Cytochrome P450 3A4-Mediated Bioactivation and Its Role in Nomilin-Induced Hepatotoxicity.

Nomilin is a furan-containing triterpenoid isolated from the medicinal plants of citrus. The aim of this study was to investigate the in vitro and in vivo bioactivation of nomilin and the role in nomilin-induced hepatotoxicity. Microsomal incubations of nomilin supplemented with NADPH and GSH or NAL resulted in the detection of six conjugates (M1-M6). The structures of the metabolites were characterized based on LC-HRMS and NMR. Nomilin was bioactivated to a reactive cis-butene-dial (BDA) intermediate dependent on NADPH, and this intermediate suffered from the reaction with the nucleophiles (GSH and NAL) to form stable adducts. M1-M4 were identified as pyrrole derivatives, and M5 and M6 were pyrrolinone derivatives. M1 was further chemically synthesized and characterized by 13C NMR spectroscopy. M1 was the major metabolite detected in mice bile. Pretreatment with ketoconazole significantly reduced the formation of M1 in mice bile, while pretreatment with rifampicin significantly increased the formation of M1. Chemical inhibition together with recombinant human CYP450 phenotyping demonstrated that CYP3A4 was the major enzyme contributing to the bioactivation of nomilin. Toxicity study suggested that nomilin displayed dose-dependent liver injury in mice, while tetrahydro-nomilin was found to be nonhepatotoxic. Pretreatment with ketoconazole prevented mice from nomilin-induced liver injury. The liver injury induced by nomilin was deteriorated when the mice were pretreated with rifampicin. These findings provide evidence that CYP3A4-mediated bioactivation was indispensable in nomilin-induced hepatotoxicity.

Nomilin protects against cerebral ischemia-reperfusion induced neurological deficits and blood-brain barrier disruption via the Nrf2 pathway.

Oxidative stress is considered to play an important role in the cerebral ischemia-reperfusion injury. The nuclear transcription factor erythroid-2-related factor 2 (Nrf2)/NAD(P)H dehydrogenase [quinone] 1 (NQO1) pathway has been considered as a potential target for neuroprotection in cerebral ischemia-reperfusion injury. Nomilin (NOM) is a limonoid compound obtained from the extracts of citrus fruits. The purpose of our study was to determine whether NOM could exert beneficial effects in cerebral ischemia-reperfusion rats. Firstly, NOM treatment significantly mitigated cell death and decreased lactate dehydrogenase (LDH) release and ROS production in SH-SY5Y cells induced by oxygen-glucose deprivation (OGD), which was almost abolished by Nrf2 knockdown. Secondly, NOM improved infarct area, brain edema and neurological deficits in an experimental stroke rat model via middle cerebral artery occlusion (MCAO). Furthermore, NOM attenuated blood-brain barrier (BBB) disruption in MCAO rats, which might be associated with alleviating the loss of tight junction proteins, including ZO-1 and occludin-5. Further results revealed that NOM treatment effectively mitigated oxidative stress and facilitated the expressions of Nrf2 and NQO1, which might confirm that the loss of tight junction proteins in the microvasculature was likely mediated by oxidative stress. In conclusion, our study provided evidence that the protective effects of NOM in cerebral ischemia-reperfusion rats were related to the Nrf2/NQO1 pathway.

Metabolic side effects induced by olanzapine treatment are neutralized by CB1 receptor antagonist compounds co-administration in female rats.

Weight gain is an important side effect of most atypical antipsychotic drugs such as olanzapine. Moreover, although many animal models with metabolic side effects have been well defined, the interaction with other pathways has to be considered. The endocannabinoid system and the CB1 receptor (CB1R) are among the most promising central and peripheral targets involved in weight and energy balance. In this study we developed a rat model based 15-days treatment with olanzapine that shows weight gain and an alteration of the blood parameters involved in the regulation of energy balance and glucose metabolism. Consequently, we analysed whether, and by which mechanism, a co-treatment with the novel CB1R neutral antagonist NESS06SM, could attenuate the adverse metabolic effects of olanzapine compared to the reference CB1R inverse agonist rimonabant. Our results showed alterations of the cannabinoid markers in the nucleus accumbens and of orexigenic/anorexigenic markers in the hypothalamus of female rats treated with olanzapine. These molecular modifications could explain the excessive food intake and the resulting weight gain. Moreover, we confirmed that a co-treatment with CB1R antagonist/inverse agonist compounds decreased food intake and weight increment and restored all blood parameters, without altering the positive effects of olanzapine on behaviour. Furthermore, rimonabant and NESS06SM restored the metabolic enzymes in the liver and fat tissue altered by olanzapine. Therefore, CB1 receptor antagonist/inverse agonist compounds could be good candidate agents for the treatment of weight gain induced by olanzapine.

Metabolism, Excretion and Pharmacokinetics of MLN3897, a CCR1 Antagonist, in Humans.

UNLABELLED: MLN3897 is a small molecule antagonist of the C-C chemokine receptor-1. Since preclinical studies showed that the molecule was metabolized into two halves, the metabolism, excretion, and pharmacokinetics of MLN3897 were investigated in humans using MLN3897 14C-radiolabeled either on the chlorophenyl (CP) or the tricyclic (TC) half of MLN3897 after an oral dose. OBJECTIVE: To evaluate the mass balance, metabolism and pharmacokinetics of MLN3897 in two cohorts of six randomized healthy subjects. METHOD: After receiving informed consent, subjects were dosed after an overnight fast of 10-hours followed by at least 4- hours after dosing on day-1. Each cohort received a single 29 mg oral dose of either the CP or the TC as an oral solution in water. Serial blood samples, urine and feces were collected over a 10-day period post-dose. RESULTS: For both radiolabeled moieties, 55-59% of the dose was recovered in feces and 32% recovered in urine. MLN3897 was metabolized extensively in humans, with minor amounts of intact MLN3897 detected in the urine and feces. N-oxidation of the tricyclic moiety (M28) and N-dealkylation of the piperidinyl moiety were the primary metabolic pathways leading to further formation of the carboxylic acid metabolite (M19) and the (4-(4-chlorophenyl)-3,3- dimethylpiperidin-4-ol) metabolite (M40). Oxidative metabolites M11, M19, M28, M44 were present at >10% of the total circulating radioactivity and also at >25% of MLN3897 exposure. Metabolites resulting from the chlorophenyl-labeled moiety (M40) had significantly more systemic exposure compared to the tricyclic-labeled moiety (M19).

Dietary obacunone supplementation stimulates muscle hypertrophy, and suppresses hyperglycemia and obesity through the TGR5 and PPARγ pathway.

Obacunone is a limonoid that is predominantly found in Citrus. Although various biological activities of limonoids have been reported, little is known about the beneficial effects of obacunone on metabolic disorders. In the present study, we examined the effects of dietary obacunone supplementation on obese KKAy mice, to clarify the function of obacunone in metabolic regulation. Mice were pair-fed a normal diet either alone or supplemented with 0.1% w/w obacunone for 28 days. Compared with the control, obacunone-fed mice had lower glycosylated hemoglobin, blood glucose, and white adipose tissue weight, although there was no significant difference in body weight. Obacunone treatment also significantly increased the weight of the gastrocnemius and quadriceps muscles. Reporter gene assays revealed that obacunone stimulated the transcriptional activity of the bile acids-specific G protein-coupled receptor, TGR5, in a dose-dependent manner. In addition, obacunone inhibited adipocyte differentiation in 3T3-L1 cells and antagonized ligand-stimulated peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity. These results suggest that obacunone stimulates muscle hypertrophy and prevents obesity and hyperglycemia, and that these beneficial effects are likely to be mediated through the activation of TGR5 and inhibition of PPARγ transcriptional activity.

Obacunone exhibits anti-proliferative and anti-aromatase activity in vitro by inhibiting the p38 MAPK signaling pathway in MCF-7 human breast adenocarcinoma cells.

Overexpression of the aromatase enzyme CYP19 has been implicated in the onset of estrogen-dependent breast carcinogenesis. Obacunone, a natural compound present in citrus fruits, has been demonstrated for various biological activities including anti-cancer and anti-inflammatory properties. In the present study, we have isolated obacunone and obacunone glucoside (OG) from lemon seeds, then fractionated these compounds using chromatographic techniques and characterized them by HPLC, LC-MS, and 2D NMR spectral analysis. To investigate the mechanism of anti-cancer and anti-aromatase activities of limonoids, their cytotoxic effect was tested on human breast cancer (MCF-7) and non-malignant (MCF-12F) breast cells. MTT assays confirmed that obacunone was strongly inhibited MCF-7 cell proliferation without affecting non-malignant breast cells. Treatment with obacunone increased apoptosis by up-regulating expression of the pro-apoptotic protein Bax and down-regulating the anti-apoptotic protein Bcl2, as well as inducing G1 cell cycle arrest. In addition, obacunone significantly inhibited aromatase activity in an in vitro enzyme assay. Exposure of MCF-7 breast cancer cells to obacunone down-regulated expression of inflammatory molecules including nuclear factor-kappa B (NF-κB) and cyclooxygenase-2 (COX-2). Furthermore, we found that obacunone inhibited COX-2 and NF-κB by activation of the p38 mitogen-activated protein kinase (MAPK). Finally, the uptake level of obacunone into MCF-7 cells was measured by HPLC and its structure was confirmed by LC-HR-MS. This study demonstrated that obacunone may have the potential to prevent estrogen-responsive breast cancer through inhibition of the aromatase enzyme and inflammatory pathways, as well as activation of apoptosis.

Simultaneous determination of limonin, dictamnine, obacunone and fraxinellone in rat plasma by a validated UHPLC-MS/MS and its application to a pharmacokinetic study after oral administration of Cortex Dictamni extract.

A rapid and selective ultra high performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of four major ingredients in Cortex Dictamni extract, including limonin, dictamnine, obacunone and fraxinellone in rats plasma. Nimodipine was used as the internal standard. Following extraction by methyl tert-butyl ether, the analytes were separated on a Thermo Syncronis C18 column by a gradient elution within a runtime of 9min. The mobile phase consisted of A (methanol) and B (2mmol/L ammonium acetate in water). The detection was accomplished by using positive ion electrospray ionization in multiple reaction monitoring mode. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.998. The lower limits of quantification were 9.18ng/mL for limonin, 12.0ng/mL for dictamnine, 16.05ng/mL for obacunone and 4.59ng/mL for fraxinellone. The intra- and inter-day precision (RSD%) was within 10% and the accuracy (RE%) ranged from -12.9% to 9.7%. This rapid and sensitive method was fully validated and successfully applied to the pharmacokinetic study of limonin, dictamnine, obacunone and fraxinellone in the rat plasma after oral administration of Cortex Dictamni extract.

Anti-obesity and anti-hyperglycemic effects of the dietary citrus limonoid nomilin in mice fed a high-fat diet.

TGR5 is a member of the G protein-coupled receptor family and is activated by bile acids (BAs). TGR5 is thought to be a promising drug target for metabolic diseases because the activation of TGR5 prevents obesity and hyperglycemia in mice fed a high-fat diet (HFD). In the present study, we identified a naturally occurring limonoid, nomilin, as an activator of TGR5. Unlike BAs, nomilin did not exhibit the farnesoid X receptor ligand activity. Although the nomilin derivative obacunone was capable of activating TGR5, limonin (the most abundant limonoid in citrus seeds) was not a TGR5 activator. When male C57BL/6J mice fed a HFD for 9 weeks were further fed a HFD either alone or supplemented with 0.2%w/w nomilin for 77 days, nomilin-treated mice had lower body weight, serum glucose, serum insulin, and enhanced glucose tolerance. Our results suggest a novel biological function of nomilin as an agent having anti-obesity and anti-hyperglycemic effects that are likely to be mediated through the activation of TGR5.

Apoptosis mediated cytotoxicity of citrus obacunone in human pancreatic cancer cells.

Obacunone is one of the oxygenated triterpenoids found in rutaceae family. It has been demonstrated for various biological activities including inhibition of cancer cells proliferation and tumor. The current study is an attempt to understand the mode of cytotoxicity of obacunone using cultured pancreatic cancer (MDA Panc-28) cells. Obacunone exhibited dose and time dependant inhibition of Panc-28 cells proliferation . This was also associated with phosphatidylserine translocation from inner surface of plasma membrane to outer surface in the treated cells, suggesting the involvement of apoptosis. Analysis of cells treated with 50 and 100 µM of obacunone suggests activation of initiator caspase-9 and effector caspase-3, indicating involvement of caspases induced apoptosis. Obacunone upregulated expression of tumor suppressor protein p53, pro-apoptotic protein Bax and downregulated anti-apoptotic protein Bcl2. Furthermore, release of cytochrome-c into cytosol was observed in the cells treated with obacunone. It also resulted in down regulation of vital inflammatory mediators such as NFκB and Cox-2 suggesting the anti-inflammatory potential. For the first time, current study provides an evidence on activation of caspase dependant, cytochrome-c mediated intrinsic apoptosis and anti-inflammatory activity in Panc-28 cells by citrus obacunone. Induction of cytotoxicity and apoptosis was also confirmed by fluorescent tagged microscopic images of obacunone treated Panc-28 cells.

Chemo- and stereoselective synthesis of benzocycloheptene and 1-benzoxepin derivatives as alpha-sympathomimetic and anorexigenic agents.

The synthesis and pharmacological evaluation of cis- and trans-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ols 4a-c and 5a-c and cis- and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ols 4d-f and 5d-f were carried out. Chemo- and stereoselective synthesis of 5a-f was achieved by reduction of corresponding alpha-amino ketones 3a-f with LiAl(t-BuO)3H. cis-4-Amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 4d and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 5d exhibited marked anorexigenic activity in mice at a dose of LD50 800 and 500 mg/kg and ED50 75 and 55 mg/kg, respectively, while the analog cis-2,3-dihydroxy-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol 8 showed typical alpha-sympathomimetic activity.

Effect of limonin and nomilin on HIV-1 replication on infected human mononuclear cells.

In the last years several plant-derived natural compounds have been screened for their anti-HIV activity in order to find lead compounds with novel structures or mechanisms of action. Among these, several triterpenoids have been found to exhibit an antiretroviral activity with different mechanisms of action. In this study the effect of two limonoids, limonin and nomilin, on the growth of human immunodeficiency virus-1 (HIV-1) in culture of human peripheral blood mononuclear cells (PBMC) and on monocytes/macrophages (M/M) is described. Limonin and nomilin were found to inhibit the HIV-1 replication in all cellular systems used. A dose-dependent inhibition of viral replication was observed in PBMC isolated from healthy donors and infected with HIV-1 strain after incubation with limonin and nomilin (EC (50) values: 60.0 microM and 52.2 microM, respectively). The two terpenoids inhibited at all concentrations studied the production of HIV-p24 antigen even when the PBMC employed were chronically infected (EC (50) values of 61.0 microM for limonin and 76.2 microM for nomilin). Moreover, these compounds inhibited the HIV-1 replication even in infected M/M. In this cellular system the inhibitory effect was significant at the concentrations of 20 microM, 40 microM and 80 microM starting from day 14 and reached the maximum effect after 18 days of incubation. As regards the mechanism of action, limonin and nomilin inhibit in vitro HIV-1 protease activity. In general, the results obtained point out a similar anti-HIV activity of limonin and nomilin indicating that this activity is not drastically influenced by the structural difference between the two compounds.

Effect of naturally occurring triterpenoids glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin on the immune system.

The effect of naturally occurring triterpenoid compounds such as glycyrrhizic acid, ursolic acid, oleanolic acid, and nomilin on the immune system was studied using Balb/c mice. Intraperitoneal treatments with 5 doses of these terpenoid compounds were found to enhance the total white blood cells (WBC) count. In ursolic acid, oleanolic acid and nomilin treated animals the maximum total WBC count was observed on the 6th day, while in glycyrrhizic acid treated animals it was observed only on the 9th day after the drug treatment. In ursolic acid, oleanolic acid and nomilin treated animals the percentage of increase in the total WBC count was to 91.48 +/- 4.6%, 135.75 +/- 6.4% and 117.33 +/- 17.9% respectively. In the glycyrrhizic acid treated animals the total WBC count was increased to 114.9 +/- 18%. Bone marrow cellularity and alpha-esterase positive cells were also enhanced by the treatment with these terpenoids. Treatment with various triterpenoids along with antigen produced an enhancement in the specific antibody titre and the number of plaque forming cells (PFC) in the spleen. Triterpenoids remarkably inhibited delayed type hypersensitivity reaction (DTH). These results indicate the immunomodulatory activity of naturally occurring triterpenoids such as glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin.

Studies on the novel antiallergic agent HSR-609: its penetration into the central nervous system in mice and guinea pigs and its selectivity for the histamine H1-receptor.

We studied the pharmacological characteristics of HSR-609 (3-[4-(8-fluoro-5,11-dihydrobenz[b]oxepino[4,3-b]pyridin-11- ylidene)- piperidino]propionic acid dihydrate), a novel amphoteric antiallergic agent, on the central nervous system (CNS). Its selectivity for the histamine H1-receptor and its ability to penetrate into the CNS were compared with those of typical antiallergic agents and the nonamphoteric basic compound PY-608 (8-fluoro-5,11-dihydro-11-(1-methyl-4-piperidylidene)benz[b]oxe pino- [4,3-b]pyridine), which has a chemical structure similar to that of HSR-609. In the in vitro study, HSR-609 had a high affinity for H1-receptors in the guinea pig cerebral cortex in comparison to affinities for muscarinic and serotonin 5-HT2-receptors in the rat cerebral cortex, while the selectivity of PY-608 for the H1-receptor was low. The inhibitory effects of these antiallergic agents on histamine-induced increase of vascular permeability in mice (ED50) were compared with the displacement of [3H]mepyramine binding to H1-receptors in mouse brain ex vivo (ID50). The ID50/ED50 ratio of HSR-609 was much larger than those of cyproheptadine, ketotifen and PY-608 and larger than those of terfenadine and cetirizine. HSR-609 was found to display selective displacement of the [3H]mepyramine binding to H1-receptors for lung vs cerebral cortex as found with terfenadine in guinea pigs ex vivo. These findings suggest that HSR-609 has high selectivity for the H1-receptor and poor ability to penetrate into the CNS in mice and guinea pigs due to its amphoteric chemical structure.

[Effect of 5-[2-(diethylamino)ethyl]amino-5,11- dihydro[1]benzoxepino[3,4-b]pyridine trihydrochloride (KW-5805) on the biosynthesis of rat gastric mucus].

The effect of 5-[2-(diethylamino)ethyl]amino-5,11- dihydro[1]benzoxepino[3,4-b]pyridine trihydrochloride (KW-5805) on the activity of an enzyme regulating gastric mucous aminosugar metabolism was studied in rats. Restraint and water-immersion stress decreased the gastric mucosal glucosamine synthetase activity and induced gastric ulcers. The decrease of the enzyme activity reached a maximum at 1 hr and then returned to the non-stressed value. Pretreatment with KW-5805 at oral doses of 3 and 30 mg/kg inhibited the decrease of the enzyme activity and the gastric adherent mucus content and gastric ulceration induced by the stress. KW-5805 (30 mg/kg, p.o.) increased the enzyme activity in normal rats and also prevented the decrease of gastric mucosal N-acethylglucosamine kinase activity in the stressed rats. KW-5805 stimulated the activities of gastric mucosal. UDP-N-acethylglucosamine 4-epimerase and UDP-galactosyltransferase in in vitro. These results suggested that the antiulcerogenic effect of KW-5805 might be due to the activation of an enzyme regulating the biosynthesis of gastric mucus resulting in the reinforcement of gastric mucosal defensive mechanisms.

Protective effect of KW-3635, a specific thromboxane A2-receptor antagonist, on experimental glomerulonephritis in mice.

We studied the effect of KW-3635, a selective thromboxane A2 (TXA2)-receptor antagonist, on experimental glomerulonephritis. The glomerulonephritis was induced in mice by the administration of rabbit anti-mouse glomerular basement membrane (GBM) antibody. It was characterized as proteinuria, changes of serum biochemical parameters and glomerular histopathological abnormalities. The administration of KW-3635 (30 mg/kg/day, p.o.) significantly ameliorated the proteinuria, elevation of serum urea nitrogen and the thickening of GBM. These data suggest that TXA2 may play an important pathogenic role in the development and progression of glomerulonephritis.

Antithrombotic effects of KW-3635, a thromboxane A2-receptor antagonist, in guinea pigs.

Antithrombotic effects of KW-3635, a newly synthesized thromboxane (TX) A2-receptor antagonist, were studied in guinea pigs. In the extracorporeal circulation thrombosis model, the shunt was filled with thrombi, and reduction of platelet count and increase in plasma TXA2 concentration were observed. KW-3635 (30 and 100 mg/kg, p.o.) inhibited the thrombus formation in the shunt and prevented the decrease in platelet count in the circulating blood without affecting the red blood cell count. BM13,505 (30, 100 mg/kg, p.o.), another TXA2-receptor antagonist, and ticlopidine (300 mg/kg, p.o.), an antiplatelet drug, also inhibited the thrombus formation, while aspirin (10, 300 mg/kg, p.o.) did not. Peripheral arterial occlusive disease was induced by injection of sodium laurate into the femoral artery in guinea pigs. Daily oral administration of KW-3635 (3-30 mg/kg) significantly prevented the progression of vascular lesions. BM13,505 (3-30 mg/kg, p.o.) and ticlopidine (100 mg/kg, p.o.) also ameliorated the vascular lesions, whereas aspirin (10, 100 mg/kg, p.o.) did not. KW-3635 at concentrations up to 10(-4) M did not affect coagulation parameters in vitro. These results suggest that TXA2 is involved in the pathogenesis of arterial thrombotic and ischemic disorders. KW-3635 may be useful for the treatment of thrombotic disease and peripheral arterial occlusive diseases.

Effects of KW-3635, a specific thromboxane A2-receptor antagonist, on the development of lupus nephritis in NZB x NZW F1 mice.

We examined the effect of KW-3635, a specific thromboxane A2 (TXA2)-receptor antagonist, on the development of lupus nephritis in NZB x NZW F1 mice. KW-3635 was orally given once a day for 33 weeks beginning at eight weeks of age. In the control group, the mice began to die at 39 weeks of age, showing severe proteinuria and histopathologic abnormality in the renal glomeruli. Administration of KW-3635 (30 mg/kg/day) significantly reduced urinary protein excretion (1.7 +/- 0.9 vs. 8.5 +/- 2.4 mg/6 hr/mouse, P < 0.01), mortality (1/18 vs. 6/19, P < 0.05) and the histopathologic score of the kidney examined at 41 weeks of age. Thus, chronic administration of KW-3635 markedly attenuated the renal disease in NZB x NZW F1 mice, suggesting that TXA2 is an important mediator of the pathogenesis in this murine model of lupus nephritis.

Inhibitory effect of KW-3635, a new thromboxane A2-receptor antagonist, on arterial thrombosis in guinea pigs.

The antithrombotic effects of the thromboxane (TX) A2-receptor antagonist and aspirin were determined using a photochemically-induced arterial thrombosis model in the femoral arteries of guinea pigs. KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]-6,11- dihydrodibenz[b,e]-oxepine-2-carboxylate monohydrate) and BM-13505, both of which are TXA2-receptor antagonists, and aspirin inhibited the thrombus formation at the doses that inhibited the ex vivo platelet aggregation induced by sodium arachidonate (100 microM) or collagen (3 micrograms/ml). These results support the notion that TXA2 is an important mediator in the present model of arterial thrombogenesis.

[Effects of KW-3635 on the diuretic action of furosemide in rats].

The effects of KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl) ethylidene]-6,11-dihydrodibenz-[b,e]oxepine-2-carboxylate monohydrate, CAS 127166-41-0), a novel thromboxane A2 (TxA2) receptor antagonist, on furosemide diuresis were examined in rats. After an overnight fast, the rats received either saline or drugs, and 6-hr urine samples were collected. Urine volume, urinary excretion of electrolytes and urine TxB2 were measured. The administration of KW-3635 (10, 30 mg/kg, p.o.) or BM-13505 (10, 30 mg/kg) significantly increased the diuretic effect of furosemide (10 mg/kg, p.o.). Neither drug had any effect on urinary excretion of TxB2. These results demonstrated that the diuretic effect of furosemide was potentiated by TxA2 receptor blockade with KW-3635 or BM-13505. It is suggested that the diuretic effect of furosemide might be modulated by renal production of TxA2 elicited by this drug.