A phase III study evaluating the safety and efficacy of NEPA, a fixed-dose combination of netupitant and palonosetron, for prevention of chemotherapy-induced nausea and vomiting over repeated cycles of chemotherapy.

BACKGROUND: Safe, effective and convenient antiemetic regimens that preserve benefit over repeated cycles are needed for optimal supportive care during cancer treatment. NEPA, an oral fixed-dose combination of netupitant, a highly selective NK1 receptor antagonist (RA), and palonosetron (PALO), a distinct 5-HT3 RA, was shown to be superior to PALO in preventing chemotherapy-induced nausea and vomiting after a single cycle of highly (HEC) or moderately (MEC) emetogenic chemotherapy in recent trials. This study was designed primarily to assess the safety but also to evaluate the efficacy of NEPA over multiple cycles of HEC and MEC. PATIENTS AND METHODS: This multinational, double-blind, randomized phase III study (NCT01376297) in 413 chemotherapy-naïve patients evaluated a single oral dose of NEPA (NETU 300 mg + PALO 0.50 mg) given on day 1 with oral dexamethasone (DEX). An oral 3-day aprepitant (APR) regimen + PALO + DEX was included as a control (3:1 NEPA:APR randomization). In HEC, DEX was administered on days 1-4 and in MEC on day 1. Safety was assessed primarily by adverse events (AEs), including cardiac AEs; efficacy by complete response (CR: no emesis, no rescue). RESULTS: Patients completed 1961 total chemotherapy cycles (76% MEC, 24% HEC) with 75% completing ≥4 cycles. The incidence/type of AEs was comparable for both groups. Most frequent NEPA-related AEs included constipation (3.6%) and headache (1.0%); there was no indication of increasing AEs over multiple cycles. The majority of AEs were mild/moderate and there were no cardiac safety concerns based on AEs and electrocardiograms. The overall (0-120 h) CR rates in cycle 1 were 81% and 76% for NEPA and APR + PALO, respectively, and antiemetic efficacy was maintained over repeated cycles. CONCLUSIONS: NEPA, a convenient single oral dose antiemetic targeting dual pathways, was safe, well tolerated and highly effective over multiple cycles of HEC/MEC.

Influence of pHi and creatine phosphate on alpha-adrenoceptor-mediated cardiac hypertrophy.

Stimulation of alpha-adrenoceptors on ventricular cardiomyocytes isolated from adult rat hearts leads to cellular alkalization, increases of creatine phosphate concentration, RNA mass, and protein synthesis. This study investigated whether the increase of creatine phosphate concentrations is causally linked to the hypertrophic response of cardiomyocytes under alpha-adrenoceptor stimulation. Cellular alkalization achieved with phenylephrine (10 microM), an alpha-adrenoceptor agonist, was abolished in the presence of the sodium-proton-exchange (NHE)-inhibitor HOE 694 (1 microM). HOE 694 inhibited also the alpha-adrenoceptor-mediated increase in cellular creatine phosphate and the increase in cellular RNA mass. The phenylephrine-induced stimulation of protein synthesis (determined by incorporation of 14C-phenylalanine) was reduced by one-third when HOE 694 was present. beta-Guanidinopropionic acid was added to cardiomyocytes to reduce cellular creatine phosphate concentrations. In these cultures, alpha-adrenoceptor stimulation activated NHE, but creatine phosphate concentrations were not increased. Protein synthesis was augmented to the same extent as in control cultures, but total RNA mass did not increase. From these results we conclude that alpha-adrenoceptor stimulation causes the increase in protein synthesis via activation of NHE, but independent of the concomitant increase in creatine phosphate contents. The effect of alpha-adrenoceptor stimulation on total RNA mass (translational capacity) is also caused by NHE activation, but depends on the changes in creatine phosphate contents as well.

Protection of rat cardiomyocytes against simulated ischemia and reoxygenation by treatment with protein kinase C activator.

The aim of this study was to investigate whether treatment with the protein kinase C (PKC) agonist 1,2-dioctanoyl-sn-glycerol (1,2DOG) can protect isolated adult Wistar rat cardiomyocytes against simulated ischemia and reoxygenation. Cytosolic Ca2+ (assessed by fura 2 fluorescence), pHi (assessed by BCECF fluorescence), and cell length were measured during 80 minutes of simulated ischemia (anoxia, pHo 6.4) and 20 minutes of reoxygenation (pHo 7.4) and compared between control cells and cells treated with 20 micromol/L 1,2DOG before anoxia (10-minute treatment and 10-minute washout), before and during anoxia (two-step treatment), or only during anoxia. Treatment before anoxia attenuated rigor contracture but did not influence anoxic Ca2+ overload. In contrast, two-step treatment before and during anoxia accelerated rigor contracture but reduced the rate of anoxic Ca2+ accumulation. During reoxygenation, control cells developed irreversible hypercontracture (reduction of cell length to 43+/-2% of the initial cell length, n=62), which was accompanied by spontaneous oscillations of cytosolic Ca2+ (19.6+/-1.6 per minute). Two-step treatment with 1,2DOG before and during anoxia significantly reduced hypercontracture (reduction of cell length to 60+/-2%, P<.01 versus control, n=41) and suppressed spontaneous Ca2+ oscillations (2.8+/-0.9 per minute, P<.01 versus control). These effects could not be reproduced by treatment with 1,2DOG before anoxia or during anoxia or by a two-step treatment with the PKC-inactive 1,3-dioctanoyl-sn-glycerol and were fully abolished with 1 micromol/L bisindolylmaleimide (PKC inhibitor). We conclude that a two-step activation of PKC before and during anoxia is required for effective protection of cardiomyocytes against anoxic Ca2+ overload and reoxygenation-induced hypercontracture.

Protection of isolated cardiomyocytes against reoxygenation-induced hypercontracture by SIN-1C.

Previous studies have shown that SIN-1C (N-morpholinoiminoacetonitrile) can protect ischemic-reperfused myocardium. The aim of the present study was to analyse on the cellular level the mechanism by which SIN-1C may exert this effect. To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in presence or absence of SIN-1C. Reoxygenation was started when intracellular Ca2+ (measured with fura-2) had increased to > or = 10(-5) mol/L and pHi (BCECF) decreased to 6.6. Development of hypercontracture was determined microscopically. In the control group reoxygenation provoked oscillations of cytosolic Ca2+ (60.9 +/- 9.6 min-1 at 5 min of reoxygenation) accompanied by development of hypercontracture (to 77.2 +/- 3.8% of end-ischemic cell length). When SIN-1C was added upon reoxygenation, Ca2+ oscillations were markedly reduced (27.0 +/- 4.5 min-1, p < 0.001) and hypercontracture virtually abolished (90.6 +/- 2.0% of end-ischemic cell length, p < 0.001). SIN-1C did not influence the recovery of pHi during reoxygenation. The results indicate that SIN-1C protects cardiomyocytes against reoxygenation-induced hypercontracture by its ability to suppress oscillations of intracellular Ca2+ during the early phase of reoxygenation.

Simulated ischemia increases the susceptibility of rat cardiomyocytes to hypercontracture.

The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca2+ after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca2+ concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Camax) was used as an index for their susceptibility to hypercontracture (SH). SH was increased in cardiomyocytes after prolonged periods of simulated ischemia; ie, these cells developed hypercontracture at significantly lower cytosolic Ca2+ levels than did normoxic cells (Camax, 0.80 +/- 0.05 mumol/L versus 1.27 +/- 0.05 mumol/L; P < .01). To find the possible cause of increased SH, the influence of Ca2+ overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca2+ overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Camax. In contrast, use of 10 mumol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Camax. Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Camax. Therefore, prolonged simulated ischemia increases susceptibility of cardio-myocytes to hypercontracture. This seems to be due to protein dephosphorylation.

Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture.

BACKGROUND: Resupply of oxygen to the myocardium after extended periods of ischemia or hypoxia can rapidly aggravate the already existing injury by provoking hypercontracture of cardiomyocytes (acute reperfusion injury). Previous studies indicated that halothane can protect ischemic-reperfused myocardium. The aim of the present study was to analyze on the cellular level the mechanism by which halothane may protect against reoxygenation-induced hypercontracture. METHODS AND RESULTS: To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in the presence or absence of 0.4 mmol/L halothane. Reoxygenation was started when intracellular Ca2+ (measured with fura 2) had increased to > or = 10(-5) mol/L and pHi (BCECF) had decreased to 6.5. Development of hypercontracture was determined microscopically. In the control group, reoxygenation provoked oscillations of cytosolic Ca2+ (72+/-9 per minute at fourth minute of reoxygenation) accompanied by development of hypercontracture (to 65+/-3% of end-ischemic cell length). When halothane was added on reoxygenation, Ca2+ oscillations were markedly reduced (4+/-2 per minute, P<.001) and hypercontracture was virtually abolished (90+/-4% of end-ischemic cell length, P<.001). Halothane did not influence the recovery of pHi during reoxygenation. Similar effects on Ca2+ oscillations and hypercontracture were observed when ryanodine (3 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ release, or cyclopiazonic acid (10 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, were applied instead of halothane. CONCLUSIONS: Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture by preventing oscillations of intracellular Ca2+ during the early phase of reoxygenation.

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes.

The inhibitors of the Na+/H+-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Nai) and calcium (Cai) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na+ and Ca2+ (pHi, Nai and Cai) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2, 7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pHi 7.2 to pHi 6.8) and an increase in Cai from about 50 nmol/l to about 1 micromol/l. The decrease in pHi began before the cells underwent hypoxic (rigor) contracture, whereas Cai only began to rise after rigor shortening had taken place. After reoxygenation, pHi returned to its control value and Cai oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 micromol/l Hoe 642 was present from the beginning of the experiment, pHi and Cai were not significantly different from control experiments. At reoxygenation, pHi did not recover, but Cai oscillated and returned to its resting level. To monitor Nai, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 micromol/l ouabain, Nai increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 micromol/l) did not prevent the increase in Nai. In contrast, the blocker of the persistent Na+ current R56865 (10 micromol/l) attenuated the CN--induced rise in Nai. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell's autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Cai overload, nor CN--induced Nai and Cai overload. Hoe 642 prevented the recovery of pHi from anoxic acidification. This low pHi maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca2+ overload in mitochondria, cannot be ruled out. The increase in Nai during anoxia is possibly due to an influx of Na+ via persistent Na+ channels.

The role of Na+/H+ exchange in ischemia-reperfusion.

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pH(i)-regulating transport systems involved is the Na+/H+ exchanger. Inhibitors of the Na+/H+ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na+ and secondary Ca2+ influx. Under reperfusion conditions Na+/H/ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na+/H+ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.