Structural model for phenylalkylamine binding to L-type calcium channels.

Phenylalkylamines (PAAs), a major class of L-type calcium channel (LTCC) blockers, have two aromatic rings connected by a flexible chain with a nitrile substituent. Structural aspects of ligand-channel interactions remain unclear. We have built a KvAP-based model of LTCC and used Monte Carlo energy minimizations to dock devapamil, verapamil, gallopamil, and other PAAs. The PAA-LTCC models have the following common features: (i) the meta-methoxy group in ring A, which is proximal to the nitrile group, accepts an H-bond from a PAA-sensing Tyr_IIIS6; (ii) the meta-methoxy group in ring B accepts an H-bond from a PAA-sensing Tyr_IVS6; (iii) the ammonium group is stabilized at the focus of P-helices; and (iv) the nitrile group binds to a Ca(2+) ion coordinated by the selectivity filter glutamates in repeats III and IV. The latter feature can explain Ca(2+) potentiation of PAA action and the presence of an electronegative atom at a similar position of potent PAA analogs. Tyr substitution of a Thr in IIIS5 is known to enhance action of devapamil and verapamil. Our models predict that the para-methoxy group in ring A of devapamil and verapamil accepts an H-bond from this engineered Tyr. The model explains structure-activity relationships of PAAs, effects of LTCC mutations on PAA potency, data on PAA access to LTCC, and Ca(2+) potentiation of PAA action. Common and class-specific aspects of action of PAAs, dihydropyridines, and benzothiazepines are discussed in view of the repeat interface concept.

Identification of human cytochrome P-450 isoforms involved in metabolism of R(+)- and S(-)-gallopamil: utility of in vitro disappearance rate.

Isoforms of cytochrome P-450 (CYP) involved in the metabolism of gallopamil enantiomers were identified by measuring the disappearance rate of parent drug from an incubation mixture with human liver microsomes and recombinant human CYPs. Mean (+/- S.D.) intrinsic clearances (CL(int)) of R(+)- and S(-)-gallopamil in human liver microsomes were 0.320 +/- 0.165 and 0.205 +/- 0.107 ml/min/mg protein, respectively. These values were highly correlated with the 6beta-hydroxylation activity of testosterone, a marker substrate of CYP3A4 (r = 0.977 and 0.900 for R(+)- and S(-)-gallopamil, respectively, p <.001). Ketoconazole and troleandomycin, selective inhibitors of CYP3A4, and polyclonal antibodies raised against CYP3A4/5 markedly reduced the CL(int) of gallopamil enantiomers in human liver microsomes. Among the 10 recombinant human CYP isoforms, CYP3A4 exhibited the highest CL(int) of gallopamil enantiomers, and CYP2C8 and CYP2D6 also exhibited appreciable activity. When the contribution of CYP3A4 to the total metabolic clearance of gallopamil enantiomers in human liver microsomes was estimated by relative activity factor, the mean (+/- S.D.) contributions were 92 +/- 18 and 68 +/- 19% for R(+)- and S(-)-gallopamil, respectively. These values were comparable to the rates of immunoinhibition by antibodies raised against CYP3A4/5 observed in human liver microsomes. The present study suggests that CYP3A4 is a major isoform involved in the overall metabolic clearance of gallopamil enantiomers in the human liver, and that the present approach based on disappearance rate may be applicable to identify major isoforms of CYP involved in the metabolism of a drug in human liver microsomes.

Mechanism of verapamil block of a neuronal delayed rectifier K channel: active form of the blocker and location of its binding domain.

1. The mechanism of verapamil block of the delayed rectifier K currents (I K(DR)) in chick dorsal root ganglion (DRG) neurons was investigated using the whole-cell patch clamp configuration. In particular we focused on the location of the blocking site, and the active form (neutral or charged) of verapamil using the permanently charged verapamil analogue D890. 2. Block by D890 displayed similar characteristics to that of verapamil, indicating the same state-dependent nature of block. In contrast with verapamil, D890 was effective only when applied internally, and its block was voltage dependent (136 mV/e-fold change of the on rate). Given that verapamil block is insensitive to voltage (Trequattrini et al., 1998), these observations indicate that verapamil reaches its binding site in the uncharged form, and accesses the binding domain from the cytoplasm. 3. In external K and saturating verapamil we recorded tail currents that did not decay monotonically but showed an initial increase (hook). As these currents can only be observed if verapamil unblock is significantly voltage dependent, it has been suggested (DeCoursey, 1995) that neutral drug is protonated upon binding. We tested this hypothesis by assessing the voltage dependence of the unblock rate from the hooked tail currents for verapamil and D890. 4. The voltage dependence of the off rate of D890, but not of verapamil, was well described by adopting the classical Woodhull (1973) model for ionic blockage of Na channels. The voltage dependence of verapamil off rate was consistent with a kinetic scheme where the bound drug can be protonated with rapid equilibrium, and both charged and neutral verapamil can unbind from the site, but with distinct kinetics and voltage dependencies.

Extracellular site of action of phenylalkylamines on L-type calcium current in rat ventricular myocytes.

The effects of the phenylalkylamines verapamil, gallopamil, and devapamil on L-type calcium currents (ICa) were studied in ventricular myocytes from rat hearts using the whole-cell patch-clamp technique. In particular, the question was addressed, whether the pharmacological binding sites for these drugs were located at the inner and/or at the outer surface of the cell membrane. Therefore, tertiary verapamil, gallopamil, and devapamil and their corresponding quaternary derivatives were applied either from the outside or the inside of the cell membrane. Extracellular application of verapamil, gallopamil and devapamil (each at 3 microM) reduced ICa to 16.1 +/- 8.6%, 11 +/- 8.9%, and 9.3 +/- 6% of control, respectively. Intracellular application of the same substances, via the patch pipette filled with 30 microM of either verapamil, gallopamil, or devapamil, failed to depress ICa. The quaternary derivatives of the phenylalkylamines (30 microM) were ineffective both when applied extracellularly or intracellularly. It is suggested that phenylalkylamines block ICa in ventricular myocytes by acting on a binding site of the calcium channel molecule located at the outer surface of the cell membrane.

Pharmacological profile of UK-74,505, a novel and selective PAF antagonist with potent and prolonged oral activity.

UK-74505, a novel 1,4-dihydropyridine PAF antagonist, exhibited highly selective, time-dependent inhibition of PAF-induced aggregation of rabbit washed platelets (IC50 = 26.3 +/- 0.88 and 1.12 +/- 0.04 nM after 0.25 and 60 min preincubation, respectively), which became irreversible within 15 min, whereas inhibition by WEB-2086 was both independent of preincubation time (IC50 = 145.7 +/- 24.7 nM) and competitive (KI = 27.5 +/- 7.7 nM; Schild slope = 0.98 +/- 0.04). The selective inhibition of specific [3H]PAF binding by UK-74,505 exhibited a slower onset, the IC50 obtained without preincubation (14.7 +/- 2.6 nM) decreasing 2-fold at 45 min. UK-74,505 was 450-fold weaker as an antagonist of [3H]nitrendipine binding to bovine brain membranes and KCl-induced contraction of rat aorta. UK-74,505 was 10-30-fold more potent than WEB-2086 in vivo as an inhibitor of PAF-induced hypotension in rats (ED50 = 35 +/- 5.8 micrograms/kg, i.v.), cutaneous vascular permeability in guinea pigs (ED50 = 0.37 +/- 0.08 mg/kg, p.o.) and lethality in mice, with oral ED50 values of 0.26 +/- 0.03 and 1.33 +/- 0.19 mg/kg at 2 and 8 h, respectively. These data demonstrate that UK-74,505 is a potent, selective, long-acting irreversible PAF antagonist.

Allosteric interaction of semotiadil fumarate, a novel benzothiazine, with 1,4-dihydropyridines, phenylalkylamines, and 1,5-benzothiazepines at the Ca(2+)-channel antagonist binding sites in canine skeletal muscle membranes.

We assessed the binding characteristics of a benzothiazine Ca(2+)-channel antagonist, semotiadil, in canine skeletal muscle membranes. Semotiadil inhibited binding of (+)-[3H]PN 200-110 (maximum inhibition 80%), and almost completely inhibited binding of both (-)-[3H]desmethoxyverapamil and D-cis-[3H]diltiazem to their specific binding sites with an IC50 value of 0.2-2 microM and a Hill slope of 0.6-0.9. Saturation isotherm and dissociation kinetic studies suggest that semotiadil acts as a noncompetitive inhibitor at the 1,4-dihydropyridine, phenylalkylamine, and benzothiazepine (BTZ) recognition sites in the L-type Ca2+ channel: (a) Scatchard analysis showed that semotiadil decreased maximum binding (Bmax) of the three classes of Ca2+ channel antagonists, while causing a slight increase in the equilibrium dissociation constant (Kd) in the case of (+)-[3H]PN 200-110 binding or no significant change in Kd values for binding of (-)-[3H]desmethoxyverapamil and D-cis-[3H]diltiazem to their specific binding sites; and (b) dissociation kinetics of the (+)-[3H]PN 200-110 and D-cis-[3H]diltiazem bindings were accelerated by semotiadil. These results suggest that semotiadil has a strong negative allosteric interaction with three classes of Ca2+ channel antagonists, including 1,4-dihydropyridines, phenylalkylamines, and BTZ at their specific binding sites.

Conformational analysis of free and Ca(2+)-bound forms of verapamil and methoxyverapamil.

In a recent experimental study (Tetreault, S. and Ananthanarayanan, V.S. (1993) J. Med. Chem. 36, 1324-1332) we showed that verapamil can bind Ca2+ in a nonpolar medium to form 1:1 and 2:1 drug:Ca2+ complexes and proposed that such complexes may represent the bioactive form of the drug. A similar suggestion has also been made earlier from theoretical considerations of the geometry of the drug (Zhorov, B. and Govyrin, V. (1983), Dokl.Akad.Nauk SSSR 273, 497-501). In order to fully understand the nature of the drug-Ca2+ complex, we present in this paper a systematic conformational analysis of the protonated and neutral forms of verapamil and one of its potent analogues, methoxyverapamil (D600). For each form of verapamil and D600, the energies and generalized coordinates of all minimum-energy conformations (MECs) with the energy less than 5 kcal/mol above the global minimum have been accumulated and sorted in the order of increasing energies. A protocol was then used to search in the files MECs meeting a set of geometrical criteria and to sum up their populations. The geometrical criteria involved the predisposition of the oxygen and nitrogen atoms of the drug molecule to form bi- tri- and tetradentate complexes with Ca2+. Use of these criteria demonstrated that both verapamil and D600 have several low-energy structural patterns that are predisposed for bi- and polydentate chelation of Ca2+. Models of various types of 1:1 drug:Ca2+ complexes as well as two models of 2:1 drug:Ca2+ "sandwich" complex were obtained. Such models may be biologically relevant in understanding the nature of the ternary complex formed by the drug, Ca2+ and the calcium channel.

Comparison of lowest energy conformations of dimethylcurine and methoxyverapamil: evidence of ternary association of calcium channel, Ca2+, and calcium entry blockers.

Verapamil and dimethylcurine are Ca2+ entry blockers of essentially different chemical structures which presumably bind to the same arylalkylamine receptor of the L-type Ca channel. A systematic conformational analysis of methoxyverapamil (D-600) and dimethylcurine has been carried out using a molecular mechanics method. The lowest minimum-energy conformations of D-600 are predisposed to chelate Ca2+ by four oxygen atoms of the stacked methoxyphenyl moieties. Comparison of the lowest energy conformations of D-600-Ca2+ and dimethylcurine revealed a similar spatial disposition of cationic groups and methoxyphenyl moieties in the two compounds. A three-dimensional model of arylalkylamine receptor was suggested which incorporates two nucleophilic areas of the Ca channel. Dimethylcurine binds to these areas by its quaternary amine functions, whereas D-600 does so by amine function and via coordinated Ca2+. The results support the hypotheses on ternary complex formation between the ligands of Ca channel, their receptors, and Ca2+.

Bis(benzylisoquinoline) analogs of tetrandrine block L-type calcium channels: evidence for interaction at the diltiazem-binding site.

Bis(benzylisoquinoline) alkaloids block Ca2+ uptake through the L-type Ca2+ channel and modulate binding of ligands to four distinct sites (dihydropyridine, benzothiazepine, aralkylamine, and (diphenylbutyl)piperidine) in the Ca2+ entry blocker receptor complex of the channel. These alkaloids are structural analogs of tetrandrine, which has previously been demonstrated to block the L-type Ca2+ channel through interaction at the benzothiazepine (diltiazem) site (King et al., 1988). Different alkaloid conformational classes display either alpha-beta, beta-alpha, alpha-alpha, or beta-beta stereochemistry at the two chiral isoquinoline carbons. Compounds from all four classes were tested for their ability to interact with Ca2+ entry blocker ligands. All analogs completely inhibit diltiazem binding, but many only partially inhibit D-600 and fluspirilene binding. For dihydropyridine binding, the compounds show either stimulation or inhibition or exhibit no effect. This profile is quite different from the interaction displayed by diltiazem or tetrandrine. Scatchard analyses show effects predominantly on Kd for diltiazem, D-600, and PN200-110 binding. Representative conformers do not effect diltiazem dissociation rates but alter dissociation kinetics of ligands which bind to the other three sites. A correlation of the ability of these compounds to inhibit Ca2+ uptake through the L-type Ca2+ channel in GH3 cells exists only with their inhibition of diltiazem binding but not with inhibition of binding of ligands representing other classes of Ca2+ entry blockers. These data, taken together, indicate that a variety of bis(benzylisoquinoline) congeners act to block the L-type Ca2+ channel by binding to the benzothiazepine site on the channel.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for estimation of drug affinity constants to the open conformational state of calcium channels.

The affinity of D600 to calcium channels in the open state has been examined in isolated smooth muscle cells of the rabbit ear artery. Calcium channel currents were measured in high external barium solution by means of the patch-clamp technique. The current inhibition in various D600 concentrations (3-100 microM) on application of trains of short test pulses (20-80 ms) has been studied in nonmodified calcium channels and in cells where the calcium channels were modified by the agonist dihydropyridine (+) 202,791 (100 nM). The kinetics of the peak current decay has been analyzed with a mathematical model which is based on the experimental finding that D600 interacts primarily with calcium channels in the open conformational state. The model approach allows the estimation of drug affinity constants of D600 to the calcium channel in the open conformation. An association rate constant to the open conformational state of D600 of 6.16 x 10(4) M-1 s-1 was estimated. The association rate of the drug was not significantly changed after the calcium channels have been modified with 100 nM (+) 202,791. A method for correction of rate constants for possible drug trapping is discussed.

Halothane depresses D600 binding to bovine heart sarcolemma.

Volatile anesthetics exert their negative inotropic effects by interfering with Ca2+ homeostasis in the myocardial cell. The mechanism of this dose-dependent action is uncertain. 3H-D600 (3H-Gallopamil), a Ca(2+)-channel antagonist, binds to the voltage-dependent Ca2+ channels (VDCC) in a specific, saturable, and reversible manner. We used this ligand to study the effect of halothane on the binding characteristics of the VDCC in purified bovine heart sarcolemma. Cardiac sarcolemmal vesicles were isolated from fresh bovine heart by differential centrifugation and filtration. 3H-D600 equilibrium binding assays were performed in the presence or absence of 1.0 mM unlabeled D600 to determine total and nonspecific binding in room air and at 0.7, 1.3, and 2.5% (vol/vol) halothane. Halothane produced a significant dose-dependent and reversible depression of 3H-D600 specific binding in bovine heart sarcolemma. Depression was completely reversed when halothane had evaporated from the samples prior to filtration. Halothane 1.3% (vol/vol) produced a 40% reduction in the maximum binding capacity. The dissociation constant was not affected by any concentration of halothane. One mechanism by which the volatile anesthetics may induce negative inotropism is through the reduction of functional VDCCs in the heart, leading to reduction of Ca2+ entry. The results of this study support this hypothesis.

Interspecies differences in the effect of pH on gallopamil protein binding to albumin and alpha 1-acid glycoprotein.

There is little information about the factors which influence drug protein binding between species. We have therefore investigated the role of pH on the binding of gallopamil, a calcium channel antagonist known to exhibit pH-sensitive binding, among four species, human, baboon, bovine, and canine. We used pure protein solutions of alpha 1 acid glycoprotein (AAG) (60 mg.l-1), albumin (45 gm.l-1), and their combination and three values of pH, 7.0, 7.4, and 8.0. Gallopamil protein binding was determined over a concentration range of 2.0 x 10(-7) mol.l-1 to 2.1 x 10(-3) mol.l-1 using equilibrium dialysis. Gallopamil binding in all solutions was best described using a two binding site model in the combination solution and a one binding site model in the pure solutions. pH did not affect the number of identical binding sites. However, the influence of pH on gallopamil binding was species specific. Increasing the pH from 7.0 to 8.0 influenced binding affinity differently between species. There were directionally similar changes in unbound fraction at a gallopamil concentration of 2 x 10(-7) mol.l-1 as pH increased, although there were species differences in the degree of change. In protein solutions containing both AAG and albumin a reduction in pH from 7.4 to 7.0 resulted in species-specific increases in the unbound fraction. Increasing the pH from 7.4 to 8.0 again resulted in species-specific reductions in the unbound fraction of gallopamil. Similar changes were seen when pure AAG or albumin solutions were used, indicating species variance in both gallopamil protein binding and the effect of pH on binding.

D600 binding sites on voltage-sensors for excitation-contraction coupling in frog skeletal muscle are intracellular.

Charge movements were measured in frog cut twitch fibres mounted in a double Vaseline gap chamber at 14 degrees C with 30 microM D600 in the external solution. TEST-minus-CONTROL current traces appear normal with a hump current component (I gamma) embedded in the decay phase of the early current component (I beta) in the ON-segment and an exponentially decaying current transient in the OFF-segment. When a conditioning depolarization to 0 mV is applied at around 6 degrees C, charge movement is greatly reduced but not completely suppressed and no hump component can be visualized in the ON-segment. In addition, an extra capacitive component is generated having a time course slower than, and a polarity opposite to, that of the usual charge movement. This extra component makes the transients in both the ON- and OFF-segments appear bisphasic. When temperature is restored to 14 degrees C, the biphasic nature is greatly enhanced. After the application of a conditioning hyperpolarization, the shape of the TEST-minus-CONTROL current trace is converted back to that before paralysis, but the total amount of charge reprimed is less than 100% of control. In general, more Q beta is reprimed than Q gamma, and the amount of Q gamma reprimed varies over a wider range from fibre to fibre than that of Q beta. Extracellularly applied D890 cannot reproduce the blocking effect of D600 whereas intracellularly applied D890 can. As D890 is permanently charged and cannot permeate through the plasma membranes, it can be concluded that the binding sites for D600/D890 on the charge movement macromolecules must be on the myoplasmic side. This adds another parallelism between the charge movement entities and L-type calcium channels. However, the specific prerequisites for the blockage of the former not shared by the latter differentiates the two physiological units.

Pathways of gallopamil metabolism. Regiochemistry and enantioselectivity of the O-demethylation processes.

The oxidative O-demethylation of pseudoracemic gallopamil by rat and human liver microsomes was studied. By comparison of GC/MS retention times and fragmentation patterns with data from authentic standards, the four possible regioisomeric monophenolic metabolites, 2-(4-hydroxy-3,5-dimethoxyphenyl)-2-isopropyl-5-[(3,4- dimethoxyphenethyl)methylamino]-valeronitrile (2), 2-(5-hydroxy-3,4-dimethoxyphenyl)-2-isopropyl-5-[(3,4- dimethoxyphenethyl)methylamino]valeronitrile (3), 2-(3,4,5-trimethoxyphenyl)-2-isopropyl-5-[(4-hydroxy-3-methoxyphenethyl) -methylamino]valeronitrile (4), and 2-(3,4,5-trimethoxyphenyl)-2-isopropyl-5-[(3-hydroxy-4- methoxyphenethyl)methylamino]valeronitrile (5), were characterized. Rat liver microsomal oxidation produced all four regioisomeric monophenols which accounted for only 10% of the oxidative metabolism, the remaining 90% being N-dealkylation metabolites. Preference for metabolism of the O-methyl ethers at p-positions on each of the aromatic ring systems was noted, with more O-demethylation of the O-methyl ethers on the aromatic ring adjacent to the chiral center than on the aromatic ring in the short side chain. Significant enantio-selectivity was noted, the S/R ratios being 2.26, 1.97, 1.87 and 1.30 for formation of 2, 3, 4 and 5, respectively. Biliary excretion of the O-demethylated metabolites as conjugates, cleaved by beta-glucuronidase, was observed in rats after administration of pseudoracemic gallopamil. Significant stereoselectivity was noted, S/R ratios being 0.62, 1.61, 1.49 and 2.19 for 2, 3, 4 and 5, respectively. Human liver microsomal oxidation produced more p- than m-O-demethylation, with 4 less than 5, and 2 less than 3, but quantitatively the pathway is a minor one compared to N-dealkylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathways of gallopamil metabolism. Regiochemistry and enantioselectivity of the N-dealkylation processes.

The N-dealkylation pathway for the metabolism of pseudoracemic gallopamil was studied in the presence of rat and human liver microsomes and in vivo in rats and man. Metabolites were characterized by comparison of their GC/MS retention times and fragmentation patterns with those of authentic compounds. In the presence of rat liver microsomes, N-dealkylation accounted for about 90% of the observed oxidative metabolism, affording a 4:1 ratio of norgallopamil (2) and, N-methyl-N-[2-methyl-3-cyano-3-(3,4,5-trimethoxyphenyl)-6-hexyl] amine (3), and about 1% of N-methyl-N-(3,4-dimethoxyphenethyl)amine (4). Secondary amines 2 and 3 arose enantioselectively from S-(-)-gallopamil, the S/R ratios being 1.36 and 1.71, respectively. The alcohols, 3,4-dimethoxyphenylethanol (6) and 2-methyl-3-cyano-3-(3,4,5-trimethoxyphenyl)-6-hexanol (8), were formed from the respective intermediate aldehydes 5 and 7, probably non-enzymatically, under the reductive conditions (NADPH) of the microsomal incubations. Incubation of gallopamil with 9,000g supernatant fraction of rat liver led to carboxylic acid metabolites arising from oxidative metabolism of the aldehydes. 3,4-Dimethoxyphenylacetic acid (12) and 4-(3,4,5-trimethoxyphenyl)-5-methyl-4-cyanohexanoic acid (11) were formed in a 3:5:1 ratio. In the presence of human liver microsomes, formation of 2 also predominated over formation of 3, with alcohols 6 and 8 being produced as well. However, 4 was not observed. Consistently, the N-dealkylation process provided slightly more R than S products with the S/R ratio being 0.7-0.9 for metabolites 2, 3, 6, and 8. The amines formed from N-dealkylation were also observed as urinary metabolites in a human subject after a single oral dose of pseudoracemic gallopamil.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and identification of the N-glucuronides of norgallopamil and norverapamil, unusual metabolites of gallopamil and verapamil.

N-Glucuronides of norgallopamil and norverapamil were found as biliary metabolites after administering the corresponding tertiary amines, gallopamil and verapamil, to rats. The structures of these unusual metabolites were established by comparison with spectral data of synthesized authentic standards and by enzymic hydrolysis of the conjugates. The N-glucuronide standards were synthesized by coupling the secondary amines to either glucuronic acid or to methyl tetra-O-acetyl-beta-D-glucopyranuronate. On i.p. dosing of rats with gallopamil or verapamil, 13 and 2% of the dose, respectively, appeared in the bile as the N-glucuronide of the secondary amine metabolite over an 8-hr period. Administration of norgallopamil resulted in approximately 25% of the dose being excreted as N-glucuronide conjugate in the bile. Substantially more of the S- than R-enantiomer of both gallopamil and verapamil was converted to the corresponding secondary amine N-glucuronide. The observed high S/R ratios suggest enantio-selectivity in this pathway could contribute to the observed stereoselectivity in other routes of metabolism of the parent tertiary amines.

Studies on Ca2+ channel antagonists. A 2-diazo-3,3,3-trifluoropropionamide derivative related to verapamil as a potential photoaffinity probe.

2-(3,4-Dimethoxyphenyl)-2-isopropyl-5-[N-[4-(N-methyl-2-diazo- 3,3,3-trifluoropropionamido)phenethyl]methyl-amino]valeronitril e (3), a potential photoaffinity probe for Ca2+ channels related to verapamil (1), was prepared from N-methyl-4-nitrophenethylamine (7) and 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-(methanesulfonoxy)valeron itrile (12). Compound 3 showed concentration-dependent negative inotropic effects in rat right myocardial ventricular strips, EC50 = (1.05 +/- 0.33) X 10(-7) M (mean +/- SD), being slightly less potent than gallopamil (2), EC50 = (2.18 +/- 0.66) X 10(-8) M. It displaced [3H]gallopamil in myocardial membranes, Ki = (3.76 +/- 1.55) X 10(-8) M, compared to 2, Ki = (1.55 +/- 0.16) X 10(-8) M. Photoactivation at 265 nm reduced the recoverable binding of [3H]gallopamil to 26% compared to no effect on 2. This agent may be a useful photoaffinity probe to aid in further characterization of Ca2+ channels.

Metabolism of the calcium antagonist gallopamil in man.

The metabolism of gallopamil (5-[(3,4-dimethoxyphenyl)methylamino]-2-(3,4,5-trimethoxyphenyl) -2- isopropylvaleronitrile hydrochloride, Procorum, G) was studied after single administration (2 mg i.v., 50 mg p.o.) of unlabelled and labelled G (14G, 2H). TLC, HPLC, GLC, MS and RIA were used for assessment of G and its metabolites in plasma, urine and faeces. G clearance is almost completely metabolic, with only minimal excretion of unchanged drug. Metabolites represent most of the plasma radioactivity after p.o. administration. They are formed by N-dealkylation and O-demethylation with subsequent N-formylation, or glucuronidation, respectively. Compound A, derived by loss of the 3,4-dimethoxyphenethyl moiety of G is the main metabolite in plasma and urine (about 20% of the dose). This metabolite is accompanied by its N-formyl derivative (C), by the N-demethylated compound (H) and the acid (F), formed by oxidative deamination of A. Only 3 unconjugated monphenoles from several O-demthylated products showed distinct plasma levels which were nevertheless lower than metabolite A. These metabolites had no relevance to the pharmacodynamic action. Conjugated monophenolic and diphenolic products represented the major part in plasma and were excreted predominantly via the bile: they represented almost the whole faecal metabolite fraction. Less than 1% of the dose was recovered unchanged in the urine. About 50% of the dose is excreted by urine and 40% by faeces.

Interactions of DPI 201-106, a novel cardiotonic agent, with cardiac calcium channels.

The interaction of DPI 201-106, a novel cardiotonic agent, with the calcium entry blocker receptor complex was studied using porcine cardiac sarcolemmal membranes. DPI 201-106 and the chemically-related calcium antagonist, cinnarizine, produce concentration-dependent inhibition of nitrendipine, gallopamil and diltiazem binding to their respective sites in these vesicles. This effect of DPI 201-106 is not stereoselective since resolved stereoisomers of this compound display equal potency in inhibiting each of the binding reactions. Equilibrium ligand binding studies revealed that DPI 201-106 and cinnarizine cause mixed inhibitory patterns at the aralkylamine and benzothiazepine sites (i.e. both Kd and Bmax values were affected) while mainly increasing Kd at the dihydropyridine site. The kinetics of ligand dissociation from the three calcium entry blocker receptors, together with measurements of dihydropyridine association kinetics, further demonstrate that DPI 201-106 interacts at a unique site in the receptor complex and allosterically modulates binding of nitrendipine, gallopamil and diltiazem. The functional consequences of the above interactions with the calcium channel were studied in isolated cardiac preparations. In guinea-pig atria, DPI 201-106 increased force of contraction. This inotropic effect is seen only with the S(-) enantiomer and is unaltered by nitrendipine-, verapamil- or diltiazem-pretreatment, indicating DPI 201-106 does not act as a stimulant of this channel. Furthermore, DPI 201-106 did not alter the inotropic action of Bay K 8644, a calcium channel stimulant. Spontaneous rate of guinea-pig right atria is decreased by both DPI 201-106 and cinnarizine. In addition, potassium-induced contractures in cat papillary muscles are reduced by both agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of tetrandrine with slowly inactivating calcium channels. Characterization of calcium channel modulation by an alkaloid of Chinese medicinal herb origin.

Tetrandrine, a bis-benzylisoquinoline alkaloid derived from the Chinese medicinal herb Stephania tetrandra, is a putative Ca2+ entry blocker whose mechanism of action is unknown. To investigate this mechanism, the effects of tetrandrine were characterized on binding of three chemical classes of Ca2+ entry blockers in cardiac sarcolemmal membrane vesicles. In the range 25-37 degrees C, tetrandrine completely blocks diltiazem binding, partially inhibits D-600 binding, and markedly stimulates nitrendipine binding, with greatest enhancement occurring at 37 degrees C. The potency of tetrandrine is increased 10-fold as temperature is raised from 25 to 37 degrees C. Scatchard analyses indicate that inhibition of diltiazem binding and stimulation of nitrendipine binding result from changes in ligand affinities while inhibition of D-600 binding is due to both an increase in KD and decrease in Bmax of aralkylamine receptors. Ligand dissociation studies reveal that tetrandrine increases D-600 off-rates, decreases nitrendipine off-rates, but has no effect on diltiazem dissociation kinetics. In addition, tetrandrine reversibly blocks inward Ca2+ currents through L-type Ca2+ channels in GH3 anterior pituitary cells. These results indicate that tetrandrine interacts directly at the benzothiazepine-binding site of the Ca2+ entry blocker receptor complex and allosterically modulates ligand binding at other receptors in this complex. These findings suggest that tetrandrine is a structurally unique natural product Ca2+ entry blocker and provide a rationale explanation for the therapeutic effectiveness of this agent.