Diltiazem Binding Research Articles

Investigations of the molecular mechanism of diltiazem binding to human serum albumin in the presence of metal ions, glucose and urea

The molecular mechanism and thermodynamic properties of the interaction between diltiazem (DTZ) and human serum albumin (HSA), has been studied in vitro using spectroscopic techniques (UV-Vis, fluorescence, FTIR), and molecular docking methods. The effect of acidic and basic pH, glucose, urea, and metal ions on the DTZ-HSA binding has been investigated as well. According to the results, there is a 1:1 interaction between DTZ and HSA, while the quenching mechanism is static up to 313 K. The apparent binding constant was 2.09 × 106 M −1 that indicates a strong binding between DTZ and HSA. DTZ binding was increased in acidic pH while its binding was slowly decreased in the presence of glucose, urea, and metal ions. Thermodynamic studies showed that DTZ binds to HSA via an exothermic and spontaneous reaction via hydrogen bonding and electrostatic interactions. The conformational alteration of HSA is obvious according to the FTIR study. The site marker competitive study confirmed the binding of DTZ to the warfarin binding site. Molecular docking studies showed that DTZ binds to subdomain IB (−9.22 kcal mol−1) and subdomain IIIA (−9.03 kcal mol−1) with a higher tendency. Also, the results showed that the oxygen and nitrogen atoms of hydroxyl and amino functional groups of DTZ facilitate hydrogen bond formation. Highlights Strong binding of diltiazem to HSA was studied and confirmed by fluorescence quenching titrations. Diltiazem binding to HSA reduces in the presence of metal ions, glucose, urea and alkaline pH. Diltiazem binding to HSA is exothermic and spontaneous.

T1143 essential for CaV1.2 inhibition by diltiazem

Careful analysis of previously published reports and some new insights into the structure activity studies revealed an important role of Threonine 1143 in drug binding. Substituting T1143 by alanine and other residues significantly reduced channel inhibition by qDil and Dil. Mutation T1143A did not affect channel activation or inactivation while almost completely diminishing channel block by Dil or qDil. These findings support the view that T1143 serves as drug binding determinant. Other mutations in this position than T1143A (T1143L/Y/S/N/C/V/E) diminished channel inhibition by qDil but additionally affected channel activation and inactivation and may therefore affect channel block allosterically.Collectively, our data suggest that T1143 is an essential diltiazem binding determinant.

Structural Basis for Diltiazem Block of a Voltage-Gated Ca2+ Channel

Diltiazem is a widely prescribed Ca2+ antagonist drug for cardiac arrhythmia, hypertension, and angina pectoris. Using the ancestral CaV channel construct CaVAb as a molecular model for X-ray crystallographic analysis, we show here that diltiazem targets the central cavity of the voltage-gated Ca2+ channel underneath its selectivity filter and physically blocks ion conduction. The diltiazem-binding site overlaps with the receptor site for phenylalkylamine Ca2+ antagonist drugs such as verapamil. The dihydropyridine Ca2+ channel blocker amlodipine binds at a distinct site and allosterically modulates the binding sites for diltiazem and Ca2+ Our studies resolve two distinct binding poses for diltiazem in the absence and presence of amlodipine. The binding pose in the presence of amlodipine may mimic a high-affinity binding configuration induced by voltage-dependent inactivation, which is favored by dihydropyridine binding. In this binding pose, the tertiary amino group of diltiazem projects upward into the inner end of the ion selectivity filter, interacts with ion coordination Site 3 formed by the backbone carbonyls of T175, and alters binding of Ca2+ to ion coordination Sites 1 and 2. Altogether, our results define the receptor site for diltiazem and elucidate the mechanisms for pore block and allosteric modulation by other Ca2+ channel-blocking drugs at the atomic level. SIGNIFICANCE STATEMENT: Calcium antagonist drugs that block voltage-gated calcium channels in heart and vascular smooth muscle are widely used in the treatment of cardiovascular diseases. Our results reveal the chemical details of diltiazem binding in a blocking position in the pore of a model calcium channel and show that binding of another calcium antagonist drug alters binding of diltiazem and calcium. This structural information defines the mechanism of drug action at the atomic level and provides a molecular template for future drug discovery.

Interaction of diltiazem with an intracellularly accessible binding site on CaV1.2

BACKGROUND AND PURPOSEDiltiazem inhibits CaV1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α1 subunit of CaV1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α1 subunits.EXPERIMENTAL APPROACHCaV1.2 composed of α1, α2-δ and β2a subunits were expressed in tsA-201 cells and barium currents through CaV1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion).KEY RESULTSQuaternary derivative d-cis-diltiazem inhibited CaV1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59 ± 4% at 300 µM) and induced only a low level of tonic (non-use-dependent) block (16 ± 2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 that have previously been shown to reduce the sensitivity of CaV1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil.CONCLUSION AND IMPLICATIONSThe data show that use-dependent block of in CaV1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.

Ligands of Diltiazem Binding Site: An Overview of Some Chemotypes

The diltiazem binding site of L-type calcium channels is the least characterized to date. In this paper, we present some of the available chemotypes that bind to the benzothiazepine binding site: natural compounds, compounds synthesized by varying the benzothiazepine scaffold, and compounds discovered by means of computational approaches.

Discovery of Novel and Cardioselective Diltiazem-like Calcium Channel Blockers via Virtual Screening

With the effort to discover new chemotypes blocking L-type calcium channels (LTCCs), ligand-based virtual screening was applied with a specific interest toward the diltiazem binding site. Roughly 50000 commercially available compounds served as a database for screening. The filtering through predicted pharmacokinetic properties and structural requirements reduced the initial database to a few compounds for which the similarity was calculated toward two template molecules, diltiazem and 4-chloro-Ncyclopropyl- N-(4-piperidinyl)benzene-sulfonamide, the most interesting hit of a previous screening experiment. For 18 compounds, inotropic and chronotropic activity as well as the vasorelaxant effect on guinea pig were studied "in vitro", and for the most promising, binding studies to the diltiazem site were carried out. The procedure yielded several hits, confirming in silico techniques to be useful for finding new chemotypes. In particular, N-[2-(dimethylamino)ethyl]-3-hydroxy-2-naphthamide, N,Ndimethyl- N'-(2-pyridin-3-ylquinolin-4-yl)ethane-1,2-diamine, 2-[(4-chlorophenyl)(pyridin-2-yl)methoxy]- N,N-dimethylethanamine (carbinoxamine), and 7-[2-(diethylamino)ethoxy]-2H-chromen-2-one revealed interesting activity and binding to the benzothiazepine site.

Up‐regulation of L‐type high voltage‐gated calcium channel subunits by sustained exposure to 1,4‐ and 1,5‐benzodiazepines in cerebrocortical neurons

The aim of this study is to examine how sustained exposure to two 1,4-benzodiazepines (BZDs) with different action period, diazepam and brotizolam, and a 1,5-BZD, clobazam, affects L-type high voltage-gated calcium channel (HVCC) functions and its mechanisms using primary cultures of mouse cerebral cortical neurons. The sustained exposure to these three BZDs increased [(45)Ca2+] influx, which was due to the enhanced [(45)Ca2+] entry through L-type HVCCs but not through of Cav2.1 and Cav2.2. Increase in [(3)H]diltiazem binding after the exposure to these three BZDs was due to the increase in the binding sites of [(3)H]diltiazem. Western blot analysis showed increase of Cav1.2 and Cav1.3 in association with the increased expression of alpha2/delta1 subunit. Similar changes in [(3)H]diltiazem binding and L-type HVCC subunit expression were found in the cerebral cortex from mouse with BZD physical dependence. These results indicate that BZDs examined here have the potential to increase L-type HVCC functions mediated via the enhanced expression of not only Cav1.2 and Cav1.3 but also alpha2/delta1 subunit after their sustained exposure, which may participate in the development of physical dependence by these BZDs.

Up-regulated l-type high voltage-gated calcium channels cause increase in diazepam binding inhibitor induced by sustained morphine exposure in mouse cerebrocortical neurons

Mechanisms of increase in diazepam binding inhibitor (DBI) mRNA expression in mouse cerebrocortical neurons after sustained morphine exposure were investigated. Increases in DBI and its mRNA expressions induced by sustained morphine (0.3 μM) exposure for 3 days were completely abolished by naloxone and nifedipine, but not by ω-agatoxin VIA and ω-conotoxin GIVA. Increase in [ 3H]diltiazem binding to the particulate fractions from the morphine-treated neurons was due to increased B max value with no changes in K d value. Western blot analysis on l-type high voltage-gated calcium channel (HVCC) subunits revealed the increased expressions of α1C, α1D, and α2/δ1 subunits and decreased of β4 subunit expression, whereas expression of N- and P/Q-type HVCC subunits was not changed. These results indicate that morphine-induced increase in DBI mRNA expression is mediated via increased Ca 2+ entry through up-regulated l-type HVCCs.

8-NH2-Boldine, an Antagonist of α1Aand α1BAdrenoceptors without Affinity for the α1DSubtype: Structural Requirements for Aporphines at α1-Adrenoceptor Subtypes

Structure-activity analysis of 21 aporphine derivatives was performed by examining their affinities for cloned human alpha (1A), alpha (1B) and alpha (1D) adrenoceptors (AR) using membranes prepared from rat-1 fibroblasts stably expressing each alpha (1)-AR subtype. All the compounds tested competed for [ (125)I]-HEAT binding with steep and monophasic curves. The most interesting compound was 8-NH (2)-boldine, which retains the selective affinity for alpha(1A)-AR (pKi = 6.37 +/- 0.21) vs. alpha(1B)-AR (pKi = 5.53 +/- 0.11) exhibited by 1,2,9,10-tetraoxygenated aporphines, but shows low affinity for alpha(1D)-AR (pKi < 2.5). Binding studies on native adrenoceptors present in rat cerebral cortex confirms the results obtained for human cloned alpha (1)-AR subtypes. The compounds selective for the alpha (1A) subtype discriminate two binding sites in rat cerebral cortex confirming a mixed population of alpha (1A)- and alpha (1B)-AR in this tissue. All compounds are more selective as inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. A close relationship was found between affinities obtained for cloned alpha (1A)-AR and inhibitory potencies on noradrenaline-induced contraction or inositol phosphate accumulation in tail artery, confirming that there is a homogeneous functional population of alpha(1A)-AR in this vessel. On the contrary, a poor correlation seems to exist between the affinity of 8-NH (2)-boldine for cloned alpha (1D)-AR and its potency as an inhibitor of noradrenaline-induced contraction or inositol phosphate accumulation in rat aorta, which confirms that a heterogeneous population of alpha (1)-AR mediates the adrenergic response in this vessel.

Ethanol physical dependence is accompanied by up-regulated expression of L-type high voltage-gated calcium channel α1 subunits in mouse brain

We investigated how functional changes in high voltage-gated calcium channels (HVCCs) occurred in the cerebral cortex of mouse with ethanol physical dependence. The continuous treatment of mice with ethanol vapor for 8 days significantly increased the expressions of α1C, α1D, and α2/δ1 subunits of L-type HVCCs in association with decreased level of β4 subunit of L-type HVCCs, although α1A and α1B subunits of P/Q- and N-type HVCCs, respectively, showed no alterations. [ 3H]Diltiazem binding to the particulate fractions increased with increased B max value and no changes of K d value. These results indicate that ethanol physical dependence accompanies the up-regulation of L- type HVCCs, which is due to increased expression of α1C, α1D, and α2/δ1 subunits.

Increased expression of L-type high voltage-gated calcium channel α1 and α2/δ subunits in mouse brain after chronic nicotine administration

We investigated the effect of chronic nicotine administration on high voltage-gated calcium channels (HVCCs) in the mouse cerebral cortex. The treatment significantly increased expression of α1C, α1D, α1F, and α2/δ1 subunits with no changes of β4 subunit of L-type HVCCs. [ 3H]Diltiazem binding to the particulate fractions increased with increased Bmax value. These results indicate that chronic nicotine treatment up-regulates L-type HVCCs, which is due to increased expression of α1 and α2/δ1 subunits.

Vascular activity of (-)-anonaine, (-)-roemerine and (-)-pukateine, three natural 6a(R)-1,2-methylenedioxyaporphines with different affinities for alpha1-adrenoceptor subtypes.

We have studied the mechanism of action of three 6a( R)-1,2-methylenedioxyaporphines as vasorelaxant compounds. The alkaloids assayed showed different affinities for the three human cloned alpha (1)-adrenoceptor (AR) subtypes stably expressed in rat-1 fibroblasts, showing lower affinity for alpha(1B)-AR with regard to the alpha(1A)- or alpha(1D)-subtypes. These three natural compounds are more potent inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. As all these alkaloids inhibited noradrenaline (NA)-induced [ (3)H]-inositol phosphate formation in cerebral cortex and rat tail artery, they may be safely viewed as alpha (1)-AR antagonists, as is demonstrated by the vasorelaxant responses observed in isolated rat tail artery and/or aorta precontracted with NA. The alkaloids also inhibited the contractile response evoked by KCl (80 mM) but with a lower potency than that shown against NA-induced contraction. We have also examined their ability to inhibit the different forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aortic smooth muscle and endothelial cells, with negative results. We conclude that N-methylation favours the interaction of (R)-aporphines with all alpha (1)-AR subtypes, and that the topography of the binding site recognizing the basic or protonated nitrogen atom is similar in all three alpha (1)-AR subtypes. The presence of a hydroxy group at C-11 has different effects on the affinity for each alpha (1)-AR subtype but decreases the affinity for Ca (2+) channels. These results confirm and extend the view that subtle changes in the hydroxylation patterns on the aromatic ring of the aporphine structure affect the interactions of these compounds with the three alpha (1)-AR subtypes in different ways, suggesting that the binding site recognizing the aporphine skeleton is different in each of the three subtypes.

Flupirtine ameliorates ischaemic-like death of rat retinal ganglion cells by preventing calcium influx

The effect of flupirtine on the loss of retinal ganglion cells following transient elevation of intraocular pressure (experimental ischaemia) or NMDA-induced excitotoxicity was studied. Ischaemia (60 min) or intravitreal injection of NMDA (20 nmol) caused a decrease in Thy-1 mRNA and Thy-1 immunoreactivity which are associated with ganglion cells. Administration of flupirtine counteracted these changes. Moreover, flupirtine dose-dependently inhibited NMDA-induced 45 Ca 2+ influx into cultured cortical neurones and retinal pieces in vitro with maximal inhibition being observed at 200 μM. A similar concentration of flupirtine failed to inhibit kainate-stimulated calcium influx into cultured cortical neurones. In addition, flupirtine had no significant effect on [ 3 H ]nitrendipine or [ 3 H ]diltiazem binding to cortical membranes. The present studies are consistent with previous findings which suggested flupirtine to act as a NMDA antagonist by a mechanism that still remains to be clarified.

Molecular modeling study of diltiazem mimics at L-type calcium channels.

A theoretical study was performed to generate a pharmacophore model for chemically diverse structures that specifically interact with the diltiazem binding site of L-type calcium channels. Via molecular mechanics and quantum chemical methods solvation energies, logP values, conformational and electronic features of classical 1,5-benzothiazepin-4(5H)-one (BTZ, e.g., diltiazem), 1-benzazepin-2-one (BZ), pyrrolo[2,1-d][1,5]benzothiazepine, pyrrolo[2,1-c][1,4]benzothiazine, and benzobicyclo[2.2.2]octyl amines derivatives were determined. Furthermore, the molecular electrostatic potentials (MEPs) and common interaction fields derived from use of the GRID programme were compared. This yielded a pharmacophore model with three crucial pharmacophoric characteristics, (1) two aromatic ring systems in a distance of about 6.7 A, (2) a basic side chain with pKa in the physiological range, and (3) a 4'-methoxy moiety. In addition, a strong negative MEP in 4-position (carbonyl oxygen) and hydrophobic electron-rich features in the position equivalent to the sulphur atom of BTZ derivatives were explored to be favourable for receptor binding and calcium antagonistic effect. Moreover, the stabilizing effect of substituents in 3-position of BZs on the bioactive "M" twist-boat conformation of the heptagonal ring could be demonstrated by molecular dynamics simulations. Based on these molecular descriptors, the quinazolinone derivative MCI-176 is predicted to be a potential ligand of the diltiazem binding site.

Molecular Mechanism of Diltiazem Interaction with L-type Ca2+ Channels

Benzothiazepine Ca2+ antagonists (such as (+)-cis-diltiazem) interact with transmembrane segments IIIS6 and IVS6 in the alpha1 subunit of L-type Ca2+ channels. We investigated the contribution of individual IIIS6 amino acid residues for diltiazem sensitivity by employing alanine scanning mutagenesis in a benzothiazepine-sensitive alpha1 subunit chimera (ALDIL) expressed in Xenopus laevis oocytes. The most dramatic decrease of block by 100 microM diltiazem (ALDIL 45 +/- 4.8% inhibition) during trains of 100-ms pulses (0.1 Hz, -80 mV holding potential) was found after mutation of adjacent IIIS6 residues Phe1164(21 +/- 3%) and Val1165 (8.5 +/- 1.4%). Diltiazem delayed current recovery by promoting a slowly recovering current component. This effect was similar in ALDIL and F1164A but largely prevented in V1165A. Both mutations slowed inactivation kinetics during a pulse. The reduced diltiazem block can therefore be explained by slowing of inactivation kinetics (F1164A and V1165A) and accelerated recovery from drug block (V1165A). The bulkier diltiazem derivative benziazem still efficiently blocked V1165A. From these functional and from additional radioligand binding studies with the dihydropyridine (+)-[3H]isradipine we propose a model in which Val1165 controls dissociation of the bound diltiazem molecule, and where bulky substituents on the basic nitrogen of diltiazem protrude toward the adjacent dihydropyridine binding domain.

RECEPTOR BINDING PROFILE OF OTILINIUM BROMIDE

The interaction of Otilonium bromide (OB) with binding sites for 63 different receptors and ion channels in appropriate preparations has been investigated. Experiments were also performed in rat colon, the preferred tissue for OB `in vivo' uptake after oral administration.Among the receptors investigated OB binds with subμmaffinity to muscarinic M1, M2, M4, M5and PAF receptors and with μmaffinity to the diltiazem binding site on L type Ca2+channels. In the rat colon OB shows competitive interaction with the verapamil binding site on L type Ca2+channels and with muscarinic M2receptors with IC50of 1020 and 1220 nm, respectively. These findings provide a molecular rationale to explain the spasmolytic action exerted by OB on intestinal smooth muscle. In particular, a combination of antimuscarinic and Ca2+channel blocker properties seems to best account for the action of this compound.

Mapping of the Benzothiazepine Binding Site on the Calcium Channel

A new pharmacophoric model of the benzothiazepine (diltiazem) binding site on the L-type Ca2+ channel was derived by using the SYBYL software package. The molecular modeling study was conducted by applying the active analogue approach in a two-step procedure. In the first stage, the bioactive conformation of (+)cis-diltiazem, used as a reference template, was found by including the most rigid and active derivatives into a preliminary MULTIFIT procedure. In the second step, aimed at mapping of the binding site, the common low-energy conformations of active and inactive analogues from various chemical classes were superimposed on the diltiazem template. It was hypothesized that the essential pharmacophoric elements are two aromatic rings and a protonated amine nitrogen. This pharmacophore hypothesis was later supported by several lines of evidence. All molecular superimpositions were performed by using a flexible fit on five dummy points built on the essential pharmacophore. Molecular mechanics and dynamics were used to determine the lowest energy conformations of all the compounds as well as the conformations that displayed the common pharmacophoric geometry. Based on the van der Waals volume manipulations of the active and inactive analogues, a three-dimensional model estimating the binding site topography was derived; various regions on the boundary of the binding domain were characterized and discussed, along with other factors, in terms of potential improvement of the drug binding affinity.

CP-060 S interacts with three principal binding sites on the L-type Ca 2+ channel

CP-060 S, (−)-( S)-2-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[ N-methyl- N-[2-(3,4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin-4-one hydrogen fumarate is a novel cardioprotective drug, which is able to prevent Na +-, Ca 2+-overload and also has Ca 2+ channel blocking activity. The latter action of CP-060 S was characterized by radioligand binding experiments with rat cardiac membranes in terms of the interaction with the three principal binding sites on the L-type Ca 2+ channel, which bind such drugs as the 1,4-dihydropyridines, phenylalkylamines and benzothiazepines. CP-060 S exhibited complete and concentration-dependent inhibition of [ 3 H ](+)-PN200-110, [ 3 H ](−)-desmethoxyverapamil and [ 3 H ]cis-(+)-diltiazem binding to their specific binding sites. Saturation studies showed that CP-060 S increased the K d of [ 3 H ](+)-PN200-110 and [ 3 H ](−)-desmethoxyverapamil without causing a significant change in the maximum binding density. The dissociation kinetics of the three radioligands were accelerated by CP-060 S. These results suggest that CP-060 S interacts with a novel binding site on the L-type Ca 2+ channel and has a negative allosteric interaction with the three principal binding sites for the 1,4-dihydropyridines, phenylalkylamines and benzothiazepines.

Binding Sites for Ca2+‐Channel Effectors and Ryanodine inPeriplaneta americana—Possible Targets for New Insecticides

The calcium channel and the 'calcium release channel' of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0.1 and 300 μg ml -1 . Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [ 3 H]isradipine (PN 200-110), the phenylalkylamine [ 3 H]verapamil and the alkaloid [ 3 H]ryanodine. Preliminary binding studies with the benzothiazepine [ 3 H]diltiazem suggest a low-affinity binding site with a IC 50 value of 3.3 μM. All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM. EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca 2+ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca 2+ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca 2+ . Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K D values of 0.95 nM (B max = 550 fmol mg -1 protein) and 0.75 nM (B max = 213 fmol mg -1 protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K D = 7.4 nM and B max = 27 fmol mg -1 protein). [ 3 H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity : nitrendipine > isradipine > Bay K8664 >> nicardipine. Displacement of [ 3 H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.

Dual calcium-channel blocker therapy in the treatment of hypertension.

To review the in vitro receptor binding data of calcium-channel blockers (CCBs) and in vivo studies in humans regarding the use of dual calcium-channel blocker therapy, with a focus on the use of this therapy for hypertension. A MEDLINE search was conducted to identify literature pertaining to CCBs. In vitro studies and investigations that evaluated CCB receptor binding and the interactions between subclasses of CCBs were chosen. All studies in humans and clinical trials that evaluated the use of dual CCB therapy in the treatment of cardiovascular diseases were selected for review. Also, case reports describing the use of dual CCB therapy were included in this article. The methodology, results, and conclusions of the selected data were evaluated. Data regarding the in vitro receptor binding kinetics of CCBs, as well as interactions, were reported. Because there is limited information on dual CCB therapy for hypertension, clinical studies using this treatment for ischemic heart disease were also reviewed. They were summarized and compared on the basis of the degree of disease control (e.g., blood pressure, exercise tolerance), adverse effects, and other clinical endpoints of pharmacologic therapy. In vitro studies have identified binding sites for the dihydropyridine (nifedipine), diphenylalkylamine (verapamil), and benzothiazepine (diltiazem) subclasses of CCBs, and indicate that they are allosterically related to each other within the voltage-sensitive calcium-channel receptor. Dihydropyridine binding affinity is decreased with concomitant verapamil binding, but is enhanced by concomitant diltiazem binding. Dual CCB therapy has been shown to be efficacious in patients with ischemic heart disease. Although this therapy is limited by dose-related adverse effects, it appears to have an important role in patients with ischemia that is refractory to conventional therapy, or for those whose therapeutic options are limited by contraindications. Theoretically, many patients with hypertension may benefit similarly from dual CCB therapy. Because data evaluating this treatment option are sparse, recommendations regarding safety, efficacy, and the role of dual CCB therapy for hypertension would be premature. Controlled data evaluating dual CCB therapy for the treatment of hypertension are lacking. This treatment modality may be beneficial in the future, but requires further investigation to determine safety and efficacy.