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Home , Acetophenazine, Deptropine, Flunarizine, Gallopamil, Lidoflazine, Nimodipine

Proc. Nati Acad. Sci. USA Vol. 80, pp. 860-864, February 1983 Medical Sciences

A unitary mechanism of calcium antagonist drug action '(dihydropyridine///neuroleptic/) KENNETH M. M. MURPHY, ROBERT J. GOULD, BRIAN L. LARGENT, AND SOLOMON H. SNYDER* Departments of Neuroscience, Pharmacology and Experimental Therapeutics, and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 72S North Wolfe Street, Baltimore, Maryland 21205 Contributed by Solomon H. Snyder, October 21, 1982 ABSTRACT [3H] binding to drug sites liquid scintillation counting were carried out as described (7). associated with calcium channels is allosterically regulated by a All experiments, performed in triplicate, were replicated at diverse group of antagonists. Verapamil, D-600 least three times with similar results. (methoxyverapamit), tiapamil, , , cinnari- Guinea pig ileum longitudinal muscles were prepared for zine, and all reduce P3H]nitrendipine binding affin- recording as described by Rosenberger et aL (13) and incubated ity. By contrast, diltiazem, a benzothiazepine calcium channel an- in a modified Tyrode's buffer (14) at 370C with continuous aer- tagonist, enhances [3H]nitrendipine binding. All these drugeffects ation with 95% '02/5% CO2. Ileum longitudinal muscles were involve a single site allosterically linked to the [3H]nitrendipine incubated in this buffer for 30 min before Ca2"-dependent con- binding site. Inhibition of t3H]nitrendipine binding by prenyl- were as described Jim et aL amine, lidoflazine, or tiapamil is reversed by D-600and diltiazem, tractions recorded by (15). which alone respectively slightlyreduceorenhance H]mnitrendipine RESULTS binding. Diltiazem reverses the inhibition of [3H]nitrendipine binding by D-600. Our prediction that drugs allosterically regu- All dihydropyridine calcium antagonists evaluated inhibit lating P[H]nitrendipine binding should be calcium antagonists is [3H]nitrendipine.binding in a competitive fashion (6-10). Ver- confirmed by calcium antagonism in guinea pig ileum observed apamil inhibits [3H]nitrendipine binding (6-10), but Yamamura with the dimethindene, the neuroleptics thiorida- and associates showed that this effect is not strictly competitive, zine and , and the . because verapamil cannot completely inhibit [3H]nitrendipine binding, the extent of inhibition of binding by verapamil de- Several distinct classes of organic calcium antagonist drugs are creases at increasing [3H]nitrendipine concentrations, and ver- important therapeutic agents in cardiovascular and numerous .apamil accelerates the dissociation of13H]nitrendipine from re- other medical conditions (1, 2). At leastfour classes ofstructures ceptor sites (9). Because diphenylalkylamines inhibit [3H]- are distinguished: the dihydropyridines exemplified by nifed- nitrendipine binding completely, it was suggested that these ipine (1-3), phenylalkylamines exemplified by verapamil (1- drugs act at the same receptor sites as dihydropyridines (11). 3), benzothiazepines such as diltiazem (1, 2, 4), and diphenylal- In our studies, the diphenylalkylamines prenylamine, flunari- kylamines such as prenylamine and lidoflazine (1, -2, 5). Re- zine, and lidoflazine and the phenylailylamine tiapamil max- ceptors for the drugs are labeled with [3H]nitrendipine (6-10), imally inhibit [3HInitrendipine.binding (Fig. 1). However, max- or [3H] (10) and regulated in a physiological fashion imal inhibition of [3H]nitrendipine binding by these drugs by calcium (7). The apparent competitive inhibition of declines with increasing [3H]nitrendipine concentration be- [3H]nitrendipine binding by diphenylalkylamines has implied tween 0.1 and 3 nM (data not shown), suggesting a similarity effects at the same site as the dihydropyridines (11). Verapamil in the action ofthese agents and verapamil. We have confirmed and diltiazem respectively inhibit and stimulate [3H]nitrendipine this possibility in dissociation experiments (Fig. 2). As pre- binding allosterically, suggesting actions at different sites (9, viously reported (9), verapamil speeds the dissociation of 12). Here we show that the phenylalkylamine, diphenylalkyl- [3H]nitrendipine (data not shown), whereas the dihydropyri- amine, and benzothiazepine drugs,-in pharmacologically rele- dine nimodipine has no effect on the dissociation rate. Like vant concentrations, interact.at a single drug recognition site verapamil, the diphenylalkylamines lidoflazine and prenyl- allosterically linked to the dihydropyridine receptor. We pre- amine and the verapamil analog tiapamil accelerate [3H]- dict and confirm by bioassay previously unknown calcium an- n-itrendipine dissociation. and flunarizine similar- tagonist activity for several drugs. ly increase the dissociation rate. Having established that the diphenylalkylamines influence MATERILS AND METHODS [3H]nitrendipine binding allosterically, we investigated whether Drugs were obtained from the following sources: nifedipine, the diphenylalkylamines and phenylalkylamines act at the same Pfizer; nimodipine, Miles; prenylamine, Hoechst-Roussel site. Ifso, then increasing concentrations ofD-600 (which max- Pharmaceuticals (Somerville, NJ); D-600 (, methoxy- imally reduces [3H]nitrendipine binding only 20-30%) (Fig. 3) verapamil) and verapamil, Knoll Pharmaceutical (Whippany, should block the ability of a diphenylalkylamine to inhibit NJ); diltiazem, Marion Laboratories (Kansas City, MO) and [3H]nitrendipine binding, with a rightward shift in concentra- Tanabe (Osaka, Japan); desmethyl-cis-diltiazem and -trans-dil- tion-response curves. Indeed, increasing concentrations of D- tiazem, Tanabe; lidoflazine, flunarizine, and cinnarizine, Jans- 600 do produce rightward shifts in the concentration-response sen Pharmaceutical (Beerse, Belgium); and , McNeil curves for inhibition of [3H]nitrendipine binding by the di- Pharmaceutical (Spring House, PA). phenylalkylamines prenylamine, flunarizine, and lidoflazine Binding assays in guineapigbrain membranes, filtration, and (Fig. 1 a-c). Tiapamil, a phenylalkylamine closely similar in structure to verapamil and D-600 (16), produces the same max- The publication costs ofthis article were defrayed in part by page charge imal inhibition of [3H]nitrendipine binding elicited by the di- payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 860 Downloaded by guest on October 1, 2021 Medical Sciences: Murphy et aL Proc. NatL Acad. Sci. USA 80 (1983) 861

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0I- 0 0 0

-logiflunafizine.J (M) ._ ._ I V0 c c 0 I z I C., IsoA S0 4 to- ; U'0 7l 6 5 4 7 6 -logilidoflazinel (M) -log [tiapamil I (M) FIG. 1. Inhibition of [3Hlnitrendipine binding by prenylamine, tiapamil, lidoflazine, and flunarizine: Interactions with D-600. Concentration- response curves for the inhibition of [3Hlnitrendipine binding were constructed for prenylamine (a), flunarizine (b), lidoflazine (c), and tiapamil (d) in the absence (e) or the presence of 2.5 nM (o), 25 nM (i), 250 nM (o), 2.5 pM (A), or 25 ptM (A) D-600. Concentration-response curves are the results of experiments that employed at least six concentrations of each drug assayed in triplicate with 0.25 nM [3Hlnitrendipine. Values are presented as a percent of control [3H]nitrendipine binding.

phenylalkylamines (Fig. Id), and D-600 produces a rightward Because D-600 maximally reduces [3H]nitrendipine binding shift for tiapamil inhibition of [3Hjnitrendipine binding, re- less than the other diphenylalkylamines and phenylalkylamines flecting similar actions ofphenylalkylamines and diphenylalkyl- and assuming that the drugs act at the same site, D-600 should amines. The degree of rightward shift differs for -the drugs overcome inhibition of [3H]nitrendipine binding exerted by evaluated, being greatest with tia-pamil and least with.flunari- these other drugs. Accordingly, we examined the ability of D- zine. By contrast with these results, the concentration-response 600 to influence [3H]nitrendipine binding to membranes si- curve for inhibition of [3H]nitrendipine binding by nifedipine multaneously incubated with prenylamine, tiapamil, flunari- is not progressively shifted in the presence of increasing con- zine, or lidoflazine (Fig.. 3). In the absence of other drugs, D- centrations of D-600 (data not shown). 600 maximally reduces [3H]nitrendipine binding only about 25% with an ECG (50% effective concentration) ofabout 10 nM. By contrast, in the presence ofconcentrations ofprenylamine, tiapamil, flunarizine, or lidoflazine that give nearly maximal inhibition of [3H]nitrendipine binding, D-600 actually stimu- lates [3H]nitrendipine binding (Fig. 3). The percentage stim- ulation of [3H]nitrendipine binding by D-6(0 diminishes with too lower concentrations of the other drugs. [3H]Nitrendipine binding is restored by D-600 to the level ofbinding that occurs 0ml with maximally inhibitory concentrations of D-600 alone. As expected for drugs competing for the same site, D-600 is some- what less potent at enhancing [3H]nitrendipine binding in the presence of increasing concentrations of the other inhibitors (Fig. 3). Whereas diphenylalkylamines and phenylalkylamines inhibit 20. [3H]nitrendipine binding, diltiazem stimulates binding when evaluated in the absence ofother drugs (Fig. 4a) (12, 17). It has been suggested that diltiazem. therefore acts at a distinct site,

0 5 10 15 20 25, specific for benzothiazepines, and different from the site at act Dissociation, timer min which the phenylalkylamines and diphenylalkylamines (12, 17). If this were the case, no direct interactions should occur FiG. 2. Influences of various calcium channel antagonists on dis- between the benzothiazepines and the phenylalkylamines or sociation of [3H]nitrendipine. [3H]Nitrendipine (0.25 nM) was incu- diphenylalkylamines. To evaluate possible interactions, we ex- bated with brain membranes for 90 min to allow equilibrium associ- plored the effect ofdiltiazenmon.[3H]nitrendipine binding in the ation of to sites. Dissociations were initiated [3H]nitrendipine receptor presence of D-600, prenylamine, and tiapamil (Fig. 4a). Dil- with 100 nM nifedipine in either the absence (l) or the presence of 2.5 reverses of ,uM nimodipine (a), 2.5 ,uM prenylamine (v), 25 PM tiapamil (n), or tiazem strikingly the inhibition [3H]nitrendipine 25 ,uM lidoflazine (a). Results are presented as a percentage of equi- binding produced by tiapamil, D-60(, and prenylamine. More- librium [3H]nitrendipine binding. over, its potency in reversing such inhibition is decreased in the Downloaded by guest on October 1, 2021 862 Medical Sciences: Murphy et al. Proc. Nad Acad.'Sci. USA 80 (1983) whereas the other drugs decrease this affinity. To ascertain whether this effect ofdiltiazem is related to the pharmacologic actions of the drug, we compared cis- and trans- diltiazem, because only the cis isomer has pharmacological ef- ficacy. We also evaluated desmethyl-cis-diltiazem, which phar- macologically is about 30-50% as potent as diltiazem (4). En- hancement of [3H]nitrendipine binding in the presence of 2.5 AuM tiapamil is elicited by cis- but not trans-diltiazem (Fig. 4b). Desmethyl-cis-diltiazem is only about half as potent as cis-dil- C 40- tiazem. These relative activities are consistent with interactions 0 at the pharmacologically relevant site of action of diltiazem. Reduction of [3H]nitrendipine at 20 binding high concentrations of cis-diltiazem occurs similarly with trans-diltiazem and des- methyl-cis-diltiazem, indicating no pharmacologic relevance C (Fig. 4b). c d The phenylalkylamines and diphenylalkylamines possess two 0 Flunarizine Lidoflazine widely separated aromatic domains with the amine located cen- . 100 . trally, whereas diltiazem possesses only one aromatic domain

C. linked to an alkylamine moiety. We predicted that some drugs with one aromatic domain and an alkylamine moiety might in- 9) teract with receptors as diltiazem does. We have found A A6- that the H1 dimethindene and chlorpheniramine, the 60 ~ muscarinic anticholinergic biperiden, the neuroleptics meso- ridazine and , and the organic calcium antagonist 40 bepridil (18) all display the diltiazem-like.action of enhancing [3H]nitrendipine binding (Fig. 5). not 20- Dimethindene only enhances [3H]nitrendipine binding in the absence of other drugs but, like diltiazem, it reverses the inhibition of [3H]nitrendipine binding elicited by D-600, pre- 0 1 109 87 6 5 10 98 7 654 nylamine, and tiapamil (Fig. Sa). Bepridil, thioridazine, biperi- den, chlorpheniramine, and mesoridazine overcome the inhi- -iog[D-600] (M) bition caused by 2.5 ,uM tiapamil, enhancing [3H]nitrendipine FIG. 3. D-600 reverses the inhibition of tH,]nitrendipine binding binding from 10% to 80% of control (Fig. 5b). Bepridil, thio- produced by prenylamine, tiapamil, lidoflazine, and flunarizine. Con- ridazine, and biperiden show biphasic effects like diltiazem, centration-response curves for the influence of D-600 on [3H]ni- causing an inhibition of [3H]nitrendipine binding at higher con- trendipine binding to membranes were obtained with at least six con- centrations. centrations of D-600 and 0.25 nM [3H]nitrendipine. t5HlNitrendipine on binding was determined at each concentration of D-600 in the absence To determine whether these effects binding validly pre- (9), or the presence of 0.07 /IM (A), 0.25 ,M (A), 0.75 uM (o), 2.5 iiM dict pharmacologic calcium channel antagonism, we explored (W), or 25 ,uM (n) of the four drugs prenylamine (a), tiapamil (b), flu- the actions of these drugs on guinea pig ileum contractions (Fig. narizine (c), or lidoflazine (d). Values are presented as the percent of 6). The Schild plot for dimethindene inhibition of Ca2"-depen- control [3Hlnitrendipine binding determined in the absence of all in- hibiting agents. dent contractions shows a pA2 of 4.95 (Fig. 6), similar to di- methindene's potency in stimulating [3H]nitrendipine binding. presence ofincreased concentrations ofother drugs. Thus, dil- Furthermore, 25 uM -thioridazine, biperiden, chlorphenir- tiazem acts at the same site as phenylalkylamines and diphenyl- amine, and mesoridazine inhibit Ca2+-dependent contractions alkylamines to enhance receptor affinity for [3H]nitrendipine, of guinea pig ileal muscle by 97%, 95%, 66%, and 51%, re-

b 1 -0

- a .0 W 0 -1 80 40- 0S 0 20 :5 60 C)Q0 O rC .0 L- 40- 30 _ 0 I4 '20

7 g 5a 4 .-log |dl~tjazeml (M) -log.[drug] (M) FIG. 4. Diltiazem reverses the effects of D-600, prenylamine, and tiapamil on [3H]nitrendipine binding.- (a) Concentration-response curves for the effects of diltiazem on I5Hlnitrendipine binding were determined in the absence of any inhibiting agent (e) or in the presence of 0.25 PM D- 600 (0), 0.75 pM prenylamine (i), or 2.5 pM tiapamil (o). Values for each condition are presented as the percent of specific nitrendipine binding determined in the absence of all inhibiting agents. Results are from one of three experiments carried out for each drug with similarresults. (b) Concentration-response curves for the effects of cis-diltiazem (0), desmethyl-ci-diltiazem (o), or transrdiltiazem (-) were determined by using at least six concentrations of each of these agents in the presence of 0.2 nM [3Hlnitrendipine and 2.5 pM tiapamil incubated with brain membranes. Values are presented as the percentage of [3Hlnitrendipine binding determined in the absence of tiapamil, diltiazem, or diltiazem analogs. Downloaded by guest on October 1, 2021 Medical Sciences: Murphy et aL Proc. Natd Acad. Sci. USA 80 (1983) 863

c : 1'20 -a .00 30

_ c 30 7 rlOjICC I0

C 7 [ 5 4 3 -log Idimethinclenel (M) -log [drug] (M) FIG. 5. Diltiazem-like actions of dimethindene and other drugs. (a) Concentration-response curves for the effects of dimethindene on (5H]nitrendipine binding were determined in the absence of any inhibiting agent (a) or in the presence of 0.25 AM D-600 (o), 0.75 pM prenylamine (m), or 2.5 pM tiapamil (o). Values are presented as the percent of specific [3H]nitrendipine binding determined in the absence of all drugs. (b) Concentration-response curves for bepridil (e), thioridazine (o), biperiden (i), chlorpheniramine (o), and mesoridazine (A) were constructed by incubating 0.2 nM [3H]nitrendipine with 2.5 pM tiapamil and brain membranes. Values are the means of triplicate determinations for each con- dition presented as the percent of [3Hlnitrendipine binding determined in the absence of tiapamil and other drugs.

spectively. This order ofpotency corresponds to the drugs' po- calcium ion channels by drugs (Fig. 7). A dihydropyridine rec- tencies in enhancing [3H]nitrendipine binding (Fig. 5b). The ognition site linked to the calcium channel mediates the phar- augmentation of [3H]nitrendipine binding by this group of macologic actions of this class of calcium channel antagonists. drugs is specific, because the following drugs fail to increase A specific site for multivalent cations influences [3H]nitrendipine binding at 25 ,uM: , chlorimipramine, , binding (7). Divalent cations that mimic calcium physiologically , clozopine, , , , stimulate [3H]nitrendipine binding, whereas cations known to , , , , pyr- block the physiologic actions of calcium prevent the enhance- ilamine, , and . ment of [3H]nitrendipine binding by calcium (7). Phenylalkyl- amines and diphenylalkylamines act at the same site and in the DISCUSSION same fashion, both decreasing [3H]nitrendipine affinity for re- ceptors. These drugs vary in the extent to which they reduce Our present findings clarify actions ofcalcium antagonist drugs the receptor affinity of [3H]nitrendipine. D-600 and verapamil ofthe known structural classes. Whereas others had suggested produce the least effect, reducing [3H]nitrendipine's receptor that the diphenylalkylamines such as prenylamine act differ- affinity to 1/2-1/3, whereas tiapamil produces a reduction in ently than the phenylalkylamines such as verapamil (11), we affinity to V/5_V/1o and flunarizine a reduction to 1/10-/2o. Dil- have demonstrated that both classes ofdrugs act via the same tiazem and bepridil act at the same site to enhance [3H]- site to allosterically regulate [3H]nitrendipine binding. It had nitrendipine affinity. been suggested that diltiazem acts at a site distinct from the On the basis of structural similarities, we identified several phenylalkylamines and diphenylalkylamines (12, 17), but we neuroleptics, antihistamines, and muscarinic find that it acts at the same site. The diphenylalkylamines and that mimic the actions ofdiltiazem on [3H]nitrendipine binding phenylalkylamines decrease the affinity of[3H]nitrendipine for and have calcium channel antagonist activity in the guinea pig receptors, but diltiazem enhances this affinity, as reflected in ileum. Thus, effects at a single domain of a bipartite receptor slowing of the [3H]nitrendipine dissociation rate (12, 17). may convey diltiazem-like actions, whereas drugs acting at both These observations suggest a unitary model for regulation of domains have verapamil-like profiles. Such binding analyses may help identify calcium antagonists. Clinical actions of the drugs we have evaluated may relate to their calcium antagonist actions. Of numerous phenothi- azines evaluated, thioridazine and mesoridazine were unique in influencing [3H]nitrendipine binding in a diltiazem-like fash- ion. Blood levels ofthioridazine and mesoridazine at therapeu- tic doses are about 2 ,uM (19, 20), a concentration atwhich these drugs display calcium antagonism. Thioridazine and mesori-

Dihydropyridine -log [dimethindene] (M) Recognition Site

FIG. 6. Dimethindene blocks calcium-dependent contractions of 2+ KCl-depolarized guinea pig ileum. Concentration-response curves for the calcium-dependent contractions of the guinea pig ileum in the absence or the presence of various concentrations of dimethindene were constructed by sequential additions of calcium chloride to a cal- Diltiazem/Verapamil cium-free high-potassium buffer. Only one dimethindene concentra- Recognition Site tion was employed in any tissue. log(dose ratio - 1) is plotted against log[dimethindene] and the pA2 (4.95) was determined by linear regres- sion (r = 0.97). pA2 is the negative logarithm of the concentration at which the dose ratio is 2; it is equivalent to -log app. Dose ratio is the ratio of , in the absence and presence of antagonist, which gives FIG. 7. A model for the sites of action and interactions for inor- the same response. ganic and organic calcium channel antagonists. Downloaded by guest on October 1, 2021 864 Medical Sciences: Murphy et aLPProc. Nad Acad. Sci. USA 80 (1983) dazine elicit more cardiac side effects with Q-T interval pro- 8. Bolger, G. T., Gengo, P. J., Luchowski, E. M., Siegel, H., Trig- longation than do other (21-23). Another gle, D. J. & Janis, R. A. (1982) Biochem. Biophys. Res. Commun. unique action of thioridazine and mesoridazine is inhibition of 104, 1604-1609. 9. Ehlert, F. J., Itoga, E., Roeske, W. R. & Yamamura, H. I. (1982) ejaculation (23). Because ejaculation involves a massive con- Life Sci. 30, 2191-2202. traction of the of the vas deferens, the calcium 10. Bellemann, P., Ferry, D., Lubbecke, F. & Glossmann, H. (1982) antagonist actions of these drugs may account for such side ef- Arzneim.-Forsch. 32, 361-363. fects. 11. Ferry, D. R. & Glossmann, H. (1982) Naunyn-Schmeideberg's Arch. PharmacoL 321, 80-83. We thank Dawn C. Hanks for manuscript preparation. This work was 12. Yamamura, H. I., Schoemaker, H., Boles, R. G. & Roeske, W. supported by U.S. Public Health Service Grants DA-00266, MH- R. (1982) Biochem. Biophys. Res. Commun. 108, 640-646. 18501, and NS-16375; a grant from the and U. S. 13. Rosenberger L. B., Ticku, M. K. & Triggle, D. J. (1979) Can. J. McKnight Foundation; Physiol PharmacoL 57, 333-341. Public Health Service Research Scientist Award DA-00074 to S.H.S., 14. Triggle, C. R., Swamy, V. C. & Triggle, D. J. (1979) Can.J. Phys- Training Grant GM-073090 to K.M.M.M., and Training Grant MH- ioL PharmacoL 57, 804-818. 15330 to R.J.G. 15. Jim, K., Harris, A., Rosenberger, L. B. & Triggle, D. J. (1981) Eur. J. Pharmacol. 76, 67-72. 1. Henry, P. D. (1980) Am. J. Cardiol 46, 1047-1058. 16. Eigenmann, R., Blaber, L., Nakamura, K., Thorens, S. & 2. Triggle, D. J. (1981) in New Perspectives on Calcium Antago- Haeusler, G. (1981) Arzneim.-Forsch. 31, 1393-1401. nists, ed. Weiss, G. B. (American Physiological Society, Be- 17. DePover, A., Matlib, M. A., Lee, S. W., Dube, G. D., Grupp, thesda, MD), pp. 1-18. I. L., Grupp, G. & Schwartz, A. (1982) Biochem. Biophys. Res. 3. Fleckenstein, A. (1977) Annu. Rev. Pharmacol Toxicol 17, 149- Commun. 108, 110-117. 166. 18. Vogel, S., Crampton, R. & Sperelakis, N. (1979) J. PharmacoL 4. Nagao, T., Sato, M., Nakajima, H. & Kiyomoto, A. (1973) Chem. Exp. Ther. 210, 378-385. Pharmn. Bull 21, 92-97. 19. Calil, H. M., Avery, D. H., Hollister, L. E., Creese, I. & Sny- 5. Vanhoutte, P. M. (1981) in New Perspectives on Calcium Antag- der, S. H. (1979) Psychiatry Res. 1, 39-44. onists, ed. Weiss, G. B. (American Physiological Society, Be- 20. Nyberg, G., Axelsson, R. & Martensson, E. (1978)J. Clin. Phar- thesda, MD), pp. 109-121. macoL 14, 341-350. 6. Murphy, K. M. M. & Snyder, S. H. (1982) Eur.J. Pharmacol 77, 21. Appleton, W. S. (1982)J. Clin. Psychol 43, 12-27. 201-202. 22. Soffer, J., Dreifus, L. S. & Michelson, E. L. (1982) Am. J. Car- 7. Gould, R. J., Murphy, K. M. M. & Snyder, S. H. (1982) Proc. dioL 49, 2021-2029. Natl Acad. Sci. USA 79, 3656-3660. 23. Wendkes, M. H. (1964)J. New Drugs 4, 322-332. Downloaded by guest on October 1, 2021