![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 30, Issue 11, 1158-1163, November 2002
Developmental Research Laboratories, Shionogi and Co., Ltd., Osaka, Japan (K.N., T.M., K.I., H.H., M.N., M.K.); and Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan (K.H.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Rosuvastatin is a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. The liver is the target organ for the lipid-regulating effect of rosuvastatin; therefore liver-selective uptake of this drug is a desirable property. The aim of this study was to investigate, and compare with pravastatin and simvastatin, the tissue-specific distribution of rosuvastatin. Bolus intravenous doses (5 mg/kg) of radiolabeled rosuvastatin, pravastatin, and simvastatin were administered to rats, and initial uptake clearance (CLuptake) in various tissues was calculated. Hepatic CLuptake of rosuvastatin (0.885 ml/min/g tissue) was significantly (p < 0.001) larger than that of pravastatin (0.703 ml/min/g tissue), and rosuvastatin was taken up by the hepatic cells more selectively and efficiently than pravastatin. Hepatic CLuptake of simvastatin (1.24 ml/min/g tissue) was significantly larger than that of rosuvastatin (p < 0.01) and pravastatin (p < 0.001). However, adrenal CLuptake of simvastatin (1.55 ml/min/g tissue) was larger than hepatic CLuptake, and simvastatin was distributed to other tissues more easily than rosuvastatin. Microautoradiography of the liver, spleen, and adrenal was undertaken 5 min after administration of the study drugs; distribution was quantified by counting the number of silver grains. After administration of rosuvastatin and pravastatin, silver grains were distributed selectively in the intracellular space of the liver, but more rosuvastatin (3.3 ± 1.0 × 105 particles/mm2) than pravastatin (2.0 ± 0.3 × 105 particles/mm2) tended to distribute to the liver. Simvastatin was less liver-specific (it also distributed to the spleen and adrenal). The results of this study indicated that rosuvastatin was taken up by hepatic cells more selectively and more efficiently than pravastatin and simvastatin.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Rosuvastatin
(Crestor), calcium
bis[(+)-(3R,5S,6E)-7-[4-(p-fluorophenyl)-6-isopropyl-2-(N-methylmethanesulfonamido)-5-pyrimidinyl]-3,5-dihydroxy-6-heptenoate], is a new and highly effective inhibitor of
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA1)
reductase (the rate-limiting enzyme in cholesterol biosynthesis). The
drug, hereafter referred to as rosuvastatin, which was originally synthesized at Shionogi and Co., Ltd. (Watanabe et al., 1997
), has
completed phase III clinical development at AstraZeneca for the
treatment of patients with dyslipidemia. In clinical trials, rosuvastatin (1 to 80 mg) produced significant dose-dependent reductions in low-density lipoprotein cholesterol (up to 65%), total
cholesterol, and apolipoprotein B (Olsson et al., 2001). Reductions in
triglycerides and increases in high-density lipoprotein cholesterol
were consistently observed with rosuvastatin, and the drug was well
tolerated (Olsson et al., 2001).
The liver is the target organ for the lipid-regulating effect of rosuvastatin; therefore liver-selective uptake of this drug is a desirable property. The aim of the present study was to investigate, and compare with pravastatin and simvastatin, the tissue-specific distribution of rosuvastatin in rats.
| |
Experimental Procedures |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Materials. [14C]Rosuvastatin (1.15 MBq/mg), [14C]pravastatin (1.17 MBq/mg), and [14C]simvastatin (1.85 MBq/mg) were synthesized at Shionogi and Co., Ltd. (Osaka, Japan). The radiochemical purities were 99.4, 99.0, and 98.6%, respectively. The chemical structure of these HMG-CoA reductase inhibitors is shown in Fig. 1. All other chemicals were of analytical grade and commercially available.
|
Animals. Male rats of Jcl:Sprague-Dawley strain were purchased from CLEA Japan, Inc. (Osaka, Japan). The rats were 10-weeks old, and their weight ranged between 317 and 405 g. Prior to study, the rats acclimatized for 1 week in the animal room at Shionogi and Co., Ltd. (room temperature 23 ± 1°C; relative humidity 55 ± 10%); free access to water and laboratory food (CA-1; CLEA Japan, Inc.) was permitted.
Preparation of the HMG-CoA Reductase Inhibitors. Dosing solutions (5 mg/ml) were prepared immediately before administration of the HMG-CoA reductase inhibitors to the rats. [14C]Rosuvastatin and [14C]pravastatin were dissolved in physiological saline. [14C]Simvastatin was dissolved in a mixture of N,N-dimethylacetamide, HCO60 (hydrogenated castor oil 60), and physiological saline (2:1:4, by volume).
In Vivo Experiment. The rats were anesthetized by intraperitoneal administration of pentobarbital sodium (50 mg/kg) and incised along the abdominal midline. A bolus intravenous dose (5 mg/kg) of [14C]rosuvastatin, [14C]pravastatin, or [14C]simvastatin dosing solution was then administered through the tail vein.
At 15, 30, 45, 60, 90, 120, 150, 180, 240, and 300 s after administration of the HMG-CoA reductase inhibitor, blood (8-10 ml) was drawn from the abdominal vena cava for exactly 60 s. The rats were killed by exsanguination and the following tissues and organs collected: eyeball, cerebrum, cerebellum, thyroid, lung, heart, adrenal, testis, prostate, spleen, kidney, liver, and ileum.Handling of Samples for Calculation of Uptake Clearance. Blood samples were centrifuged at 13,000g for 30 s, and the plasma harvested. A 100-µl aliquot of the plasma sample was weighed in a counting vial. Pico-Fluor 40 (PerkinElmer Life Sciences, Boston, MA) 10 ml was then added, and the radioactivity measured by a liquid scintillation counter (Tri-Carb 2200-CA; PerkinElmer Life Sciences).
A sample of each tissue/organ (blotted on filter paper to remove as much blood as possible) was weighed in a counting vial. Soluene-350 (2 ml; PerkinElmer Life Sciences) was added to solubilize the sample, which was then bleached with 200 µl of hydrogen peroxide solution. Pico-fluor 40 (10 ml) was added, and the radioactivity measured by a liquid scintillation counter (Tri-Carb 2200-CA).Calculation of Uptake Clearance.
The uptake clearance was calculated according to the following
differential equation (Kim et al., 1988; Yanai et al., 1990).
|
|
, Yanai et al., 1990). Thus, the
AUC0-t value was calculated by the
trapezoidal rule, and the
AUC0-t/Cp,t values were plotted against the
Xtissue,t/Cp,t values. The CLuptake value was then calculated by
linear regression analysis. The data points actually employed in the
calculation of the CLuptake values occurred
15-240 s after administration of the HMG-CoA reductase inhibitor. If
the number of data points on the line was small, saturation of tissue
uptake was considered to have occurred early after administration.
Handling of Samples for Microautoradiography. Samples of the liver and spleen and the whole adrenal were excised and rapidly frozen by liquid nitrogen. Sections (10 µm) were then prepared, lyophilized, and mounted on glass slides coated with photosensitive emulsion (NTB-3; Kodak, Rochester, NY). After exposure in a shaded box for 1 month at 4°C, the glass slides were developed for 2 min at 17°C with Dektol (Kodak) and stained with methyleneblue-basic fuchsin for microscopic observation.
Quantitative Microautoradiography.
Input of image data and software for imaging analysis For quantitative measurement, three different visible fields of the tissue picture (170 × 220 µm2 in size; object lens, ×40) were taken using a digital camera (HC-2000; Fuji Photo Film Co., Ltd., Tokyo, Japan) connected to a light microscope. For imaging analysis, Optimas version 6 (Optimas Corporation, Bothell, WA) software was used.
Measurement of the outer and inner areas of cell. The unstained area was regarded as the outer area of the cell (cellular interstitial part and blood vessel). By setting the threshold level of the stained color, separation of the outer area of the cell from the inner area of the cell was achieved and each area (Scell) was measured.
Measurement of the total area of silver grains. To discriminate the silver grains, the threshold level of the color was set again for each area (outer and inner) of the cell, and the total area of silver grains (Sgtotal) in each was measured.
Measurement of the mean area of each silver grain. In the case that more than two particles of silver grains are present as a mass, this mass is counted as one particle. Therefore, the mean area of each silver grain (Sgmean) was calculated according to the following procedure.
Using the outer area of the liver cells showing good separation of each silver grain and having negligible mass of silver grains, the total number of the silver grains (Ntotal) present in the outer parts of the cells as well as Sgtotal were measured, and Sgmean was calculated according to eq. 3.
|
Measurement of the number of silver grains in each unit area of
the outer and inner parts of the cell.
The number of silver grains in each unit area (N) was
calculated (particles/mm2) for the outer and
inner parts of the cell according to eq. 4.
|
Statistical Analysis. An analysis of variance was performed to determine the statistical significance between groups using Bartlett's test. Significant differences between the means were examined using Tukey's method. Comparison of regression lines was performed by calculating the standard error of the difference in slope according to Student's t test. Statistical significance between observed data and data calculated by linear regression were determined by Pearson's method, which is used to estimate the significance for the linear correlation.
| |
Results |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Uptake Clearance. Figure 2 shows the integration plots for uptake by the liver, kidney, spleen, and adrenal following administration of [14C]rosuvastatin (Fig. 2A), [14C]pravastatin (Fig. 2B), and [14C]simvastatin (Fig. 2C). The CLuptake values (i.e., the slopes of the integration plots) are summarized in Fig. 3. The linear correlation was statistically significant in all tissues examined except for kidney for pravastatin, in which the correlation coefficient was relatively low (r = 0.767).
|
|
Microautoradiography. The microautoradiograms obtained from the liver, spleen, and adrenal are shown in Fig. 4. In the liver (Fig. 4a), a large number of silver grains were observed for all drugs. In the spleen (Fig. 4b), silver grains were only observed for simvastatin. In the adrenal (Fig. 4c), silver grains were also only observed for simvastatin (to at least the same extent as in the liver). The number of silver grains distributed to each tissue is shown in Table 1.
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
The liver is the target organ for the lipid-regulating effect of HMG-CoA reductase inhibitors; therefore liver-selective uptake of these drugs is a desirable property. In the present study, we investigated, and compared with pravastatin and simvastatin, the tissue-specific distribution of rosuvastatin in rats.
The hepatic CLuptake of rosuvastatin (0.885 ml/min/g tissue) was significantly (p < 0.001)
larger than that of pravastatin (0.703 ml/min/g tissue). [The hepatic
CLuptake value for pravastatin is comparable to
values reported previously, 0.44 ml/min/g tissue (Yamazaki et al.,
1993) and 0.59 ml/min/g tissue (Yamazaki et al., 1996).] Furthermore,
the CLuptake values of pravastatin in the kidney,
testis, thyroid, eyeball, prostate, and spleen were 2- to 4-times
larger than those of rosuvastatin, indicating that rosuvastatin was
taken up by the hepatic cells more selectively and more efficiently
than pravastatin. The microautoradiographic data showed that both
rosuvastatin and pravastatin were distributed selectively in the
intracellular space of the liver, but more rosuvastatin than
pravastatin tended to distribute to the liver.
The hepatic CLuptake of simvastatin (1.24 ml/min/g tissue) was significantly larger than that of rosuvastatin (p < 0.01) and pravastatin (p < 0.001). However, the CLuptake values of simvastatin in the other tissues were much larger than those of rosuvastatin, indicating that simvastatin distributed to other tissues more easily than rosuvastatin. The microautoradiographic data also showed that simvastatin distributed to several tissues and was less liver-specific.
The results of this study support the previous finding that pravastatin
has a higher liver-selectivity than simvastatin (Koga et al., 1995; van
Vliet et al., 1995). However, the number of silver grains in the spleen
following administration of pravastatin was much less than that of
simvastatin, and this differs from previous findings (Koga et al.,
1992). Transport of pravastatin to hepatocytes is proposed to occur
mainly via a Na+-independent anion transporter in
the sinusoidal membrane, due to the negative charge and hydrophilicity
of pravastatin (Komai et al., 1992; Yamazaki et al., 1993). In
contrast, passive diffusion may contribute largely to the transport of
simvastatin because of its high lipophilicity (Komai et al., 1992).
Therefore, pravastatin might be distributed less than simvastatin to
tissues/organs without that putative transport mechanism (e.g., the
spleen). The results of this study highlighted quite large differences
in distribution to the extrahepatic tissues between these two compounds.
It has been speculated that rosuvastatin is selectively taken up by the
liver via the organic anion transport system (possibly a
Na+-independent anion transporter), due to the
negative charge of rosuvastatin. A recent study involving oocytes
expressing OATP-C or OATP-A suggested that rosuvastatin is a substrate
for OATP-C but not for OATP-A (Brown et al., 2001).
The n-octanol-water partition coefficient of rosuvastatin is
1.46 at pH 7.0, which is closer to that of pravastatin (0.59) than that
of simvastatin (48400) (Serajuddin et al., 1991). The high
lipophilicity of simvastatin has been linked with side effects such as
myopathy (Pierno et al., 1999). Furthermore, compared with pravastatin,
a hydrophilic compound, the risk of myopathy with simvastatin was shown
to be higher when using a urethane infusion method (Matsuyama et al.,
2002). Generally, the specific distribution of drugs to the target
organ contributes to the increase of clinical effect and the decrease
of toxicity in the other organs. In the clinical dose-ranging trial
(1-80 mg) of rosuvastatin for 6 weeks, myopathy, which can be
associated with elevated creatine kinase levels, is relatively
infrequent (Olsson et al., 2001). However, there are serious events
associated with statin therapy that appear to be more common at higher
doses of the other statins (Maron et al., 2000). The high
hydrophilicity of rosuvastatin may explain the minor nonspecific
distribution to extrahepatic tissues and may be consistent with low toxicity.
In the clinical trial for 12-week dosing, 5 and 10 mg of
rosuvastatin reduced low-density lipoprotein cholesterol by 42 and 49%, respectively, compared with a 28% reduction with 20 mg of pravastatin and 37% with 20 mg of simvastatin, and the effect of
rosuvastatin was significantly larger than either pravastatin or
simvastatin (Paoletti et al., 2001). It has been reported that rosuvastatin exhibited inhibition of cholesterol synthesis with an
IC50 of 0.16 nM in rat hepatocytes and was
significantly more potent than both pravastatin and simvastatin
(McTaggart et al., 2001). In addition to this higher potency, our
findings, the more selective distribution to the liver, also contribute
to a greater clinical effect of rosuvastatin over both pravastatin and simvastatin.
In conclusion, the results of this study indicated that rosuvastatin was taken up by the hepatic cells more selectively and more efficiently than pravastatin and simvastatin.
| |
Acknowledgments |
|---|
We thank T. Nagasaki, Y. Katsuyama, and M. Segawa for synthesizing and purifying [14C]rosuvastatin, [14C]pravastatin, and [14C]simvastatin.
| |
Footnotes |
|---|
Received March 3, 2002; accepted July 31, 2002.
Address correspondence to: Ken-ichi Nezasa, Developmental Research Laboratories, Shionogi and Co., Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka 561-0825, Japan. E-mail: kenichi.nezasa{at}shionogi.co.jp
| |
Abbreviations |
|---|
Abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CLuptake, clearance uptake; Cp,t, plasma concentration of total radioactivity at time t; AUC, area under the curve; Sgtotal, total area of silver grains; Sgmean, mean area of each silver grain; OATP, organic anion transporters.
| |
References |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
