D6 to poor metabolisers of CYP2D6. The 21-day washout period was adequate to have complete recovery from quinidine inhibition of CYP2D6. The analysis of variance demonstrated that the mean maximum plasma concentration (Cmax) for cilostazol, both adjusted and unadjusted for the free fraction, was higher in the control group than in the quinidine group (p = 0.023). However, the time to Cmax (p = 0.669), the area under the plasma concentration-time curve from time zero to infinity (AUC infinity; p = 0.133), and the apparent oral clearance (p = 0.135) were unchanged. The geometric mean ratios (90% confidence interval) comparing with quinidine (test) and without quinidine (reference) coadministration for Cmax and AUC infinity are 0.86 (0.77, 0.95) and 0.92 (0.84, 1.00), respectively. Similar patterns were observed for OPC-13015 and OPC-13213 with regard to Cmax, area under the plasma concentration-time curve from time zero to the last measurable concentration at time t, and AUC infinity (where determinable). The slight decrease in the systemic availability of cilostazol and its metabolites was thought to be a result of the increased gastrointestinal motility secondary to quinidine., Conclusions: Administration of quinidine sulfate 200 mg profoundly inhibited CYP2D6-mediated metabolism. The effects of quinidine inhibition of CYP2D6 metabolism were completely reversible during the 21-day washout period. Coadministration of quinidine with cilostazol had no substantial effect on cilostazol or its metabolites (OPC-13015 and OPC-13213). Hence, CYP2D6 does not have a significant contribution in the metabolic elimination of cilostazol.

Objective: The pharmacokinetics of cilostazol were studied in patients with mild, moderate and severe renal impairment and in healthy volunteers after administration of 50 mg single and multiple doses of cilostazol., Design: This was an open-label, single and multiple dose study administering 50 mg cilostazol every 12 hours to healthy volunteers and patients with varying degrees of renal impairment., Participants: 6 normal volunteers [creatinine clearance (CLCR) > or = 90 ml/min]; 6 patients with mild (CLCR 50 to 89 ml/min), 5 with moderate (CLCR 26 to 49 ml/min) and 6 with severe (CLCR 5 to 25 ml/min) renal impairment., Outcome Measures: Noncompartmental pharmacokinetic parameters were determined for each study participant., Results: At steady state, in the severe renal disease group, cilostazol and OPC-13015 peak concentrations (Cmax) were 29 and 41% lower and the areas under the concentration-time curve over the dosage interval (AUC tau) 39 and 47% lower than in the healthy volunteers. Cmax and AUC tau of OPC-13213 were significantly higher, 173 and 209%, respectively, than those in the healthy volunteers. The accumulation ratios were not significantly different between the various renal function groups for cilostazol and its metabolites. The estimated pharmacological activity of cilostazol and its metabolites was similar between the normal volunteers and those with severe renal impairment., Conclusions: A dosage reduction in renally impaired patients is not supported by the pharmacokinetics of cilostazol and its metabolites in this patient group.

Arachidonic acid metabolites mediate inflammatory responses at a cellular level. The affinity of the newly synthesized compound YM158, 3-[(4-tert-butylthiazol-2-yl)methoxy]-5'-[3-(4-chlorobenzenesuf onyl)propyl-2'-(1H-tetrazol-5-ylmethoxy)benzanilide monosodium salt monohydrate, for leukotriene D4 and thromboxane A2 receptors was examined in radioligand binding assays. YM158 inhibited [3H]leukotriene D4 and [3H]U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F2alpha) binding to guinea pig lung membrane preparations, with Ki values of 0.64+/-0.06 nM for leukotriene D4 and 5.0+/-0.88 nM for thromboxane A2 receptors. The Hill coefficients (nH) did not significantly differ from unity, indicating that this antagonism is competitive. In contrast, YM158 showed no affinity for several other receptors, including neurotransmitter-related (alpha1-, alpha2-, beta-adrenoceptors, histamine, 5-HT, muscarinic, sigma), C5a, opioid, Ca2+ channel, K+ channel, protein kinase C, bradykinin, endothelin, neurokinin and platelet activating factor receptors. These studies indicate that YM158 is a highly selective dual antagonist for leukotriene D4 and thromboxane A2 receptors, and this has potential clinical and research applications.

Mammalian endothelin-converting enzyme is a membrane-bound metalloprotease; its C-terminal domain contains sequence motifs characteristic of zinc metalloproteases. We examined residues expected from molecular modelling to be important for substrate binding using selectively mutated recombinant rat ECE-1alpha expressed in CHO cells. A conserved N-A-Ar-Ar (Ar = aromatic) motif is likely to be important for substrate binding. Mutating N550 to Gln or Y552 to Phe reduces Vmax/Km by 8- and 18-fold, respectively. The equivalent residue to Y553 in thermolysin binds the inhibitor through its NH group. Removing this putative interaction by mutating Tyr to Pro destroys activity, but mutating it to Ala or Phe also removes most activity. Mutating G583 (in a conserved GGI motif N-terminal of the zinc-binding helix) to Ala has no measurable effect, but mutating G584 to Ala destroys activity. Changing V583 in the zinc-binding helix to Met, to mimic the sequence pattern in bovine ECE-2, increases Vmax/Km to 1.7-fold that of the wild-type. Assays of phosphoramidon binding follow the pattern of those of substrate binding, but the IC50 of the more potent ECE inhibitor CGS 26303 was not significantly altered by any of these mutations, suggesting that this compound may bind to ECE in a different mode from phosphoramidon.

A high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the quantitation of cilostazol and four of its principal metabolites (i.e. OPC-13015, OPC-13213, OPC-13217 and OPC-13326) in human liver microsomal solutions was developed and validated. Cilostazol, its metabolites, and the internal standard (OPC-3930), were analyzed by protein precipitation followed by reverse-phase HPLC separation on a TSK-Gel ODS-80TM (150 x 4.6 mm, 5 microm) column and a Cosmil C-18 column (150 x 4.6 mm, 5 microm) in tandem and UV detection at 254 nm. An 80 min gradient elution of mobile phase acetonitrile in acetate buffer (pH = 6.50) was used to obtain quality chromatography and peak resolution. All the analytes were separated from each other, with the resolution being 2.43-17.59. The components of liver microsomal incubation mixture and five metabolic inhibitor probes (quinidine sulfate, diethyl dithiocarbamate (DEDTC), omeprazole, ketoconazole and furafylline) did not interfere with this analytical method. The LOQ was 1000 ng ml(-1) for cilostazol and 100 ng ml(-1) for each of the metabolites. This method has been validated for linear ranges of 100-4000 ng ml(-1) for OPC-13213, OPC-13217 and OPC-13326; 100-2000 ng ml(-1) for OPC-13015; and 1000-20000 ng ml(-1) for cilostazol. The percent relative recovery of this method was established to be 81.2-101.0% for analytes, with the precision (% coefficient of variation (CV)) being 2.8-7.7%. The autosampler stability of the analytes was evaluated and it was found that all analytes were stable at room temperature for a period of at least 17 h. This assay has been shown to be precise, accurate and reproducible.

Described in this paper is the synthesis and pharmacological activity of five metabolites of the angiotensin II antagonist tasosartan (1). Of particular interest is the effect of the additional acidic group of the enol metabolite (8) on activity. As suggested by the structural-activity relationship of other angiotensin II antagonist series, a second acidic group can improve receptor binding activity but decrease in vivo activity after oral dosing. The metabolic introduction of a second acidic group in tasosartan bypasses this problem and contributes to the excellent profile of the compound. A molecular modeling study provides a rationale for the role of the enol group of 8 in AT1 receptor binding.

The pharmacokinetics of losartan and its active metabolite, EXP3174, were investigated after intravenous and oral administration of the drug, 5 mg/kg, to control rats and streptozotocin-induced diabetes mellitus rats (SIDRs). After 1-min intravenous infusion, the mean arterial plasma concentrations and the resultant area under the plasma concentration-time curve from zero to infinity (AUC) of both losartan and EXP3174 were not significantly different between control rats and the SIDRs. However, the renal clearance (CLR) of losartan (0.181 versus 0.0815 ml/min/kg) and EXP3174 (0.0677 versus 0.0277 ml/min/kg) were significantly faster in SIDRs than in control rats due to significant increase in glomerular filtration rate. After oral administration, the mean arterial plasma concentrations and the resultant AUC of losartan (97 versus 166 micrograms min/ml) and EXP3174 (244 versus 423 micrograms min/ml) were significantly lower in SIDRs than in control rats. The absolute extent of oral bioavailability of losartan, F, (32.5 versus 55.1%) decreased considerably in SIDRs and it was possibly due to the reduced gastrointestinal absorption of losartan by gastrointestinal disorders occurring in the diabetic state. The low F in both groups of rats was at least partially due to the increase in first-pass effects. The CLR of losartan (0.207 versus 0.101 ml/min/kg) and EXP3174 (0.0615 versus 0.0196 ml/min/kg) were significantly faster in SIDRs than in control rats after oral administration of losartan.

Purpose: To study the relationship between angiotensin II (AII) receptor occupancy ex vivo in tissues plasma concentration and hypotensive effect of a novel AII receptor antagonist, TH-142177 and losartan in rats., Methods: At 2, 8 and 24 hr after oral administration of TH-142177 and losartan in rats, AII receptors in myocardium, adrenal cortex and cerebral cortex were determined by radioligand binding assay using [125I]Sar1,Ile8-AII. Plasma concentrations of both drugs and metabolite in rats were also measured using validated HPLC assays. Further, systolic blood pressure (SBP) in conscious renal hypertensive rats treated orally with TH-142177 and losartan were measured by using a tail cuff plethysmographic method., Results: Oral administration of TH-142177 (1.8 and 5.5 micromol/kg) and losartan (6.5 and 21.7 micromol/kg) in rats brought about dose-dependent decreases in [125I]Sar1,Ile8-AII binding sites (Bmax) in myocardium and adrenal cortex. The extent of receptor occupancy by both drugs in adrenal cortex was maximal at 2 hr later but that in myocardium at 8 hr later. Further, the receptor occupancy was more sustained in myocardium than adrenal cortex. The ex vivo binding affinity of TH-142177 for AII receptors in these tissues was roughly three times higher than that of losartan. Also, cerebral cortical [125I]Sar1,Ile8-AII binding was significantly reduced by oral administration of losartan but not by TH-142177. The time course of AII receptor occupancy by both drugs in adrenal cortex appeared to be in parallel with that of their plasma concentrations, while the time course in myocardium correlated with that of their hypotensive effects rather than plasma concentrations., Conclusions: TH-142177 produced a relatively selective and sustained occupancy ex vivo of AII receptors in myocardium and adrenal cortex of rats with approximately three times greater potency than losartan. Its time course of myocardial receptor occupancy was in parallel with that of hypotensive effect rather than plasma concentration.

A reliable and sensitive radioreceptor assay based on rat lung homogenate as receptor preparation was developed to determine the angiotensin-II antagonistic profile of losartan and its main active metabolite EXP 3174 as well as its congeners exemplified by UP 269-6 and SL 91.0102-90 DL. This method proved to be precise with an intra- and interday variability of less than 10% and a limit of quantification < or = 1 ng ml-1. The analysis of the Ki values in protein-free Hepes-buffer versus blank human or rat plasma revealed the distinct high plasma-protein binding of EXP 3174 which consequently caused a dramatic drop of potency from 10-15-fold in the buffer to only about 2-fold in control plasma, when compared to the parent compound losartan and the two congeners investigated. Upon evaluation of clinical samples by both the reported radioreceptor assay (RRA) and the established high-performance liquid chromatography (HPLC), the correlation of the normalized data pairs (concentration equivalents) suggested the contribution of active metabolites to the angiotensin-II antagonistic effect of SL 91.0102-90 DL, but not to the effect of UP 269-6. In the context of an extended preclinical study in rats, the correlation of RRA with the respective HPLC concentration equivalents of losartan and its main active metabolite EXP 3174 confirmed previous findings that only losartan and EXP 3174 exert the angiotensin-II-AT1 receptor blockade without the contribution of other metabolites (P.C. Wong, W.A. Price, A.T. Chiu et al., J. Pharmacol. Exp. Ther. 255 (1990) 211-217).

Site-directed interspecies amino acid exchange was used to compare the binding determinants of a novel dual endothelial-angiotensin receptor ligand, L-746,072, with type-1 angiotensin receptor (AT1) selective antagonists on AT receptors expressed in COS cells. These studies suggest that residues on AT receptors which are non-conserved between amphibian and mammalian species play a greater role in subtype selective ligand recognition than for dual receptor ligands. These data also support the hypothesis that a common non-peptide binding site exists within transmembrane domains on peptidergic receptors.

Optimization of an aminophosphonic acid series of compounds for inhibition of endothelin-converting enzyme (ECE) has led to the discovery of CGS 31447. This compound reversibly inhibited the activity of recombinant human ECE-1 with an IC50 value of 21 nM. The effect of CGS 31447 was not due to nonspecific chelation of the zinc ion at the catalytic center of ECE-1 by the phosphonic acid of the inhibitor. Determination of kinetic parameters of ECE-1 in the presence of 5-15 nM CGS 31447 revealed the competitive nature of the compound; a K1 of 7 nM was obtained. CGS 31447 infused at concentrations of 0.01, 0.1, and 1.0 microM inhibited the mean increase in big ET-1-induced pressor responses in isolated and perfused rat kidneys by 7, 39, and 68%, respectively, compared with the controls. These results demonstrate that CGS 31447 is a potent, reversible, and competitive inhibitor of ECE-1.

In order to study the roles of the AMPA and kainate subtypes of non-NMDA glutamate receptors in the processing of persistent nociceptive information, compounds with varying activities at these receptors were examined for effects on the formalin-induced paw-licking behavior in rats. The selective AMPA antagonist, LY300164 and the mixed AMPA/kainate antagonist, NBQX, were compared for their effects on formalin-induced pain behavior. NBQX (3, 10, 20 mg/kg, i.p.), caused antinociception as well as ataxia whereas the selective AMPA antagonist, LY300164 (3,5,10 mg/kg, i.p.), did not cause antinociception at doses that did not produce ataxia. In view of the well documented distribution of kainate receptors on C fibres and of the kainate-preferring iGluR5 subtype on dorsal root ganglia (DRG), we tested a series of three decahydroisoquinolines with different profiles of activity between iGluR5 and AMPA receptors and all without activity on iGluR6, iGluR7 or KA2 subtypes. LY293558 (0.1, 1, 3, 5 mg/kg, i.p.), which had low micromolar affinity for both iGluR5 and 2 caused, like NBQX, both antinociceptive and ataxic effects. However, the selective iGluR5 antagonist LY382884 (5, 10, 30, 100 mg/kg, i.p.), exhibited antinociceptive actions without ataxia while the iGluR2 preferring antagonist LY302679 (5 mg/kg, i.p), caused ataxia but did not produce antinociceptive effects at that dose. These actions were stereoselective since the enantiomeric compounds, LY293559 and LY302680, were ineffective in these tests. The data strongly suggest an involvement of iGluR5 in the processing of nociceptive information.

Objectives: Valsartan (CGP 48933), an orally active angiotensin II antagonist, is eliminated mainly by hepatic clearance. To characterize the compound(s) excreted in the bile, biliary excretion of valsartan was investigated by collection of bile after an intravenous dose of valsartan. In addition, to determine the exposure to valsartan when liver function is impaired, a pharmacokinetic study (open, single dose) was performed in patients with mild and moderate impairment of liver function., Patients: Biliary excretion of valsartan (after intravenous administration of 20 mg valsartan) was assessed in a patient who underwent a hepaticojejunostomy with subsequent bile drainage. Exposure to valsartan in patients with mild (n = 6) or moderate (n = 6) impaired liver function (Child's-Pugh classification) and matching (sex, age, and weight) healthy volunteers (n = 12) was studied after oral administration of a single dose of 160 mg valsartan., Results: After intravenous administration, valsartan was eliminated mainly as unchanged drug in the bile. Mean exposure (measured as area under the plasma valsartan concentration-time curve) to valsartan was increased about twofold in both the mild and the moderate groups compared with matched (age, sex, and weight) healthy volunteers., Conclusion: These data are consistent with the pharmacokinetics of valsartan in that biliary excretion is the main route of elimination.

1. The present studies were designed to measure the affinity of UP 269-6, a newly developed angiotensin AT1 receptor antagonist, for vascular AT1 receptors from normotensive and hypertensive rats and to investigate in vitro, its effects on angiotensin II (AII)-induced hyperplasia and hypertrophy of vascular smooth muscle cells (VSMC). In addition the in vivo effects of UP 269-6 on neointimal proliferation in a carotid artery balloon injury in normotensive rats were also investigated. 2. UP 269-6 selectively inhibited [125I]-Sar1-Ile8-AII binding to vascular AT1 receptors present on VSMC derived from normotensive Wistar rat and from SHR (Ki = 16.6 +/- 3.6 nM and 7.5 +/- 2.0 nM, respectively). In comparison, losartan and its metabolite, EXP 3174, inhibited [125I]-Sar1-Ile8-AII binding to vascular AT1 receptors derived from both cell models with Ki values slightly lower (losartan) and higher (EXP 3174), respectively, than that of UP 269-6. 3. AII (1 microM) induced a weak and variable hyperplastic response (4 to 32% increase in cell number) in Wistar rat VSMC after 96 h. 4. AII (1 microM) induced a time-dependent increase in cell number in VSMC from SHR. UP 269-6 inhibited concentration-dependently this effect with an IC50 value of 159 +/- 58 nM. Losartan was clearly less potent and EXP 3174 showed nearly the same inhibitory potency, compared to UP 269-6. UP 269-6 (1 microM) inhibited nearly completely the action of AII. 5. AII (500 nM) caused maximal stimulation of protein synthesis in Wistar rat VSMC (117 +/- 36%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 28 +/- 6 nM, 3504 +/- 892 nM and 21 +/- 3 nM, respectively. 6. AII (50 nM) induced maximal stimulation of protein synthesis in SHR VSMC (237 +/- 67%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 16 +/- 3 nM, 282 +/- 122 nM and 3.3 +/- 1.0 nM, respectively. 7. UP 269-6 (75 mg kg-1 day-1) administered orally in the diet for 20 days induced a 38% reduction in neointimal area and a 36% reduction in neointima/media ratio associated with the intimal thickening induced by carotid artery balloon injury. 8. In conclusion, UP 269-6 was shown to be a potent antiproliferative agent both in vitro on AII-induced hyperplasia and hypertrophy of VSMC derived from normotensive and hypertensive rats, and in vivo upon intimal thickening induced by carotid artery balloon injury in the rat.

The mechanisms of the insurmountable antagonism of 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]1H-benzimid azole -7-carboxylic acid, candesartan (CV-11974), an angiotensin AT1 receptor antagonist, on angiotensin II-induced rabbit aortic contraction were examined in contraction and binding studies. Preincubation of the rabbit aorta with CV-11974 (0.1 nM) for 30 min reduced the maximal contractile response to angiostensin II by approximately 50%. This insurmountable antagonism of CV-11974 was reversed in the presence of losartan (1 microM), a surmountable angiotensin AT1 receptor antagonist. The inhibitory effect of CV-11974 on angiotensin II-induced contraction persisted longer after washing than did that of losartan but was irreversible. Scatchard analysis of [3H]CV-11974 binding in bovine adrenal cortical membranes indicated the existence of a single class of binding sites (Kd = 7.4 nM). Competition binding studies using angiotensin II receptor agonists and antagonists have demonstrated that [3H[CV-11974 binding sites may be identical to angiotensin AT1 receptors. The dissociation rate of [3H]CV-11974 binding (t1/2 = 66 min) was 5 times slower than that of [125I]angiotensin II binding (t1/2 = 12 min). These results suggest that the insurmountable antagonism by CV-11974 is due to its slow dissociation from angiotensin AT1 receptors.

The pharmacological properties of TH-142177 (N-n-butyl-N-[2'-(1-H-tetrazole-5-yl) biphenyl-4-yl]-methyl-(N-carboxymethyl-benzylamino)-acetamide), a novel antagonist of the angiotensin II (AII) AT1 receptor, were studied in vitro and in vivo, and compared to those of losartan. In the rat isolated aorta, TH-142177 produced parallel shifts to the right of the concentration-response curves for AII-induced contractions without affecting the maximal response (pA2 = 9.07). The inhibitory potency of TH-142177 in the aorta was about three times greater than that of losartan. TH-142177 completely inhibited the specific binding of [125I]AII to AT1 receptor in rat aortic membranes (Ki = 1.6 x 10(-8) M), whereas specific [125I]AII binding to AT2 receptor in bovine cerebellum and human myocardium was not affected by concentrations of TH-142177 up to 10(-5) M. Losartan also inhibited the [125I]AII binding to rat aortic membranes (Ki = 2.2 x 10(-8) M). Following the intravenous administration to anesthetized normotensive rats, TH-142177 dose-dependently inhibited the increase in systolic blood pressure induced by an intravenous bolus injection of AII that was 1.5 times less potent than losartan. Furthermore, the oral administration of TH-142177 to conscious renal hypertensive rats exerted a dose-dependent reduction of systolic blood pressure without significantly effecting the heart rate. TH-142177 was at least three times more potent than losartan. These results demonstrate that TH-142177 is a potent and selective antagonist of AT1 receptors and by oral administration has a long-lasting antihypertensive activity.

Objectives: The angiotensin type 1 (AT1) receptor antagonist, losartan (orally administered), decreases vasoconstrictor effects of angiotensin II (Ang II). Oral losartan is converted into the active metabolite, Exp3174, which causes most of the antagonistic effects. Effects of losartan as such have not been studied after its intra-arterial administration in humans. Therefore, we investigated the effects of both intra-arterially and orally administered losartan on AT1-receptor-mediated vasoconstriction., Methods: Forearm vascular resistance (FVR) was determined by venous occlusion plethysmography in 24 healthy subjects. Ang II (0.01, 0.1, 1.0, and 10.0 ng/kg/min) was infused into the brachial artery, before and after losartan, administered intra-arterially (dose range 100-3000 ng/kg/min) or orally (50 mg once daily for 5 days)., Results: Ang II concentration-dependently increased FVR (P < 0.05); tachyphylaxis did not occur. Losartan alone did not change FVR. Intra-arterially infused losartan dose-dependently inhibited Ang-II-induced vasoconstriction. At a concentration of 10(-8) M Ang II, losartan reduced FVR, as a percentage of baseline values, from 287 +/- 30 to 33 +/- 8% (mean +/- s.e.m.; P < 0.05). Orally given losartan reduced FVR from 297 +/- 40 to 73 +/- 19% (P < 0.05)., Conclusions: Losartan, intra-arterially administered, causes no effect on baseline vascular resistance, but markedly inhibits Ang-II-induced vasoconstriction in the human forearm vascular bed. Relatively high doses of intra-arterial losartan were required when compared to the antagonism by the orally administered drug. These data indicate that Ang-II-induced vasoconstriction is mediated by AT1-receptors, which are blocked by losartan. The more effective antagonism exerted by oral losartan is presumably explained by the formation of Exp3174. Endogenous Ang II does not contribute to baseline vascular tone in healthy, sodium-replete, subjects.

Specific, high-affinity angiotensin II (A II) receptors were observed on granulosa and thecal cells of preovulatory ovarian follicles from immature PMSG-treated rabbits. Scatchard analysis of 125I-[Sar1,Ile8]A II binding to freshly prepared cells was indicative of only one class of binding sites. Kd values were 0.26 +/- 0.11 nM and 0.18 +/- 0.02 nM, densities of A II receptors were 0.06 +/- 0.02 fmol/10(5) cells and 0.08 +/- 0.01 fmol/10(5) cells for granulosa and thecal cells, respectively. When cells were incubated for 48 h with hCG, Kd values were of the same order of magnitude, but the amount of A II receptors was increased 2-fold in granulosa and 4-fold in theca. Using subtype specific ligands (Losartan for AT1 and PD 123319 for AT2) in competitive binding experiments, A II receptors were found to be of the AT1 type on both granulosa and thecal cells freshly prepared or incubated 48 h in vitro. These results establishing the existence of high affinity AT1 receptors on the two cell types of the rabbit preovulatory follicles contrast with previous observations showing the presence of AT2 receptors on granulosa or theca from several species.

From evidence based on the use of specific receptor subtype antagonists, it has generally been assumed that human uterine tissue contains only type 2 (AT2) angiotensin II (AII) receptor subtype. Using a monoclonal antibody, 6313/G2, directed against a specific sequence in the extracellular domain of the type 1 AII receptor (AT1), in immunocytochemical studies, we show here that AT1 receptor is expressed in human endometrium. In particular, positive staining was seen in the endometrial glandular epithelium, and in the vascular endothelium, while the myometrium and endometrial stroma were negative. The most intense staining was observed during the late proliferative phase and less in the luteal phase. The ligand binding assay, using [125I]-angiotensin II, revealed high concentrations of AII receptors both in the endometrium and in the myometrium. Competition studies using losartan (AT1 specific) and CGP42112B (AT2 specific) showed that both AT1 and AT2 receptor subtypes were present in the endometrium, though only the AT2 receptor subtype was detected in the myometrium. Immunoblotting confirmed that the antibody 6313/G2 detected a single protein with a molecular weight of approximately 60 kDa. These data clearly demonstrate the presence of endometrial AT1 receptors whose expression appears to be under hormonal control.

Imidazoline/guanidinium receptive sites (IGRS) are shown to be present in the subfornical organ and hypothalamic arcuate nucleus by a derivative of cirazoline, 2-(3-amino-4-[125I]iodophenoxy)methylimidazoline ([125I]AMIPI). Because many of the nonpeptide angiotensin II (Ang II) receptor antagonists contain imidazole ring structures, they may interact with IGRS. Therefore, we studied competitive activity of Ang II and several nonpeptide Ang II receptor antagonists [DuP 753 (losartan), EXP 3174, CV11974, and PD123319] at IGRS in rat forebrain. The results showed specific binding of 944 +/- 169 fmol/mg protein in the subfornical organ (n = 11) and of 367 +/- 27 fmol/mg protein in the arcuate nucleus (n = 6) at 0.4 nM [125I]AMIPI, as defined by competition with 10 microM cirazoline. Specific [125I]AMIPI binding was competed for completely by 10 microM idazoxan or clonidine as further characterization of IGRS. Ang II and the nonpeptide AT1 and AT2 antagonists did not significantly compete for specific [125I]AMIPI binding in either brain region at concentrations of 10 microM (< 20% competition with each compound), which is 10- to 100-fold higher than the concentration necessary to compete completely for their respective Ang II receptor subtypes. Only at the highest concentration (100 microM) did losartan compete significantly for binding (56 +/- 8%). Therefore, Ang II receptor antagonists interact with IGRS in rat forebrain cardiovascular areas only at high concentrations.

It has been demonstrated recently that antagonists of the tachykinin NK1 receptor, specifically CP-99,994 and GR203040, possess anti-emetic activity in a range of species. To optimise this activity, a series of analogues based around the structure of GR203040 have been synthesised and their affinity at the human tachykinin NK1 receptor determined. In addition, the potency of these analogues to inhibit emesis induced in the ferret by whole-body X-irradiation has been examined. A range of substitution at the C-1 position of the tetrazole moiety in GR203040 were explored in vitro and in vivo. The trifluoromethyl compound, GR205171, was the most potent antagonist with regard to the ability to inhibit emesis induced by X-irradiation. This compound was demonstrated to have a broad spectrum of anti-emetic activity, inhibiting emesis in the ferret induced by cisplatin, cyclophosphamide, morphine, ipecacuanha and copper sulphate. Furthermore, emesis was also inhibited in the house-musk shrew, Suncus murinus, when induced by either motion or cisplatin, and in the dog when induced by ipecacuanha. GR205171 has the most potent anti-emetic activity of any tachykinin NK1 receptor antagonist described to date. The compound is orally active in the ferret and dog, long-lasting, and warrants further investigation as a potential broad-spectrum anti-emetic agent.

The potassium salt of a chemically stabilized dipeptide, {1-[4-(1 H-tetrazol-5-yl)butyl]indol-3-yl}carbonyl-Hyp-Nal-N(methyl)-Bzl , (Hyp = (R)-4-hydroxy-L-proline; Nal = 3-L-(beta-naphthyl)-alanine), S18523, is described as a new water-soluble, potent and selective NK1 receptor antagonist. The low molecular weight antagonist (M(r) = 736) displays nanomolar potency (pA2 = 9.6) in the rabbit vena cava (NK1) bioassay and nanomolar affinity (pKi = 9.1) on the human NK1 receptor expressed by lymphoblastoma cells. It is devoid of mu-opiate affinity (Ki > 10(-4) M with respect to tritiated Tyr-DAla-Gly-MePhe-Gly-ol), has negligible calcium-channel affinity (estimated Ki = 2.6 x 10(-5) M, with respect to isradipine) and does not cause peritoneal mast-cell degranulation. S18523 has strong antinociceptive effects in three classical pain tests in vivo both by i.v. and p.o. routes. The dipeptide potently antagonizes bronchoconstriction provoked by exogenous substance P in the guinea-pig and acts longer than the non-peptide antagonist CP99994, when administered as aerosol. Finally, S18523 displays antiinflammatory properties, since it dose-dependently inhibits substance P-induced plasma extravasation both in the bladder (ID50 = 0.18 mg/kg i.v.) and bronchi (ID50 = 0.14 mg/kg i.v.) of the guinea-pig.

Using an antibody (6313/G2) directed against a specific sequence in the extracellular domain of the type 1 angiotensin II receptor (AT1), we demonstrated the presence of angiotensin II (AII) receptors in human fallopian tube. Immunoperoxidase staining for AT1 receptor showed positive staining in the epithelium of the tubal mucosa. The intensity of staining varied depending upon the hormonal status at the time of salpingectomy, being strongest in the proliferative phase of the ovarian cycle and weakest after menopause. Ligand binding assay confirmed that the AII receptor concentration was highest in the mucosa of fallopian tubes from premenopausal women. Mucosa from the ampullary segment had higher concentrations of AII receptor than the fimbrial and isthmic segments in both premenopausal and postmenopausal women. Displacement studies using specific AII receptor subtype antagonists showed that approximately 60% of the total activity could be displaced by CGP42112B (type 2 specific) and 40% by losartan (AT1 specific). Immunoblotting confirmed that the antibody detected a protein of approximately 60 kDa. Functional studies showed that AII had a stimulatory action on tubal ciliary beat frequency, but had no significant effect on myosalpingseal activity. This effect was achieved at nanomolar concentrations of AII; further increases in the AII concentration were without additional effect. The stimulatory effect of AII was inhibited by the specific AT1 antagonist losartan, whereas the type 2 antagonist, CGP42112B, had no effect. The data demonstrate that AII may play an important role in ovum transport and fertility.

Catalysis by glycogen phosphorylase involves a mechanism in which binding of one substrate tightens the binding of the other substrate to produce a productive ternary enzyme-substrate complex. In this work the molecular basis for this synergism is probed in crystallographic studies on ternary complexes in which the glucosyl component is substituted by the putative transition state analogue nojirimycin tetrazole, a compound which has been established previously as a transition state analogue inhibitor for a number of glycosidases. Kinetic studies with glycogen phosphorylase showed that nojirimycin tetrazole is a competitive inhibitor with respect to glucose 1-phosphate and uncompetitive with respect to phosphate. Ki values for the phosphorylase-AMP-glycogen complex and the phosphorylase-AMP-glycogen-phosphate complexes are 700 microM and 53 microM, respectively, indicating that by itself norjirimycin tetrazole has poor affinity for glycogen phosphorylase but that phosphate substantially improves the binding of norjirimycin tetrazole. X-ray crystallographic binding studies to 2.4 A resolution with T state phosphorylase b crystals showed that nojirimycin tetrazole binds at the catalytic site and promotes the binding of phosphate through direct interactions. Phosphate binding is accompanied by conformational changes that bring a crucial arginine (Arg569) into the catalytic site. The positions of the phosphate oxygens were definitively established in X-ray crystallographic binding experiments at 100 K to 1.7 A resolution using synchrotron radiation. X-ray crystallographic binding studies at 2.5 A resolution with R state glycogen phosphorylase crystals showed that the protein atoms and water molecules in contact with the nojirimycin tetrazole and the phosphate are similar to those in the T state. In both T and R states the phosphate ion is within hydrogen-bonding distance of the cofactor pyridoxal 5'-phosphate group and in ionic contact with the N-1 atom of the tetrazole ring. The results are consistent with previous time-resolved structural studies on complexes with heptenitol and phosphate. The structural and kinetic results suggest that nojirimycin tetrazole in combination with phosphate exhibits properties consistent with a transition state analogue and demonstrate how one promotes the binding of the other.

Paralleling the classic circulating system, recent evidence has demonstrated the presence of a cardiac renin-angiotensin system, as well as the synthesis of angiotensin II in the heart. Two receptors for angiotensin II have been identified and classified as AT1 and AT2. The proportions of these receptor subtypes vary with the tissues, species and stage of development. From the results of other studies, it might be generalized that the expression of angiotensin II receptors and the proportion of AT2 receptor subtype are much higher in fetal and neonatal tissues than in the same tissues from an adult. The aim of this study was to specifically evaluate the AT1/AT2 ratio in the neonatal and adult conduction systems of rat hearts by means of quantitative autoradiogrphy. In the neonatal hearts, angiotensin II binding sites were highly concentrated in the vasculature, arterial duct, and conduction system, whereas their concentrations were barely detectable in the myocardium. Incubation with selective angiotensin II receptor ligands (losartan and CGP 42112) revealed that AT2 was the major subtype in vasculature (86 +/- 3%) and conduction system (73 +/- 4%). In the adult conduction system, the total expression of angiotensin II receptors was greatly reduced meanwhile the AT1 receptors represented the major proportion of the binding sites (80 +/- 3%). Our results demonstrated that the pattern of angiotensin II receptor expression in the conduction system of the rat heart is developmentally regulated. We suggest, as others have already, that the renin-angiotensin system plays a role during the early stage of cardiac development.

The effects of the new anti-platelet agent KBT-3022, ethyl 2-[4,5-bis(4-methoxyphenyl)-thiazol-2-yl]pyrrol-1-ylacetate, and its metabolite desethyl KBT-3022 on rabbit neutrophil function were investigated in comparison with the effects of acetylsalicylic acid (ASA), ticlopidine hydrochloride (TP), cilostazol (CIL) and indomethacin (IM). The adhesion and migration of neutrophils induced by formyl-methionyl-leucyl-phenylalanine (fMLP) were inhibited by all the compounds tested, their rank order of potency being KBT-3022 = desethyl KBT-3022 > TP = CIL = IM > ASA. KBT-3022, desethyl KBT-3022, CIL and IM all suppressed fMLP-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) in neutrophils, their potencies correlating with their inhibitory effects on fMLP-induced adhesion and migration. KBT-3022 (1 microM), desethyl KBT-3022 (1-10 microM) and CIL (10 microM) but not IM significantly inhibited both neutrophil migration and the increase in [Ca2+]i induced by leukotriene B4 (LTB4). KBT-3022 (1 microM) and desethyl KBT-3022 (1 microM) suppressed the increase in [Ca2+]i induced by complement C5a. Although KBT-3022 and desethyl KBT-3022 did not influence [3H]LTB4 and [125I]C5a specific binding, [3H]fMLP specific binding was inhibited by desethyl KBT-3022 (IC50: 1.9 microM). Neutrophil adhesion and superoxide anion production stimulated by phorbol 12-myristate 13-acetate were partially inhibited by KBT-3022 (1 microM) and desethyl KBT-3022 (1-10 microM). These results suggest that KBT-3022 and desethyl KBT-3022 have a wider spectrum of action and are more potent inhibitors of neutrophil activation than ASA, TP, CIL and IM.

Differentiation of NG108-15 neuroblastoma cells following exposure to either 1.5% dimethyl sulfoxide (DMSO)/0.5% fetal bovine serum (FBS) or serum starvation resulted in significant differences in angiotensin (AT) receptor levels and the AT1/AT2 receptor ratio. When NG108 cells were differentiated for 4 days with DMSO/low serum, the number of AT binding sites increased 30-fold compared with the binding levels on undifferentiated (blast) cells. However, cells differentiated by serum starvation for 4 or 14 days resulted in only a modest 2.5- and fivefold increase in AT receptor levels, respectively, over the levels seen with the undifferentiated cells. KD values for all treatment conditions were not significantly different (0.71 +/- 0.11 nM, p = 0.06). Using the AT1 and AT2 isoform-specific receptor antagonists losartan and PD123319, the relative numbers of AT receptor subtypes on undifferentiated and differentiated cells were determined by competitive inhibition against 125I-[Sar1,Ile8]-angiotensin II (sarile). A majority of the AT receptors on undifferentiated NG108 cells were the AT1 subtype (AT1/AT2 receptor ratio of 8:3). Differentiation by serum starvation and DMSO/low serum treatment resulted in fivefold and 30-fold increases in AT receptor levels, respectively, compared with the levels seen with the undifferentiated cells. Although serum starvation increased the total number of AT1 and AT2 receptors, it did not significantly alter the AT1/AT2 receptor ratio. In contrast, differentiation with DMSO/low serum both increased the total number of AT1 and AT2 receptors and reversed the AT1/AT2 receptor ratio (1:3). The increase in AT receptors following differentiation with DMSO/low serum for 4 days was largely accounted for by an 80-fold increase in the AT2 receptor level. Previous studies by Tallant at al. (1991) and Bryson et al. (1992) reported increased AT2 receptor expression following neuroblastoma differentiation with dibutyryl cyclic AMP and DMSO/low serum, respectively, and suggested a role for the AT2 receptor in neuronal differentiation. In the present study, we have extended these earlier observations by demonstrating that the method of differentiation significantly affects both the AT receptor level and the ratio of AT1 to AT2 receptor expression. Finally, our findings indicate that the AT2 receptor is expressed as a consequence of neuronal maturation and dose not mediate morphological differentiation.

To investigate the effect of hypophyseal transection (HST) on GH secretagogue activity of the non-peptidyl GH secretagogue L-692,585 in the conscious pig, male castrated swine were randomly assigned to either a hypophyseal stalk transection group (HST; n = 3) or to a sham-operated control group (SOC; n = 3). Treatments administered were L-692,585 (100 micrograms/kg), human GH-releasing factor(1-29)NH2 (GRF; 20 micrograms/kg) or L-692,585 (100 micrograms/kg) + GRF (20 micrograms/kg) on days -7 to -3 before surgery and days +3 to +8 after surgery. To evaluate the integrity of the pituitary gland, the animals were challenged with corticotropin-releasing hormone (CRH; 150 micrograms) or GnRH (150 ng/kg) both before and after surgery. Blood was collected from -60 to +180 min post treatment and assayed for GH, cortisol and LH. Before surgery, no significant difference (P > 0.05) in peak GH response (ng/ml) was present between the two groups (SOC vs HST) in response to L-692,585 (101 +/- 12 vs 71 +/- 9) or L-692,585 + GRF (171 +/- 21 vs 174 +/- 21). Only two out of three SOC vs three out of three HST pigs responded to GRF (13 +/- 2 vs 25 +/- 3) resulting in a significant difference between groups. Following surgery, significant differences were present in peak GH response (ng/ml) between SOC and HST groups following L-692,585 (79 +/- 6 vs 13.8 +/- 1.0); however, the response to L-692,585 + GRF was similar (115 +/- 8 vs 94 +/- 7). All animals responded to GRF; however, a significant difference was present between groups due to the magnitude of the responses. Whereas the cortisol responses (ng/ml) to L-692,585 in the SOC and HST groups were similar before surgery, a significant difference was present after surgery (44.4 +/- 6.4 vs 14.6 +/- 2.1). No significant difference was noted between the HST and SOC groups in response to CRH or GnRH either before or after surgery. These results indicated that L-692,585 induced an immediate GH response in the intact animal in contrast to GRF where the GH release was variable. L-692,585 also stimulated an immediate increase in cortisol levels. Transection of the hypophyseal stalk dramatically decreased but did not ablate the GH or cortisol response to L-692,585. Co-administration of L-692,585 + GRF induced an immediate GH response of similar magnitude in the intact and HST animal. We conclude that L-692,585 has a direct but limited action at the level of the pituitary and that an intact hypophyseal stalk is required for a maximal GH and cortisol response. L-692,585 acts with GRF at the level of the pituitary to induce a maximal GH response. These findings suggest that L-692,585 stimulates GH secretion by acting in combination with GRF and interrupting the inhibitory tone of somatostatin on the somatotroph.

Type 1 angiotensin receptors (AT1) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT1A receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser105, Ser107, and Ser109 are not essential for maintenance of the angiotensin II binding site. Replacement of Asn111 or Ser115 does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT1 (DuP753)- or AT2 (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser115 mutant suggests that this residue, in addition to previously identified residues, Asp74 and Tyr292, participates in the receptor activation mechanism. Finally, Lys102 (third helix) and Lys199 (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation.