Department of Biopharmacy, School of Pharmaceutical Sciences,
University of Shizuoka, Shizuoka, Japan (S.Y., T.O., R.K.);
Department of Biopharmaceutics, Hokkaido College of
Pharmacy (Y.D.); and Central Pharmaceutical Research Institute, Japan
Tobacco, Inc., Osaka, Japan (Y.S., T.K., K.A.)
 |
Introduction |
1-Adrenoceptor
antagonists are effective therapeutic agents for urinary obstruction in
patients with benign prostatic hypertrophy (BPH1). However, prazosin often produces
orthostatic hypotension as a side effect, due to a reduction in
peripheral resistance mediated by blockade of the vascular
1-adrenoceptors. Previous studies have shown
that the 1A-adrenoceptor subtype mediates the
contractile response to noradrenaline in prostatic smooth muscles
(Lepor et al., 1993
; Price et al., 1993; Forray et al., 1994; Chapple,
1996). In addition, it has been shown that the
1L-adrenoceptor subtype is involved in
contraction of the prostate (Muramatsu et al., 1994; Takahashi et al.,
1999). On the other hand, the 1B-adrenoceptor subtype mediates the contraction of vascular tissues produced by
noradrenaline (Hatano et al., 1994).
JTH-601 (3-{N-[2-(4-hydroxy-2-isopropyl-5-methylphenoxy)ethyl]- N-methylaminomethyl}-4-methoxy-2,5,6-trimethylphenol
hemifumarate) is a novel 1-adrenoceptor
antagonist having a relatively higher affinity for both
1L- and
1A-adrenoceptors than
1B-adrenoceptors (Muramatsu et al., 1996;
Suzuki et al., 1999, 2000a). Among several metabolites of JTH-601,
JTH-601-G1 (JTH-601 
-D-glucopyranosyl uronic
acid) and JTH-601-S1 (JTH-601 hydrogen sulfate) (Fig.
1) have been shown to be
pharmacologically active metabolites mainly by in vitro functional
assays (Takahashi et al., 1999; Suzuki et al., 2000b). However, the
disposition and in vivo binding characteristics of JTH-601 and its
metabolites in the prostate have not been investigated in detail. The
in vivo receptor binding characteristics of JTH-601 and its metabolites
in relation to their pharmacokinetics may be important for a thorough
understanding of the pharmacological effects of JTH-601. The aim of the
present study was to characterize the disposition and
1-adrenoceptor binding of JTH-601 and its metabolites in the rat prostate and other tissues under in vivo conditions.
| |
Materials and Methods |
Chemicals.
[3H]Tamsulosin
([3H]YM617, 2.08 TBq/mmol),
[3H]JTH-601 (1.04 TBq/mmol), and
[3H]JTH-601-G1 (1.37 TBq/mmol) were synthesized
by Amersham International PLC (Buckinghamshire, England). JTH-601 and
its metabolites were chemically synthesized by Japan Tobacco Inc.
(Osaka, Japan). All other chemicals were purchased from commercial sources.
Animals.
Male Sprague-Dawley rats weighing about 200 g were obtained from
Japan SLC Inc. (Shizuoka, Japan) and housed three to four per cage in
the laboratory with free access to food and water and maintained on a
12-h dark/light cycle in a room with controlled temperature (24 ± 1°C) and humidity (55 ± 5%).
In Vitro Binding Assay.
The binding of [3H]tamsulosin in rat tissues
was measured using a previously described method (Yamada et al., 1994).
Rat prostate, submaxillary gland, and spleen were homogenized using a
Kinematica (Lucerne, Switzerland) Polytron homogenizer in 20 to
30 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.5). The homogenates
were then centrifuged at 40,000g for 20 min; the pellets
were resuspended in the ice-cold buffer; and the suspension was
centrifuged at 40,000g for 20 min. The resulting pellet was
suspended in the buffer for the binding assay. All steps were performed
at 4°C. The tissue homogenates (5-10 mg wet tissue weight) were
incubated with [3H]tamsulosin in 50 mM Tris-HCl
buffer. Incubation was carried out for 30 min at 25°C. The reaction
was terminated by rapid filtration (Cell harvester; Brandel,
Gaithersburg, MD) through a Whatman GF/B glass filter, 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 the 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 by liquid scintillation counter. Specific
binding of each ligand was determined experimentally from the
difference between counts in the absence and presence of 10 µM
phentolamine. All assays were conducted in duplicate.
In Vivo [3H]Tamsulosin Binding.
In vivo measurement of specific [3H]tamsulosin
binding in rat tissues was performed as described previously (Yamada et
al., 1999). Rats were anesthetized with diethyl ether, and JTH-601 (6.5-2176 nmol/kg), JTH-601-G1 (168-1678 nmol/kg), and JTH-601-S1 (204-2038 nmol/kg) were injected together with
[3H]tamsulosin (555 kBq, 1.3 nmol/kg) into the
femoral vein of rats. The animals were allowed to recover and were then
killed by taking blood from the descending aorta under temporary
anesthesia with diethyl ether 10 min after injection. The prostate,
aorta, submaxillary gland, and spleen were rapidly removed, and each
tissue was homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.5), to
give a final tissue concentration of 10 mg/ml, using a Kinematica
Polytron homogenizer. Particulate-bound radioactivity was determined by
rapid filtration over Whatman GF/C glass filters, which were washed
subsequently in 2 ml of ice-cold buffer. The particulate-bound
radioactivity was measured by liquid scintillation counter after
addition of scintillation fluids. Similarly,
[3H]tamsulosin was injected into rats given
vehicle and phentolamine (62.9 µmol/kg i.p. 0.5 h of
pretreatment) to determine total and nonspecific binding, respectively,
and the difference was taken to represent the in vivo specific
[3H]tamsulosin binding.
Measurement of [3H]JTH-601 and
[3H]JTH-601-G1 in Plasma and Prostate.
To determine the concentration of [3H]JTH-601
and [3H]JTH-601-G1 in plasma and prostate, rats
received [3H]JTH-601 (555 kBq, 2.4 nmol/kg) and
[3H]JTH-601-G1 (555 kBq, 2.0 nmol/kg). Then,
10, 60, and 120 min later, the blood was taken from the descending
aorta, and the prostate was removed. Plasma was isolated from blood by
centrifugation. The plasma and prostate were stored at 
80°C until
analysis. Concentrations of [3H]JTH-601 and
[3H]JTH-601-G1 in plasma and prostate were
determined by the high performance liquid chromatography (HPLC) method.
Briefly, the plasma and prostate homogenate, after the addition of
methanol, were centrifuged at 3000 rpm for 10 min at 4°C. The
supernatant was evaporated to dryness under reduced pressure. In the
case of prostatic homogenate, the pellet after centrifugation at 3000 rpm was suspended with methanol and then centrifuged at 10,000 rpm for
10 min. The supernatant was combined with the initial supernatant.
After evaporation, the residue was dissolved in 100 µl of solvent A
(20 mM phosphate buffer:methanol = 9:1), filtered by a hydrophilic
polytetrafluoroethylene membrane (samprep-LCR4(T)LH, pore size: 0.5 µm, Nihon Millipore Ltd., Tokyo, Japan), and 50 µl of the
solution was injected into the HPLC system. The HPLC system consisted
of a pump (880-PU, Jasco, Tokyo, Japan), a 7125 syringe loading
injector (Rheodyne Inc., Cotati, CA), a UV detector (875-UV, Jasco), a
171 radioisotope detector (Beckman Instruments Inc., Fullerton, CA),
and a stainless steel column. The column consisted of STR ODSII
(Shimadzu, 250- × 4.6-mm, i.d.) and Guard Cartridge CAPCELL C18 UG120
(Shinseido, 10- × 4-mm, i.d.). The column temperature was maintained
at 40°C. Gradient elution was performed using mobile phases
consisting of solvent A and solvent B (1:9) of 20 mM phosphate buffer
(pH 7.0) and methanol. After eluting with solvent A for 5 min, linear
gradient elution was performed going from solvent A to solvent B over
60 min. The solvent flow rate was 1.0 ml/min. The UV absorbance at 285 nm and the radioactivity were monitored.
Total Radioactivity and in Vivo Specific Binding of
[3H]JTH-601 and [3H]JTH-601-G1.
Measurement of the in vivo specific binding of
[3H]JTH-601 and
[3H]JTH-601-G1 was performed as described above
for the in vivo measurement of specific binding of
[3H]tamsulosin (Yamada et al., 1999). At 10 and
60 min after i.v. injection of [3H]JTH-601 (555 kBq, 2.4 nmol/kg) and [3H]JTH-601-G1 (555 kBq,
2.0 nmol/kg), rat prostate, vas deferens, aorta, cerebral cortex,
submaxillary gland, spleen, heart, lung, liver, and kidney were rapidly
removed. Each tissue was homogenized in ice-cold 50 mM Tris-HCl buffer
to give a final concentration of 10 mg/ml. Aliquots of homogenate (1 ml) were used to measure the total radioactivity. Also,
particulate-bound radioactivity was determined by rapid filtration of 1 to 3 ml of homogenate over Whatman GF/C glass filters, which were
washed subsequently with 2 ml of ice-cold buffer. Total and
particulate-bound radioactivity were measured by liquid scintillation
counter after addition of scintillation fluid. In this case, the
particulate-bound radioactivity of [3H]JTH-601
and [3H]JTH-601-G1 in each tissue from rats
given vehicle and phentolamine (62.9 µmol/kg i.p. 0.5-h pretreatment)
was defined as the total and nonspecific binding, respectively, and the
difference was determined to be the in vivo specific binding of each radioligand.
Data Analysis.
Analysis of the in vitro binding data was performed as described
previously (Yamada et al., 1980). The ability of JTH-601 and its
metabolites to inhibit specific [3H]tamsulosin
(0.3 nM) binding in rat prostate, submaxillary gland, and spleen was
estimated by the IC50 values, which are the molar concentration of unlabeled drug able to displace 50% of the specific binding (estimated probit analysis). A value for the inhibition constant, Ki, was calculated from the
equation Ki = IC50/(1 + L/Kd), where L
equals the concentration of radioligands. The dose (ID50) of JTH-601 and its metabolites that
inhibited specific [3H]tamsulosin binding by
50% was determined by fitting curve of specific binding (expressed as
a percentage of the control-specific binding without treatment of
1-adrenoceptor antagonists) in each tissue
versus the dose of injected antagonists using the nonlinear least-squares program and the single site receptor model as follows: Bi = B0 B0 × [D]/(ID50 + [D]), where
Bi is the specific binding in the presence
of 1-adrenoceptor antagonists,
B0 is the curve fitted estimate of the
maximal specific binding of [3H]tamsulosin in
rat tissues, and [D] is the injected dose of antagonists. Statistical analysis of data was performed by Student's t
test and a value of P < .05 was considered significant.
| |
Results |
In Vitro and in Vivo Inhibitory Effect on
1-Adrenoceptor Binding in Rat Tissues.
JTH-601 (1-100 nM), JTH-601-G1 (1-100 nM), and JTH-601-S1 (1-100 nM)
competed in a concentration-dependent manner with specific [3H]tamsulosin binding in the prostate,
submaxillary gland, and spleen of rats. As shown in Table
1, the Ki
values for JTH-601-G1 and JTH-601-S1 in each tissue were 2.5 to 6.4 times greater than the value for JTH-601. The
Ki value for JTH-601 in the prostate was
similar to that in the submaxillary gland, and the values for JTH-601
and JTH-601-G1 were 2.0 and 3.4 times, respectively, lower in the
prostate than in the spleen.
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TABLE 1
In vitro inhibition by JTH-601, JTH-601-G1, and JTH-601-S1 of specific
[3H]tamsulosin binding in rat tissues
The competitive inhibition by JTH-601 and its metabolites of specific
[3H]tamsulosin binding was examined in rat tissues in vitro.
Each value represents mean ± S.E. of three to four rats.
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A constant amount of [3H]tamsulosin (1.3 nmol/kg, i.v.) was coinjected with increasing doses of JTH-601,
JTH-601-G1, and JTH-601-S1 in rats. Intravenous injection of these
compounds inhibited in a dose-dependent manner the in vivo specific
[3H]tamsulosin binding in particulate fractions
of the prostate, aorta, submaxillary gland, and spleen. Figure
2 illustrates the dose-dependent
inhibition curves in the rat prostate. The ID50 values for JTH-601, JTH-601-G1, and JTH-601-S1 differed both in terms
of the drugs and tissues studied (Table
2). The ID50 values for JTH-601-G1, compared with that for JTH-601, were 1.9 to 2.9 times
smaller in rat prostate, aorta, submaxillary gland, and spleen.
Conversely, the ID50 values for JTH-601-S1 were
1.3 to 3.2 times greater than those for JTH-601 in each tissue except spleen, which was 1.5 times smaller. To examine tissue selectivity or
1-adrenoceptor subtype selectivity of JTH-601
and its metabolites in vivo, we compared the ratios of their
ID50 values in rat tissues. The ratios of
ID50(aorta) to
ID50(prostate) of JTH-601, JTH-601-G1, and
JTH-601-S1 were 1.10, 0.80, and 0.53, respectively, and the ratios of
ID50(spleen) to
ID50(prostate) were 0.48, 0.36, and 0.12, respectively. The ratios of ID50(spleen) to
ID50(submaxillary gland) were 0.63, 0.44, and
0.13, respectively.
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TABLE 2
In vivo inhibition by JTH-601, JTH-601-G1, and JTH-601-S1 of specific
[3H]tamsulosin binding in rat tissues
JTH-601 (6.5-2176 nmol/kg), JTH-601-G1 (168-1678 nmol/kg), and
JTH-601-S1 (204-2038 nmol/kg) were injected together with
[3H]tamsulosin (555 kBq, 1.3 nmol/kg) into the femoral vein
of rats, and 10 min later, specific [3H]tamsulosin binding
was measured. Each value represents mean ± S.D. of 24 to 30 rats.
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Concentrations of [3H]JTH-601 and
[3H]JTH-601-G1 in Plasma and Prostate.
At various times (10, 60, and 120 min) following i.v. injection of
[3H]JTH-601 and
[3H]JTH-601-G1 at similar doses (2.4 and 2.0 nmol/kg, respectively), the radioactivity in plasma and prostate of
rats was identified exclusively as the unchanged form of each
radioligand, except for the appearance of a low concentration of
[3H]JTH-601-G1 in the plasma 10 min after
injection of [3H]JTH-601 (Table
3). When measured 10 min after i.v.
injection of each radioligand, the plasma concentration of
[3H]JTH-601-G1 was 3 times higher than that of
[3H]JTH-601. At 60 min, the plasma
concentrations of both radioligands were markedly reduced. In contrast,
the concentration of [3H]JTH-601 in the
prostate at 10 min was 3 times higher than that of
[3H]JTH-601-G1.
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TABLE 3
Concentrations of [3H]JTH-601 and [3H]JTH-601-G1 in
plasma and prostate of rats after i.v. injection of each radioligand
[3H]JTH-601 (555 kBq, 2.4 nmol/kg) and
[3H]JTH-601-G1 (555 kBq, 2.0 nmol/kg) were injected into the
femoral vein of rats, and 10, 60, and 120 min later, concentrations of
both radioligands in plasma and prostate were determined. Each value
represents mean ± S.E. of three to four rats.
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Total Radioactivity and in Vivo Specific Binding of
[3H]JTH-601 and [3H]JTH-601-G1 in Rat
Tissues.
At 10 and 60 min after i.v. injection of
[3H]JTH-601 (2.4 nmol/kg) and
[3H]JTH-601-G1 (2.0 nmol/kg), the total
radioactivity and in vivo specific binding in rat tissues were
measured. In the prostate, cerebral cortex, submaxillary gland, spleen,
heart, lung, and liver, the total radioactivity at 10 min after i.v.
injection of [3H]JTH-601-G1 was significantly
lower than that after [3H]JTH-601 (Fig.
3). The radioactivity at 60 min after the
injection of both radioligands was considerably reduced in all tissues, and the radioactivity after [3H]JTH-601-G1 was
much lower than that after [3H]JTH-601.
We showed previously that the difference in the particulate-bound
radioactivity of tissues from rats pretreated with vehicle and
phentolamine (62.9 µmol/kg i.p.) after i.v. injection of
[3H]tamsulosin represented the in vivo specific
binding of the radioligand to 1-adrenoceptors
(Yamada et al., 1999). Thus, the in vivo specific binding of
[3H]JTH-601 and
[3H]JTH-601-G1 in tissues was measured at 10 and 60 min after i.v. injection of each radioligand (2.4 and 2.0 nmol/kg, respectively) in the rats pretreated with vehicle and
phentolamine (62.9 µmol/kg i.p.). As shown in Fig.
4, in vivo specific binding of
[3H]JTH-601 at 10 min was observed in each
tissue except the aorta, which exhibited little specific binding, and
the degree of binding was relatively higher in the heart, lung, and
kidney. In vivo specific binding of
[3H]JTH-601-G1 was also observed in each tissue
except the cerebral cortex, and the degree of binding in the
submaxillary gland, heart, lung, and kidney was significantly less than
that of [3H]JTH-601. Interestingly, there was a
similar degree of specific binding of both radioligands in the
prostate, vas deferens, and liver. Sixty minutes later, the in vivo
specific binding of [3H]JTH-601 was
considerably reduced in all tissues except the vas deferens and
prostate, both of which showed no or only a relatively small reduction.
Specific binding of [3H]JTH-601-G1 was more
markedly reduced than that of [3H]JTH-601.
| |
Discussion |
The 1-adrenoceptor binding of JTH-601 and
its metabolites in rat tissues was investigated. JTH-601, JTH-601-G1,
and JTH-601-S1 inhibited specific
[3H]tamsulosin binding in the rat prostate,
submaxillary gland, and spleen in vitro, and the inhibitory effect of
JTH-601 was greater than that of JTH-601-G1 and JTH-601-S1. Moreover,
the inhibitory effects of JTH-601 and JTH-601-G1 were more potent in
the prostate than in the spleen. Inasmuch as JTH-601-G1 and JTH-601-S1,
albeit with a lower affinity than JTH-601, bind to 1-adrenoceptors in rat tissues, it is
considered that both metabolites may contribute to the pharmacological
effect of JTH-601 in vivo.
We have previously shown that [3H]tamsulosin is
a useful radioligand for evaluating novel
1-adrenoceptor antagonists in terms of tissue
selectivity and 1-adrenoceptor subtype
selectivity under in vivo conditions (Yamada et al., 1999). Intravenous
injection of JTH-601, JTH-601-G1, and JTH-601-S1 inhibited in vivo
specific [3H]tamsulosin binding in particulate
fractions of rat prostate, aorta, submaxillary gland, and spleen.
Compared with the values for JTH-601, the ID50
values for JTH-601-G1 in these tissues were smaller and those for
JTH-601-S1 were greater in tissues except the spleen. Thus, it appears
that JTH-601, JTH-601-G1, and JTH-601-S1 bind to
1-adrenoceptors in rat tissues in vivo and the
binding affinity of JTH-601-G1 is higher than that of JTH-601 and
JTH-601-S1. Such in vivo data appear to contrast with the in vitro
situation where JTH-601 has higher
1-adrenoceptor binding affinity than JTH-601-G1. Although we have no precise explanation for this
discrepancy, there may be some difference between these compounds in
terms of their pharmacokinetics and
1-adrenoceptor binding characteristics under
in vivo conditions.
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).
It has been reported that the prostate and submaxillary gland of rats contain predominantly 1A subtype, whereas the
spleen and liver contain the 1B subtype (Han
et al., 1987; Michel et al., 1989; Han and Minneman, 1991; Testa et
al., 1993). Therefore, to evaluate the in vivo tissue selectivity or
1-adrenoceptor subtype selectivity of JTH-601
and its metabolites, it may be useful to compare the ratio of
ID50 values for both agents with the value for
prazosin among different tissues. Our previous study showed that the
ID50 values for prazosin inhibition of in vivo
[3H]tamsulosin binding were 72.8, 12.6, 45.6, and 4.87 nmol/kg, respectively, in rat prostate, aorta, submaxillary
gland, and spleen (Yamada et al., 1999). Based on the ratios of
ID50(spleen) to
ID50(prostate) or
ID50(submaxillary gland), JTH-601, JTH-601-G1, and JTH-601-S1 were shown to exhibit 5.7 to 6.9, 4.0 to 5.1, and 1.2 to
1.7 times greater 1-adrenoceptor selectivity
than prazosin in the prostate and submaxillary gland versus the spleen.
Similarly, they exhibited 6.5, 4.7, and 3.1 times greater
1-adrenoceptor selectivity than prazosin in
the prostate versus the aorta. Consequently, these data are probably
the first in vivo evidence that JTH-601 and JTH-601-G1 bind to the
1-adrenoceptor subtype with higher affinity in
the prostate and submaxillary gland than in the spleen and aorta; thus,
they provide a rationale for the pharmacological specificity showing
that JTH-601 and JTH-601-G1 are more effective antagonists of the
1-agonist-induced increase in urethral
pressure compared with blood pressure in anesthetized rabbits and dogs (Suzuki et al., 1999, 2000a).
The disposition and in vivo 1-adrenoceptor
binding of JTH-601 and JTH-601-G1 in rat tissues were further examined
by using radioligands with high specific activity. The total
radioactivity after i.v. injection of
[3H]JTH-601 and
[3H]JTH-601-G1 differed among tissues, and the
radioactivity in most rat tissues at 10 and 60 min after
[3H]JTH-601-G1 was considerably lower than that
after [3H]JTH-601. The lower tissue
radioactivity seems to be due mainly to the relatively higher
hydrophilicity of the metabolite. In contrast, the plasma concentration
of [3H]JTH-601-G1 was 3 times higher than that
of [3H]JTH-601 10 min after i.v. injection in
rats, and 60 min later, the plasma concentration of JTH-601-G1 had
fallen to one-fourth the concentration of
[3H]JTH-601. The fast elimination of
[3H]JTH-601-G1 from the circulation may be
ascribable to its high clearance. Specific binding of
[3H]JTH-601 and
[3H]JTH-601-G1 was observed in particulate
fractions of rat prostate and other tissues 10 min after i.v. injection
of each radioligand. Although the in vivo specific binding of
[3H]JTH-601-G1 was significantly lower than
that of [3H]JTH-601 in most rat tissues, there
was comparable binding between these radioligands in the prostate and
vas deferens. This observation is of interest because the concentration
of [3H]JTH-601-G1 in the prostate after i.v.
injection was 3 times lower than that of
[3H]JTH-601. Taken together, these findings
allow us to speculate that JTH-601-G1, compared with the parent
compound, exhibits a very high affinity to prostatic
1-adrenoceptors in vivo. This coincides with
the higher potency of JTH-601-G1 than that of JTH-601 in competitive
inhibition of in vivo [3H]tamsulosin binding in
the prostate. Specific binding of [3H]JTH-601
at 60 min after i.v. injection, compared with that at 10 min, was
considerably reduced in all rat tissues except the prostate and vas
deferens, both of which showed relatively sustained binding. This
observation is in reasonable agreement with the ex vivo binding data
showing that oral administration of JTH-601 produces a long-lasting
blockade of 1-adrenoceptors in the rat prostate (Ohkura et al., 1999).
In conclusion, the present study has shown that JTH-601 and its
metabolites bind to 1-adrenoceptors in rat
tissues in vivo and that JTH-601-G1 retains the prostatic
1-adrenoceptor subtype selectivity of the
parent compound.
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1-Adrenoceptor
Binding Characteristics of JTH-601 and Its Metabolites in Rat Tissues
Top
Abstract
Introduction
),
JTH-601-G1 (
) and JTH-601-S1 (
) of specific
[3H]tamsulosin binding in the rat prostate.
).
