A unitary mechanism of calcium antagonist drug action '(dihydropyridine/nifedipine/verapamil/neuroleptic/diltiazem)

Transcription

1 Pro. Nati Aad. Si. USA Vol. 8, pp , February 1983 Medial Sienes A unitary mehanism of alium antagonist drug ation '(dihydropyridine/nifedipine/verapamil/neurolepti/diltiazem) KENNETH M. M. MURPHY, ROBERT J. GOULD, BRAN L. LARGENT, AND SOLOMON H. SNYDER* Departments of Neurosiene, Pharmaology and Experimental Therapeutis, and Psyhiatry and Behavioral Sienes, Johns Hopkins University Shool of Mediine, 72S North Wolfe Street, Baltimore, Maryland 2125 Contributed by Solomon H. Snyder, Otober 21, 1982 ABSTRACT [3H]Nitrendipine binding to drug reeptor sites assoiated with alium hannels is allosterially regulated by a diverse group of alium hannel antagonists. Verapamil, D-6 (methoxyverapamit), tiapamil, lidoflazine, flunarizine, innarizine, and prenylamine all redue P3H]nitrendipine binding affinity. By ontrast, diltiazem, a benzothiazepine alium hannel antagonist, enhanes [3H]nitrendipine binding. All these drug effets involve a single site allosterially linked to the [3H]nitrendipine binding site. nhibition of t3h]nitrendipine binding by prenylamine, lidoflazine, or tiapamil is reversed by D-6 and diltiazem, whih alone respetively slightly redue or enhane H]mnitrendipine binding. Diltiazem reverses the inhibition of [3H]nitrendipine binding by D-6. Our predition that drugs allosterially regulating P[H]nitrendipine binding should be alium antagonists is onfirmed by alium antagonism in guinea pig ileum observed with the antihistamine dimethindene, the neuroleptis thioridazine and mesoridazine, and the antiholinergi biperiden. Several distint lasses of organi alium antagonist drugs are important therapeuti agents in ardiovasular and numerous other medial onditions (1, 2). At least four lasses ofstrutures are distinguished: the dihydropyridines exemplified by nifedipine (1-3), phenylalkylamines exemplified by verapamil (1-3), benzothiazepines suh as diltiazem (1, 2, 4), and diphenylalkylamines suh as prenylamine and lidoflazine (1, -2, 5). Reeptors for the drugs are labeled with [3H]nitrendipine (6-1), or [3H]nimodipine (1) and regulated in a physiologial fashion by alium (7). The apparent ompetitive inhibition of [3H]nitrendipine binding by diphenylalkylamines has implied effets at the same site as the dihydropyridines (11). Verapamil and diltiazem respetively inhibit and stimulate [3H]nitrendipine binding allosterially, suggesting ations at different sites (9, 12). Here we show that the phenylalkylamine, diphenylalkylamine, and benzothiazepine drugs, -in pharmaologially relevant onentrations, interat. at a single drug reognition site allosterially linked to the dihydropyridine reeptor. We predit and onfirm by bioassay previously unknown alium antagonist ativity for several drugs. MATERLS AND METHODS Drugs were obtained from the following soures: nifedipine, Pfizer; nimodipine, Miles; prenylamine, Hoehst-Roussel Pharmaeutials (Somerville, NJ); D-6 (gallopamil, methoxyverapamil) and verapamil, Knoll Pharmaeutial (Whippany, NJ); diltiazem, Marion Laboratories (Kansas City, MO) and Tanabe (Osaka, Japan); desmethyl-is-diltiazem and -trans-diltiazem, Tanabe; lidoflazine, flunarizine, and innarizine, Janssen Pharmaeutial (Beerse, Belgium); and bepridil, MNeil Pharmaeutial (Spring House, PA). Binding assays in guinea pig brain membranes, filtration, and The publiation osts ofthis artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U. S. C solely to indiate this fat. 86 liquid sintillation ounting were arried out as desribed (7). All experiments, performed in tripliate, were repliated at least three times with similar results. Guinea pig ileum longitudinal musles were prepared for reording as desribed by Rosenberger et al (13) and inubated in a modified Tyrode's buffer (14) at 37C with ontinuous aeration with 95% '2/5% CO2. leum longitudinal musles were inubated in this buffer for 3 min before Ca2"-dependent ontrations were reorded as desribed by Jim et al (15). RESULTS All dihydropyridine alium antagonists evaluated inhibit [3H]nitrendipine.binding in a ompetitive fashion (6-1). Verapamil inhibits [3H]nitrendipine binding (6-1), but Yamamura and assoiates showed that this effet is not stritly ompetitive, beause verapamil annot ompletely inhibit [3H]nitrendipine binding, the extent of inhibition of binding by verapamil dereases at inreasing [3H]nitrendipine onentrations, and ver-.apamil aelerates the dissoiation of13h]nitrendipine from reeptor sites (9). Beause diphenylalkylamines inhibit [3H]- nitrendipine binding ompletely, it was suggested that these drugs at at the same reeptor sites as dihydropyridines (11). n our studies, the diphenylalkylamines prenylamine, flunarizine, and lidoflazine and the phenylailylamine tiapamil maximally inhibit [3Hnitrendipine.binding (Fig. 1). However, maximal inhibition of [3H]nitrendipine binding by these drugs delines with inreasing [3H]nitrendipine onentration between.1 and 3 nm (data not shown), suggesting a similarity in the ation ofthese agents and verapamil. We have onfirmed this possibility in dissoiation experiments (Fig. 2). As previously reported (9), verapamil speeds the dissoiation of [3H]nitrendipine (data not shown), whereas the dihydropyridine nimodipine has no effet on the dissoiation rate. Like verapamil, the diphenylalkylamines lidoflazine and prenylamine and the verapamil analog tiapamil aelerate [3H]- n-itrendipine dissoiation. Cinnarizine and flunarizine similarly inrease the dissoiation rate. Having established that the diphenylalkylamines influene [3H]nitrendipine binding allosterially, we investigated whether the diphenylalkylamines and phenylalkylamines at at the same site. f so, then inreasing onentrations of D-6 (whih maximally redues [3H]nitrendipine binding only 2-3%) (Fig. 3) should blok the ability of a diphenylalkylamine to inhibit [3H]nitrendipine binding, with a rightward shift in onentration-response urves. ndeed, inreasing onentrations of D- 6 do produe rightward shifts in the onentration-response urves for inhibition of [3H]nitrendipine binding by the diphenylalkylamines prenylamine, flunarizine, and lidoflazine (Fig. 1 a-). Tiapamil, a phenylalkylamine losely similar in struture to verapamil and D-6 (16), produes the same maximal inhibition of [3H]nitrendipine binding eliited by the di- * To whom reprint requests should be addressed.

2 Medial Sienes: Murphy et al Pro. NatL Aad. Si. USA 8 (1983) 861.5z -._._ V z -logiflunafizine.j C., soa S 4 to- ; U' 7l 6 5 -logilidoflazinel log [tiapamil FG. 1. nhibition of [3Hlnitrendipine binding by prenylamine, tiapamil, lidoflazine, and flunarizine: nterations with D-6. Conentrationresponse urves for the inhibition of [3Hlnitrendipine binding were onstruted for prenylamine (a), flunarizine (b), lidoflazine (), and tiapamil (d) in the absene (e) or the presene of 2.5 nm (o), 25 nm (i), 25 nm (o), 2.5 pm (A), or 25 ptm (A) D-6. Conentration-response urves are the results of experiments that employed at least six onentrations of eah drug assayed in tripliate with.25 nm [3Hlnitrendipine. Values are presented as a perent of ontrol [3H]nitrendipine binding. phenylalkylamines (Fig. d), and D-6 produes a rightward shift for tiapamil inhibition of [3Hjnitrendipine binding, refleting similar ations ofphenylalkylamines and diphenylalkylamines. The degree of rightward shift differs for -the drugs evaluated, being greatest with tia-pamil and least with.flunarizine. By ontrast with these results, the onentration-response urve for inhibition of [3H]nitrendipine binding by nifedipine is not progressively shifted in the presene of inreasing onentrations of D-6 (data not shown). ml too , Dissoiation, timer min FiG. 2. nfluenes of various alium hannel antagonists on dissoiation of [3H]nitrendipine. [3H]Nitrendipine (.25 nm) was inubated with brain membranes for 9 min to allow equilibrium assoiation of [3H]nitrendipine to reeptor sites. Dissoiations were initiated with 1 nm nifedipine in either the absene (l) or the presene of 2.5,uM nimodipine (a), 2.5,uM prenylamine (v), 25 PM tiapamil (n), or 25,uM lidoflazine (a). Results are presented as a perentage of equilibrium [3H]nitrendipine binding. Beause D-6 maximally redues [3H]nitrendipine binding less than the other diphenylalkylamines and phenylalkylamines and assuming that the drugs at at the same site, D-6 should overome inhibition of [3H]nitrendipine binding exerted by these other drugs. Aordingly, we examined the ability of D- 6 to influene [3H]nitrendipine binding to membranes simultaneously inubated with prenylamine, tiapamil, flunarizine, or lidoflazine (Fig.. 3). n the absene of other drugs, D- 6 maximally redues [3H]nitrendipine binding only about 25% with an ECG (5% effetive onentration) ofabout 1 nm. By ontrast, in the presene of onentrations of prenylamine, tiapamil, flunarizine, or lidoflazine that give nearly maximal inhibition of [3H]nitrendipine binding, D-6 atually stimulates [3H]nitrendipine binding (Fig. 3). The perentage stimulation of [3H]nitrendipine binding by D-6( diminishes with lower onentrations of the other drugs. [3H]Nitrendipine binding is restored by D-6 to the level ofbinding that ours with maximally inhibitory onentrations of D-6 alone. As expeted for drugs ompeting for the same site, D-6 is somewhat less potent at enhaning [3H]nitrendipine binding in the presene of inreasing onentrations of the other inhibitors (Fig. 3). Whereas diphenylalkylamines and phenylalkylamines inhibit [3H]nitrendipine binding, diltiazem stimulates binding when evaluated in the absene of other drugs (Fig. 4a) (12, 17). t has been suggested that diltiazem. therefore ats at a distint site, speifi for benzothiazepines, and different from the site at whih the phenylalkylamines and diphenylalkylamines at (12, 17). f this were the ase, no diret interations should our between the benzothiazepines and the phenylalkylamines or diphenylalkylamines. To evaluate possible interations, we explored the effet ofdiltiazenmon.[3h]nitrendipine binding in the presene of D-6, prenylamine, and tiapamil (Fig. 4a). Diltiazem strikingly reverses the inhibition of [3H]nitrendipine binding produed by tiapamil, D-6(, and prenylamine. Moreover, its poteny in reversing suh inhibition is dereased in the

3 862 Medial Sienes: Murphy et al. C 2 Flunarizine Lidoflazine. 1. C. 9) 4 A 6 ~ C A iog[d-6] FG. 3. D-6 reverses the inhibition of th,]nitrendipine binding produed by prenylamine, tiapamil, lidoflazine, and flunarizine. Conentration-response urves for the influene of D-6 on [3H]nitrendipine binding to membranes were obtained with at least six onentrations of D-6 and.25 nm [3H]nitrendipine. t5hlnitrendipine binding was determined at eah onentration of D-6 in the absene (9), or the presene of.7 /M (A),.25,M (A),.75 um (o), 2.5 iim (W), or 25,uM (n) of the four drugs prenylamine (a), tiapamil (b), flunarizine (), or lidoflazine (d). Values are presented as the perent of ontrol [3Hlnitrendipine binding determined in the absene of all inhibiting agents. presene of inreased onentrations of other drugs. Thus, diltiazem ats at the same site as phenylalkylamines and diphenylalkylamines to enhane reeptor affinity for [3H]nitrendipine, d Pro. Nad Aad.'Si. USA 8 (1983) whereas the other drugs derease this affinity. To asertain whether this effet of diltiazem is related to the pharmaologi ations of the drug, we ompared is- and transdiltiazem, beause only the is isomer has pharmaologial effiay. We also evaluated desmethyl-is-diltiazem, whih pharmaologially is about 3-5% as potent as diltiazem (4). Enhanement of [3H]nitrendipine binding in the presene of 2.5 AuM tiapamil is eliited by is- but not trans-diltiazem (Fig. 4b). Desmethyl-is-diltiazem is only about half as potent as is-diltiazem. These relative ativities are onsistent with interations at the pharmaologially relevant site of ation of diltiazem. Redution of [3H]nitrendipine binding at high onentrations of is-diltiazem ours similarly with trans-diltiazem and desmethyl-is-diltiazem, indiating no pharmaologi relevane (Fig. 4b). The phenylalkylamines and diphenylalkylamines possess two widely separated aromati domains with the amine loated entrally, whereas diltiazem possesses only one aromati domain linked to an alkylamine moiety. We predited that some drugs with one aromati domain and an alkylamine moiety might interat with reeptors as diltiazem does. We have found that the H1 antihistamines dimethindene and hlorpheniramine, the musarini antiholinergi biperiden, the neuroleptis mesoridazine and thioridazine, and the organi alium antagonist bepridil (18) all display the diltiazem-like.ation of enhaning [3H]nitrendipine binding (Fig. 5). Dimethindene not only enhanes [3H]nitrendipine binding in the absene of other drugs but, like diltiazem, it reverses the inhibition of [3H]nitrendipine binding eliited by D-6, prenylamine, and tiapamil (Fig. Sa). Bepridil, thioridazine, biperiden, hlorpheniramine, and mesoridazine overome the inhibition aused by 2.5,uM tiapamil, enhaning [3H]nitrendipine binding from 1% to 8% of ontrol (Fig. 5b). Bepridil, thio C) Q O L- _ a 7 g 5a 4.-log dl~tjazeml 1 -. W 8 S :5 6 rc. b '2 ridazine, and biperiden show biphasi effets like diltiazem, ausing an inhibition of [3H]nitrendipine binding at higher onentrations. To determine whether these effets on binding validly predit pharmaologi alium hannel antagonism, we explored the ations of these drugs on guinea pig ileum ontrations (Fig. 6). The Shild plot for dimethindene inhibition of Ca2"-dependent ontrations shows a pa2 of 4.95 (Fig. 6), similar to dimethindene's poteny in stimulating [3H]nitrendipine binding. Furthermore, 25 um -thioridazine, biperiden, hlorpheniramine, and mesoridazine inhibit Ca2+-dependent ontrations of guinea pig ileal musle by 97%, 95%, 66%, and 51%, re- -log.[drug] FG. 4. Diltiazem reverses the effets of D-6, prenylamine, and tiapamil on [3H]nitrendipine binding.- (a) Conentration-response urves for the effets of diltiazem on 5Hlnitrendipine binding were determined in the absene of any inhibiting agent (e) or in the presene of.25 PM D- 6 (),.75 pm prenylamine (i), or 2.5 pm tiapamil (o). Values for eah ondition are presented as the perent of speifi nitrendipine binding determined in the absene of all inhibiting agents. Results are from one of three experiments arried out for eah drug with similarresults. (b) Conentration-response urves for the effets of is-diltiazem (), desmethyl-i-diltiazem (o), or transrdiltiazem (-) were determined by using at least six onentrations of eah of these agents in the presene of.2 nm [3Hlnitrendipine and 2.5 pm tiapamil inubated with brain membranes. Values are presented as the perentage of [3Hlnitrendipine binding determined in the absene of tiapamil, diltiazem, or diltiazem analogs.

4 Medial Sienes: Murphy et al Pro. Natd Aad. Si. USA 8 (1983) 863 : 1' 2 -a. _ CC rloj C 7 [ log dimethinlenel -log [drug] FG. 5. Diltiazem-like ations of dimethindene and other drugs. (a) Conentration-response urves for the effets of dimethindene on (5H]nitrendipine binding were determined in the absene of any inhibiting agent (a) or in the presene of.25 AM D-6 (o),.75 pm prenylamine (m), or 2.5 pm tiapamil (o). Values are presented as the perent of speifi [3H]nitrendipine binding determined in the absene of all drugs. (b) Conentration-response urves for bepridil (e), thioridazine (o), biperiden (i), hlorpheniramine (o), and mesoridazine (A) were onstruted by inubating.2 nm [3H]nitrendipine with 2.5 pm tiapamil and brain membranes. Values are the means of tripliate determinations for eah ondition presented as the perent of [3Hlnitrendipine binding determined in the absene of tiapamil and other drugs. spetively. This order of poteny orresponds to the drugs' potenies in enhaning [3H]nitrendipine binding (Fig. 5b). The augmentation of [3H]nitrendipine binding by this group of drugs is speifi, beause the following drugs fail to inrease binding at 25,uM: promazine, hlorimipramine, nortriptyline, fluphenazine, lozopine, deptropine, protriptyline, antazoline, spiperone, orphenadrine, promethazine, aetophenazine, pyrilamine, histamine, and atropine. DSCUSSON Our present findings larify ations of alium antagonist drugs of the known strutural lasses. Whereas others had suggested that the diphenylalkylamines suh as prenylamine at differently than the phenylalkylamines suh as verapamil (11), we have demonstrated that both lasses of drugs at via the same site to allosterially regulate [3H]nitrendipine binding. t had been suggested that diltiazem ats at a site distint from the phenylalkylamines and diphenylalkylamines (12, 17), but we find that it ats at the same site. The diphenylalkylamines and phenylalkylamines derease the affinity of [3H]nitrendipine for reeptors, but diltiazem enhanes this affinity, as refleted in slowing of the [3H]nitrendipine dissoiation rate (12, 17). These observations suggest a unitary model for regulation of -log [dimethindene] FG. 6. Dimethindene bloks alium-dependent ontrations of KCl-depolarized guinea pig ileum. Conentration-response urves for the alium-dependent ontrations of the guinea pig ileum in the absene or the presene of various onentrations of dimethindene were onstruted by sequential additions of alium hloride to a alium-free high-potassium buffer. Only one dimethindene onentration was employed in any tissue. log(dose ratio 1) is plotted against log[dimethindene] and the pa2 (4.95) - was determined by linear regression (r =.97). pa2 is the negative logarithm of the onentration at whih the dose ratio is 2; it is equivalent to -log app. Dose ratio is the ratio of agonist, in the absene and presene of antagonist, whih gives the same response. alium ion hannels by drugs (Fig. 7). A dihydropyridine reognition site linked to the alium hannel mediates the pharmaologi ations of this lass of alium hannel antagonists. A speifi site for multivalent ations influenes [3H]nitrendipine binding (7). Divalent ations that mimi alium physiologially stimulate [3H]nitrendipine binding, whereas ations known to blok the physiologi ations of alium prevent the enhanement of [3H]nitrendipine binding by alium (7). Phenylalkylamines and diphenylalkylamines at at the same site and in the same fashion, both dereasing [3H]nitrendipine affinity for reeptors. These drugs vary in the extent to whih they redue the reeptor affinity of [3H]nitrendipine. D-6 and verapamil produe the least effet, reduing [3H]nitrendipine's reeptor affinity to 1/2-1/3, whereas tiapamil produes a redution in affinity to V/5_V/1o and flunarizine a redution to 1/1-/2o. Diltiazem and bepridil at at the same site to enhane [3H]- nitrendipine affinity. On the basis of strutural similarities, we identified several neuroleptis, antihistamines, and musarini antiholinergis that mimi the ations ofdiltiazem on [3H]nitrendipine binding and have alium hannel antagonist ativity in the guinea pig ileum. Thus, effets at a single domain of a bipartite reeptor may onvey diltiazem-like ations, whereas drugs ating at both domains have verapamil-like profiles. Suh binding analyses may help identify alium antagonists. Clinial ations of the drugs we have evaluated may relate to their alium antagonist ations. Of numerous phenothiazines evaluated, thioridazine and mesoridazine were unique in influening [3H]nitrendipine binding in a diltiazem-like fashion. Blood levels of thioridazine and mesoridazine at therapeuti doses are about 2,uM (19, 2), a onentration at whih these drugs display alium antagonism. Thioridazine and mesori- Dihydropyridine Reognition Site FG. 7. A model for the sites of ation and interations for inorgani and organi alium hannel antagonists. 2+ Diltiazem/Verapamil Reognition Site

5 864 Medial Sienes: Murphy et alp Pro. Nad Aad. Si. USA 8 (1983) dazine eliit more ardia side effets with Q-T interval prolongation than do other phenothiazines (21-23). Another unique ation of thioridazine and mesoridazine is inhibition of ejaulation (23). Beause ejaulation involves a massive ontration of the smooth musle of the vas deferens, the alium antagonist ations of these drugs may aount for suh side effets. We thank Dawn C. Hanks for manusript preparation. This work was supported by U.S. Publi Health Servie Grants DA-266, MH- 1851, and NS-16375; a grant from the MKnight Foundation; and U. S. Publi Health Servie Researh Sientist Award DA-74 to S. H.S., Training Grant GM-739 to K.M.M.M., and Training Grant MH to R.J.G. 1. Henry, P. D. (198) Am. J. Cardiol 46, Triggle, D. J. (1981) in New Perspetives on Calium Antagonists, ed. Weiss, G. B. (Amerian Physiologial Soiety, Bethesda, MD), pp Flekenstein, A. (1977) Annu. Rev. Pharmaol Toxiol 17, Nagao, T., Sato, M., Nakajima, H. & Kiyomoto, A. (1973) Chem. Pharmn. Bull 21, Vanhoutte, P. M. (1981) in New Perspetives on Calium Antagonists, ed. Weiss, G. B. (Amerian Physiologial Soiety, Bethesda, MD), pp Murphy, K. M. M. & Snyder, S. H. (1982) Eur.J. Pharmaol 77, Gould, R. J., Murphy, K. M. M. & Snyder, S. H. (1982) Pro. Natl Aad. Si. USA 79, Bolger, G. T., Gengo, P. J., Luhowski, E. M., Siegel, H., Triggle, D. J. & Janis, R. A. (1982) Biohem. Biophys. Res. Commun. 14, Ehlert, F. J., toga, E., Roeske, W. R. & Yamamura, H.. (1982) Life Si. 3, Bellemann, P., Ferry, D., Lubbeke, F. & Glossmann, H. (1982) Arzneim.-Forsh. 32, Ferry, D. R. & Glossmann, H. (1982) Naunyn-Shmeideberg's Arh. PharmaoL 321, Yamamura, H.., Shoemaker, H., Boles, R. G. & Roeske, W. R. (1982) Biohem. Biophys. Res. Commun. 18, Rosenberger L. B., Tiku, M. K. & Triggle, D. J. (1979) Can. J. Physiol PharmaoL 57, Triggle, C. R., Swamy, V. C. & Triggle, D. J. (1979) Can.J. PhysioL PharmaoL 57, Jim, K., Harris, A., Rosenberger, L. B. & Triggle, D. J. (1981) Eur. J. Pharmaol. 76, Eigenmann, R., Blaber, L., Nakamura, K., Thorens, S. & Haeusler, G. (1981) Arzneim.-Forsh. 31, DePover, A., Matlib, M. A., Lee, S. W., Dube, G. D., Grupp,. L., Grupp, G. & Shwartz, A. (1982) Biohem. Biophys. Res. Commun. 18, Vogel, S., Crampton, R. & Sperelakis, N. (1979) J. PharmaoL Exp. Ther. 21, Calil, H. M., Avery, D. H., Hollister, L. E., Creese,. & Snyder, S. H. (1979) Psyhiatry Res. 1, Nyberg, G., Axelsson, R. & Martensson, E. (1978)J. Clin. PharmaoL 14, Appleton, W. S. (1982)J. Clin. Psyhol 43, Soffer, J., Dreifus, L. S. & Mihelson, E. L. (1982) Am. J. CardioL 49, Wendkes, M. H. (1964)J. New Drugs 4,