The potencies and selectivities of four calcium antagonists as inhibitors of uterine contractions in the rat in vivo.

The potencies of four calcium antagonists (nifedipine, gallopamil, verapamil and diltiazem) at inhibiting uterine contractions in vivo have been assessed in the conscious ovariectomized, post-partum rat. Their selectivities for this action, relative to their effects on blood pressure and heart rate, have been compared with salbutamol. All compounds produced a dose-dependent inhibition of intra-uterine pressure cycles. The rank order of potency was salbutamol greater than nifedipine greater than diltiazem = gallopamil greater than verapamil. All compounds produced a dose-dependent fall of mean blood pressure. The rank order of potency was salbutamol greater than nifedipine greater than gallopamil greater than verapamil greater than diltiazem. Salbutamol and nifedipine produced a tachycardia, which was very marked with salbutamol. Gallopamil, verapamil and diltiazem induced a moderate tachycardia at low doses but temporary cessation of heart beat occurred at high doses. Nifedipine and diltiazem, like salbutamol, exhibited some selectivity for inhibition of uterine contractions relative to their cardiovascular actions. Gallopamil and verapamil showed no selectivity for the uterus.     

Regulation of the renal basolateral transport system for organic anions by calcium channel blockers.

Calcium channel blockers have been reported to exert multiple effects on renal tubular function. Therefore, the effects of the calcium channel blockers nifedipine and verapamil on the cellular uptake of tritiated para -aminohippuric acid (PAH) into microdissected non-perfused rabbit kidney S2 proximal tubule segments were investigated to study a possible influence of calcium channel blockers on renal PAH transport. Since the tubules were entirely collapsed, the accumulated radioactivity overwhelmingly reflects the transport across the basolateral membrane. Tubular PAH accumulation was increased by 1 and 10 microm verapamil, and by 1 microm nifedipine, but it was unaffected by lower or higher concentrations of these calcium channel blockers. The increase in PAH accumulation caused by 1 microm nifedipine or 10 microm verapamil was independent from changes in intracellular Ca(2+) and inhibited by staurosporine (1 and 10 n m) a potent inhibitor of protein kinase C (PKC). Our results indicate that the calcium channel blockers nifedipine and verapamil increase the transport of organic anions across the basolateral membrane of proximal tubules, probably by an activation of PKC. Furthermore, the increased tubular PAH transport may disturb the estimation of the renal PAH-clearance.     

Combination of calcium channel blockers and beta-blockers for patients with exercise-induced angina pectoris: beneficial effect of calcium channel blockers largely determined by their effect on heart rate.

The combination of calcium channel blockers and beta-blockers is more effective for the treatment of exercise-induced angina pectoris than beta-blocker monotherapy. As ischemia in exercise-induced angina is essentially preceded by an increase in heart rate, calcium channel blockers with a negative chronotropic property may perform better for this purpose than nonchronotropic compounds. A 335-patient, 10-week, double-blind, parallel-group comparison of amlodipine 5 mg and 10 mg, diltiazem 200 mg and 300 mg, and mibefradil 50 mg and 100 mg treatment added to baseline beta-blocker treatment was performed. Exercise testing (ETT) was performed by bicycle ergometry. All of the calcium channels blockers significantly delayed the onset of 1 mm ST-segment depression on ETT (p < 0.001 for any treatment vs. baseline). In addition, mibefradil, in both low- and high-dose treatments, produced the largest delays (low dose: different from diltiazem and amlodipine by 24.1 and 29.8 seconds, respectively, p < 0.003 and < 0.001; high dose: different from diltiazem and amlodipine by 33.7 and 37.0 seconds, respectively, p < 0.001 and < 0.001). A stepwise logistic regression analysis revealed that this beneficial effect of calcium channel blockers was largely dependent on their effect on heart rate. Serious symptoms of dizziness likewise occurred significantly more frequently on mibefradil (p < 0.05 vs. diltiazem) and urged no fewer than 19 patients on mibefradil to withdraw from the trial. The authors conclude that calcium channel blockers with a negative chronotropic property provide a better delay of ischemia in patients with exercise-induced angina, but the concomitant risk of intolerable dizziness may reduce this benefit.     

2-aminoethoxydiphenyl borate as a prototype drug for a group of structurally related calcium channel blockers in human platelets

We have synthesized a series of 2-aminoethoxydiphenyl borate (2-APB, 2,2-diphenyl-1,3,2-oxazaborolidine) analogs and tested their ability to inhibit thrombin-induced Ca(2+) influx in human platelets. The analogs were either synthesized by adding various substituents to the oxazaborolidine ring (methyl, dimethyl, tert-butyl, phenyl, methyl phenyl, and pyridyl) or increasing the size of the oxazaborolidine ring to seven- and nine-membered rings. NMR analysis of the boron-containing analogs suggests that each of them exist as a ring structure through the formation of an N-->B coordinate bond (except for the hexyl analog). The possibility that these boron-containing compounds formed dimers was also considered. All compounds dose-dependently inhibited thrombin-induced Ca(2+) influx in human platelets, with the 2,2-diphenyl-1,3,2-oxazaborolidine-5-one derivative having the weakest activity at 100 microM, whereas the (S)-4-benzyl and (R)-4-benzyl derivatives of 2-APB were approximately 10 times more potent than the parent 2-APB. Two nonboron analogs (3-methyl and 3-tert-butyl 2,2-diphenyl-1,3-oxazolidine) were synthesized; they had approximately the same activity as 2-APB, and this implies that the presence of boron was not necessary for inhibitory activity. All of the compounds tested were also able to inhibit thrombin-induced calcium release. We concluded that extensive modifications of the oxazaborolidine ring in 2-APB can be made, and Ca(2+)-blocking activity was maintained.     

Augmentation by calcium channel antagonists of general anaesthetic potency in mice.

The effects of three kinds of calcium channel antagonists on the anaesthetic potencies of ethanol, pentobarbitone and argon were examined in mice. Ethanol and pentobarbitone anaesthetic potencies in mice were significantly increased by verapamil 10 mg kg-1, flunarizine 40 mg kg-1 and nitrendipine 100 mg kg-1. Argon anaesthetic potency was significantly increased by nitrendipine 50 mg kg-1 and 100 mg kg-1 in a dose-related fashion. Even at very high doses the calcium channel antagonists did not produce anaesthesia by themselves. At the doses used the calcium channel antagonists did not affect the blood concentrations of ethanol, 2 h, or pentobarbitone, 15 min, after anaesthetic administration.     

Modulation of intestinal P-glycoprotein function by polyethylene glycols and their derivatives by in vitro transport and in situ absorption studies

We examined the effect of polyethylene glycols (PEGs) with different molecular weights and their derivatives on the intestinal absorption of rhodamine123, a P-glycoprotein (P-gp) substrate, across the isolated rat intestinal membranes by an in vitro diffusion chamber system. The serosal to mucosal (secretory) transport of rhodamine123 was greater than its mucosal to serosal (absorptive) transport, indicating that the net movement of rhodamine123 across the intestinal membranes was preferentially secretory direction. The secretory transport of rhodamine123 was inhibited by the addition of PEGs with average molecular weights of 400, 2000 and 20,000, irrespective of its molecular weight. The inhibitory effects of these PEGs for the intestinal P-gp function were concentration dependent over the range 0.1-20% (v/v or w/v). Similar inhibitory effect for the intestinal P-gp function was observed when PEG derivatives including PEG monolaurate, PEG monooleate and PEG monostearate were added to the mucosal site of the chambers. Furthermore, we also examined effect of PEG20,000 on the intestinal absorption of rhodamine123 by an in situ closed loop method. The intestinal absorption of rhodamine123 was enhanced in the presence of PEG20,000. These findings suggest that PEGs and their derivatives are useful excipients to inhibit the function of intestinal P-gp, thereby improving the intestinal absorption of P-gp substrates, which are secreted by a P-gp-mediated efflux system.     

Transport of beta-blockers and calcium antagonists by diffusion in cat myocardium.

Beta-blockers and calcium antagonists have been claimed to possess cardioprotective properties. This study addresses the question of whether a significant amount of these drugs will reach the cardiac myocytes during no-flow ischemia, where solute transport depends solely on diffusion. In anesthetized cats the hearts were excised. Apparent diffusion coefficients in cat myocardium at 37 degrees C (D'37) for 14C-verapamil (protein bound), 3H-metoprolol (lipophilic), 3H-atenolol (hydrophilic), and 3H-propranolol (lipophilic and protein bound) were determined by means of a "true transient diffusion" method and compared with the free diffusion coefficients in water (D37). D'37 of 14C-verapamil, 3H-metoprolol, 3H-atenolol, and 3H-propranolol (in cm2 s-1 10(5)) were (mean +/- SEM) 0.025 +/- 0.002, 0.055 +/- 0.003, 0.041 +/- 0.007, and 0.025 +/- 0.002, respectively. The mean diffusive progression of the concentration profile of 3H-metoprolol and 3H-atenolol into the tissue during 20 min was calculated to be 0.36 and 0.31 mm, respectively. The protein binding of 14C-verapamil and 3H-propranolol caused a significant fall in the progression to 0.24 mm for both drugs. These results indicate that, by diffusion, these drugs traverse the tissue too slowly to reach a significant amount of myocardium before myocyte necrosis occurs during conditions of noflow. Cardioprotective drugs are probably most effective, provided sufficient amounts are present in the tissue prior to the ischemic episode or sufficient supply via collateral blood flow is achieved.     

Comparative potencies of CGP 47654A and CGP 46165A as GABA(B) receptor antagonists in rat brain.

In rat neocortical slices maintained in Mg2+-free Krebs medium, the gamma-aminobutyric acid (GABAB) receptor agonist baclofen concentration-dependently depressed the frequency of spontaneous discharges (EC50 = 6.1 microM). This was reversibly antagonised by 3-aminopropyl-(1,1-difluoro-n-butyl)-phosphinic acid (25, 100, 500 microM) (CGP 47654A) and 3-aminopropyl-P-(alpha-hydroxybenzyl)-phosphinic acid (CGP 46165A) (50, 100, 400 microM) which produced rightwards shifts of the baclofen concentration-response curves, with respective pA2 values of 4.9+/-0.2 and 4.6+/-0.15. Although relatively potent on GABAB heteroreceptors studied here, CGP 47654A and CGP 46165A were 5 and 50 times weaker, respectively, as GABAB autoreceptor antagonists [Froestl, W., Mickel, S.J., Von Sprecher, G., Diel, P.J., Hall, R.G., Maier, L., Strub, D., Melillo, V., Baumann, P.A., Bernasconi, R., Gentsch, C., Hauser, K., Jaekel, J., Karlsson, G., Klebs, K., Maitre, L., Marescaux, C., Pozza, M.F., Schmutz, M., Steinmann, M.W., Van Riezen, H., Vassout, A., Mondadori, C., Olpe, H.R., Waldmeier, P.C., Bittiger, H., 1995. Phosphinic acid analogues of GABA. 2. Selective, orally active GABAB antagonists. J. Med. Chem. 38: 3313-3331.], representing potentially useful ligands for differentiating GABA hetero- and autoreceptors.     

P-glycoprotein drug efflux pump involved in the mechanisms of intrinsic drug resistance in various colon cancer cell lines. Evidence for a saturation of active daunorubicin transport.

We studied the resistance of colon tumors to anticancer agents in vitro. Using daunorubicin (DN), a number of cellular parameters which normally indicate acquired or multidrug resistance (MDR), were compared for several human wild-type colon cell lines, i.e. HT29, SW1116 and COLO 320, and the murine colon cell line C-26. The sensitive/MDR human ovarian cancer cell line couple A2780/2780AD was used as a reference. The amount of P-glycoprotein (P-gp) was in the order HT29, A2780 less than or equal to SW1116 less than C26 less than or equal to COLO 320 less than 2780AD. The MDR modifiers verapamil, Cremophor EL, cyclosporin A and Ro 11-2933/001 had significant effects on DN cytotoxicity, total DN accumulation and efflux, only if P-gp was present. A flow-through system was used to study the mechanism of DN transport. For the first time, evidence for saturation of an active transport of DN from the cells is reported. We discussed the possible presence of cooperative activity between at least two binding sites on the protein responsible for DN efflux, likely to be P-gp.     

Inhibition of cholesteryl ester deposition in macrophages by calcium entry blockers: an effect dissociable from calcium entry blockade.

The effects of calcium entry blockers on stimulated cholesteryl [3H]-oleate deposition in cultured macrophages were characterized in order to elucidate mechanisms underlying possible antiatherosclerotic effects. Stimulation of intracellular cholesteryl [3H]-oleate deposition was initiated by incubation of macrophages with beta-very low density lipoproteins (beta-VLDL). Nifedipine (Class I) markedly reduced cholesteryl [3H]-oleate deposition at all concentrations tested. However, Bay K 8644, a dihydropyridine which is known to stimulate calcium entry, also reduced cholesteryl [3H]-oleate deposition with a similar potency to nifedipine. The effects of three Class II calcium entry blockers were evaluated: verapamil, methoxyverapamil, and diltiazem. Verapamil inhibited cholesteryl [3H]-oleate deposition in a concentration-dependent manner. Similarly, methoxyverapamil reduced cholesteryl [3H]-oleate deposition in a concentration-dependent manner although the reduction was not as great as that produced by verapamil. In contrast, diltiazem at any concentration tested did not inhibit cholesteryl [3H]-oleate deposition. Flunarizine (a Class III calcium entry blocker) produced a modest stimulation of cholesteryl [3H]-oleate deposition at the lowest concentration used (10(-7)M) but marked depression at the highest concentration (10(-5)M). The results indicate calcium entry blockers may exert protective effects on the development of atherosclerosis in animal models of diet-induced hyperlipidaemia by inhibiting intracellular cholesteryl ester deposition, but this effect may not be related to their calcium entry-blocking effects.     

Inhibition of neurally-evoked transmitter release by calcium channel antagonists in rat parasympathetic ganglia.

1. Excitatory postsynaptic potentials (e.p.s.ps) were recorded from the submandibular parasympathetic ganglia of newborn rats (10-20 days old), by intracellular microelectrode recording and a suction electrode to deliver stimulus trains to the lingual nerve (15 stimuli at 0.1, 0.3, 1, 3, and 10 Hz, 22 degrees C). Only evoked responses without voltage-dependent action potentials were analyzed (observed at membrane potentials negative to -70 mV), and e.p.s.p. amplitudes were determined for the plateau responses during each train (5-15th response). 2. Cadmium, an inorganic calcium channel antagonist, reduced e.p.s.p. amplitudes in a dose-dependent manner (Kd 74 microM, P less than 0.01). Nickel (1-300 microM) did not attenuate the amplitude of evoked responses. 3. Verapamil (0.1-30 microM), a phenylamine, had no significant effects upon e.p.s.p. amplitudes at any frequency examined. Higher concentrations of verapamil (100 microM) blocked neurally evoked responses in a manner consistent with the antagonism of voltage-sensitive sodium currents. 4. Diltiazem, a benzothiazepine, reduced e.p.s.p. amplitudes in a dose-dependent manner, the depression being accentuated at high stimulation frequencies (80% block at 30 microM and 10 Hz). The pure (-)-cis enantiomer of diltiazem (10-30 microM) was without effect. 5. Amlodipine, a 1,4-dihydropyridine, did not antagonize synaptic transmission at any stimulus frequency examined (10-30 microM, 0.1-10 Hz, n = 3). 6. Amiloride, a potassium-sparing diuretic, depressed the amplitudes of evoked responses in a dose-dependent manner (one-site Kd 31 microM, P less than 0.005), although the extent of the block was alleviated with high stimulus frequencies. The effects of 30 microM amiloride were unlikely to be of post-synaptic origin as both the amplitudes of miniature e.p.s.ps, and the iontophoretic potentials induced by exogenous acetylcholine, were not attenuated by treatment with this compound. The amiloride derivative, 3',4'-dichlorobenzamil was ineffective in reducing the amplitude of e.p.s.ps (30-100 microM). 7. omega-Conotoxin GVIA, a marine neurotoxin, which depressed whole cell calcium currents recorded from cultured rat parasympathetic cardiac neurones (up to 90% block at 10 nM), was ineffective at blocking synaptic transmission in submandibular ganglia (0.1-1 microM). 8. The differential effects of these calcium channel antagonists upon synaptic transmission in rat parasympathetic ganglia, suggest that either more than one type of calcium channel may be involved in transmitter release, or that the presynaptic calcium channels possess pharmacological sensitivities different from those of channel types described in ne     

A study of the mechanism of resistance to Adriamycin in vivo. Glutathione metabolism, P-glycoprotein expression, and drug transport

A spontaneously originated murine mammary adenocarcinoma (16C), selected for its sensitivity to agents active against breast cancer in women, and one of the very few experimental solid tumor models responsive to Adriamycin (ADR) was used to study the mechanism of induced ADR resistance in vivo. A resistant variant of the tumor was obtained from the explant of a regrown tumor following a dose of ADR (12 mg/kg) that caused complete tumor repression but not cure. Progressive refractoriness to ADR was observed following up to six repeated cycles of treatment, regression and regrowth. However, beyond the sixth treatment, no further degree of resistance could be obtained. The cell line so established, designated 16C/ADRR, has a glutathione (GSH) content 1.67 times greater than the parent 16C line. Depletion of GSH by buthionine sulfoximine (BSO) enhanced the cytoxicity of ADR in both cell lines. The sensitization effect appeared to be dependent on the degree of GSH depletion, requiring a threshold level of depletion to approximately 30% of control. The resistance of 16C/ADRR, however, appeared not to be directly related to the increased absolute GSH level per se since reduction of the GSH content of the 16C/ADRR line to levels similar to that of the parent 16C line did not restore the original sensitivity to ADR. However, the activities of two important elements in the GSH detoxification system, GSH peroxidase and S-transferase, were found to be elevated in resistant cells by factors of 2.4 and 4.7-5.6 respectively. In vivo studies with a diverse spectrum of antineoplastic drugs revealed a pattern of cross-resistance consistent with the idea that elevated GSH S-transferase and peroxidase activities may be responsible for the decreased (2.8- to 5.3-fold) sensitivity to ADR. 16C/ADRR exhibited cross-resistance with melphalan (MEL), but none with vincristine (VCR), vinblastine (VBL) or etoposide (VP-16). These results clearly demonstrate non-adherence by the 16C/ADRR tumors to the well characterized multidrug resistance (mdr) phenotype. Further affirmation of this conclusion was obtained by immunochemical and pharmacological studies. When a monoclonal antibody prepared against the mdr associated, 170 kD P-glycoprotein (170 P-gp), was used, the presence of the 170 kD P-gp in both the sensitive and resistant 16C lines could not be detected, although the presence of a lower molecular weight form of P-gp could not be ruled out entirely. High performance liquid chromatographic measurement of ADR accumulation and elimination also failed to reveal any significant differences between the sensitive and resistant variants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective modulation by membrane potential of the interaction of some calcium entry blockers with calcium channels in rat mesenteric artery.

1. The effects of flunarizine, (+)-PN 200-110 and nifedipine on [3H]-(+)-PN 200-110 specific binding were investigated in intact rat mesenteric arteries bathed in physiological solution or in KCl-depolarizing solution, and in a membrane fraction from rat mesenteric arteries. 2. Unlabelled dihydropyridines, (+)-PN 200-110 and nifedipine, inhibited [3H]-(+)-PN 200-110 specific binding concentration-dependently in polarized as well as in depolarized intact arteries. The Ki value of (+)-PN 200-110 was decreased in arteries bathed in KCl-depolarizing solution compared to arteries bathed in physiological solution, while the Ki value of nifedipine was not significantly changed. Ki values measured in depolarized arteries were close to the IC50 values (concentrations inhibiting by 50% the KCl-contraction of rat mesenteric artery). 3. Flunarizine (10(-6) M) was unable to displace the specific binding of [3H]-(+)-PN 200-110 in intact arteries bathed in physiological solution. At 10(-7) M-10(-6) M, it inhibited the binding in depolarized arteries, suggesting that prolonged depolarization is required for the interaction of flunarizine with the dihydropyridine receptor. 4. In a membrane fraction isolated from rat mesenteric arteries, (+)-PN 200-110, nifedipine and flunarizine were all able to displace completely the specific binding of [3H]-(+)-PN 200-110. Displacement curves were parallel and Hill coefficients were close to unity. Ki values were close to the values obtained in depolarized intact arteries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and biological characterization of 6-aryl-7-isopropylquinazolinones as novel TRPV1 antagonists that are effective in models of chronic pain

Vanilloid receptor 1 (VR1, TRPV1) is a cation-selective ion channel that is expressed on primary afferent neurons and is upregulated following inflammation and nerve damage. Blockers of this channel may have utility in the treatment of chronic nociceptive and neuropathic pain. Here, we describe the optimization from a high throughput screening hit, of a series of 6-aryl-7-isopropylquinazolinones that are TRPV1 antagonists in vitro. We also demonstrate that one compound is active in vivo against capsaicin-induced hyperalgesia and in models of neuropathic and nociceptive pain in the rat.     

Actions of prostaglandin F2 alpha and noradrenaline on calcium exchange and contraction in rat mesenteric arteries and their sensitivity to calcium entry blockers.

1 The actions of prostaglandin F2 alpha (PGF2 alpha) and noradrenaline on contraction and 45Ca exchange have been studied in rat mesenteric arteries. 2 PGF2 alpha and noradrenaline contracted rat isolated mesenteric artery preparations to about the same extent. The PGF2 alpha-stimulated contractions, unlike those produced by noradrenaline, were completely inhibited in calcium-free physiological solution. 3 The calcium entry blocking drugs, cinnarizine and flunarizine, had little effect on the resting exchange of calcium in the arterial smooth muscle, but inhibited PGF2 alpha-stimulated contractions and 45Ca uptake to a similar extent. 4 Flunarizine was about 7 fold more potent as an inhibitor of noradrenaline- than of PGF2 alpha-mediated contraction and 45Ca uptake and this ratio was about 50 for cinnarizine. 5 EGTA (1.25 mM) produced a relaxation of noradrenaline and PGF2 alpha-induced maximal contractions. Measured over the first 2 min of EGTA contact, the rate of relaxation was much faster in noradrenaline than in PGF2 alpha-stimulated preparations. 6 Turnover of cellular calcium (influx plus efflux) during the first 2 min of noradrenaline contact was much greater than that produced by PGF2 alpha, largely due to a greater effect of noradrenaline on calcium efflux. 7 The results suggest that PGF2 alpha-but not noradrenaline-induced contractions are entirely dependent on the influx of extracellular calcium and that the agonists may stimulate calcium gating mechanisms differently.     

The effects of calcium antagonists on calcium overload contractures in embryonic chick myocytes induced by ouabain and veratrine.

1. The protective effects of some calcium antagonists against different forms of calcium overload contracture were investigated in embryonic chick cardiac myocytes. 2. Tetrodotoxin-sensitive sodium currents were recorded from the myocytes by the whole-cell voltage-clamp technique. Although the peak current was attenuated by veratrine, the inactivation process was markedly inhibited, resulting in a large increase in the total inward current. Action potentials were prolonged by veratrine, automaticity was inhibited and the membrane potential depolarized from -79 to around -45 mV. 3. Measurements of contraction were made from aggregates of myocytes using a video edge detection technique which quantified edge movement. Veratrine caused an initial positive inotropism then inhibited automaticity of aggregates with subsequent development of a tonic contracture to around 300% of the twitch contraction. 4. Veratrine-induced contractures were not significantly affected by 10 microM diltiazem or verapamil. Nifedipine (5 microM), nimodipine (5 microM) and ryanodine (5 microM) also had little effect whilst nicardipine and flunarizine caused a concentration-dependent inhibition of veratrine-induced contractures with IC50s of 3 microM and 2 microM respectively. 5. Veratrine-induced contractures were found to be very sensitive to extracellular calcium concentration with an EC50 of 32 microM. Edge movement associated with beating of the myocytes was much less sensitive to calcium (EC50 = 1 mM). Submaximal veratrine contractures in 20-50 microM extracellular calcium were not potentiated by 1 microM Bay K 8644. 6. Tetrodotoxin also inhibited veratrine-induced contractures but did not affect contractions induced by ouabain in the presence of 10 microM diltiazem. 7. Ouabain-induced contractures were also inhibited by nicardipine and flunarizine indicating that these drugs can protect against calcium overload in embryonic chick heart by a mechanism independent of the normal form of voltage-sensitive sodium or calcium channels.