Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Preterm labor still remains one of the most serious problems in obstetric practice. For preventing or treating premature labor, 2-adrenoceptor (AR) stimulants, such as ritodrine and terbutaline, that inhibit contractions of uterine smooth muscle by stimulating the production of cytosolic cAMP via 2-AR, have been used extensively. However, the 2-AR agonists used at present are not particularly selective for uterine smooth muscle, and side effects on the cardiovascular and metabolic systems are frequent.

In recent years, the presence of 3-AR, an additional -AR subtype, has been confirmed in various human tissues, including smooth muscle such as colon (Ponti et al., 1996), ureter (Park et al., 2000), and urinary bladder (Igawa et al., 1999). Moreover, a number of other investigators have reported that 1-AR preferentially exists in human and various rodent hearts (Juberg et al., 1985; Jones et al., 1989). Although it is not certain whether 1- or 3-AR is present and has functional roles in uterine smooth muscle cells, in human uterine smooth muscle 2-AR is the most abundant subtype in the later stages of gestation (Hayashida et al., 1982; Legrand et al., 1987).

In the present study, we characterized the pharmacological profile of a putative -AR agonist, KUR-1246 (Fig. 1), paying special attention to its 2-AR selectivity over 3-AR, as well as over 1-AR. We evaluated both the efficacy and selectivity of this compound in the pregnant rat and rabbit. In addition, its 2-AR selectivity was confirmed in a receptor-binding assay using membrane preparations obtained from Sf9 cells expressing human 1- or 2-AR, and from SK-N-MC neuroblastoma cells expressing human 3-AR.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Chemical structure of KUR-1246, ()-bis(2-{[(2S)-2-({(2R)-2-hydroxy-2-[4-hydroxy-3-(2-hydroxyethyl)phenyl]ethyl}amino)-1,2,3,4-tetrahydronaphthalen-7-yl]oxy}-N,N-dimethyl-acetamide) monosulfate.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animal and Animal Care

Albino rats of the Sprague-Dawley strain (SLC Co., Ltd., Hamamatsu, Japan) and rabbits of the New Zealand White strain (Kitayama Labes Co., Ltd., Ina, Japan) were used. They were housed in a constant-temperature room with a 12-h light/dark cycle. Virgin female rats were placed in separate cages with one male each and left overnight. Pregnancy was dated by taking the morning sperm-plug detection as day 0 of gestation. In the case of rabbits, the day of mating was taken as day 0 of gestation. The experiments in this study were conducted in accordance with institutional guidelines.

Drugs

The following drugs were obtained from commercial sources: ICI-118551 ((±)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl) oxy]-3-[(1-methylethyl) amino]-2-butanol hydrochloride) (Funakoshi, Tokyo, Japan), terbutaline hemisulfate and atenolol (Sigma, St. Louis, MO), ()-isoproterenol hydrochloride (Nikken-Chemical, Tokyo, Japan), [³H]CGP-12177 (()-[5,7-³H]-[4-[3[(1,1-dimethylethyl) amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one]) and [¹²⁵I]iodocyanopindolol (PerkinElmer Life Science Products, Boston, MA), dl-propranolol hydrochloride (Nacalei tesque, Kyoto, Japan), oxytocin (Teikoku-zoki, Tokyo, Japan), and prostaglandin F₂ (PGF₂; Pharmacia-Upjohn, Tokyo, Japan).

KUR-1246 ()-bis(2-{[(2S)-2-({(2R)-2-hydroxy-2-[4-hydroxy-3-(2-hydroxyethyl)phenyl]ethyl} amino)-1,2,3,4-tetrahydronaphthalen-7-yl]oxy}-N,N-dimethyl-acetamide)monosulfate, ritodrine hydrochloride, (±)-bupranolol hydrochloride, and CGP-20712A (2-hydroxy-5(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl)1H-imidazole-2-yl) phenoxy)propyl)amino)ethoxy)benzamide monomethane sulfonate) were all synthesized in our laboratory.

Each drug was dissolved in buffer solutions (in vitro experiments) or physiological saline (in vivo experiments) just before the experiment.

In Vitro Functional Study

Effects of -AR Agonists on Spontaneous or Drug-Induced Myometrial Contractions in Pregnant Rats and Rabbits. Rats. The experiments were carried out by the method of Kawarabayashi et al. (1996). Myometrial contractions were quantified as the sum of the amplitudes of all the contractions recorded over a 5-min period, and the percentage change (after versus before drug application) was calculated. Drug potency was expressed as the pD₂ value, which is the negative logarithm of the EC₅₀ value (the 50% effective concentration of the -AR agonist).

Rabbits. The experiments were performed using essentially the same method as that described above for the evaluation of spontaneous contractions in rats. Uterine tissues were obtained from rabbits on pregnancy day 29. Modified Krebs' solution (same as Krebs' solution contents except for KCl: 10.0 mM) and Krebs' solution were used in the experiments carried out to evaluate the effects on spontaneous myometrial contractions and oxytocin (10 mU/ml)-induced myometrial contractions, respectively. Myometrial contractions were quantified as the sum of the amplitudes of the contractions recorded over a 10-min period.

Effects of -AR Agonists on Isolated Atria and Proximal Colon from Pregnant Rats. The chronotropic effects of KUR-1246 and other -AR agonists were determined using atria isolated from pregnant rats on gestational day 21 by the method of Mattsson et al. (1982). Each drug was applied cumulatively to the bath solution, with the drug being added only when the chronotropic response had reached maximum at the previous concentration. Intrinsic activity was calculated as the ratio between the maximal increment in heart rate for a given drug and isoproterenol.

7

7

Receptor Binding Study

-AR binding assays were carried out using membrane preparations obtained from Sf9 cells expressing human cloned 1- or 2-AR (PerkinElmer Life Science Products) or from SK-N-MC neuroblastoma cells expressing human nonrecombinant 3-AR (Receptor Biology, Inc., Manor Road, VA).

In the 1- and 2-AR binding experiments, the membrane preparation was suspended at protein concentration of 40 µg/ml in incubation buffer (75 mM Tris-HCl, 12.5 mM MgCl₂, and 2 mM EDTA; pH 7.4). In a test tube, 500 µl of incubation buffer containing the membrane preparation were incubated for 60 min at 27°C with 20 µl [³H]CGP-12177 (0.4 nM) and 20 µl of incubation buffer (for total binding), KUR-1246 (0.1 nM-100 µM) or dl-propranolol hydrochloride (for nonspecific binding; 1 µM). Then, the medium was filtered (GF/C filter; Whatman, Maidstone, UK) and washed three times with 1 ml of ice-cold Tris-HCl buffer (75 mM; pH 7.4).

In the 3-AR binding experiment, the membrane preparation was suspended at a protein concentration of 7.5 µg/ml in incubation buffer (50 mM HEPES, 4.0 mM MgCl₂, and 0.04% bovine serum albumin; pH 7.5). In a test tube, 500 µl of incubation buffer containing the membrane preparation were incubated for 90 min at 37°C with 100 µl of [¹²⁵I]iodocyanopindolol (0.93 nM), 100 µl of atenolol (selective 1-AR antagonist; 1 µM), 100 µl of ICI-118551 (selective 2-AR antagonist; 0.1 µM), and 200 µl of incubation buffer (for total binding), KUR-1246 (0.3 µM-100 µM) or bupranolol (for nonspecific binding; 0.1 mM). Then, the medium was filtered (GF/C filter; Whatman) and washed three times with 1 ml of ice-cold Tris-HCl buffer (50 mM, pH 7.4). The radioactivity trapped on the filter was measured using a liquid scintillation counter (Packard 1900C; Downers Grove, IL) or gamma counter (Packard COBRA). The displacement of [³H]CGP-12177 or [¹²⁵I]iodocyanopindolol binding by KUR-1246 was calculated. The inhibition constant (K_i value) for KUR-1246 was obtained by fitting the data to the following equation:
where IC₅₀ is the 50% inhibitory molar concentration of the drug tested, [L] is the molar concentration of [³H]CGP-12177 or [¹²⁵I]iodocyanopindolol present in the tube, and K_d is the dissociation constant of [³H]CGP-12177 or [¹²⁵I]iodocyanopindolol (obtained from Scatchard plot analysis). The pK_i is the negative logarithm of K_i. The 2-AR selectivity of KUR-1246 ([IC₅₀ for 1-AR]/[IC₅₀ for 2-AR] or [IC₅₀ for 3-AR]/[IC₅₀ for 2-AR]) was calculated from three separate experiments.

In Vivo Functional Study

The experiments were carried out by the method of Kawarabayashi et al. (1996). Spontaneous uterine activity over 15-min periods was calculated by measuring the area under the intrauterine pressure curve using an integrator (NEC San-ei; 1322), and the percentage change (after versus before drug administration) was calculated. Another cannula was inserted into the carotid artery for the measurement of blood pressure and heart rate. Each drug was administered by a sequential intravenous infusion, with the dose increased every 15 min.

Statistics

The results were expressed as the mean ± S.E.M. A one-way analysis of variance was used for the statistical analysis of multiple comparisons within each group. When a significant difference was detected by one-way analysis of variance, data were further analyzed with Tukey-Kramer's test, and a P value less than 0.05 was considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Inhibitory Effects of KUR-1246 on Myometrial Contractions in Vitro

Spontaneous and Drug-Induced Contractions of the Pregnant Rat Myometrium. Table 1 summarizes the inhibitory effects of KUR-1246 and other -AR agonists on spontaneous and drug-evoked myometrial contractions in the pregnant rat. All the drugs tested concentration-dependently inhibited spontaneous myometrial contractions (Fig. 2). The pD₂ value for KUR-1246 was similar to that of isoproterenol. The potency of KUR-1246 was about 20 times that of terbutaline and 50 times that of ritodrine.

View this table: [in this window] [in a new window]   TABLE 1 pD2 values for the inhibitory effects of KUR-1246 and other 2-adrenoceptor agonists on contractions of myometrium isolated from pregnant rats Values are mean ± S.E.M. of 10 to 15 separate experiments. Numbers in parenthesis indicate potency of each drug relative to that of KUR-1246.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of KUR-1246 and other -adrenoceptor agonists on spontaneous myometrial contractions in tissues obtained from pregnant rats on gestational day 21. Concentration-response curves for KUR-1246 (), isoproterenol (), ritodrine (), and terbutaline (). Data represent the mean ± S.E.M. of 10 experiments.

2

2

Spontaneous Contractions of the Pregnant Rabbit Myometrium. The inhibitory effects of KUR-1246 and other -AR agonists were also evaluated in myometrial strips isolated from the pregnant rabbit. All the drugs tested elicited concentration-dependent inhibitions of both the spontaneous and oxytocin-induced contractions within the same concentration range for a given drug as observed in pregnant rat myometrium. The potency of KUR-1246 was 33 to 50 times that of ritodrine and 16 to 33 times that of terbutaline, and it was almost equal to that of isoproterenol (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 pD2 values for the inhibitory effects of KUR-1246 and other 2-adrenoceptor agonists on contractions of myometrium isolated from pregnant rabbits Values are mean ± S.E.M. of 8 to 10 separate experiments. Numbers in parenthesis indicate potency of each drug relative to that of KUR-1246.

Determination of the -AR Subtype Mediating the Inhibitory Effect of KUR-1246 on Myometrial Contractions, Using Selective -AR Antagonists. Neither the selective 1-AR antagonist, CGP-20712A (10⁹ to 10⁸ M), nor the 3-AR antagonist, SR-58894A (3 × 10⁹ to 3 × 10⁸ M), modified the concentration-response curves for the inhibitory effect of KUR-1246 on spontaneous contractions in myometrium isolated from pregnant rats (data not shown). In contrast, ICI-118551 (3 × 10⁹ to 3 × 10⁸ M), the selective 2-AR antagonist, produced a parallel rightward shift in this concentration-response curve for KUR-1246 (Fig. 3A). The slope and pA₂ values obtained from the Schild plot analysis were 1.094 and 8.97, respectively, indicating a competitive antagonism by ICI-118551 against the KUR-1246-induced response (Fig. 3B).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Antagonism by ICI-118551 (A) of KUR-1246-induced inhibition of spontaneous contractions in myometrial strips isolated from pregnant rats on gestational day 21. ICI-118551 was added 15 min before KUR-1246 treatment. Data represent the mean ± S.E.M. of 11 to 15 experiments. (B) Corresponding Schild plot to (A).

Selectivity of KUR-1246 for 2-AR in Vitro

Functional Study Using Atria and Colon Isolated from Pregnant Rats. All drugs increased the heart rate in a concentration-dependent manner. Isoproterenol was the strongest stimulant of heart rate, with its potency being about 570, 1030, and 1630 times that of ritodrine, KUR-1246, and terbutaline, respectively. With regard to the maximal positive chronotropic effects produced by these drugs, KUR-1246 produced only a 15.8% increase in rate over the basal level, which was less than that of isoproterenol (81.4%), terbutaline (81.8%), and ritodrine (54.6%). The intrinsic activity of each drug, relative to that of isoproterenol, was 0.20 for KUR-1246, 0.69 for ritodrine, and 0.96 for terbutaline (Table 3).

View this table: [in this window] [in a new window]   TABLE 3 Selectivity of KUR-1246 and other -adrenoceptor agonists for myometrium in pregnant rats Numbers in parenthesis indicate the intrinsic activity of each drug relative to that of isoproterenol.

Receptor-Binding Study Using Human -AR. Scatchard analysis of the receptor-binding data provided K_d values of 0.4 and 0.16 nM for [³H]CGP12177 binding to 1- and 2-ARs, respectively (data not shown). The K_d value obtained for [¹²⁵I]iodocyanopindolol binding to the 3-AR was 1.05 nM. KUR-1246 displaced radioligand binding to 1-, 2-, and 3-ARs, each in a concentration-dependent manner, with pK_i values of 5.75 ± 0.03, 7.59 ± 0.08, and 4.75 ± 0.03, respectively. The affinity of KUR-1246 for the human 2-AR was 39.2 times that for the 1-AR and 198.2 times that for the 3-AR (Table 4).

View this table: [in this window] [in a new window]   TABLE 4 Inhibition constant (pKi) and 2-adrenoceptor selectivity of KUR-1246 for the displacement of [3H]CGP12177 or [125I]iodocyanopindolol-binding to membrane preparations expressing human 1-, 2-, and 3-adrenoceptors The pKi values are the mean ± S.E.M. of three separate experiments.

Effectiveness and Selectivity of KUR-1246 in Pregnant Rats in Vivo: Comparative Evaluation of Effects on Uterine Motility and the Cardiovascular System. Figure 4 shows a representative recording of the effect of an intravenous infusion of KUR-1246 (0.1-10.0 µg/kg/min) on spontaneous oscillation of the intrauterine pressure, heart rate, and blood pressure in the pregnant rat. KUR-1246, reduced the frequency of the spontaneous oscillation of the intrauterine pressure in a dose-dependent manner, without any detectable change in blood pressure and heart rate. The basal tone of the intrauterine pressure was also inhibited by administration of high doses of KUR-1246 (3-10 µg/kg/min). As shown in Fig. 5A, ritodrine and terbutaline as well as KUR-1246, reduced the spontaneous uterine activity with ED₃₀ (30% effective dose) values of 51.29 (ritodrine), 0.76 (terbutaline), and 0.13 (KUR-1246) µg/kg/min, respectively. The inhibitory potency of KUR-1246 for this effect was about 6 times that of terbutaline, and 400 times that of ritodrine. Each of the drugs tested caused a dose-dependent increase in heart rate; however, the positive chronotropic effect of KUR-1246 was weaker than that of terbutaline and ritodrine (Fig. 5B). The maximal increase in heart rate was only 20 beats per min, even at the highest dose of KUR-1246 (10 µg/kg/min), whereas terbutaline and ritodrine produced maximal increases of about 50 to 55 beats per min over the basal level. KUR-1246, terbutaline, and ritodrine all decreased mean blood pressure (Fig. 5C). The KUR-1246-induced decrease in blood pressure was similar to that produced by terbutaline and ritodrine.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Representative recording of the effects of KUR-1246 on intrauterine pressure, blood pressure, and heart rate in an anesthetized pregnant rat on gestational day 21.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effects of KUR-1246 (), terbutaline (), and ritodrine () on spontaneous uterine activity (A), heart rate (B), and change in mean blood pressure (C) in anesthetized pregnant rats on gestational day 21. Each drug was administered by sequential intravenous infusion, the dose being increased every 15 min. The ED30 (30% effective dose) values were: KUR-1246, 0.13 µg/kg/min; terbutaline, 0.76 µg/kg/min; and ritodrine, 51.29 µg/kg/min. Values are mean ± S.E.M. of 10 experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study was designed to evaluate the efficacy and 2-AR selectivity of KUR-1246, a new uterine relaxant, and to compare this drug to other 2-AR agonists. The results obtained indicated that KUR-1246 is a highly selective 2-AR agonist for relaxing uterine muscle. In addition, we have previously found by using receptor-binding assays that, KUR-1246 did not bind to other receptors, except for -ARs, including -adrenergic, histamine, muscarine, and dopamine receptors (data not shown).

It is well known that rodent studies estimating the efficacy of 2-AR agonists have been good predictors of the clinical utility of these drugs. In our experiments, we compared the inhibitory effects of KUR-1246 and other -AR agonists on spontaneous or drug-induced contractions of myometrium isolated from pregnant rats and rabbits. KUR-1246 inhibited both types of myometrial contraction in both species, and it was the most potent myometrial relaxant among the -AR agonists clinically used as tocolytics. However, all the -AR agonists tested exhibited a significantly lower inhibitory potency (one-tenth or less) for PGF₂-induced myometrial contractions than for oxytocin- and KCl-induced contractions. A number of investigators have reported that oxytocin and PGF₂ contribute to myometrial contractions in preterm labor (Romero et al., 1989; Kobayashi et al., 1999). An increase in the intracellular Ca²⁺ concentration plays an essential role in the generation of smooth muscle contractions. An influx of extracellular Ca²⁺ into the intracellular space via voltage-dependent Ca²⁺ channels and the subsequent release of Ca²⁺ from intracellular sites may be both involved in this process. Activation of voltage-dependent Ca²⁺ channels is thought to be mainly responsible for the generation of both spontaneous and KCl-induced myometrial contractions, whereas release of Ca²⁺ from intracellular sites is also involved in the increase in the intracellular Ca²⁺ concentration underlying oxytocin-induced contractions (Kawarabayashi et al., 1997). In contrast, PGF₂ produces myometrial contractions primarily by stimulating intracellular sites to release Ca²⁺ (Davis et al., 1987; Yang et al., 1997). It is well established that -AR agonists act by hyperpolarizing the myometrial plasma membrane, thus interfering with the influx of extracellular Ca²⁺ (Huszar and Walsh, 1989). On this basis, it seems most likely that the lower potency shown by the -AR agonists against PGF₂-induced contractions might reflect differences in the site of action between -AR agonists and PGF₂. KUR-1246 and the other -AR agonists all completely inhibited PGF₂-induced contractions when their concentration was increased further. Since -AR agonists, at high concentrations, also facilitate incorporation of intracellular Ca²⁺ into storage sites, it may be possible to use KUR-1246 effectively against PGF₂-related myometrial contractions if it indeed has a higher selectivity for the uterus than for other tissues.

Therefore, we analyzed the mode of action of KUR-1246 by using specific -AR antagonists. Neither CGP-20712A nor SR-58894A, at concentrations that occupy virtually all 1- or 3-AR (Kaumann, 1986; Manara et al., 1995), antagonized the relaxing effect of KUR-1246 on the pregnant rat myometrium. On the other hand, ICI-118551, a selective 2-AR antagonist, produced an effective competitive antagonism against the KUR-1246-induced inhibition of myometrial contractions. The pA₂ value and the slope of the Schild plot for ICI-118551 are quite similar to those reported by Bilski et al. (1983). These data clearly demonstrate that KUR-1246 produced its inhibitory effect on myometrial contractions by stimulating the 2-AR subtype. With regard to the 3-AR, there are as yet no reports in the literature supporting a possible functional role in the myometrium, although many species, including the rat and human, have been examined. Our experiments provided no evidence in favor of a functional 3-AR in the myometrium in the pregnant rat.

We examined the 2-AR selectivity of KUR-1246 and other -AR agonists in two sets of experiments: namely, functional and receptor-binding experiments. In the functional experiment, using myometrium, atria, and proximal colon, isolated from the pregnant rat, our main aim was to compare the inhibitory effect of KUR-1246 on the myometrium with its effects on the other tissues. All the drugs tested increased the heart rate (1-AR-mediated response), but the maximal positive chronotropic effect of KUR-1246 was definitely much lower than that of the other three drugs, indicating that KUR-1246 acted on the rat atria only as a partial agonist. This is one of the most important pharmacological characteristics of KUR-1246 that suggest that it has potential as a clinically beneficial drug with few side effects on the heart. Additional studies will be needed to clarify the behavior of KUR-1246 as a partial agonist on 1-AR. KUR-1246 also inhibited spontaneous contractions of the isolated rat colon in the presence of both specific 1-AR- and 2-AR antagonists. But its potency was very low, compared with that of isoproterenol (one-thousandth). Since 3-AR mediates such responses as thermogenesis (Goldberg and Frishman, 1995), lipolysis (Umekawa et al., 1999), and relaxation of the colon (Ponti et al., 1996) in humans, this low potency should be another benefit of KUR-1246 in the treatment of pregnant women. This functional experiment clearly demonstrated that KUR-1246 has excellent selectivity for the myometrial 2-AR over both 1-AR (1630 times) and 3-AR (540 times).

In the receptor-binding assay, we used membrane preparations obtained from cells expressing each of the cloned human -AR subtypes. The affinity of KUR-1246 for the human 2-AR was higher than that for 1-AR (39.2 times) and 3-AR (198.2 times). However, the selectivity ratios were somewhat lower in the binding assay than in the functional assay, possibly a reflection of the different methods and/or species used. Indeed, other researchers have reported that a given compound does not necessarily produce an identical result in a binding assay and a functional assay (Maguire et al., 1976; Minneman et al., 1981; Ohashi et al., 1996).

In the last in vivo experiment, we evaluated both the efficacy and selectivity of KUR-1246 to try to confirm the results obtained in the functional in vitro experiment, and we made comparisons with ritodrine and terbutaline. KUR-1246 strongly suppressed spontaneous uterine contractions without causing severe cardiovascular side effects, such as tachycardia and hypotension. These results were fully in accord with those obtained in the in vitro experiments. Thus, we confirmed the potent and selective inhibitory effects of KUR-1246 on uterine contractions in the pregnant rat in vivo.

In conclusion, we have clearly demonstrated in the present study that KUR-1246 is a potent and highly selective 2-AR agonist. On the basis of the evidence available so far, it shows great potential as a drug for the treatment of preterm labor in humans.