Anti-Inflammatory and Analgesic Potency of Carboxyamidotriazole, a Tumorostatic Agent

Carboxyamidotriazole (CAI) is a calcium influx inhibitor that is undergoing clinical trials for the treatment of various human cancers following the identification of its antiproliferative and antimetastatic activities. The exact mechanism of its action is not clearly understood, and whether it has other functions besides the established antitumor activity has not been reported either. In the present study, we demonstrate for the first time that CAI possesses anti-inflammatory and analgesic activities using a variety of animal models, including croton oilinduced ear edema, cotton-induced granuloma, rat adjuvantinduced arthritis, acetic acid-induced writhing, and the formalin test. We also show that CAI significantly inhibits local vascular permeability stimulated by vascular endothelial growth factor or histamine and decreases tumor necrosis factorand interleukin-1 levels at the site of inflammation and in serums, which may contribute to the anti-inflammatory effect. These data suggest that CAI is a promising anti-inflammatory and analgesic agent, and they provide new insight into the biological activity of the drug. Carboxyamidotriazole (CAI), initially developed as a coccidiostatic agent and then identified for its anti-invasive capacity, has been shown to inhibit tumor and endothelial cell proliferation by inhibition of calcium uptake and calcium-mediated signal transduction. Up to now, many studies have been conducted mainly focusing on the cancer fighting properties of CAI, which inhibits the proliferation and invasive characteristics of several tumor cell lines in vitro (Wasilenko et al., 1996; Jacobs et al., 1997; Lambert et al., 1997; Moody et al., 2003; Enfissi et al., 2004; Perabo et al., 2004). In addition, the antiangiogenic effect of CAI has also been well established. In vitro studies have shown that CAI reduces proliferation, adhesion, motility, and vascular tube formation of human umbilical vein endothelial cells (Kohn et al., 1995). Local administration of CAI inhibits capillary expansion in the chick chorioallantoic membrane assay. Furthermore, inhibition of angiogenesis by orally administered CAI has been observed in animal models and in tumor xenografts (Luzzi et al., 1998). Herein, CAI is also described as a new nonendothelial cellspecific inhibitor of angiogenesis (or angiopreventive agent). In an earlier study (Felder et al., 1991), CAI was shown to have an immediate inhibitory effect on carbachol-stimulated release of arachidonic acid in muscarinic acetylcholine receptor m2-transfected Chinese hamster ovary cells. Another noticeable finding is that pretreatment of human T cells with CAI inhibits the nuclear accumulation of c-Rel and p65, causing a selective repression of nuclear factorB DNA binding and a near complete inhibition of calcium-regulated mitogen-induced transcription from the human immunodeficiency virus long terminal repeat (Yasui et al., 1997). According to our current knowledge, the above-mentioned events, including angiogenesis, release of arachidonic acid, and nuclear factorB activation, modulated by CAI are involved in many pathological processes, such as cancer and chronic inflammation (Griffioen and Molema, 2000; Lu et al., 2006). Thus, we put forward the hypothesis that CAI, currently recognized as a tumoristatic and antiangiogenic agent, may have more pharmacological activities in addition to what it already presents. In the present work, we evaluate the possible beneficial effect of CAI in the treatment of acute and chronic inflammatory processes, such as croton oil-induced ear edema, cotThis work was supported by the National High Technology Research and Development Program of China (2005AA2Z3G41) and a grant of international cooperation from the Ministry of Science and Technique of China (2005DFA31110). L.G. and C.Y. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.131888. ABBREVIATIONS: CAI, carboxyamidotriazole; TNF, tumor necrosis factor; IL, interleukin; PEG, polyethylene glycol; ELISA, enzyme-linked immunosorbent assay; NS, 0.9% NaCl; VEGF, vascular endothelial growth factor; PLC, phospholipase C. 0022-3565/08/3251-10–16$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 325, No. 1 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 131888/3316256 JPET 325:10–16, 2008 Printed in U.S.A. 10 at A PE T Jornals on Jne 0, 2017 jpet.asjournals.org D ow nladed from ton-induced granuloma, and rat adjuvant-induced arthritis. Local vascular permeability in mouse modulated by CAI has also been determined. In addition, we have assessed the effect of CAI on the levels of proinflammatory cytokines TNFand IL-1 at the site of inflammation and in sera. Moreover, antinociceptive effect of CAI on inflammatory pain has been assessed using acetic acid-induced writhing model and the formalin test. This study is, to our knowledge, the first showing that CAI possesses anti-inflammatory and analgesic activities. Materials and Methods Materials. CAI was synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences. Polyethylene glycol (PEG) 400, acetic acid, and formaldehyde solution 37% (w/w) were provided by Beijing Chemical Reagents Company (Beijing, China). Dexamethasone sodium phosphate injection was from Tianjin Jinyao Amino Acid Co., Ltd. (Tianjin, China). Morphine hydrochloride was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for TNFand IL-1 were from R&D Systems (Minneapolis, MN). Animals. Female Wistar rats (180–200 g) or male ICR mice (18–22 g; 6–8 weeks old) were obtained from Beijing Vital River Laboratory Animal Co., Ltd. (Beijing, China). They were housed in an air-conditioned room (22 2°C and 40–70% humidity), with a controlled 12-h light/dark cycle (lights on 8:00 AM). Animals had free access to standard chow and water. All animal studies and procedures were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College. Croton Oil-Induced Ear Edema. The croton oil ear test was performed as described previously (Olajide et al., 2000). Male ICR mice were randomly divided into five groups, with 15 animals in one group. CAI (10 and 20 mg/kg), dexamethasone (0.8 mg/kg), or vehicle (PEG 400 and 0.9% NaCl) was administered once daily (orally for CAI and PEG 400; i.p. for dexamethasone and saline). One hour after the third administration, 100 l of croton oil solution (croton oil/ ethanol/distilled water/ether, 2:20:5:73) was applied to both sides of the right ear of each mouse. The left ear remained untreated. The animals were sacrificed 4 h later, and a plug (6.5 mm in diameter) was removed from both the treated and untreated ear. The difference in weight between the two plugs was taken as a measure of edema intensity. The anti-inflammatory activity was expressed as percentage of the edema reduction in treated mice compared with control mice. Induction of Granulomatous Tissue. Subacute inflammation was produced in female Wistar rats (180–200 g) by cotton pelletinduced granuloma (Swingle and Shideman, 1972). Sterile cotton (20 1 mg) soaked in 0.4 ml of 5% ampicillin solution was implanted s.c. bilaterally in groin under anesthesia. The following was administered daily: 0.9% NaCl (NS; i.p.), 0.8 mg/kg dexamethasone (i.p.), PEG 400 (p.o.), and 10 and 20 mg/kg CAI (p.o.). On the 7th day, the animals were sacrificed. The granulomatous tissue with cotton pellet was removed and dried at 60°C to constant weight. Increment in the dry weight of the pellets was taken as measure of granuloma formation. Inhibition of Adjuvant-Induced Arthritis. Female Wistar rats (180–200 g) were divided into five groups. On day 0, animals in groups 1 to 4 were induced adjuvant arthritis by a single intradermal injection of 0.1 ml of Freund’s complete adjuvant into the plantar region of the right hind paw. NS (i.p.), 0.5 mg/kg dexamethasone (i.p.), PEG 400 (p.o.), and 20 mg/kg CAI (p.o.) were administered once a day from day 0 to day 27. As a nonarthritic control, animals in group 5 were free of Freund’s complete adjuvant injection, and they received injections with 0.9% NaCl daily. Right hind paw volume was measured before adjuvant injection and on days 1 to 14 with a water displacement volume meter to assess the primary inflammatory response, and left hind paw volume was measured on days 21, 24, and 27 to assess the secondary inflammatory response. The antiinflammatory effect was expressed as the difference in paw edema compared with that of saline-treated arthritic rats (Newbould, 1963; Walz et al., 1971). Histopathology Evaluation. Rat synovial membrane samples were obtained from the knee joint 14 days after adjuvant injection. The specimens were fixed in buffered 10% formalin and embedded in paraffin. They were serially sectioned onto microscope slides at a thickness of 4 m and then deparaffinized, stained with hematoxylin and eosin, and evaluated for morphological changes and cellular