collagen-induced arthritis

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1 Pro. Natl. Aad. Si. USA Vol. 92, pp , January 1995 Medial Sienes Leukotriene B4 plays a ritial role in the progression of ollagen-indued arthritis R. J. GRIFFITHS*, E. R. PETrIPHER, K. KOCH, C. A. FARRELL, R. BRESLOW, M. J. CONKLYN, M. A. SMITH, B. C. HACKMAN, D. J. WIMBERLY, A. J. MILICI, D. N. SCAMPOLI, J. B. CHENG, J. S. PILLAR, C. J. PAZOLES, N. S. DOHERTY, L. S. MELVIN, L. A. REITER, M. S. BIGGARS, F. C. FALKNER, D. Y. MITCHELL, T. E. LISTON, AND H. J. SHOWELL Central Researh Division, Pfizer In., Groton, CT 634 Communiated by George E. Palade, University of California, San Diego, La Jolla, CA, Otober 3, 1994 ABSTRACT Leukotriene B4 (LTB4) is a produt of the 5-lipoxygenase pathway of arahidoni aid metabolism. LTB4 is a potent hemotati fator for neutrophils and has been postulated to play an important role in a variety of pathologial onditions inluding rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. The role of LTB4 in suh diseases has not yet been defined but in this paper we provide diret evidene that LTB4 plays a ritial role in a murine model of RA. CP-15,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)- 4-hydroxyhroman-7-yl]ylopentane arboxyli aid, is an LTB4 reeptor antagonist that inhibits LTB4 binding to human neutrophil membranes with an IC5 of 3.7 nm and inhibits LTB4-indued hemotaxis of these ells with an ICso of 5.2 nm. CP-15,696 inhibits LTB4-indued neutrophil influx in mouse skin when administered orally with an ED5 of 4.2 mg/kg. CP-15,696 had a dramati effet on both the linial symptoms and histologial hanges of murine ollagen-indued arthritis when administered at doses of.3-1 mg/kg. Inhibition was not assoiated with suppression of the humoral immune response to ollagen and was equally effetive if drug treatment was ommened just prior to the onset of arthritis or throughout the experiment. These results suggest that LTB4 reeptor antagonists may be effetive therapeuti agents for the treatment of RA. Rheumatoid arthritis (RA) is a hroni inflammatory polyarthritis that is inadequately treated with urrently available drugs (1). One potential strategy to better treat this disease is to redue the influx of leukoytes into the joint, sine reent studies have shown that the extent of neutrophil infiltration into the joints of RA patients preedes linial signs of inflammation and is preditive of pain (2). There are a number of mediators of the inflammatory response that ould potentially be responsible for neutrophil aumulation, but leukotriene B4 (LTB4) is an attrative target sine it is a potent hemotati agent for human neutrophils (3), is produed in large amounts by these ells, and is found in the synovial fluid of patients with RA (4). In this paper, we desribe experiments to assess the role of LTB4 in a murine model of RA, ollagen-indued arthritis. The immunologial and histologial features of this model resemble those seen in RA patients. The strategy we employed was to use a LTB4 reeptor antagonist to blok the biologial effets of endogenously produed LTB4. Several potent and seletive LTB4 reeptor antagonists, with a variety of strutural types, have been reported (reviewed in ref. 5). However, there are no data on the effiay of these agents in models of arthritis. CP-15,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)-4- hydroxyhroman-7-yl]ylopentane arboxyli aid, is a newly disovered LTB4 reeptor antagonist that has a high affinity The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U.S.C solely to indiate this fat. OH for human and mouse LTB4 reeptors and has a long plasma half-life in the mouse, whih allows the maintenane of pharmaologially relevant onentrations of the drug with a one daily dosing protool. Here we report the use of CP- 15,696 to demonstrate the importane of LTB4 as a ritial mediator in the pathogenesis of murine ollagen-indued arthritis. MATERIALS AND METHODS In Vitro LTB4 Reeptor Ligand Binding Assays. The proedure for [3H]LTB4 binding was adapted from the method of Cheng and o-workers (6). Binding was performed in 15,ul in a buffer ontaining 5 mm Tris HCl (ph 7.3), 1 mm MgCl2, 9% methanol,.7 nm [3H]LTB4 ( GBq/ mmol; New England Nulear), and either.83 mg (murine spleen) or.13 mg (human neutrophil) of membrane per ml. Unlabeled LTB4 was added at a onentration of 5,uM to determine nonspeifi binding. Inubations were arried out in mirotiter plates at 4 C for 3 min and the bound ligand was separated from the free ligand with a Betaplate apparatus (Pharmaia LKB) with double-thikness glass fiber filter mats. In Vitro Chemotaxis Assay. Chemotaxis assays were performed as desribed by Harvath and o-workers (7). Neutrophils were isolated aording to the proedure of Ferrante and Thong (8); resuspended at a density of 3 x 16 ells per ml in Hanks' balaned salt solution ontaining alium, magnesium, and 2 mg of bovine serum albumin (BSA) per ml; and plaed in the upper wells of a multiwell hemotaxis hamber (Neuroprobe, Cabin John, MD). Serial dilutions of CP-15,696 were present in both wells of the hemotaxis hamber, and LTB4 (5 nm) was present in the bottom hamber. After a 1-hr inubation at 37 C the ellulose nitrate filters (pore size, 3,m) were removed from the hambers and fixed, stained, and assayed. The total number of ells (observed mirosopially at X4) migrating from 2,um beneath the monolayer to the leading front (measured at 2-,Lm intervals) were summed and provided an index of ell migration. In Vitro Adhesion Moleule Expression Assay. Human neutrophils were isolated as desribed above and resuspended at a density of x 17 ells per ml in phosphate-buffered saline ontaining 1 mm EDTA. In test tubes, serial dilutions Abbreviations: LTB4, leukotriene B4; RA, rheumatoid arthritis; IL-la, interleukin la. *To whom reprint requests should be addressed. 517

2 518 Medial Sienes: Griffiths et at of CP-15,696 were added to the ells and inubated for 5 min at 37 C followed by LTB4 to ahieve a final onentration of 5 nm. After a further inubation for 1 min at 37 C, ells were washed and then labeled with fluoresein isothioyanatelabeled anti-cd11b (Gen-Trak Systems, Framingham, MA), washed and then ontaminating red blood ells were lysed with fluoresene-ativated ell sorting lysing solution (Beton Dikinson), rewashed, and then resuspended for quantifiation of fluoresent staining with a Beton Dikinson FACSan flow ytometer. LTB4-Indued Neutrophil Influx in Mouse Skin. Male BALB/ mie were anesthetized with methoxyflurane and injeted intradermally with LTB4 (1 ng in 2,ul of saline ontaining 1 mg of BSA per ml), and neutrophil infiltration was measured after 4 hr by the myeloperoxidase ontent of the skin. Known numbers of mouse neutrophils (harvested from the peritoneal avity after injetion of oyster glyogen) were inluded in eah assay as a standard urve and data are expressed as numbers of neutrophil equivalents per site. Drug was dosed orally 1 hr prior to injetion of LTB4 (9). Indution and Assessment of Collagen-Indued Arthritis. Male DBA/lLaJ (9-13 weeks old) mie were immunized at the base of the tail with 1,ug of hiken type II ollagen in Freund's omplete adjuvant on days and 21. Severity of the symptoms of arthritis was assessed by inspetion of the paws ( m. Co I- o -J E E x O C. m -o -J E ' x O A L -1 B I Competing Ligand Conentration (log M) = normal paw, 1 = swelling and/or redness of one toe or finger joint, 2 = two or more joints involved, and 3 = severe arthritis in the entire paw; maximum sore for eah animal = 12). In some experiments, administration of interleukin la (IL-la) was used to ause a severe flare of the arthritis;.3 jig of reombinant murine IL-la, diluted in phosphate-buffered saline ontaining 1 mg of BSA per ml, was administered s.. on the days indiated. This protool auses all of the ontrol animals to exhibit a severe form of the disease and is valuable for pharmaologial studies sine it is more reproduible and allows the number of animals in eah group to be redued. Changes in body weight over the ourse of the experiment were also monitored. Nonimmunized mie treated with IL-1 lose body weight, but by day 7 this has returned to normal. The immunized mie show a more profound and persistent fall in body weight so that at day 7 the weight loss hanges are seen only in arthriti mie. CP-15,696 or vehile [methylellulose in water (5 mg/ml)] were administered orally in a dose volume of 1 ml/kg one daily. Treatment ommened either the day before the first immunization or the day before the first injetion of IL-1. Groups of 15-2 mie were used for the standard protool, and groups of 7-9 mie were used for the IL-1-stimulated protool. Eah experiment has been repeated on at least three oasions. Histopathology. Knee joints were dealified in Kristensen's solution, embedded in Paraplast plus, setioned, and stained - o o 1 8 8o 6-, E 2. U 4 o 1 ) f _ h. o E 6 'D 2 mi- J o C D Pro. NatL Aad Si USA 92 (1995). 9 - a CP (log M) FIG. 1. In vitro antagonism of LTB4 reeptors by CP-15,696. (A and B) Displaement of speifi [3H]LTB4 binding to human neutrophil (A) and murine (C3H/HEJ) spleen (B) membranes by LTB4 (-) and CP-15,696 (). Values depited are means ± SEM of three independent experiments. (C) Inhibition of LTB4-mediated human neutrophil hemotaxis by CP-15,696. Values depited are means ± SEM of five independent experiments. (D) Inhibition of LTB4-mediated upregulation of the a subunit of the 32-integrin CD11b/CD18 by CP-15,696. Values depited are means ± SEM of four independent experiments. 5

3 B C w LuI ei I LU Co wco Medial Sienes: Griffiths et at A CP-15,696 (mg/kg) TIME (DAYS) 1 ' 1 CP-15,696 (mgtkg) FIG. 2. In vivo effets of CP-15,696 in murine models of inflammation. (A) Inhibition of LTB4-mediated neutrophil infiltration by CP-15,696. Values depited are means ± SEM of three independent experiments. CP-15,696 doses of 3 mg/kg and greater produed statistially signifiant effets ompared to ontrols (P <.5). (B and C) Inhibition of the symptoms of ollagen-indued arthritis by CP- 15,696. (B) Mie were dosed with vehile (, ), 1 mg (U, o), or 1 mg (-, A) of CP-15,696 per kg daily throughout the experiment. Any animal that had not developed arthritis by day 5 was given an s.. injetion of reombinant murine IL-la (.3 gg) on days 5 and 51. Results show a typial experiment representative of four performed. Effets of both doses of CP-15,696 were statistially signifiant ompared to ontrol (P <.5). (C) Drug treatment ommened on day 25, IL-1 was administered on days 26 and 27, and severity of arthritis () and hange in body weight (A) were measured on day 34. Pro. Natl. Aad Si USA 92 (1995) 519 with Safranin ; approximately mathed setions were examined by light mirosopy on a Nikon FXA mirosope. Quantitation of the proteoglyan staining in the femoral ondyle was performed as desribed (1). Anti-Collagen Antibody Levels. IgG antibody levels against the immunizing antigen were measured by standard ELISA methodology. Mirotiter plates were oated with 2.5 jig of hiken ollagen per well, bloked with BSA, and then inubated with dilutions of the test sera. An alkaline phosphatase rabbit anti-mouse IgG was then added, followed by substrate (p-nitrophenyl phosphate). Results are expressed as the reiproal of the dilution ausing 5% of the maximum absorbane hange at 45 nm. Statistis. Results of the in vivo experiments were analyzed by Student's t test with a Bonferroni orretion fator for multiple omparisons. RESULTS Struture. The struture of CP-15,696 is shown in Fig. 1. In Vitro Ativity in Ligand Binding Assays. CP-15,696 is a highly potent inhibitor of the binding of [3H]LTB4 to human neutrophil and mouse spleen membranes with IC5 values at 3.7 and 3.3 nm, respetively (Fig. 1 A and B). Respetive IC5 values for LTB4 obtained in the same ligand binding assays (Fig. 1A and B) were 2.1 nm (human neutrophil) and 1.3 nm (mouse spleen). In Vitro Ativity in Funtional Assays. LTB4-indued hemotaxis and upregulation of the adhesion moleule CD1lb in human neutrophils have been used to demonstrate the antagonist ativity of CP-15,696 in funtional assays. Chemotaxis (a funtion mediated by the high-affinity form of the LTB4 reeptor) was inhibited by CP-15,696 with an IC5 value of 5.2 nm (Fig. 1C) and adhesion moleule upregulation (a funtion mediated by the low-affinity form of the reeptor) with an ICso of 43 nm (Fig. ID). CP-15,696 did not demonstrate any agonist ativity in these assays, and at 1,uM it did not inhibit hemotaxis in response to CSa, platelet ativating fator, or IL-8 (data not shown), demonstrating the seletivity of the ompound. In Vivo Inhibition of LTB4-Indued Neutrophil Infiltration. To demonstrate the ability of CP-15,696 to inhibit the biologial ativity of LTB4 in vivo, LTB4 was injeted intradermally into mouse skin and neutrophil aumulation was measured. Oral administration of CP-15,696 inhibited this response with an ED5 value of 4.2 mg/kg (Fig. 2A). To demonstrate the seletivity of CP-15,696, it was tested for the ability to inhibit IL-1-indued neutrophil aumulation. Neutrophil equivalents (as measured by myeloperoxidase ontent of the skin) at IL-1-injeted sites in vehile-treated mie were 7.7 ± 1.3 x 15. In mie treated with 1 mg of CP-15,696 per kg the orresponding value was 11.2 ± 1.1 x 15, whih was not signifiantly different from the response in vehile-treated mie. In addition, CP-15,696 has been tested for the ability to inhibit zymosan-stimulated leukotriene and prostaglandin prodution in the mouse peritoneal avity. Doses of up to 3 mg/kg do not affet the prodution of either of these produts (data not shown). Inhibition of the Development of Collagen Arthritis. The pharmaokineti profile of CP-15,696 in the mouse was studied before ommening experiments in hroni models. The drug is well absorbed after oral administration (>5%) and has a plasma elimination half-life of 33 hr after i.v. administration. This means that a single daily dosing protool Values depited are means ± SEM of three independent experiments. CP-15,696 doses of 3 mg/kg and greater produed statistially signifiant effets ompared to ontrols (P <.5). Eah experimental group onsisted of 7-2 mie. All experiments with animals were in aordane with institutional guidelines.

4 52 Medial Sienes: Griffiths et al FIG. 3. Effet of CP-15,696 on histologial hanges in the joints of mie with ollagen-indued arthritis. (A) Sham-immunized animal treated with vehile showing the normal artiular artilage proteoglyan staining (arrows) and typial ellularity of the subsynovial onnetive tissue (arrowheads). (B) In ontrast, in the ollagenimmunized mie treated with vehile there was destrution of the artiular artilage (arrows), erosion of bone (E), and a massive influx of inflammatoryi ells into the subsynovial onnetive tissue (arrowheads). (C) In immunized mie treated with 1 mg of CP-15,696 per kg artiular artilage (arrows) and subsynovial onnetive tissue (arrowheads) were indistinguishable from ontrols. (X5.) an be used to provide adequate plasma levels to antagonize the LTB4 reeptor for 24 hr per day. The inidene of the disease in this model varies (in our hands, the proportion of mie showing any sign of arthritis varies from 6% to 9%) and the severity of the disease within a partiular group of mie an also be extremely variable. Administration of the ytokine IL-1 as an adjunt in this model inreases the inidene and severity of the disease, whih is useful for pharmaologial studies (11). The mehanism of this exaerbation is not lear but it may relate to the neutrophilia this ytokine auses. In the experiment shown in Fig. 2B, daily dosing of CP-15,696 starting the day before immunization Pro. Natt Aad SL USA 92 (1995) dereased the severity of arthritis in immunized animals at doses of 1 and 1 mg/kg. There was no evidene of any toxi effets of the ompound even after this extensive period of dosing. Fifty days into the experiment, animals that had not demonstrated any sign of arthritis were given two s.. injetions of IL-1 24 hr apart. This aused all the animals in the vehile-treated group to rapidly develop a severe arthritis. However, the animals treated with 1 mg of CP-15,696 per kg developed a muh less severe form of the disease, and at 1 mg/kg the disease was almost ompletely suppressed (Fig. 2B). CP-15,696 did not affet IgG antibody titers to type II ollagen in this model [titer in vehile-treated mie was 1/(3732 ± 252) ompared to 1/(3818 ± 295) in mie treated with CP-15,696 at a dose of 1 mg/kg throughout the experiment], suggesting that it does not interfere with the humoral immune response to the immunizing antigen. Inhibition of the Progression of Collagen Arthritis. The ativity of CP-15,696 in this model was explored further by using the IL-i-exaerbated protool. We found that if dosing of CP-15,696 was started 1 day before the first injetion of IL-1 (day 26 after immunization) it was just as effiaious as when dosed throughout the experiment (Fig. 2C). Vehiletreated animals lose body weight as the arthritis develops. Nonimmunized mie treated with IL-1 lose body weight, but by day 7 this has returned to normal. The immunized mie show a more profound and persistent fall in body weight so that at day 7 the weight loss hanges are seen only in arthriti mie. Body weight loss at early times after IL-1 administration was not affeted by CP-15,696. However, the weight loss observed at day 7 was redued, in parallel with the inhibition of arthritis development (Fig. 2C). This suggests that the persistent weight loss seen in the arthriti mie may be seondary to an impairment in feeding due to the pain and disability of the arthritis. A histologial examination of the joints of vehile-treated animals revealed a massive inflammatory ell influx with extensive artilage and bone damage. Although not obviously affeted by marosopi inspetion, the knee joints proved to be severely affeted when assessed histologially. Sine reproduible setions an be more easily prepared from this joint, it was used for quantitative histologial studies. Treatment with CP-15,696 prevented not only the inflammatory ell influx but also the artilage and bone destrution (Fig. 3). Quantitation of the proteoglyan ontent of the femoral ondyle by image analysis onfirms this observation. The extent of proteoglyan staining in the artiular artilage of the femoral ondyle was expressed as a perentage of that in the growth plate of eah animal, whih ated as an internal ontrol. In normal knees, this value was 65.9% ± 3.4%, whih dereased to 26.6% ± 6.8% in immunized mie treated with vehile. In mie treated with CP-15,696 (1 mg/kg), this was inreased to 48.3% ± 4.1% (P <.5 ompared to vehile group, mean ± SEM; n = 3 experiments). DISCUSSION Although there is no perfet animal model of RA, murine ollagen-indued arthritis does share ertain features with the human disease. Neutrophil infiltration into the joints is a prominent feature of mie with ollagen-indued arthritis, and it has been shown that antibody depletion of irulating neutrophils is antiinflammatory in this model (12, 13). Clinial data have shown that neutrophil infiltration into the joints of patients with RA is preditive of pain and preedes linial signs of inflammation (2). The histologial features of this model losely approximate those observed in rheumatoid joints, and a proportion of patients with RA display humoral and ellular immunity to type II ollagen. Furthermore, the importane of immunity to type II ollagen in the pathogenesis of RA has reently been demonstrated by the finding that

5 Medial Sienes: Griffiths et at ingestion of type II ollagen an suppress disease, presumably by induing a state of immunologial tolerane (14). Several reent studies have shown that biologial reagents that neutralize the effets of the ytokines tumor nerosis fator (TNF) (15-17) and IL-1 (18-2) are effiaious in this murine model of RA. These experiments led to trials of anti-tnf antibodies in RA patients, where striking therapeuti effiay has been reported (21). The studies reported here diretly impliate LTB4 as a ritial mediator of inflammatory arthritis and suggest that trials of suh agents in RA are warranted. Clearly there would be a great advantage in terms of ost and onveniene in treating human subjets with an orally ative, low moleular weight agent rather than i.v. administration of an antibody or soluble reeptor. CP-15,696 inhibits disease expression in this model by targeting the effetor arm of the immune/inflammatory response. This is supported by the fat that the drug had no effet on antibody levels to ollagen, and it was equally effiaious when dosed either throughout the experiment, or just prior to disease onset. The effiay of a LTB4 antagonist in the standard, non-il-1-boosted version of this model is at least equivalent if not superior to antiytokine interventions. The fat that the therapeuti effet of CP-15,696 was evident even in immunized animals treated with IL-1 is remarkable, sine these animals exhibit a partiularly rapid and severe flare response. This may at first sight seem surprising. However, the likely explanation is that LTB4 exerts its effets distal to those of ytokines suh as IL-1 and TNF. This suggests that there is a limited redundany of mediators of the inflammatory response and holds out the hope that new and effetive treatments for hroni inflammatory disease in humans may be on the horizon. Drugs suh as CP-15,696 will allow us to test the hypothesis that LTB4 plays a similar role in human inflammatory joint disease. 1. Brooks, P. (1993) Lanet 341, Jones, A., Al-Janabi, M., Solanki, K., Sobnak, R., Greenwood, A., Doyle, D., Britton, K. & Huskisson, E. (1991)ArthritisRheum. 34, Pro. NatL Aad Si. USA 92 (1995) Ford-Huthinson, A. W., Bray, M.A., Doig, M. V., Shipley, M. E. & Smith, M. J. H. (198) Nature (London) 286, Klikstein, L. B., Shapleigh, C. & Goetzl, E. J. (198) J. Clin. Invest. 66, Cohen, N. & Yagaloff, K. A. (1994) Curr. Opin. Invest. Drugs 3, Cheng, J. B., Cheng, E. I. P., Kohi, F. & Townley, R. G. (1986) J. Pharmaol. Exp. Ther. 236, Harvath, L., MCall, C. E., Bass, D. A. & MPhail, L. C. (1987) J. Immunol. 139, Ferrante, A. & Thong, Y. H. (1978) J. Immunol. Methods 26, Pettipher, E. R., Salter, E. D., Breslow, R., Rayroft, L. & Showell, H. J. (1993) Br. J. Pharmaol. 11, Otterness, I. G., Bliven, M. L., Milii, A. J. & Poole, A. R. (1994) Am. J. Pathol. 144, Hom, J. T., Bendele, A. M. & Carlson, D. G. (1988) J. Immunol. 141, Courtenay, J. S., Dallman, M. J., Dayan, A. D., Martin, A. & Mosedale, I. (198) Nature (London) 283, Shrier, D., Gilbertsen, R., Lesh, M. & Fantone, J. (1984) Am. J. Pathol. 117, Trentham, D., Dynesius-Trentham, A., Orav, E., Combithi, D., Lorenzo, C., Sewell, K, Hafler, D. & Weiner, H. (1993) Siene 261, Williams, R. O., Feldmann, M. & Maini, R. N. (1992) Pro. Natl. Aad. Si. USA 89, Piguet, P. F., Grau, G. E., Vesin, C., Loetsher, H., Gentz, R. & Lesslauer, W. (1992) Immunology 77, Wooley, P. H., Duther, J., Widmer, M. B. & Gillis, S. (1993) J. Immunol. 151, Wooley, P. H., Whalen, J. D., Chapman, D. L., Berger, A. E., Rihard, K. A., Aspar, D. G. & Staite, N. D. (1993) Arthritis Rheum. 36, Van Den Berg, W. B., Joosten, L. A. B., Helsen, M. & Van de Loo, F. A. J. (1994) Clin. Exp. Immunol. 95, Geiger, T., Towbin, H., Cosenti-Vargas, A., Zingel, O., Arnold, J., Rordorf, C., Glatt, M. & Vosbek, K. (1993) Clin. Exp. Rheum. 11, Elliot, M. J., Maini, R. N., Feldman, M., Long-Fox, A., Charles, P., Katsikis, P., Brennan, F. M., Walker, J., Bijl, H., Ghrayeb, J. & Woody, J. N. (1993) Arthritis Rheum. 36,