Multiple symmetric lipomatosis: a defect in adrenergic-stimulated lipolysis.

The cellularity of normal and lipomatous adipose tissue and its response to different lipolytic agents have been studied in a group of 10 patients with multiple symmetric lipomatosis (MSL). In MSL patients, fat cells from lipomatous tissue are smaller than normal, uninvolved adipocytes. Fat cells from lipomata show minimal variations in size following conspicuous increase of lipomatous masses. These findings suggest that the growth of lipomata can be attributed to the neoformation of adipocytes rather than to an enlargement in the single fat cells. The incidence of reduced glucose tolerance and of hyperlipoproteinemia is similar in MSL patients and in controls. A significant reduction in plasma free fatty acids was observed in MSL patients after a 24-h fast as well as after noradrenaline infusion. A specific insensitivity of lipomatous tissue to the lipolytic effect of noradrenaline and isoprenaline was observed in vitro, as indicated by glycerol release in the medium, whereas response to theophylline and to dibutyryl cyclic AMP was retained. The lipolytic response to catecholamines was retained. The lipolytic response to catecholamines was normal in the nonlipomatous adipose tissue of MSL patients. In basal conditions ATP concentrations were similar in normal and in lipomatous adipose tissue. However, incubation with noradrenaline induced a significant fall in intracellular ATP levels in normal tissue, whereas no variations were observed in lipomatous tissue. Theophylline, instead, induced a prompt and significant decrease in intracellular ATP levels in lipomatous tissue. These observations indicate that the block in catecholamine-stimulated lipolysis in lipomatous tissue of MSL patients can be localized at a level preceding the formation of cyclic AMP.

A pharmacological, crystallographic, and quantum chemical study of new inotropic agents.

The cardiac activity of a series of milrinone analogues, 2-substituted 3-acyl-1,6-dihydro-6-oxo-5-pyridinecarbonitriles, 1,6,3,2,11,12-hexahydro-6,3-dioxo-5-quinolinecarbonitriles, the correlated carboxylic acids, 2-substituted 3-acyl-6(1H)-pyridones, and 7,8-dihydro-2,5(1H,6H)-quinolinediones, was evaluated in spontaneously beating and in electrically driven atria from reserpine-treated guinea pigs. Their effects were compared with those induced by amrinone and milrinone in both the atria preparations. Compounds SF28 (3-acetyl-1,6-dihydro-2-methyl-6-oxo-5-pyridinecarbonitrile) and SF40 (7,8-dihydro-7-methyl-2,5(1H,6H)-quinolinedione) were the most effective positive inotropic agents. An inhibition of the negative influence exerted by endogenous adenosine on heart preparations seems to be involved in their contractile activity. SF38 (3-benzoyl-2-phenyl-6(1H)-pyridinone), on the contrary, reduced the contractile force and the frequency rate of guinea pig atria with a mechanism not related to an activation of cholinergic or purinergic inhibitory receptors on the heart. X-ray analysis carried out on the three model compounds, SF28, SF40 (positive inotropic agents), and SF38 (negative inotropic agent), and molecular modeling evidenced that the change from phenyl (SF38) to methyl (SF28) or the introduction of a side cyclic aliphatic chain (SF40) results in a variation of conformational preference and topography which may address the different molecules toward distinct receptor pockets according to the resulting inotropic effect.

Synthesis and cardiotonic activity of novel pyrimidine derivatives: crystallographic and quantum chemical studies.

The synthesis of ethyl or methyl 4-substituted or unsubstituted 2-(dimethylamino)-5-pyrimidinecarboxylates 10-20, which is mainly carried out by reaction of ethyl or methyl 2-[(dimethylamino)methylene]-3-oxoalkanoates with 1,1-dimethylguanidine, is described. The above esters were hydrolyzed to the relative carboxylic acids 21-30, which were decarboxylated to the corresponding 2,4-disubstituted pyrimidines 31-40. All the new synthesized pyrimidines were evaluated in spontaneously beating and electrically driven atria from reserpine-treated guinea pigs. Their effects were compared to those induced by milrinone in both atria preparations. Compound 28 (4-benzyl-2-(dimethylamino)-5-pyrimidinecarboxylic acid) was the most effective positive inotropic agent, while the corresponding methyl ester 17 reduced both the contractile force and the frequency of guinea pig atria. An antagonism toward the negative influence exerted by endogenous adenosine on the heart seems to be involved in the contractile activity of compound 28. By contrast, compound 17 might be partial agonist at the purinergic inhibitory (A1) receptor. X-ray analysis carried out on 17 and 28 and molecular modeling investigations extended also to related derivatives allowed a possible rationalization between structure and inotropic activity for this series of compounds.

Interaction of amrinone with endogenous adenosine in guinea-pig atria.

In spontaneously beating atria from reserpine-treated guinea-pigs, amrinone (10 microM to 2 mM) induced a positive inotropic and chronotropic effect that was preceded by a transient reduction in contractile force and in frequency. Both the positive and negative effects were concentration-dependent. The inotropic action of amrinone was antagonized by low concentrations of 8-phenyltheophylline that compete with adenosine at R-receptors on plasma membrane without significantly influencing phosphodiesterase activity. Cumulative concentrations of amrinone (1 mM) antagonized the reduction of rate of contraction and amplitude induced by dipyridamole 1 microM in spontaneously beating atria and restored the maximum contractile effect reached in the absence of dipyridamole. In spontaneously beating preparations incubated in the presence of adenosine deaminase (1 u ml-1), amrinone lost its positive effects on the atria and only reduction of rate and contractile force was evident. Both effects were antagonized by scopolamine 1 mM thus indicating their cholinergic nature. Adenosine at 0.1 microM and 0.5 microM significantly inhibited the inotropic effect induced by amrinone (0.03 to 3 mM) and the concentration-effect curves of amrinone obtained in the absence and presence of adenosine clearly indicate a competitive antagonism between the two drugs. Thus the contractile activity of amrinone in spontaneously beating atria from reserpine-treated guinea-pigs originates from a displacement of adenosine from its R-receptor sites in the cardiac cell.

Impairment of endothelium-dependent but not of endothelium-independent dilatation in guinea-pig aorta rings incubated in the presence of elevated glucose.

1. Purine compounds such as ATP and adenosine, respectively endothelium-dependent and- independent vasodilators, are largely involved in the control of vascular tone and vascular reactivity to contracting stimuli. We investigated the relaxing activity of ATP and adenosine in guinea-pig aorta rings exposed for 6 h to elevated glucose concentration (50 mM), in order to mimic hyperglycaemic conditions. Guinea-pigs were reserpine-treated (2 mg kg-1, i.p., 48 and 24 h before death). 2. Rings of aortae incubated in 50 mM glucose, contracted submaximally by 1 microM noradrenaline, lost endothelium-dependent relaxation in response to acetylcholine (10 nM to 10 microM). Aortae incubated with 50 mM mannose, as a hyperosmotic control, relaxed to acetylcholine normally. Rings of aortae incubated in 50 mM glucose, contracted submaximally by 3 mM 4-aminopyridine, lost endothelium-dependent relaxation in response to ATP (30 microM) whereas endothelium-independent relaxation in response to adenosine (0.3 mM) was well preserved. 4. The relaxation induced by A23187 or sodium nitroprusside (10 nM to 0.1 microM) did not differ between rings exposed to control (5.5 mM) or elevated glucose (50 mM) and contracted submaximally by 3 mM 4-aminopyridine. 5. When incubated with aortic tissue in the presence of elevated glucose, the cyclo-oxygenase inhibitors, indomethacin (10 microM) and mefenamic acid (30 microM), or the scavenger of superoxide anions, superoxide dismutase (150 u ml-1), prevented the impairment of ATP-mediated relaxation. 6. The present results indicate that endothelium-dependent, receptor-induced relaxation in response to acetylcholine and ATP is impaired in guinea-pig aorta rings exposed to elevated glucose. The endothelial dysfunction caused by glucose might be located at a step between receptor activation and intracellular calcium increase, and might be related to an increased metabolism of arachidonic acid coupled to an increased production, or to a reduced inactivation of superoxide anions.

Endothelium is required in the vascular spasm induced by tetraethylammonium and endothelin-1 in guinea-pig aorta.

1. To investigate the role of endothelium in vascular spasm, we studied the influence of endothelin-1 (ET-1) on the contracting and spasmogenic effect of the K+-channel blocker, tetraethylammonium (TEA), in aorta rings of reserpine-treated guinea-pigs, perfused with either control (5.5 mM) or elevated (50 mM) glucose concentration. 2. Endothelium-dependent relaxation induced by acetylcholine was lost in rings contracted by noradrenaline in the presence of elevated glucose. In control medium, TEA (1-20 mM) induced a sustained tonic contraction, followed by a phasic spasm, characterized by rhythmic contractions. Elevated glucose, ET-1 (3 nM), or both, reduced the EC50 of TEA-induced tonic contraction, without modifying the maximum contractile effect. 3. In control medium, ET-1 reduced the time before TEA-induced spasm and increased the rate of rhythmic contractions. TEA-induced spasm was abolished by elevated glucose, and restored by ET-1. The spasm induced by TEA and ET-1 was amplified by the ETA antagonist, EMD94246, and suppressed by the ET(A)-ET(B) antagonist, bosentan. In endothelium-denuded vessels incubated with high glucose and ET-1, TEA evoked only a tonic contraction. 4. In control medium, L-NAME (N(G)-nitro-L-arginine methyl ester) abolished TEA-induced rhythmic contractions. L-arginine, but not D-arginine, prevented the effect of L-NAME. In the presence of elevated glucose and ET-1, TEA-induced spasm was not affected by L-NAME, whereas verapamil, indomethacin, metyrapone, glybenclamide or apamin abolished the phasic spasm, unmasking the tonic contracture. 5. In conclusion, endothelium plays a regulatory role in the genesis and maintenance of TEA-induced rhythmic contractions, through the release endothelium derived relaxing factor and vasodilating eicosanoids.

An analysis of the mechanism of the inotropic action of some milrinone analogues in guinea-pig isolated atria.

1. It has been reported previously that the milrinone analogues, ethyl 5-cyano-1,6-dihydro-2-methyl-6-oxo-3 pyridine carboxylate (I) and ethyl 5-cyano-1,6-dihydro-2-ethyl-6-oxo-3 pyridine carboxylate (II) exert a positive inotropic effect (EC50 = 15.6 +/- 0.2 microM and 40.3 +/- 0.1 microM) both on spontaneously beating and on electrically driven atria from reserpine-treated guinea-pigs. In the present study the mechanism of the inotropic action of these two agents was investigated. 2. In electrically driven left atrium from reserpine-treated guinea-pigs the EC50 values for inotropic activity for compounds (I) and (II) corresponded to that of milrinone (EC50 = 25 +/- 0.1 microM) but compound (I) induced a greater maximum effect. This corresponded to a percentage increase in developed tension over control of 63 +/- 0.3 whereas the maximum inotropic effect of milrinone was 48 +/- 0.3 and that of compound (II) was 47 +/- 0.2. 3. The inotropic activity of compounds (I) and (II) (10-100 microM) was resistant to propranolol (0.1 microM), thus excluding the involvement of beta-adrenoceptors. 4. Since the inotropism induced by compounds (I) and (II) was not reduced by carbachol (1 nM-0.5 microM), an action involving changes in adenosine 3':5'-cyclic monophosphate (cyclic AMP) can be excluded. 5. The inotropic action of compounds (I) and (II) was blocked selectively by 8-phenyltheophyline (10 microM) or adenosine deaminase (2 u ml-1). 6. Both (I) and (II) inhibited, in an apparently competitive manner, the negative inotropic effect induced by N6-(L-phenylisopropyl) adenosine (L-PIA), a stable adenosine agonist. The pA2 values for (I) and (II) were 4.79 and 4.36, respectively.7. In rat brain compounds (I) and (II) inhibited the specific binding of N6-cyclohexyl[3H]-adenosine- ([3H]-CHA) with an IC50 of 0.18 + 0.01 mM and 0.25 + 0.02 mm, respectively, which were similar to their IC50 values for blocking the PIA-induced negative inotropic effect and which are also in the range of concentrations that are effective in inducing positive inotropism in guinea-pig atria.8. The results from the present study suggest that antagonism of endogenous purines causes positive inotropism without affecting intracellular cyclic AMP levels.

A reexamination of the effects induced by adenosine and its degradation products on rat fat cell lipolysis.

The effects of adenosine and of some products of its metabolic degradation on lipolysis were studied in rat fat cells isolated from epididymal adipose tissue. Basal glycerol release was not affected by adenosine and by uric acid, but it was significantly increased by inosine (1-100 microM) and by hypoxanthine (10-100 microM). Adenosine was more effective than inosine in antagonizing the lipolytic response of fat cells to theophylline. Also hypoxanthine and uric acid exerted a very potent, noncompetitive antagonism towards theophylline. Norepinephrine-induced lipolysis was inhibited by adenosine, hypoxanthine and uric acid approximately to the same extent, while inosine was ineffective at this level. Adenosine deaminase (0.5 U/ml) increased basal as well as theophylline- and norepinephrine-induced lipolysis. Moreover, adenosine deaminase enhanced the lipolytic rate in cells incubated with low (0.1, 1 microM) and, to a lesser extent, with high (10, 100 microM) inosine concentrations. These results suggest that inosine is the adenosine metabolite that may accumulate in the incubation medium following fat cell treatment with adenosine deaminase, thus contributing to the stimulatory effects of this enzyme on lipolysis.

Prostacyclin and lipolysis in rat fat cells.

In rat fat cells incubated for 15 min at 37 degrees and pH 8.5, glycerol release was highly stimulated both by norepinephrine and by theophylline. Prostacyclin (PGI2) (10(-8)-10(-7)M) did not alter the basal rate of glycerol release but potentiated the lipolytic effect of 2 X 10(-6)M norepinephrine. The rate of norepinephrine-induced glycerol release was increased by PGI2 during 10 min of incubation and then maintained for the next 5 min. Lipolysis induced by concentrations of norepinephrine which produced maximal effects was not altered by PGI2. PGI2 (10(-7)-10(-6)M) also potentiated the effect of 5 X 10(-4)M theophylline on glycerol release, but antagonized the stimulation induced by a maximally effective concentration of the methylxanthine (2 X 10(-3)M). Incubation of the cells with norepinephrine in the presence of 2 X 10(-4) or 5 X 10(-4)M theophylline caused a loss of the potentiating effect of PGI2 on norepinephrine-induced lipolysis. In the presence of 10(-3)M theophylline, the lipolytic action of norepinephrine was inhibited by PGI2. In fat cells incubated with adenosine deaminase (0.5 U/ml), 2.5 X 10(-7)M PGI2 did not alter the response to 5 X 10(-4)M theophylline and inhibited the effect of norepinephrine both in the absence and in the presence of theophylline. The present results show that, under appropriate experimental conditions, PGI2 may act as a lipolytic agent in isolated fat cells and that some kind of interaction exists between stimulation of methylxanthine-sensitive adenosine receptors and stimulation of PGI2 receptors.

New milrinone analogues: in vitro study of structure-activity relationships for positive inotropic effect, antagonism towards endogenous adenosine, and inhibition of cardiac type III phosphodiesterase.

Two mechanisms are responsible for the positive inotropic effect of the cardiotonic drug milrinone, i.e., inhibition of type III cAMP phosphodiesterase (PDE III), and displacement of endogenous adenosine from A(1) inhibitory receptor. Since PDE III inhibition may increase the likelihood of cardiac arrhythmias by increasing cAMP content, our attention focused on the synthesis of new compounds with more pronounced characteristics as adenosine antagonists. In this work, four new milrinone analogues were studied, in comparison with the parent drug, for their effects on the contractility of guinea pig isolated atrial preparations, their ability to antagonize endogenous adenosine at the level of A(1) receptor, and to inhibit the activity of PDE III partially purified from guinea pig heart. The new compounds present various chemical substitutions with respect to the parent drug: in compounds SF397 (methyl 5-cyano-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate) and SF399 (benzyl 5-cyano-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate), the 4-pyridil moiety of milrinone was replaced with a methoxycarbonyl and a benzyloxycarbonyl group, respectively; the same structural modifications were also associated with the replacement of the cyano-group in 5-position with an acetyl group in compounds SF416 (methyl 5-acetyl-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate) and SF419 (benzyl 5-acetyl-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate). All the new compounds had a marked positive inotropic effect, most of them also being more active and more potent than milrinone. When their affinity for A(1) receptor was assessed as the displacement of [(3)H] 8-cyclopentyl-1,3-dipropylxanthine ([(3)H]DPCPX) from cardiac membranes, SF397 and SF399 showed affinity (K(i) of about 600 nM) similar to that of milrinone (K(i) 550 nM). By contrast, SF416 and SF419 had very low (K(i) of about 10000 nM) or scarce (K(i) of about 2000 nM) anti-adenosine component, respectively. All the new compounds inhibited PDE III activity, their K(i) values proceeding in the following order: milrinone (3.80 microM)

Synthesis and cardiotonic activity of 2-substituted 5-cyano-1,6-dihydro-6-oxo-3-pyridinecarboxylic acids and their methyl or ethyl esters.

The synthesis of ethyl or methyl esters of 5-cyano-1,6-dihydro-6-oxo-3- pyridinecarboxylic acids carrying as 2-substituent a polar group such as CO2C2H5, (CH2)2CO2CH3, (CH2)3CO2C2H5, CH2OCH3, or CF3 group is described. Also 2-[5-cyano-1,6-dihydro-2-(1,1-dimethylethyl)-6-oxo-3-pyridyl]-2- oxoacetic acid and 2,5,6,8-tetrahydro-2,5-dioxo-1H-thiopyrano[3,4-b]pyridine-3-carbon itrile were prepared. Nearly all the above esters gave routinely the corresponding carboxylic acids by alkaline hydrolysis followed by acidification. As milrinone analogues, the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria from reserpine-treated guinea-pigs. 5-Cyano-2-trifluoromethyl-1,6- dihydro-6-oxo-3-pyridinecarboxylic acid and, in a lesser degree, the relative ethyl ester showed an appreciable positive inotropic activity, although inferior to that of milrinone.

Inotropic agents. Synthesis and structure-activity relationships of new milrinone related cAMP PDE III inhibitors.

The synthesis of 6-substituted 5-acyl-1,2-dihydro-2-oxo-3-pyridinecarbonitriles 1b,c, 1,2,5,6,7,8-hexahydro-2,5-dioxo-3-quinolinecarbonitriles 1d-g and esters of 5-cyano-1,6-dihydro-2-methyl-6-oxo-3-pyridinecarboxylic acid 2b-e is described. In the case of 1e and 1f, a careful elucidation of the reaction mechanism is discussed. As milrinone analogues, the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria from reserpine-treated guinea pigs. The methyl and the benzyl esters 2b and 2e showed an appreciable positive inotropic activity when compared to milrinone. A fitting study with the DISCO (Distance Comparison) model has been carried out on 2e. This modeling approach allowed for the improvement of the pharmacophoric requirements for a better interaction with the cAMP-specific PDE (PDE III), thought to be the final biological target of these cardiotonic agents.

3-Acetyl-5-acylpyridin-2(1H)-ones and 3-acetyl-7,8-dihydro-2,5(1H,6H)-quinolinediones: synthesis, cardiotonic activity and computational studies.

A series of milrinone analogues, namely 6-substituted 3-acetyl-5-acylpyridin-2(1H)-ones 4a-c, e, f and 7-substituted or unsubstituted 3-acetyl-7,8-dihydro-2,5(1H,6H)-quinolinediones 4g-j, in which the cyano group was replaced by the acetyl function, was prepared. In a preliminary pharmacological investigation on spontaneously beating atria from reserpine-treated guinea-pigs, all new compounds did not induce any inotropic effect equivalent or higher than that of the milrinone chosen as the reference compound. In order to rationalise how the structure modifications influence the activity and the selectivity of the title compounds, a computational study has been performed. The important role of the substituents in positions-3 and -6 on the pyridone nucleus has been highlighted.

Synthesis and pharmacological activities of ethyl 5-cyano-1,6-dihydro-6-oxo-2-(2,3,4-pyridyl)-3-pyridinecarboxylates and derivatives.

The synthesis of ethyl esters of 5-cyano-1,6-dihydro-6-oxo-3-pyridinecarboxylic acids carrying as 2-substituent the 2-,3- or 4-pyridyl group is described. By alkaline hydrolysis followed by acidification, these esters gave the corresponding carboxylic acids, which were decarboxylated to 1,2-dihydro-2-oxo-6-(2,3,4-pyridyl)-3-pyridinecarbonitriles. As milrinone analogues, the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria from reserpine-treated guinea-pigs. Ethyl 5-cyano-1,6-dihydro-6-oxo-2-(2-pyridyl)-3-pyridinecarboxylate showed an appreciable positive inotropic activity, although inferior to that of milrinone; moreover, some other compounds bearing the above 2-substitution pattern showed interesting antiinflammatory, analgesic and hypotensive activity.

Pharmacological characterization of a new milrinone analogue.

In a new series of milrinone analogues (esters of 2-substituted 5-acetyl-1,6-dihydro-6-oxo-3-pyridinecarboxylic acids), ethyl 5-acetyl-1,6-dihydro-6-oxo-2-phenyl-3-pyridinecarboxylate (compound 2f) has been found to be more potent and more effective than milrinone as a positive inotropic agent while affecting only marginally the frequency rate of guinea-pig isolated atria. This finding prompted us to study the mechanism of cardiac action of compound 2f in electrically driven left atrium from reserpine-treated guinea pigs. Compound 2f induced a statistically significant increase in the contractile force at a concentration as low as 1 microM, while the minimum effective concentration of milrinone was 10 microM. The beta-blocker propranolol (0.1 microM) caused a marked inhibition of the inotropic effect of compound 2f. Adenosine deaminase (1 and 2 U/ml) inhibited significantly and in a concentration-dependent manner the increase in inotropism induced by compound 2f and the adenosine deaminase-resistant response was abolished by 0.1 microM propranolol. In the presence of 0.1 microM propranolol, compound 2f (5 to 30 microM) antagonised in competitive manner the negative inotropic effect induced by N6-(R-phenylisopropyl) adenosine (R-PIA) (0.01-1.0 microM), a stable adenosine receptor agonist. Schild regression analysis gave in fact a slope of 1.02 +/- 0.06 and the pA2 value for compound 2f was 5.41 +/- 0.28. Compound 2f also inhibited phosphodiesterase (PDE) III isolated from calf heart, this inhibition being quantitatively significant only at the highest concentrations tested (0.5 M to 1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

"Metabolic reprogramming" in ovarian cancer cells resistant to cisplatin.

The way cancer cells escape cisplatin-induced apoptosis has not been completely elucidated yet. We questioned the relevance of "metabolic reprogramming" in cisplatin-resistance by studying mitochondrial function and metabolism in human ovarian carcinoma cell lines, both cisplatin-sensitive (2008) and resistant (C13). C13 cells, in comparison to 2008 cells, showed lower apoptotic response to cisplatin exposure, lower basal oxygen consumption (4.2±0.2 vs 6.5±0.7 fmol/cell/min, p<0.005) and a lower basal transmembrane mitochondrial potential (Δψm) (18.7±1.5 vs 32.2±1 MFI p<0.001). Moreover, C13 cells showed a lower sensitivity to rotenone and oligomycin, two mitochondrial respiratory chain inhibitors. To further investigate the impact of mitochondria on cisplatin-resistance, 2008 and C13 cells were depleted of their mitochondrial DNA (rho(0)-clones). The cytotoxicity of cisplatin was lower in 2008-rho(0)clones than in 2008 cells (IC(50) of 3.56 µM and 0.72 µM, respectively) but similar between C13-rho(0) and C13 cells (IC(50) of 5.49 µM and 6.49 µM, respectively). The time-course of cell viability in glucose-free galactose medium indicated that C13 cells are more strictly dependent on glucose than 2008 cells. (1)H-NMR spectroscopy showed a higher basal content of intracellular glutathione (GSH) and mobile lipids (MLs) in C13 cells as compared to 2008 cells, with higher lipid accumulation mainly within cytoplasmic droplets of the C13 cells. These findings allow us to propose a "metabolic remodelling" of ovarian carcinoma cells to a lipogenic phenotype, which includes alteration of mitochondrial function, as an advantageous mechanism to escape cisplatin-induced apoptosis. This hypothesis is of interest to exploit new pharmacological targets to improve the clinical impact of platinum drugs.