Journal t~{ Cardim·a.H·nlar PharmacoloJ{\'
`10:627-635 <iJ 19H7 Raven Press, Ltd .. New York
`
`Effects of Bay k 8644 on the Coronary Vascular Selectivity
`of the Dihydropyridine Ca Antagonists in the Canine
`Isolated, Blood-Perfused Papillary Muscle Preparation
`
`Shigeru Motomura, Keitaro Hashimoto, and *Koroku Hashimoto
`
`Department ofPharmacolo!{y, Yamanashi Medical Colle!{e. Tamalw, Yamanashi; and *Hatano Research Institute,
`Food and Dru~: Sq(ety Center. Hadano. Kww~:m1'a, Japan
`
`Summary: Antagonism by Bay k 8644 of the coronary
`vasodilator and negative inotropic effects of Ca antago(cid:173)
`nists was simultaneously investigated in the canine iso(cid:173)
`lated, blood-perfused papillary muscle electrically driven
`at 2 Hz. Drugs were given intraarterially. Continuously
`infused intraarterially, Bay k 8644 inhibited more pre(cid:173)
`dominantly the coronary vasodilator effects of the dihy(cid:173)
`dropyridine Ca antagonists (nifedipine, nitrendipine, and
`nicardipine) than those of diltiazem and vcrapamil, or the
`negative inotropic effects of nifedipine and nitrendipine.
`The negative inotropic effects of nicardipine, diltiazem,
`and verapamil were not significantly affected by Hay k
`8644. The dose ratio for producing a 50% decrease in de(cid:173)
`veloped tension of the papillary muscle over the dose for
`
`producing a 50% increase in coronary blood flow (which
`reflects vascular selectivity) were 41 for nitrendipine. 25
`for nifedipine. 21 for nicardipine. 9 for diltiazem. and 7
`for verapamil. During infusion by Bay k 8644. the ratio
`was small (2 to 6) and almost similar among all five Ca
`antagonists. These results indicate that Bay k 8644 antag(cid:173)
`onizes more effectively the coronary vasodilating effects
`of the dihydropyridine Ca antagonists, suggesting that
`there is a specific type of Ca channel on coronary vas(cid:173)
`cular smooth muscles, through which the dihydropyri(cid:173)
`dine Ca antagonists act. Key Words: Bay k 8644-Coro(cid:173)
`nary vascular selectivity-Dihydropyridine calcium an(cid:173)
`tagonists- N ifedipine- N icardipine- Nitrendipine.
`
`The mechanism of high vascular selectivity of the
`dihydropyridine Ca antagonists such as nifedipine,
`as compared to other Ca antagonists such as vera(cid:173)
`pamil and diltiazem, has not been clearly explained
`(1-3). Two possibilities have been proposed for ex(cid:173)
`plaining the discrepancy between the equilibrium
`dissociation constants (Kd) of 3 H-nitrendipine
`binding with high affinity (4- 7) and the pharmaco(cid:173)
`logical IC 50 of nitrendipine for inhibiting the con(cid:173)
`traction or slow inward Ca currents in cardiac
`muscle (8,9). First, it is possible that a second type
`of Ca channel with low affinity exists in cardiac
`muscle. Indeed, the low affinity binding site in car(cid:173)
`diac muscle with the Kd being close to the IC50 for
`depression of cardiac contractility has been re(cid:173)
`ported ( 10-14), while it has not been found in vas(cid:173)
`cular smooth muscle (7). In vascular smooth
`
`muscle, the Kd is almost equal to the IC 50 for de(cid:173)
`pressing the contraction (6-8). In this regard, Bay
`k 8644, a new dihydropyridine compound, was
`found to have opposite effects to nifedipine (15,16)
`as the cardiac and vascular effects of Bay k 8644
`were competitively antagonized by nifedipine
`(15,16) and vice versa (17-19). However, the pA2
`values for competitive antagonism were two to
`three orders of magnitude larger in smooth muscle
`(17,18) than in cardiac muscle (19). Bay k 8644
`bound to the dihydropyridine binding sites in car(cid:173)
`diac muscles with either high or low affinity
`(13,14,20-22). These findings might suggest that
`the vascular selectivity of the dihydropyridine Ca
`antagonists is based on their direct action on the
`vascular smooth muscle cells through a specific di(cid:173)
`hydropyridine acting site related to high affinity
`
`Received July 28, 1986; revision accepted March 13. 1987.
`Address correspondence and reprint requests to Dr. S.
`
`Motomura at Department of Pharmacology, Yamanashi Medical
`College, Tamaho, Yamanashi 409-38, Japan.
`
`627
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`628
`
`S. MOTOMURA ET AL.
`
`binding. Second, the unitary Ca channel with high
`affinity exists in both vascular smooth and cardiac
`muscles. but it is modified by factors known to reg(cid:173)
`ulate the state and function of the Ca channel, such
`as membrane potential and protein phosphorylation
`(23,24). In this connection, depolarization of car(cid:173)
`diac cell membrane decreased the IC 50 of nitrendi(cid:173)
`pine for Ca current blocking (25), but did not
`change the Kd of 3H-nitrendipine binding (26).
`However. it has been reported that block of
`voltage-dependent Ca channel in cardiac muscle by
`the Ca antagonists are depending on their hydro(cid:173)
`philicity (27) and that the vascular selectivity of the
`dihydropyridine Ca antagonists might be attributed
`to their intracellular effects after entering across
`the lipid plasma membrane depending on the lipo(cid:173)
`philicity (28). In other words, it is possible that the
`pKa value (i.e., the degree of ionization) may be
`related to vascular selectivity of Ca antagonists.
`In the present experiments, we attempted to
`ascertain the above two possibilities for the vas(cid:173)
`cular selectivity of the dihydropyridine Ca antago(cid:173)
`nists. The antagonisms by Bay k 8644 of the coro(cid:173)
`nary vasodilator and negative inotropic effects of
`nifedipine, nitrendipine, and nicardipine were com(cid:173)
`pared with those of diltiazem and verapamil in the
`isolated, blood-perfused papillary muscle prepara(cid:173)
`tion of the dog, which permits simultaneous and
`quantitative comparison of the potencies (29,30).
`Bay k 8644 seems to antagonize more susceptibly
`the selective coronary vasodilator effects of the di(cid:173)
`hydropyridine Ca antagonists than those of the
`nondihydropyridines [since verapamil and dil(cid:173)
`tiazem allosterically modify the high affinity dihy(cid:173)
`dropyridine binding (31 ,32)) or the negative ino(cid:173)
`tropic effects of the dihydropyridine Ca antagonists
`[since the low atfinity binding might be responsible
`for the effects (7,13.14.33)). It is uncertain. how(cid:173)
`ever, whether the antagonisms by Bay k 8644 of the
`effecb of nicardipine and nitrendipine are identical
`to that of nifedipine, since nicardipine (pKa = 7.0)
`is about 28% ionized, but nifedipine and nitrendi(cid:173)
`pine (pKa < 3.5) exist almost entirely in the neutral
`form at physiological pH 7.4 (34).
`
`METHODS
`
`Experiments were performed on 30 isolated, blood(cid:173)
`perfused papillary muscle preparations of the dog. Mon(cid:173)
`grel dogs of either sex, weighing 8-12 kg, were anesthe(cid:173)
`tized with sodium pentobarbital (30 mg/kg i.v.), given so(cid:173)
`dium heparin (500 U/kg i. v .), and exsanguinated. The
`heart was excised and immediately plunged into cold
`Tyrode's solution at about 4"C. Then the papillary muscle
`preparation was prepared by the same methods as de(cid:173)
`scribed by Endoh and Hashimoto (29). The preparation
`essentially consists of the anterior papillary muscle of the
`right ventricle attached to the interventricular septum.
`and the anterior septal artery, the nutrient artery of the
`papillary muscle, was cannulated. The papillary muscle
`
`J Cardiol'ti.H' Plwrmaml, Vol. /0, No. 6, /Y87
`
`was fixed at the base to a plastic plate with a hole and
`placed in a double-walled glass jacket maintained at 380C
`by circulating warm water. The preparation was perfused
`through the cannulated anterior septal artery with arterial
`blood conducted from the right carotid artery of a sup(cid:173)
`port dog at a constant perfusion pressure of 100 mm Hg
`using a Cole-Parmer Masterflex pump and a Starling
`pneumatic resistance placed parallel to the perfusion
`system. Venous blood from the papillary muscle prepara(cid:173)
`tion and excess blood passing through the pneumatic re(cid:173)
`sistance were collected in a blood-reservoir and returned
`to the support dog through the jugular vein. The rate of
`blood flow through the anterior septal artery was mea(cid:173)
`sured using an electromagnetic flowmeter (Nihon
`Kohden, MFV-1100) and a 2-mm cannulating flow probe.
`The papillary muscle was electrically driven (MEC,
`ME-6012, ME-6217) with rectangular pulses of 1-4 V
`(about 20% above threshold voltage and of 5 ms duration
`at a fixed rate of2 Hz through bipolar silver-silver stimu(cid:173)
`lating electrodes in contact with the base of the papillary
`muscle. Developed tension of the papillary muscle pre(cid:173)
`loaded with the weight at which maximum tension devel(cid:173)
`oped was measured isometrically using a force displace(cid:173)
`ment transducer (Oia Medical. DRM-T200) and an ampli(cid:173)
`fier (Oia Medical, DRM-T20J.
`Adult mongrel dogs of either sex, weighing 14-23 kg,
`were used as support dogs. which were anesthetized ini(cid:173)
`tially with pentobarbital sodium, 30 mg/kg i. v .. and given
`an additional 4-5 mg/kg/h. The animals received an ini(cid:173)
`tial dose of 500 U/kg heparin sodium, followed by 200
`U/kg/h. Respiration was controlled using an animal respi(cid:173)
`rator (Harvard Apparatus, model 607). The systemic
`blood pressure at the femoral artery and heart rate trig(cid:173)
`gered by R wave of the lead II EKG were monitored con(cid:173)
`tinuously with a polygraph (San-ei Instruments, 361-6).
`Drugs used were Bay k 8644 [methyl 1,4-dihydro-2,6-
`dimethyl-3-nitro-4-(2-trifluoromethyl-phenyl)-pyridine-5-
`carboxylate: kindly supplied by Dr. Kazda, Bayer AG,
`Wuppertal, F.R.G.]. nifedipine (Bayer AG), nitrendipine
`(Bayer AG through Yoshitomi Pharmac. Co .. Osaka,
`Japan). nicardipine (Yamanouchi Pharmac. Co., Tokyo,
`Japan), diltiazem hydrochloride (Tanabe Pharmac, Co.,
`Osaka, Japan), and verapamil (Eisai, Tokyo, Japan). Bay
`k 8644 and nitrendipine were dissolved in 96% ethanol
`and polyethyleneglycol 500 ( 150 mg/L) to obtain a con(cid:173)
`centration of 10 mg/ml and I mg/ml, respectively. Nifed(cid:173)
`ipine has a concentration of 100 f.Lg/ml in an ampoule.
`Nicardipine, diltiazem, and verapamil were dissolved in
`water to give a concentration of I mg/ml. The desired
`concentrations of the drugs were obtained by diluting the
`stock solution with 0.9% saline. A volume of 30 f.l-1 was
`injected intraarterially over a period of 4 s by using a mi(cid:173)
`crosyringe (Terumo, Japan) through the rubber tubing
`connected to the cannula. To analyze influences of Bay k
`8644 on the effects of Ca antagonists, Bay k 8644 was
`continuously infused into the rubber tube at a rate of 0.1
`ml/min using an infusion pump (Harvard Apparatus,
`model 600-900). Bay k 8644 and nifedipine were kept in
`the dark during experiments.
`Experimental protocols are as follows. After I h equili(cid:173)
`bration, a Ca antagonist was injected intraarterially in a
`bolus fashion to obtain the dose-response curves for its
`negative inotropic and coronary vasodilator effects at
`control. After returning the values of the developed ten(cid:173)
`sion and the blood flow to those before injection of the
`
`CRTXCAR000687 4
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`
`

`

`VASCULAR SELECTIVITY OF THE DIHYDROPYRIDINES
`
`629
`
`drug. Bay k 8644 was continuously infused intraarterially
`at a rate of I !J-g/min. After getting the steady levels of the
`parameters during Bay k 8644 infusion, the Ca antagonist
`was repeated. When the values returned to control after
`cessation of Bay k 8644 infusion, 10 flog/min Bay k 8644
`infusion started and then the Ca antagonist was repeated
`again. The peak changes in the developed tension and
`blood flow obtained by i.a. bolus injection of the drugs
`were expressed as percentages of the predrug values ei(cid:173)
`ther before or during infusion of Bay k 8644. The doses of
`Ca antagonists. which produce a 50% increase in blood
`flow. ED50(CVD). and which induce a 50% decrease in
`the developed tension. ID 50(NIE). were calculated in
`each experiment before and during infusion of Bay k
`8644. Although Taira (2) and Himori et al. (30) used
`ED 100(CVD) (i.e .. the dose required to double the blood
`flow) instead of ED50(CVD). for comparison of coronary
`vasodilator potencies it is difficult to obtain the
`ED 100(CVD) during infusion of Bay k 8644 and there is no
`qualitative difference between the ED50 and ED 100 for
`quantitative comparison of the potencies. The ratio of
`ID50(NIE)/ED50(CVD). which indicates the vascular se(cid:173)
`lectivity of the drugs. was compared either among the
`drugs or between. before. and during infusion of Bay k
`8644. Statistical comparisons of mean values were evalu(cid:173)
`ated by t-test for paired data.
`
`RESULTS
`
`Stability of the preparation during Bay k
`8644 infusion
`After I h equilibration. the basal developed ten(cid:173)
`sion of the papillary muscle was 3.2 ± 0.2 g (n =
`30) and the basal blood flow through the anterior
`septal artery was 6.6 ± 0.3 ml/min (n = 30) at con(cid:173)
`trol. After getting the dose-response curves for the
`negative inotropic and coronary vasodilator effects
`of the Ca antagonist, both values returned to con(cid:173)
`trol. The developed tension and the blood flow
`were 3.0 ± 0.3 g and 6.5 ± 0.3 ml/min (n = 30)
`before I J-lg/min Bay k 8644 infusion, and 2.8 ± 0.3
`g and 6.2 ± 0.3 ml/min (n = 30) before 10 J-lg/min
`Bay k 8644 infusion. respectively. which are not
`significantly different from those at control. When
`Bay k 8644 was continuously infused intraarterially,
`the developed tension was gradually increased and
`reached a steady level about 10 min after the begin(cid:173)
`ning of the infusion. The increased steady levels of
`the developed tension were 64 ± 7% and 104 ± 7%
`(n = 30) of the corresponding basal developed ten(cid:173)
`sion at infusion rates of I and 10 J-lg/min, respec(cid:173)
`tively, which are significantly different from each
`other (p < 0.01). On the other hand, the blood flow
`was maximally decreased about I min after the be(cid:173)
`ginning of Bay k 8644 infusion and then returned
`toward the basal value and reached the steady level
`about 10 min after the beginning of the infusion.
`The maximal decreases in the blood flow were by
`33 ± 2% and 48 ± 2% (n = 30) of the corre(cid:173)
`sponding basal blood flow at infusion rates of I and
`10 J-lg/min, respectively (p < 0.01). The recovered
`
`FIG. 1. A typical experiment of the influence of Bay k 8644
`on the negative inotropic and coronary vasodilator effects of
`nifedipine. DT, developed tension of the papillary muscle;
`BF, blood flow through the anterior septal artery; NF, nifed(cid:173)
`ipine injected i.a. in a bolus fashion. A: Before Bay k 8644
`infusion. Nifedipine (0.03 and 0.1 f.l.g) caused a slight in(cid:173)
`crease in DT accompanied with significant increase in BF.
`Nifedipine (1
`f.l.g) almost doubled BF and decreased DT by
`about 20%. B: During infusion of Bay k 8644 at rate of 1 tJ.g/
`min. BF decreased transiently to about 60% and returned to
`steady level of about 90% of basal blood flow. DT increased
`and reached steady level of 180% of basal developed ten(cid:173)
`sion. At this steady level, 1 f.l.g nifedipine increased BF by
`about 30%, but decreased DT by about 15%. C: During infu(cid:173)
`sion of Bay k 8644 at rate of 10 f.l.g/min. BF decreased to
`about 50% and returned to steady level of about 70% of basal
`blood flow. DT increased and reached steady level of about
`180% of basal developed tension. Nifedipine (1
`tJ.9) caused
`only slight changes in BF and DT.
`
`1 Cardiol'li.H' Plwrmacol, Vol. 10. No. 6, 1987
`
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`

`

`630
`
`S. MOTOMURA ET AL.
`
`NIFEDIPINE
`
`)
`
`I
`
`'1:
`
`0
`0
`
`1:
`
`0 u: ..,
`iii ..,
`" 1:
`0 ·;;;
`
`1: ., ,_ .., .,
`0 -: .,
`.: .. ..
`"' 1: .. .r:
`
`Q.
`
`0
`
`u
`
`0.03
`
`0.1
`
`0.3
`
`10
`
`30
`
`Niledlpine (~g)
`
`FIG. 2. Dose-response curves for the coronary vasodilator
`(open symbols) and the negative inotropic effects (closed
`symbols) of nifedipine before (circles) and during infusion of
`Bay k 8644 at the rates of 1 (triangles) and 10 (squares) fig/
`min, obtained from 8 papillary muscle preparations. Hori(cid:173)
`zontal fine dotted lines at +50% and
`50% on ordinate indi(cid:173)
`cate the levels of a 50% increase in blood flow (BF) and a
`50% decrease in developed tension (DT), respectively. Ver(cid:173)
`tical fine dotted lines from the cross points of +50 and
`-50% changes and each dose- response curve indicate the
`ED50(CVD) and the ID50(NIE) before and during Bay k 8644
`infusion. The ED50(CVD), the ID50(NIE), and the ratio of the
`ID5afED50 of nifedipine before and during Bay k 8644 infusion
`at rates of 1 and 10 fig/min are presented in Table 1. Before
`Bay k 8644 infusion, 0.03 fig nifedipine caused a slight in(cid:173)
`crease in DT despite a significant increase in BF.
`
`steady levels of the blood flow were 83 ± 2o/c and
`72 ± 2% (n = 30) of the basal blood flow, respec(cid:173)
`tively (p < 0.01).
`
`Influences of Bay k 8644 on the negative inotropic
`and coronary vasodilator effects of nifedipine
`A typical experiment of the interaction of Bay k
`8644 with nifedipine is shown in Fig. I. In the ab(cid:173)
`sence of Bay k 8644 (Fig. lA), nifedipine, 0.03 - 10
`f.lg, caused dose-dependent increases in the blood
`flow, whereas the developed tension first slightly
`increased at low doses and then decreased dose-re(cid:173)
`latedly at higher doses of nifedipine. During infu(cid:173)
`sion of Bay k 8644 at a rate of I f.lg/min (Fig. IB),
`coronary vasodilation induced by lower doses of ni(cid:173)
`fedipine was abolished and the increases in blood
`tlow by its higher doses were suppressed. On the
`other hand, the negative inotropic ctTccts of nifed-
`
`1 Cardiol'll.\C Phurmacol, Vol. 10, Nu. 6, /WO
`
`ipine were little affected by I f.lg/min Bay k 8644,
`although the slight increases in the developed ten(cid:173)
`sion induced by lower doses of nifedipine were
`abolished. In the presence of 10 f.lg/min Bay k 8644
`(Fig. I C). the coronary vasodilator effect of nifed(cid:173)
`ipine was further suppressed and the negative ino(cid:173)
`tropic effect was also inhibited. In Fig. 2 are shown
`the dose-response curves for the coronary vasodi(cid:173)
`lator and negative inotropic effects of nifedipine
`obtained in the absence and presence of I and 10
`f.lg/min Bay k 8644. The dose-response curve for
`coronary vasodilation shifted to the right more than
`that for negative inotropic effects. The ED50(CVD)
`of nifedipine, the dose which produced a 50% in(cid:173)
`crease in blood flow. was 10 to 25 times larger
`during Bay k 8644 infusion at rates of I and 10 f.lg/
`min, respectively (Table 1). In comparison, the
`ID50(NIE), the dose which induced a 50% decrease
`in the developed tension, was only 1.2 and 4 times
`larger in the presence of I and 10 f.lg/min of Bay k
`8644 than in its absence, respectively (Table 1). Fur(cid:173)
`ther, as shown in Table I, the ratio of ID50(NIE)/
`ED50(CVD) became small and similar during infu(cid:173)
`sion of Bay k 8644, independent of the infusion
`rate.
`
`Influence of Bay k 8644 on the coronary vasodilator
`and the negative inotropic effects of nitrendipine
`and nicardipine
`Like nifedipine, two other dihydropyridines, ni(cid:173)
`trendipine and nicardipine, caused pronounced cor(cid:173)
`onary vasodilation and less potent negative ino(cid:173)
`tropic effects. Nitrendipine had an even larger ratio
`of ID50(NIE)/ED50(CVD) than nifedipine as shown
`in Table I. Figure 3 shows that the dose-response
`curves for the coronary vasodilator and negative
`inotropic effects of nitrendipine and nicardipine
`were diversely influenced by Bay k 8644. The
`dose-response curves of nitrendipine were in(cid:173)
`fluenced by Bay k 8644 in much the same way as
`those to nifedipine (Fig. 3A); the ED50(CVD) was
`12 and 35 times larger and the ID50(NIE) was 1.2
`and 5 times larger in the presence of Bay k 8644 at
`infusion rates of I and 10 f.lg/min, respectively
`(Table 1). On the other hand, the dose-response
`curves for the coronary vasodilator effect of nicar(cid:173)
`dipine clearly shifted to the right, although the shift
`was smaller than those seen with nifedipine and ni(cid:173)
`trendipine. Meanwhile, the dose-response curves
`for the negative inotropic effect of nicardipine were
`little affected by Bay k 8644 (Fig. 3B); the
`ED50(CVD) was 7 and 10 times larger and the
`ID50(NIE) was only 1.0 and 1.3 times larger during
`Bay k 8644 infusion at rates of I and 10 f.lg/min,
`respectively (Table I). Nevertheless, the ratio of
`ID50(NIE)/ED50(CVD) of both nitrendipine and ni(cid:173)
`cardipine, as well as that of nifedipine, had almost
`similar values in the presence of Bay k 8644, as
`shown in Table I.
`
`CRTXCAR0006876
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`

`

`VASCULAR SELECTIVITY OF THE DIHYDROPYRIDJNES
`
`631
`
`TABLE l. The dose.~ of calcium antaKonists that produced a 5()o/,:
`decrease in del'l'loped ten.1·ion, JD50(N/E), those that produced a
`50% increase in hlood flow, ED50(CVD), and the ratio of/D5JED50
`hej(Jre and durinK inji1sion of Bay k 8644 at rates of I and 10
`ILK! min
`
`tl)
`
`Nifedipine (n =
`Control
`Bay k 8644 (I IJ.g/min)
`Bay k 8644 ( 10 fJ.g/min)
`Nitrendipine (n = 6)
`Control
`Bay k 8644 (I fJ.g/min)
`Bay k 8644 ( 10 fJ.g/min)
`Nicardipine In = 6)
`Control
`Bay k 8644 (I IJ.gfmin)
`Bay k 8644 ( 10 fJ.g/min)
`Diltiazem (n = 5)
`Control
`Bay k 8644 (I fJ.g/min)
`Bay k 8644 ( 10 fJ.g/min)
`Verapamil (n = 5)
`Control
`Bay k 8644 (I IJ.g/minl
`Bay k 8644 ( 10 IJ.gfmin)
`
`1Dl01NIE)
`
`EDl0(CVD)
`
`IDsr/EDlo
`
`4.2 :+: O.tl
`5.5 ± 0.8•
`19.0 ± 0.7b
`
`75 :!: 9
`104:!: 19•
`441 ± I07b
`
`54± II
`59± II'
`790 ± 24'
`
`226 ± 41
`169 ± 42'
`164 ± 2K'"
`
`40 ± 5
`42 ± II'
`50± K'"
`
`0.19 :!: 0.02
`1.9 ± O.Jb
`5.3 :!: 0.2b
`
`2.7 ± 1.2
`33 ± Xb
`84 ± 19b
`
`2.9 ± 0.6
`18 ± :Jb
`29 ± 4b
`
`27 ± 9
`83 ± 24a
`97 ± na
`
`6.5 ± 1.2
`24 ± 6b
`21 ± 4b
`
`25 ± 7
`4 ± ::!b
`I b
`4 ±
`
`41 ± II
`5 :!: 2b
`6 ± 2b
`
`21 ± 5
`4 :!: (b
`3 ± I b
`
`9 ±
`2 ± ()"
`2 ± I a
`
`7 ±
`± I"
`3 ± I a
`
`"p<0.05.
`b p < 0.01, significantly different from each control value.
`'No significant difference compared with each control value.
`
`Influence of Bay k 8644 on the coronary vasodilator
`and negative inotropic effects of diltiazem
`and verapamil
`Diltiazem and verapamil caused marked coro(cid:173)
`nary vasodilator and negative inotropic effects. Un-
`
`like nifedipine and the other dihydropyridines,
`however, the ratios of ID50(NIE)/ED50(CVD) were
`relatively low for diltiazem and verapamil as shown
`in Table I. Furthermore, the antagonism by Bay k
`8644 of the coronary vasodilator effects of dil-
`
`A
`
`NITRENDIPINE
`
`+100
`
`+50
`
`:;;
`J
`0
`iL
`" 0
`0
`iii
`~
`c
`0
`~ ..
`;;
`" . a.
`0 : . Q
`.5 .. . ~
`
`-so
`
`~
`u
`
`-100
`
`0.3
`
`B
`
`NICARDIPINE
`
`+100
`
`+50
`
`0 ;;
`
`D
`
`"' J
`0 u:
`" 0
`" c . c
`0
`ii
`c . ..
`" . a.
`0 : . a
`.!: . . i ~ "
`
`-60
`
`-100
`
`i
`
`I
`
`10
`
`30
`
`100
`
`300
`
`100
`
`0.3
`
`10
`
`30
`
`100
`
`Nlcordlplne (~g)
`Nltrendlplne ("g)
`FIG. 3. Dose- response curves for the coronary vasodilator and the negative inotropic effects of nitrendipine (A) and nicardipine
`(B) before and during infusion of Bay k 8644 at rates of 1 and 10 IJ.Q/min, obtained from 6 papillary muscle preparations,
`respectively. All symbols and supplementary lines are the same as those in Fig. 2. Before Bay k 8644 infusion, 0.3 IJ.g nitrendipine
`and nicardipine caused a slight increase in DT, despite a significant increase in BF. The ED50(CVD), the ID50(NIE), and the ratio of
`the IDsoJED50 before and during Bay k 8644 infusion at rates of 1 and 10 fJ.g/min are presented in Table 1.
`
`J CurdiomH· Plwrmuml. Vol. 10. No. 6, 1987
`
`CRTXCAR0006877
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`

`632
`
`,\'. MOTOMURA ET AL.
`
`tiazem and verapamil was less marked than the an(cid:173)
`tagonism of the dihydropyridines. The negative ino(cid:173)
`tropic effects of diltiazem and verapamil were not
`affected by Bay k 8644. A typical experiment of the
`interaction between diltiazcm and Bay k 8644 is
`shown in Fig. 4. Even during a high rate of infusion
`of 10 f.Lg/min Bay k 8644. the coronary vasodilation
`was slightly suppressed, whereas the negative ino(cid:173)
`tropic effect was unaffected and even potentiated.
`although not significantly. The dose-response
`curves for the coronary vasodilator and the nega(cid:173)
`tive inotropic effects of diltiazem and verapamil in
`the absence and presence of I and 10 f.Lg/min Ray k
`8644 were shown in Fig. 5A and B. The ED50(CYD)
`of diltiazem was 3.5 and 3.8 times larger in the pres(cid:173)
`ence of I and I 0 f.Lg/min Bay k 8644, while
`ID50(NIE) was only 0.9 and 0.8 times larger during
`infusion of Bay k 8644 at rates of I and 10 f,Lg/min.
`respectively (Table I). Like diltiazem. the coronary
`vasodilation by verapamil was slightly suppressed,
`
`but the negative inotropic effect was little affected
`by Bay k 8644; the ED~0(CVD) of verapamil was
`3.5 and 3 times larger and the ID 50(NIE) was only
`1.0 and 1.2 times larger in the presence of I and 10
`f.Lg/min Bay k 8644, respectively. The ratio of
`ID50(N IE)/ED50(CVD) of diltiazem and verapamil
`also became small and similar to those values of the
`dihydropyridines in the presence of Bay k 8644
`(Table 1).
`
`DISCUSSION
`
`In the present experiments. when Bay k 8644 was
`intraarterially infused, the developed tension was
`increased and maintained at steady levels during
`the infusion in relation to infusion rates. The coro(cid:173)
`nary blood flow maximally decreased about I min
`after the beginning of the infusion. when it returned
`toward the basal values, and was then maintained
`at steady levels in relation to the infusion rates (Fig.
`
`FIG. 4. A typical experiment of influences of
`Bay k 8644 on the coronary vasodilating and
`the negative inotropic effects of diltiazem.
`Diltiazem (D) injected i.a. in a bolus fashion
`caused dose-dependent increases in blood
`flow (BF) and decreases in developed ten(cid:173)
`sion (DT), before (A) and during infusion of
`Bay k 8644 at a rate of 10 ~g/min (B). Even
`during high rate of Bay k 8644 infusion, in(cid:173)
`creases in BF induced by lower doses of dil(cid:173)
`tiazem were slightly suppressed, while de(cid:173)
`creases in DT were unaffected.
`
`J Cardi<ll'll,\T Phamwco/, Vol. /0, No. 6. /'JX7
`
`CRTXCAR0006878
`
`EKR Therapeutics, LLC Exhibit 2016 Page 6
`
`

`

`VASCULAR SELECTIVITY OF THE DJHYDROPYRJDJNES
`
`633
`
`A
`
`OIL TIAZEM
`
`B
`
`VERAPAMIL
`
`+100
`
`0
`
`~
`0
`Li:
`-g
`0 a; ,
`c:
`"' c:
`~
`" ....
`~
`c.
`0
`~
`" c
`.: ..
`2 u
`
`~
`
`+50
`
`0
`
`-50
`
`z
`
`J
`0
`Li:
`g
`0
`iii
`~
`"' c:
`0 ·;;;
`c: " .... ,
`" c.
`~
`" Q
`..
`.s
`" "' 2 u
`
`0
`
`+50
`
`-50
`
`).
`I 1,.
`T
`
`10
`
`30
`
`100
`
`300
`
`-100
`
`0.3
`
`10
`
`30
`
`100
`
`Dlltlazem (~g)
`FIG. 5. Dose-response curves for the coronary vasodilator and the negative inotropic effects of diltiazem (A) and verapamil (B)
`before and during Bay k 8644 infusion at the rates of 1 and 10 tJ.g/min, obtained from 5 papillary muscle preparations, respec(cid:173)
`tively. All symbols and supplementary lines are the same as those in Fig. 2. The ED50(CVD), the ID50(NIE), and the ratio of the
`ID5JED50 before and during Bay k 8644 infusion at rates of 1 and 10 tJ.g/min are presented in Table 1.
`
`Verapamil (.,g)
`
`IB and C). These observations might suggest that
`the vascular effects of Bay k 8644 are metabolically
`modified secondary to the increased developed ten(cid:173)
`sion. A similar observation was reported by Wada
`et al. (35) at the case of bolus intraarterial injections
`of Bay k 8644. In their observations, the time
`course of the coronary vasoconstrictor effect was
`shorter than that of the positive inotropic effect.
`but not significantly different from those of the pos(cid:173)
`itive chronotropic and dromotropic effects. Thus,
`they concluded that a relatively short duration of
`action on coronary vasculature may be not due to
`metabolic compensation. However, since a quick
`return of the vascular response was marked in ven(cid:173)
`tricular muscle preparation, where energy demand
`is large, metabolic modification of the vascular ef(cid:173)
`fects of Bay k 8644 could he possible. In any case,
`the steady levels of coronary blood flow after re(cid:173)
`turning were also dependent on the infusion rates in
`the present experiments. Thus, it seems to be com(cid:173)
`patible to compare the effects of Ca antagonists
`during infusion of Bay k 8644. In this model. how(cid:173)
`ever, it is very difficult to analyze the precise antag(cid:173)
`onism with the Schild plot, competitive or not,
`since it is hard to obtain four stable dose-response
`curves at control and in the presence of three dif(cid:173)
`ferent concentrations of the antagonists. It is also
`difficult to obtain the maximum response of coro(cid:173)
`nary vasodilation to the intraarterially injected
`drugs, particularly during infusion of Bay k 8644, as
`mentioned in the Methods section. Thus, in the
`present results, we chose the doses for producing a
`
`50% increase in blood flow and a 507c decrease in
`developed tension. by which the potency and then
`the vascular selectivity of the drugs were com(cid:173)
`pared.
`Nevertheless, it was clearly demonstrated that
`Bay k 8644 inhibited the coronary vasodilator ef(cid:173)
`fects of the dihydropyridine Ca antagonists (nifed(cid:173)
`ipine, nitrendipine. and nicardipine) more than
`those of diltiazem and verapamil. and also more
`than the negative inotropic effects induced by ni(cid:173)
`fedipine and nitrendipine. The negative inotropic
`effects of nicardipine. diltiazem, and verapamil
`were not significantly affected by Bay k 8644. As
`shown in Table I, the dihydropyridine Ca antago(cid:173)
`nists had higher vascular selectivity [i.e., larger
`ratio of ID50(NIE)/ED50(CVD)] in the order nitren(cid:173)
`dipine > nifedipine > nicardipine. than those of dil(cid:173)
`tiazem and verapamil: however, this ratio became
`small and almost similar among all five Ca antago(cid:173)
`nists during infusion of Bay k 8644 independent of
`the infusion rate. These results arc very suitable for
`the concept that there is a specific Ca channel for
`the dihydropyridine Ca antagonists in vascular
`smooth muscles. High affinity dihydropyridine
`binding sites in vascular smooth muscles may be
`related to this specific Ca channel (6-8). Thus, the
`specific Ca channel for the dihydropyridines in vas(cid:173)
`cular smooth muscles was specifically blocked by
`nifedipine, nitredipine, and nicardipine (resulting in
`highly selective vasodilation), while it was allosteri(cid:173)
`cally blocked by verapamil and diltiazem (31 ,32)
`(resulting in relatively selective vasodilation), both
`
`1 Cardiol'li.\C Plwrmacol, Vol. 10. No. 6. 1987
`
`CRTXCAR0006879
`
`EKR Therapeutics, LLC Exhibit 2016 Page 7
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`

`

`634
`
`S. MOTOMURA ET AL.
`
`of which were readily antagonized by Bay k 8644,
`although the latter is less selective. On the other
`hand, the high affinity binding sites in cardiac
`muscles do not contribute to cardiac functions
`(7,33). but a second type of dihydropyridine
`binding sites with low affinity in cardiac muscle
`plays a role for the negative inotropic effects of the
`Ca antagonists (7,13,14). Thus, the negative ino(cid:173)
`tropic effects of the dihydropyridine Ca antagonists
`were less sensitively antagonized by Bay k 8644.
`However, since the negative inotropic effect of ni(cid:173)
`cardipine was not affected by Bay k 8644, it is prob(cid:173)
`able that the second type of dihydropyridine
`binding sites might not be specific for the dihydro(cid:173)
`pyridine, or that the site of interaction of Bay k
`8644 with nifedipine and nitrendipine might be dif(cid:173)
`ferent from that with nicardipine. In any case, as a
`result, vascular selectivity of the dihydropyridine
`Ca antagonists was removed by infusions of Bay k
`8644. then similar selectivity was observed among
`all Ca antagonists used during infusion of Bay k
`8644.
`It is of interest that the negative inotropic effect
`of nicardipine was scarcely affected by Bay k 8644,
`although the coronary vasodilator effect was
`readily antagonized. Recently, Sanguinetti and
`Kass (27) reported that in cardiac cells the Ca
`channel blocking activity of nicardipine was inter(cid:173)
`mediate between nitrendipine and verapamil, de(cid:173)
`pending on the hydrophilicity (i.e .. the degree of
`ionization, which is determined by the pK 3 value).
`Nicardipine has the pKa value of 7.0 and is about
`28% ionized at physiological pH 7 .4, while nifed(cid:173)
`ipine and nitrendipine (pKa < 3.5) exist almost en(cid:173)
`tirely