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Introduction
Male erectile dysfunction (MED), the persistent inability to achieve or maintain an erection for satisfactory sexual
performance, is a common and important medical
problem. Sildenafil is a potent and selective inhibitor of the human
cGMP-specific phosphodiesterase type 5 (PDE5), the predominant isozyme responsible for the metabolism of cGMP in the corpus
cavernosum of the penis[1_4]. The development of sildenafil citrate (Viagra) as an effective and orally active agent for the
treatment of MED revolutionized the treatment of this
disease[5,6]. Despite the efficacy of sildenafil as a treatment for MED,
there are some notable drawbacks associated with its
use. Clinically significant adverse effects, such as headache, facial
flushing, dyspepsia, and visual disturbance have been
reported[1,7,8].
5-Ethyl-2-{5-[4-(2-hydroxy-ethyl)-piperazine-1-sulfonyl]-
2-propoxy-phenyl}-7-propyl-3,5-dihydro-pyrrolo(3,2-
d)pyrimidin-4-one (SK-3530; Figure 1) is a novel PDE5
inhibitor that was synthesized to alleviate the drawbacks of
sildenafil, and is now being developed as a new drug
candidate. Herein, we describe the pharmacokinetics and
tissue distribution of SK-3530 in rats using the
radioisotope-labeled compound as part of the pharmacological preclinical
study of SK-3530.
Materials and methods
Chemicals and reagents 14C-labeled SK-3530 (specific
activity 93.52 µCi/mg) was synthesized at Korea
Radiochemicals Center (Suwon, Korea) with radiochemical
purity ³98% as judged by HPLC-radiochromatography.
Unlabeled SK-3530 was provided by SK Chemical Ltd (Gunpo,
Korea) with a chemical purity of 99%. Polyethylene glycol
(PEG, average molecular weight 300), sodium hydroxide
(NaOH), and ammonium formate were obtained from Sigma
Chemical Co (St Louis, MO, USA). Ethyl acetate (EtOAc),
acetonitrile, and methanol were of HPLC grade and purchased
from Mallinckrodt Baker (Phillipsburg, NJ, USA).
Animals Male Sprague-Dawley rats weighing 210_250 g
were purchased from DaeHan Laboratory Animal Research
Center (Taejeon, Korea). The animals were housed at 23±
2 ºC temperature and moisture [55%±10%]-controlled
room, with a 12 h light/dark cycle, and allowed free access
to food and water.
Preparation of dosing solution Appropriate
quantities of 14C-labeled SK-3530 were diluted with cold
SK-3530 to adjust the specific activity required for the dose
preparation (6 µCi for pharmacokinetics and 2 µCi for tissue
distribution). SK-3530 was dissolved in PEG solution (50%
in water).
Pharmacokinetic experiment Two days before
adminis-tration, the femoral artery and vein intravenous (iv) only
were cannulated using polyethylene-50 and
polyethylene-10 tubing (Becton Dickinson, Lincoln Park, NJ, USA). The
cannulae were fixed to the back of the neck. The rats were
fasted overnight before drug administration and until 6 h
after dosing. For the iv experiment, the rats
(n=4) were given a single 5 mg/kg bolus of
14C-labeled SK-3530. Heparinized samples of blood (0.4 mL) were collected at 1, 5, 10, 30, 60, 90,
120, 180, 240, 360, 480, and 600 min post-dose. For the oral
experiments, the rats (n=4) were given a single dose of 10, 20,
and 40 mg/kg, respectively. Then the blood samples were
collected at 10, 20, 30, 60, 90, 120, 180, 240, 360, 480, 600, and
720 min post-dose. Plasma was harvested after
centrifugation and stored frozen at -20 ºC until analyzed.
Distribution The rats (n=4 per group) were dosed with
[14C]SK-3530 orally at 40 mg/kg. At 1, 4, and 24 h after dosing,
each animal was lightly anaesthetized with diethyl ether.
Blood was collected by heart puncture and the rats were
then killed by cervical dislocation. The representative
tissues or organs were rapidly dissected, washed with saline,
weighed, and patted dry on a combustion pad in preparation
for sample oxidation.
Radioactivity measurements Plasma was mixed with 10
mL scintillation fluid (Insta Gel XF, Packard, Meriden, CT,
USA) and counted directly for radioactivity.
Aliquots of tissue samples were weighed and combusted in a sample
oxidizer (Tri-Carb Model 307, Packard, Meriden, CT,
USA). The resulting
14CO2 was absorbed on Carbosorb (Packard,
Meriden, CT, USA) and then mixed with Permafluor V
scintillation fluid (Packard, Meriden, CT, USA) The radioactivity
of samples was counted using a liquid scintillation counter
(Packard, USA).
LC-MS/MS analysis To the 100 µL plasma sample we
added 5 µL internal standard solution (sildenafil 2 mg/L in
methanol), 100 µL of 50 mmol/L NaOH, and 700 µL
EtOAc. After voltex-mixing and centrifugation, the supernatant was
dried under nitrogen evaporation. The residue was dissolved
in HPLC initial buffer and then analyzed by
LC-MS/MS. The LC-MS/MS system consisted of a LC-10ADvp binary pump
system with an API2000 triple quadruple mass spectrometer
(Applied Biosystems-SCIEX, Concord, Canada) equipped
with a turboionspray source. The HPLC mobile phases
consisted of 5 mmol/L ammonium formate buffer (pH 6.0) and
5 mmol/L ammonium formate in 90% acetonitrile.
Chromatographic separation was achieved on a Capcellpak
Phenyl (1.5×150 mm, 5 µm, Shiseido, Tokyo, Japan ) using a
isocratic elution of 80% solvent at a flow rate of 0.15
mL/min. The run time was 3.5 min. Electrospray
ionization was performed in the positive mode with nitrogen as
the nebulizing turbo spray and curtain gas, with optimum
values set at 40, 80, and 40 (arbitrary units).
Multiple reaction monitoring (MRM) detection was employed using
nitrogen as the collision gas (4 arbitrary units) with a dwell
time of 150 ms for each transition; the transitions
monitored were 532®99 for SK-3530 and
475®58 for sildenafil (IS). The representative MRM chromatograms for SK-3530
and sildenafil in the rat plasma sample are presented in
Figure 2.
The calibration standards for SK-3530 were prepared by
adding various amounts of SK-3530 to 100 µL rat blank
serum and analyzed in the same manner as the plasma samples
from the dosed rats. The calibration curves were prepared
by plotting peak area ratios of SK-3530/sildenafil (IS) against
the SK-3530 concentration and analyzed by the linear
least-squares regression analysis. The calibration curve for
SK-3530 was linear over the concentration range of
5_5000 µg/L with correlation coefficients
(r2) more than 0.99.
For the validation of the LCMS/MS method, precision
and accuracy were determined by repeated analysis of 3
concentration levels of quality control samples (5, 200, and 5000
µg/L, n=3) on 3 separate days. The limit of quantification
was 5 µg/L. The accuracy of the method was more than 90%.
The inter-day precisions (as the percentage relative
standard deviation) were 10.2%, 0.7%, and 4.2% at 5, 200, and
5000 µg/L, respectively).
Pharmacokinetic analysis The area under the plasma
concentrationtime curve (AUC), total plasma clearance,
terminal half-life, and volume of distribution at steady-state
(Vdss) were determined by a non-compartmental method
using WinNonlin (Scientific Consulting, Lexington, KY,
USA). Maximum plasma concentration
(Cmax) and the corresponding
Tmax were reported as observed. Oral bioavailability
was calculated as follow:
Bioavailability
(%)=(AUCoral/doseoral)×(dose
iv/AUCiv)×100.
Protein binding assay Plasma protein binding was
determined by ultrafiltration using Amicon centrifuge
micropartition devices (Amicon, Beverly, MA, USA;
molecular weight cut-off, 30 000). [14C]SK-3530 was added
to the blank rat and human plasma samples
(n=4) to yield final concentrations of 1, 5, 50, and 100 µmol/L and
incubated for 10 min at 37 °C. After incubation, the mixtures
were added to the ultrafiltration units and centrifuged at
37 °C for 30 min at 14 000×g. The concentrations of
[14C]SK-3530 in the plasma and ultrafiltrate were determined
by liquid scintillation spectrometry. For the ex vivo
determination of plasma protein binding, a 40 mg/kg dose of
[14C]SK-3530 was orally administered and blood samples
were drawn at 1, 4, and 24 h after dosing. The plasma
samples were then prepared and protein binding was
analyzed as described earlier.
Results
Plasma concentrations of total radioactivity and SK-3530
The mean arterial plasma concentration-time profiles of total
radioactivity and SK-3530 after iv administration at a dose of
5 mg/kg in the rats are shown in Figure 3. After iv
administration of [14C]SK-3530, the parent SK-3530 was rapidly cleared
with a terminal half-life of 0.37 h and was no longer
detected at 4 h after dosing,
whereas the radioactive equivalents in the plasma declined more slowly. Total
radioactivity was eliminated in a biphasic fashion with a terminal half-life of
4.7 h. The AUC of the parent SK-3530 was only 17%
compared with the AUC of the total radioactivity. The
Vdss was higher than the volume of body water at 5.7 and 3.2 L/kg for
the total radioactivity and parent SK-3530, respectively.
Clearance was measured as 21.1 and 102
mL·min-1·kg-1 for the
total radioactivity and parent SK-3530, respectively.
The mean arterial plasma concentration-time profiles of
the total radioactivity and SK-3530 following oral
administration at doses of 10, 20, and 40 mg/kg in the rats are shown
in Figure 4. After the oral administration of
[14C]SK-3530, the drug was absorbed; the peak concentration of the total
radioactivity occurred within 1.3_1.7
h. The parent
SK-3530 peaked at 1.2_1.7 h, which was similar to the total
radioactivity profile, but declined more
rapidly. Increases in the
Cmax and AUC of the total radioactivity were almost
proportional to the administered dose, suggesting that SK-3530
has linear pharmacokinetics. However, in the case of the
arent SK-3530, the AUC increased at disproportionately
higher rates compared to the administered doses.
These phenomena seem to be due to metabolic saturation at the
higher SK-3530 doses. The AUC of the
parent compound was 6.8%_10.6% compared with that of the
total radioactivity profile. The
oral bioavailabilities based on the total
radioactivity were estimated to be 60.4%, 62.2%, and 69.9% for 10,
20, and 40 mg/kg doses, respectively. The
oral bioavailabi-lities of the parent SK-3530 were
estimated to be 24.1%,
30.1%, and 43.4% for 10, 20, and 40 mg/kg doses,
respectively. The pharmacokinetic parameters of the total radioactivity
and parent SK-3530 are summarized in Tables 1 and 2.
Plasma binding The in vitro incubation of
[14C]SK-3530 with rat and human plasma samples resulted in high protein
binding rates of more than 97%; binding
was concentration-independent over the range tested (1_100
µmol/L), with no difference between the species show. An
in vivo plasma analysis revealed that more than 98% of the radioactivity (98.3%,
99.0%, and 98.5% for samples collected at 1, 4, and 24 h
post-dose, respectively) was associated with the plasma protein.
Tissue distribution The tissue distribution of the total
radioactivity after a single oral administration of
[14C]SK-3530 at a dose of 40 mg/kg is shown in Table
3. Radioactivity was widely
distributed into all tissues. The tissue/plasma
radioactivity ratio at 1 h after administration was calculated
to be 0.15_5.4 with the exception of the excretory
organs. The concentrations of the total radioactivity for several
representative tissues are as follows: 13.0 µg equivalents/g in
the adrenal glands, 20.0 µg equivalents/g in the
lungs, 53.1 µg equivalents/g in the liver, 6.2 µg equivalents/g in the
heart, and 12.5 µg equivalents/g in the
kidneys. At 4 h post-dose, the radioactivity of several tissues slightly increased or
decreased, but on the whole, no significant changes of
radioactivity were shown compared to the radioactivity profile
at 1 h post-dose. The radioactivity decreased in most
tissues over a 24 h period, but was still detectable with
relatively high penetration shown in the thyroid, liver, and
brain. Notably, the radioactivity of the brain gradually increased
with time up to 24 h post-dose.
Discussion
After the oral administration of [14C]SK-3530 in the rats,
the maximum plasma concentration of the total radioactivity
was achieved around 1 h post-dose, showing a moderate
absorption rate of SK-3530 from the gastrointestinal
tract. The Cmax and AUC of the total radioactivity increased almost
proportionally to the dose, indicating that SK-3530 has
linear kinetics over the dose range of 10_40
mg/kg. However, the AUC of the parent SK-3530 showed a hyperbolic pattern
with increasing doses, suggesting that metabolic saturation
occurred with high orally administered doses.
The oral bioavailability, based on the total radioactivity,
was estimated to be between 60% and 70%, whereas that of
the parent SK-3530 was calculated to be between 20% and
40%, suggesting a considerable amount of SK-3530 was
eliminated through an extensive first-pass metabolism, such as
sildenafil[9,10]. The low bioavailability of sildenafil after oral
administration has been reported to be mainly due to a
considerable intestinal first-pass
effect[11], which can consequently be considered as a major cause for the low
bioavaila-bility of SK-3530 considering their structural similarity.
SK-3530 is modified from sildenafil at N- or
O-side chain and has a dihydropyrrole ring instead of a pyrazole ring. It was
reported that the chemical modifications in SK-3530 enhanced
specific activity for PDE5 inhibition and consequently
reduced adverse effects[12]. The principle metabolic
pathways of sildenafil is known to be piperazine
N-demethylation, pyrazole N-demethylation, piperazine
N,N'-deethylation,
oxidation of the piperazine ring, and aliphatic hydroxyla-
tion[10]. In the metabolism study of SK-3530 using
LC-MS/MS (unpublished data), SK-3530 exhibited almost a similar
metabolic pattern to that of sildenafil, except for pyrazole
N-dealkylation and conjugates formation. Significant
differences, especially, the enhancement of bioavailability,
were not observed in SK-3530 pharmacokinetic data
compared with that of sildenafil. It seems that the
pharmacokinetic properties of SK-3530 make little contribution to the
enhanced compliance of SK-3530.
SK-3530 exhibits a volume of distribution (Tables 1 and
2) in excess of the volume of body water (~0.8 L/kg), indicating
the high tissue affinity of SK-3530, which is in keeping with
the weak, basic nature of SK-3530. The tissue distribution
data showed the [14C]SK-3530 radio equivalents were well
distributed in all the tissues examined. The tissue/plasma
radioactivity ratio at 1 h after administration was calculated
to be 0.15_5.4, with the exception of the excretory organs,
indicating that [14C]SK-3530 radio equivalents have relatively
high tissue affinity. The highest concentration of radio
equivalents was found in the gastrointestinal tract, resulting
from either the unabsorbed drug following oral
administration or biliary elimination of drug radio
equivalents. Notably, the radioactivity of brain tissue gradually increased with the
incubation time up to 24 h post-dose, suggesting
[14C]SK-3530 radio equivalents cross the blood_brain barrier and
potentially exert an inhibitory effect on PDE5 in the cerebral
blood vessels.
[14C]SK-3530 exhibited a high protein binding
rate. Nevertheless, the actual effect of protein binding on either
the total drug distribution to tissues or the drug levels in
tissues is considered minimal, as at least 97% of the dose
resides were outside the circulation, that is, within the tissues,
based on the volume of SK-3530 distribution (~3.2 L/kg) and
the blood volume of the rats (~0.1
L/kg)[13].
In conclusion, the present study has demonstrated that
the administered SK-3530 was relatively well absorbed in the
gastrointestinal tract and showed linear pharmacokinetics
over the investigated dose range. SK-3530 had low oral
bioavailability due to a high first-pass
metabolism. The moderate lipophilic and weak, basic natures of SK-3530 result in
extensive tissue distribution and clearance due to
metabolism.
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