Dilazep analogues for the study of equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2).

As ENT inhibitors including dilazep have shown efficacy improving oHSV1 targeted oncolytic cancer therapy, a series of dilazep analogues was synthesized and biologically evaluated to examine both ENT1 and ENT2 inhibition. The central diamine core, alkyl chains, ester linkage and substituents on the phenyl ring were all varied. Compounds were screened against ENT1 and ENT2 using a radio-ligand cell-based assay. Dilazep and analogues with minor structural changes are potent and selective ENT1 inhibitors. No selective ENT2 inhibitors were found, although some analogues were more potent against ENT2 than the parent dilazep.

Protective effects of dilazep and its derivative K-7259 on the haemolysis induced by amphiphiles in rat erythrocytes.

The effects of dilazep and K-7259, a dilazep derivative, on the haemolysis (as evidenced by release of haemoglobin) induced by palmitoyl-L-carnitine (PAL-CAR) or palmitoyl 1-alpha-lysophosphatidylcholine (PAL-LPC) have been determined in rat erythrocytes. At concentrations above the critical micelle concentration both PAL-CAR and PAL-LPC induced haemolysis; the concentrations of PAL-CAR and PAL-LPC producing 50% haemolysis were approximately 13 and 14 microM, respectively. The 50% haemolysis induced by PAL-CAR or PAL-LPC was attenuated by dilazep (1, 10 or 100 microM) but not at the highest concentration used (1 mM). K-7259 attenuated the 50% haemolysis induced by PAL-CAR or PAL-LPC at concentrations ranging from 1 microM to 1 mM. Similarly, dilazep (1 to 100 microM) and K-7259 (1 microM to 1 mM) significantly or insignificantly attenuated the 25% and 75% haemolysis induced by PAL-CAR or PAL-LPC. Neither dilazep nor K-7259 affected micelle formation by PAL-CAR or PAL-LPC, nor, at concentrations of 1 and 10 microM, did they attenuate the haemolysis induced by osmotic imbalance (hypotonic haemolysis). These results suggest that both dilazep and K-7259 protect the erythrocyte membrane from the damage induced by PAL-CAR or PAL-LPC. The protective effects of dilazep and K-7259 are mediated by some mechanism other than prevention of micelle formation or protection of the erythrocyte membrane against osmotic imbalance.

Cardioprotective effect of K-7259, a novel dilazep derivative, against ischemia-reperfusion damage in isolated, working rat hearts.

Global ischemia (15 min) followed by reperfusion (10, 20 or 30 min) was performed in isolated, working rat hearts. Ischemia depressed mechanical function, which was not restored by reperfusion of 20 min. Preischemic administration of K-7259 (N,N'-bis[4-(3,4,5-trimethoxyphenyl)butyl]homopiperazine dihydrochloride) (1, 5 or 10 microM) decreased the function before ischemia, but it attenuated the ischemia-induced dysfunction during reperfusion (20 min). Postischemic administration of K-7259 (10 microM) or dilazep (20 microM) also attenuated the ischemia-induced dysfunction during reperfusion (30 min). Ischemia-reperfusion (10 min) increased the tissue malondialdehyde level, and postischemic administration of K-7259 (10 microM) or dilazep (20 microM) attenuated the malondialdehyde accumulation. K-7259 has a cardioprotective effect when given either before or after ischemia.

A new approach to the development of anti-ischemic drugs. Substances that counteract the deleterious effect of lysophosphatidylcholine on the heart.

Lysophosphatidylcholine (LPC) is an amphiphilic metabolite that can be produced from membrane-phospholipids by activation of phospholipase A2 (PLA2), and it accumulates in the heart during ischemia and reperfusion. It is known that LPC is an arrhythmogenic substance. Recent studies have revealed that LPC produces mechanical and metabolic derangements in perfused working rat hearts, and Ca(2+)-overload in isolated cardiac myocytes. Thus, LPC possesses an ischemia-like effect on the heart. LPC accumulated in the myocardium activates phospholipase A2, establishing a vicious circle; i.e. LPC itself has an ability to produce another LPC. Therefore, a drug that has an anti-LPC effect would protect or improve ischemia/reperfusion damage. This article will review the effect of LPC in relation to ischemia, and consider a possibility of developing new anti-ischemic drugs on the basis of the anti-LPC action.

Dilazep and its derivative, K-7259, attenuate mechanical derangement induced by palmitoyl-L-carnitine in the isolated, perfused rat heart.

The effect of dilazep, a potentiator of the adenosine-mediated effects, on the palmitoyl-L-carnitine (PALCAR)-induced mechanical derangement was studied in the isolated rat heart and compared with that of K-7259, a dilazep derivative having less potentiating action on the adenosine-mediated effects. The heart was perfused aerobically by the Langendorff's technique at a constant flow and driven electrically. PALCAR (5 microM) decreased the left ventricular developed pressure and increased the left ventricular end diastolic pressure in the heart (i.e., mechanical dysfunction). These mechanical alterations induced by PALCAR were attenuated by dilazep (1 microM) and K-7259 (1 microM). In contrast, adenosine (10 or 100 microM) did not attenuate the PALCAR-induced mechanical derangement. On the other hand, neither dilazep nor K-7259 modified the mechanical function of the normal (PALCAR-untreated) heart. These results suggest that dilazep and K-7259 attenuate the PALCAR-induced mechanical derangement and that the protective action of both drugs is not due to potentiation of adenosine-mediated effects.

[A new approach to the understanding of the mechanism of ischemia/reperfusion damage in the heart and the effects of anti-ischemic drugs].

The classical understanding of the mechanism of anti-anginal or anti-ischemic drugs is an increase in blood supply to the heart and/or a decrease in oxygen consumption of the heart, maintaining energy balance in the heart between supply and demand and hence maintaining the tissue levels of high-energy phosphates. This scheme is reasonable. During reperfusion following ischemia, however, there is more serious damage to the heart, although the tissue levels of high-energy phosphates increase. This is probably because toxic substances are generated in the heart during ischemia/reperfusion. We propose that both lysophosphatidylcholine and palmitoyl-L-carnitine that accumulate in the myocardium during ischemia/reperfusion are candidates for the toxic substances that accelerate ischemia/reperfusion damage to the heart. Therefore, drugs that have anti-lysophosphatidylcholine and/or anti-palmitoyl-L-carnitine effects are promising for the treatment of ischemic heart diseases. We found that K-7259, a novel derivative of dilazep having a minimal effect on the normal heart, is a drug that attenuates the deleterious effects of both lysophosphatidylcholine and palmitoyl-L-carnitine on the heart, and therefore attenuates the ischemia/reperfusion damage.

Protective effects of dilazep and its novel derivative, K-7259, on mechanical and metabolic derangements induced by hydrogen peroxide in the isolated perfused rat heart.

The effect of dilazep, a potentiator of the adenosine-mediated effects, on the hydrogen peroxide (H2O2)-induced mechanical and metabolic derangements was studied in the isolated rat heart, and compared with that of K-7259, a dilazep derivative having less potentiating action on the adenosine-mediated effects. The heart was perfused aerobically by Langendorff's technique at a constant flow and driven electrically. H2O2 (600 microM) decreased the left ventricular developed pressure and increased the left ventricular end diastolic pressure in the heart (i.e, mechanical dysfunction), decreased the tissue ATP level and increased the tissue AMP level (i.e., metabolic change) and increased the tissue level of malondialdehyde (i.e., lipid peroxidation). These mechanical and metabolic alterations induced by H2O2 were attenuated by dilazep (1 microM), and the effect of dilazep was not modified by 8-(p-sulfophenyl)-theophylline (20 microM), a nonselective adenosine receptor antagonist. K-7259 (1 microM) also attenuated the H2O2-induced mechanical and metabolic derangements. Nevertheless, neither dilazep nor K-7259 modified the tissue malondialdehyde level, which was increased by H2O2, and the mechanical function and energy metabolism of the normal (H2O2-untreated) heart. These results suggest that both dilazep and K-7259 attenuate mechanical and metabolic derangements induced by H2O2. The protective action of dilazep and K-7259 on the H2O2-induced derangements is not due to potentiation of adenosine-mediated effects, reduction of lipid peroxidation or preservation of energy.

K-7259, a novel dilazep derivative, and d-propranolol attenuate H2O2-induced cell damage.

We studied the effects of dilazep, K-7259 (a novel derivative of dilazep) and d-propranolol on the change in cell shape and accumulation of nonesterified fatty acids (NEFA) induced by hydrogen peroxide (H2O2) in isolated rat cardiac myocytes. Myocytes were incubated in a Krebs-Ringer bicarbonate buffer containing 2 mM diethyltriamine pentaacetic acid (DETAPAC) and 2mM FeSO4 for 10 min, and then treated with 2mM H2O2 for 50 min. Before the treatment with H2O2, the percentage of the number of rod-shaped cells to that of total cells was 66 +/- 2%, and decreased to 35 +/- 3%, 25 +/- 4% and 14 +/- 2%, after 30, 40 and 50 min of the H2O2 treatment, respectively. The levels of NEFA (lauric, myristic, palmitoleic, arachidonic, linoleic, palmitic, oleic and stearic acids) increased after the treatment with H2O2. In the absence of FeSO4 and DETAPAC, however, H2O2 did not have these effects, and therefore all the experiments with drugs were performed in the presence of Fe2SO4 and DETAPAC. K-7259 (30 microM) and d-propranolol (50 microM) attenuated both the changes in cell shape and accumulation of NEFA induced by H2O2, whereas dilazep (30 or 50 microM) did not. N-(2-mercaptopropionyl)glycine (2 mM), an .OH scavenger, inhibited the H2O2-induced changes completely. These results suggest that K-7259 and d-propranolol attenuate the H2O2-induced changes in cell shape and accumulation of NEFA, probably because of their .OH-scavenging effect.

Suppression of age-related changes in mouse hippocampal CA3 nerve cells by a free radical scavenger.

This study was designed to evaluate the relationship between oxygen free radicals and age-related morphological changes in hippocampal nerve cells using K-7259 (N,N' bis[4-(3,4,5-trimethoxyphenyl)butyl] homopiperazine dihydrochloride), a known neuro-protective agent. A chemiluminescence assay has shown that this agent is a potent free radical scavenger with an IC50 of 1.6 x 10(-5)M. Mice fed diets containing 10, 20, 40 mg/kg/day of K-7259, for periods ranging from 25 to 40 or 50 weeks of age were used as test groups, and 10-, 20-, 30-, 40-, and 50-week-old mice fed a standard diet were used as controls. We measured the number and area of pyramidal nerve cells within a defined frame in the hippocampal CA3 field using an image analyzer and the density of nerve cells by the disector method. These values decreased gradually in controls as expected, and the number and area yielded a significant difference between control mice at the ages of 10 and 30 weeks. As compared with the corresponding controls, all test groups had greater cell numbers (statistical significance at 40 weeks in the 40 mg/kg/day group) and density, while cell areas were greater in all but a 10 mg/kg/day group (statistical significance at 50 weeks). In summary, the free radical scavenger K-7259 forestalled an age-related decrease in the number and size of hippocampal CA3 nerve cells, thus suggesting that free radicals play an important role in the cellular morphological changes which appear with age.

Effect of K-7259, a novel derivative of dilazep, on cardiovascular actions of adenosine: comparison with dilazep.

The effect of K-7259, a novel derivative of dilazep, on the cardiovascular action of adenosine was studied in the aortic ring and isolated perfused heart in guinea-pigs, and compared with that of dilazep. Adenosine produced a concentration-dependent relaxation in phenylephrine (2 x 10(-6) M)-contracted aortic rings and exhibited a negative chronotropic effect in the isolated perfused heart. Dilazep (10(-8), 10(-7) and 10(-6) M) potentiated significantly the relaxing action of adenosine on the aortic ring in a concentration-dependent way. K-7259 (10(-6) M), however, did not potentiate the relaxing action of adenosine, although the high concentration of K-7259 (10(-5) M) potentiated it slightly but significantly. The negative chronotropic effect of adenosine was also potentiated by dilazep (10(-7) and 10(-6) M), but not by K-7259 (10(-6) M). These results suggest that the potentiating action of K-7259 on the cardiovascular effects of adenosine is very weak when compared with that of dilazep.

Effects of K-7259 on neuronal activity and synaptic transmission in the rat dorsolateral septal nucleus.

K-7259 is a dilazep dihydrochloride derivative that minimizes the damaged area from middle cerebral artery hemiocclusion in the rat (Yamauchi et al. 1992a, b). The effects of K-7259 on the electrophysiological properties of neurons in the rat dorsolateral septal nuclei (DLSN) were examined. K-7259 (100 microM-3 mM) depolarized the membrane with a decrease in input resistance in 36% of the DLSN cells. K-7259 (100 microM) depressed the inhibitory postsynaptic potential (IPSP) and the late hyperpolarizing potential (LHP). The magnitudes of the depressions of the IPSP and LHP with 100 microM K-7259 were 50 +/- 25% (n = 5) and 52 +/- 15% (n = 4), respectively. The amplitudes of the excitatory postsynaptic potentials (EPSPs) were augmented during the inhibition of the IPSP and LHP. However, a voltage-clamp analysis showed that K-7259 did not affect the isolated excitatory postsynaptic current (EPSC). The outward current produced by pressure application of gamma-aminobutyric acid (GABA) to the recording cell was not inhibited by K-7259. These results indicate that K-7259 presynaptically inhibits the IPSP and LHP through a GABAergic pathway.