Involvement of angiotensin II in progression of renal injury in rats with genetic non-insulin-dependent diabetes mellitus (Wistar fatty rats).

Wistar fatty (WF) rats have a genetic predisposition to hyperglycemia, polyuria, hyperinsulinemia, hyperlipidemia, obesity and nephropathy. These phenotypic characteristics are similar to those observed in obese patients with non-insulin-dependent diabetes mellitus (NIDDM) nephropathy. In this study, the effects of two types of renin-angiotensin system inhibitors, an angiotensin II type 1-receptor antagonist (AT1A) and an angiotensin I-converting enzyme inhibitor (ACEI), on renal injury in WF rats were studied during the progressive phase of diabetic nephropathy. An AT1A, candesartan cilexetil (1 mg/kg), and an ACEI, enalapril (10 mg/kg), were administered orally once a day for 12 weeks, beginning when the rats were 27-week-old and already showed diabetic nephropathy and obesity. Both drugs prevented an increase in proteinuria during the experimental period. Furthermore, after 4-week intervention, the levels of proteinuria were markedly lower in drug-treated rats. At the end of the experiment, both drugs prevented the development of glomerular lesions without affecting glucose metabolism and obesity. In conclusion, the inhibition of angiotensin II activity ameliorated both existing proteinuria and the progression of proteinuria, resulting in preservation of glomerular structure. Thus angiotensin II plays important roles in the development and the progression of nephropathy in genetically obese diabetic WF rats.

Effect of candesartan cilexetil (TCV-116) in rats with chronic renal failure.

BACKGROUND: Inhibition of the renin-angiotensin system by both angiotensin II type 1 receptor antagonists (AT1As) and angiotensin I-converting enzyme inhibitors (ACEIs) shows renoprotective effects in rats with chronic renal failure when treatment is started in the early phase of renal injury. In this study, we examined the renal protective effects of candesartan cilexetil (TCV-116), an AT1A, and enalapril, an ACEI, in the progressive phase of renal injury in 5/6 nephrectomized rats. METHODS: Candesartan cilexetil (1 mg/kg/day) and enalapril (10 mg/kg/day) were orally administered once a day for 4 weeks (the short-term experiment) or 16 weeks (the long-term experiment) to 5/6 nephrectomized rats beginning 15 weeks after the nephrectomy, that is, after they had already showed marked proteinuria. RESULTS: In vehicle-treated rats, proteinuria, glomerulosclerosis, and interstitial fibrosis developed. Moreover, enhanced expression of transforming growth factor-beta1 (TGF-beta1) in the injured glomeruli was observed. These adverse changes progressed with time, and in the short-term experiment, both drugs inhibited them. In the long-term experiment, the progressive proteinuria and the elevation of blood pressure were similarly attenuated by both drugs. However, candesartan cilexetil significantly inhibited the progression of glomerulosclerosis, the expression of TGF-beta1, and interstitial fibrosis, whereas enalapril did not. CONCLUSION: These results indicate that candesartan cilexetil shows potent and long-term preventive effects against the progression of previously developed renal injury.

Protective effects of candesartan cilexetil (TCV-116) against stroke, kidney dysfunction and cardiac hypertrophy in stroke-prone spontaneously hypertensive rats.

The effects of chronic treatment with an angiotensin II receptor antagonist, candesartan cilexetil (TCV-116, 0.1, 1, 10 mg/kg), and an angiotensin converting enzyme inhibitor, enalapril maleate (enalapril, 10 mg/kg), on the development of end-organ damage were examined in stroke-prone spontaneously hypertensive rats (SHRSP). The control SHRSP developed severe hypertension with stroke signs and increased urinary protein excretion. TCV-116 (0.1 mg/kg) reduced the stroke incidence and urinary protein excretion without affecting the blood pressure. TCV-116 (1 and 10 mg/kg) and enalapril reduced blood pressure, the stroke incidence, the urinary indices and left ventricular weight. Circulating renin-angiotensin system (RAS) and renal renin mRNA expression were significantly accelerated or tended to be accelerated in the control SHRSP with end-organ damages. A low dose of TCV-116 tended to reduce the RAS indices in plasma by improving the damages, whereas a high dose (10 mg/kg) increased them by the reflexes with blocking RAS. The present results indicate that chronic All blockade reduces the increase in blood pressure, end-organ damages and RAS related to the damages in SHRSP.

Candesartan (CV-11974) dissociates slowly from the angiotensin AT1 receptor.

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.

Effects of candesartan cilexetil (TCV-116) and enalapril in 5/6 nephrectomized rats.

The renal protective properties of candesartan cilexetil (TCV-116), an angiotensin II type 1 receptor antagonist (AT1A), and enalapril, an angiotensin I converting enzyme inhibitor (ACEI), were investigated in 5/6 nephrectomized (NX) rats. Candesartan cilexetil (1 mg/kg/day) and enalapril (10 mg/kg/day) were administered orally to 5/6 NX rats for four weeks (during the early phase of disease development) or 16 weeks (through the late phase). In vehicle-treated rats, proteinuria, glomerulosclerosis, interstitial mononuclear cell (MNC) infiltration and interstitial fibrosis developed. Moreover, immunohistological studies showed enhanced expression of transforming growth factor-beta 1 (TGF-beta 1) in the injured glomeruli. Both drugs inhibited these adverse changes in the early phase. In the late phase, the progressive proteinuria, interstitial MNC infiltration were attenuated by both drugs. However, candesartan cilexetil significantly inhibited the progression of glomerulosclerosis, the expression of TGF-beta 1 and the interstitial fibrosis, while enalapril did not. Candesartan cilexetil and enalapril showed comparable hypotensive effects after the 16-week administration. These results indicate that candesartan cilexetil shows a more potent protective effect than enalapril against the progression of renal injury in the late phase. Thus, an AT1A might be more useful than an ACEI for the treatment of patients with chronic renal failure.

TCV-116 inhibits renal interstitial and glomerular injury in glomerulosclerotic rats.

TCV-116 and enalapril were given in two stages: (early phase) for 6 to 10 weeks to 5/6 nephrectomized (NX) rats two weeks after nephrectomy, 12-week-old Wistar fatty (WF) rats and 7-week-old spontaneously hypercholesterolemic (SHC) rats; and (late phase) for 6 to 16 weeks to 5/6 NX rats 11 weeks after nephrectomy, 27-week-old WF rats and 10-week-old SHC rats. Urinary albumin, blood pressure (BP), glomerular filtration rate (GFR) and renal histology were examined. In the early phase, both agents inhibited proteinuria and tended to inhibit glomerulosclerosis. TCV-116 also inhibited interstitial inflammation. The antiproteinuric effects did not necessarily correlate with the BP-lowering effects. In the late phase, both agents showed equal antiproteinuric and antihypertensive effects. In 5/6NX and WF rats, TCV-116 inhibited tubulointerstitial inflammation/fibrosis, glomerulosclerosis and renal dysfunction, but enalapril had little effect on these parameters. In the SHC rats, TCV-116 inhibited renal tubulointerstitial inflammation and glomerulosclerosis, but enalapril inhibited only glomerulosclerosis. After drug administration, there was a positive correlation between proteinuria and BP, and a negative correlation between the severity of tissue damage and GFR, but not BP. These findings suggest that initial BP-independent tubulointerstitial inflammation may enhance glomerulosclerosis in the late phase, and TCV-116 might prevent the development of glomerulosclerosis through inhibition of angiotensin II-mediated tubulointerstitial damage in these models.

Comparison of the antihypertensive effects of the new angiotensin II (AT1) receptor antagonist candesartan cilexetil (TCV-116) and the angiotensin converting enzyme inhibitor enalapril in rats.

Antihypertensive effects of an angiotensin (Ang) II receptor antagonist, candesartan cilexetil (TCV-116), were compared with those of an angiotensin converting enzyme (ACE) inhibitor, enalapril, in spontaneously hypertensive rats (SHR), 2-kidney, 1-clip hypertensive rats (2K, 1C-HR) and 1-kidney, 1-clip hypertensive rats (1K, 1C-HR). CV-11974, the active form of TCV-116, had no inhibitory activity for plasma ACE. In rats, TCV-116 inhibited the pressor responses to Ang I, Ang II, and Ang III without an effect on the bradykinin (BK)-induced depressor response. Enalapril inhibited only the Ang I-response and potentiated the BK-response. In SHR, the antihypertensive effect of TCV-116 (10 mg/kg) was larger than the maximum antihypertensive effect of enalapril and was not intensified by combination with enalapril. Administration of CV-11974 potentiated the maximum antihypertensive effect of enalapril. Although both agents reduced blood pressure in 2K, 1C-HR, only TCV-116 had a marked antihypertensive effect in 1K, 1C-HR. These findings indicate that TCV-116 is more effective than enalapril in reducing blood pressure in SHR and 1K, 1C-HR, and that the BK- and/or prostaglandin-potentiating effect of enalapril contributes little to its antihypertensive mechanism in SHR.

[Nonpeptide angiotensin II-receptor antagonists].

The renin-angiotensin system (RAS) is known to play a very important role in cardiovascular diseases. The present state of the research on orally active nonpeptide angiotensin II (A II)-receptor antagonists and their pharmacological characterization will be reviewed. In the late 1970s, the first nonpeptide A II-receptor antagonists were discovered among some derivatives of imidazoleacetic acid, and this was followed by the development of losartan in 1989. TCV-116, synthesized in our laboratories, is a prodrug that is converted in vivo to the active form CV-11974. TCV-116 and CV-11974 were demonstrated to be effective antagonists for many A II-induced cardiovascular actions and are effective antihypertensive agents in several animal models of hypertension. TCV-116 also demonstrated secondary benefits in treating congestive heart failure (CHF), preventing stroke, delaying the progression of renal disease and preventing the intimal thickening of vascular injury in animal models. Clinical studies confirmed the efficacy of TCV-116 in the treatment of essential hypertension. The utility of A II antagonist may extend beyond that of hypertension and CHF, as suggested by the potential usefulness of ACE inhibitors in the treatment or prevention of many other cardiovascular diseases. The A II antagonists will help to determine the role of the RAS in the physiologic regulation and in the pathophysiology of various cardiovascular diseases.

Antihypertensive effects of a highly potent and long-acting angiotensin II subtype-1 receptor antagonist, (+-)-1-(cyclohexyloxycarbonyloxy)ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylate (TCV-116), in various hypertensive rats.

The antihypertensive effects of (+-)-(cyclohexyloxycarbonyloxy)ethyl2-ethoxy-1-[[2'-(1H- tetrazol-5- yl)biphenyl-4-yl]methyl]-1-H-benzimidazole-7-carboxylate (TCV-116), an angiotensin II (AII) subtype-1 receptor antagonist, were studied in various hypertensive and normotensive rats, using 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bip hen yl-4- yl)methyl]-imidazole, potassium salt (losartan) as a reference compound. TCV-116 is a prodrug, which is converted in vivo to the active component, 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl)]methyl]-1H- benzimidazole-7-carboxylic acid (CV-11974). In spontaneously hypertensive rats (SHR) p.o. TCV-116 (0.1 mg/kg) demonstrated a sustained antihypertensive effect that lasted for more than 10 hr and the dose that reduced the blood pressure by an average of 25 mm Hg for 24 hr (ED25), was 0.68 mg/kg. Intravenous CV-11974 reduced the blood pressure with an ED25 of 0.0027 mg/kg. Repeated p.o. administration of TCV-116 (1 mg/kg) to SHR once daily for 2 weeks reduced the blood pressure by 30 to 50 mm Hg over 24 hr without any heart rate changes. The antihypertensive effects of TCV-116 correlated well with the inhibition of angiotensin II-induced contractile responses of aortic strips prepared ex vivo after p.o. administration of TCV-116. Oral TCV-116 had a sustained antihypertensive effect with ED25 of 0.03 and 0.23 mg/kg in two-kidney, one-clip and one-kidney, one-clip hypertensive rats, respectively, and was much more potent in SHR and renal-hypertensive rats than losartan.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of an angiotensin II receptor antagonist, CV-11974, and its prodrug, TCV-116, on production of aldosterone.

In dispersed rabbit adrenocortical glomerulosa cells, a non-peptide angiotensin II (AT1) receptor antagonist, CV-11974 (10(-10)-10(-5) M), competitively inhibited angiotensin II- or angiotensin III-stimulated aldosterone production, whereas PD123177, an angiotensin AT2 receptor antagonist, did not. CV-11974 inhibited aldosterone production induced by 4 mM K+ but not by 12 mM K+. CV-11974 had no effect on adrenocorticotropic hormone-stimulated aldosterone or corticosterone production, but inhibited angiotensin II-stimulated corticosterone production. In the rat, TCV-116, the prodrug of CV-11974, (0.1 and 1 mg/kg, p.o.) markedly reduced the elevation of both plasma aldosterone concentration and blood pressure induced by i.v. infusion of angiotensin II. In spontaneously hypertensive rats, TCV-116 at daily p.o. doses of 0.1, 1 and 10 mg/kg for 2 weeks caused a dose-dependent reduction of blood pressure and plasma aldosterone concentration without affecting plasma corticosterone. Thus, TCV-116 inhibited the induction of aldosterone production by not only exogenous but also endogenous angiotensin II.

Pharmacological profile of a novel nonpeptide angiotensin II subtype 1 receptor antagonist, TCV-116.

TCV-116, an angiotensin II (AII) receptor antagonist, is a prodrug that is converted in vivo to the active form, CV-11974. CV-11974 selectively and competitively inhibited the specific binding of [125I]AII-(Sar1,lle8) to All subtype 1 (AT1) receptors in rabbit aortic membranes (Ki = 0.64 nM) and insurmountably inhibited the AII-induced maximal contractile response of rabbit aortic strips (pD'2 = 9.97). TCV-116 inhibited the AII-induced pressor response in rats (ID50 = 0.069 mg/kg. p.o.). In spontaneously hypertensive rats (SHR), TCV-116 had a sustained antihypertensive effect (ED25 = 0.68 mg/kg, p.o.). Repeated oral administration of TCV-116 (1 mg/kg) to SHR once daily for 2 weeks reduced blood pressure by 30-50 mmHg over 24 h. The antihypertensive effects of TCV-116 correlated well with the inhibition of AII-induced contractile responses of aortic strips prepared ex vivo after administration of TCV-116. TCV-116 had sustained effects in both 2 kidney, 1 clip hypertensive rats and in 1 kidney, 1 clip hypertensive rats, but had no effect in DOCA/salt hypertensive rats. Unlike enalapril, TCV-116 had no potentiating effect on the incidence of cough induced by citric acid in guinea pigs. These results suggest that TCV-116 is a promising antihypertensive agent with once daily administration.

Nonpeptide angiotensin II receptor antagonists. Synthesis and biological activity of potential prodrugs of benzimidazole-7-carboxylic acids.

In order to improve the oral bioavailability (BA) of 2-butyl-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimid azole - 7-carboxylic acid (3: CV-11194) and 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4- yl]methyl]-1H-benzimidazole-7-carboxylic acid (4: CV-11974), novel angiotensin II (AII) receptor antagonists, chemical modification to yield prodrugs has been examined. After selective tritylation of the tetrazole rings in 3 and 4, treatment of N-tritylated benzimidazole-7-carboxylic acids (6, 7) with a variety of alkyl halides, followed by deprotection with hydrochloric acid, afforded esters of 3 and 4. Mainly 1-(acyloxy)alkyl esters and 1-[(alkoxycarbonyl)oxy]alkyl esters, double ester derivatives, were synthesized. Their inhibitory effect on AII-induced pressor response in rats and oral BA were investigated. (Pivaloyloxy)methyl and (+/-)-1-[[(cyclohexyloxy)-carbonyl]oxy]ethyl esters of 3 and 4 showed marked increases in oral bioavailability which significantly potentiated the inhibitory effect of the parent compounds on AII-induced pressor response. Among them, (+/-)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl 2- ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimida zole- 7-carboxylate (10s, TCV-116) was selected as a candidate for clinical evaluation.

Inhibition of rabbit aortic angiotensin II (AII) receptor by CV-11974, a new nonpeptide AII antagonist.

The angiotensin II (AII) antagonistic action of CV-11974 (2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl] benzimidazole-7-carboxylic acid) was investigated in an AII-receptor binding assay using rabbit aortic membranes and an AII-induced contraction assay using rabbit aortic strips. A single class of [125I]AII-(Sar1,Ile8) binding sites was found in the membranes with a dissociation constant (Kd) of 0.15 nM and a receptor concentration (Bmax) of 86.9 fmol/mg protein. CV-11974 markedly reduced Kd without affecting Bmax. The specific binding of [125I]AII-(Sar1,Ile8) in this preparation was inhibited completely by CV-11974 [the inhibition constant (Ki) = 0.64 nM], DuP 753 [an angiotensin II type I (AT1) receptor-selective antagonist] (Ki = 51 nM) and EXP3174 (an active metabolite of DuP 753) (Ki = 6.8 nM), but was not affected by PD123177 (an AT2 receptor-selective antagonist). These results suggest that the single binding site in rabbit aortic membranes is an AT1 receptor subtype. The affinity of CV-11974 to these AT1 receptors was approximately 80 and 10 times higher than that of DuP 753 and EXP3174, respectively. CV-11974 showed no appreciable affinity for the AT2 receptors found in bovine cerebellum. In the in vitro functional study, CV-11974 markedly reduced the AII-induced maximal contractile response of rabbit aortic strips (pD'2 = 9.97). In contrast, Compound 7-H, which lacks the carboxyl group at the benzimidazole ring of CV-11974, inhibited the contraction in a competitive manner. The inhibition by CV-11974 was long lasting. These results suggest that CV-11974 is a potent and long-acting AT1 receptor-selective, competitive antagonist. The carboxyl group at the benzimidazole ring plays an important role in the interaction between CV-11974 and the AT1 receptor.

Pharmacological profile of a highly potent and long-acting angiotensin II receptor antagonist, 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4- yl]methyl]-1H-benzimidazole-7-carboxylic acid (CV-11974), and its prodrug, (+/-)-1-(cyclohexyloxycarbonyloxy)-ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5- yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylate (TCV-116).

The angiotensin II (AII) antagonistic action of 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl-1H-benzi mid azole-7 - carboxylic acid (CV-11974) was examined in in vitro assay systems, including AII receptor binding assay using membrane fractions of bovine adrenal cortex or rabbit aorta and AII-induced contraction assay using rabbit aortic strips, and CV-11974 and its prodrug, (+/-)1-(cyclohexyloxycarbonyloxy)ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylate (TCV-116), were examined in an in vivo system of AII-induced pressor response in conscious rats. DuP 753 or EXP3174 (the main active metabolite of DuP 753) was used as the reference compound. CV-11974 inhibited the binding of [125I] AII to the bovine adrenal cortical membrane and rabbit aortic membrane with IC50 values of 1.12 x 10(-7) and 2.86 x 10(-8) M, respectively. Similar results were obtained with EXP3174. CV-11974 interacted with AII in these membrane fractions with subtype 1 receptor in a competitive manner. CV-11974 at 10(-5) M did not affect the binding of [125I]AII to subtype 2 (AT2) receptor in bovine cerebellum. CV-11974 selectively inhibited the AII-induced contraction of rabbit aortic strips in a noncompetitive manner (pD' 2, 9.97); it had no effects on the contraction induced by norepinephrine, KCl, serotonin, prostaglandin F2 alpha or endothelin. EXP3174 showed a pD'2 value of 8.95 for the AII-induced contraction. CV-11974 given intravenously and TCV-116 given orally inhibited the AII-induced pressor response in rats with ID50 values of 0.033 mg/kg and 0.069 mg/kg, respectively. These effects of CV-11974 and TCV-116 were 12 and 48 times more potent than those of EXP3174 and DuP 753, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological activities of new 1,4-benzothiazine derivatives.

New 2H-1,4-benzothiazin-3(4H)-one derivatives possessing (4-phenyl-1-piperazinyl)alkyl moieties at the 2-position were synthesized and tested for calcium antagonistic and calmodulin antagonistic activities. Antihypertensive effects in spontaneously hypertensive rats were also evaluated. In general, these compounds were rather weak calcium channel blockers, although, in contrast, many of them had moderate to potent calmodulin antagonistic activity, and 2-[3-(4-(4-fluorophenyl)-1-piperazinyl]propyl]-2H-1,4-benzothiazin -3 (4H)-one derivatives 45, 74 and 75 showed potent antihypertensive effects.

Synthesis and biological activities of new 2-substituted 1,4-benzoxazine derivatives.

A series of new 1,4-benzoxazine derivatives (XI, XII) possessing (4-phenyl-1-piperazinyl)alkyl moieties at the 2-position and related compounds (XIII) were synthesized and tested for calcium antagonistic, calmodulin antagonistic and antihypertensive activities. Various compounds had in vitro calmodulin antagonistic activity superior or comparable to that of trifluoperazine. Among these compounds, tetrahydronaphtho[2,3-b] [1,4]oxazine derivatives such as 51, 53, 54, 58, 59, 60, 73 and 75 showed potent antihypertensive effects in spontaneously hypertensive rats. Optical isomers of 51 were also synthesized and evaluated biologically. No differences in biological activities were seen between the enantiomers.

Involvement of thromboxane A2, leukotrienes and free radicals in puromycin nephrosis in rats.

Thromboxane A2 (TXA2), leukotrienes (LTs) and free radicals are considered to be possible mediators in the induction of glomerular injury and proteinuria. In this study, we examined the involvement of these three mediators and the protective effect of simultaneous inhibition of all three in puromycin aminonucleoside (PAN) nephrosis in rats. A single intraperitoneal injection of PAN (100 mg/kg) induced massive proteinuria and enhanced production of TXA2 and LTs from arachidonic acid in renal cortical slices and renal glomeruli, and increased malondialdehyde levels in plasma, urine and renal cortex. Oral administration of CV-6504(HCl) (3 to 20 mg/kg/day, for 1 to 2 weeks), a novel treble inhibitor of TXA2 synthetase, 5-lipoxygenase and lipid peroxidation, dose-dependently attenuated PAN-induced proteinuria and the increases in these three mediators. Any single specific inhibitor (CV-4151, a TXA2 synthetase inhibitor; AA-861, a 5-lipoxygenase inhibitor; or CV-3611, a radical scavenger) or a combination of two inhibitors showed no or only a slight antiproteinuric effect, but the combination of all three inhibitors significantly reduced PAN-induced proteinuria. These results suggest that, these three mediators may be involved in the pathogenesis of PAN nephrosis and that CV-6504(HCl), which can simultaneously inhibit all three, may be a useful therapeutic agent for nephrosis.

Dual inhibitors of thromboxane A2 synthase and 5-lipoxygenase with scavenging activity of active oxygen species. Synthesis of a novel series of (3-pyridylmethyl)benzoquinone derivatives.

A novel series of (3-pyridylmethyl)benzoquinone derivatives was molecular designed and synthesized for the dual purpose of inhibiting thromboxane A2 and leukotriene biosynthesis enzymes and scavenging active oxygen species (AOS). They were evaluated for inhibition of TXA2 synthase, inhibition of 5-lipoxygenase, and for their scavenging activity of AOS using the thiobarbituric acid method. 2,3,5-Trimethyl-6-(3-pyridylmethyl)-1,4-benzoquinone (24, CV-6504) was the most promising derivative since it showed efficient AOS scavenging activity (inhibition of lipid peroxidation in rat brain homogenates: IC50 = 1.8 x 10(-6) M) as well as potent, specific, and well-balanced inhibitory effects on both enzymes (inhibitory effect on TXA2 synthase in human blood, IC50 = 3.3 x 10(-7) M; inhibitory effect on 5-lipoxygenase in human blood, IC50 = 3.6 x 10(-7) M). In adriamycin-induced proteinuria in a rat model, compound 24 at 10 mg/kg per day (po) suppressed proteinuria by more than 50%. The proteinuria, however, could not be reduced by single administration of an inhibitor specific for thromboxane A2 synthase [(E)-7-phenyl-7-(3-pyridyl)-6-heptenoic acid (2, CV-4151)] or for 5-lipoxygenase [2-(12-hydroxy-5,10-dodecadiynyl)-3,5,6-trimethyl-1,4-benzoquinone (1, AA-861)]. The proteinuria was also not reduced by administration of an AOS scavenger, 2-O-octadecylascorbic acid (4, CV-3611). Triple function compounds such as compound 24 that specifically inhibit both enzymes as well as scavenge AOS possess a variety of pharmacologically beneficial effects.

Antagonistic action of AA-2414 on thromboxane A2/prostaglandin endoperoxide receptor in platelets and blood vessels.

AA-2414, (+/-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptanoi c acid, inhibited the aggregation of guinea pig platelets induced by a prostaglandin endoperoxide (PGH2) analogue, U-44069 and the specific binding of another analogue, [3H]U-46619 to washed guinea pig platelets with IC50 values of 3.1 x 10(-7) and 8.2 x 10(-9) M, respectively. AA-2414 competitively inhibited the contraction of rabbit aorta and pig coronary arteries induced by U-44069 with pA2 values of 8.3 and 9.0, respectively. AA-2414 also inhibited the contraction of rabbit aorta induced by PGF2 alpha (pA2: 7.8) and the contraction of pig coronary arteries induced by PGF2 alpha, PGD2 and 9 alpha,11 beta-PGF2 with pA2 values of 7.8, 8.6 and 7.8, respectively. But, AA-2414 had no effect on the antiaggregatory effect of PGD2 on the aggregation of guinea pig platelets. In experiments with guinea pigs ex vivo, AA-2414 (0.1-1 mg/kg, p.o.) dose-dependently inhibited the platelet aggregation induced by U-44069; the inhibition at a dose of 1 mg/kg was 100% at 1 hr and was 89% even at 24 hr after the administration. The thromboxane (TX) A2/PGH2 receptor antagonistic action of AA-2414 was stereospecific. These results show that AA-2414 is a potent, orally active and long acting TXA2/PGH2 receptor antagonist. In addition, AA-2414 has PGF2 alpha, PGD2 and 9 alpha,11 beta-PGF2 antagonistic effects.