Cardiovascular Profile of Xanthone-Based 1,4 Dihydropyridines Bearing a Lidoflazine Pharmacophore Fragment.

As a follow-up to our previous studies on differently substituted 1,4-dihydropyridines endowed with a peculiar cardiac selectivity, in this paper, a small series of hybrid compounds bearing the pharmacophore fragment of lidoflazine in position 2 or 3 on a 4-(xanthen-9-one)-dihydropyridine core was reported. Lidoflazine was selected due to our promising previously reported data, and the xanthen-9-one substituent was introduced in position 4 of the dihydropyridine scaffold based on the cardiac selectivity observed in several of our studies. The new hybrid compounds were tested to assess cardiac and vascular activities, and the data were evaluated in comparison with those previously obtained for 4-(xanthen-9-one)-dihydropyridines and lidoflazine⁻nifedipine hybrid compounds. The functional studies indicated an interesting peculiar selectivity for the cardiac parameter inotropy, in particular when the lidoflazine fragment was introduced in position 2 of the dihydropyridine scaffold (4a⁻e), and thus a possible preferential binding with the Cav 1.2 isoform of l-type calcium channels, which are mainly involved in cardiac contractility.

Lidoflazine combined with nucleotide precursors increases ATP content and adenosine production in cardiomyocytes.

We have previously identified that the nucleoside transport blocker dipyridamole increases adenosine production but may cause depletion of the nucleotide pool in cardiomyocytes during extended exposure and that this effect was abolished by co-administration of adenine and ribose. The present study aimed to establish whether lidoflazine, a newer generation of nucleoside transport inhibitor with calcium antagonist properties, would cause a similar effect. We conclude that lidoflazine did not affect the nucleotide pool while the combined application of lidoflazine with precursors of nucleotide resynthesis increased ATP concentration and further enhanced adenosine production.

Lidoflazine is a high affinity blocker of the HERG K(+)channel.

Lidoflazine is an antianginal calcium channel blocker that carries a significant risk of QT interval prolongation and ventricular arrhythmia. We investigated whether or not lidoflazine inhibits current through the rapid delayed rectifier K(+) channel alpha subunit (encoded by HERG - human ether-a-go-go-related gene), since this channel has been widely linked to drug-induced QT-prolongation. Lidoflazine inhibited potently HERG current (I(HERG)) recorded from HEK 293 cells stably expressing wild-type HERG (IC(50) of approximately 16 nM). It was approximately 13-fold more potent against HERG than was verapamil under similar conditions. On membrane depolarization, I(HERG) inhibition developed gradually, ruling out closed-channel state dependent inhibition. The effect of command voltage on the drug's action suggested that lidoflazine preferentially inhibits activated/open HERG channels. The S6 mutation Y652A largely eliminated the inhibitory action of lidoflazine, whilst the F656A mutation also reduced blocking potency. We conclude: first, that lidoflazine produces high affinity blockade of the alpha subunit of the HERG channel by binding to aromatic amino acid residues within the channel pore and, second, that this is likely to represent the molecular mechanism of QT interval prolongation by this drug.

Effects of lidoflazine on left ventricular function in patients.

OBJECTIVE: The present study evaluated the effects of the nucleoside transport inhibitor, lidoflazine, at a dose of 1 mg/kg, on left ventricular function. DESIGN: Patients were randomly assigned to receive either lidoflazine or saline in a double-blind manner. SETTING: A university hospital. PARTICIPANTS: The study was performed in 32 patients scheduled for elective coronary artery bypass surgery. INTERVENTIONS: Left ventricular pressures were measured with fluid-filled catheters. Data were digitally recorded during pressure elevation induced by tilt-up of the legs. Transgastric short-axis echocardiographic views of the left ventricle were simultaneously recorded on videotape. Systolic function was evaluated with the slope (Ees, mmHg/mL) of the systolic pressure-volume relationship. Diastolic function was evaluated with the chamber stiffness constant (Kc, mmHg/mL) of the diastolic pressure-volume relationship. Cardiac function was assessed at baseline and after administration of either lidoflazine (group A [n = 16]) or placebo (group B [n = 16]). Data were compared using two-factor analysis of variance. MEASUREMENTS AND MAIN RESULTS: At baseline, diastolic and systolic function were comparable in both groups. Lidoflazine increased Kc from 0.079 +/- 0.015 to 0.125 +/- 0.017 mmHg/mL and decreased Ees from 2.481 +/- 0.213 to 1.217 +/- 0.211 mmHg/mL (p = 0.009 and p = 0.004, respectively). None of these changes occurred when placebo was administered. CONCLUSIONS: Administration of lidoflazine before the start of cardiopulmonary bypass impaired left ventricular systolic function but also increased diastolic stiffness.

1,4-Dihydropyridines bearing a pharmacophoric fragment of lidoflazine.

A series of 1,4-dihydropyridines bearing a pharmacophoric fragment of lidoflazine was synthesized. The compounds were evaluated for inotropic, chronotropic, and calcium antagonist activities. All compounds behave as inotropic and chronotropic agents, except for compounds 4b, 5a, and 5b, which exhibit a rather weak calcium antagonism in vascular smooth muscle (like aorta). Compound 5b is about twofold more potent in decreasing both chronotropy and inotropy, while compound 5c is about fivefold more potent in decreasing inotropy than nifedipine. Moreover, compound 5b is the most potent calcium antagonist derivative of the series.

Nucleoside influx and efflux in guinea-pig ventricular myocytes. Inhibition by analogues of lidoflazine.

Adenosine influx and formycin B influx and efflux were characterized in guinea-pig ventricular myocytes at 22 degrees. Transport by both modes was saturable and inhibited by nitrobenzylthioinosine (NBMPR), indicating the presence of an equilibrative NBMPR-sensitive nucleoside transporter in the cardiomyocytes. The kinetic constants for influx and efflux of formycin B, a non-metabolized nucleoside, were similar, suggesting that the nucleoside transporter exhibits symmetrical kinetics (apparent Km 490 +/- 160 and 700 +/- 140 microM; Vmax 6.5 +/- 1.7 and 3.5 +/- 0.3 nmol/10(6) cells per min for influx and efflux, respectively). No evidence was found of either NBMPR-insensitive equilibrative nucleoside transport or sodium-dependent concentrative nucleoside transport. Inhibition of adenosine influx (apparent Km100 +/- 33 microM), by lidoflazine and the analogues mioflazine, soluflazine and R73-335, gave average Ki values of 730, 100, 64 and 2.9 nM, respectively. These compounds also inhibited formycin B efflux with a similar potency to that of adenosine influx. NBMPR-sensitive nucleoside transport was associated with high affinity binding of NBMPR (apparent Kd approximately 1 nM; 9.6 x 10(5) sites/cell). Specific binding of NBMPR was also inhibited by lidoflazine and its analogues. Mioflazine and soluflazine were 20-30-fold more potent at inhibiting NBMPR-sensitive nucleoside influx in guinea-pig erythrocytes than ventricular myocytes, indicating that the potency of some of the compounds studied is tissue dependent.

The cardioprotective effects of R 75231 and lidoflazine are not caused by adenosine A1 receptor activation.

The goal of this study was to determine the cardioprotective profile for the nucleoside transport inhibitor 2-(aminocarboxyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis(4- fluorophenyl)pentyl]-1-piperazinylacetamide trihydrochloride-2,5 hydrate (R 75231) in isolated rat hearts and whether its protective effects are caused by adenosine A1 activation. R 75231 increased time to contracture during global ischemia in a concentration-dependent manner (EC25 = 2.6 microM) that was comparable to the structurally related compound lidoflazine (EC25 = 1.2 microM). R 75231 caused only modest improvements in reperfusion contractile function, whereas it profoundly reduced LDH release. The cardioprotective effects of R 75231 were accompanied by preischemic negative inotropy with modest bradycardic effects. Adenosine also increased time to contracture, although it was not very potent (EC25 > 300 microM), and this effect was accompanied by significant preischemic bradycardia without measurable negative inotropic activity. Both the preischemia bradycardia and increase in ischemic time to contracture with adenosine were abolished completely by the A1 blocker 8-cyclopentyl-1,3-dipropylxanthine. The adenosine-induced increase in time to contracture was reversed partially by glybenclamide. Neither the pre- nor postischemic effects of R 75231 were abolished by 8-cyclopentyl-1,3-dipropylxanthine or glybenclamide, except for the preischemic bradycardia. Similar results were observed for lidoflazine. Thus, the cardioprotective effects of R 75231 are not mediated by adenosine A1 receptor activation and, thus, probably are not caused by its activity as a nucleoside transport inhibitor. It may be acting as a calcium antagonist in this model.

K(+)-stimulated 45Ca2+ flux into rat neocortical mini-slices is blocked by omega-Aga-IVA and the dual Na+/Ca2+ channel blockers lidoflazine and flunarizine.

High-threshold neuronal voltage-sensitive Ca2+ channels (VSCCs) have been classified into at least three subtypes, including L, N, and P, based on biophysical and pharmacological criteria. We examined K(+)-induced 45Ca2+ flux into rat neocortical mini-slices to determine which of these subtype(s) might be involved in this phenomenon. Neither the L-type Ca2+ channel antagonist isradipine at 10 microM nor the N-type antagonist omega-conotoxin GVIA at 1 microM were effective antagonists of 45Ca2+ flux in this model. However, the P-type Ca2+ channel antagonist, omega-Aga-IVA, blocked 70% of flux at 200 nM, with an IC50 of 17 nM, strongly implicating P-type Ca2+ channel involvement in K(+)-stimulated Ca2+ entry into mammalian nerve terminals. About 30% of the flux response was resistant to the action of omega-Aga-IVA, suggesting that a still uncharacterized subtype of VSCC is involved in Ca2+ entry into mammalian nerve terminals. Both the omega-Aga-IVA sensitive and insensitive components of 45Ca2+ flux were blocked by the diphenylalkylpiperazines, lidoflazine and flunarizine (IC50 = 6.4 microM and 11 microM, respectively), which have dual Na+/Ca2+ channel blocking actions.

Protective effects of the lazaroid U74500A and lidoflazine on liver preservation with UW solution.

The effect of adding a 21-aminosteroid, U74500A, and a Ca2+ antagonist, lidoflazine, alone and together to UW solution was assessed in a rat liver preservation model. Following preservation, the livers were reperfused using a closed circuit, and the release of hepatocellular enzymes (ASAT, ALAT, and LDH) into the perfusate was determined with increasing time. Both drugs reduced the amount of enzymes lost from the liver. The combination of the two drugs was better than either drug alone. These data suggest that both agents may be of value in organ preservation for clinical liver transplantation.

Nucleoside transport inhibition mediates lidoflazine-induced cardioprotection during intermittent aortic crossclamping.

The effects of pretreatment with the nucleoside transport inhibitor lidoflazine on repeated ischemia-reperfusion injury induced by normothermic intermittent aortic crossclamping were studied in canine hearts. Eighteen mongrel dogs were allocated to three groups: placebo (n = 6), lidoflazine (1 mg/kg) (n = 6), and lidoflazine (1 mg/kg) plus the adenosine receptor blocker aminophylline (7 mg/kg) (n = 6). Pretreatment was performed intravenously during 15 minutes before extracorporeal circulation. All hearts were subjected to four intervals of 15 minutes of global ischemia each followed by 10 minutes of reperfusion. After weaning from extracorporeal circulation, functional recovery was followed for 1 hour. In the lidoflazine group, myocardial adenosine content (0.25 +/- 0.06 mumol/gm dry weight) was 3.5 times higher than that in the control group (0.07 +/- 0.03 mumol/gm dry weight; p < 0.05) at the end of the last aortic crossclamping. The release of adenosine from the myocardium during each reperfusion period was significantly higher than that in the control group (p < 0.05). Myocardial extraction of lactate was normalized at every reperfusion interval in the lidoflazine group but not in the control group (p < 0.05). In the lidoflazine group functional recovery was significantly better than that in the control group. Positive rate of rise of pressure, negative rate of rise of pressure, and cardiac output recovered to, respectively, 150% +/- 19%, 82% +/- 8%, and 131% +/- 15% in the lidoflazine group versus, respectively, 37% +/- 9%, 23% +/- 7%, and 29% +/- 8% in the control group (p < 0.001) at 1 hour after extracorporeal circulation. When the adenosine receptor blocker aminophylline was administered in association with lidoflazine, protection dropped significantly: positive and negative rate of rise of pressure and cardiac output were, respectively, 58% +/- 8%, 46% +/- 9%, and 67% +/- 16% at 1 hour after extracorporeal circulation (p < 0.05 versus lidoflazine alone). These results suggest that the cardioprotective effects of lidoflazine are at least in part mediated by adenosine receptor stimulation via nucleoside transport inhibition-induced accumulation of endogenous adenosine in the myocardium.

Protection of human, rat, and guinea-pig atrial muscle by mioflazine, lidoflazine, and verapamil against the destructive effects of high concentrations of Ca2+.

In right atrial trabeculae from humans and in left atria from rat and guinea-pig hearts, the protective effects of mioflazine, lidoflazine, and verapamil against the accumulation of cellular calcium were investigated. Two consecutive, cumulative increases in the extracellular calcium concentration, [Ca2+]o (1-25 mmol/l), were induced, in between which the muscles were exposed for at least 30 minutes to solvent or drug. When using solvent in the 30-minute interval, the force of contraction was much lower during the second Ca2+ challenge, while the aftercontractions and the increase in passive tension at high [Ca2+]o tended to be larger. These signs of functional impairment were prevented by exposure to mioflazine or lidoflazine (3 mumol/l each) but not to verapamil (3 mumol/l). Muscles were fixed with glutaraldehyde at the end of the second Ca2+ challenge for morphological and cytochemical examination. After solvent treatment, more than half of the cells were severely damaged, showing cellular edema, contraction-band necrosis, mitochondrial swelling, and nuclear pyknosis; the sarcolemma was devoid of calcium deposits, damaged mitochondria contained either large deposits of calcium or flocculent densities, and in some cells, the cytoplasm was filled with calcium deposits. Following exposure to mioflazine and lidoflazine, but not to verapamil, the number of intact cells after the second Ca2+ challenge was not different from time-matched controls (80-90%). Furthermore, the shifts in cellular calcium distribution were prevented with mioflazine and lidoflazine, whereas verapamil was less effective. There were no species differences with respect to either morphological or contractile changes. In conclusion, exposure of atria to high [Ca2+]o induced similar ultrastructural and cytochemical changes as seen after ischemia-reperfusion induced damage. Indeed, under the mentioned conditions the sarcolemma lost its capacity to exclude Ca2+ after a challenge with high [Ca2+]o and allowed excessive Ca2+ entry. The pathway for this extra Ca2+ remains to be elucidated. L-type calcium channels are probably not involved, since verapamil cannot prevent the Ca2+ overload.

Caffeine-induced contractions in rabbit isolated renal artery are differentially inhibited by calcium antagonists.

Caffeine (1-60 mM) induced concentration-dependent, endothelium-independent phasic contractile responses in isolated rabbit renal artery ring preparations. For concentrations of caffeine over 2 mM, responses were mainly the result of intracellular calcium ion mobilization since they were relatively resistant to removal of calcium ions from the bathing medium. The L-type slow calcium channel blocker, nifedipine (10 microM), had no effect and high concentrations of verapamil and diltiazem (10-30 microM) only slight and inconsistent effects (not concentration-dependent) upon these caffeine responses. Likewise, the highly lipophilic calcium antagonists flunarizine and lidoflazine (3-30 microM) only slightly displaced caffeine concentration-response curves to the right and reduced the maximum response. These small inhibitory effects of flunarizine and lidoflazine were not augmented in a calcium-free medium. In contrast, the other lipophilic calcium antagonists, bepridil and fendiline (3-30 microM), produced marked, non-competitive type inhibition of caffeine responses, completely inhibiting responses to the alkaloid at the highest concentration. Furthermore, the inhibitory effects of bepridil and fendiline were markedly augmented in calcium-free medium. These results clearly differentiate bepridil and fendiline from the other calcium antagonists studied. In addition they provide further evidence for effects other than at the cell membrane which could theoretically contribute to the efficacy of bepridil and fendiline as anti-anginal agents.

Risk monitoring of randomized trials in emergency medicine: experience of the Brain Resuscitation Clinical Trial II.

Risk monitoring for the Brain Resuscitation Clinical Trial II, a multicenter, placebo-controlled trial to evaluate the efficacy of the calcium-entry blocker lidoflazine in the amelioration of brain damage in comatose cardiac-arrest survivors, posed unexpected challenges. Concern arose when monitoring of adverse reactions showed an excess of dangerous cardiac arrhythmias, including rearrest, in the lidoflazine group. To ascertain the cause of this problem and determine whether it was ethical for the trial to continue, an in-depth review of data was conducted, outside experts were consulted, and additional data were collected. These efforts suggested possible causes for the problem. Existing drug administration protocols for blood pressure control were reinforced, resulting in lower subsequent arrhythmia rates. Thus, through an efficient monitoring system, an important problem was uncovered and resolved, allowing the trial to be completed without major changes.

Inhibition of nucleoside uptake in human erythrocytes by a new series of compounds related to lidoflazine and mioflazine.

The zero-trans influx of uridine in human erythrocytes is inhibited by lidoflazine and analogs thereof. The concentrations required for inhibition of nucleoside transport were higher when the compounds were simultaneously added with uridine than upon preincubation of the inhibitors with the erythrocytes. R70380 proved to be the most active compound in this respect, its IC50 value being 13 nM after preincubation. Even the reference compounds nitrobenzylthioinosine and dilazep were remarkably more potent with preincubation; dipyridamole, however, was not.

Differential inhibition of nucleoside transport systems in mammalian cells by a new series of compounds related to lidoflazine and mioflazine.

The sensitivity of facilitated-diffusion and Na(+)-dependent nucleoside transporters to inhibition by a series of novel compounds related to lidoflazine and mioflazine was investigated. Uridine transport by rabbit erythrocytes, which proceeds solely by the nitrobenzylthioinosine (NBMPR)-sensitive facilitated-diffusion system, was inhibited with apparent Ki values of less than 10 nM by lidoflazine, mioflazine, soluflazine and R73-335. These compounds also blocked site-specific [3H]NBMPR binding to rabbit erthrocyte membranes in a competitive fashion. The NBMPR-sensitive system in rat erythrocytes was also inhibited by lidoflazine, mioflazine, soluflazine and R73-335 but was two to three orders of magnitude less sensitive to inhibition than the system in rabbit erythrocytes (apparent Ki 7.3, 2.4, 5.7 and 0.1 microM, respectively). Lidoflazine, mioflazine and R73-335 exhibited a similar potency for the NBMPR-sensitive and -insensitive nucleoside transporters in rat erythrocytes. In contrast, soluflazine was 20- to 100-fold more potent as an inhibitor of the NBMPR-insensitive nucleoside transport component in rat erythrocytes (IC50 of 0.08-0.2 microM) compared to the NBMPR-sensitive nucleoside carrier in these cells (IC50 approximately 10 microM). None of the test compounds were potent inhibits of Na(+)-dependent uridine transport in bovine renal brush-border membrane vesicles. These results indicate that lidoflazine, mioflazine, soluflazine and R73-335 are selective inhibitors of nucleoside transport in animal cells and that the potency of these compounds as nucleoside transport inhibitors is species dependent.

Renal function during reperfusion after warm ischaemia in rabbits: an experimental study on the possible protective effects of pretreatment with oxygen radical scavengers or lidoflazine.

Survival and renal function were studied after 60 min of renal ischaemia and contralateral nephrectomy in four groups of French loop rabbits. One group served as untreated control animals. The other groups were pretreated with superoxide dismutase (SOD) and catalase, lidoflazine or a buffered albumin solution containing mannitol, L-methionine and MgCl2. Six out of nine rabbits died during the 14-day follow-up period in the untreated control group, while the corresponding figure in each of the three treatment groups was one out of nine. Five of the rabbits that died exhibited azotaemia or hyperpotassaemia that could explain the death, while four died of non-renal related causes. In the surviving animals, no differences in serum creatinine, potassium and sodium concentration or urinary output of osmoles was observed between the four groups. The increase in serum creatinine of the French loop rabbits observed after 60 min of ischaemia was considerably less pronounced than the corresponding increase observed in New Zealand White rabbits, indicating that the kidneys of the former strain are more tolerant to ischaemia. A cardiomyodepressant factor could be demonstrated in the venous effluent from previously ischaemic kidneys. This release could not be prevented by pretreatment with SOD and catalase.

Class IV Ca2+ antagonists do not affect lipid peroxidation in singlet oxygen challenged cardiomyocytes.

The effects of various Ca2+ antagonists on lipid peroxidation in singlet O2-challenged isolated cardiomyocytes from adult rat heart were investigated. Singlet O2-challenged untreated cells all hypercontracted as a consequence of Ca2+ overload and produced 463.6 +/- 143.6 nM malondialdehyde (MDA; mean +/- SD, n = 8). Protective Ca2+ antagonists reduced the amount of damaged cells, but did generally not affect MDA production. On the other hand, free radical scavengers and antioxidants displayed a good correlation between number of protected cells and MDA produced. It is concluded that flunarizine-like Ca2+ antagonists protect cells against Ca2+ overload without, however, interfering with peroxidative processes.

Myocardial protective effects of lidoflazine during ischemia and reperfusion.

The myocardial protective effect of intravenous (i.v.) lidoflazine with potassium cardioplegia and hypothermia (28 degrees C) was investigated in 21 greyhounds. Animals were injected a single dose of cardioplegia (30 ml/kg body weight) and subjected to 120 minutes of ischaemia and 60 minutes of reperfusion. Ten dogs served as controls (Group C) and 11 dogs received i.v. lidoflazine (1.25 mg/kg b.w.) (Group L). Myocardial drill biopsies for the adenosine triphosphate (ATP) and the creatine phosphate (CP) levels were obtained. Hemodynamic measurements were made at intervals. In Group C, no dog could be weaned from bypass, whereas all 11 dogs in Group L came off bypass and maintained their circulation for 15 minutes. After a 120 minute ischemic period, the ATP and CP contents diminished significantly in both groups. Following reperfusion, the ATP level was 28% of the control level in Group C (p less than 0.005) and 38% in Group L (p less than 0.01). The CP levels showed an overshoot in both groups. There was no significant difference between the groups. In Group L animals, cardiac output (CO) and mean aortic pressure (MAP) were significantly reduced after bypass; from 5 +/- 1/min to 3.2 +/- 1, from 156 +/- 26 mmHg to 82 +/- 11 mmHg respectively (p less than 0.005). Left ventricular minute work (LVMW) also deteriorated markedly from 9.7 +/- 2 kg-m to 3.2 +/- 1 (p less than 0.005). The use of lidoflazine achieved considerable protection in terms of survival, but did not prevent the severe loss of high-energy phosphates in this experimental model.