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Recently, it is suggested that the use of nonsteroidal anti-inflammatory drugs (NSAID) may contribute to the occurrence of cardiovascular events, while the formation of atherosclerotic lesions is related to inflammation. Loxoprofen sodium, a non-selective NSAID, becomes active after metabolism in the body and inhibits the activation of cyclooxygenase. We fed apoE−/− mice a western diet from 8 to 16 weeks of age and administered loxoprofen sodium. We measured atherosclerotic lesions at the aortic root. We examined serum levels of cholesterol and triglycerides with HPLC, platelet aggregation, and urinary prostaglandin metabolites with enzyme immune assay. Atherosclerotic lesion formation was reduced to 63.5% and 41.5% as compared to the control in male and female apoE−/− mice treated with loxoprofen sodium respectively. Urinary metabolites of prostaglandin E2, F1α, and thromboxane B2, and platelet aggregation were decreased in mice treated with loxoprofen sodium. Serum levels of cholesterol and triglycerides were not changed. We conclude that loxoprofen sodium reduced the formation of early to intermediate atherosclerotic lesions at the proximal aorta in mice mediated by an anti-inflammatory effect.
As a result of progress in therapy for rheumatoid arthritis (RA), cardiovascular events have become the major cause of death in these patients. RA is associated with an increased and accelerated vascular risk that results in lifespan reduction [1–3]. There is an association between disease activity and cardiovascular risk in RA and other inflammatory disorders, such as systemic lupus erythematosus (SLE) [1, 4, 5]. Several factors may be associated with accelerated arteriosclerosis in patients with RA or SLE. One factor may be the disease itself, while others may include the pharmacological therapies that patients with RA or SLE receive.
Rofecoxib, a highly selective cyclooxygenase(COX)-2 inhibitor, has recently been voluntarily withdrawn from the market due to evidence from the APPROVe trial demonstrating increased cardiovascular events after 18 months of use. Combination therapy with the selective COX-2 inhibitors, valdecoxib and parecoxib increased the risk by 3.7-fold . Concerns have been raised that a highly selective COX-2 inhibition may promote cardiovascular events by inhibiting prostacyclin and inducing a prothrombotic state . In contrast, inhibition of COX-1 mediated production of platelet thromboxane (Tx) by aspirin reduces the risk of myocardial infarction and stroke .
Selective COX-2 inhibitors are the major anti-pain drugs used in western countries. Loxoprofen sodium is a pro-drug of phenyl mefenamic acid and a non-selective COX inhibitor. It is the dominant anti-pain drug in Japan and most frequently prescribed in East Asian countries . In view of the reports related to COX-2 selective inhibitors in the USA, the adverse effects of nonsteroidal anti-inflammatory drugs (NSAIDs) associated with cardiovascular disease were surveyed in Japan in June 2005. However, no evidence was found of any adverse effects of loxoprofen sodium associated with cardiovascular events. The Pharmaceutical and Medical Devices Agency in Japan concluded that it was not necessary to perform safety measures for loxoprofen sodium against cardiovascular events [www.info.pmda.go.jp].
It has been reported that selective inhibition of COX-1 attenuates atherosclerosis in apoE−/− mice . However, reports on the effect of selective COX-2 inhibition on the development of atherosclerosis in murine models have been mixed with decreased, increased or unchanged atherosclerotic lesions all noted [11, 12]. The divergence in the results may be the consequence of differences in experimental design, including efficacy and selectivity of the inhibitors, gender of the mice and stage of atherosclerotic lesions.
The surveillance study of adverse effects of loxoprofen sodium in Japan and these studies of non-selective inhibition of COX inhibition on the development of atherosclerosis in animal models demonstrate the ability of non-selective COX inhibition with loxoprofen sodium to reduce early and intermediate atherosclerotic lesion formation in apoE−/− mice, supporting the efficacy of anti-inflammatory approaches in the prevention of atherosclerosis.
Three healthy men participated in this study. They received blood examinations and collected urine for 24 h before administration of loxoprofen sodium. After receiving loxoprofen sodium 60 mg/body three times a day for 4 days, they received blood examinations and collected urine again. The ethics committee of Kyoto Prefectural University of Medicine approved the study.
ApoE−/− mice (C57BL/6J-Apoetm1Unc) were originally purchased from Jackson Laboratories (Bar Harbor, ME). ApoE−/− mice were at the 10th backcross into the C57BL/6 background. Mice were maintained on a rodent chow diet containing 4.5% fat (Oriental BioService, Kyoto, Japan) and autoclaved acidified (pH 2.8) water before 8 weeks of age. From 8 (day 57) to 16 (day 113) weeks of age, these mice were maintained on a “Western” high-fat diet (0.2% cholesterol, 21% saturated fat; Oriental BioService, Kyoto, Japan). The atherosclerosis studies were designed to examine the effect of administration of loxoprofen sodium during early atherosclerotic lesion formation in apoE−/− mice. The mice were divided into 3 groups (vehicle group, 7 day loxoprofen sodium group, 56 day loxoprofen sodium group) at 8 weeks of age (day 57), randomly. Animal care and experimental procedures were carried out in accordance with the regulations and with the approval of Kyoto University’s Animal Care Committee. The mice were continued on this regimen until 16 weeks of age, when they were killed with an overdose of sodium pentobarbital.
Randomly selected mice were treated with loxoprofen sodium (4 mg/kg/day) added to the drinking water from 8 (day 57) to 16 (day 113) weeks of age or from 15 (day 106) to 16 (day 113) weeks of age. The dose of loxoprofen sodium used in our study was chosen based on oral dosing in acute studies of carageenan induced footpad edema plethysmometry in rats in which the oral ED50 for this assay in rats is 2 mg/kg, twice a day . The dose of the drug was calculated on the basis of the average consumption of water and the body weight, determined weekly. Loxoprofen sodium was kindly provided by Daiichi-Sankyo, Co. (Tokyo, Japan).
As platelet TxA2 metabolite, prostaglandin (PG) I metabolite, and PGE2 metabolites, we measured urinary 2,3 dinor TxB2, urinary 2,3 dinor 6 keto PGF1α, urinary PGE2 metabolites. From 15 to 16 weeks of age, 24 h urine samples of mice were collected with metabolic cages (Tecniplast, Italy). Mouse and human samples were analyzed by a commercial assay according to the manufacturer’s procedure (Cayman Chemical, MI). Urine creatinine (CRE) was measured with dry chem system (SRL, Tokyo, Japan).
ApoE−/− mice were maintained on the “Western” high-fat diet from 8 to 16 weeks of age and were treated with vehicle or 4 mg/kg/day of loxoprofen sodium from 15 to 16 weeks of age. At 90 min after final dose administration, blood was collected from five mice in each group. Blood was collected in 12.9 mM sodium citrate and gently agitated at 37°C for 30 min, and centrifuged at 250 g for 15 min to obtain plasma . Blood samples were also collected from healthy volunteers in 12.9 mM sodium citrate and centrifuged at 150 g for 15 min to obtain platelet rich plasma (PRP) before loxoprofen sodium administration and after receiving loxoprofen sodium 60 mg/body three times a day for 4 days. After separation of PRP, tubes were centrifuged again at 1,200 g for 15 min to obtain platelet poor plasma.
To measure changes in the light transmission rate, PRP samples (200 µl) were incubated for 2 min at 37°C, then with 22.2 µl of adenosine diphosphate (ADP) or collagen solution for 5 min at 37°C. The intensity of light transmitted over 5 min was measured using MCM hematoracer 313 (MC medical. Tokyo, Japan). ADP and collagen solution were purchased from MC medical (MC medical. Tokyo, Japan). ADP added at 1, 2, 4, 8 µM in humans and at 0.5, 1, 2, 4 µM in mice. Similarly, collagen was added at 0.25, 0.5, 1, 2 µg/mL in humans and 0.5, 1, 2, 4 µg/mL in mice. The pressure rate was standardized using a grading curve produced by plotting data with four concentrations of agonist on the x-axis and pressure rate (%) on the y-axis. The concentration of agonist causing a 50% maximum aggregation rate was calculated and applied as the platelet aggregatory threshold index (PATI) .
ApoE−/− mice were maintained on a “Western” high-fat diet from 8 to 16 weeks of age and were treated with vehicle or 4 mg/kg/day of loxoprofen sodium from 8 to 16 weeks of age. The sera were obtained from mice fasted overnight (11 animals from each group). Serum albumin, CRE, and free fatty acids (FFA) were analyzed with dry chem system (SRL, Tokyo, Japan). A high performance liquid chromatograph (HPLC) system with two tandem gel permeation columns was used to evaluate the size distribution of plasma lipoprotein particles (Skylight Biotech, Akita, Japan) [15, 16]. Fast performance liquid chromatography was performed on an HPLC system model 600 (Waters) with a Sepharose 6 column (Skylight Biotech, Akita, Japan). Samples were diluted 20 times and analyzed at a flow rate of 350 µl/min by monitoring the concentrations of total cholesterol, and triglycerides with absorbance at 550 nm.
The hearts were flushed through the left ventricle, and the aortic root was obtained for analysis. The hearts were removed immediately after the mice were killed, rinsed in cold phosphate buffered saline to remove traces of blood, and placed in 4% paraformaldehyde overnight. The hearts were sliced with a scalpel on a plane parallel to the tips of the atria at the base of the aortic root, according to a procedure described by Paigen et al. . The tissue was processed and embedded in OTC compound for histological sectioning by conventional methods. Fifteen alternate cryosections of 10-mm thickness were collected from the proximal aorta starting from the beginning of the aortic sinus and extending 300 µm distally. The sections were stained with Oil-Red-O and photographed at a magnification of 400 with an Olympus U-CMAD3 camera connected to an Olympus BX50 microscope and DXC-S500/OL digital capture (Olympus, Tokyo, Japan). The vessel structures were traced and areas calculated in the computer-digitized images with the Image J program (National Institutes of Health, MD). Sections from the aortic arch were used to quantify atherosclerosis (8 animals from each group). An observer who was blinded to all other data analyzed the lesions.
Sections (10 µm) of OCT embedded tissue were acetone fixed, and endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide. Serial sections were subjected to immunostaining with 2 µg/mL goat anti-COX-1 antibody (Santa Cruz Biotechnology, CA), 2 µg/mL goat anti-COX-2 antibody (Santa Cruz Biotechnology, CA), 5 µg/mL goat monoclonal antibodies to macrophages (MOMA2) antibody (AbD Serotec, Oxford, UK), followed by avidin-biotin rabbit anti-goat antibody (Nichirei, Tokyo, Japan). The slides were then incubated with 3,3-Diaminobenzidine tetrahydrochloride (Sigma, MO) and counterstained with hematoxylin (Wako Pure Chemical Industries, Osaka, Japan). Isotype-matched controls were stained in parallel and in all cases showed no significant reactivity (data not shown). Intensity of COX-1, COX-2, and MOMA2 staining was measured by determining the maximum density of DAB stained cells using the Image J program (National Institutes of Health, MD). Sections from the aortic arch were used to quantify atherosclerosis (8 animals from each group). A blinded observer analyzed the lesions.
Data are expressed as mean ± SEM. Statistical significance was calculated in the SPSS statistical package, version 11.0.1 J (SPSS, IL) for Windows. Student’s t test was used for comparisons between two groups and Dunnett test was used to for comparisons among more than three groups. Probability values <0.05 were regarded as significant.
To test the effect and the COX-2 selectivity of loxoprofen sodium, we measured platelet Tx, PGI2, and PGE2 production in apoE−/− mice and healthy men. Loxoprofen sodium inhibited the platelet production of TxA2 metabolite, urinary TxB2, by 29.0% compared to vehicle in apoE−/− mice (269.55 ± 77.48 ng/mg CRE vs 929.17 ± 196.29 ng/mg CRE, p = 0.012), and inhibited the production of urinary TxB2 by 26.2% compared to pre medication in men (0.53 ± 0.10 ng/mg CRE vs 2.04 ± 0.12 ng/mg CRE, p = 0.001) (Fig. 1A and D). Loxoprofen sodium inhibited the production of PGE2 metabolite in apoE−/− mice by 9.0% compared to vehicle (1.29 ± 0.29 ng/mg CRE vs 14.49 ± 3.60 ng/mg CRE, p = 0.003, respectively), and inhibited the production of urinary PGE2 metabolite by 29.7% compared to pre medication in men (0.06 ± 0.02 ng/mg CRE vs 0.20 ± 0.03 ng/mg CRE, p = 0.028) (Fig. 1C and F). Loxoprofen sodium also inhibited the production of PGI2 metabolite, urinary 2,3 dinor 6 keto PGF1α, in apoE−/− mice by 26.9% compared to vehicle (107.0 ± 17.83 ng/mg CRE vs 397.2 ± 122.1 ng/mg CRE, p<0.001), and inhibited the production of urinary 2,3 dinor 6 keto PGF1α by 44.0% compared to pre medication in men (1.11 ± 0.04 ng/mg CRE vs 2.52 ± 0.09 ng/mg CRE, p<0.001) (Fig. 1B and E).
Fig. 2 A, B, C and D shows representative results of mouse platelet aggregation stimulated by ADP or collagen solutions. Although PATIADP was 1.96 ± 0.66 µM and PATIcollagen was 2.17 ± 0.44 µg/ml in mice with vehicle, they were suppressed to >4 µM (n = 15, p = 0.033), and >4 µg/ml (n = 15, p<0.001) after administration of loxoprofen sodium. Fig. 2 E, F, G and H show representative results of healthy human platelet aggregation stimulated by ADP or collagen solutions, respectively. After administration of loxoprofen sodium, platelet aggregation was suppressed. Although PATIADP was 1.52 ± 0.39 µM and PATIcollagen was 0.41 ± 0.03 µg/ml in humans before administration of loxoprofen sodium, they were suppressed to 3.18 ± 1.50 µM (n = 5, p = 0.004), and 1.28 ± 0.50 µg/ml (n = 5, p = 0.034) after its administration.
Body weights were unchanged in mice treated with both loxoprofen sodium and vehicle (Table 1). Dry chem system demonstrated that the serum levels of albumin, CRE, and FFA were unchanged in mice treated with loxoprofen sodium and vehicle. HPLC analysis demonstrated that the serum levels of total cholesterol and serum triglycerides were unchanged (Table 1).
Atherosclerotic lesion area of the aortic root was significantly decreased to 41.5% (308,545 ± 36,892 µm2, p<0.001) in female mice receiving 4 mg/kg/day of loxoprofen sodium for 56 days and was decreased to 75.6% (487,455 ± 29,623 µm2, p = 0.001) in female mice receiving 4 mg/kg/day of loxoprofen sodium for 7 days compared to mice treated with vehicle (644,372 ± 28,314 µm2) (Fig. 3D). Similarly, atherosclerotic lesion area was significantly decreased to 63.5% (372,145 ± 38,149 µm2, p = 0.015) in male mice receiving loxoprofen sodium for 56 days and 84.8% (432,789 ± 39,236 µm2, p = 0.015) in male mice receiving it for 7 days compared to mice treated with vehicle (vs 510,117 ± 34,409 µm2) (Fig. 3E).
Representative Oil-Red-O and hematoxylin stained sections from the proximal aorta of female mice treated with vehicle (Fig. 3A), female mice receiving 4 mg/kg/day loxoprofen sodium for 7 days (Fig. 3B), and female mice receiving 4 mg/kg/day loxoprofen sodium for 56 days (Fig. 3C), indicate fatty streak lesions consisting predominantly of foam cells. The prevalence of Oil-Red-O stained lesions among arteriosclerotic lesions in male and female mice with loxoprofen sodium was equal to that in male and female mice receiving vehicle (Fig. 4A).
The prevalence of positive staining lesions of MOMA2, COX-1 and COX-2 in atherosclerotic lesions in female mice treated with loxoprofen sodium for both 7 days and 56 days was similar to that in female mice treated with vehicle (Fig. 4B, C and D).
Treatment of apoE−/− mice with 4 mg/kg/day loxoprofen sodium was well tolerated. The histological examination of liver, kidney and stomach showed no abnormalities even at a dose of 8 mg/kg of loxoprofen sodium.
Atherosclerotic lesion formation was reduced to 63.5% and 41.5%, in male and female apoE−/− mice with loxoprofen sodium compared to control, respectively. However, the composition of atherosclerotic lesions in mice treated with loxoprofen sodium differed from that in the mice receiving vehicle. The extent of Oil-Red-O, COX-1 and COX-2 expression was the same in mice receiving loxoprofen sodium and vehicle. Loxoprofen sodium reduced the production of TxB2 and PGI2, and also reduced platelet aggregation in vivo. Loxoprofen sodium has anti-inflammatory effects and reduced the production of PGE2. Thus, loxoprofen sodium, a non-selective NSAID, may reduce atherosclerotic lesion formation in apoE−/− mice by an anti-inflammatory effect. It is widely accepted that aspirin prevent the occurrence of myocardial infarction and/or ischemic stroke primarily through the inhibition of platelet aggregation, although their role in the development of fatty streak lesions remains a matter of debate.
Belton et al. demonstrated that the profile of PG generation and expression of COX were remarkably similar in the apoE−/− murine model and human atherosclerosis. In the apoE−/− murine model and human atherosclerosis, there is an increase in the biosynthesis of TXA2 and PGI2, with induction of COX-1 and COX-2 . The COX-2 selectivity of loxoprofen sodium active metabolite (loxoprofen-SRS) in vitro (the ratio of COX-1 IC50 /COX-2 IC50) was 0.35 . Our study indicated that administration of loxoprofen sodium for apoE−/− mice (4 mg/kg/day) reduced biosynthesis of PEG2 metabolite to 9.0%, TXA2 metabolite to 29.0%, and PGI2 metabolite to 26.9%. Moreover, administration of loxoprofen sodium to healthy men (60 mg/body, three times a day) reduced the biosynthesis of PGE2 metabolite to 29.7%, TXA2 metabolite to 26.2%, and PGI2 metabolite to 44.0%. Thus, loxoprofen sodium may be a well-balanced COX-1 and COX-2 inhibitor, which contributes to a reduction of atherosclerotic lesion formation.
On the other hand, loxoprofen sodium did not change the serum levels of total cholesterol or serum triglycerides. Some COX inhibitors might affect lipid metabolism. Rofecoxib has been reported to increase the susceptibility of human LDL-c and cell membrane lipids to oxidative modification, a hallmark feature of atherosclerosis. Rofecoxib promoted the non-enzymatic formation of isoprostanes from biological lipids, which act as important mediators of inflammation in the atherosclerotic plaque . Celecoxib does not have these effects. Thus, each NSAID has different pharmacologic actions [20–22].
In conclusion, loxoprofen sodium, a non-selective NSAID, reduces atherosclerotic lesion formation in apoE−/− mice by suppression of PGE2, TxB2 and PGI2. The influence of COX-2 selective inhibition in atherosclerosis has been controversial, but non-selective NSAID could reduce atherosclerotic lesion formation in a murine model. Our data may provide insights into the mechanisms underlying the cardiovascular safety of NSAIDs.