Na+-H+交换及其抑制剂研究进展

Na^ -H^ 交换（NHE）普遍存在于包括人在内的哺乳动物组织细胞膜，对生物有着重要的作用。本文综述了NHE对于生物体内维持细胞恒定的pH值，调节电解质的含量，控制上皮细胞物质的的转运等多种生理作用，介绍了其在心肌缺血中的病理作用，报道了近年来NHE抑制剂的最新研究进展。     

In vitro pharmacology of R 80122, a novel phosphodiesterase inhibitor.

The cardiac in vitro effects of R 80122, a novel phosphodiesterase (PDE) inhibitor, were investigated and compared with those of the reference compound milrinone and of the calcium-sensitizer adibendan. In guinea pig left atria, both milrinone and R 80122 increased contractile force; 10 microM milrinone was equieffective to 1 microM R 80122. The rate of spontaneously beating atria was not altered by R 80122 in the concentration range of 0.01-0.3 microM. Higher concentrations (1-10 microM) led to a statistically insignificant increase of 20%. Milrinone's effect on frequency was more pronounced and amounted to 21% at 10 microM and to 40% at 100 microM. Adibendan increased heart rate (HR) by 10% at a concentration of only 0.03 microM. This effect was not enhanced any further by increasing the concentration. In papillary muscle, the positive inotropic effects of both milrinone and R 80122 were inhibited by carbachol, indicating involvement of cyclic AMP. Further indications for a cyclic AMP-dependent action were obtained by induction of slow action potentials and synergism with isoprenaline. In electrophysiologic measurements, milrinone reduced action potential duration (APD) in a high concentration whereas R 80122 had no effect. Action potential changes elicited by a toxic concentration of ouabain were reduced by R 80122. Relaxation of rat aortic rings contracted by KCl and relaxation of guinea pig aortic rings contracted by norepinephrine (NE) was comparable for both milrinone and R 80122. R 80122 also caused relaxation of canine coronary arteries constricted with prostaglandin F2 alpha (PGF2 alpha) both with and without endothelium. NE-induced contractions in canine gastrosplenic arteries were not affected by R 80122. Cardiac contractility that had been impaired to various degrees by pentobarbital or by aging was restored to control values by both milrinone and R 80122. R 80122 enhanced cardiac contractility at lower concentrations than milrinone with no concomitant increase in frequency or shortening of the action potential, which may be advantageous for treatment of heart failure.     

Potent and selective inhibition of the human Na+/H+ exchanger isoform NHE1 by a novel aminoguanidine derivative T-162559.

We isolated Na+/H+ exchanger (NHE)-deficient Chinese hamster ovary (CHO-K1) cells stably expressing human NHE isoforms (hNHE1, hNHE2 and hNHE3) and established an assay system for measuring their Na+/H+ exchange activity by monitoring intracellular pH alterations. Using this assay system, we demonstrated that the acylguanidine derivatives, cariporide and eniporide, cause selective inhibition of hNHE1 (IC50 value of 30 nM for cariporide, IC50 value of 4.5 nM for eniporide). Furthermore, we found that a novel synthetic aminoguanidine derivative, T-162559 ((5E,7S)-[7-(5-fluoro-2-methylphenyl)-4-methyl-7,8-dihydro-5(6H)-quinolinylideneamino] guanidine dimethanesulfonate), causes a selective inhibition of hNHE1 with more potent activity than cariporide and eniporide (IC50 value of 0.96 nM). This compound did not affect Na+/HCO3- cotransport and Na+/Ca2+ exchange.     

Effect of Hoe 694, a novel Na(+)-H+ exchange inhibitor, on intracellular pH regulation in the guinea-pig ventricular myocyte.

1. Hoe 694 (3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride) is a Na+/H+ exchange (NHE) inhibitor exhibiting cardioprotective properties during ischaemia and reperfusion in animal hearts. We have (i) tested the selectivity of Hoe 694 for NHE over other pHi-regulating mechanisms in the myocardium, and (ii) tested if the functionally important NHE isoform contributing to intracellular pH regulation in heart is NHE-1, as suggested from molecular biology studies of this protein. 2. pHi was recorded by fluorescence microscopy with carboxy SNARF-1, AM-loaded into single ventricular myocytes of guinea-pig. 3. In nominally HCO3-free media, recovery of pHi from an intracellular acid load is mediated by NHE, and was inhibited by Hoe 694, amiloride (an NHE inhibitor) or dimethyl amiloride (DMA, a high affinity NHE inhibitor) with potency values of 2.05, 87.3 and 1.96 microM respectively, giving the potency series: Hoe 694 congruent to DMA > > amiloride. This potency series, and the potency values (corrected for drug competition with extracellular Na+) match those determined previously for cloned NHE-1 expressed in mutant fibroblasts. In the absence of extracellular Na+ (to inhibit NHE), Hoe 694 had no effect on pHi. 4. In 5% CO2/HCO3(-)-buffered solution containing DMA, pHi recovery from acidosis is mediated by Na(+)-HCO3- symport and was unaffected by Hoe 694. The drug also had no effect on pHi recovery from an alkali-load, a process largely mediated by Cl(-)-HCO3- exchange. Finally, the fall of pHi upon adding extracellular Na-lactate is assisted by H(+)-lactate symport, and this too was unaffected by Hoe 694. 5. We conclude (i) Hoe 694 has no detectable inhibitory potency for pH-regulating carriers in heart other than NHE. (ii) native NHE functioning during pHi-regulation in the cardiomyocyte is the NHE-1 isoform. These data strengthen the case for NHE-1 being the receptor for mediating the cardioprotective effects of Hoe 694.     

Influence of a Na+-H+ exchange inhibitor ethylisopropylamiloride, a Na+-Ca2+ exchange inhibitor KB-R7943 and their combination on the increases in contractility and Ca2+ transient induced by angiotensin II in isolated adult rabbit ventricular myocytes

In rabbit, ventricular myocytes loaded with indo-1/AM, angiotensin II (0.1 nM-0.1 microM) exerted a positive inotropic effect with a significant increase in the amplitude of Ca2+ transients. For a given increase in cell shortening, the increase in Ca2+ transients induced by angiotensin II was less than that induced by elevation of extracellular Ca2+ concentration ([Ca2+]0) or isoprenaline, an indication that both the increase in mobilization of intracellular Ca2+ ions and myofibrillar sensitivity to Ca2+ ions contribute to the positive inotropic effect of angiotensin II. The effects of angiotensin II on Ca2+ transients and cell shortening were inhibited by the AT1 receptor antagonist losartan. A Na+ -H+ exchange inhibitor EIPA [5-(N-ethyl-N-isopropyl)amiloride] at 1 and 3 microM did not affect the Ca2+ transients and cell shortening, but it inhibited the angiotensin-II-induced responses in a concentration-dependent manner more effectively than the responses to elevation of [Ca2+]0, indicating that EIPA elicited a selective inhibitory action on the effects of angiotensin II. The observation that EIPA at 10 microM abolished the positive inotropic effect of angiotensin II without a significant depression of the inotropic response to elevation of [Ca2+]0 supports the selective action of EIPA at the high concentration on the response to angiotensin II. A novel selective Na+ -Ca2+ exchange (reverse mode) inhibitor KB-R7943, 2-[2-[4-(-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulphonate, at 0.3 and 1 microM inhibited also the responses to angiotensin II more effectively than the response to elevation of [Ca2+]0; however, over the same concentration range it suppressed significantly the amplitude of Ca2+ transients and cell shortening. Combination of EIPA (3 microM) and KB-R7943 (0.3 microM), each of which attenuated partially the angiotensin-II-induced responses, abolished the positive inotropic effect and the increase in Ca2+ transients induced by angiotensin II with much less depressant effect on the responses to elevation of [Ca2+]0. Thus, these ion exchange inhibitors exerted selective actions on the respective targets. The results with these selective inhibitors indicate that the activation of Na+ -H+ exchanger and subsequent modulation of the activity of Na+ -Ca2+ exchanger may be responsible for the increase in [Ca2+]i and the myofilament Ca2+ sensitization induced by stimulation of AT1 receptors by angiotensin II in rabbit ventricular myocytes.     

Effects of MS-31-038, a novel Na(+)-H+ exchange inhibitor, on the myocardial infarct size in rats after postischemic administration.

The abilities of 2-(2-methylphenyl)-5,7-dimethoxy-4-quinolyl carbonylguanidine dihydrochloride (CAS 181048-29-3, MS-31-050) and 2-phenyl-8-(2-methoxyethoxy)-4-quinolyl carbonylguanidine bismethanesulfonate (CAS 181048-36-2, MS-31-038) in inhibiting Na(+)-H+ exchange, ischemia- and reperfusion-induced injury were determined and compared with those of 4-isopropyl-3-methylsulfonylbenzoyl guanidine methanesulfonate (CAS 159138-81-5, IMGM), a selective inhibitor of Na(+)-H+ exchange. MS-31-050 and IMGM exhibited comparable inhibitory effects on Na(+)-dependent pH recovery and antiarrhythmic effects during ischemia in anesthetized rats. In rats subjected to ischemia and reperfusion, MS-31-050 (10 mg/kg i.v.) significantly reduced the infarct size when given prior to the onset of ischemia. However, postischemic treatment with either MS-31-050 or IMGM failed to protect reperfused hearts. In contrast, MS-31-038 reduced the infarct size dramatically from 65.4 +/- 7.4% in control to 29.9 +/- 11.6% at 3 mg/kg and 9.8 +/- 3.4% at 10 mg/kg even when administered before the onset of reperfusion. These results suggest the beneficial effects of Na(+)-H+ exchange inhibitors on myocardial ischemia/reperfusion injury.     

In vitro and in vivo antithrombotic activity of PD-198961, a novel synthetic factor Xa inhibitor

PD-198961, 3-(4-5-[(2R,6S)-2,6-dimethyltetrahydro-1(2H)-pyridinyl]pentyl-3-oxo-3,4-dihydro-2-quinoxalinyl)-4-hydroxybenzenecarboximidamide, is a novel, synthetic factor Xa inhibitor with a Ki of 2.7 nM against human factor Xa. The aim of the present study was to evaluate the pharmacokinetic profile and antithrombotic efficacy of PD-198961 in rabbits. When tested in vitro, PD-198961 doubled prothrombin time (PT) and activated partial thromboplastin time (aPTT) at concentrations of 0.13 and 0.32 microM in human plasma, 0.2 and 0.09 microM in rabbit plasma, 0.3 and 0.4 microM in dog plasma, respectively. Intravenous administration of PD-198961 at 1 mg/kg over 30 minutes resulted in a maximal prolongation in PT and aPTT of 4.9 +/- 0.4 and 4.1 +/- 0.9-fold of baseline, respectively. The peak plasma concentration of PD-198961 was 977 +/- 96 ng/ml. The anticoagulant effect of PD-198961 was readily reversible; coagulation parameters and plasma concentration returned to near baseline 15 minutes after cessation of infusion. There was a good correlation between PT prolongation and plasma concentration of PD-198961 (r = 0.93). In an FeCl3-induced model of arterial thrombosis in rabbits, the antithrombotic effects of PD-198961 were compared with that of LB-30057, a direct thrombin inhibitor, and enoxaparin, a low molecular weight heparin (LMWH). PD-198961 dose dependently increased the time to occlusion (TTO), reduced thrombus weight (TW), and decreased the incidence of occlusion. When administered at 3.0 microg/kg/min IV, PD-198961 prolonged TTO from 28 +/- 5 minutes (control) to 120 +/- 0 minutes (P < 0.001) and reduced TW from 9.9 +/- 1.5 mg (control) to 2.8 +/- 0.9 mg (P < 0.01). PD-198961 also dose dependently inhibited ex vivo plasma FXa activity. At the highest dose tested, PD-198961 increased aPTT to 1.4 +/- 0.1-fold of baseline (compared with 1.5 +/- 0.1 and 2.8 +/- 0.3-fold of baseline for LB-30057 [CI-1028] and enoxaparin, respectively), and had modest effects on bleeding time (< or = 2-fold). These results indicate that PD-198961 is a potent FXa inhibitor and an effective antithrombotic agent at doses that produce only modest changes in normal hemostasis.     

Reduction of myocardial infarct size by SM-197378, a novel Na+/H+ exchange inhibitor, in rabbits

The effects of SM-197378, 2-[[[amino(imino)methyl]amino]carbonyl]-1-methyl-4-trifluoromethyl-1H-indol-7-yl=hydrogen=sulfate monohydrate, a novel potent Na+/H+exchange inhibitor, on heart injury were studied using a rabbit model involving 30 min of myocardial ischemia and 5 h of reperfusion. Intravenous administration of SM-197378 before ischemia reduced the infarct size by approximately 30-50% in a dose-dependent manner. This anti-necrotic effect was achieved without significant hemodynamic changes. Moreover, administration of SM-197378 before reperfusion also resulted in a significant, approximately 30-40%, reduction in the infarct size. The anti-necrotic effect of pre-ischemic bolus treatment with SM-197378 was compared with that of nicorandil, a K+channel opener with nitrate-like activity, and ischemic preconditioning. With 30 and 60 min of ischemia, the anti-necrotic effects of SM-197378 and ischemic preconditioning were similar and superior to that of nicorandil. With 90 min of ischemia, the anti-necrotic effect of SM-197378 was superior to that of ischemic preconditioning.     

In vivo pharmacology and antidiarrheal efficacy of a thiazolidinone CFTR inhibitor in rodents

A small-molecule inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172), reduces enterotoxin-induced intestinal fluid secretion in rodents. Here, we study CFTR(inh)-172 pharmacology and antidiarrheal efficacy in rodents using (14)C-labeled CFTR(inh)-172, liquid chromatography/mass spectrometry, and a closed intestinal loop model of fluid secretion. CFTR(inh)-172 was cleared primarily by renal glomerular filtration without chemical modification. CFTR(inh)-172 accumulated in liver within 5 min after intravenous infusion in mice, and was concentrated fivefold in bile over blood. At 30-240 min, CFTR(inh)-172 was found mainly in liver, intestine, and kidney, with little detectable in the brain, heart, skeletal muscle, or lung. Pharmacokinetic analysis in rats following intravenous bolus infusion showed a distribution volume of 770 mL with redistribution and elimination half-times of 0.14 h and 10.3 h, respectively. CFTR(inh)-172 was stable in hepatic microsomes. Closed-loop studies in mice indicated that a single intraperitoneal injection of 20 microg CFTR(inh)-172 inhibited fluid accumulation at 6 h after cholera toxin by >90% in duodenum and jejunum, approximately 60% in ileum and <10% in colon. No toxicity was seen after high-dose CFTR(inh)-172 administration (3 mg/kg/day in two daily doses) in mice over the first 6 weeks of life. The metabolic stability, enterohepatic recirculation, slow renal elimination, and intestinal accumulation of CFTR(inh)-172 account for its efficacy as an antidiarrheal.     

In vivo pharmacology of BIIR 561 CL, a novel combined antagonist of AMPA receptors and voltage-dependent Na(+) channels.

Glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype and voltage-gated Na(+) channels are associated with diseases of the central nervous system characterized by neuronal over-excitation as in epilepsy or cerebral ischaemia. In animal models, AMPA receptor antagonists and Na(+) channel blockers provide protection in these conditions. Dimethyl-[2-[2-(3-phenyl-[1,2,4]oxadiazol-5-yl)-phenoxyl]-ethyl]-amine hydrochloride (BIIR 561 CL) combines both, AMPA receptor - and Na(+) channel blocking properties in one molecule. Here, BIIR 561 CL was investigated in vivo. BIIR 561 CL protected mice against AMPA-induced toxicity with an ED(50) value of 4.5 mg kg(-1) following subcutaneous (s.c.) administration. A 0.1% solution of BIIR 561 CL provided local anaesthesia in the corneal reflex test in rabbits. In mice, the compound prevented tonic seizures in the maximal electroshock (MES) model with an ED(50) value of 3.0 mg kg(-1) s.c. In amygdala-kindled rats, BIIR 561 CL inhibited seizures at doses of 3 and 11 mg kg(-1) following intraperitoneal (i.p.) injection. The data show that the combination of blocking AMPA receptor- and voltage-gated Na(+) channels in one molecule induces effective protection in animal models of neuronal over-excitation.     

Effects of SEA0400, a Na+/Ca2+ exchange inhibitor, on ventricular arrhythmias in the in vivo dogs

SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline), a novel and selective inhibitor of Na+/Ca2+ exchanger, was investigated for its possible antiarrhythmic effects on arrhythmias of Ca2+ overload induced by coronary ligation/reperfusion and by digitalis in the dog. SEA0400 (1.0 mg/kg) did not change the hemodynamics but slightly prolonged the QRS duration (P<0.05). Pre-ischemic administration (10 min before coronary occlusion) of SEA0400 (1.0 mg/kg) and post-ischemic administration (1 min before reperfusion) of SEA0400 (0.3, 1.0 and 3.0 mg/kg) had no effects on the incidence of ventricular fibrillation induced by coronary ligation/reperfusion. On the other hand, SEA0400 (3.0 mg/kg) decreased the arrhythmic ratio in the digitalis arrhythmias (P<0.01). However, atrioventricular block and cardiac standstill were induced in two digitalized dogs. In conclusion, SEA0400 has no significant antiarrhythmic effect on arrhythmias induced by coronary ligation/reperfusion, but has an obvious suppressing effect on tachyarrhythmias induced by digitalis in in vivo canine models.     

Characterization of SN-6, a novel Na+/Ca2+ exchange inhibitor in guinea pig cardiac ventricular myocytes

We examined the effect of SN-6, a new benzyloxyphenyl Na(+)/Ca(2+) exchange (NCX) inhibitor on the Na(+)/Ca(2+) exchange current (I(NCX)) and other membrane currents in isolated guinea pig ventricular myocytes using the whole-cell voltage-clamp technique. SN-6 suppressed I(NCX) in a concentration-dependent manner. The IC(50) values of SN-6 were 2.3 microM and 1.9 microM for the outward and inward components of the bi-directional I(NCX), respectively. On the other hand, SN-6 suppressed the outward uni-directional I(NCX) more potently (IC(50) value of 0.6 microM) than the inward uni-directional I(NCX). SN-6 at 10 microM inhibited the uni-directional inward I(NCX) by only 22.4+/-3.1%. SN-6 and KB-R7943 suppressed I(NCX) more potently when intracellular Na(+) concentration was higher. Thus, both drugs inhibit NCX in an intracellular Na(+) concentration-dependent manner. Intracellular application of trypsin via a pipette solution did not change the blocking effect of SN-6 on I(NCX). Therefore, SN-6 is categorized as an intracellular-trypsin-insensitive NCX inhibitor. SN-6 at 10 microM inhibited I(Na), I(Ca), I(K) and I(K1) by about 13%, 34%, 33% and 13%, respectively. SN-6 at 10 microM shortened the action potential duration at 50% repolarization (APD(50)) by about 34%, and that at 90% repolarization (APD(90)) by about 25%. These results indicate that SN-6 inhibits NCX in a similar manner to that of KB-R7943. However, SN-6 at 10 microM affected other membrane currents less potently than KB-R7943.     

Pharmacology of KB-R7943: a Na+-Ca2+ exchange inhibitor

The Na+-Ca2+ exchange (NCX) system plays a pivotal role in regulating intracellular Ca2+ concentration in cardiomyocytes, neuronal cells, kidney and a variety of other cells. It performs a particularly important function in regulating cardiac contractility and electrical activity. One of the leading NCX inhibitors is KB-R9743 (KBR) that appears to exhibit selectivity for Ca2+-influx-mode NCX activity (reverse mode of NCX). In this article we reviewed pharmacology of KBR and provide a brief summary of studies with other NCX inhibitors, such as SEA0400 (SEA) and SN-6 (SN). Potential clinical usefulness of KBR and other NCX inhibitors is still controversial but the reviewed findings may be helpful in designing more selective and clinically useful NCX inhibitors for the treatment of cardiac, neuronal and kidney diseases.     

A Na+-H+ exchange inhibitor (SM-20550) protects from microvascular deterioration and myocardial injury after reperfusion.

Na+-H+ exchange inhibitors may reduce myocardial damage after reperfusion. However, their effects on microvascular deterioration are not known. We examined the potency of a novel Na+-H+ exchange inhibitor, SM-20550 [ N-(Aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate], and its effects on microvascular damage after reperfusion. In an in vitro study, the Na+-H+ exchange inhibiting activity of SM-20550 was about 10 times greater than that of ethylisopropyl amiloride. In in vivo experiments, we occluded the left circumflex coronary artery in 29 dogs for 2 h and then reperfused for 5 h. SM-20550 was administered either before ischemia (n = 11) or before reperfusion (n = 7). Another 11 dogs served as controls. We found that SM-20550 not only improved coronary vasodilator responses to acetylcholine and adenosine after reperfusion, but also reduced infarct size (P < 0.01). Intramyocardial bleeding, which should reflect microvascular damage, was not found in dogs with SM-20550 treatment. Infarct size was correlated inversely with collateral blood flow in control (both, P < 0.01) but not in SM-20550-treated animals. Furthermore, SM-20550 significantly suppressed ventricular fibrillation during both ischemia and reperfusion. These results suggest that protective effects of Na+-H+ exchange inhibitors on reperfused myocardium are due at least in part to microvascular protection.     

In vitro and in vivo characterization of a novel naphthylamide ATP-sensitive K+ channel opener, A-151892

1. Openers of ATP-sensitive K(+) channels are of interest in several therapeutic indications including overactive bladder and other lower urinary tract disorders. This study reports on the in vitro and in vivo characterization of a structurally novel naphthylamide N-[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl]-acetamide (A-151892), as an opener of the ATP-sensitive potassium channels. 2. A-151892 was found to be a potent and efficacious potassium channel opener (KCO) as assessed by glibenclamide-sensitive whole-cell current and fluorescence-based membrane potential responses (-log EC(50)=7.63) in guinea-pig bladder smooth muscle cells. 3. Evidence for direct interaction with KCO binding sites was derived from displacement of binding of the 1,4-dihydropyridine opener [(125)I]A-312110. A-151892 displaced [(125)I]A-312110 binding to bladder membranes with a -log Ki value of 7.45, but lacked affinity against over 70 neurotransmitter receptor and ion channel binding sites. 4. In pig bladder strips, A-151892 suppressed phasic, carbachol-evoked and electrical field stimulus-evoked contractility in a glibenclamide-reversible manner with -log IC(50) values of 8.07, 7.33 and 7.02 respectively, comparable to that of the potencies of the prototypical cyanoguanidine KCO, P1075. The potencies to suppress contractions in thoracic aorta (-log IC(50)=7.81) and portal vein (-log IC(50)=7.98) were not substantially different from those observed for suppression of phasic contractility of the bladder smooth muscle. 5. In vivo, A-151892 was found to potently suppress unstable bladder contractions in obstructed models of unstable contractions in both pigs and rats with pED(35%) values of 8.05 and 7.43, respectively. 6. These results demonstrate that naphthylamide analogs exemplified by A-151892 are novel K(ATP) channel openers and may serve as chemotypes to exploit additional analogs with potential for the treatment of overactive bladder and lower urinary tract symptoms.     

In vitro and in vivo characterization of AS2643361, a novel and highly potent inosine 5'-monophosphate dehydrogenase inhibitor

Inosine 5'-monophosphate (IMP) dehydrogenase is a critical target in solid organ transplantation. To this end, the development of mycophenolate mofetil (MMF) represents a major advance in transplant medicine. Here, we investigated the in vitro and in vivo pharmacological effects of a novel IMP dehydrogenase inhibitor, AS2643361, in several immunological and non-immunological models. The in vitro inhibitory activity of AS2643361 on immune cell and endothelial cell proliferation and on antibody production from lipopolysaccharide-stimulated B cells, was significantly more potent than that of mycophenolic acid, the active form of MMF, despite the similar potency of these compounds on IMP dehydrogenase. In a rat heterotopic cardiac transplant model, monotherapy using orally administered AS2643361 at 10 or 20mg/kg/day prolonged the median graft survival time from 6 to 16 and 19days, respectively. In dinitrophenol-lipopolysaccharide stimulated rats, oral administration of AS2643361 at 2.5, 5 or 10mg/kg/day resulted in suppression of antibody production. In vivo antibody production against alloantigen was also suppressed by AS2643361 treatment at 5 or 10mg/kg/day. Furthermore, treatment with AS2543361 effectively inhibited balloon injury induced-intimal thickening, which is a major cause of late allograft loss. Overall, the in vivo activity of AS2643361 was over two-fold more potent than that of MMF. In addition, gastrointestinal toxicity, considered a dose-limiting factor for MMF, was reduced with AS2643361 treatment. These results suggest AS2643361 has higher potency and less toxicity than MMF, making it a potential candidate for treatment of acute and chronic rejection in transplant medicine.     

Protective effect of KB-R7943, a novel Na+/Ca2+ exchange inhibitor, on ischemic acute renal failure in rats

The effects of KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), a novel Na+/Ca2+ exchange inhibitor, on ischemic acute renal failure (ARF) in rats were examined. ARF was induced by clamping the left renal pedicle for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function was markedly diminished in ARF rats. Pretreatment with KB-R7943 (10 mg/kg, i.v.) markedly attenuated the ARF-induced renal dysfunction. Histopathological examination of the kidney of ARF rats revealed severe renal damage, which was suppressed by KB-R7943. Activation of the reverse mode of Na+/Ca2+ exchange seems to play an important role in the pathogenesis of ARF. A selective Na+/Ca2+ exchange inhibitor may be useful in cases of ARF.     

In vitro and in vivo characterization of XR11576, a novel, orally active, dual inhibitor of topoisomerase I and II

XR11576, a novel phenazine, was developed as an inhibitor of both topoisomerase I and II. This study characterized the ability of XR11576 to inhibit both enzymes, and determined its in vitro and in vivo antitumor efficacy against a number of murine and human tumor models. XR11576 was a potent inhibitor of purified topoisomerase I and IIalpha, and exhibited similar potency for both enzymes. The compound stabilized enzyme-DNA cleavable complexes indicating that it acted as a topoisomerase poison. The DNA cleavage patterns obtained with XR11576 were different from those induced by camptothecin and etoposide, which are topoisomerase I and II poisons, respectively. XR11576 demonstrated potent cytotoxic activity against a variety of human and murine tumor cell lines (IC50=6-47 nM). Its activity profile was comparable to or better than that of many widely used anticancer drugs. Moreover, XR11576 was unaffected by multidrug resistance (MDR) mediated by overexpression of either P-glycoprotein or MDR-associated protein, or by down-regulation of topoisomerase II. The latter property supports the dual inhibitory mechanism of action of the compound. XR11576 exhibited a similar pharmacokinetic profile in mice and rats after either i.v. or p.o. administration. In vivo XR11576 showed marked efficacy against a number of tumors including sensitive (H69/P) and multidrug-resistant (H69/LX4) small cell lung cancer and the relatively refractory MC26 and HT29 colon carcinomas following i.v. and p.o. administration. The efficacy of XR11576 was at least comparable to that of TAS-103, originally proposed as a dual inhibitor of topoisomerase I and II. These results suggest that XR11576 is a promising new antitumor agent with oral and i.v. activity, and warrants further development.     

TY-12533, a novel Na(+)/H(+) exchange inhibitor, prevents myocardial stunning in dogs

Anesthetized open-chest dogs were subjected to 15-min myocardial ischemia followed by 2-h reperfusion to induce myocardial stunning. A novel Na(+)/H(+) exchange inhibitor 6,7,8,9-tetrahydro-2-methyl-5H-cyclohepta[b]pyridine-3-carbonylguanidine maleate (TY-12533), administered 10 min before or 10 min after start of ischemia (3 mg/kg/10 min, i.v.), did not affect reductions in regional myocardial wall thickening, blood flow and pH during ischemia, but it significantly improved recovery of the wall thickening and blood flow after reperfusion. These results indicate that TY-12533, even when administered during ischemia, could prevent myocardial stunning without affecting myocardial dysfunction or acidosis induced by brief ischemia.     

Protective effect of SM-20550, a selective Na+ - H+ exchange inhibitor, on ischemia-reperfusion-injured hearts

The protective effects of Na+ - H+ exchange inhibitors SM-20550 (SM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) against ischemia-reperfusion injury were investigated in guinea pig Langendorff hearts. The changes in intracellular pH (pHi), high-energy phosphates, and biologic intracellular active ions ([Na+]i and [Ca2+]i) were regarded using the 31P-NMR and specific fluorescent signals from the heart tissues together with simultaneous recordings of the left ventricular developed pressure (LVDP). The recovery rate of LVDP from ischemia (40 min) by reperfusion was 36.8% in the control experiments, whereas in the presence of SM 10(-7) M, a gradual increase to 75.9% (55.5% with 10(-8) M), in contrast to EIPA (10(-7) M), 47.5% was observed. SM 10(-7) M restored the ATP level by 70% in 40-min reperfusion, which was already higher than the control in the latter half (20-40 min) of the ischemic period. The recovery rate of phosphocreatine by pretreatment of the heart with SM 10(-7) M was 75% in 40 min reperfusion. The pHi estimated from Pi/phosphocreatine chemical shift became highly acidic in ischemic heart so that SM 10(-7) M caused slight but significant pHi reduction from control pHi of 5.89 to 5.75. The level returned to pHi at around 7.38 during 30-40 min reperfusion, and the recovery was significantly greater than the control pHi of 7.24. The fura-2 Ca2+ or SBFI-Na+ signals during Langendorff ischemia heart increased, and rapidly returned to the control level after the reperfusion. SM suppressed the [Na+]i or [Ca2+]i elevation induced in the late stage during ischemia, resulting in LVDP restoration after reperfusion; Diastolic Ca2+ in the end period of ischemia, SM 10(-7) M 194% versus drug-free 220.7%. Na+: SM 10(-7) M 121.6% versus drug-free 128.0%. The present results suggest that the selective Na+ - H+ exchange inhibitor SM is promising as a potent and specific protective agent against ischemia-reperfusion injuries with Ca2+ overload induced via Na+ - H+, Na+ - Ca2+ exchange.