Abstract
Rosuvastatin is a relatively new member of the statin family (HMG-CoA reductase inhibitors), with superior lipid-lowering effects and a pattern of clinical side effects, including a low incidence of myopathy, similar to other widely prescribed statins. This article describes investigations of myopathy in the rat following administration of very high doses of rosuvastatin. The nature of the changes were found to be entirely consistent with those seen with other statins, including a differential sensitivity of muscle fibers (with glycolytic fibers [type IIB] the most sensitive and oxidative fibers [type I] the least), a delay of approximately 10 days after the start of oral dosing before necrosis was apparent, and ultrastructural alterations appearing first in mitochondria. In addition, the development of myopathy was prevented by coadministration of mevalonate, the product of HMG-CoA reductase. The findings illustrate a pattern of induced myopathy in the rat directly attributable to inhibition of HMG-CoA reductase that is entirely consistent between the various statins, with the oral dose required to produce the changes being a differentiating feature (based on these new data and a previously reported study from the same laboratory): cerivastatin dose less than simvastatin, and simvastatin dose less than rosuvastatin.
Introduction
Rosuvastatin is a relatively new member of the statin family of drugs used to treat hypercholesterolemia. It has been approved in the United States and many other countries at doses from 5 mg to 40 mg daily. As with other agents in this class, it is an inhibitor of the enzyme 3-hydroxy-methylglutaryl CoA (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate, the major rate-limiting step of the sterol pathway (Tobert et al., 1982; Goldstein and Brown, 1990) responsible for cholesterol biosynthesis. However, rosuvastatin has lipid-lowering effects that are superior to other statins and an overall incidence of side effects similar to other widely prescribed statins (Jones et al., 2003; Brewer, 2003; Olsson et al., 2002; Scott et al., 2004). Although statins are well tolerated by most people, the association of their use with a low incidence of myopathy is well established (Farmer and Torre-Amione, 2000; Pasternak et al., 2002; Rosenson, 2004); however, the risk of rhabdomyolysis with the currently marketed statins is very low (White, 2002; Graham et al., 2004; Rosenson, 2004). We previously developed an experimental model in the rat to characterize the development of statin-induced myopathy (Westwood et al., 2005). In those investigations, we showed that even with very high doses of simvastatin and cerivastatin, myopathy did not occur until approximately 10 days after the start of oral dosing, even when the dosing was suspended because of morbidity. We also found that there was a differential “muscle-fiber–type” sensitivity that matched their oxidative/glycolytic nature (least sensitive →
Materials And Methods
Test Materials
Rosuvastatin calcium salt of purity >95% was obtained from AstraZeneca Research and Development, Macclesfield, and formulated for dosing as a suspension in water containing 0.5% hydroxypropyl methylcellulose and 0.1% w/v polysorbate 80. Mevalonic acid lactone was supplied by Sigma Aldrich, UK, and dosed as a solution in the same vehicle.
Animals and Treatments
The studies were planned in accordance with the standards of animal care and ethics described in “Guidance on the Operations of the Animals (Scientific Procedures) Act 1986” issued by the UK Home Office. It was conducted so that any clinical expression of toxicity remained within a moderate severity limit as described in the guidelines included in the project license issued by the Home Office.
Female Wistar Hannover rats, substrain Crl:WI(Glx/BRL/ Han)BR, were obtained from Charles River, UK. They were multiple-housed appropriate to each study and were acclimatized for at least 6 days before treatment was started. Animal rooms were illuminated in a 12-hour light/dark cycle, and temperature and humidity were controlled within the limits 21 ± 2°C and 55 ± 15% RH. Pelleted RM1 (E) SQC rodent diet and drinking water were freely available. The animals used were within an age range of 6 to 8 weeks at the start of dosing.
Two separate studies were performed: a preliminary study to define the extent and distribution of myopathy throughout the muscular system (doses 120 to 160 mg/kg/day) and a second study (dose 150 mg/kg/day) in which the time course of muscle changes could be defined. Details of dosing and necropsy schedules for the studies are included in Tables 1 and 2, respectively. The formulated test materials were dosed by oral gavage. Rosuvastatin was given once daily using a standard dose volume of 5 ml/kg body weight, but the daily dose of mevalonic acid was given in 2 equal fractions, the first at about 1 hour before administration of the statin dose and the second at about 6 hours after administration of the statin. The standard fractional dose volume for mevalonic acid was 2.5 ml/kg body weight. The animals were observed and weighed daily, and as necessary, the degree of toxicity expressed in individual animals was contained within predefined limits of severity by withholding dosing when the body weight loss from day 1 exceeded 10%. Dosing was then restarted when the body weight gain showed recovery. Blood sampling into lithium heparin for an evaluation of plasma creatinine kinase (CK) activity (spectrophotometric assay) was carried out for the time course study as detailed later.
Necropsy and Histology
In the preliminary study, heart, kidneys, liver, stomach, and a range of muscle tissues were sampled from at least 3 rats per group, including all premature decedents. The muscle tissues sampled were biceps femoris, extensor digitorum longus, gastrocnemius, semimembranosus, semitendinosus, soleus, tibialis cranialis, and vastus medialis from the left hind limb; biceps brachii, extensor carpi radialis longus, flexor carpi ulnaris, supraspinatus, triceps brachii caput laterale, and triceps brachii caput longum from the left forelimb; and abdominal peritoneal, diaphragm, longissimus lumborum, masseter superficialis, panniculus carnosus (skin), tongue, and trapezius. In the time-course study, kidneys, liver, stomach, and a selected range of muscle tissues (comprising biceps femoris, extensor digitorum longus, gastrocnemius, soleus, tibialis cranialis from the left hind limb, and longissimus lumborum) were sampled from all animals.
Tissues were fixed in buffered 10% formalin, processed to wax blocks, and then sectioned and stained with hematoxylin and eosin for examination by light microscopy. During the histopathological examination of the muscle sections, necrosis was graded subjectively: minimal (*)—up to 10 single fibers affected in the whole section; mild (**)—up to 20% of fibers in the section affected; moderate (***)—up to 50% of fibers in the section affected; or severe (****)—more than 50% of fibers in the section affected.
For the time-course study, further samples of a selection of muscles were also sampled and processed for ultrastructural or histochemical assessment as described below.
Electron Microscopy
Samples for ultrastructural assessment were immersion fixed in 2.5% glutaraldehyde fixative, postfixed in 1% osmium tetroxide, and processed to Araldite resin blocks. Thin, 70- to 90-nm resin sections were cut and stained using uranyl acetate and lead citrate. Ultrastructural morphology was examined on a Hitachi H7100 transmission electron microscope using a 75kV accelerating voltage.
Muscle Histochemistry
Muscle samples were trimmed, orientated on a cork disk, and frozen in isopentane (Fisher Scientific) precooled with liquid nitrogen. Serial cryosections of 7-μm thickness were cut from each sample for fiber typing. Sections were stained for mATPase activity following preincubation at high and low pH. One section was placed in an incubating solution at pH 9.4 consisting of 0.5% ATP (Sigma) in 0.1-M glycine/NaC1 buffer with 0.75 M CaC12 for 45 minutes at 37°C. A further section was preincubated in 0.1-M sodium acetate buffer with 10 mM ethylenediaminetetracetic acid (EDTA, pH 4.15) for 10 minutes at 4°C before placing in the incubation solution noted above. Following incubation, the slides were transferred to 2% CoC12 for 5 minutes followed by 30 seconds in 10% ammonium sulphide solution. Sections were washed thoroughly in distilled water between each step. Sections were lightly counterstained with Carazzi’s hematoxylin before being dehydrated, cleared, and mounted in Histomount. Using this method, type I, IIC, IIA, IID, and IIB fibers (see results below—Fiber Typing in Muscle Showing Low-Grade Necrosis—for description of fiber types) could be discriminated.
Muscle Immunohistochemistry
4-μm sections of formalin-fixed, paraffin-embedded muscle were stained for fiber type, using the dual-labeling immunohistochemical technique described by Behan et al. (2002). Dewaxed sections were subjected to 2 minutes full pressure in a microwave pressure cooker containing 0.01-M citrate buffer at pH 6.0 and then 5 minutes digestion at room temperature by proteinase K (Dako S3020). Endogenous peroxidase activity was blocked by incubation in Dako’s Peroxidase Block (K401111) for 20 minutes, followed by 15 minutes in 20% normal rabbit serum. Mouse monoclonal antibody to slow myosin (Sigma M-8421) at a dilution of 1:2,000 was applied for 30 minutes, followed by 30 minutes in peroxidase-conjugated rabbit antimouse antibody (Dako P0260) at 1:100. Vector Laboratory’s SG peroxidase substrate kit (SK4700) was then applied for 10 minutes. Following a further 15 minutes in 20% normal rabbit serum, Sigma’s alkaline phosphatase-conjugated mouse monoclonal antibody to fast myosin (A4335) was applied at a dilution of 1:50 for 60 minutes. This was visualized using Vector Red alkaline phosphatase substrate kit (Vector Labs SK5100) for 10 minutes. All incubations were at room temperature, and sections were washed thoroughly in tris-buffered saline between each step. Sections were dehydrated, cleared, and mounted in Histomount.
Results
Clinical Observations
In the preliminary study, 1 rat dosed with 120 mg/kg/day showed body weight loss. Two of 3 rats dosed with 140 and all 3 rats dosed with 160 mg/kg/day showed elements of hunched posture, piloerection, and thin appearance with body-weight loss. In the time-course study (150 mg/kg/day), 27/30 rats showed elements of hunched posture, piloerection, thin and pale appearance, “cold to touch,” and decreased activity (3 of 5 rats dosed to day 4 and killed for scheduled termination on day 5 showed no adverse clinical observations). Based on the severity of these changes, animals were excluded from dosing for a period of time or killed as detailed in Tables 1 (preliminary study) and 2 (time-course study). These findings indicate that the treatments used in this study were the maximum tolerated dose (MTD)/moderate severity limit, and if administered without periods of rest, would be above MTD. There were no noteworthy clinical observations from control rats or those dosed with 150 mg/kg/day rosuvastatin plus mevalonic acid.
Plasma Chemistry
Daily administration of rosuvastatin at 150 mg/kg/day was associated with increases in plasma CK activities as detailed in Table 3. Clear increases in CK levels were not apparent until day 10, although on days 14 and 16, they had returned to control values.
General Pathology
All changes were consistent with those seen with other statins when administered to the rat at or above maximum tolerated dose (Westwood et al., 2005). In the liver, hepatocyte single-cell necrosis, increased mitotic figures, and cytoplasmic basophilia occurred following rosuvastatin administration at the high dose levels used in this study. Also, the forestomach showed epithelial hyperplasia, with some hyper-keratosis and subepithelial mixed inflammatory cell infiltration. Coadministration of mevalonic acid did not abolish these changes in the liver and stomach.
There were no changes in the hearts of any animal resulting from administration of rosuvastatin.
Muscle Necrosis, Temporal Development, Distribution, and Histopathology
Control Muscles
The incidence of fiber necrosis in control rats was low and consistent with that reported previously (Westwood et al., 2005). It was characterized by minimal necrosis of a single or a few isolated individual muscle fibers, generally with an associated mononuclear cell infiltration. In muscles from the 5 control rats examined (time-course study—Table 2), the soleus was the only muscle affected.
Preliminary Studies
Table 1 details the incidence and severity of muscle necrosis seen during the preliminary study. The incidence and severity of myofiber necrosis was variable between animals and muscles. Only 1 of 3 rats dosed with 120 mg/kg/day and 2 dosed with 140 mg/kg/day showed clear evidence of induced muscle necrosis. All 3 rats given 160 mg/kg/day were affected. Overall, most sampled muscles were affected, although certain specific muscles were consistently spared. These were the soleus, masseter, and tongue. Other muscles predominantly spared included the flexor carpi ulnaris and the diaphragm.
Time-course Studies
Table 2 shows the incidence and severity of myofiber necrosis in the time-course study. Muscle necrosis in animals dosed to and including day 8 was restricted to a minimal change in the soleus of 1 animal. This was not considered a result of administration of rosuvastatin at 150 mg/kg/day. From day 10, a proportion of the animals sacrificed at each time point showed compound-induced myofiber necrosis. The incidence and severity in individual muscles was variable, but the soleus muscle was generally spared, showing no more than a typical control incidence and severity of necrosis. There was no evidence of induced muscle necrosis in animals dosed with rosuvastatin and mevalonic acid. The minimal change seen in 2 muscles was entirely consistent with background incidence levels (Westwood et al., 2005).
The muscle necrosis, when acute, was segmental, affected individual fibers, and was characterized by cytoplasmic eosinophilia with loss of cytoplasmic structure, vacuolation, and little or no inflammatory infiltrate. Often, the necrosis was more widespread, and there was infiltration by mononuclear and polymorphonuclear cells, edema, and vacuolation with fragmentation and loss of cytoplasm. The basement membrane was commonly retained. In more advanced lesions, there was a more profound infiltration by mononuclear cells and some regeneration with myotube formation (Figure 1).
Ultrastructure
The following muscles from control animals on the time-course study were assessed ultrastructurally: the biceps femoris, extensor digitorum longus, and gastrocnemius muscles from animal 1 and the biceps femoris muscle from animal 4. Changes were consistent with those seen in control animals from the previous studies (Westwood et al., 2005). Occasional mitochondrial vacuolation with some minor accumulation of concentric myelinoid bodies was present. These findings were not accompanied by any further notable ultrastructural changes.
Muscles from animals given rosuvastatin in the time-course study showing early histological changes of low severity and with only minor or no frank inflammation were selected for further ultrastructural assessment. These were the biceps femoris from animals 33, 42, 43, 46, and 50; the extensor digitorum longus from animals 43 and 46; and the gastrocnemius from animal 29.
Changes in excess of those seen in control samples were restricted to glycolytic type II fibers showing few mitochondria and abundant sarcoplasmic glycogen deposits. Two findings that occurred in isolation of any other ultrastructural changes were increased vacuolation of mitochondria and accumulation of aberrant organelles throughout the sarcoplasm but accumulating particularly in the subsarcolemmal regions. These organelles (Figure 2) were variable in size and characterized by accumulation of concentric, multilaminated membranous whorls and spheroids, often with retained mitochondrial cristae and occasional lysosome-like dark-staining inclusions. Many appeared clearly to be degenerate mitochondria or derived from degenerate mitochondria.
Fiber Typing in Muscles Showing Low-Grade Necrosis
The muscle fiber type of mammals is determined by the particular myosin heavy chain (MHC) isoform expressed. The limb muscles of the adult rat express 1 slow and 3 fast MHC isoforms (Schiaffino and Reggiani, 1994). Most fibers normally express only 1 isoform and are referred to as “pure,” although fibers are present that contain more than 1 (Staron and Pette, 1993). The pure and mixed fiber types constitute a continuum from the slowest twitch type I fibers to the fastest twitch type IIB (Staron et al., 1999):
From the time-course study (Table 2), a selection of control muscles and those showing early histological changes of low severity and with little or no apparent inflammation were selected for further histochemical assessment including type I and II fiber typing by immunohistochemistry and myosin ATPase histochemistry. These were the biceps femoris from animals 1, 3, 4, 29, 33, 42, 43, 46, and 50; the gastrocnemius from animals 1 and 29; the cranial tibial from animals 1 and 46; and the extensor digitorum longus from animals 1, 43, and 46. In addition to immunohistochemistry and myosin ATPase stability, fiber diameter was used as an indicator of fiber type for early necrotic fibers (Westwood et al., 2005). Immunohistochemistry for type I and II fibers clearly showed that in muscles containing mixtures of these fibers, when early necrosis was present, the type I fibers were spared. Even when a substantial proportion of the type II fibers were necrotic, the type I fibers retained their normal histological and immunohistochemical appearance (Figure 3). Myosin ATPase staining and fiber-diameter assessment also indicated sparing of type I and IIA fibers relative to Type IID and IIB fibers (Figure 4).
Discussion
Since statins have been used in clinical practice and myopathy has been recognized as an adverse event, there have been numerous investigations into the pathogenesis of the myopathy. These studies have resulted in a diverse range of hypotheses including (1) preferential uptake of statins in skeletal muscle cells by monocarboxylate transporters (Sirvent, Bordenave, et al., 2005), (2) decreased membrane cholesterol resulting in alterations in fluidity with destabilization and degeneration (Pierce et al., 1990; Waclawik et al., 1993), (3) reduced chloride channel conductance (Baker, 2004), and (4) alterations in mitochondrial function leading to cytoplasmic Ca2+ overload (Sirvent, Mercier, et al., 2005). A number of hypotheses have focused on nonsterol products of mevalonate (Johnson et al., 2005), particularly the isoprenoids, with a resulting effect on products, including (1) reduced coenzyme Q/ubiquinone, a powerful antioxidant and membrane stabilizer that is used in mitochondria for electron transport (Krum and McMurray, 2002; De Pinieux et al., 1996; Carvalho et al., 2004; Bliznakov, 2002; Rosenson, 2004); (2) reduced enzymatic isopentylation of selenocysteine-tRNA, with prevention of its maturation to a functional tRNA molecule resulting in a fall in available selenoproteins (Moosmann and Behl, 2004); (3) inhibition of the prenylation/geranylgeranylation of proteins by reductions in farnesyl and geranylgeraniol pyrophosphates (Thompson et al., 2003; Johnson et al., 2004; Carvalho et al., 2004; Baker, 2004; Flint et al., 1997a, 1997b); and (4) perturbation of N-linked glycosylation of proteins (Baker, 2004). However, all of these theoretical mechanisms remain unproven, and it can be concluded that the pathogenesis of statin-induced myopathy in humans and animals is still uncertain. However, previous data (Westwood et al., 2005) and findings here confirm that the myopathy is a direct result of inhibition of HMG Co-A reductase, as coadministration of mevalonic acid (mevalonate), the product of the enzyme, precludes its occurrence.
The character, muscle fiber sensitivity, temporal development, and earliest subcellular changes during statin myopathy, particularly for the rat, have been well characterized. We previously reported, using simvastatin (80 mg/kg/day) and cerivastatin (0.5 mg/kg/day), a reproducible and robust in vivo model of statin-induced muscle necrosis (Westwood et al., 2005). It is with such a model system that more conclusive mechanistic investigations may be performed. During these investigations, statin-induced muscle necrosis in the rat occurred at or above the MTD. This is also illustrated by current investigations in which muscle necrosis occurred above the MTD of rosuvastatin, as evidenced by the need to suspend dosing for periods of time at all the doses used (see Tables 1 and 2) for the majority of rats to reduce morbidity and allow completion of the studies. Using high doses on this basis, we achieved a high incidence of muscle necrosis with a limited number of animals. It is notable that any early dose-limiting morbidity seen at the levels used was not a result of muscle necrosis, as this did not occur until at least day 10. As with simvastatin and cerivastatin, the preliminary study with rosuvastatin showed that at doses above MTD, the incidence of muscle necrosis in the rat was dose related and widespread, although there was some variability in severity between muscles and there was sparing of particular muscles. It is also of note that a greater dose of rosuvastatin was required to achieve a consistent incidence of muscle necrosis than was observed in the earlier study with simvastatin (80 mg/kg/day), and particularly, cerivastatin (0.5 mg/kg/day; Westwood et al., 2005).
The character of the muscle necrosis with rosuvastatin was also entirely consistent with that seen with other statins, including histological appearance and development and occurrence only after approximately 10 days of dosing (Reijneveld et al., 1996; Schaefer at al., 2004; Smith et al., 1991; Waclawik et al., 1993; Westwood et al., 2005). Also, the muscle fiber type primarily affected was the same. There is a continuum of muscle fiber types in adult mammalian skeletal muscles from the slow-twitch type I to the fastest twitch type IIB:
In conclusion, our findings illustrate a pattern of induced myopathy in the rat directly attributable to inhibition of HMG-CoA reductase that is entirely consistent between the various statins, with a differentiating feature’s being the oral dose required to produce the changes: cerivastatin dose less than simvastatin, and simvastatin dose less than rosuvastatin.