Introduction

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Urinary outlet obstruction in patients with benign prostatic hypertrophy (BPH) is attributed to a mechanical component, which is the urethral compression produced by the hypertrophied prostatic tissue, and a dynamic component, which is related to the tone of urethral and prostatic smooth muscles. Stimulation of ₁-adrenoceptors (ARs) of urethral and prostatic smooth muscles has been shown to cause bladder outlet obstruction in patients with BPH (Chapple et al., 1989). Furthermore, a significant increase in the number of ₁-ARs in the hypertrophied prostate was reported (Yamada et al., 1987). Hence, ₁-AR antagonists such as prazosin and terazosin are used in the treatment of BPH. These drugs, however, frequently induce orthostatic hypotension as a side effect, which is due to a reduction in peripheral arterial resistance mediated by a blockade of vascular ₁-ARs. Therefore, it is highly desirable to develop an ₁-AR antagonist that can selectively suppress the tone of the lower urinary tract without vascular effects (uroselectivity) for the treatment of urinary outlet obstruction in patients with BPH.

Currently, the ₁-AR is classified into three cloned subtypes, _1a-, _1b-, and _1d-ARs, and three native subtypes, _1A, _1B, and _1D (formerly termed _1c, _1b, and _{1a, a/d}, respectively; reviewed by Hieble et al., 1995). There are a number of reports indicating that _1a-AR is the predominant ₁-AR subtype in the prostate (Price et al., 1993; Moriyama et al., 1996; Nasu et al., 1996). Moreover, the contractile response of human prostate to norepinephrine is mediated by the _1A-AR (Marshall et al., 1995), whereas _1B-AR is predominant in human peripheral arteries (Hatano et al., 1994). These findings suggest a possibility for the development of an _1a-AR antagonist specific for the prostate and useful in the treatment of BPH. Thus, the _1a-AR subtype selectivity of ₁-AR antagonists receives great deal of attention.

To obtain a uroselective ₁-AR antagonist, an efficient screening system that can evaluate the suppressive effects of many compounds on the increase in urethral pressure mediated by ₁-ARs is essential. Although in vitro screenings of many compounds for their antagonistic activities toward ₁-AR in the lower urinary tract has usually been carried out with the use of isolated prostatic or urethral muscular strips (Honda et al., 1985; Cohen and Drey, 1989; Testa et al., 1993; Yamagishi et al., 1996) or radioligand receptor-binding studies (Morita and Kondo, 1992; Yamada et al., 1994), suitable in vivo methods for this purpose have not been established yet. Previously, the dog (Poirier et al., 1988; Breslin et al., 1993; Kenny et al., 1994), cat (Lefèvre-Borg et al., 1993), and rabbit (Yamaguchi et al., 1993) have been used to examine the effect of ₁-AR antagonists on ₁-AR-mediated urethral pressure responses in vivo. However, these models are useful only for evaluating the effect of a certain ₁-AR antagonist on the response of the lower urinary tract; it is impossible to screen a large number of ₁-AR antagonists requiring the use of such large animals.

KMD-3213 is a novel _1a-AR-selective antagonist (Shibata et al., 1995) and is under development for the treatment of urinary outlet obstruction in patients with BPH. Some in vitro experiments using isolated tissues from rabbit and rat revealed that KMD-3213 more potently inhibited the ₁-AR-mediated contraction of the prostate than that of the aorta (Yamagishi et al., 1996). KMD-3213 also potently inhibited norepinephrine-induced contraction of human isolated prostatic tissue with a potency similar to that of tamsulosin (Moriyama et al., 1997). These data suggest a therapeutic usefulness of KMD-3213 for the treatment of urinary outlet obstruction in patients with BPH.

In the present study, we sought to measure the prostatic intraurethral pressure (IUP) directly in rats, instead of dogs, cats, or rabbits, so as to establish a rat model for in vivo evaluation of the inhibitory effects of ₁-AR antagonists on the ₁-AR-mediated increase in IUP. We confirmed the adequacy of this model to determine the potency of certain ₁-AR antagonists. Using our novel rat model, we confirmed uroselectivity, absorption from the digestive tract, and distribution to the prostatic tissue of KMD-3213, and we compared these data with those on some other ₁-AR antagonists. Moreover, we also evaluated the duration of action of KMD-3213 in the lower urinary tract. In this way, we were able to determine the relative merit of KMD-3213 for the treatment of urinary outlet obstruction in patients with BPH.

	Materials and Methods

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General Procedure for Experiments

Male Sprague-Dawley rats (Japan SLC Inc., Shizuoka, Japan), 9 to 11 weeks old and weighing 250 to 350 g, were anesthetized with an i.p. administration of urethane (1.25 g/kg). After intubation of the trachea, the urinary bladder, prostate, and urethra were exposed through an abdominal midline and symphysis pubica incision. A polyethylene catheter (5-Fr; Hibiki, Tokyo, Japan) was placed into the prostatic urethra through the bladder dome and secured at the bladder neck (vesico-urethral junction) with a silk suture. The distal side of the urethra under the pubic bone was also closed. The other side of the catheter was connected to a pressure transducer (DX-312; Nihon-Kohden, Tokyo, Japan) to measure the IUP. Change in IUP was recorded with a pen recorder (Rectihoriz 8K-23; NEC-Sanei, Tokyo, Japan). The saphenous vein on both sides was cannulated: one for administration of ₁ agonists, and the other for the administration of test compounds. After all surgical preparations, the urethral pressure was equilibrated at approximately 10 cm H₂O by injecting a small volume (0.1-0.2 ml) of glucose-free Tyrode's solution (137.9 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 1.1 mM NaH₂PO₄, 11.9 mM NaHCO₃) or physiological saline. During the entire procedure, care was taken to avoid damaging the nerves and vessels. An increase in IUP was evoked by the injection of appropriate concentrations of l-phenylephrine hydrochloride (PHE) solution into one of the saphenous veins at a bolus injection rate of 0.6 ml/min for 100 s/kg · rat by the use of a syringe pump (Terumo STC-525; Terumo, Tokyo, Japan). Throughout the experiments, the submaximal dose of 30 µg/kg (i.e., 30 µg/ml PHE) was injected (see Results), except for the generation of a dose-response curve for PHE.

Role of Prostatic Tissue in Increase in IUP Response to PHE

To investigate the role of the prostatic tissue in the increase in IUP, the IUP responses to PHE (30 µg/kg i.v.) in "prostate-lacking" rats were compared with those in "prostate-intact" rats as follows.

Prostate-Ablated Rat. After all preparations for the measurement of IUP had been made, both dorsal and ventral prostatic tissues were partially ablated from the urethral wall by gentle rubbing with a cotton bud. The IUP responses to PHE in these rats were compared with those in prostate-intact male rats. During this experiment, rats that exhibited bleeding in the area of ablation were excluded.

Female Rats. The IUP response to PHE at the proximal urethra (between vesicourethral junction and distal urethra under the pubic bone) was also measured in 9- to 10-week-old female Sprague-Dawley rats (Japan SLC Inc., Shizuoka, Japan) weighing 200 to 250 g and compared with those in prostate-intact male rats.

Castrated Rat. Castration was performed on 5-week-old male rats. Four weeks after the castration, the IUP response to PHE (30 µg/kg) was measured by the same procedure as described above, and the results were compared with those for sham-operated rats. Both ventral and dorsal prostatic weights were also determined after the measurement of urethral pressure responses to PHE had been completed.

Role of Urethral Tissue and Effects of Urethral Ligations in Increase in IUP Response to PHE

IUP Response to PHE in In Vivo No-Ligation Model. The IUP was detected using 4-Fr microtipped catheter (Millar Instruments) that was inserted from the bladder and was placed in the prostatic urethra in the male rats or in the proximal urethra in the female rats, without ligation at the bladder neck and distal urethra. All other procedures, such as the application of PHE, were the same as the double-ligation model.

Isolated Urethral Muscle Response to PHE. Urethra was isolated from the prostate-intact (9-11 weeks old, 336-376 g), castrated male (9-11 weeks old, 316-333 g), and female (9-11 weeks old, 209-217 g) rats, and connective tissues, prostate, and other urogenital tissues were carefully removed. Then, each urethra was spirally cut and was vertically suspended in an organ bath containing 10 ml of Krebs' solution bubbled with a gas mixture of 95% O₂/5% CO₂. PHE was cumulatively applied into each bath to construct a concentration-response curve. The maximum contractile forces were compared for all pair combinations using Tukey's multiple comparison test.

Effects of i.v. ₁-AR Antagonists on IUP Response

After a reproducible control response to 30 µg/kg PHE i.v. had been obtained, a control response to PHE was recorded; after the urethral pressure recovered to the baseline, the lowest dose of a given ₁-AR antagonist was administered (1 ml/kg i.v.) 5 min before an additional application of the PHE. The next higher dose of ₁-AR antagonist was applied 5 min before the next IUP response was evoked. By linear regression analysis, we evaluated the correlation between the binding affinity of the tested compounds for human or animal ₁-AR subtypes (affinities taken from previous reports by Foglar et al., 1995; Shibata et al., 1995; Testa et al., 1995) and their inhibitory potencies in the present i.v. study.

Effects of Intraduodenal (i.d.) ₁-AR Antagonists on IUP Response

After all preparations described above had been finished, a polyethylene catheter was inserted into the duodenum from the stomach to administrate the tested compound; the pylorus was ligated to prevent retrograde flow of the suspension or solution of the compound. After a stable response to 30 µg/kg PHE i.v. had been obtained, a control IUP response was recorded. Then, 5 min and 0.5, 1, 2, 3, and 4 h after the i.d. administration, the PHE-induced increase in IUP was evoked. The IUP value with each dose of compound was compared with the predosing value and plotted against time after administration. Dunnett's multiple comparison test was used to compare the extent of blockade of the increase in IUP response with the control group at each time point during the course of the i.d. study. The maximal effect with each dose of compound was extracted to estimate uroselectivity.

Duration of Inhibitory Effect on PHE-Induced Increase in IUP

At 12, 18, or 24 h after the oral administration of KMD-3213 or tamsulosin, PHE-induced IUP responses were measured and compared with the response of the vehicle-control group. Preparations of lower urinary tract to measure the urethral pressure were made approximately 2 h before each IUP response evoked. Dunnett's multiple comparison test was used to compare the extent the increase in IUP response with the control group.

Measurement of Blood Pressure

In a separate experiment, the basal mean blood pressure (MBP) was continuously measured from a carotid artery in male rats by a routine method. In the i.v. study, a solution of tested compounds were administered into a saphenous vein every 0.5 to 1 h in increasing doses. The maximal effect observed with each dose was compared with the predosing value. In the i.d. study, data were extracted at 5 and 15 min and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h after administration into the duodenum and then each value was compared with the predosing value. The maximal hypotensive effect observed with each dose was collected to estimate the uroselectivity of each compound. Dunnett's multiple comparison test was used to compare the extent of lowering the basal MBP with the control group at each time point during the course of the i.d. study.

Data Analysis

Inhibitory potency of the compound against an increase in IUP was expressed as the ID₅₀ (µg/kg), the value of the dose required to produce a 50% reduction of the control value. Hypotensive potency was expressed as the ED₁₅ (µg/kg) value, the dose required to produce a 15% decrease in MBP. Uroselectivity for the compound was determined as ED₁₅ value of blood pressure/ID₅₀ value of urethral pressure.

The methods of statistical analysis of each experiment were described in each part.

Compounds and Solutions

KMD-3213, tamsulosin hydrochloride, and terazosin hydrochloride were synthesized in the chemical section of our laboratory. WB4101 hydrochloride and prazosin hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). Urapidil, 5-methyl urapidil, and yohimbine hydrochloride were purchased from Funakoshi (Tokyo, Japan). PHE was purchased from Wako (Osaka, Japan). All other chemicals were of reagent grade.

PHE was dissolved in physiological saline and diluted with the same solution to the appropriate concentrations. In the study of i.v. administration, KMD-3213 was dissolved in Hartmann's solution of the following composition (w/v %): 0.60 NaCl, 0.03 KCl, 0.02 CaCl₂, and 0.31 lactic acid, containing hydrobromide of 2-fold equivalent of KMD-3213. Tamsulosin, WB4101, and urapidil were dissolved in physiological saline and diluted with the same solution to the appropriate concentrations. Prazosin, 5-methyl urapidil, and yohimbine were dissolved in distilled water and diluted with physiological saline to the appropriate concentrations. In the study with i.d. or oral administration, KMD-3213, tamsulosin, and prazosin were suspended in 0.5% methylcellulose solution and diluted with same solution to the appropriate concentrations.

	Results

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PHE-Induced Increase in IUP Response

To obtain a stable IUP response to agonists, it is necessary to maintain the i.v. injection of PHE at a constant rate. We found the optimum rate to be 0.6 ml/min (data not shown). Figure 1A shows a profile of a typical tracing of the IUP response induced by 30 µg/kg PHE. IUP was transiently increased up to 20 to 30 cm H₂O after the administration of PHE and gradually decreased to the baseline within 30 min. PHE increased the IUP in a dose-dependent manner (Fig. 1B), with an ED₅₀ value of 9.8 µg/kg. Because the response was saturated at over 100 µg/kg of this agonist, the following experiments were performed at a submaximal dose, 30 µg/kg, of PHE. Stable and reproducible responses were obtained when the IUP response was evoked at 0.5, 1, 2, 3, and 4 h after the control response had been evoked (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Effect of PHE on the IUP in an anesthetized male rat. A, typical tracing of the IUP response to 30 µg/kg PHE. PHE was administered i.v. at the time indicated () at a bolus injection rate of 0.6 ml/min for 100 s/kg. B, dose-response curve for PHE. Each point represents the percent of the IUP response found at a dose of 300 µg/kg.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Reproducibility of the IUP response to PHE. The increase in IUP was evoked by i.v. administration of 30 µg/kg PHE at 0, 0.5, 1, 2, 3, and 4 h. Data are presented as a percentage of the increase in IUP at time 0 (n = 4).

IUP Responses in Prostate-Lacking Rats

Figure 3A shows the comparison of IUP responses in male prostate-intact, male prostate-ablated, and female rats. The ablation of the prostate resulted in a significant decrease in the urethral pressure response to PHE. IUP responses in female rats were also significantly weaker than those in male prostate-intact rats. As shown in Fig. 3B, the IUP responses to PHE in the castrated rats were significantly weaker than those in sham-operated rats. Both ventral and dorsal prostatic weights, which were measured after the evaluation of IUP responses, of the castrated rats were also significantly smaller than those of sham-operated ones (Table 1).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Comparison of the IUP responses to i.v. administered 30 µg/kg PHE in (A) prostate-intact male, prostate-ablated male, and female rats and (B) sham-operated and castrated rats. Values shown are the mean ± S.E. (n = 5-7). a, significantly different from prostate-intact male rats at p < .01. b, significantly different from sham-operated male rats at p < .01 (Student's t test).

Effects of Urethral Ligations and Role of Urethral Tissue on Increase in IUP Response

To investigate the effects of double-ligation on the IUP response and the participation of the urethral tissue in the IUP response, the next two experiments were performed.

Figure 4 shows the IUP responses to PHE in prostate-intact, prostate-ablated, castrated, and female rats with no-ligation of urethra. The ablation of the prostate resulted in a significant decrease in the IUP response to PHE by approximately 90%. Similarly, responses in the castrated and female rats were significantly smaller than in the prostate-intact male rats. The magnitudes of the increase in IUP responses of these prostate-lacking rats were essentially identical. These results were consistent with the results of the double-ligation model (Fig. 3, A and B).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   PHE-induced increase in IUP responses in prostate-intact male, prostate-ablated, castrated male, and female rats. The IUP was detected using a microtipped catheter placed in the prostatic urethra in male rats or proximal urethra in female rats without ligation at the bladder neck and distal urethra. Data represent the mean ± S.E. of four or five rats. a, significantly different from prostate-intact male rats at p < .05 (paired t test). b, significantly different from prostate-intact male rats at p < .01 (Student's t test).

The contractile responses of urethral preparations isolated from prostate-intact male, castrated, and female rats are shown in Fig. 5 and Table 2. PHE produced concentration-dependent contraction in all isolated urethra. The maximum contractile forces in castrated rats and female rats were approximately 60 and 20% of that in prostate-intact rats, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   PHE-induced contractile responses of isolated urethra in prostate-intact male (), castrated male (), and female () rats. Each urethral tissue was spirally cut from the bladder neck (vesico-urethral junction) to the distal urethra and vertically suspended in an organ bath containing 10 ml of Krebs' solution. PHE were cumulatively applied to construct the concentration-response curve. Each plot represents the mean ± S.E. of absolute contractile force (milligrams) of each tissue (n = 5).

In Vivo Uroselectivity

Intravenous Administration and Correlation Study. The dose-effect relationship of the cumulative i.v. administration of KMD-3213, tamsulosin, prazosin, and terazosin in the suppression of the increase in the IUP response to PHE and that in decreasing the basal MBP in male anesthetized rats are shown in Fig. 6. These compounds potently inhibited the IUP response, and decreased the basal MBP, in a dose-dependent manner. KMD-3213 had more substantial effect on the PHE-induced IUP response than on the basal MBP. In contrast to the profile of KMD-3213, tamsulosin exhibited nearly equivalent effects on both IUP response and MBP, whereas prazosin and terazosin were more efficacious in lowering MBP than on the IUP response to PHE. Rank order of potency for inhibition of the increase in IUP was tamsulosin > KMD-3213 > prazosin > terazosin, whereas rank order of hypotensive potency was tamsulosin > prazosin > terazosin > KMD-3213. Inhibitory effects of i.v. doses of WB4101, 5-methyl urapidil, urapidil, and yohimbine on IUP response to PHE were also determined (Fig. 7). These compounds, except yohimbine, potently inhibited the PHE-induced IUP response in a dose-dependent manner with almost complete inhibition of the IUP response at higher doses. Yohimbine, an ₂-AR antagonist, was not able to completely inhibit this response.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Comparison of the inhibitory effects on the increase in IUP response to PHE () and effects on MBP () of i.v. doses of KMD-3213 (A), prazosin (B), tamsulosin (C), and terazosin (D) in anesthetized male rats. Values shown are the mean ± S.E. of five to eight rats. Both parameters were determined in separate experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Inhibitory effects of i.v. bolus doses of WB4101 (), 5-methyl urapidil (), urapidil (), and yohimbine () on the i.v. administered 30 µg/kg PHE-induced increase in IUP. Each compound was administered 5 min before the injection of PHE. Values shown are mean ± S.E. mean (n = 4 or 5).

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Correlation between the binding affinities (pKi) of the different compounds tested for the recombinant human or animal (A) 1a, (B) 1b, and (C) 1d and their inhibitory effects on the increase in IUP response to PHE (pID50). Binding affinity of each compound was taken from Shibata et al. (1995), Foglar et al. (1995), and Testa et al. (1995).

View this table: [in this window] [in a new window]   TABLE 4 Affinity of KMD-3213 and reference compounds for the recombinant 1-AR subtypes Data represent the mean pKi (log10 Ki) values taken from Shibata et al. (1995), Foglar et al. (1995), and Testa et al. (1995) and pID50 [log10 (ID50)] values of the present i.v. study.

Intraduodenal Administration. Inhibitory activity and hypotensive effect of KMD-3213, prazosin, and tamsulosin were immediately manifested after i.d. administration (Fig. 9). All three compounds potently and dose-dependently inhibited IUP response, and their effects lasted at least for 4 h. Although the hypotensive effect of higher doses of prazosin and tamsulosin lasted for 4 h, that of KMD-3213 recovered within 4 h. Moreover, the MBP at a dose of KMD-3213 required to completely inhibit the IUP response (300 µg/kg) decreased less than 10% and recovered to the initial level within 1 h after administration.

View larger version (48K): [in this window] [in a new window]   Fig. 9.   Effects of i.d. doses of KMD-3213, prazosin, and tamsulosin on the increase in IUP response to PHE and the basal MBP in male anesthetized rats. PHE was administered at a dose of 30 µg/kg i.v. periodically up to 4 h after administration of each compound. In a separate experiment, MBP was monitored continuously. Values shown are the mean ± S.E. (n = 5-8). Data were analyzed by Dunnett's multiple comparison test at each time point to determine significant inhibition of PHE-induced IUP responses. Significantly different from the control at *p < .05 and **p < .01, respectively. Dose (mg/kg i.d.): , vehicle control; , 0.003; , 0.01; , 0.03; , 0.1; , 0.3; , 1; , 3; and ×, 10.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Comparison of inhibitory effects on the increase in the IUP response to PHE and on the basal MBP in male anesthetized rats. Each value was taken from the most effective point in the i.d. study.

Duration of Inhibitory Effect of KMD-3213 and Tamsulosin on PHE-Induced IUP Responses

KMD-3213 was orally administered to conscious rats and then IUP response to PHE was measured at the prescribed times, with the animals anesthetized, by the same procedure described above. IUP responses to PHE after various doses of oral administration of KMD-3213 or tamsulosin are revealed in Fig. 11. At 12 h after the oral administration of 0.03 to 1 mg/kg KMD-3213, the IUP responses were significantly and dose-dependently smaller than those of control rats. The ID₅₀ value of KMD-3213 at this time point was 0.12 mg/kg, suggesting that the half-life of the inhibitory potency of KMD-3213 was approximately 12 h. At 18 h, 1 mg/kg KMD-3213 still significantly inhibited the IUP response; and at doses of 0.1 and 0.3 mg/kg, KMD-3213 tended to inhibit the response. In contrast to KMD-3213, although at doses of 0.3 and 0.1 mg/kg, tamsulosin could not inhibit IUP responses. The ID₅₀ value of tamsulosin at 12 h could not be calculated because more than 50% inhibition was not obtained. At 18 h after tamsulosin administration up to 1 mg/kg, no inhibition of IUP responses was observed.

View larger version (43K): [in this window] [in a new window]   Fig. 11.   Effects on the increase in IUP response to PHE 12, 18, and 24 h after oral doses of KMD-3213 (A-C) and tamsulosin (D and E), respectively. Preparation for the measurement of IUP was made approximately 2 h before evoking the IUP response to PHE. Data were analyzed by Dunnett's multiple comparison test. Significantly different from the control at *p < .05 and **p < .01, respectively (n = 3-8 rats). ND, not determined.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

KMD-3213 is a novel _1a-AR-selective antagonist (Shibata et al., 1995; Yamagishi et al., 1996; Moriyama et al., 1997) having high prostate selectivity, and is under development for the treatment of urinary outlet obstruction in patients with BPH. This is the first report of in vivo evaluation of the uroselectivity of KMD-3213 in a novel rat model.

In the rat model described here, stable increases in the IUP responses were induced by i.v. administration of PHE, an ₁-AR-selective agonist, in a dose-dependent manner, and the responses were stable and reproducible in the same animal for at least 4 h after the initial (control) response. This allows the construction of an inhibitory dose-response curve to evaluate the potency for ₁-AR antagonists. Intravenously administered higher doses of ₁-AR-selective antagonists tested almost completely inhibited the PHE-induced IUP response, whereas the inhibitory potency for yohimbine was markedly less than that of ₁-AR-selective antagonists. These data indicate that urethral pressure responses to PHE in this model are primarily mediated by ₁-ARs.

As shown in Fig. 3, reduced IUP responses were observed in the prostate-lacking (prostate-ablated, castrated, and female) rats. These results suggest that the existence of intact prostate is necessary to induce the sufficient increase in IUP response. However, we could not rule out that the ischemia occurred in the proximal urethra between ligatures and resulted in lower values among IUP responses that were due to urethral muscular contraction in prostate-lacking rats than in the prostate-intact rats. To verify this problem, we investigated the effect of the ligations on the IUP response by using a microtipped catheter without ligating the urethra. In those experiments, as well as the double-ligation model (Fig. 3, A and B), the increase in IUP in the prostate-lacking rats were approximately 10-fold less than that in prostate-intact rats (Fig. 4). In addition, the fact that IUP responses were reproducible at least for 4 h (Fig. 2) indicates that no ischemia in prostatic tissue occurred and that double ligation of the urethra had little influence on the IUP response in the prostate-intact rat. In the macroscopic findings, no edema or swelling was observed in the prostatic and urethral tissues throughout the experiment (data not shown).

We also verified the participation of urethral smooth muscle contraction in the increase in IUP response using isolated proximal urethral preparations from prostate-intact, castrated, and female rats in in vitro (Fig. 5 and Table 2). Androgen deprivation (castration) caused 40% decrease and estrogen (female) caused subsequent 40% decrease in the maximum contractile force to PHE. However, there was little correlation between the in vitro urethral contractile responses and the in vivo IUP responses (in both double-ligation and no-ligation model) in the three type of prostate-lacking rats. These results suggest that the effects of androgen and estrogen are not involved in the in vivo IUP response, although the hormones affect the urethral contraction. Therefore, the PHE-induced IUP response is thought to depend primarily on intact-prostatic muscular contraction, and that urethral muscular contraction is only a minor component in this response in the prostate-intact rats. Because the prostatic muscle tone mediated by ₁-ARs is reported to be one of the important components of urinary outlet obstruction in patients with BPH and therapeutic strategy based on ₁-antagonism in lower urinary tract has been added to the recent management of BPH (Caine, 1986; Kawabe et al., 1990), this rat model could be applied for use as a BPH model.

Hancock et al. (1998) reported that uroselectivity of ₁-AR antagonist depended on its _1A-subtype selectivity in a conscious dog model. Our data on reference compounds in the rat are in good agreement with such a dog model. In comparison with other in vivo models using dogs (Poirier et al., 1988; Imagawa et al., 1989; Somers et al., 1989; Breslin et al., 1993; Kenny et al., 1994), cats (Lefèvre-Borg et al., 1993), and rabbits (Yamaguchi et al., 1993), the present rat model facilitates the treatment and handling of animals and simultaneous measurement, as well as improving economy. In addition, because rats of identical species, strains, age, and similar body weight are readily available, a uniform response of the urethral pressure with less deviation of data can be obtained. Therefore, this rat model could be applied to evaluate the antagonistic activities of many ₁-AR antagonists in the lower urinary tract in vivo. For these reasons, this rat model offers an advantage over dog and rabbit models as an in vivo screening system in estimating uroselectivity of a large number of compounds.

Intravenously administered KMD-3213 exhibited excellent uroselectivity in comparison with tamsulosin, prazosin, and terazosin in this model. This finding is of clinical importance. This functional uroselectivity of KMD-3213 in rats is supported by the in vivo binding study of Yamada et al. (1998). In that study, i.v. administered KMD-3213 selectively inhibited in vivo [³H]tamsulosin binding to prostate, and the prostate specificity of KMD-3213 was significantly greater than that of prazosin and tamsulosin in rats. Moreover, the amount of specific binding of i.v. administered [³H]KMD-3213 in prostate was significantly greater than that of [³H]prazosin, but that in aorta was much smaller. These data indicate that KMD-3213 exhibits selectively higher affinity for ₁-ARs in prostate than in vascular tissues in vivo. A tamsulosin-, prazosin-, or terazosin-induced decrease in basal MBP can cause adverse cardiovascular effects in some patients, probably resulting from blockade of vascular ₁-ARs.

By linear regression analysis, we evaluated the correlation between the binding affinity for each of the human or animal _1a-, _1b-, and _1d-AR subtypes of the compounds tested (Foglar et al., 1995; Shibata et al., 1995; Testa et al., 1995) and their inhibitory potency in the present i.v. study for IUP in this model. All compounds, including KMD-3213, tamsulosin, and prazosin, fell on the regression line for the _1a subtype (R² = .81, p < .01). Many reports have presented correlation between ₁ subtypes and in vivo efficacy toward urogenital tissues. Both prazosin and terazosin are nonsubtype-selective antagonists, and tamsulosin has similar affinities for both _1a and _1d subtypes but has 10- to 20-fold lower affinity for _1b than for the other subtypes (Shibata et al., 1995; Noble et al., 1997). Recently, the _1a-AR subtype was shown to be predominant in the rat prostate (Couldwell et al., 1993; Yazawa and Honda, 1993; Scofield et al., 1995) as well as in human prostatic tissues (Price et al., 1993; Forray et al., 1994; Marshall et al., 1995; Tseng-Crank et al., 1995; Nasu et al., 1996). Thus, uroselectivity of ₁-AR antagonists should depend on _1a-AR subtype specificity. The data obtained from the present study agree well with those reports, suggesting that PHE-induced increases in IUP responses are mediated by the _1a-AR subtype. Thus, the potency of ₁-AR antagonists in this model appears to correlate with those obtained in in vitro experiments.

On the other hand, Muramatsu et al. (1994) reported that contraction of human isolated prostate evoked by norepinephrine is mediated by the putative _1L-AR, which shows lower affinity for prazosin. Testa et al. (1997) and Leonardi et al. (1997) indicated the ₁-AR agonist-induced IUP response was mediated via _1L-AR because of lower potencies of SNAP 5089, RS 17053 (Ford et al., 1996), and Rec 15/2615, which are selective for _1A-AR subtype with having lower affinities for _1L-AR. In the present study, we could not confirm the participation of _1L-AR on the IUP response in this rat model because those antagonists were not available. It will be interesting to investigate the participation of _1L-AR in this model in a future study.

Intraduodenally administered compounds tested, including KMD-3213, inhibited PHE-induced IUP responses up to 4 h in a dose-dependent manner. These compounds also decreased the basal blood pressure in a dose-dependent manner. However, unlike prazosin and tamsulosin, the dose of KMD-3213 required to decrease the basal MBP was much higher than that required to inhibit PHE-induced IUP responses. Although the hypotensive effects of KMD-3213 were lost within 4 h after administration, those of prazosin and tamsulosin continued 4 h, even at lower doses. These observations indicate that KMD-3213 is absorbed and is distributed to the prostatic tissue sufficiently via the oral route in rats to inhibit the IUP response to PHE. In addition, the uroselectivity of KMD-3213 was well preserved even when this compound was administered orally. The results of the rat model indicate a prolonged duration of action of KMD-3213 for IUP, extending many hours after administration, which suggests that KMD-3213 reaches concentrations in the rat prostate that are of sufficient magnitude to significantly block ₁-ARs in this tissue for many hours. These results indicate a clinical advantage of KMD-3213 (i.e., that it could be administered only once daily).

In conclusion, this novel in vivo rat model allows the investigation of the pharmacokinetics of many ₁-AR antagonists when these drugs are administered i.d. or orally. For example, dose-potency relationship could indicate relationship to the absorption, tissue distribution, metabolism, and/or excretion of certain compounds. Indeed, KMD-3213 was selected from many KMD series compounds and was shown to have good uroselectivity, good availability of action, and long duration of action by use of our in vivo rat model. Thus, the studies detailed in this report demonstrate that KMD-3213 is a compound that can potently inhibit an ₁-AR-stimulated increase in IUP response after either i.v. or i.d. (p.o.) administration with a prolonged duration of action and with far weaker and more transient effects on cardiovascular ₁-AR function. KMD-3213 should have use in the improvement of the symptoms of BPH and represents a useful pharmacological tool for the study of the pharmacology of ₁-ARs.