Introduction

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₁-Adrenoceptors participate in the regulation of physiological responses in numerous tissues, and these receptors are clinically important targets for therapeutic manipulation as well as a possible site of pathological etiology. Benign prostatic hyperplasia (BPH) is a condition that causes micturition problems in a relatively high proportion of elderly men. A number of previous studies have demonstrated the functional importance of ₁-adrenoceptors in the human prostate, the "target tissue" in BPH. Clinical studies have demonstrated the efficacy of ₁-adrenoceptor antagonists such as prazosin in ameliorating bladder outlet obstruction in patients with BPH and, thus, novel ₁-adrenoceptor antagonists that exhibit high selectivity to ₁-adrenoceptors in the prostate have been screened as potentially effective therapeutic agents for bladder outlet obstruction in BPH (Hedlund et al., 1983; Shibata et al., 1995; Testa et al., 1995; Ford et al., 1996; Muramatsu et al., 1996; Wilde and McTavish, 1996). Among these agents, tamsulosin has been shown to improve urinary obstruction in patients with BPH with less incidence of orthostatic hypotension (Kawabe et al., 1990; Chapple, 1996; Wilde and McTavish, 1996).

Currently, ₁-adrenoceptors are classified into several subtypes (Hieble et al., 1995; Michel et al., 1995; Muramatsu et al., 1995). KMD-3213 is a highly selective antagonist of the _1A-adrenoceptor subtype having prostatic selectivity (Shibata et al., 1995; Yamagishi et al., 1996; Moriyama et al., 1997; Akiyama et al., 1999; Murata et al., 1999) and it is now being developed as a therapeutic agent to treat urinary outlet obstruction in patients with BPH. Although relatively high selectivity of KMD-3213 to the _1A-adrenoceptor subtype, compared with the _1B- and _1D-adrenoceptor, has been demonstrated by in vitro radioligand binding studies in recombinant ₁-adrenoceptor subtypes and native tissue membrane preparations, these data may not necessarily reflect the tissue selectivity or subtype selectivity in vivo because various pharmacokinetic and pharmacodynamic factors are not taken into account under in vitro condition. Recent studies by ourselves and others emphasize the necessity for characterizing drug-receptor binding under physiological conditions to obtain more practical information for the evaluation of novel drugs (Beauchamp et al., 1995; Yamada et al., 1998, 1999). In vivo measurement of ₁-adrenoceptor binding makes it possible to evaluate simultaneously receptor binding properties of ₁-adrenoceptor antagonists in a variety of organs with different proportions of ₁-adrenoceptor subtypes (Yamada et al., 1999). Therefore, the aim of present study was to characterize the in vivo ₁-adrenoceptor binding specificities of KMD-3213 in rat tissues by using tritiated ligand with high specific activity under physiological conditions.

	Experimental Procedures

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Materials. [³H]KMD-3213 (1.92 TBq/mmol) was synthesized by Amersham International plc (Buckinghamshire, England) and kindly provided by Kissei Pharm. Co. Ltd. (Matsumoto, Japan). [³H]Prazosin (2.75 TBq/mmol) was purchased from New England Nuclear (Boston, MA). The following drugs were kindly donated by the companies indicated: prazosin hydrochloride, Pfizer Pharm. Co. Ltd. (Tokyo, Japan); KMD-3213, Kissei Pharm. Co. Ltd.; tamsulosin hydrochloride, Yamanouchi Pharm. Co. Ltd. (Tokyo, Japan); and terazosin hydrochloride, Mitsubishi Chemical Industries (Tokyo, Japan). Phentolamine hydrochloride, yohimbine hydrochloride, and nifedipine hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). All other drugs and materials were obtained from commercial sources.

Animals. Male Sprague-Dawley rats (Japan SLC Inc., Shizuoka, Japan) weighing approximately 200 g were used. Rats were housed with a 12-h light/dark cycle and fed laboratory food and water ad libitum.

In Vitro Binding of [³H]KMD-3213 and [³H]Prazosin. Rat tissues (prostate, submaxillary gland, and spleen) were minced with scissors and homogenized in a Kinematica Polytron homogenizer in 20 to 80 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.5). The homogenates were centrifuged at 40,000g for 20 min. The pellet was resuspended in the ice-cold buffer, and the suspension was centrifuged again at 40,000g for 20 min. The resulting pellet was resuspended in the buffer for the binding assay. All steps were performed at 4°C. The tissue homogenates (5-10 mg of wet weight tissue) were incubated with [³H]KMD-3213 (0.01-1.5 nM) or [³H]prazosin (0.01-1.0 nM), in 50 mM Tris-HCl buffer (pH 7.5). Incubation was carried out for 60 min (30 min in the case of the [³H]prazosin assay) at 25°C. The reaction was terminated by rapid filtration (Cell Harvester; Brandel, Gaithersburg, MD) through Whatman GF/B glass fiber filters, and the filters were rinsed three times with 3 ml of ice-cold buffer. The tissue-bound radioactivity was extracted from the filters by placing them overnight in scintillation fluid {2 liters of toluene, 1 liter of Triton X-100, 15 g of 2,5-diphenyloxazole, and 0.3 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene} and the radioactivity was determined in a liquid scintillation counter. The specific binding of [³H]KMD-3213 and [³H]prazosin was determined experimentally from the difference between counts in the absence and presence of 10 µM phentolamine. All assays were conducted in duplicate. Protein concentrations were measured by the method of Lowry et al. (1951).

In Vivo Binding of [³H]KMD-3213 and [³H]Prazosin. The in vivo measurement of the specific binding of [³H]KMD-3213 and [³H]prazosin in rat tissues was performed as described for the in vivo measurement by [³H]tamsulosin in rat tissues (Yamada et al., 1999). Rats were anesthetized with diethyl ether, and [³H]KMD-3213 (1.4 nmol/kg, 555 kBq in 150 µl of saline) or [³H]prazosin (1.2 nmol/kg, 555 kBq in 150 µl of saline) was injected into the femoral vein. The animals were allowed to recover, and then they were sacrificed by taking blood from the descending aorta under temporary anesthesia with diethyl ether 10 min after the injection to minimize metabolism of the radioligand. In the experiment examining the time course of specific binding of each radioligand, rats were sacrificed at 10, 60, 120, and 240 min (10, 60, and 120 min in the case of [³H]prazosin assay). A blood sample was taken from the descending aorta, and tissues (prostate, vas deferens, aorta, cerebral cortex, spleen, liver, submaxillary gland, heart, lung, and kidney) were rapidly removed. After dissection on ice, each tissue was homogenized in ice-cold 50 mM Tris-HCl buffer to give a final tissue concentration of 10 mg/ml using a Kinematica Polytron homogenizer. Particulate-bound radioactivity was determined by rapid filtration of 1 to 3 ml of homogenate over Whatman GF/C filters, which were washed subsequently with 2 ml of ice-cold buffer. The particulate-bound radioactivity was measured in a liquid scintillation counter after the addition of scintillation fluids {2 liters of toluene, 1 liter of Triton X-100, 15 g of 2,5-diphenyloxazole, and 0.3 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene}. In this case, the particulate-bound radioactivity of [³H]KMD-3213 was determined in each tissue after administration of phentolamine (3.14, 31.4, and 62.9 µmol/kg i.p., 0.5-h pretreatment) or nifedipine (28.9 µmol/kg p.o., 1-h pretreatment) to rats. Based on the data on pharmacological specificity, the particulate-bound radioactivity from vehicle- and phentolamine (62.9 µmol/kg i.p.)-pretreated rats was defined as total binding and nonspecific binding, respectively, and the difference was taken as the in vivo specific binding of [³H]KMD-3213 or [³H]prazosin. In a preliminary experiment, it was shown that there was no significant difference in the amount of in vivo specific binding of these radioligands between one and two washouts with 2 ml of ice-cold buffer of the Whatman GF/C filters after the filtration of tissue homogenates. Thus, we considered that the nonspecifically bound radioactivity was almost completely removed by one washout with 2 ml of buffer under the present assay conditions. The data were expressed as femtomoles per milligram of tissue (wet weight). Plasma was separated from the blood of rats receiving [³H]KMD-3213 or [³H]prazosin and frozen at 20°C until needed to determine the plasma concentration of these radioligands.

Determination of [³H]KMD-3213 and [³H]Prazosin. Determination of the concentration of [³H]KMD-3213 and [³H]prazosin in plasma using HPLC was performed as described previously for the measurement of [³H]tamsulosin and [³H]prazosin (Yamada et al., 1999). Methanol (1.1-2.3 ml) was added to the plasma (0.3-0.6 ml) from rats receiving [³H]KMD-3213 (1.4 nmol/kg, 555 kBq) and acetonitrile (0.4 ml) was added to the plasma (0.05-0.4 ml) from rats receiving [³H]prazosin (1.2 nmol/kg, 555 kBq). After being stirred, the mixtures were centrifuged at 8500g for 5 min. The supernatant was dried by centrifugation and the residue was dissolved in 100 µl of mobile phase and 50 µl of this solution was injected into the HPLC system. The HPLC system consisted of a pump (880-PU; Jasco, Tokyo, Japan) and a stainless steel column packed with STR ODSII (250 × 4.6 mm, i.d.) for the determination of [³H]KMD-3213 or a stainless steel column packed with nucleosil 5C₁₈ (Marchery-Nagel, 150 × 4.0 mm, i.d.) for the determination of [³H]prazosin. The mobile phase for the determination of [³H]KMD-3213 was 0.05 M phosphate buffer (pH 6.5) and propanol (3:1, v/v) at a flow rate of 1.0 ml/min. The mobile phase for [³H]prazosin was 0.05 M phosphate buffer and acetonitrile (3:7, v/v). The column elute was collected in vials, and the radioactivity was measured in a liquid scintillation counter.

Analysis of Data. The analysis of binding data was performed as described previously (Yamada et al., 1980). The K_d and B_max values for [³H]KMD-3213 and [³H]prazosin were estimated by Rosenthal analysis of the saturation data (Rosenthal, 1967). The ability of ₁-adrenoceptor antagonists to inhibit specific [³H]KMD-3213 binding (0.15 nM) was estimated from the IC₅₀ values, which are the molar concentrations of unlabeled drug necessary to displace 50% of the specific binding of [³H]KMD-3213 (determined by log probit analysis). The inhibition constant, K_i, was calculated from the equation K_i = IC₅₀/(1 + L/K_d), where L is the concentration of [³H]KMD-3213. The Hill coefficients for the inhibition by ₁-adrenoceptor antagonists were obtained by Hill plot analysis. The rate constants were determined from the association and dissociation velocities. The areas under the curve (AUC_0-120) from 0 to 120 min of the in vivo specific binding of [³H]KMD-3213 and [³H]prazosin in each tissue versus time were calculated by the trapezoidal method. The K_d and B_max values for in vivo [³H]KMD-3213 binding were estimated as described previously for in vivo [³H]tamsulosin binding (Yamada et al., 1999). Statistical analysis of the data was performed by Student's t test and by one-way analysis of variance followed by Dunnett's test for single and multiple comparisons, respectively.

	Results

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In Vitro Binding of [³H]KMD-3213 and [³H]Prazosin. The specific [³H]KMD-3213 binding in the rat prostate and submaxillary gland was saturable and reversible. The K_d values for [³H]KMD-3213 in the prostate and submaxillary gland were 49.3 ± 4.8 and 55.0 ± 16.9 pM, respectively, and the B_max values were 17.8 ± 1.2 and 111 ± 16 fmol/mg of protein (mean ± S.E., n = 3), respectively. These binding parameters in both tissues were not significantly different from those for [³H]prazosin (Table 1). On the other hand, the specific [³H]KMD-3213 binding in the spleen was extremely low; thus, K_d and B_max values could not be estimated. This contrasted with the result that there was significant amount of specific [³H]prazosin binding in the rat spleen. The rate constants for association (k₊₁) and dissociation (k₁) of [³H]KMD-3213 in the rat prostate were calculated to be 0.447 ± 0.016/nM/min and 0.008 ± 0.001/min, respectively. The corresponding values for [³H]prazosin were 0.670 ± 0.134/nM/min and 0.030 ± 0.002/min, respectively. The k₊₁ for [³H]KMD-3213 was similar to that for [³H]prazosin, whereas the k₁ was significantly (P < 0.001) lower.

In Vivo Binding of [³H]KMD-3213 and [³H]Prazosin. The particulate-bound radioactivity was measured in rat tissues (prostate, vas deferens, aorta, cerebral cortex, spleen, liver, submaxillary gland, heart, lung, and kidney) 10 min after i.v. injection of [³H]KMD-3213 (1.4 nmol/kg). Pretreatment with phentolamine at doses of 3.15 and 31.5 µmol/kg (i.p.) reduced in a dose-dependent manner (41.3-62.9% and 65.4-86.4%, respectively) the [³H]KMD-3213 binding in particulate fractions of the prostate, submaxillary gland, heart, lung, and kidney, but no further significant reductions, compared with the reduction at 31.5 µmol/kg, were seen at the higher dose (62.9 µmol/kg) of phentolamine (Fig. 1). Therefore, as shown in Fig. 2, the difference in particulate-bound radioactivity of [³H]KMD-3213 in each tissue between vehicle- and phentolamine (62.9 µmol/kg)-pretreated rats could be defined as the in vivo specific binding of the radioligand. Ten minutes after i.v. injection of [³H]KMD-3213 to rats, a significant degree of specific binding occurred in all tissues, except the cerebral cortex, spleen, and liver, which showed a little or no specific binding. The degree of specific [³H]KMD-3213 binding differed markedly among tissues. The rank order of specific [³H]KMD-3213 binding was kidney > heart, lung > submaxillary gland > prostate > aorta. Administration (p.o.) of nifedipine (28.9 µmol/kg), a potent vasodilator, had little inhibitory effect on the particulate-bound radioactivity in rat tissues after i.v. injection of [³H]KMD-3213, except the liver and submaxillary gland, which showed a significant increase (Fig. 2).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of pretreatment with different doses of phentolamine on [3H]KMD-3213 binding in rat tissues (, aorta; , prostate; , kidney; , submaxillary gland; , heart) 10 min after i.v. injection of the ligand. Rats received different doses (3.15-62.9 µmol/kg i.p.) of phentolamine 30 min before i.v. injection of [3H]KMD-3213. The [3H]KMD-3213 (555 kBq, 1.4 nmol/kg) was injected into the femoral vein, and rats were sacrificed at 10 min. [3H]KMD-3213 binding in particulate fraction of each tissue was determined, and expressed as percentage of the control total binding of [3H]KMD-3213 in each tissue from vehicle-pretreated rats. Each point represents the mean ± S.E. of three rats.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effects of pretreatment with phentolamine and nifedipine on the [3H]KMD-3213 binding in rat tissues. Rats received vehicle (control), phentolamine (62.9 µmol/kg i.p.) at 30 min, and nifedipine (28.9 µmol/kg p.o.) at 60 min before i.v. injection of [3H]KMD-3213. [3H]KMD-3213 (555 kBq, 1.4 nmol/kg) was injected into the femoral vein, rats were sacrificed at 10 min, and [3H]KMD-3213 binding in the particulate fractions of each tissue was determined. Each column represents the mean ± S.E. of three rats. Asterisks show a significant difference from control values, *P < 0.05, **P < 0.01, ***P < 0.001.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Time course of plasma free concentration of [3H]KMD-3213 () and [3H]prazosin () after i.v. injection in rats. [3H]KMD-3213 (555 kBq, 1.4 nmol/kg) and [3H]prazosin (555 kBq, 1.2 nmol/kg) were injected into the femoral vein. Blood samples were taken from the femoral artery at 1 to 120 min. Each point represents the mean ± S.E. of three rats.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Time course of in vivo specific binding of [3H]KMD-3213 in rat tissues (A: , vas deferens; , prostate; , aorta; , spleen and B: , kidney; , lung; , heart, , submaxillary gland) after i.v. injection of the ligand. [3H]KMD-3213 (555 kBq, 1.4 nmol/kg) was injected into the femoral vein, and rats were sacrificed 10 to 240 min later. The specific binding of [3H]KMD-3213 was experimentally defined as the difference in binding in particulate fractions of each tissue from vehicle (total binding)- and phentolamine (62.9 µmol/kg i.p.) (nonspecific binding)-pretreated rats. Each point represents the mean ± S.E. of three to six rats.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Time course of in vivo specific binding of [3H]prazosin in rat tissues (A: , liver; , aorta; , submaxillary gland; , vas deferens; , prostate and B: , heart; , kidney; , lung; , spleen) after i.v. injection of the ligand. [3H]prazosin (555 kBq, 1.2 nmol/kg) was injected into the femoral vein, and rats were sacrificed 10 to 120 min later. The specific binding of [3H]prazosin was experimentally defined as the difference in binding in particulate fractions of each tissue from vehicle (total binding)- and phentolamine (62.9 µmol/kg i.p.) (nonspecific binding)-pretreated rats. Each point represents mean ± S.E. of three to four rats.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   In vivo specific binding of [3H]KMD-3213 (A) and [3H]prazosin (B) in the particulate fraction from the rat submaxillary gland (), prostate (), aorta (), and spleen () as a function of increasing dose of the ligand. A mixture of [3H]KMD-3213 (555 kBq, 1.4 nmol/kg) and unlabled KMD-3213 at doses of 1.4 to 13.6 nmol/kg or [3H]prazosin (555 kBq, 1.2 nmol/kg) and unlabeled prazosin at doses of 1.2 to 10.6 nmol/kg was injected into the femoral vein of vehicle (total binding) and phentolamine (62.9 µmol/kg i.p.) (nonspecific binding)-pretreated rats, and the binding in the particulate fraction of each tissue was measured at 10 min. Each point represents the mean ± S.E. of three to six rats.

Competition Studies. A constant amount of [³H]KMD-3213 (1.4 nmol/kg) was coinjected with increasing amounts of prazosin and yohimbine in rats. The i.v. injection of low doses of prazosin (23.9-239 nmol/kg) reduced the in vivo specific [³H]KMD-3213 binding in particulate fractions of the rat prostate, aorta, submaxillary gland, heart, lung, and kidney in a dose-dependent manner (Fig. 7). In contrast to the marked inhibition by prazosin, i.v. injection of a relatively high dose of yohimbine (256 nmol/kg) had little inhibitory effect on the in vivo specific [³H]KMD-3213 binding in rat tissues, including the prostate.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Effects of prazosin and yohimbine on specific [3H]KMD-3213 binding in rat tissues. Prazosin (23.9-239 nmol/kg) and yohimbine (256 nmol/kg) were injected into the femoral vein with [3H]KMD-3213 (555 kBq, 1.4 nmol/kg), and rats were sacrificed at 10 min. Each column represents the mean ± S.E. of three rats. Asterisks show a significant difference from control values, *P < 0.05, **P < 0.01, ***P < 0.001.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The present study was carried out to characterize in vivo ₁-adrenoceptor binding of [³H]KMD-3213 in rat tissues after i.v. injection of the radioligand, compared with that of [³H]prazosin. The in vitro specific binding of [³H]KMD-3213 in the rat prostate and submaxillary gland was saturable and reversible, and their K_d and B_max values were comparable with those for [³H]prazosin binding. Although there was a considerable degree of specific binding of [³H]prazosin (Table 1) and [³H]tamsulosin (Yamada et al., 1999) in the rat spleen, the specific [³H]KMD-3213 binding in this tissue was extremely low. Thus, KMD-3213 was shown to have a higher affinity for ₁-adrenoceptors in the prostate and submaxillary gland than in the spleen in vitro. Tamsulosin, prazosin, KMD-3213, and terazosin were competitive inhibitors of the [³H]KMD-3213 binding sites in rat tissues and, among these tissues, tamsulosin was the most potent.

The particulate-bound radioactivity was measured in homogenates of rat tissues 10 min after i.v. injection of [³H]KMD-3213 (1.4 nmol/kg). Pretreatment with phentolamine at doses of 3.15 and 31.5 µmol/kg caused a dose-dependent reduction in [³H]KMD-3213 binding in particulate fractions of the rat prostate, submaxillary gland, heart, lung, and kidney, but no further reductions were seen at a higher dose (62.9 µmol/kg) of phentolamine. Thus, these tissues exhibited a significant degree of in vivo specific binding of [³H]KMD-3213, which was defined as the difference in particulate-bound radioactivity in each tissue between vehicle- and phentolamine (62.9 µmol/kg i.p.)-pretreated rats. There is a possibility that i.p. administration of a high dose of phentolamine in the measurement of the nonspecific binding of [³H]KMD-3213 may produce vasodilation and thus affect the distribution of [³H]KMD-3213 and the coinjected drugs. Also, the high doses of KMD-3213 and prazosin in the dose-dependence and competition studies could have some cardiovascular effects that might alter the distribution of agents to various tissues. However, pretreatment with nifedipine, a potent vasodilator, at a p.o. dose (28.9 µmol/kg) that causes marked and sustained hypotension in rats (Yamanaka et al., 1991), produced little inhibition of the particulate-bound radioactivity in each rat tissue after i.v. injection of [³H]KMD-3213. Thus, it seems unlikely that hypotension due to the blockade of vascular ₁-adrenoceptors has a significant effect on the tissue distribution of [³H]KMD-3213 and the coinjected drugs, and also on the subsequent ₁-adrenoceptor binding. Although the in vivo specific binding of [³H]KMD-3213 in rat tissues was inhibited by the coinjection of low doses of prazosin with the radioligand in a dose-dependent manner, it was unaffected by the relatively high dose of ₂- adrenoceptor-selective antagonist yohimbine. Furthermore, a dose-dependent specific binding of [³H]KMD-3213 and [³H]prazosin in the rat prostate was seen after i.v. injections of KMD-3213 (1.4-13.6 nmol/kg) and prazosin (1.2-10.6 nmol/kg). Recently, Akiyama et al. (1999) have shown that KMD-3213 and prazosin, at the i.v. doses examined in the present study, attenuated the phenylephrine-induced increases in intraurethral pressure of anesthetized rats in a dose-dependent manner. Therefore, as shown in Fig. 8, there was a close correlation in i.v. dose ranges between the in vivo ₁-adrenoceptor binding activities of KMD-3213 and prazosin in the rat prostate and their functional activities in the lower urinary tract. A similar correlation was seen between the aortic in vivo specific binding and hypotensive effects of both agents (data not shown). Taken together, these data strongly suggest that the specific binding of [³H]KMD-3213 and [³H]prazosin in rat tissues after i.v. injection reflects the in vivo selective labeling of the pharmacologically relevant ₁-adrenoceptors.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Correlation between the in vivo 1-adrenoceptor binding in the prostate () and inhibition of phenylephrine (phe)-induced increases in intraurethral pressure (IUP) () of rats after i.v. injection of KMD-3213 (0.46-46.4 nmol/kg) (A) of prazosin (0.72-71.6 nmol/kg) (B). The in vivo specific binding of KMD-3213 and prazosin in the rat prostate were obtained from Fig. 6, and the pharmacological data were derived from the literature (Akiyama et al., 1999).

The in vivo specific binding of [³H]KMD-3213 10 min after i.v. injection was widely distributed in various tissues. A relatively high degree of in vivo specific binding of [³H]KMD-3213 was observed in the rat kidney, lung, heart, and submaxillary gland, followed by the vas deferens, prostate, and aorta. On the other hand, the spleen, cerebral cortex, and liver exhibited only a small degree of specific binding of [³H]KMD-3213, or even none at all, 10 min after i.v. injection of the radioligand. The absence of in vivo specific binding of [³H]KMD-3213 in the cerebral cortex may be due to poor permeability through the blood-brain barrier. Also, there was a tissue difference in the time course of the in vivo [³H]KMD-3213 binding. The specific [³H]KMD-3213 binding in the lung, kidney, and spleen was greatest 10 min after i.v. injection, and it declined rapidly with the disappearance of [³H]KMD-3213 from the plasma. On the other hand, [³H]KMD-3213 binding in the submaxillary gland, vas deferens, and prostate peaked at 60 min, with a considerable degree of specific binding in these tissues persisting up to 240 min post injection. These data are consistent with our ex vivo observation that p.o. administration of KMD-3213 in rats, despite a rapid decline in the plasma concentration, brought about sustained occupancy of the ₁-adrenoceptors in the rat prostate and submaxillary gland (T. Okura, S. Yamada, and R. Kimura, unpublished observation). Furthermore, the in vitro dissociation of [³H]KMD-3213 from prostatic ₁-adrenoceptors was significantly slower than that of [³H]prazosin. Sustained blockade by KMD-3213 of prostatic ₁-adrenoceptors was suggested by the functional study; that is, a prolonged inhibition by KMD-3213 of phenylephrine-induced intraurethral pressure in rats (Akiyama et al., 1999). Accordingly, the constant level of in vivo specific binding as a function of time for [³H]KMD-3213 in the rat prostate and submaxillary gland may be ascribable to the relatively slow dissociation rate of this ligand from the receptor sites, possibly due to a high affinity for the receptors. Also, pharmacokinetic factors such as blood flow rate and volume of distribution in each organ may be responsible largely for the observed difference among tissues in the amount and time course of in vivo specific binding of the radioligands. Tissue blood flow may be a critical determinant for in vivo binding of drugs to receptors. In fact, the level of blood flow rate measured by [¹⁴C]iodoantipyrine was greater in the heart, lung, and kidney than in other tissues (Yamada et al., 1999).

There was some difference in the time course and degree of specific binding between [³H]KMD-3213 and [³H]prazosin in the submaxillary gland, spleen, and prostate after i.v. injection of similar amounts of each radioligand. The in vivo specific binding of [³H]prazosin in the submaxillary gland was greatest 10 min after i.v. injection of similar dose as [³H]KMD-3213, and it then fell rapidly. On the other hand, the specific [³H]prazosin binding in the spleen attained a peak level at 60 min, which was maintained even at 120 min. The prostate showed a constant degree of [³H]prazosin binding from 10 to 120 min. Furthermore, the AUC_0-120 values for the specific binding of [³H]KMD-3213, compared with those for [³H]prazosin, were markedly (70.1-98.7%) lower in the rat aorta, spleen, and liver, whereas those in the prostate, submaxillary gland, and lung were 1.7- to 3.1-fold higher. Prazosin is known generally as a nonselective antagonist of ₁-adrenoceptor subtypes both in vitro and in vivo (Hanft and Gross, 1989; Aboud et al., 1993; Martin et al., 1997). Thus, the degree and time course of specific binding of [³H]prazosin in rat tissues may represent the difference in ₁-adrenoceptor density and/or pharmacokinetics rather than ₁-adrenoceptor subtype affinity. It is known that the _1A-subtype exists predominantly in the rat submaxillary gland and prostate (Han et al., 1987; Michel et al., 1989; Testa et al., 1993; Yazawa and Honda, 1993; Lepor et al., 1994; Shibata et al., 1995), whereas the _1B-subtype is predominant in the spleen and liver (Han et al., 1987; Han and Minneman, 1991; Michel et al., 1993; Shibata et al., 1995). In other tissues, both subtypes coexist in similar or slightly different ratios. Based on these in vitro observations, the observed difference between [³H]KMD-3213 and [³H]prazosin in the degree and time course of in vivo specific binding in the rat prostate, submaxillary gland, spleen, and liver suggests strongly that KMD-3213, unlike prazosin, exhibits considerably higher affinity for the _1A-subtype compared with the _1B-subtype in rat tissues under in vivo conditions. In other words, the in vivo data presented here agree with previous in vitro data showing that [³H]KMD-3213 does not bind specifically to the recombinant _1b-adrenoceptor subtype and to ₁-adrenoceptors in the rat liver (Murata et al., 1999).

[³H]KMD-3213 exhibited essentially similar characteristics of in vivo specific binding to those of [³H]tamsulosin previously reported (Yamada et al., 1999), and the only difference was that there was less binding capacity for [³H]KMD-3213 than [³H]tamsulosin in the aorta and spleen. The AUC_0-120 values for [³H]KMD-3213 in both tissues were 4- and 6-fold, respectively, smaller than those for [³H]tamsulosin, indicating that [³H]KMD-3213 may exhibit considerably lower affinity for the ₁-subtype in the aorta and spleen compared with [³H]tamsulosin. Previous studies have shown that ₁-agonist-induced contraction of the rat aorta is mediated primarily via the _1D-subtype (Aboud et al., 1993; Kenny et al., 1995) and that tamsulosin, but not KMD-3213, exhibits high affinity for the _1D-subtype (Noble et al., 1997; Taguchi et al., 1997; Murata et al., 1999). In addition, the _1B-adrenoceptor binding affinity of KMD-3213 was shown to be much lower than that of tamsulosin in vitro (Shibata et al., 1995; Murata et al., 1999). These observations have been clearly confirmed by the present in vivo findings for [³H]KMD-3213 involving ₁-adrenoceptors in rat tissues.

In conclusion, the present study provides the first in vivo evidence that KMD-3213 binds to the _1A-adrenoceptor subtype with higher affinity than to the _1B- and _1D-subtypes, thus resulting in prostate selectivity.