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Objective: Disruptions in pH regulation are known to cause impairments in cognitive processes such as learning and memory, leading to various neurological disorders. Na+/ H+ exchangers (NHEs), embedded in the membranes of neurons, are crucial for regulating intracellular and extracellular pH. This project aims to investigate the effect of cariporide, a potent inhibitor of NHE1, on spatial learning performance. Materials and Methods: A total of 20 male Wistar rats weighing between 240 g and 360 g were divided randomly into two groups: control (n=10) and cariporide (n=10). The cariporide group received a dosage of 10 mg/kg of cariporide added to the drinking water of the rats for three weeks. The Morris Water Maze (MWM) test was conducted on all groups. Distance moved (DM), escape latency (EL), swimming speed (SS), mean distance to the platform (MdtP), and time spent in the target quadrant (TSTQ) of the rats were recorded using the monitoring and recording system. Results: The DM to find the platform and EL time did not show significant differences between the groups (p>0.05). The SS of the rats in the cariporide group significantly decreased compared to the control group on all days (p<0.05). The MdtP was statistically significantly increased in the cariporide group on the 2nd and 4th days. Conclusion: The findings of this study indicate that systemic inhibition of NHE1 with cariporide affects hippocampal plasticity, leading to impairments in learning and memory. To elucidate its role in neurodegenerative diseases, further research should investigate cariporide's effect on hippocampal synaptic plasticity. [ABSTRACT FROM AUTHOR]

To date, recanalization interventions are the only available treatments for ischemic stroke patients; however, there are no effective therapies for reducing stroke-induced neuroinflammation. We recently reported that H+ extrusion protein Na+/H+ exchanger-1 (NHE1) plays an important role in stroke-induced inflammation and white matter injury. In this study, we tested the efficacy of two potent NHE1 inhibitors, HOE642 and Rimeporide, with a delayed administration regimen starting at 24 h post-stroke in adult C57BL/6J mice. Post-stroke HOE642 and Rimeporide treatments accelerated motor and cognitive function recovery without affecting the initial ischemic infarct, neuronal damage, or reactive astrogliosis. However, the delayed administration of NHE1 blockers after ischemic stroke significantly reduced microglial inflammatory activation while enhanced oligodendrogenesis and white matter myelination, with an increased proliferation and decreased apoptosis of the oligodendrocytes. Our findings suggest that NHE1 protein plays an important role in microglia-mediated inflammation and white matter damage. The pharmacological blockade of NHE1 protein activity reduced microglia inflammatory responses and enhanced oligodendrogenesis and white matter repair, leading to motor and cognitive function recovery after stroke. Our study reveals the potential of targeting NHE1 protein as a therapeutic strategy for ischemic stroke therapy. [ABSTRACT FROM AUTHOR]

Acidification of cancerous tissue induced pharmacologically may slow tumor growth and can be detected using magnetic resonance imaging. Numerous studies have shown that pharmacologically inhibiting specific transporters, such as the Na+/H+ exchanger 1 (NHE1), can alter glycolitic metabolism and affect tumor acidosis. The sodium proton exchanger inhibitor Cariporide can acidify U87MG gliomas in mice. This study aimed to determine whether Cariporide could acidify C6 glioma tumors in rats with an intact immune system. C6 glioma cells were implanted in the right brain hemisphere of ten rats. Chemical exchange saturation transfer (CEST) MRI (9.4T) was acquired on days 7–8 and 14–15 after implantation to measure in vivo tissue intracellular pH (pHi) within the tumors and on the contralateral side. pHi was basic relative to contralateral tissue at both time points assessed using the amine and amide concentration-independent detection (AACID) value. On day 14–15, measurements were made before and up to 160 min after Cariporide injection (N = 6). Twenty minutes after drug injection, the average AACID value in the tumor significantly increased by ∼6.4% compared to pre-injection, corresponding to 0.31 ± 0.20 lower pHi, while in contralateral tissue, AACID value increased significantly by ∼4.3% compared to pre-injection, corresponding to 0.22 ± 0.19 lower pHi. Control rats without tumors showed no changes following injection of Cariporide dissolved in 10% or 1% DMSO and diluted in PBS. This study demonstrates the sensitivity of CEST-based pH-weighted imaging for monitoring the response of tumors to pharmacologically induced acidification. [ABSTRACT FROM AUTHOR]

Defense against intracellular acidification of breast cancer tissue depends on net acid extrusion via Na+,HCO 3 −-cotransporter NBCn1/Slc4a7 and Na+/H+-exchanger NHE1/Slc9a1. NBCn1 is increasingly recognized as breast cancer susceptibility protein and promising therapeutic target, whereas evidence for targeting NHE1 is discordant. Currently, selective small molecule inhibitors exist against NHE1 but not NBCn1. Cellular assays—with some discrepancies—link NHE1 activity to proliferation, migration, and invasion; and disrupted NHE1 expression can reduce triple-negative breast cancer growth. Studies on human breast cancer tissue associate high NHE1 expression with reduced metastasis and—in some molecular subtypes—improved patient survival. Here, we evaluate Na+/H+-exchange and therapeutic potential of the NHE1 inhibitor cariporide/HOE-642 in murine ErbB2-driven breast cancer. Ex vivo , cariporide inhibits net acid extrusion in breast cancer tissue (IC 50 = 0.18 μM) and causes small decreases in steady-state intracellular pH (pH i). In vivo , we deliver cariporide orally, by osmotic minipumps, and by intra- and peritumoral injections to address the low oral bioavailability and fast metabolism. Prolonged cariporide administration in vivo upregulates NBCn1 expression, shifts pH i regulation towards CO 2 /HCO 3 −-dependent mechanisms, and shows no net effect on the growth rate of ErbB2-driven primary breast carcinomas. Cariporide also does not influence proliferation markers in breast cancer tissue. Oral, but not parenteral, cariporide elevates serum glucose by ∼1.5 mM. In conclusion, acute administration of cariporide ex vivo powerfully inhibits net acid extrusion from breast cancer tissue but lowers steady-state pH i minimally. Prolonged cariporide administration in vivo is compensated via NBCn1 and we observe no discernible effect on growth of ErbB2-driven breast carcinomas. [Display omitted] • Enhanced acid extrusion protects breast cancer cells in the acidic tumor environment. • We use cariporide to target the Na+/H+-exchanger NHE1 and lower acid extrusion. • Cariporide acutely attenuates acid extrusion but minimally affects steady-state pH i. • Alternative acid extrusion mechanisms compensate during chronic cariporide application. • Cariporide does not influence growth of ErbB2-driven breast carcinomas. [ABSTRACT FROM AUTHOR]

The inhibition of Na+/H+ Exchangers (NHEs) has shown efficacy in the pathology of several diseases like tumors, cardiovascular, and neurological disorders. The role of guanidine ligands such as amiloride, cariporide, and rimeporide as NHE inhibitors is very well documented but their interaction studies with genomic DNA are still unexplored. In this study, a combination of various biophysical and molecular docking studies was employed to investigate their binding aspects.UV–Visible, fluorescence, and circular dichroism (CD) studies indicated that guanidine ligands bind to the grooves of Calf Thymus DNA (ctDNA). Fluorescence titration studies depict that amiloride binds to ctDNA with a binding constant in the order of 102 M−1 and free energy change (ΔG0) of −14.05 KJ mol−1. Competitive fluorescence studies indicated the minor groove binding property of amiloride, whereas major groove binding mode was deduced for rimeporide and cariporide. Molecular docking studies were also found to be in accordance with the experimental results, revealing the information about the binding energy of the guanidine ligand-ctDNA complex. The docked structures depicted binding energy of −6.4 kcal mol−1 for amiloride and − 6.6 kcal mol−1 for rimeporide and cariporide. Such physicochemical studies of DNA-ligand interactions may facilitate the understanding of the mechanisms of NHE inhibition. [Display omitted] • The interaction studies of guanidine ligands (amiloride, rimeporide and cariporide) with ctDNA is still unexplored. • DNA-drug interaction. • Several biophysical and molecular docking techniques are used to study DNA-guanidine ligands interaction. • Amiloride interacts with the minor groove of ctDNA • Rimeporide and cariporide both interact with the major groove of ctDNA. [ABSTRACT FROM AUTHOR]

Background: Doxorubicin (DOX) is a potent chemotherapeutic agent with limited usage due to its cumulative cardiotoxicity. The Na+/H+ exchanger isoform 1 (NHE1) is a known regulator of oxidative stress, inflammation, and apoptosis. The present study was designed to investigate the possible protective effect of cariporide (CAR), a selective inhibitor of NHE1, against DOX-induced cardiotoxicity in rats. Methods: Male Sprague-Dawley rats were intraperitoneally injected with DOX to induce cardiac toxicity and CAR was given orally for treatment. The injured H9c2 cell model was established by incubation with DOX in vitro. Echocardiography, as well as morphological and ultra-structural examination were performed to evaluate cardiac function and histopathological changes. The biochemical parameters were determined according to the manufacturer's guideline of kits. ROS were assessed by using an immunofluorescence assay. The serum levels and mRNA expressions of inflammatory cytokines were measured by using ELISA or qRT-PCR. Cardiac cell apoptosis and H9c2 cell viability were tested by TUNEL or MTT method respectively. The protein expressions of Cleaved-Caspase-3, Bcl-2, Bax, Akt, GSK-3β, and Sirt1 were detected by western blot. Results: Treatment with CAR protected against DOX-induced body weight changes, impairment of heart function, leakage of cardiac enzymes, and heart histopathological damage. In addition, CAR significantly attenuated oxidative stress and inhibited the levels and mRNA expressions of inflammatory cytokines (TNF-α, IL-6, IL-18, and IL-1β), which were increased by DOX treatment. Moreover, CAR significantly suppressed myocardial apoptosis and Cleaved-Caspase-3 protein expression induced by DOX, which was in agreement with the increased Bcl-2/Bax ratio. Also, DOX suppressed phosphorylation of Akt and GSK-3β, which was significantly reversed by administration of CAR. Furthermore, CAR treatment prevented DOX-induced down-regulation of Sirt1 at the protein level in vitro and in vivo. Finally, Sirt1 inhibitor reversed the protective effects of CAR, as evidenced by reduced cell viability and Sirt1 protein expression in vitro. Conclusion: Taken together, we provide evidence for the first time in the current study that CAR exerts potent protective effects against DOX-induced cardiotoxicity in rats. This cardio-protective effect is attributed to suppressing oxidative stress, inflammation, and apoptosis, at least in part, through regulation of Akt/GSK-3β and Sirt1 signaling pathway, which has not been reported to date. [ABSTRACT FROM AUTHOR]

Background: Doxorubicin (DOX) is a potent chemotherapeutic agent with limited usage due to its cumulative cardiotoxicity. The Na+/H+ exchanger isoform 1 (NHE1) is a known regulator of oxidative stress, inflammation, and apoptosis. The present study was designed to investigate the possible protective effect of cariporide (CAR), a selective inhibitor of NHE1, against DOX-induced cardiotoxicity in rats.Methods: Male Sprague-Dawley rats were intraperitoneally injected with DOX to induce cardiac toxicity and CAR was given orally for treatment. The injured H9c2 cell model was established by incubation with DOX in vitro. Echocardiography, as well as morphological and ultra-structural examination were performed to evaluate cardiac function and histopathological changes. The biochemical parameters were determined according to the manufacturer’s guideline of kits. ROS were assessed by using an immunofluorescence assay. The serum levels and mRNA expressions of inflammatory cytokines were measured by using ELISA or qRT-PCR. Cardiac cell apoptosis and H9c2 cell viability were tested by TUNEL or MTT method respectively. The protein expressions of Cleaved-Caspase-3, Bcl-2, Bax, Akt, GSK-3β, and Sirt1 were detected by western blot.Results: Treatment with CAR protected against DOX-induced body weight changes, impairment of heart function, leakage of cardiac enzymes, and heart histopathological damage. In addition, CAR significantly attenuated oxidative stress and inhibited the levels and mRNA expressions of inflammatory cytokines (TNF-α, IL-6, IL-18, and IL-1β), which were increased by DOX treatment. Moreover, CAR significantly suppressed myocardial apoptosis and Cleaved-Caspase-3 protein expression induced by DOX, which was in agreement with the increased Bcl-2/Bax ratio. Also, DOX suppressed phosphorylation of Akt and GSK-3β, which was significantly reversed by administration of CAR. Furthermore, CAR treatment prevented DOX-induced down-regulation of Sirt1 at the protein level in vitro and in vivo. Finally, Sirt1 inhibitor reversed the protective effects of CAR, as evidenced by reduced cell viability and Sirt1 protein expression in vitro.Conclusion: Taken together, we provide evidence for the first time in the current study that CAR exerts potent protective effects against DOX-induced cardiotoxicity in rats. This cardio-protective effect is attributed to suppressing oxidative stress, inflammation, and apoptosis, at least in part, through regulation of Akt/GSK-3β and Sirt1 signaling pathway, which has not been reported to date.

Abstract Background The Na+/H+ exchanger (NHE1) plays a crucial role in cancer cell proliferation and metastasis. However, the mechanism underlying chemotherapeutic resistance in cancer cells has not been completely elucidated. The NHE1 inhibitor cariporide has been demonstrated to inhibit human cancer cell lines. The goal of this study was to provide new sights into improved cancer cell chemosensitivity mediated by cariporide with activation of the apoptosis pathway. Methods The NHE1 expression levels were first evaluated using the online database Oncomine and were determined by RT-PCR and western blot in vitro and in vivo. Cell proliferation was assessed In vitro through a CCK-8 assay, and apoptosis was analyzed by flow cytometry. An in vivo analysis was performed in BALB/c nude mice, which were intraperitoneally injected with MCF-7/ADR cells. Results NHE1 levels were significantly higher in breast cancer tissue than adjacent tissue, as well as in resistant cancer cells compared to sensitive cells. Cariporide induced the apoptosis of MCF-7/ADR cells and was associated with the intracellular accumulation of doxorubicin and G0/G1 cell cycle arrest. Moreover, cariporide decreased MDR1 expression and activated cleaved caspase-3 and caspase-9, promoting caspase-independent apoptosis in vitro. In vivo, cariporide significantly improved doxorubicin sensitivity in a xenograft model, enhancing tumor growth attenuation and diminishing tumor volume. Conclusions Our results demonstrate that cariporide significantly facilitates the sensitivity of breast cancer to doxorubicin both in vitro and in vivo. This finding suggests that NHE1 may be a novel adjuvant therapeutic candidate for the treatment of resistant breast cancer.

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.

This paper presents the hands-on modeling toolbox, HBV-Ensemble, designed as a complement to theoretical hydrology lectures, to teach hydrological processes and their uncertainties. The HBV-Ensemble can be used for inclass lab practices and homework assignments, and assessment of students’ understanding of hydrological processes. Using this modeling toolbox, students can gain more insights into how hydrological processes (e.g., precipitation, snowmelt and snow accumulation, soil moisture, evapotranspiration and runoff generation) are interconnected. The educational toolbox includes a MATLAB Graphical User Interface (GUI) and an ensemble simulation scheme that can be used for teaching uncertainty analysis, parameter estimation, ensemble simulation and model sensitivity. HBV-Ensemble was administered in a class for both in-class instruction and a final project, and students submitted their feedback about the toolbox. The results indicate that this educational software had a positive impact on students understanding and knowledge of uncertainty in hydrological modeling.

Increasing evidence indicates that ion channels and transporters cooperate in regulating different aspects of tumor pathophysiology. In cancer cells, H+/HCO3- transporters usually invert the transmembrane pH gradient typically observed in non-neoplastic cells, which is thought to contribute to cancer malignancy. To what extent the pH-regulating transporters are functionally linked to K+ channels, which are central regulators of cell membrane potential (Vm), is unclear. We thus investigated in colorectal cancer cells the implication of the pH-regulating transporters and KV11.1 (also known as hERG1) in the pH modifications stimulated by integrin-dependent cell adhesion. Colorectal cancer cell lines (HCT 116 and HT 29) were seeded onto β1 integrin-dependent substrates, collagen I and fibronectin. This led to a transient cytoplasmic alkalinization, which peaked at 90 min of incubation, lasted approximately 180 min, and was inhibited by antibodies blocking the β1 integrin. The effect was sensitive to amiloride (10 µM) and cariporide (5 µM), suggesting that it was mainly caused by the activity of the Na+/H+ antiporter NHE1. Blocking KV11.1 with E4031 shows that channel activity contributed to modulate the β1 integrin-dependent pHi increase. Interestingly, both NHE1 and KV11.1 modulated the colorectal cancer cell motility triggered by β1 integrin-dependent adhesion. Finally, the β1 integrin subunit, KV11.1 and NHE1 co-immunoprecipitated in colorectal cancer cells seeded onto Collagen I, suggesting the formation of a macromolecular complex following integrin-mediated adhesion. We conclude that the interaction between KV11.1, NHE1, and β1 integrin contributes to regulate colorectal cancer intracellular pH in relation to the tumor microenvironment, suggesting novel pharmacological targets to counteract pro-invasive and, hence, pro-metastatic behavior in colorectal cancer.

Hypoxia often leads to severe cardiac malfunctions. It is assumed that intracellular calcium overload is -inter alia- responsible for left ventricular (LV) deterioration. Inhibition of the sodium-proton exchanger (NHE), which finally inhibits/slows calcium overload, may ameliorate cardiac function. Our aim was to evaluate cariporide, an inhibitor of NHE1 in a Langendorff-perfused heart model. To discriminate a potentially different impact of extracellular acidosis and hypoxia we examined 48 Chinchilla Bastard rabbits divided into 8 experimental groups: control group (pH = 7.4, O2 = 100%) without or with cariporide (1 μM), acidosis group (pH = 7.0, O2 = 100%) without or with cariporide (1 μM), hypoxia group (pH = 7.4, O2 = 40%) without or with cariporide (1 μM) and hypoxia+acidosis group (pH = 7.0, O2 = 40%) without or with cariporide (1 μM). Hearts were subjected to acidotic/hypoxic conditions for 90 min followed by 60 min of reperfusion. Hypoxia and hypoxia+acidosis led to a severe deterioration of LV function with a decrease in LV pressure by about 70% and an increase of end-diastolic pressure from 6.7 ± 0.6 to 36.8 ± 5.4 (hypoxia) or from 7.0 ± 0.2 to 18.6 ± 4.1 (hypoxia+acidosis). Moreover, maximum contraction velocity decreased from about 1,800 mmHg/s to 600 mmHg/s during hypoxia ± acidosis and maximum relaxation velocity deteriorated from −1,500 mmHg/s to about −600 mmHg/s. During reperfusion hearts subjected to hypoxia+acidosis recovered faster than hearts subjected to hypoxia alone, reaching control levels after 5 min of reperfusion. Electrophysiologic analysis revealed an 1.2 fold increase in both dispersion of activation-recovery interval and in total activation time in the hypoxia ± acidosis group. Cariporide application significantly improved LV hemodynamics and electrophysiology in the hypoxia group but not in the group subjected to hypoxia+acidosis. Immunohistologic analysis of cardiac specimen revealed a significant increase of factors involved in hypoxia/reperfusion injury like nitrotyrosine and poly-ADP-ribose as well as apoptosis-inducing factors like AIF or cleaved-caspase 3 in LV after hypoxia ± acidosis. ATP was reduced by hypoxia but not by acidosis. Again, cariporide mitigated these processes only in the hypoxia alone group, but not in the group with additional acidosis. Acidosis without hypoxia only marginally disturbed LV function and electrophysiology, and was not affected by cariporide. Thus, our study demonstrated that several detrimental effects of hypoxia were mitigated or abrogated by acidosis and that NHE-inhibition improved only hypoxia-induced cardiac dysfunction.

Objective: We aimed to study the effect of cariporide (CP) on protecting the saphenous vein and the role of acetaldehyde dehydrogenase 2 (ALDH2) in coronary artery bypass grafting (CABG). Background: The saphenous vein is the main graft material used in CABG. Recent studies suggested that CP is effective in protecting against various cardiovascular diseases. Methods: Segments of a surgically removed saphenous vein were used to examine the vascular response to CP. The ALDH2 genotype and expression of related proteins were assessed by Western blotting and immunohistochemistry. Results: Among the conditions tested, the University of Wisconsin solution with CP (4°C, 5 min) treatment showed the best protective effect on the saphenous vein. The incidence of major adverse cardiac events was higher in the ALDH2-GA (heterozygous mutant) genotype population after CABG. Conclusion: CP plays a role in reducing the production of reactive oxygen species and apoptosis by ALDH2-mediated mitochondrial function improvement. The ALDH2 mutant genotype might be one of the risk factors for coronary atherosclerotic heart disease. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that ion channels and transporters cooperate in regulating different aspects of tumor pathophysiology. In cancer cells, H+/HCO3- transporters usually invert the transmembrane pH gradient typically observed in non-neoplastic cells, which is thought to contribute to cancer malignancy. To what extent the pH-regulating transporters are functionally linked to K+ channels, which are central regulators of cell membrane potential (Vm), is unclear. We thus investigated in colorectal cancer cells the implication of the pH-regulating transporters and KV11.1 (also known as hERG1) in the pH modifications stimulated by integrin-dependent cell adhesion. Colorectal cancer cell lines (HCT 116 and HT 29) were seeded onto β1 integrin-dependent substrates, collagen I and fibronectin. This led to a transient cytoplasmic alkalinization, which peaked at 90 min of incubation, lasted approximately 180 min, and was inhibited by antibodies blocking the β1 integrin. The effect was sensitive to amiloride (10 µM) and cariporide (5 µM), suggesting that it was mainly caused by the activity of the Na+/H+ antiporter NHE1. Blocking KV11.1 with E4031 shows that channel activity contributed to modulate the β1 integrin-dependent pHi increase. Interestingly, both NHE1 and KV11.1 modulated the colorectal cancer cell motility triggered by β1 integrin-dependent adhesion. Finally, the β1 integrin subunit, KV11.1 and NHE1 co-immunoprecipitated in colorectal cancer cells seeded onto Collagen I, suggesting the formation of a macromolecular complex following integrin-mediated adhesion. We conclude that the interaction between KV11.1, NHE1, and β1 integrin contributes to regulate colorectal cancer intracellular pH in relation to the tumor microenvironment, suggesting novel pharmacological targets to counteract pro-invasive and, hence, pro-metastatic behavior in colorectal cancer. [ABSTRACT FROM AUTHOR]

Hypoxia often leads to severe cardiac malfunctions. It is assumed that intracellular calcium overload is -inter alia- responsible for left ventricular (LV) deterioration. Inhibition of the sodium-proton exchanger (NHE), which finally inhibits/slows calcium overload, may ameliorate cardiac function. Our aim was to evaluate cariporide, an inhibitor of NHE1 in a Langendorff-perfused heart model. To discriminate a potentially different impact of extracellular acidosis and hypoxia we examined 48 Chinchilla Bastard rabbits divided into 8 experimental groups: control group (pH = 7.4, O2 = 100%) without or with cariporide (1 μM), acidosis group (pH = 7.0, O2 = 100%) without or with cariporide (1 μM), hypoxia group (pH = 7.4, O2 = 40%) without or with cariporide (1 μM) and hypoxia+acidosis group (pH = 7.0, O2 = 40%) without or with cariporide (1 μM). Hearts were subjected to acidotic/hypoxic conditions for 90 min followed by 60 min of reperfusion. Hypoxia and hypoxia+acidosis led to a severe deterioration of LV function with a decrease in LV pressure by about 70% and an increase of end-diastolic pressure from 6.7 ± 0.6 to 36.8 ± 5.4 (hypoxia) or from 7.0 ± 0.2 to 18.6 ± 4.1 (hypoxia+acidosis). Moreover, maximum contraction velocity decreased from about 1,800 mmHg/s to 600 mmHg/s during hypoxia ± acidosis and maximum relaxation velocity deteriorated from −1,500 mmHg/s to about −600 mmHg/s. During reperfusion hearts subjected to hypoxia+acidosis recovered faster than hearts subjected to hypoxia alone, reaching control levels after 5 min of reperfusion. Electrophysiologic analysis revealed an 1.2 fold increase in both dispersion of activation-recovery interval and in total activation time in the hypoxia ± acidosis group. Cariporide application significantly improved LV hemodynamics and electrophysiology in the hypoxia group but not in the group subjected to hypoxia+acidosis. Immunohistologic analysis of cardiac specimen revealed a significant increase of factors involved in hypoxia/reperfusion injury like nitrotyrosine and poly-ADP-ribose as well as apoptosis-inducing factors like AIF or cleaved-caspase 3 in LV after hypoxia ± acidosis. ATP was reduced by hypoxia but not by acidosis. Again, cariporide mitigated these processes only in the hypoxia alone group, but not in the group with additional acidosis. Acidosis without hypoxia only marginally disturbed LV function and electrophysiology, and was not affected by cariporide. Thus, our study demonstrated that several detrimental effects of hypoxia were mitigated or abrogated by acidosis and that NHE-inhibition improved only hypoxia-induced cardiac dysfunction. [ABSTRACT FROM AUTHOR]

Background: The Na+/H+ exchanger (NHE1) plays a crucial role in cancer cell proliferation and metastasis. However, the mechanism underlying chemotherapeutic resistance in cancer cells has not been completely elucidated. The NHE1 inhibitor cariporide has been demonstrated to inhibit human cancer cell lines. The goal of this study was to provide new sights into improved cancer cell chemosensitivity mediated by cariporide with activation of the apoptosis pathway.Methods: The NHE1 expression levels were first evaluated using the online database Oncomine and were determined by RT-PCR and western blot in vitro and in vivo. Cell proliferation was assessed In vitro through a CCK-8 assay, and apoptosis was analyzed by flow cytometry. An in vivo analysis was performed in BALB/c nude mice, which were intraperitoneally injected with MCF-7/ADR cells.Results: NHE1 levels were significantly higher in breast cancer tissue than adjacent tissue, as well as in resistant cancer cells compared to sensitive cells. Cariporide induced the apoptosis of MCF-7/ADR cells and was associated with the intracellular accumulation of doxorubicin and G0/G1 cell cycle arrest. Moreover, cariporide decreased MDR1 expression and activated cleaved caspase-3 and caspase-9, promoting caspase-independent apoptosis in vitro. In vivo, cariporide significantly improved doxorubicin sensitivity in a xenograft model, enhancing tumor growth attenuation and diminishing tumor volume.Conclusions: Our results demonstrate that cariporide significantly facilitates the sensitivity of breast cancer to doxorubicin both in vitro and in vivo. This finding suggests that NHE1 may be a novel adjuvant therapeutic candidate for the treatment of resistant breast cancer. [ABSTRACT FROM AUTHOR]

Background: Na+/H+ exchanger-1 (NHE-1) is involved in pH regulation and is up-regulated in different malignancies. Activation of NHE-1 is one way for allowing cells to avoid intracellular acidification and protect them against apoptosis. Inhibitors of NHE-1 are able to decrease intracellular pH and induce apoptosis. Some statins can also act by partial inhibition of NHE-1. This review presents progress in understanding the mechanisms of action of these inhibitors, connections with certain genetic mutations and acquired treatment resistance, as well as new patents on them. Methods: A MEDLINE search for original and review articles using key terms, Na+/H+ exchanger, leukemia, cariporide, and amiloride. Recent patents with NHE-1 inhibitors published by United States Patent and Trademark Office are also presented. Results and Conclusions: Sorafenib is used for the treatment of acute myeloid leukemia patients carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation. 5-(N, N-hexamethylene)-amiloride can increase the suppression of FLT3 signaling by sorafenib. NHE-1 inhibitors are able to increase the sensitivity of chronic myeloid leukemia cells to tyrosine kinase inhibitors, including through the inhibition of P-glycoprotein. NHE-1 inhibitors are promising adjuvant drugs for overcoming acquired resistance to treatment in various malignant hemopathies.

Glioblastoma is an aggressive brain cancer that is very difficult to treat. Clinically, it is important to be able to distinguish aggressive from non-aggressive brain tumors. Previous studies have shown that some drugs can induce a rapid change in intracellular pH that could help to identify aggressive cancer. The sodium proton exchanger (NHE1) plays a significant role in maintaining pH balance in the tumor microenvironment. Cariporide is a sodium proton exchange inhibitor that is well tolerated by humans in cardiac applications. We hypothesized that cariporide could selectively acidify brain tumors. The purpose of this study was to determine whether amine/amide concentration-independent detection (AACID) chemical exchange saturation transfer (CEST) MRI measurement of tumor pHi could detect acidification after cariporide injection. Using a 9.4T MRI scanner, CEST spectra were acquired in six mice approximately 14 days after implanting 105 U87 human glioblastoma multiforme cells in the brain, before and after administration of cariporide (dose: 6 mg/kg) by intraperitoneal injection. Three additional mice were studied as controls and received only vehicle injection (DMSO + PBS). Repeated measures t test was used to examine changes in tumor and contralateral tissue regions of interest. Two hours after cariporide injection, there was a significant 0.12 ± 0.03 increase in tumor AACID value corresponding to a 0.48 decrease in pHi and no change in AACID value in contralateral tissue. A small but significant increase of 0.04 ± 0.017 in tumor AACID value was also observed following vehicle injection. This study demonstrates that acute CEST MRI contrast changes, indicative of intracellular acidification, after administration of cariporide could help localize glioblastoma. [ABSTRACT FROM AUTHOR]

This study aims to investigate the utilization of The Warburg Effect, cancer’s “sweet tooth” and natural greed for glucose to enhance the effect of monocarboxylate transporter inhibition on cellular acidification. By simulating hyperglycemia with high glucose we may increase the effectiveness of inhibition of lactate and proton export on the dysregulation of cell pH homeostasis causing cell death or disruption of growth in cancer cells. MCT1 and MCT4 expression was determined in MCF7 and K562 cell lines using RT-PCR. Cell viability, growth, intracellular pH and cell cycle analysis was measured in the cell lines grown in 5 mM and 25 mM glucose containing media in the presence and absence of the MCT1 inhibitor AR-C155858 (1 μM) and the NHE1 inhibitor cariporide (10 μM). The MCT1 inhibitor, AR-C155858 had minimal effect on the viability, growth and intracellular pH of MCT4 expressing MCF7 cells. AR-C155858 had no effect on the viability of the MCT1 expressing K562 cells, but decreased intracellular pH and cell proliferation, by a glucose-dependent mechanism. Inhibition of NHE1 on its own had a no effect on cell growth, but together with AR-C155858 showed an additive effect on inhibition of cell growth. In cancer cells that only express MCT1, increased glucose concentrations in the presence of an MCT1 inhibitor decreased intracellular pH and reduced cell growth by G1 phase cell-cycle arrest. Thus we propose a transient hyperglycemic-clamp in combination with proton export inhibitors be evaluated as an adjunct to cancer treatment in clinical studies. [ABSTRACT FROM AUTHOR]

Background. Na+/H+ exchanger-1 (NHE-1) is involved in pH regulation and is up-regulated in different malignancies. Activation of NHE-1 is one way for allowing cells to avoid intracellular acidification and protect them against apoptosis. Inhibitors of NHE-1 are able to decrease intracellular pH and induce apoptosis. Some statins can also act by partial inhibition of NHE-1. This review presents progress in understanding the mechanisms of action of these inhibitors, connections with certain genetic mutations and acquired treatment resistance, as well as new patents on them. Methods. A MEDLINE search for original and review articles using key terms, Na+/H+ exchanger, leukemia, cariporide, and amiloride. Recent patents with NHE-1 inhibitors published by United States Patent and Trademark Office are also presented. Results and Conclusions. Sorafenib is used for the treatment of acute myeloid leukemia patients carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation. 5-(N, N-hexamethylene)-amiloride can increase the suppression of FLT3 signaling by sorafenib. NHE-1 inhibitors are able to increase the sensitivity of chronic myeloid leukemia cells to tyrosine kinase inhibitors, including through the inhibition of P-glycoprotein. NHE-1 inhibitors are promising adjuvant drugs for overcoming acquired resistance to treatment in various malignant hemopathies. [ABSTRACT FROM AUTHOR]

Synopsis Background Direct myocardial cell protection in patients with unstable angina or evolving myocardial infarction (MI) could prevent cell necrosis or reduce its extent, and minimize the risk of MI and death associated with percutaneous coronary interventions (PCIs) and coronary artery bypass surgery. The myocardial NHE plays a critical role in mediating the progression of ischemia to necrosis by promoting intracellular accumulation of sodium and calcium in exchange for hydrogen. Blockage of the system in various experimental models of ischemia and reperfusion had a strong antinecrotic effect. The present paper describes a trial that was intended to investigate the potential clinical benefit of cariporide, a potent and selective inhibitor of the NHE, in a large spectrum of at-risk patients. Trial design The GUARDIAN trial was a multicenter, double-blind, randomized, four-arm trial that compared three cariporide dosages with placebo in patients with unstable angina and non-ST-segment elevation myocardial infarction (UA/NSTEMI) and in patients undergoing a high-risk PCI or coronary artery bypass surgery. A total of 11 590 patients with one of the three possible entry diagnoses were enroled in 23 countries. The trial was designed as a combined phase 2/phase 3 study. The primary objective was to evaluate the efficacy of cariporide in reducing all-cause mortality and/or MI across the various entry populations 36 days after randomization. Three different doses of cariporide were compared with placebo. Secondary end-points were death or non-fatal MI at 10 days and 6 months, and cardiac events related to left ventricular dysfunction. The extent of MI was also assessed by peak elevation in creatinine kinase (CK)-MB and a ratio of peak elevation to normal values. The sample size was driven by a total event rate of 1200 patients experiencing a primary end-point, powered to detect a 25% risk reduction in any of the three treatment groups compared with placebo at a significance level of 0.02, accounting for the three pair wise comparisons.

Platelets are anucleated cell fragments derived from mature megakaryocytes and function in hemostasis when the endothelium is injured. Hemostasis involving platelets can be divided into four phases: adhesion, activation, secretion, and aggregation. Platelet activation requires a rise in intracellular Ca 2 + concentrations and results in both a morphological change and the secretion of platelet granule contents. Na + /H + exchanger isoform 1 (NHE1) regulates the intracellular pH (pH i ) and the volume of platelets. In addition, NHE1 plays a large role in platelet activation. Thrombus generation involves NHE1 activation and an increase in [Ca 2 + ] i , which results from NHE1-mediated Na + overload and the reversal of the Na + /Ca 2 + exchanger. Cariporide (HOE-642), a potent NHE1 inhibitor, has inhibitory effects on the degranulation of human platelets, the formation of platelet–leukocyte-aggregates, and the activation of the GPIIb/IIIa receptor (PAC-1). However, despite the demonstrated protection against myocardial infarction as mediated by cariporide in patients undergoing coronary artery bypass graft surgery, the EXPEDITION clinical trial revealed that cariporide treatment increased mortality due to thromboembolic stroke. These findings suggest that a better understanding of NHE1 and its effect on platelet function and procoagulant factor regulation is warranted in order to develop therapies using NHE inhibitors. [ABSTRACT FROM AUTHOR]

Cardiac arrest requires immediate treatment, in order to prevent patient death. Cardiac arrest outcomes still remain very poor, especially when the patient requires vasopressor treatment. Vasopressors have been advocated, in order to increase the coronary and cerebral perfusion pressure during cardiopulmonary resuscitation (CPR). Recent data suggest an epinephrine-related benefit with respect to short- and long-term outcomes, only when epinephrine is administered within the first 10 min of collapse. Also, increasing the epinephrine dosing interval from 3-5 to 6-10 min during CPR may be associated with improved long-term outcomes. In the in-hospital setting, the combination of vasopressin, epinephrine, and corticosteroid supplementation during and after CPR (in the presence of post-resuscitation shock) may be superior to epinephrine alone during CPR. The use of new formulations of amiodarone, potentially devoid of serious hypotensive effects, may contribute to increased rates of sustained return of spontaneous circulation in patients with ventricular fibrillation/ pulseless ventricular tachycardia cardiac arrest. Encouraging preliminary results have been reported on the use of beta blockers in patients with shockable cardiac arrest. Other potentially promising pharmacological interventions include the use of cariporide, nitrates (and particularly inhaled nitric oxide), noble gases, levosimendan, and erythropoietin. The purpose of the current paper is to review the clinical and laboratory evidence that support new and potentially useful pharmacological interventions during CPR. [ABSTRACT FROM AUTHOR]

Aims To examine the effects of cariporide, a Na + /H + exchanger-1 inhibitor, on cardiac norepinephrine (NE) and myoglobin release during myocardial ischemia/reperfusion by applying a microdialysis technique to the rabbit heart. Main methods In anesthetized rabbits, two dialysis probes were implanted into the left ventricular myocardium and were perfused with Ringer's solution. Cariporide (0.3 mg/kg) was injected intravenously, followed by occlusion of the left circumflex coronary artery. During 30-min coronary occlusion followed by 30-min reperfusion, four consecutive 15-min dialysate samples (two during ischemia and two during reperfusion) were collected in vehicle and cariporide-treated groups. Dialysate myoglobin and NE concentrations were measured by immunochemistry and high-performance liquid chromatography, respectively. Key findings Dialysate myoglobin and NE concentrations increased significantly during myocardial ischemia/reperfusion in both vehicle and cariporide-treated groups (P < 0.01 vs. baseline). In cariporide-treated group, dialysate myoglobin concentrations were significantly lower than those in vehicle group throughout ischemia/reperfusion (P < 0.01 at 0–15 min of ischemia, P < 0.05 at 15–30 min of ischemia, P < 0.01 at 0–15 min of reperfusion, and P < 0.01 at 15–30 min of reperfusion). However, dialysate NE concentrations in cariporide-treated group were lower than those in vehicle group only during ischemia (P < 0.01 at 0–15 min of ischemia, and P < 0.05 at 15–30 min of ischemia). Significance When administered before ischemia, cariporide reduces myoglobin release during ischemia/reperfusion and decreases NE release during ischemia. [ABSTRACT FROM AUTHOR]

Aims: Recent studies have reported that intracellular calcium (Ca2+) mobilization is involved in homocysteine (Hcy)-induced endothelial dysfunction and the Na+/H+ exchanger (NHE) is responsible for an increase in the intracellular Ca2+ concentration in cardiovascular disease. We hypothesized that inhibition of the NHE had protective effects on Hcy-induced endothelial dysfunction. Methods: Acetylcholine-induced endothelium-dependent relaxation (EDR) and biochemical parameters were measured in the rat isolated aorta. The level of reactive oxygen species (ROS) was designed by a specific fluorescent probe. The phosphorylation of the nuclear factor-κB (NF-κB) system was studied by Western blot. Results: Cariporide significantly prevented Hcy-impaired EDR and increased nitric oxide (NO) release; endothelial NO synthase activity simultaneously decreased ROS production. We also found that cariporide blocked Hcy-induced NF-κB activation and inhibitor-κB degradation, thus inhibiting the production of tumor necrosis factor-α and intercellular adhesion molecule-1. Conclusions: The mechanisms of protective effects of cariporide may be related to the inhibition of NHE and the decrease in oxidative stress and inflammatory injury. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Sodium–hydrogen exchangers (NHE) are among the main regulators of cell volume and intracellular concentration of hydrogen and sodium ions. By indirectly affecting sodium/calcium exchange across the plasma membrane, NHE can also influence the intracellular concentration of calcium. Excess activation of NHE or inappropriate sodium extrusion due to failure of ATP-dependent Na+/K+ transport system can be deleterious during cardiac or peripheral organ ischemia. Besides being responsible for the regulation of intracellular pH and sodium–calcium inward currents, NHE isoform 1 (NHE-1), which is predominantly expressed in the cardiovascular system, influences the tone of the vessel wall in response to a variety of stimuli, including hypertonic stress. Because of the extensive involvement of NHE-1 in cardiac myocyte contracture and necrosis, stunning, reperfusion arrhythmias, as well as hypertension and myocardial diseases such as diabetic cardiomyopathy, efforts have been made in developing inhibitors of this transporter. We here review the biology and regulation of NHE, focusing on current knowledge of the role of NHE-1 as a potential target in the development of novel compounds that could play a role in cardiovascular homeostasis, both in physiological and pathological conditions. [Copyright &y& Elsevier]

Background: Cangrelor, a P2Y receptor blocker, administered just prior to reperfusion reduced but did not eliminate myocardial infarction in rabbits. Combining cangrelor with ischemic postconditioning offered no additional protection suggesting they protected by a similar mechanism. To determine if cangrelor's protection might be additive to other cardioprotective interventions we tested cangrelor in combination with ischemic preconditioning, cariporide, a sodium-hydrogen exchange blocker, and mild hypothermia. Methods: Open-chest rats underwent 30-min coronary occlusion/2-h reperfusion. Results: Cangrelor, administered as a bolus (60 μg/kg) 10 min before reperfusion and continued as an infusion (6 μg/kg/min) for the duration of the experiment, decreased infarction from 45.3 % of risk zone in control hearts to 25.0 %. Combining cangrelor and ischemic preconditioning offered no additional protection. Mild hypothermia (32-33 °C) instituted by peritoneal lavage with cold saline just prior to coronary occlusion resulted in 25.2 % infarction, and combining cangrelor and hypothermia nearly halved infarction to 14.1 % of risk zone. Cariporide (0.5 mg/kg) just prior to occlusion resulted in 27.2 % infarction and 15.8 % when combined with cangrelor. Combining cangrelor, hypothermia and cariporide further halved infarction to only 6.3 %. We also tested another P2Y inhibitor ticagrelor which is chemically similar to cangrelor. Ticagrelor (20 mg/kg) fed 1 h prior to surgery reduced infarct size by an amount similar to that obtained with cangrelor (25.6 % infarction), and this protective effect was abolished by chelerythrine and wortmannin, thus implicating participation of PKC and PI3-kinase, resp., in signaling. Conclusions: Cardioprotection from a P2Y receptor antagonist can be combined with at least 2 other strategies to magnify the protection. Combining multiple interventions that use different cardioprotective mechanisms could provide powerful protection against infarction in patients with acute coronary thrombosis. [ABSTRACT FROM AUTHOR]

Abstract: The concept of Na H exchange (NHE) involvement in cardiac pathology has been espoused for decades and supported by a plethora of experimental studies demonstrating salutary effects of NHE inhibition in protecting the myocardium against ischemic and reperfusion injury as well as attenuating myocardial remodelling and heart failure. NHE is actually a family of sodium and proton transporting proteins of which 10 isoforms have been identified. Myocardial NHE is represented primarily by the ubiquitous NHE-1 subtype which is expressed in most tissues. The robust positive results seen with NHE-1 inhibitors in experimental studies have led to relatively rapid development of these pharmacological agents for clinical assessment especially as potential cardioprotective therapies. Yet clinical studies have revealed, at best, inconsistent results as evidenced by poor efficacy and serious side effects, the latter revealed with the use of the NHE-1 inhibitor cariporide in high-risk patients undergoing coronary artery bypass grafting and evidenced by an increased incidence of cerebrovascular events of thromboembolic origin. The lack of success in clinical trials coupled with potential for toxicity has had a negative impact on development of cardiac therapeutic agents which have been developed based on the concept of NHE-1 inhibition. Whether this response is justified is open for discussion although a close scrutiny of clinical trial outcomes suggests that it may not be and that NHE-1 inhibition, if applied appropriately continues to represent an effective, if not the most effective approach for myocardial salvage following ischemic insult. Moreover, in addition to its cardioprotective effects, emerging evidence further suggests that NHE-1 inhibition is an effective strategy to minimize myocardial remodelling as well as a potentially effective strategy to improve efficacy of resuscitation following cardiac arrest. Thus, NHE-1 inhibition continues to represent a potentially highly effective therapeutic approach for the treatment of heart disease. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”. [Copyright &y& Elsevier]