S35972, a direct-acting thrombin inhibitor with high oral bioavailability and antithrombotic efficacy.

OBJECTIVES: Dabigatran etexilate is the first oral thrombin inhibitor to demonstrate superior efficacy to warfarin for stroke prevention in patients with atrial fibrillation. This study describes the in vitro, ex vivo anticoagulant and in vivo antithrombotic effects of an oral thrombin inhibitor, S35972, in comparison with dabigatran etexilate. METHODS: Enzyme assays with thrombin and related serine proteases were performed. Clotting times, including activated partial thromboplastin time (APTT) and thrombin time (TT), were measured in vitro in different species and ex vivo in dogs and rats to determine pharmacologic bioavailabilities. The formation of occlusive venous and arterial thrombi in the rat vena cava and aorta was induced with stasis plus thromboplastin or ferrous chloride, respectively. RESULTS: S35972 inhibited human thrombin with an IC(50) of 3.7 nm, and did not inhibit other serine proteases. The anticoagulant activities of S35972 in vitro were comparable in dog and human plasmas, and the sensitivity of the clotting times to S35972 was TT > APTT > prothrombin time. In the fasted dog, oral administration of 3 mg kg(-1) S35972 increased TT rapidly and for at least 8 h, and its pharmacologic bioavailability was 75.4% ± 0.1%. In the rat venous thrombosis model, 3 mg kg(-1) oral S35972 or dabigatran etexilate significantly decreased the thrombus weight. In the rat aortic thrombosis model, oral S35972 at 10mg kg(-1) significantly decreased thrombus weight, by approximately 50%, whereas, at this dose, no effect was obtained with dabigatran etexilate. CONCLUSIONS: S35972 is a non-prodrug thrombin inhibitor with high selectivity, oral bioavailability, and antithrombotic efficacy.

Psychotropic profile of S 17092, a prolyl endopeptidase inhibitor, using quantitative EEG in young healthy volunteers.

The central activity of S 17092, a prolyl endopeptidase (PEP) inhibitor, was investigated by quantitative electroencephalography (qEEG) in 48 young healthy men participating in a double-blind, randomized, placebo-controlled, cross-over study. S 17092 (100, 200, 400 or 600 mg) and placebo were administered once daily for 10 days in a rising multiple-dose scheme. EEG recordings were performed before and repeatedly from 0.5 to 24 h after dose on day 1 and day 10. PEP activity in plasma was also measured for the same periods. S 17092 appeared as a potent inhibitor of PEP activity at all doses, after both single and repeated administrations. EEG changes after acute doses were slight and of short duration, mainly characterized by increased relative alpha 1 power, suggesting a vigilance-promoting EEG profile. After repeated doses and more strikingly after a superimposed dose, increases in relative alpha 1 power were still present with additional increase in relative delta power and decreases in absolute fast alpha, fast beta, theta powers and total power at all doses. These EEG findings suggest that S 17092 might possess some mood-stabilizing potential in addition to its cognition-enhancing properties.

Effects of ceramide on aggrecanase activity in rabbit articular cartilage.

Ceramide participates in signal transduction of IL-1 and TNF, two cytokines likely involved in cartilage degradation in osteoarthritis. We previously showed that ceramide stimulates proteoglycan degradation, mRNA expression of matrix metalloproteinase (MMP)-1, -3, and -13, and pro-MMP-3 production in rabbit cartilage. Since aggrecan, the main cartilage proteoglycan, can be cleaved by metalloproteinases both of MMP and aggrecanase type, the aim of this study was to determine if ceramide stimulates aggrecanase action and, if that is the case, in which measure aggrecanase mediates the degradative effect of ceramide. To this end, antibodies were used against the C terminal aggrecan neoepitopes generated by aggrecanases (NITEGE(373)) and MMPs (DIPEN(341)). Ceramide C(2) at 10(-5) to 10(-4) M dose-dependently increased NITEGE signal, without changing that of DIPEN, in cultured explants of rabbit cartilage. The effects of 10(-4) M C(2) on NITEGE signal and proteoglycan degradation were similarly antagonized by the metalloproteinase inhibitor batimastat, with return to the basal level at 10(-6) M. These results show that, similarly to IL-1 and TNF, ceramide-induced aggrecan degradation is mainly due to aggrecanases. That no increase of MMP activity was detected, despite stimulation of MMP expression, was probably due to lack of proenzyme conversion to mature form, since addition of a MMP activator to C(2)-treated cartilage increased both DIPEN signal and proteoglycan degradation. These findings support the hypothesis that cytokine-induced ceramide could play a mediatory role in situations of increased degradation of cartilage matrix.

Disease-modifying anti-osteoarthritic drugs: current therapies and new prospects around protease inhibition.

Although osteoarthritis is commonly found in the elderly, the pathophysiological mechanisms of this degenerative disease are still poorly understood. Among the many factors leading to cartilage degradation, the proteolytic activity of a panel of enzymes seems to play a major role, leading to the cleavage of collagen and proteoglycans, the two main components of cartilagenous matrix. Aspartic, cysteine, serine and metalloproteases have been detected in or around the osteoarthritic articulation and their enzymatic activity is reviewed here. The cartilage-sparing properties of the respective inhibitors are listed, giving rise to the hypothesis that some of these compounds could be developed as chondroprotective agents.

Selective blockade of neurokinin (NK)(1) receptors facilitates the activity of adrenergic pathways projecting to frontal cortex and dorsal hippocampus in rats.

The selective NK(1) receptor antagonist, GR205,171 (2.5-40.0 mg/kg, i.p.), dose-dependently elevated dialysate levels of noradrenaline (NA), but not serotonin (5-HT), in the frontal cortex of freely moving rats. This action was exerted stereospecifically inasmuch as its less active isomer, GR226,206, was ineffective. In the dorsal hippocampus, GR205,171 (but not GR226,206) also significantly increased dialysate levels of NA, whereas levels of 5-HT were unaffected. Further, in anaesthetized rats, GR205,171 dose-dependently (1.0-4.0 mg/kg, i.v.) increased the firing rate of adrenergic perikarya in the locus coeruleus. In contrast, their activity was not modified by GR226,206. These findings indicate that selective blockade of NK(1) receptors enhances the activity of ascending adrenergic pathways in rats. Adrenergic mechanisms may, thus, be involved in the potential antidepressant and other functional properties of NK(1) receptor antagonists.

Pharmacodynamic and pharmacokinetic profile of S 17092, a new orally active prolyl endopeptidase inhibitor, in elderly healthy volunteers. A phase I study.

AIMS: The aim of this study was to characterize the pharmacodynamics and the pharmacokinetics of S 17092, a new orally active prolyl endopeptidase inhibitor following single and repeated administration in elderly healthy volunteers. METHODS: This was a double-blind, randomized, placebo-controlled, single and multiple dose study in elderly healthy male and female volunteers (n = 36). Four doses were investigated in sequential order: 100, 400, 800 and 1200 mg. Each dose was administered orally once a day in single administration and then, after a 1 week washout period, during 7 days. Pharmacodynamics were assessed by measurement of plasmatic prolyl endopeptidase (PEP) activity, quantitative electroencephalogram (EEG) and psychometric tests. S 17092 concentrations in plasma were quantified by high performance liquid chromatography with tandem mass spectrometric detection. RESULTS: PEP activity in plasma was dose-dependently inhibited both after administration of a single dose and after repeated doses of S 17092. The mean maximal inhibition was obtained within 0.5-2 h after dosing, while inhibition lasted at least 12 h after dose administration. S 17092 appeared to be a centrally active substance as it induced statistically significant modifications in EEG compared with placebo. S 17092 at 100 mg exerted an acute increase in alpha band following single administration at 4 h and 8 h postdosing. When administered repeatedly over 7 days S 17092 did not appear to induce significant lasting central nervous system (CNS) effects. In psychometric tests, response times in the numeric working memory were significantly reduced compared with placebo, following the 800 mg dose. There were some beneficial residual effects of the 1200 mg dose on day 13: delayed word recall and word recognition sensitivity improved compared with the declines noted under placebo. Maximum measured concentration (Cmax) and area under the curve (AUC) parameters increased in proportion to the dose. The terminal half-life (t(1/2)) values ranged between 9 and 31 h on day 1 and between 7 and 18 h on day 14. A high interindividual variability was observed at all dose levels. S 17092 was well tolerated with no clinically significant changes in laboratory or physical parameters observed at any dose. CONCLUSIONS: S 17092 had a potent, dose-dependent inhibitory effect on plasmatic PEP, increased alpha band EEG at the 100 mg dose and improved performance in two verbal memory tests at the 1200 mg dose while there were disruption to the vigilance task. The results obtained in elderly healthy subjects indicated that S 17092 is suitable for once-daily dosing without any serious adverse events.

Further evidence for a dissociation between different forms of mnemonic expressions in a mouse model of age-related cognitive decline: effects of tacrine and S 17092, a novel prolyl endopeptidase inhibitor.

It has been demonstrated previously on the radial maze that the emergence of an age-related mnemonic impairment is critically dependent on the form which the discrimination problems took. Hence, when the arms were presented one by one (i.e., successive go-no-go discrimination), both adult and aged mice learned to distinguish between positive (baited) and negative (unbaited) arms readily, as evidenced by their increased readiness to enter positive relative to negative arms (i.e., by a differential in arm-entry latencies). A selective impairment in the aged mice was seen when these arms were presented subsequently as pairs, such that the mice were confronted with an explicit choice (i.e., simultaneous 2-choice discrimination). When discriminative performance was measured by the differential run speed between positive and negative arms, aged mice were also impaired. This was particularly pronounced in the 2-choice discrimination condition. We examined the effects of tacrine (3mg/kg, subcutaneously) or S 17092 (10mg/kg, orally) in aged mice on the three behavioral indices of this 2-stage spatial discrimination paradigm. The results indicated that: (1) Tacrine, but not S 17092, enhanced the acquisition of go-no-go discrimination as reflected in arm-entry latencies; (2) both drugs improved choice accuracy in simultaneous discrimination, although the effect of tacrine was less striking and, in particular, far from statistical significance in the very first 2-choice responses; and (3) neither drugs significantly affected run-speed performance. We conclude further that the specific patterns of drug effects on the three indices of discriminative performance might suggest that each index is associated with a distinct form of mnemonic expression relying on separate neural systems.

Effects of ceramide on apoptosis, proteoglycan degradation, and matrix metalloproteinase expression in rabbit articular cartilage.

Cartilage loss in osteoarthritis is characterized by matrix degradation and chondrocyte death. The lipid messenger ceramide is implicated in signal transduction of the catabolic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as in apoptosis. The aim of this study was to examine the in vitro effects of ceramide on proteoglycan degradation, matrix-metalloproteinase (MMP) expression and activity, and chondrocyte apoptosis in rabbit articular cartilage. Cell-permeant ceramide C(2) stimulated proteoglycan degradation in cartilage explants starting from 3 x 10(-5) M, with 100% increase at the dose of 10(-4) M. This effect was probably due to MMPs since it was blocked by the MMP inhibitor batimastat. Furthermore, in isolated chondrocytes, C(2) stimulated the expression of MMP-1, 3, and 13 at the mRNA level, MMP activity, and MMP-3 production. Ceramide also caused chondrocyte apoptosis at doses ranging from 10(-5) to 10(-4) M. This study supports the hypothesis that ceramide might play a mediatory role in both matrix degradation and apoptosis in processes of cartilage loss such as those observed in osteoarthritis.

S 17092-1, a highly potent, specific and cell permeant inhibitor of human proline endopeptidase.

Several lines of evidence indicate that proline endopeptidase (PE) could participate to the symptomatology and/or etiology of Alzheimer's disease. Thus, proline endopeptidase appears to contribute to the degradation of neuropeptides involved in learning and memory and could also control the production of the amyloidogenic peptide Abeta. Therefore the design of potent, selective and permeant inhibitors of human PE should lead to potential probes to assess the genuine contribution of this enzyme in Alzheimer's pathology. A novel perhydroindol carboxylic derivative, S17092-1 inhibits the hydrolysis of Z-Gly-Pro-7AMC-hydrolysing activity present in human brain nuclei with a high affinity (Ki = 1 nM) and behaves as a highly potent (Ki = 1.5 nM) inhibitor of partially purified human PE. By contrast, S17092-1 is unable to affect a series of other peptidases including aminopeptidases B and M, dipeptidylaminopeptidase IV, endopeptidases 3.4.24.11, 3.4.24.15, 3.4.24.16, calpains and angiotensin-converting enzyme. Furthermore, we show that the embryonic human kidney 293 cell line displays an intracellular PE-like activity that is blocked after preincubating cells with S17092-1, indicating that this inhibitor penetrates in HEK293 cells and could affect intracellular human PE. Altogether, we establish that S17092-1 behaves as a highly potent, specific and cell permeant inhibitor of human proline endopeptidase and can be seen as a probe to examine PE contribution in Alzheimer's disease.

S 18326: a thrombin inhibitor that shows potential for oral treatment of thromboembolic disorders.

Antivitamin K agents are currently the only orally available anticoagulant compounds. During the past two decades, important research has focused on the discovery of a direct, orally active thrombin inhibitor. In 1991, DuP 714, a boro arginine derivative, was shown to possess anticoagulant activity in different animal species after oral administration. S 18326, structurally related to DuP 714, is a further potent thrombin inhibitor. Moreover, its improved selectivity profile, associated with potent anticoagulant and antithrombotic properties, favours potential development of this compound for venous, as well as arterial, thromboembolism.

Selection of S18326 as a new potent and selective boronic acid direct thrombin inhibitor.

Using enzymatic microassays, the potency of a series of new boroarginine tripeptides was determined versus thrombin and a panel of serine-proteases implicated in the coagulation and fibrinolysis pathways. The inhibition of the serine-protease complement factor I was also studied. Factor I regulates the alternate pathway of the complement and its inhibition appears to be responsible for the toxic effects of the orally available thrombin inhibitor Ac-D-Phe-Pro-boroArg (DuP-714). The structure of the new boronic acid derivatives tested was modified from that of DuP-714 by replacing the proline in the P2 position by N-cycloalkyl-glycine residues of increasing size (S18989: cyclopropyl; S18563: cyclobutyl; S18326: cyclopentyl; S18229: cyclohexyl). All compounds were found to be slow-tight binding inhibitors of thrombin versus purified human fibrinogen. Replacement of proline by N-cycloalkyl-glycines did not decrease the anti-thrombin potency of the substances up to the cyclopentyl size and this result was confirmed by classical coagulation assays with human plasma in vitro. In contrast, the inhibitory activities of the four new boronic acids were found to be lower than those of DuP-714 versus plasmin, urokinase (u-PA), plasmatic kallikrein, activated protein C (aPC) and complement factor I. The cyclopentyl derivative S18326 is a slightly more active inhibitor of thrombin than DuP-714 (initial IC50 values 3.99 +/- 0.18 nM versus 4.73 +/- 0.27 nM, respectively). Moreover S18326 was identified as the most selective compound of the series with relative potencies being 2 to 29 fold higher than that of DuP-714 versus the panel of serine-proteases tested; the rank order of potency versus the other serine-proteases for S18326 was t-PA>kallikrein>aPC>factor I>plasmin>fXa>u-PA. These results indicate that the size of the thrombin hydrophobic pocket S2 is sufficient to accept larger residues than proline in the P2 position of Ac-D-Phe-X-boroArg derivatives while this is not the case for other important serine-proteases of the fibrinolysis, coagulation and complement pathways. The N-cyclopentyl glycine containing derivative S 18326, which is the most potent and the most selective anti-thrombin compound of the series, currently undergoes major preclinical testing.

Dual inhibition of human leukocyte elastase and lipid peroxidation: in vitro and in vivo activities of azabicyclo[2.2.2]octane and perhydroindole derivatives.

A series of potent and selective human leukocyte elastase (HLE) inhibitors of the Val-Pro-Val type has been developed. Initially, the central proline residue was replaced by nonnatural amino acids Phi ((2S,3aS,7aS)-perhydroindole-2-carboxylic acid) and Abo ((3S)-2-azabicyclo-[2.2.2]octane-3-carboxylic acid), and secondly several groups able to confer antioxidant properties to the molecule were introduced at the lipophilic N-terminal side chain. When compared to reference inhibitors, in vitro HLE inhibitory potency was maintained (10-100 nM) both with compounds containing the antioxidant moiety at the end of the N-terminal side chain and with compounds in which the N-terminal valine of the tripeptidic sequence had been replaced by a epsilon-substituted lysine. The lipidic peroxidation inhibitory potency of this series of inhibitors was found to be similar to that of the reference antioxidant compounds (around 1 microM). Moreover, HLE-induced hemorrhage in the hamster lung was effectively prevented (40-60% at 15 micrograms/kg) by most of the inhibitors tested when administered intratracheally 3 h before instillation of elastase. Among the most active analogs, compounds 11a,c,g were still active when administered 18 h before elastase. Interestingly, compound 14a was able to prevent HLE-mediated lung damage when administered 72 h prior to enzymatic challenge, indicating exceptional stability and retention in the lung. Finally, in a 14-day chronic model of emphysema in the hamster, 14a significantly conserved alveolar spaces, a marker of lung tissue destruction, and was more potent than reference inhibitor ICI 200 880. This indicates that addition of peroxidation inhibitory properties to an HLE inhibitor can provide a powerful in vivo inhibitor of pulmonary tissue destruction.

Activation of MMP-9 by neutrophil elastase in an in vivo model of acute lung injury.

The effect of neutrophil elastase on the functional status of gelatinases was studied in an hamster model developed by intratracheal administration of lipopolysaccharide followed by in situ cell activation with phorbol myristate acetate. This resulted in the production in bronchoalveolar lavage fluids, in addition to the matrix metalloproteinase MMP-9, of a 75 kDa gelatinase associated with collagenolytic activity. Treatment in vivo with an elastase inhibitor abolished the latter activity. Since, in addition, elastase activates in vitro purified MMP-9 gelatinase into a similar 75 kDa entity, these data suggest that elastase may be a physiological activator of MMP-9 in vivo.

New prolyl endopeptidase inhibitors: in vitro and in vivo activities of azabicyclo[2.2.2]octane, azabicyclo[2.2.1]heptane, and perhydroindole derivatives.

A series of potent and selective prolylendopeptidase (PEP) inhibitors of the alpha-keto heterocyclic type has been obtained by replacing the classical central proline of 1-[1-(4-phenylbutanoyl)-L-prolyl]pyrrolidine (SUAM 1221,3) by non-natural amino acids PHI, ABO, and ABH. These 4-phenylbutanoyl side-chain-containing inhibitors exhibited potent in vitro inhibitory potencies with IC50 around 30 nM (compounds 24 and 25). Modulation of the side chain by replacement of the terminal phenyl ring by the dicyclopropyl moiety afforded derivatives 30 and 32 with improved potencies (IC50 between 10 and 20 nM). Furthermore, replacing the linear 4-phenylbutanoyl side chain by the (2-phenylcyclopropyl)carbonyl entity provided potent inhibitors with IC50 culminating at 0.9 nM on a rat cortex enzymatic preparation (compound 70). The configuration of the cyclopropyl ring had to be R,R in order to obtain not only a strong PEP inhibition in vitro but also a good activity in vivo, exemplified by inhibitor 68, which gave ID50 ip and po of 0.3 and 1 mg/kg, respectively. Finally, demonstration of the cognition-enhancing properties of compound 54 was given in the passive avoidance test using scopolamine-induced amnesia in the rat, where it dose dependently inhibited the scopolamine-induced decrease in avoidance response.

Euglycaemic and biological activities of novel thiazolidine-2,4-dione derivatives.

A new series of thiazolidine-2,4-dione derivatives was obtained by incorporating one or the other of the two carbons of the central chain into different rings. These compounds lower blood glucose levels in the genetically obese and insulin-resistant ob/ob mouse. Moreover, they decreased insulin and triglyceride levels in the Zucker fa/fa rat. Incorporation of the left hand carbon of the chain afforded compounds among which pyrrolidino derivatives 5, 9 and 13 were the most potent. The same carbon atom was used to elaborate different types of rings (benzocyclobutane, benzodioxane), giving rise to compounds 14 and 19 with moderate to good activity. Finally, cyclization using the right hand carbon of the chain gave rise to highly potent benzofurane 24.

New tripeptidic thrombin inhibitors. Influence of P2 and P3 residues on activity and selectivity.

Structural variations of P2 and P3 residues in tripeptidic boroarginine thrombin inhibitors led to compounds with similar potency than reference compound DuP 714, but with enhanced selectivity for thrombin compared to plasmin.

A screening procedure to evaluate the anticoagulant activity and the kinetic behaviour of direct thrombin inhibitors.

The development of a fibrin clot microassay to define both the kinetic behaviour and the anticoagulant activity of direct thrombin inhibitors targeting various domains of thrombin (catalytic site, anion binding exosite or both) is described. Since classical kinetics studies are difficult to perform in a fibrin-clot assay, methodological conditions were selected in order to obtain a linear relationship between fibrin formation and the thrombin concentration i.e. 0.67 nM thrombin, 6 microM fibrinogen, 5 minutes reaction. Under those conditions, the concentration of the complex thrombin-inhibitor can easily be calculated from a standard curve performed with increasing concentrations of thrombin and fitted versus the total inhibitor concentration using adapted equations. To detect the slow establishment of the thrombin inhibition, results obtained with a protocol in which the inhibitor is pre-incubated with thrombin before the addition of fibrinogen is compared to a protocol in which the inhibitor is pre-incubated with fibrinogen before thrombin is added. Our assay which is validated using different types of thrombin inhibitors (classical competitive: NAPAP and hirudin 55-65; tight binding: r-hirudin; slow tight binding: DUP-714), provides a rapid screening protocol allowing to evaluate the biochemical and anticoagulant properties of any direct thrombin inhibitor.

S 17162 is a novel selective inhibitor of big ET-1 responses in the rat.

Endothelin-1 (ET-1) is a powerful renal vasoconstrictor peptide that may be implicated in acute renal failure. The aim of the present study was to test the effects of the novel endothelin-converting enzyme inhibitor S 17162 (N-(2,3 dihydroxy propyl phosphonyl)-(S)-Leu-(S)-Trp-OH, disodium salt) on pressor responses to ET-1 and its precursor, big ET-1, in isolated perfused rat kidneys and in pithed rats. In both models, phosphoramidon selectively inhibited the pressor responses to big ET-1 without influencing those to ET-1, angiotensins (AT-I and AT-II) or norepinephrine. S 17162 was active against big ET-1 in both test systems. It selectively inhibited the pressor responses to big ET-1 with ID50 values of 160 micrograms/kg/min (phosphoramidon: 120 micrograms/kg/min) in the spinal rat and 6 microM (phosphoramidon: 5 microM) in the perfused rat kidney. In the nonanesthetized rat, S 17162 at 20 mg/kg p.o. inhibited the pressor responses to big ET-1, demonstrating its oral bioavailability. Therefore, S 17162 is a potential candidate for development as an orally active anti-endothelin drug.

Biguanide derivatives: agonist pharmacology at 5-hydroxytryptamine type 3 receptors in vitro.

The effects of 24 biguanide and four guanidine derivatives on 5-hydroxytryptamine (5-HT)3 receptors in N1E-115 neuroblastoma cells were examined using radioligand binding and whole-cell voltage-clamp techniques. Displacement of the selective 5-HT3 receptor antagonist [3H]BRL 43694 by phenylbiguanide (PBG) derivatives revealed Ki values ranging from 3.4 x 10(-4) to 4.4 x 10(-10) M. The rank order of potency of agonists was 2,3,5-trichloro-PBG > 2,3-dichloro-PBG = 2,5-dichloro-PBG = 3,5-dichloro-PBG > 3,4-dichloro-PBG = 3-chloro-PBG > 2-chloro-PBG = 4-chloro-PBG = 2-methyl-PBG = 2,4-difluoro-PBG > PBG = 2-trifluoro-5-chloro-PBG > 4-fluoro-PBG = 3-trifluoromethyl-PBG > 4-nitro-PBG = 1,5-bis-4-chloro-PBG = 3,5-ditrifluoromethyl-PBG > 4-ethoxy-PBG >> 4-sulfonic acid-PBG. All of the benzylbiguanides and indanylbiguanide were inactive on [3H]BRL 43694 binding or displaced it only weakly. The four guanidine derivatives were quite inactive. In the PBG series, all antagonist competition curves were steep (pseudo-Hill coefficients ranging from 1.05 to 1.58), monophasic, and best fit with a one-site model. Among PBG derivatives, the chlorinated compounds exhibited a good degree of selectivity for 5-HT3 receptors versus other 5-HT receptor subtypes and other neurotransmitter binding sites. Electrophysiological studies showed that the PBG derivatives tested produced rapid inward currents, at a holding potential of -65 mV, that showed rapid desensitization. The current induced by the 2,3,5-trichloro-PBG derivative was inhibited by the specific 5-HT3 receptor antagonist ICS 205-930 but was unaffected by the 5-HT2 receptor antagonist ketanserin. Analysis of concentration-response curves for the PBG derivatives gave EC50 values ranging from 2.2 x 10(-5) to 2.7 x 10(-8) M and Hill slopes ranging from 1.02 to 2.10. The rank order of potency was similar to that obtained from the binding data, and a good correlation was found between Ki and EC50 values. It is concluded that the triple-chloro substitution yielded a compound that is 30-fold more potent than 3-chloro-PBG and approximately 10-fold more potent than dichloro-PBG derivatives, making 2,3,5-trichloro-PBG the most potent 5-HT3 agonist described thus far.

Phosphoramidon inhibits the conversion of big ET-1 into ET-1 in the pithed rat and in isolated perfused rat kidneys.

Endothelin-1 (ET-1) is a powerful renal vasoconstrictor peptide that could be implicated in acute renal failure. The aim of this study was to test the effects of the endothelin-converting enzyme (ECE) inhibitor phosphoramidon on pressor responses to ET-1 and its precursor, big ET-1, in isolated perfused rat kidneys and in pithed rats. In Tyrode-perfused rat kidneys, both big ET-1 (0.2-0.4 nmol) and ET-1 (0.01-0.03 nmol) evoked dose-dependent constrictions. Phosphoramidon (10 microM) selectively inhibited the pressor responses to big ET-1 without altering those to ET-1, norepinephrine, angiotensin I (AT-I), or angiotensin II (AT-II). The metalloprotease inhibitor thiorphan, but not the angiotensin-converting enzyme (ACE) inhibitor perindoprilate, also selectively inhibited the renal constrictions caused by big ET-1 but not those induced by ET-1. In vivo, both big ET-1 and ET-1 (0.5-2 nmol/kg) evoked pressor responses that were augmented by indomethacin (15 mg/kg) and L-NNA (1 mg/kg/min). Phosphoramidon selectively inhibited the pressor responses to big ET-1 (ID50: 78 micrograms/kg/min) without affecting those to ET-1, AT-I, or AT-II. These data illustrate that the pressor responses to big ET-1 in the rat, both in vivo and in vitro, are due to its conversion into ET-1 by a phosphoramidon-sensitive ECE. In the rat, phosphoramidon selectively inhibits ECE but not ACE both in vitro and in vivo.