Sodium/hydrogen exchange inhibition with cariporide reduces leukocyte adhesion via P-selectin suppression during inflammation

Background and purpose:

The Na⁺/H⁺ exchange (NHE) inhibitor cariporide is known to ameliorate ischaemia/reperfusion (I/R) injury by reduction of cytosolic Ca²⁺ overload. Leukocyte activation and infiltration also mediates I/R injury but whether cariporide reduces I/R injury by affecting leukocyte activation is unknown. We studied the effect of cariporide on thrombin and I/R induced leukocyte activation and infiltration models and examined P-selectin expression as a potential mechanism for any identified effects.

Experimental approach:

An in vivo rat mesenteric microcirculation microscopy model was used with stimulation by thrombin (0.5 μ ml⁻¹) superfusion or ischaemia (by haemorrhagic shock for 60 min) and reperfusion (90 min).

Key results:

Treatment with cariporide (10 mg kg⁻¹ i.v.) significantly reduced leukocyte rolling, adhesion and extravasation after thrombin exposure. Similarly, cariporide reduced leukocyte rolling (54±6.2 to 2.4±1.0 cells min⁻¹, P<0.01), adherence (6.3±1.9 to 1.2±0.4 cells 100 μm⁻¹, P<0.01) and extravasation (9.1±2.1 to 2.4±1.1 cells per 20 × 100 μm perivascular space, P<0.05), following haemorrhagic shock induced systemic ischaemia and reperfusion. The cell adhesion molecule P-selectin showed a profound decrease in endothelial expression following cariporide administration in both thrombin and I/R stimulated groups (35.4±3.2 vs 14.2±4.1% P-selectin positive cells per tissue section, P<0.01).

Conclusions and implications:

The NHE inhibitor cariporide is known to limit reperfusion injury by controlling Ca²⁺ overload but these data are novel evidence for a vasculoprotective effect of NHE inhibition at all levels of leukocyte activation, an effect which is likely to be mediated at least in part by a reduction of P-selectin expression.     

Is timing everything? Therapeutic potential of modulators of cardiac Na+ transporters

Sodium ion (Na⁺) transporters have roles in the modulation of cardiomyocyte pH and Na⁺ and Ca²⁺ handling. Activation of the cardiac Na⁺-H⁺ exchanger 1 (NHE1) during ischaemia induces arrhythmias, myocardial stunning and irreversible cell injury. As the benefits of NHE1 inhibitors (e.g., amiloride, cariporide) in models of myocardial infarction are usually much greater when used as pretreatment, rather than during or after ischaemia, it is probably not surprising that clinical trials with cariporide in ischaemia have shown little shortterm benefit. NHE1 inhibitors have been shown to be beneficial in animal models of ventricular fibrillation and resuscitation, cardioplegia, hypertrophy and heart failure, and their therapeutic potential in these conditions should be further developed. The Na⁺-HCO₃^- cotransporter (NBC) is also stimulated by intracellular acidification, and part of the benefit of angiotensin-converting enzyme inhibitors after myocardial infarction may be due to inhibition of the NBC. Selective inhibitors of the NBC are required to determine the therapeutic potential of this mechanism. The Na⁺-Ca²⁺ exchanger (NCX) has a major role in cardiac Na⁺ and Ca²⁺ homeostasis and influences cardiac electrical activity. The NCX also has a role in ischaemia/infarction, arrhythmias, hypertrophy and heart failure. NCX inhibitors may have beneficial effects in animal models of ischaemia and reperfusion injury and the therapeutic benefit of these should be further studied in animal models.     

Therapeutic potential of Na-H exchange inhibitors for the treatment of heart failure

The Na-H exchanger (NHE) represents a family of transporters which regulate intracellular pH by removing protons in exchange for sodium influx in an electroneutral 1:1 stoichiometric relationship. Six isoforms have thus far been identified with the NHE-1 subtype representing the primary isoform in the cardiac cell. It is well-established that NHE-1 contributes to cardiac injury produced by ischaemia and reperfusion and inhibitors of the antiporter exert excellent cardioprotection. More recent evidence suggests that NHE-1 may also be important for cell growth and may contribute to the maladaptive remodelling which contributes to heart failure particularly the early hypertrophic responses. Evidence from in vitro studies suggest that NHE-1 inhibitors attenuate cardiomyocyte hypertrophy in response to various stimuli whereas in vivo studies report substantial attenuation of both hypertrophy and heart failure by these agents, especially after myocardial infarction. Accordingly, NHE-1 inhibitors could emerge as important therapeutic tools for the attenuation and treatment of heart failure.     

Blood cardioplegic protection in profoundly damaged hearts: role of Na+-H+ exchange inhibition during pretreatment or during controlled reperfusion supplementation

BACKGROUND: Inhibition of the Na+/H+ exchanger before ischemia protects against ischemia-reperfusion injury, but use as pretreatment before blood cardioplegic protection or as a supplement to controlled blood cardioplegic reperfusion was not previously tested in jeopardized hearts. METHODS: Control studies tested the safety of glutamate-aspartate-enriched blood cardioplegic solution in 4 Yorkshire-Duroc pigs undergoing 30 minutes of aortic clamping without prior unprotected ischemia. Twenty-four pigs underwent 30 minutes of unprotected normothermic global ischemia to create a jeopardized heart. Six of these hearts received normal blood reperfusion, and the other 18 jeopardized hearts underwent 30 more minutes of aortic clamping with cardioplegic protection. In 12 of these, the Na+/H+ exchanger inhibitor cariporide was used as intravenous pretreatment (n = 6) or added to the cardioplegic reperfusate (n = 6). RESULTS: Complete functional, biochemical, and endothelial recovery occurred after 30 minutes of blood cardioplegic arrest without preceding unprotected ischemia. Thirty minutes of normothermic ischemia and normal blood reperfusion produced 33% mortality and severe left ventricular dysfunction in survivors (preload recruitable stroke work, 23% +/- 6% of baseline levels), with raised creatine kinase MB, conjugated dienes, endothelin-1, myeloperoxidase activity, and extensive myocardial edema. Blood cardioplegia was functionally protective, despite adding 30 more minutes of ischemia; there was no mortality, and left ventricular function improved (preload recruitable stroke work, 58% +/- 21%, p < 0.05 versus normal blood reperfusion), but adverse biochemical and endothelial variables did not change. In contrast, Na+/H+ exchanger inhibition as either pretreatment or added during cardioplegic reperfusion improved myocardial recovery (preload recruitable stroke work, 88% +/- 9% and 80% +/- 7%, respectively, p < 0.05 versus without cariporide) and comparably restored injury variables. CONCLUSIONS: Na+/H+ exchanger blockage as either pretreatment or during blood cardioplegic reperfusion comparably delays functional, biochemical, and endothelial injury in jeopardized hearts.     

Improved Preservation of the Rat Heart with Celsior Solution Supplemented with Cariporide Plus Glyceryl Trinitrate

Our aim was to investigate whether the addition of glyceryl trinitrate (GTN), a source of nitric oxide, and/or cariporide, a Na/H exchange inhibitor, to a commercial preservation solution (Celsior) improved and extended cardiac preservation. After baseline indices of cardiac function (aortic flow, coronary flow, heart rate, cardiac output) were measured in an isolated working rat heart model, hearts were arrested and stored at 2-3 degrees C for 6 or 10 h in Celsior solution alone, Celsior supplemented with either 0.1 mg/mL GTN or 10 microM cariporide or both. After storage, functional measurements were repeated and recovery of each parameter was expressed as a percentage of its pre-storage baseline. After 6 h storage, recovery of cardiac function was significantly better in hearts stored in GTN- or cariporide-supplemented Celsior solution compared with Celsior solution alone. The beneficial effect of GTN was significantly abrogated in hearts perfused with glibenclamide prior to storage. Significant recovery of cardiac function after 10 h storage was only observed in hearts stored in Celsior solution supplemented with both GTN and cariporide. Combined supplementation with GTN and cariporide extends the safe period of storage of the rat heart and may be a useful approach to enhancing preservation of the donor heart.     

心肌钠氢交换抑制药在心血管疾病治疗中的应用

心肌钠氢交换抑制药通过抑制心肌细胞内外钠氢交换,从而防止或减少心肌细胞内钙超负荷。研究表明心肌钠氢交换抑制药对心肌缺血、再灌注损伤、心肌肥厚和心力衰竭有明确的治疗作用。     

Na^＋／H^＋交换抑制剂cariporide抗心肌缺血再灌注损伤研究进展

Na^ /H^ 交换抑制剂cariporide能减少细胞内Na^ 和Ca^2 的超负荷，减轻心肌缺血再灌注损伤，延缓心肌从缺血到坏死的发展进程，这种保护人单个细胞，离体心脏到在体模型均有实验依据。缺血前使用效果明显，再灌注时使用仍有一定效益，采用cariporide治疗心肌缺血高危患者的第一项大规模临床试验已经完成，结果不及想象中好，但仍有进一步研究的必要。     

卡立泊来德对K562细胞诱导的血管生成的影响

目的研究卡立泊来德(cariporide)处理对K562细胞诱导的血管生成能力的影响。方法应用MTT检测K562细胞上清液对脐静脉内皮细胞增殖能力的影响;transwell检测K562细胞上清液对脐静脉内皮细胞迁移能力的影响;基质胶血管形成法检测K562细胞上清液对脐静脉内皮细胞体外血管形成能力的影响;激光扫描共聚焦显微镜测定K562细胞的细胞内的pH;酶联免疫吸附实验(ELISA)检测K562细胞上清中血管内皮生长因子的表达水平。结果cariporide处理可以明显降低K562细胞上清液对脐静脉内皮细胞增殖,迁移和体外成管能力的诱导;cariporide处理后K562细胞的细胞内pH明显下降,分泌VEGF能力也受到抑制。结论 cariporide能抑制K562细胞的血管生成诱导能力,这种抑制是通过细胞内pH下降以及VEGF分泌减少引起的。     

卡立泊来德的合成工艺改进

以对异丙基苯甲酸为原料,经过氯磺化、还原、甲基化、胍解得到卡立泊来德,总收率为45·3%。改进后的工艺路线,降低了成本,简化了操作和反应条件。     

Cytostatic potential of novel agents that inhibit the regulation of intracellular pH

Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH (pHi) through the activity of membrane-based ion exchange mechanisms including the Na(+)/H(+) antiport and the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger. Inhibition of these regulatory mechanisms has been proposed as an approach to tumour therapy. Previously available inhibitors of these exchangers were toxic (e.g. 4,4-diisothiocyanstilbene-2,2-disulphonic acid), and/or non-specific (e.g. 5-N-ethyl-N-isopropyl amiloride). Using two human (MCF7, MDA-MB231) and one murine (EMT6) breast cancer cell lines, we evaluated the influence of two new agents, cariporide (an inhibitor of the Na(+)/H(+) antiport) and S3705 (an inhibitor of the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger) on the regulation of intracellular pH (pHi). The cytotoxicity of the two agents was assessed by using clonogenic assays. Our results suggest that cariporide has similar efficacy and potency to 5-N-ethyl-N-isopropyl amiloride for inhibition of Na(+)/H(+) exchange while S3705 is more potent and efficient than 4,4-diisothiocyanstilbene-2,2-disulphonic acid in inhibiting Na+-dependent Cl(-)/HCO3(-) exchange. The agents inhibited the growth of tumour cells when they were incubated at low pHe (7.0-6.8), but were non-toxic to cells grown at doses that inhibited the regulation of pHi. Our results indicate that cariporide and S3705 are selective cytostatic agents under in vitro conditions that reflect the slightly acidic microenvironment found in solid tumours.