Novel (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids: peroxisome proliferator-activated receptor γ selective agonists with protein-tyrosine phosphatase 1B inhibition.

A novel series of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and (S)-2-[(2E,4E)-hexadienoyl]-7-(2-{5-methyl-2-[(1E)-5-methylhexen-1-yl]oxazol-4-yl}ethoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (14i) was identified as a potent human peroxisome proliferator-activated receptor γ (PPARγ) selective agonist (EC(50)=0.03 µM) and human protein-tyrosine phosphatase 1B (PTP-1B) inhibitor (IC(50)=1.18 µM). C(max) after oral administration of 14i at 10mg/kg was 2.2 µg/ml (4.5 µM) in male SD rats. Repeated administration of 14i and rosiglitazone for 14 days dose-dependently decreased plasma glucose levels, ED(50)=4.3 and 23 mg/kg/day, respectively, in male KK-A(y) mice. In female SD rats, repeated administration of 14i at 12.5-100mg/kg/day for 28 days had no effect on the hematocrit value (Ht) and red blood cell count (RBC), while rosiglitazone significantly decreased them from 25mg/kg/day. In conclusion, 14i showed about a fivefold stronger hypoglycemic effect and fourfold or more weaker hemodilution effect than rosiglitazone, indicating that 14i is 20-fold or more safer than rosiglitazone. Compound 14i is a promising candidate for an efficacious and safe anti-diabetic drug targeting PPARγ and PTP-1B.

A novel series of (S)-2,7-substituted-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids: peroxisome proliferator-activated receptor α/γ dual agonists with protein-tyrosine phosphatase 1B inhibitory activity.

Novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and (S)-7-(2-{2-[(E)-2-cyclopentylvinyl]-5-methyloxazol-4-yl}ethoxy)-2-[(2E,4E)-hexadienoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (14c) was identified as a peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist. The transactivation activity of 14c was comparable to that of rosiglitazone in human PPARγ (EC50=0.14 µM) and was much higher than in human PPARα (EC50=0.20 µM). In addition, 14c, but not rosiglitazone, showed human protein-tyrosine phosphatase 1B (PTP-1B) inhibitory activity (IC50=1.85 µM). 14c showed about 10-fold stronger hypoglycemic and hypotriglyceridemic effects than rosiglitazone by repeated application for 14 d in male KK-Ay mice. Furthermore, 14c, but not rosiglitazone, increased hepatic peroxisome acyl CoA oxidase activity at 30 mg/kg/d for 7 d in male Syrian hamsters, probably due to its PPARα agonist activity. 14c did not affect plasma volume at 100 mg/kg/d for 14 d in male ICR mice, while rosiglitazone significantly increased it. In conclusion, 14c is a promising candidate for an efficacious and safe anti-diabetic drug with triple actions as a PPARα/γ dual agonist with PTP-1B inhibitory activity.

Computational design and selections for an engineered, thermostable terpene synthase.

Terpenoids include structurally diverse antibiotics, flavorings, and fragrances. Engineering terpene synthases for control over the synthesis of such compounds represents a long sought goal. We report computational design, selections, and assays of a thermostable mutant of tobacco 5-epi-aristolochene synthase (TEAS) for the catalysis of carbocation cyclization reactions at elevated temperatures. Selection for thermostability included proteolytic digestion followed by capture of intact proteins. Unlike the wild-type enzyme, the mutant TEAS retains enzymatic activity at 65°C. The thermostable terpene synthase variant denatures above 80°C, approximately twice the temperature of the wild-type enzyme.

Novel prodrugs of meropenem with two lipophilic promoieties: synthesis and pharmacokinetics.

To improve the oral absorption of meropenem (MEPM), we synthesized and evaluated a series of its double-promoiety prodrugs, in which lipophilic promoieties were introduced into carboxyl and pyrrolidinyl groups. Among these prodrugs, pivaloyloxymethyl (1R,5S,6S)-2-[(3S,5S)-5-(N,N-dimethylcarbamoyl)-1-(isobutyryloxymethyloxycarbonyl)pyrrolidin-3-ylthio]-6-[(1R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate (4) and 1-ethylpropyloxycarbonyloxymethyl (1R,5S,6S)-2-[(3S,5S)-5-(N,N-dimethylcarbamoyl)-1-(isobutyryloxymethyloxycarbonyl)pyrrolidin-3-ylthio]-6-[(1R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate (8) were chosen for further evaluation. Compounds 4 and 8 were well absorbed after oral administration to rats and beagles (bioavailability 18.2-38.4%), and expected to show potent therapeutic efficacy in patients infected with various pathogens, such as penicillin-resistant S. pneumoniae and ß-lactamase-negative ampicillin-resistant H. influenzae.

Novel acyl-CoA: cholesterol acyltransferase inhibitor: indoline-based sulfamide derivatives with low lipophilicity and protein binding ratio.

To find a novel acyl-CoA: cholesterol acyltransferase inhibitor, a series of sulfamide derivatives were synthesized and evaluated. Compound 1d, in which carboxymethyl moiety at the 5-position of Pactimibe was replaced by a sulfamoylamino group, showed 150-fold more potent anti-foam cell formation activity (IC(50): 0.02 microM), 1.6-fold higher log D(7.0) (4.63), and a slightly lower protein binding ratio (93.2%) than Pactimibe. Compound 1i, in which the octyl chain at the 1-position in 1d was replaced by an ethoxyethyl, showed markedly low log D(7.0) (1.73) and maintained 3-fold higher anti-foam cell formation activity (IC(50): 1.0 microM), than Pactimibe. The plasma protein binding ratio (PBR) of 1i was much lower than that of Pactimibe (62.5% vs. 98.1%), and its partition ratio to the rabbit atherosclerotic aorta after oral administration was higher than that of Pactimibe. Compound 1i at 10 microM markedly inhibited cholesterol esterification in atherosclerotic rabbit aortas even when incubated with serum, while Pactimibe had little effect probably due to its high PBR. In conclusion, compound 1i is expected to more efficiently inhibit the progression of atherosclerosis than Pactimibe.

Novel tetrahydroisoquinoline derivatives with inhibitory activities against acyl-CoA: cholesterol acyltransferase and lipid peroxidation.

To find a novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor with anti-lipid peroxidative activity, a series of tetrahydroisoquinoline derivatives were synthesized and evaluated. A compound with a N-(4-hydroxy-2,3,5-trimethylphenyl)carbamoyl moiety at the 3-position and an octanoyl moiety at the 2-position (7) was demonstrated to show anti-foam cell formation activity stronger than and anti-lipid peroxidative activity comparable to those of Pactimibe, while it was hardly absorbed orally. To increase its bioavailability, the acyl chain at the 2-position was shortened and various polar or basic moieties were introduced at the 7-position of 7. Among the synthesized derivatives, (S)-7-dimethylamino-N-(4-hydroxy-2,3,5-trimethylphenyl)-2-isobutyryl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide hydrochloride (21) showed about 16-fold stronger anti-foam cell formation activity, 3-fold stronger hepatic ACAT inhibitory activity, similar anti-low density lipoprotein (LDL) oxidative activity and 2-fold more potent protective activity against macrophage cell death by oxidative stress in comparison with Pactimibe. Compound 21 was efficiently absorbed after oral administration at 10 mg/kg in rats and dogs and its C(max) values were higher than its IC(50) values for in vitro activities. In conclusion, a tetrahydroisoquinoline structure is a useful scaffold for designing a phenolic anti-oxidative ACAT inhibitor, and compound 21 is expected to effectively prevent atherosclerosis.

Lipopolysaccharide-induced impairment of nitric oxide-mediated vasorelaxation and protective effects of nitric oxide synthesis inhibitors in isolated rat mesenteric arteries.

Isolated rat mesenteric arteries were incubated with lipopolysaccharide (LPS) for 6 h and then mounted in an organ bath to investigate their responses to various relaxants. Exposure to LPS moderately reduced acetylcholine (ACh)-induced endothelium-dependent relaxation (EDR), and markedly reduced sodium nitroprusside (SNP)-induced endothelium-independent relaxation (EIR). It did not affect ACh-induced EDR under treatment with a nitric oxide synthase (NOS) inhibitor, which is mediated by an endothelium-derived hyperpolarizing factor (EDHF), and forskolin-induced EIR. N-(3-(Aminomethyl)benzyl)acetamidine (1400 W), an inducible nitric oxide synthase (iNOS) inhibitor, actinomycin D, an RNA polymerase inhibitor, cycloheximide, a protein synthesis inhibitor, and dexamethazone reduced the nitric oxide (NO) production and reversed the reduced ACh-induced EDR and SNP-induced EIR. In LPS-treated mesenteric artery, L-arginine-induced relaxation was not affected by removal of endothelium, indicating muscular inducible nitric oxide synthase (iNOS) induction. Pre-exposure to SNP (NO donor) also moderately reduced ACh-induced EDR and markedly reduced SNP-induced EIR with little effect on ACh-induced EDHF-mediated EDR. In conclusion, in vitro exposure to LPS desensitized vascular smooth muscle cells to endogenous and exogenous NO by overproduction of muscular iNOS-derived NO, and an iNOS inhibitor and iNOS induction inhibitors prevented the LPS-induced desensitization.

Novel indoline-based acyl-CoA: cholesterol acyltransferase inhibitor: Effects of introducing a methanesulfonamide group on physicochemical properties and biological activities.

A novel series of indoline-based acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors with a methanesulfonamide group at the 5-position were synthesized and their lipophilicity and biological activities were evaluated. Hepatic ACAT inhibitory and anti-foam cell formation activity increased dependent on lipophilicity of derivatives with various alkyl chains at the 1-position. The logD(7.0)-biological activity curve of the derivatives with a methanesulfonamide group shifted leftward compared to that of Pactimibe derivatives with a carboxymethyl group, and derivatives with no substituent, suggesting that a methanesulfonamide group plays an important role in the interaction with ACAT protein. Among derivatives, N-(1-ethyl-5-methanesulfonylamino-4,6-dimethylindolin-7-yl)-2,2-dimethylpropanamide (1b) had about twofold lower logD(7.0) than Pactimibe, while it showed twofold higher hepatic ACAT inhibition than and the same anti-foam cell formation as Pactimibe, respectively. The C(max) of 1b (10mg/kg, po) was higher than that of Pactimibe in rats. The plasma protein binding ratio of 1b was lower than that of Pactimibe: 64.8% and 97.9%, respectively. Compound 1b showed greater inhibitory effects on hepatic cholesterol secretion in mice than Pactimibe. In conclusion, the introduction of a methanesulfonamide group is effective to design less lipophilic, more efficacious and more bioavailable indoline-based ACAT inhibitors than previous indoline-based inhibitors.

NK1 receptor-mediated endothelium-dependent relaxation and contraction with different sensitivity to post-receptor signaling in pulmonary arteries.

In rabbit intrapulmonary arteries, substance P (SP) has been reported to induce endothelium-dependent relaxation (EDR) and endothelium-dependent contraction (EDC) via tachykinin NK(1) receptors, and endothelium-independent contraction (EIC) via tachykinin NK(2) receptors. The present study pharmacologically examined whether these opposite responses (EDR and EDC) are mediated by the same NK(1) receptor. Five tachykinin agonists, including septide, a reportedly atypical NK(1) agonist, caused concentration-dependent EDR in the presence of NK2 antagonist (SR-48968) + TXA2 synthetase inhibitor (ozagrel), which blocked EIC and EDC, in pre-contracted arteries, and concentration-dependent EDC in the presence of NK2 antagonist (SR-48968) + nitric oxide synthase inhibitor (l-N(G)-nitro-arginine methyl ester), which blocked EIC and EDR, in non-contracted arteries. The EC(50) values of these agonists for EDR were smaller than those for EDC, indicating that the affinities of NK(1) agonists to NK(1) receptors are different between EDR and EDC. However, the rank order of their potency for EDR and EDC was the same: SP = septide > SP methyl ester (SPME) > neurokinin A > neurokinin B. [Ala(5), beta-Ala(8)]-alpha-neurokinin fragment 4-10 (NK2 agonist) and senktide (NK3 agonist) caused no responses. Two structurally different NK(1) antagonists, CP-99994 and SR-140333, shifted the concentration-EDC and -EDR curves of SPME, a selective NK(1) agonist, and septide rightward and suppressed their maximal responses in a similar concentration-dependent manner, indicating that the affinities of NK(1) antagonists to NK1 receptors are similar between EDR and EDC. U-73122, a phospholipase C inhibitor, and thapsigargin, 2,5-di-tert-butylhydroquinone, and ruthenium red, all intracellular Ca2+ release blockers, inhibited SP-induced EDR and EDC. Effective concentrations of ionomycin (Ca2+ ionophore) causing EDR were also lower than those causing EDC. Taken together, SP-induced EDR and EDC are mediated by activation of the same NK1 receptor followed by an increase in intracellular Ca2+, and sensitivity to Ca2+ may be higher in the EDR than EDC pathway.

Regional heterogeneity of substance P-induced endothelium-dependent contraction, relaxation, and -independent contraction in rabbit pulmonary arteries.

AIMS: The present study examined whether substance P (SP)-induced endothelium-dependent TXA(2)-mediated contraction (EDC), nitric oxide (NO)-mediated relaxation (EDR), and endothelium-independent contraction (EIC) are different between the rabbit proximal and distal intrapulmonary arteries. MAIN METHODS: The helically cut strips of isolated proximal and distal arteries were fixed vertically between hooks in organ bath, and changes in isometric tension were measured. KEY FINDINGS: SP-induced EDC was greater in the distal than proximal arteries, and EDR was greater in the proximal than distal arteries. However, under the complete blockade of NK(2) receptors and NO production, SP (10(-9)-3x10(-7) M)-induced EDC did not differ between proximal and distal arteries. Under the complete blockade of NK(2) receptors and TXA(2) production, SP (3x10(-10)-3x10(-8) M)-induced EDR was greater in the proximal than distal arteries. Neither contraction induced by U-46619, a TXA(2) agonist, nor relaxation by sodium nitroprusside, an NO donor, was different between both portions of the arteries. Both ionomycin (10(-8) M)- and l-arginine (1 mM)-induced EDRs were also significantly greater in the proximal than distal arteries. Under the blockade of NK(1) receptors and NO and TXA(2) production, SP (10(-7) M)-induced EIC was greater in the distal than proximal arteries. In summary, the capacity for NO production is higher in the proximal than distal arteries, resulting in SP-induced higher EDR and lower EDC in the proximal arteries. SIGNIFICANCE: These regional differences in responses to SP may play important roles in maintaining the homogenous distribution of blood flow in the lung.

Novel indoline-based acyl-CoA:cholesterol acyltransferase inhibitor with antiperoxidative activity: improvement of physicochemical properties and biological activities by introduction of carboxylic acid.

A series of novel indoline derivatives with an ionizable moiety were synthesized to find a bioavailable acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor with antiperoxidative activity. [7-(2,2-Dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate (2, pactimibe sulfate) with low lipophilicity and high water solubility showed good oral absorption and inhibitory activity against foam cell formation in THP-1 cells exposed to acetyl-LDL after differentiation (IC50: 0.3 microM) and an antiperoxidative effect in LDL of hypercholesterolemic rabbits (IC50: 1.0 microM). 2 inhibited macrophage, hepatic, and intestinal ACAT activity (IC50: 1.9, 0.7, and 0.7 microM, respectively). Maximal plasma concentration after oral administration of 2 at 10 mg/kg was 0.9 microg/mL in rats, 3.0 microg/mL in rabbits, and 11.2 microg/mL in dogs. Repeated administration of 2 lowered plasma LDL/VLDL cholesterol in hypercholesterolemic rabbits at 1 mg/kg/day, rats and dogs at 3 mg/kg/day, and in normocholesterolemic hamsters at 3 mg/kg/day. 2 is a promising candidate for antihyperlipidemic and antiatherosclerotic drugs.

Synthesis and biological evaluation of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids: a novel series of PPAR gamma agonists.

A novel series of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and biologically evaluated. (S)-2-Benzyl-7-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (10, KY-021) was identified as a novel peroxisome proliferators-activated receptor (PPAR) gamma agonist, which showed potent activity in human PPAR gamma (EC50=11.8 nM). KY-021 reduced plasma glucose and triglyceride levels at 3 mg/kg/d for 7 d in male KK-Ay mice. KY-021 also decreased plasma triglyceride levels at 0.3-3 mg/kg/d for 6 d, and improved oral glucose tolerance at 1 and 3 mg/kg/d for 7 d in male Zucker fatty rats. Maximal plasma concentration of KY-021 after oral administration at 10 mg/kg was 6.6 microg/ml and 2.1 microg/ml in male ICR mice and male SD rats, respectively. Repeated oral administration of KY-021 at 30 mg/kg/d for 10 weeks had little toxicity in male SD rats. These results demonstrated that KY-021 has great potential as an efficacious and safe drug for diabetes.

Impairment of endothelium-dependent ACh-induced relaxation in aorta of diabetic db/db mice--possible dysfunction of receptor and/or receptor-G protein coupling.

Diabetes is a risk factor of ischemic heart disease, cerebral ischemia, and atherosclerosis, in which endothelial dysfunction plays a role in the pathogenesis. We examined vascular responses in the aorta of pre-diabetic db/db mice with normoglycemia, hyperlipidemia, and hyperinsulinemia (6 weeks old), and diabetic db/db mice with hyperglycemia, hyperlipidemia, and hyperinsulinemia (11 weeks old) in comparison with age-matched non-diabetic db/+ mice. Prostaglandin F2alpha (PGF2alpha)-induced contraction was significantly enhanced in the aorta of diabetic but not pre-diabetic db/db mice compared to age-matched non-diabetic db/+ mice. Acetylcholine (ACh), adenosine-5'-diphosphate (ADP), NaF, a G protein activator and A-23187, a Ca-ionophore, caused endothelium-dependent and nitric oxide (NO)-mediated relaxation, and sodium nitroprusside (SNP), an NO donor, caused endothelium-independent relaxation in the pre-contracted aorta of db/db mice. Maximal endothelium-dependent ACh-induced relaxation was reduced in diabetic but not pre-diabetic db/db mice compared to age-matched db/+ mice, while maximal SNP-induced relaxation was not different between diabetic and non-diabetic mice. ACh-induced relaxation in diabetic db/db mice was not affected by ozagrel, a thromboxane A2 (TXA2) synthetase inhibitor, or acetylsalicylic acid (aspirin), a cyclooxygenase inhibitor, suggesting no involvement of endogenous TXA2 or prostanoids in the reduction of relaxation. Maximal endothelium-dependent ADP-, A-23187-, and NaF-induced relaxation was not reduced in diabetic db/db mice. EC50 values for ACh- and SNP-induced relaxation were increased in diabetic but not pre-diabetic db/db mice, suggesting decreases in sensitivity to NO in diabetic mice. Two-week treatment with KV-5070, a PPARgamma agonist, lowered plasma glucose, triglyceride (TG), and insulin but not cholesterol, and reversed the reduced ACh-induced relaxation. In conclusion, ACh-induced endothelium-dependent relaxation is impaired in diabetic db/db mice, probably due to the dysfunction of ACh receptors and/or receptor-G protein coupling. Endothelial dysfunction was not genetic and was considered to be initiated primarily by hyperglycemia, and was improved by anti-diabetic treatment with a PPARgamma agonist.

Relationships between lipophilicity and biological activities in a series of indoline-based anti-oxidative acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors.

A novel series of 1-alkyl-7-amido-indoline-based anti-oxidative acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors have been reported and are expected to lower plasma cholesterol levels due to the inhibition of intestinal and hepatic ACAT, and to inhibit cholesterol accumulation in macrophages due to the inhibition of low density lipoprotein (LDL) oxidation. In the present study, relationships between lipophilicity and biological activities were examined in 13 derivatives. Lipophilicity (logP) increased and water solubility decreased with dependence on the number of carbons in the 1-alkyl chain. Inhibitory activity against both in vitro intestinal ACAT and LDL oxidation positively correlated with logP; however, the optimum logP, at which the level of activity is maximal, differed between these two effects. Inhibitory activity against in vitro plasma oxidation was weakly dependent on logP. Plasma concentrations of the derivatives after oral administration at 10 mg/kg correlated negatively with logP and positively with water solubility. Hypocholesterolemic activity in rats fed a high-cholesterol diet, and the ratio of Cmax and IC50 values for ACAT inhibition, an index of effective plasma concentration, positively and highly correlated with logP, while ex vivo inhibitory activity against plasma oxidation in rats, and the ratio of Cmax and IC50 values for the inhibition of plasma oxidation negatively correlated with logP. In conclusion, in vitro ACAT inhibitory and anti-oxidative activity were differently dependent on logP, and intestinal absorption was inversely dependent on lipophilicity in indoline-based anti-oxidative ACAT inhibitors. The hypocholesterolemic effect positively correlated and the ex vivo anti-oxidative effect negatively correlated with lipophilicity. Optimum logP as a bioavailable dual inhibitor against in vivo ACAT and lipid peroxidation was estimated to be 3.8 (1-pentyl and 1-isopentyl derivatives) in the present series of derivatives.

Effects of an anti-oxidative ACAT inhibitor on apoptosis/necrosis and cholesterol accumulation under oxidative stress in THP-1 cell-derived foam cells.

THP-1 cell-derived foam cells were exposed to oxidative stress through combined treatment with acetylated LDL (acLDL) and copper ions (Cu2+). The foam cells showed caspase-dependent apoptotic changes on exposure to oxidative stress for 6 h, and necrotic changes with the leakage of LDH after 24 h. KY-455, an anti-oxidative ACAT inhibitor, and ascorbic acid (VC) but not YM-750, an ACAT inhibitor, prevented apoptotic and necrotic changes. These preventive effects of KY-455 and VC were accompanied by the inhibition of lipid peroxidation in culture medium containing acLDL and Cu2+, suggesting the involvement of oxidized acLDL in apoptosis and necrosis. Foam cells accumulated esterified cholesterol (EC) for 24 h in the presence of acLDL without Cu2+, which was suppressed by KY-455 and YM-750. Foam cells showed necrotic changes and died in the presence of acLDL and Cu2+. KY-455 but not YM-750 prevented cell death and reduced the amount of EC accumulated. The foam cells treated with VC further accumulated EC without necrotic changes for 24 h even in the presence of acLDL and Cu2+. YM-750 as well as KY-455 inhibited lipid accumulation when co-incubated with VC in foam cells exposed to oxidative stress. It is concluded that an anti-oxidative ACAT inhibitor or the combination of an antioxidant and an ACAT inhibitor protects foam cells from oxidative stress and effectively reduces cholesterol levels, which would be a promising approach in anti-atherosclerotic therapy.

Effect of combination of nitric oxide synthase and cyclooxygenase inhibitors on carrageenan-induced pleurisy in rats.

In the present study, we examined the effects of L-nitroarginine methylester (L-NAME), a non-selective nitric oxide synthase (NOS) inhibitor, indomethacin (IND), a non-selective COX inhibitor and a combination of these agents (L-NAME+IND) on carrageenan-induced pleurisy in rats. Exudate volume, albumin leakage, leukocyte influx, exudate and plasma nitrite/nitrate (NO(x)) levels and exudate PGE(2) levels increased markedly 6 h after an intrapleural injection of 2% carrageenan. First, the effects of L-NAME and IND alone were investigated. L-NAME non-significantly reduced exudate volume by 26% at 10 mg/kg (i.p.), and significantly by 45% at 30 mg/kg. IND dose-dependently decreased the exudate volume at 0.3-10 mg/kg (p.o.) and the effect reached the maximal level at 1 mg/kg (33%). Second, the effects of L-NAME (10 mg/kg, i.p.), IND (1 mg/kg, p.o.) and L-NAME+IND were examined. L-NAME and IND alone at the dose employed significantly reduced the exudate volume and albumin levels by 21-26%. L-NAME but not IND tended to reduce the increased exudate and plasma NO(x) by 18% and 19%, respectively. IND but not L-NAME significantly reduced leukocyte numbers and PGE(2) levels in the exudates by 25% and 77%, respectively. L-NAME+IND significantly reduced exudate volume, albumin leakage, leukocyte number, PGE(2) and NO(x) by 43%, 41%, 31%, 80% and 37%, respectively. The inhibitory effects of L-NAME+IND on exudate volume, albumin leakage and NO(x) levels were greater than those of L-NAME and IND alone. In conclusion, a non-selective NOS inhibitor and COX inhibitor showed anti-inflammatory effects at the early phase of carrageenan-induced pleurisy, and a combination of both inhibitors had a greater effect than each alone probably via the potentiation of NOS inhibition. The simultaneous inhibition of NOS and COX could be a useful approach in therapy for acute inflammation.

Synergistic effect of nitric oxide synthase and cyclooxygenase inhibitors on carrageenan-induced paw edema in rats.

The present study examined the effects of L-nitroarginine methylester (L-NAME, CAS 50903-99-6), a non-selective nitric oxide synthase (NOS) inhibitor, indometacin (IND, CAS 3305-29-1), a non-selective cyclooxygenase (COX) inhibitor, and a combination of these agents (L-NAME + IND) on carrageenan-induced paw edema for 4 h after the injection of carrageenan in rats. L-NAME at 10 and 30 mg/ kg but not 3 mg/kg (i.p.) decreased paw volume slightly but significantly only at 1 h after the carrageenan injection. IND reduced paw volume slightly at 1 and 3 mg/kg, and markedly at 10 mg/kg (p.o.). A combination of L-NAME and IND at a subthreshold dose (3 mg/kg, i.p. and 1 mg/kg, p.o., respectively) caused a marked reduction of paw edema, which was also confirmed by histopathological examinations. A combination of N-(3-(aminomethyl)benzyl)acetamidine (1400W, CAS 180001-34-7), a selective inhibitor of inducible NOS, and IND at 3 mg/kg, i.p., and 1 mg/kg, p.o., respectively, did not show synergistic anti-inflammatory effects. In conclusion, the combination of non-selective NOS and COX inhibitors had synergistic anti-inflammatory effects on carrageenan-induced paw edema at an early stage, suggesting negative crosstalk between the endogenous NOS-NO and COX-PG pathways in the early stages of acute inflammation.