Introduction

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Alpha-1 adrenoceptor antagonists are used in the symptomatic treatment of benign prostatic hyperplasia (Oesterling, 1995; Chapple, 1995). It is believed that their beneficial therapeutic effect results from the antagonism of noradrenaline-induced contraction of prostatic smooth muscle that occurs via the alpha-1 adrenoceptors (Hieble et al., 1985). In recent years it has become clear that at least three subtypes of the alpha-1 adrenoceptors exist, which are now designated as alpha-1A (formerly alpha-1c), alpha-1B and alpha-1D (formerly alpha-1a/d) (Hieble et al., 1995; Michel et al., 1995). Among these subtypes the alpha-1A adrenoceptor dominates in the human prostate at the mRNA level (Price et al., 1993; Tseng-Crank et al., 1995), the protein level (Lepor et al., 1993; Michel et al., 1996) and may also be most important for the mediation of contraction (Forray et al., 1994; Marshall et al., 1995). Therefore, it has been suggested that drugs with selectivity for the alpha-1A adrenoceptors may be efficacious in benign prostatic hyperplasia, but may be more tolerable than the nonselective alpha adrenoceptor antagonists (Chapple, 1995).

Tamsulosin is the only alpha-1 adrenoceptor antagonist used clinically in benign prostatic hyperplasia, which is selective for the alpha-1A relative to alpha-1B adrenoceptors, and has an intermediate affinity for the alpha-1D adrenoceptors (Testa et al., 1995; Foglar et al., 1995; Michel et al., 1996). Because in vivo drug effects may also involve the metabolites, we have compared the affinities of tamsulosin and its metabolites M1, M2, M3, M4 and AM1 (fig. 1) at the alpha-1 adrenoceptor subtypes by using competition binding studies with rat tissues (Michel et al., 1993) and the cloned subtypes (Michel and Insel, 1994) as well as functional measurements in the rabbit prostate (Honda et al., 1985a) and the aorta (Honda et al., 1985b).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Chemical structure of tamsulosin and its metabolites. Throughout the study, M4 was used in its racemic form whereas pure optical (minus)-isomers were used for all other compounds.

	Materials and Methods

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Membrane preparations from the rat liver and the kidney were prepared from male Wistar rats (Lippische Versuchstierzucht, Extertal, Germany) (200-300 g) as described previously in detail (Michel et al., 1993). The expression vector plasmids pCMValpha-1a containing the EcoR1-Pst1 2520 b.p. fragment of the rat alpha-1D adrenoceptor cDNA and pcDV1Ralpha-1b containing a 2573 b.p. fragment including the entire coding region of the rat alpha-1B adrenoceptor cDNA (Lomasney et al., 1991) were obtained from Dr. R. J. Lefkowitz (Durham, NC). The plasmid pMT2alpha-1c that contains the entire coding region of the rat alpha-1A adrenoceptor (Perez et al., 1994) was obtained from Dr. R. M. Graham (Sydney, Australia). All three constructs were transfected into COS-1 cells for transient expression by using the diethylaminoethyl-dextran method with the addition of chloroquine and dimethylsulfoxide steps as described previously (Suryanarayana and Kobilka, 1991; Michel and Insel, 1994). Four days after transfection, the cells were harvested, resuspended into ice-cold binding buffer (50 mM Tris and 0.5 mM EDTA, pH 7.5) and homogenized by a Tissuemizer for 10 sec at full speed followed twice for 20 sec at speed. The homogenate was centrifuged for 20 min at 50,000 × g and the resulting pellet was resuspended in the binding buffer at a concentration of 0.6 to 2 mg/ml.

[³H]Prazosin binding to the membrane preparations from the rat tissue or transfected COS-1 cells was performed in the binding buffer (see above) as described previously (Michel et al., 1993). Briefly, 100 µl of membrane suspension were incubated in a total volume of 1 ml with the indicated [³H]prazosin concentrations for 45 min at 25°C. The incubations were terminated by rapid vacuum filtration over Whatman GF/C filters. Nonspecific binding was defined as binding in the presence of 10 µM phentolamine. In competition experiments, a [³H]prazosin concentration of  200 pM was used.

For the functional experiments, male albino rabbits (weight, 2.3-4.8 kg) were obtained from Kitayama Labes Co. (Nagano, Japan). Experiments were performed as described previously (Honda et al., 1985a,b) at 37°C in 30-ml organ baths containing Krebs-Henseleit solution of the following composition (millimolar): NaCl, 118.4; KCl, 4.7; KH₂PO₄, 1.2; MgSO₄, 1.2; CaCl₂, 2.5; NaHCO₃, 25.0; and glucose, 11.1. For experiments on the rabbit aorta, helical strips of 2 × 30 mm were used. For the experiments on the prostate, tissue strips (3 mm wide and 15 mm long) were prepared in the transverse direction. Aortic and prostatic specimens were equilibrated under a resting tension of 2 and 1 g, respectively, for 1 to 2 hr; these resting tensions were chosen because they allow maximum tension development (Honda et al., 1985a,b). Phenylephrine (3 µM) was administered repeatedly until responsiveness became stable. After vigorous washout, cumulative phenylephrine concentration-response curves were generated with half-logarithmic concentration increments. After the washout, the tissues were equilibrated with the antagonists for 30 min and another concentration-response curve was constructed. For each antagonist, except for AM-1, three to four (rabbit aorta) or two to four (rabbit prostate) concentrations were tested. Apparent pA₂ values were determined at each antagonist concentration from the shift of the concentration-response curve by the Furchgott equation where EC₅₀ and EC₅₀ are the half-maximally effective agonist concentrations in the absence and presence of the antagonist, respectively, and [A] is the concentration of the antagonist. pA₂ values from all antagonist concentrations were then averaged. Schild analysis was not performed routinely in the present study, but we have shown previously that tamsulosin as well as prazosin, yohimbine and phentolamine yield Schild-regressions with slopes not significantly different from unity when large numbers of preparations are tested under these conditions (Honda et al., 1985a,b, 1987).

[³H]Prazosin (specific activity, 70-80 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Tamsulosin and its metabolites were synthesized by Yamanouchi. Phentolamine was a gift of Ciba Geigy (Basel, Switzerland). Phenylephrine HCl was obtained from Tokyo Kasei (Tokyo, Japan).

Data are the means ± S.E.M. of the number (n) of experiments. Statistical significance of drug affinity differences at the alpha-1 adrenoceptor subtypes was determined in two ways: first, competition binding experiments were analyzed by fitting mono- and biphasic sigmoidal curves to the experimental data; a biphasic fit was accepted only if it resulted in a significant improvement of the fit as judged by an F test. Second, drug affinities at the cloned alpha-1 adrenoceptor subtypes were compared by a one-way analysis of variance; if this indicated that the variance between groups was significantly greater than that within groups, individual groups were compared by the Tukey-Kramer multiple comparison tests. In all tests, a P value < .05 was considered significant. Statistical analysis was performed by the InStat program (GraphPAD Software, San Diego, CA). All curve fitting procedures were performed by using the InPlot program (GraphPAD Software).

	Results

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[³H]Prazosin was bound to the rat liver membranes with a K_d of 132 ± 36 pM and a B_max of 123 ± 13 fmol/mg of protein (n = 3). Except for the AM1, all test compounds competed for [³H]prazosin binding to the rat liver membranes with steep and monophasic competition curves (fig. 2; table 1). The order of potency in the rat liver was tamsulosin  M4 > M1 > M2  M3  AM1. Thus, AM1 in concentrations up to 100 µM competed for less than 50% of the [³H]prazosin binding. Similarly high concentrations of AM1 also competed for only a small fraction of [³H]prazosin binding in the rat kidney or with any of the cloned alpha-1 adrenoceptor subtypes. Thus, the AM1 appears to have very low affinity for all subtypes of the rat alpha-1 adrenoceptors and will not be discussed any further.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Competition of tamsulosin and its metabolites for the [3H]prazosin binding to the rat liver membranes. Data are the means of three experiments. Error bars are not shown for clarity of presentation, but usually were 5 to 10%. A numerical analysis of these data is shown in table 1.

View this table: [in this window] [in a new window]   TABLE 1 Affinity of tamsulosin and its analogs for rat tissue alpha-1 adrenoceptor subtypes Data are the means ± S.E.M. of three to five experiments. Hill-slopes for the rat kidney are derived from a monophasic fit, whereas log Ki high, and log Ki low and percentage of high-affinity sites in this tissue were estimated from a biphasic fit. In the rat kidney, the biphasic fit was significantly better than the monophasic fit (P < .05) as judged by an F test. A graphical representation of the data is shown in figures 1 and 2. Data for 5-methylurapidil, (+)-niguldipine and WB 4101 generated in our laboratory with identical methods are taken from (Michel et al., 1993, 1996; Büscher et al., 1996; Chess-Williams et al., 1996) and are shown for reference purposes.

[³H]Prazosin was bound to the rat kidney membranes with a K_d of 110 ± 15 pM and a B_max of 31 ± 3 fmol/mg of protein (n = 3). In the rat renal membranes, all test compounds competed for the [³H]prazosin binding with shallow competition curves that were explained much better by a two- rather than a one-site model (fig. 3; table 1). The order of potency in the renal membranes at both the high and the low affinity sites was similar to that in the rat liver, and all compounds recognized a similar percentage of the high affinity sites, i.e., approximately 35 to 50%.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Competition of tamsulosin and its metabolites for the [3H]prazosin binding to the rat kidney membranes. Data are the means of three to five experiments. Error bars are not shown for clarity of presentation, but usually were 5 to 10%. A numerical analysis of these data is shown in table 1.

[³H]Prazosin was bound to the cloned rat alpha-1A, alpha-1B and alpha-1D adrenoceptors with K_d values of 263 ± 36, 176 ± 22 and 137 ± 51 pM and B_max values of 3012 ± 180, 2625 ± 400 and 135 ± 27 fmol/mg of protein (n = 3 each), respectively. All test compounds competed for the [³H]prazosin binding to the cloned rat alpha-1 adrenoceptor subtypes (table 2). The order of potency at each subtype was similar to that observed in the rat liver or the kidney. All compounds were subtype-selective, having their lowest affinity at the alpha-1B adrenoceptor. Most compounds recognized the cloned alpha-1 adrenoceptor subtypes with the order of potency alpha-1A  alpha-1D > alpha-1B. The metabolite M4, however, had the highest affinity at the alpha-1D adrenoceptor.

In the rabbit aorta and the prostate phenylephrine elicited contractions with potencies (EC₅₀) of approximately 0.3 and 4 µM, respectively. The metabolite AM1 did not affect the phenylephrine-induced contraction in either tissue in concentrations up to 1 µM. In contrast, tamsulosin and its other metabolites caused concentration-dependent parallel shifts of the phenylephrine concentration-response curve to the right toward higher concentrations without affecting its maximal response. From these right shift, drug affinities (apparent pA₂ values) could be calculated, which are depicted in table 3.

	Discussion

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Presently, tamsulosin is the only alpha-1 adrenoceptor antagonist in clinical use that discriminates alpha-1 adrenoceptor subtypes (Testa et al., 1995; Foglar et al., 1995; Michel et al., 1996). Inasmuch as drug effects in vivo may result in part from their metabolites, it is important to know whether tamsulosin metabolites also are subtype-selective alpha-1 adrenoceptor antagonists. Studies with [¹⁴C]tamsulosin have suggested that unchanged tamsulosin accounts for 91% of the recovered radioactivity from plasma at C_max and for 74% of the area under the curve_0-, indicating that the metabolites do not play a major role for the in vivo effects of tamsulosin; the compounds M1, M2, M3, M4 and AM1 have been identified as major tamsulosin metabolites (Soeishi et al., 1996). To evaluate further a possible role of metabolites in the in vivo effects of tamsulosin, the present study has determined the potency of tamsulosin metabolites at alpha-1 adrenoceptors and their selectivity for alpha-1 adrenoceptor subtypes in the radioligand binding and functional assay system in comparison with the parent compound, tamsulosin. In the radioligand binding experiments, we have used the rat liver as a tissue containing a homogeneous population of the alpha-1B adrenoceptors (Han and Minneman, 1991; Büscher et al., 1996) and the rat kidney as a tissue containing a mixed population of multiple alpha-1 adrenoceptor subtypes (Michel et al., 1993); additionally, the cloned rat alpha-1A, alpha-1B and the alpha-1D adrenoceptors (Schwinn et al., 1990; Lomasney et al., 1991; Perez et al., 1994) were studied upon transient expression in COS cells. Whereas we have reported previously an excellent correlation between drug affinities at the rat liver and the cloned alpha-1B adrenoceptors for a large number of compounds (Büscher et al., 1996), in the present study drug affinities at the cloned alpha-1B adrenoceptor were generally somewhat lower than in the liver. Moreover, in the present study tamsulosin affinities at all cloned subtypes were somewhat lower than in our previous studies (Michel and Insel, 1994). Although we have no good explanation for these discrepancies, it should be noted that reported affinity estimates at the cloned subtypes underly a surprisingly large variation that considerably exceeds that in the native tissues (Michel et al., 1995). For the functional tests, phenylephrine-induced contractions were studied in a model of alpha-1A adrenoceptors, rabbit prostate (Testa et al., 1993, 1995). Additionally, we have used the rabbit aorta in which phenylephrine-induced contraction mainly occurs via an alpha-1A adrenoceptor in our hands (Honda et al., 1985b, 1987), but which has been demonstrated by other investigators to contain the multiple alpha-1 adrenoceptor subtypes (Vargas and Gorman, 1995).

The biochemically or functionally determined affinities of tamsulosin in the various models in the present study are consistent with values obtained in our laboratories in previous studies (Honda et al., 1985a,b; Michel et al., 1993; Büscher et al., 1996). Overall, the affinities observed at the cloned alpha-1 adrenoceptor subtypes are well within the range of values obtained in other laboratories (Perez et al., 1994; Horie et al., 1994; Foglar et al., 1995; Testa et al., 1995). Thus, in a balanced view of published data, tamsulosin appears to be 10- to 20-fold-selective for the alpha-1A relative to the alpha-1B adrenoceptors with intermediate affinities at the alpha-1D adrenoceptors.

The tamsulosin metabolites generally showed the rank order of potency tamsulosin  M4 > M1 > M2  M3  AM1 in the radioligand binding and functional assays. Thus, the metabolite M4 has an affinity at the alpha-1 adrenoceptors similar to that of tamsulosin itself and therefore might contribute to the sympatholytic effect of tamsulosin; this contribution, however, is unlikely to be large due to the low abundance of the metabolite. The metabolites M1, M2 and M3 have somewhat lower affinity for the alpha-1 adrenoceptors than tamsulosin; therefore, it may be expected that these metabolites contribute even less to the pharmacological in vivo profile in humans. The metabolite AM1 has only a negligible alpha-1 adrenoceptor affinity and is highly unlikely to contribute to the pharmacological tamsulosin effects in vivo.

Due to the high alpha-1 adrenoceptor affinity of some tamsulosin analogs, it is interesting to know whether these metabolites retain the subtype-selectivity profile of tamsulosin. Our data in the rat kidney demonstrate that indeed all tested tamsulosin analogs (except for the very low affinity AM1) are sufficiently subtype-selective to yield biphasic competition curves and to allow discrimination of the alpha-1 adrenoceptors in this tissue. However, it should be noted that the rat kidney most likely contains more than two alpha-1 adrenoceptor subtypes (Michel et al., 1993), and thus the high- and low-affinity sites in the rat kidney may not exactly reflect the alpha-1A and alpha-1B adrenoceptor affinities.

Our studies on the cloned alpha-1 adrenoceptor subtypes confirm that all tamsulosin analogs (except AM1) have significantly higher affinity for the alpha-1A relative to the alpha-1B adrenoceptors, and that the degree of selectivity for all of them is similar to that of tamsulosin itself. High-potency functional antagonism of the metabolites was also confirmed in two functional models, the rabbit aorta and prostate, which at least in our hands mainly involve the alpha-1A adrenoceptors (Honda et al., 1985b, 1987); however, the rabbit aorta may also involve other subtypes according to published data (Vargas and Gorman, 1995). Most tamsulosin analogs, similar to tamsulosin itself, have intermediate affinity for the alpha-1D adrenoceptors. A notable exception is M4, that has a higher affinity for the alpha-1D than for the alpha-1A adrenoceptors. M4 also differs from the other compounds of this study, because it is the only compound in which the benzenesulfonamide rather than the phenoxyring has been modified. In particular, in the M4 the methoxy group of the benzenesulfonamide ring has been replaced by a hydroxy group, yielding a more catecholamine-like structure. Thus, the M4 has certain similarities with the endogenous catecholamines adrenaline and noradrenaline that also are somewhat selective for the alpha-1D relative to the alpha-1A and alpha-1B adrenoceptors among the rat or the human alpha-1 adrenoceptor subtypes (Forray et al., 1994; Laz et al., 1994; Michel and Insel, 1994; Schwinn et al., 1995). From these data, it can be hypothesized that the alpha-1A/alpha-1B adrenoceptor selectivity of tamsulosin is encoded in the phenoxy ring moiety of the molecule and that this selectivity is not affected by the additional hydroxylation or substitution of the ethoxy by a methoxy goup. In contrast, hydroxylation of the benzenesulfonamide moiety selectively increases the alpha-1D adrenoceptor affinity of the molecule. Whether tamsulosin and its metabolites functionally behave as an alpha-1D adrenoceptor antagonist has not been tested directly to our knowledge. However, tamsulosin is a high-potency antagonist for the contraction of the rat aorta (Eltze, 1994; van der Graaf et al., 1996), a bona fide model of alpha-1D adrenoceptors (Vargas and Gorman, 1995.

In conclusion, we have demonstrated that most tamsulosin metabolites are high-affinity antagonists at the alpha-1 adrenoceptors and retain the subtype-selectivity profile of their parent compound, tamsulosin.