Pharmacology and clinical assessment of cariporide for the treatment coronary artery diseases

Myocardial protection through pharmacological approaches represents a large therapeutic challenge and is an important therapeutic strategy in patients with coronary artery disease, particularly after myocardial infarction. Extensive animal experiments have repeatedly demonstrated the efficacy of sodium-hydrogen exchange (NHE) inhibition as a potent cardioprotective approach. The heart possesses primarily the NHE1 isoform which has led to the development of NHE1 specific inhibitors for cardiovascular therapeutics. Cariporide (HOE 642) is the first of such agents to have been developed and subjected to clinical trial. Preclinical studies with cariporide revealed excellent protection against necrosis, apoptosis, arrhythmias and mechanical dysfunction in hearts subjected to ischaemia and reperfusion. Cariporide has recently been evaluated in a large dose-finding Phase II/Phase III clinical trial (GUARDIAN) to assess its efficacy in patients with acute coronary syndromes. Overall results failed to demonstrate protection but sub-group analysis revealed significant risk reductions with the highest cariporide dose (120 mg t.i.d.) especially in high risk patients undergoing coronary artery bypass surgery. This suggests that insufficient dosage may have accounted, at least in part, for the less than optimum results. Another NHE1 inhibitor, eniporide, is currently in Phase II clinical trial (ESCAMI) in patients with acute myocardial infarction (MI) who are given angioplasty or thrombolysis. Although the study has not been completed interim findings appear positive. Both drugs were well-tolerated and produced no excess side effects compared with placebo. Further studies are needed to confirm the efficacy of NHE1 inhibitors for the treatment of coronary heart disease, even so initial results are encouraging.     

Colorectal cancer extracellular acidosis decreases immune cell killing and is partially ameliorated by pH-modulating agents that modify tumor cell cytokine profiles

Tumor cells upregulate myriad proteins that are important for pH regulation, resulting in the acidification of the extracellular tumor microenvironment (TME). Abnormal pH is known to dampen immune function, resulting in a worsened anti-tumor immune response. Understanding how extrinsic alterations in pH modulate the interactions between immune cells and tumors cells will help elucidate opportunities for new therapeutic approaches. We observed that pH impacts the function of immune cells, both natural killer (NK) and T cells, which is relevant in the context of a highly acidic TME. Decreased NK and T cell activity was correlated with decreasing pH in a co-culture immune cell-mediated tumor cell-killing assay. The addition of pH-modulating drugs cariporide, lansoprazole, and acetazolamide to the co-culture assay was able to partially mitigate this dampened immune cell function. Treatment of colorectal cancer (CRC) cells with NHE1 inhibitor cariporide increased CRC cell-secreted cytokines involved in immune cell recruitment and activation and decreased cytokines involved in epithelial-mesenchymal transition (EMT). Cariporide treatment also decreased CRC cell shed TRAIL-R2, TRAIL-R3, and PD-L1 which is relevant in the context of immunotherapy. These experiments can help inform future investigations into how the pH of the tumor microenvironment may be extrinsically modulated to improve anti-tumor immune response in solid tumors such as colorectal cancer.     

Mechanisms of Protection of the Ischemic and Reperfused Myocardium by Sodium-Hydrogen Exchange Inhibition

Sodium-hydrogen exchange (Na-H exchange) is a major regulator of intracellular pH and is one of the major mechanisms for restoring pH after ischemia-induced intracellular acidosis. However, activation of Na-H exchange during ischemia and reperfusion is also involved in paradoxical induction of cell injury. This likely reflects the fact that activation of the exchanger is closely coupled to sodium influx and, as a consequence, to elevation in intracellular calcium concentrations through sodium-calcium exchange. In addition to intracellular acidosis, other factors can also stimulate the exchanger, including various autocrine and paracrine factors, such as endothelin-1, angiotensin II, ₁-adrenergic agonists, as well as toxic agents, such as hydrogen peroxide and lysophosphatidylcholine. Although at least six Na-H exchange isoforms have thus far been identified, it appears that the 1 subtype, termed NHE1, is the predominant isoform in the mammalian myocardium. Effective pharmacological inhibitors of Na-H exchange, including those that are NHE1 specific, have been extensively demonstrated to protect the ischemic and reperfused myocardium in terms of improved systolic and diastolic function, preservation of cellular ultrastructure, attenuation of the incidence of arrhythmias, and reduction of apoptosis. Moreover, the salutary effects of these agents have been demonstrated using a variety of experimental models as well as animal species, suggesting that the role of Na-H exchange in mediating injury is not species specific. Thus, Na-H exchange represents an important target for pharmacological intervention in attenuation of ischemia and reperfusion-induced cardiac injury. Coupled with the low potential for toxicity of the agents, Na-H exchange inhibition could emerge as an effective therapeutic strategy in cardiac disorders, particularly involving conditions associated with ischemia and reperfusion.     

Cariporide对热缺血大鼠停跳心脏颈部移植术后bcl-2和bax基因表达的影响

OBJECTIVE: To detect the expression of bcl-2 and bax genes after heterotopic heart transplantation in rats that died of warm ischemia, and to explore the effect of cariporide on the protection of the ratos non heart-beating donors. METHODS: One hundred and twelve clearing Sprague-Dawley male rats were divided into 7 groups at random (each group contained 16 rats): the control group (Group C), the groups of transplanted hearts after 10, 30, and 45 min of asystolia (Group S10,S30,and S45), and the groups of transplanted hearts after 10,30, and 45 min of asystolia and infused with cariporide(Group SH10,SH30, and SH45).The experimental groups were sacrificed totally by warm ischemia, and heterotopic heart transplantation was processed by the Cuff method. The heart samples of S10,SH10,S30, and SH30 groups were taken at 48 hours after the transplantation, and the heart samples of S45, and SH45 groups were taken just after transplantation. The expression of bcl-2 and bax genes were detected by RT-PCR. RESULTS: The death of rats was affirmed when cardiac electric waves vanished after 9~11 minutes of transsection of abdominal aorta. On the RT-PCR test, the expression of bcl-2 gene was the highest and ROD value was maximum in the control group. The expression of bax gene was the lowest and ROD value was minimum in the control group. The ROD value of bcl-2 genes in S10 and S30 groups was less than that in SH10 and SH30 group. The ROD value was just the opposite, and there was stastistical difference (P<0.05).There was no statistical difference between Group S45 and Group SH45 (P>0.05). CONCLUSION: The model of heteroto-pic neck heart transplantation is a convenient animal model for the cardiac muscle protection. Cariporide can suppress the apoptosis of cardiac muscle cells in rats (within 30 min) after death caused by warm ischemia.     

Intracellular pH reduction prevents excitotoxic and ischemic neuronal death by inhibiting NADPH oxidase

Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H⁺ along with extracellular superoxide, and the intracellular H⁺ must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDA-induced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na⁺/H⁺ exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia–reperfusion was likewise prevented by inhibiting Na⁺/H⁺ exchange and by reduced expression of the Na⁺/H⁺ exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na⁺/H⁺ exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production.Many metabolic processes generate hydrogen ions, and hydrogen ions in turn influence cell metabolism and survival (1). Cerebral ischemia in particular produces acidosis of variable degree, depending upon blood glucose levels, degree of blood flow reduction, and other factors. Severe acidosis, below pH 6.4, exacerbates ischemic injury (2) by mechanisms involving protein denaturation, acid-sensing calcium channels, and release of ferrous iron (3–5). Conversely, lesser degrees of acidosis, in the range of 7.0–6.5, reduce both ischemic injury (6) and glutamate-induced neuronal death (7). These neuroprotective effects have been attributed to an inhibitory effect of hydrogen ions on NMDA receptor activation (8–10), but a causal link has not been demonstrated.Excessive activation of N-methyl-D-aspartate (NMDA) type glutamate receptors leads to excitotoxic cell death in stroke and other neurological disorders (11, 12). Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic cell death (13–19). NADPH oxidase exists as several isoforms, of which NOX2 is the one most abundantly expressed in CNS neurons. NOX2 is also the isoform most abundantly expressed in phagocytes, microglia, and other immune cells, in which its regulation and function have been extensively characterized (20). NOX2 is composed of three cytosolic subunits, p47^phox, p67^phox, and p40^phox, which when phosphorylated bind with two membrane-bound subunits, p22^phox and gp91^phox (the catalytic unit), to form an active transmembrane enzyme complex. The transmembrane complex generates superoxide in the extracellular space and hydrogen ions in the intracellular space: 2O₂ + NADPH → 2O₂⁻ + NADP⁺ + H⁺. In immune cells, H⁺ concentration influences the phosphorylation status of the NOX2 p47^phox subunit, and the H⁺ generated by NOX2 must be transferred to the extracellular space to sustain NOX2 activity (21–24).Together, the pH sensitivity of NOX2 and the role of NOX2 in NMDA receptor-mediated cell death suggest the possibility that reduced intracellular pH might limit neurotoxicity by dissociating NMDA receptor activation from superoxide production. Findings presented here confirm that both the superoxide production and cell death resulting from neuronal NMDA receptor activation are highly pH sensitive. We show that neurons use Na⁺/H⁺ exchange as a major route of proton efflux during NOX2 activation, and either genetic or pharmacologic inhibition of neuronal Na⁺/H⁺ exchange prevent both excitotoxic superoxide production and cell death.     

Cariporide对热缺血大鼠停跳心脏颈部移植术后bcl-2和bax基因表达的影响

目的:对热缺血死亡大鼠停跳心脏异位移植术后的供体进行有关bcl-2和bax基因表达的研究,以观察cariporide对大鼠停跳心脏供体的保护作用.方法:取清洁级Sprague-Dawley雄性大鼠共112只,随机分成7组,每组16只,即正常心脏对照组(C组);心脏停搏后10,30,45min取心组(S10,S30,S45组);心脏停搏后10,30,45 min取心并用cariporide灌注组(SH10,SH30,SH45组);对实验组大鼠均采用热缺血死亡处理,再用颈部Cuff法进行异位心脏移植.S10,SH10,S30,SH30组于移植后48 h取供心标本,S45,SH45组于移植术后取供心标本.然后采用RT-PCR法检测标本心肌细胞的bcl-2和bax基因的表达.结果:大鼠腹主动脉横断放血后9～11(10.11±0.59)min死亡;RT-PCR检测bcl-2基因的电泳图显示实验C组的表达最强,相对光密度ROD值最大,bax基因的表达最弱,ROD值最小;S10组、S30组bcl-2基因ROD值小于SH10组、SH30组;而bax基因的ROD值正好相反,差异具有统计学意义(P<0.05);S45和SH45组比较ROD值结果无统计学差异(P>0.05).结论:热缺血死亡大鼠颈部异位心脏移植模型是进行NHBD的心肌保护研究的理想模型;cariporide能抑制热缺血死亡后(30 min内)大鼠心脏心肌细胞凋亡的发生.     

Verapamil prevents stretch-induced shortening of atrial effective refractory period in langendorff-perfused rabbit heart

INTRODUCTION: Atrial dilation and rapid pacing reduce atrial effective refractory periods (AERPs), thereby increasing the susceptibility to sustained atrial fibrillation (AF) in Langendorff-perfused rabbit hearts. It is unclear whether similar pathophysiologic mechanisms are operative in short-term electrophysiologic changes caused by dilation and rapid pacing. Therefore, we analyzed whether both forms of short-term electrophysiologic changes are similarly affected by pharmacologic interventions acting on different potential mechanisms underlying these changes. METHODS AND RESULTS: Thirty Langendorff-perfused rabbit hearts underwent a protocol with stepwise increase of intra-atrial pressure from 0 to 12 cm H2O followed by 10 minutes of rapid pacing at 4 cm H2O. The protocol was repeated after addition of glibenclamide (10 micromol/L, n = 7), cariporide (1 micromol/L, n = 7), or verapamil (1 micromol/L, n = 9). In the basal state, increase of intra-atrial pressure from 0 to 12 cm H2O decreased AERPs from 85 +/- 11 to 55 +/- 9 msec (P < 0.01), rapid pacing at low intra-atrial pressure (4 cmH2O) decreased AERP to a similar extent, from 81 +/- 11 to 60 +/- 10 (P < 0.01). At higher intra-atrial pressure, decrease of AERP was more pronounced (10 cm H2O: 37 +/- 2 msec) (n = 7). Addition of verapamil decreased basal AERP from 86 +/- 10 msec to 68 +/- 11 msec (P < 0.05). Short-term electrophysiologic changes due to atrial dilation were abolished; changes due to rapid pacing were reduced but still present. Glibenclamide and cariporide had no significant effect. CONCLUSION: Langendorff-perfused rabbit heart is a suitable model for studying short-term electrophysiologic changes due to both rapid pacing and atrial dilation. AERPs are shortened to a similar extent by both mechanisms, whereas a combination of the two leads to more pronounced AERP reduction. Calcium overload plays a crucial role in short-term electrophysiologic changes caused by atrial dilation, whereas atrial ischemia or acidosis has no significant impact.     

抑制钠氢交换蛋白下调IL-8表达和促进p38磷酸化

我们前期的研究证明,抑制钠氢交换蛋白1(NHE1)可以减少VEGF表达从而抑制K562细胞诱导的血管生成。本研究探讨抑制NHE1后其他可能的血管生成因子变化及相关机制。用NHE1特异性抑制剂卡立泊来德10μmol/L处理K562细胞;蛋白芯片筛选NHE1抑制后血管生成因子的表达变化,实时定量PCR验证卡立泊来德处理后白介素-8(IL-8)的表达水平;构建K562稳定干扰NHE1细胞株,实时定量PCR验证干扰NHE1后IL-8的表达变化,Western blot检测卡立泊来德处理后细胞内p38磷酸化水平;p38抑制剂SB203580处理K562细胞,实时定量检测IL-8表达变化。蛋白芯片筛选结果显示,卡立泊来德处理后K562细胞中IL-8表达显著下调;实时定量结果进一步验证了这种抑制效应;卡立泊来德处理后p38磷酸化水平显著上调;卡立泊来德处理后加入SB203580抑制p38,IL-8表达可以部分恢复。结论:抑制NHE1可能通过促进p38磷酸化,下调促血管生成因子IL-8的表达。     

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.