Strategies for the development of new antiarthritic agents

Strategies for the development of new antiarthritic agents

0192-0561/92 $5.00 + .00 Pergamon Press plc. International Society for Immunopharmacology. Int. J. lmmunopharmac., Vol. 14, No. 3, pp. 497-504, 1992...

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0192-0561/92 $5.00 + .00 Pergamon Press plc. International Society for Immunopharmacology.

Int. J. lmmunopharmac., Vol. 14, No. 3, pp. 497-504, 1992. Printed in Great Britain.


Therapeutic advances in rheumatoid arthritis (RA) have largely focused on the development of non-steroidal antiinflammatory drugs (NSAIDs) with improved characteristics compared with aspirin [Brooks & Day, New Engl. J. Med., 324, 1716-1725 (1991)]. For example, greater potency, safety, improved tolerance in the elderly and reduced frequency of dosing have been achieved. However, these agents are generally considered to be palliative treating of the symptoms of the disease. The development of disease modifying drugs (DMD), also known as second line drugs, for RA has not been very successful. Most of the agents that are currently used in this category were originally used to treat other diseases such as malignancy (cyclophosphamide, methotrexate), Wilson's disease (d-penicillamine) and tuberculosis (gold salts) [Pullar, Br. J. clin. Pharmac., 30, 501-510 (1990)]. Unfortunately, none of the agents is ideal and each has potentially serious side-effects. There have been several attempts to develop agents with new mechanisms of action that hopefully will greatly improve these current therapies.

Abstract --



The arachidonic acid cascade has proven to be a fertile research ground for biologists and medicinal chemists alike. The discovery that NSAIDs inhibit the cyclooxygenase pathway that produces prostaglandins and thromboxanes was rapidly followed by numerous attempts to inhibit other enzymes in the cascade. Recent attention has been paid to modulating 5-1ipoxygenase (5-LO), the enzyme responsible for oxygenation of arachidonic acid to the leukotrienes (includng LTB4) in several cell types including PMNs. LTB4 is a potent chemokinetic, chemotactic and proaggregant for a variety of leukocytes and is thought to be an important mediator of inflammatory disease (Belch, 1989). There are a number of mechanisms thought to be responsible for 5-LO inhibition including acting as an antioxidant and/or radical scavenger, iron chelation and interference with translocation mechanisms. Zileuton (A-64,077) is one of the most advanced 5-LO inhibitors thought to work by iron chelation (5-LO contains a non-heme iron at its active site) (Carter et al., 1989). It has apparent clinical efficacy in asthma, allergic rhinitis, ulcerative colitis and, recently, RA. It reduces

swollen joints when administered orally at 800 mg b.i.d. A back-up compound (A-69,412) with lower protein binding and a greater half-life and bioavailability is also under development. The 5-LO enzyme is normally cytosolic and segregated from its substrate, arachidonic acid, which is a component of the cell membrane. Upon activation by hydroperoxides, ATP and calcium, the 5-LO enzyme is activated and translocates, possibly a result of conversion to a hydrophobic conformation, to the cell membrane where it complexes with the docking protein, FLAP (5-LO activating protein) (Ford-Hutchinson, 1991). Once docked to the cell membrane, 5-LO oxidizes free arachidonic acid to leukotrienes and other 5-LO products. MK 886 has been shown to be a FLAP inhibitor that prevents 5-LO binding to FLAP and has been referred to as a translocation inhibitor (Ford-Hutchinson, 1991). MK 886 has been recently withdrawn from clinical development. Phospholipase A2 is the rate limiting enzyme responsible for the cleavage of arachidonic acid from the sn-2 position of membrane phospholipids (Chang, Musser & McGregor, 1987; Mobilio & Marshall, 1989). Arachidonic acid and the remaining lysophospholipid are then converted to several 497


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proinflammatory mediators [prostaglandins, leukotrienes, lipoxins, hydroxy fatty acids and platelet activating factor (PAF), respectively]. Two major forms of PLA2 exist - - an intracellular, high molecular weight ( 7 0 - 110 kd) form and an extracellular, low molecular weight (14 kd) form. Attempts to inhibit PLA_, have proved difficult. There are over 50 PLA2 enzymes identified and evaluation of a popular source, porcine pancreatic PLA~, has not proven predictive in inflammatory models. Human PLA2 is now available for examination and is the preferred source. PLA2 does not conform to classical enzyme kinetics and the choice of substrate, ionic strength and [Ca 2+] all determine experimental outcome. Although several classes of PLA2 inhibitors have been described they are often weakly efficacious, non-specific and the precise mechanism of action is unclear (Mobilio & Marshall, 1989; Wilkerson, 1990). In vivo efficacy has also been difficult to demonstrate. This approach is nevertheless a very attractive way to modulate the arachidonic acid cascade. This cascade remains of continued interest and now that the cDNAs for several enzymes have been isolated and characterized (including cyclooxygenase, 5-LO, LTA~ hydrolase, 15-LO, 12-LO, FLAP), efforts to examine enzyme localization in disease and control of gene expression of the enzymes are underway. Furthermore, the cloning of eicosanoid receptors, including thromboxane A2 and PAF, will continue to focus attention on these important inflammatory mediators.


An ever-increasing number of cytokines that play an essential role as mediators of immunoinflammatory responses are being discovered. Many cytokines including IL-1, IL-6, IL-8, M-CSF, a-IFN, GM-CSF, PDGF and TNF have been detected in synovial fluid and synovial tissue from patients with RA (Arend & Dayer, 1990; Firestein & Zvaifler, 1990). In particular IL-lfl and TNF-a genes are expressed by large numbers of synovial cells. IL-I exists in two different forms, termed IL-l~ and IL-lp, which are synthesized from distinct precursor proteins, each of 31.5 kd mol.wt. It has numerous systemic (fever, synthesis of acute phase proteins, decreased appetite) and local activities (chemotaxis of PMN, lymphocytes, and monocytes; leukocyte

adherence to endothelial cells; fibroblast proliferation; PGE2, MMP-I and neutral protease production; stimulation of T- and B-lymphocytes) (Dinarello, 1991). TNF-a (17 kd) is produced primarily by monocytes and macrophages. IL-1 and TNF-a are usually synthesized and secreted simultaneously although they are regulated and controlled by different mechanisms. Both IL-1 and TNF-a act in an autocrine manner to stimulate their own transcription as well as the production of each other. TNF is less 'inflammatory" than IL-1 but synergizes with IL-1. A connection between eicosanoids and cytokine formation has been suggested. Prostaglandins have been shown to suppress formation of various cytokines (including IL-1), and leukotrienes may play a role in the activation of cytokine synthesis (including IL-1 and TNF). There is some evidence, although controversial, that 5-LO inhibitors may suppress IL-1 and TNF release (Hoffman et al., 1991). Our own results demonstrated that the selective 5-LO inhibitors Wy-50,295, MK 886 and zileuton failed to inhibit 1L-I production from LPS-stimulated mouse peritoneal cells (Adams, Baeder, Yonno & Chang, 1990). The selective cyclooxygenase inhibitors indomethacin and ibuprofen also failed to modulate IL-1 in this assay although dual 5-LO/cyclooxygenase inhibitors did inhibit the IL-1 production. The latter observations confirm those of others using other cell sources. The precise mechanism by which 5-LO/cyclooxygenase inhibitors modulate IL-1 synthesis is unknown. Tenidap is a potent cyclooxygenase inhibitor that also inhibits 5-LO (Blackburn, Heck, Loose, Eskra & Carty, 1991). In addition to its abilty to inhibit IL-1 production in vitro, it reduced IL-I activity in the synovial fluid of RA patients. Tenidap inhibits the release of activated neutral protease and IL-1 responses in chondrocytes. It is currently under clinical development in RA (Robinson, 1990). Several other compounds have been reported to inhibit IL-I production and they include the DMDs, glucocorticoids, probucol, ciprofloxacin, 5-aminosalicyclic acid, 3-deazadenosine and pentamidine. Production may also be inhibited by affecting the conversion of the 31.5 kd precursors of IL-I~ and IL-1/3 to the secreted 17.5 kd forms, which is accomplished by a variety of enzymes including elastase, plasmin, cathepsin G, collagenase and serine protease. Monocytes but not fibroblasts contain an IL-l-fl converting enzyme (ICE) that may also be responsible for IL-lfl production intracellularly (Kostura et al., 1989). Thus, there is more

Development of New Antiarthritic Agents than one mechanism to cleave the precursor into active IL-1 peptides, making this a difficult target to modulate. I L - l a and /3 interact with Type I (80 kd; T-cell, fibroblast type) and Type II ( 6 0 - 6 8 kd; B-cell/ macrophage type) IL-1 receptors. A soluble IL-1 receptor has been synthesized by cloning and expression of the extracellular, IL-1 binding domain of the Type I receptor (Dower et al., 1989). This protein can inhibit the rejection of heterotopic heart allografts in mice, and block immune response to injected allogeneic cells (Fanslow et al., 1990). Clinical trials of the human soluble IL-1 receptor in RA and transplantation are planned for later this year. Recently, the IL-1 receptor antagonist (IL-lra) has been identified and shown to inhibit IL-I binding to these receptors (Hannum et al., 1990). Human I L - l r a counteracts the acute effects of IL-1 in in vivo animal models if administered in 100-1000 fold excess (McIntyre et al., 1991). This excess is probably a requirement to swamp IL-1 receptors. Functional inhibitors of IL-1 responses have also been reported and include pentoxifylline and romazarit. Less is known about modulation of TNF-a release/effects. Two TNF-a receptors exist, Type I (55 kd) and Type II (75 kd) and soluble extracellular fragments of these receptors are known to bind to TNF and, consequently, act as inhibitors. These TNF-a inhibitors do not bind to the TNF receptor.


This family of zinc containing enzymes is responsible for the degradation of components of the extracellular matrix, such as collagen and proteoglycans, that occurs in RA (Woessner, 1991). These enzymes are secreted in zymogen form and are activated by proteinases which results in a loss of peptides of mol. wt approximately 10,000. A nomenclature for these enzymes has been proposed and includes MMP-1 (interstitial collagenase), MMP-2 (72 kd gelatinase) and MMP-3 (stromelysin). The activity of the MMPs is inhibited by tissue inhibitor of metalloproteinases (TIMP). TIMP-1 (tool. wt 28,500) is found in many tissues and forms a 1 : 1 stoichiometric complex with MMP-1 and MMP-3. The TIMP family also consists of IMP or TIMP-2 (20 kd) and a larger TIMP-like inhibitor (LIMP; 76 kd). Most cells capable of secreting MMP-1 and MMP-3 also secrete TIMP. However, IL-1 and TNF preferentially induce MMP-1 and MMP-2 expression


in rheumatoid synovial fibroblasts without inducing TIMP (Marte!-Pelletier, Zafarullah, Kodama & Pelletier, 1991). This suggests an important role for the MMPs (and IL-1 and TNF) in the pathogenesis of RA and cartilage destruction. Several synthetic inhibitors of the MMPs have been prepared including SC-40827, Ro30-7467 and Ro30-4724. Furthermore, MMP production can be inhibited by retinoids, glucocorticoids and TGF-/3. Efforts to inhibit these enzymes or their production could prove to be very effective in RA.


T-lymphocytes orchestrate the antigen-specific response to external pathogens, abnormal cells, and autoimmune antigens. In contrast to antibodies which are the B-cells' antigen receptor, T-cells recognize antigen only when presented by antigen presenting cells in the context of the major histocompatibility complex (MHC) determinants via the T-cell receptor complex. The T-cell receptor (TCR) complex of most mature T-lymphocytes is comprised of the following components: the a and/3 chains which bind antigen - MHC complex, the CD3 complex which contains three distinct polypeptide chains, the ~ and ~ chains involved in signal transduction and either the CD4 or the CD8 molecule which restrict the TCR to recognize antigen in the context of MHC class II or class I molecules, respectively. Strategies to inhibit the disease-causing T-lymphocytes include blocking and/or deleting T-cell populations or various subpopulations, blocking antigen presentation and/or stimulation, and inducing or stimulating suppressor cells. Most therapies alter the function of T-lymphocytes, independent of the specific antigen recognized by the disease-causing T-cells. However, many programs are evolving towards the development of agents which will selectively affect only disease-causing T-lymphocytes, based on their antigenic specificity.

Antigen independent strategies First, the population of T-cells targeted by various approved immunosuppressive agents range from the whole T-lymphocyte populaton (antilymphocyte serum (ALS) or OKT3) to the T-helper/inducer subpopulation characterized by the presence of the CD4 + surface molecule. For example, monoclonal


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antibodies specific for CD4 or CD5 are being tested in the clinic for their ability to prevent transplantation rejection and RA. Second, the subpopulation of activated T-lymphocytes characterized by CD44 IL-2R ~ markers have been targeted for immunosuppression by agents including Seragen's diptheria t o x i n - I L - 2 fusion protein (DAB4~6 IL-2) and antiIL-2 receptor antibodies. A preliminary report of uncontrolled studies described a 25% improvement in 12/13 RA patients undergoing treatment with the DAB,s6 IL-2 molecule although numerous sideeffects were observed. Third, the variable regions of the a and/3 chains of the TCR have been shown to be very polymorphic, as expected. However, T-cells which recognize myelin basic protein and induce EAE utilize a very restricted repertoire of the variable region of the /3 chain of the TCR (V/3) (Herber-Katz & Acha-Orbea, 1989). Paliard et al. (1991) have reported that some RA patients have a higher proportion of VpH+ T-cells in their synovial fluids in comparison with their blood. Howell reported that activated T-lymphocytes in synovial fluids of RA patients primarily use a restricted V/3~;,, V{3~ and V/3~4+ repertoire in comparison with activated peripheral T-lymphocytes (IRA Meeting, April 1991). Strategies are being developed to induce non-responsiveness of these T-cell subpopulations. As a step towards antigen-specific therapies, antigen presentation and stimulation can be impaired by agents which block the binding of the antigenic peptide to the MHC. RA is associated with HLADR4 and DRI in multiple ethnic groups and with Dwl6, but not in the Dwl0 and Dwl3 allele, in the Yakima nation of the North American Indians (Willkens, Nepom, Marks, Nettles & Nepom, 1991). Comparison of the encoded amino acid sequence in Dw4, Dwl4 and Dwl6, the alleles associated with RA susceptibility, revealed identity in amino acids (AA) 6 7 - 7 4 . Divergence was found in these same AA 67 - 74 in the closely related MHC alleles Dwl0, Dwl3, DR6a and DR6b, which are not associated with RA (Willkens et al., 1991). Several groups are designing molecules which have such high affinity for the binding groove of DR1 that other antigenic peptides cannot compete (Lamont, Sette, Fujinami, Col6n, Miles & Grey, 1990). The ideal molecules will be specific for the region of the haplotype involved in antigenic presentation to the disease-inducing Tlymphocyte, presumably A A 6 7 - 7 4 of the susceptible loci. Since most humans are heterozygous for their DR, DQ, and DP alleles, the blocking of antigen presentation by one allele will not impair the patient's ability to present and thus respond to most antigens.

A ntigen-specific strategies"

Oral administration of Type II collagen prior to conventional parenteral arthrogenic challenge of collagen in collagen-induced arthritis in mice delayed the onset and decreased the incidence of the disease (Thompson & Staines, 1990). Phase I toxicity/ efficacy trials involving feeding chicken collagen Type I1 to arthritis patients have commenced at the Beth Israel Hospital in Boston, MA. In a second strategy, antibodies specific for the autoantig e n - M H C complex have been shown to inhibit proliferation of the disease-causing T-cells in EAE in vitro, and were able to block the development of EAE in vivo by prophylactic administration (Aharoni, Teitelbaum, Arnon & Puri, 1991). A similar approach may be feasible in the prophylactic treatment of individuals genetically predisposed to RA. Third, molecules which resemble the diseasecausing antigen, but bind with higher affinity to the associated MHC determinant and thereby block binding of the arthrogenic antigen, are being pursued by several companies. Fourth, although the results are controversial, prophylactic immunization with peptides from the predominant TCR Va and Vp subtypes which are associated with EAE in mice were reported to decrease the incidence of EAE (Howell, Winters, Olee, Powell, Carlo & Brostoff, 1989). These strategies may yield very specific immunosuppressants in the future. SELECTIVE I M M U N O S U P P R E S S A N T D R U G S

Recent studies with new immunosuppressant drugs are providing clues to the future design of selective inhibitors of T-cell activation. The effectiveness of cyclosporin A in treating T-cell-mediated organ allograft rejection is well documented and its beneficial effects in autoimmune diseases including RA are established. Unfortunately, its use in RA is associated with nephrotoxicity which is more pronounced and less reversible than the loss of renal function seen when it is used in transplantation (Van Rijthove, Dijkmans, Goeithe, Boers & Cats, 1991). The main reason for its increased nephrotoxicity in RA is the concurrent use of NSAIDs. Additionally, an implied subclinical nephropathy in RA patients may be associated with this observation. The discovery that two macrolides, rapamycin and FK-506, selectively inhibit T-cell proliferation and are generally more potent than cyclosporin A in prolonging organ allografts and preventing autoimmune diseases in animals has created much interest (Morris, 1991). With regard to their

Development of New Antiarthritic Agents mechanism of action, rapamycin and FK-506 appear to modulate different steps of T-cell activation (Chang, Sehgal & Bansbach, 1991). For example, FK-506, like cyclosporin A, counteracts mitogenic or antigenic stimulation by inhibiting IL-2 biosynthesis, whereas rapamycin intervenes in events more closely related to the transduction of cytokine signals. This difference in functional effects is not reflected at the molecular level since both FK-506 and rapamycin bind to the same intracellular c i s - t r a n s prolyl isomerase, FKBP (Harding, Galat, Uehling & Schreiber, 1989). Although recent data discount the possibility that inhibition of isomerase activity is related to immunosuppressive activity (Schreiber, 1991), it is nevertheless intriguing that both these macrolides and cyclosporin A complex with enzymes that catalyse protein folding. Whether non-renal-toxic doses of cyclosporin A, FK-506 or rapamycin are effective in RA remains to be established. Our knowledge of the mechanism of T-cell activation and its inhibition has been greatly enhanced by examination of these three molecules and it seems likely that additional immunosuppressants will emerge from these research efforts.

CELL ADHESION MOLECULES Cell adhesion is involved in most events surrounding the generation of immune and inflammatory responses, and in regulating immune cell trafficking throughout the body. This system enables leukocytes to interact with each other or with foreign antigen, transiently adhere to the endothelium, migrate along the endothelial surface, diapedese between the endothelial cell junctions, and migrate through the extracellular matrix to participate in immune/inflammatory reactions. Three families of cell surface adhesion molecules (the integrins, the selectins, and members of the immunoglobulin superfamily) have been identified which mediate these interactions (Springer, 1990). The integrins are a supergene family of heterodimeric molecules present on cell surfaces which are divided into three major subfamilies. One subfamily, the leukocyte cell adhesion molecules (LeuCAMs), is most relevant to immunological diseases. Three LeuCAMs are presently identified: lymphocyte function-associated antigen (LFA-1), MAC-1 and p150,95. The common 95 kd/32 subunit in this subfamily is designated CD18. The three distinct a subunits are designated C D I l a (for LFA-1), C D l l b (for M A C - l ) and C D l l c (for


p150,95). While LFA-1 can be identified on all leukocytes, MAC-I and p150,95 distribution is limited to phagocytes and large granular lymphocytes (Kurzinger et al., 1981; Rothlein, Czajkowski, O'Neill, Marlin, Mainolfi & Merluzzi, 1988). The major ligand for LFA-I is intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is a member of the immunoglobulin gene superfamily and has a wide tissue distribution (Kishimoto, Larson, Corbi, Dustin, Staunton & Springer, 1989). It is present only at low levels in normal tissues but at higher levels in inflamed tissue. It is upregulated by IFN-y, IL-I and TNF-a. ICAM-2 is also a ligand for LFA- 1 and has only two immunoglobulin domains in contrast to ICAM-1 which has five extracellular immunoglobulin domains. Vascular cell adhesion molecule-1 (VCAM-I) is another member of the immunoglobulin gene superfamily and binds to the /31 integrin, VLA-4, on lymphocytes. Modulation of ICAM-l-dependent functions has been reported using monoclonal antibodies directed against this adhesion molecule (Wegner, Gundel, Reilly, Haynes, Letts & Rothlein, 1990). The selectins are also involved in leukocyte-endothelial cell adhesion. E n d o t h e l i a l leukocyte adhesion molecule-1 (ELAM-1) is the most well known member of this family of glycoproteins, and is located on vascular endothelial cells. There is no basal expression of ELAM-1 and it is only present when endothelial cells are activated by cytokines or endotoxin (Bevilacqua, Stengelin, Gimbrone & Seed, 1989). The ligand for ELAM-1 has been elucidated recently by several groups (Lowe, Stoolman, Nair, Larson, Berhand & Marks, 1990; Phillips et al., 1990) to be a leukocyte surface oligosaccharide, sialyl Lewis x, thus providing impetus to the theory that cell surface carbohydrates are involved in specific c e l l - c e l l recognition events (Brandley, Swiedler & Robbins, 1990). The precise role(s) of adhesion molecules in the histopathology of RA has not yet been extensively studied, although the patterns of leukocyte emigration into the inflamed rheumatoid tissues may depend on the selective induction and expression of leukocyte/endothelial cell adhesion molecules (Carlos & Harlan, 1990). Koch, Burrows, Haines, Carlos, Harlan & Leibovich (1991) have observed elevations in C D l l c , ELAM-1, VCAM-I and ICAM-1 leukocyte and endothelial cell adhesion molecules in synovial tissues from RA patients and to a lesser extent in synovial tissue from osteoarthritics. However, the proportion of cells expressing adhesion molecules did not correlate with the extent of tissue inflammation. Allen, Highton &


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P a l m e r (1989) have also reported that C D l l c was elevated in r h e u m a t o i d synovial m e m b r a n e s . Interestingly, Jasin, Lightfoot, Kavanaugh, Rothlein, Faanes & Lipsky (1990) have reported preliminary findings that t r e a t m e n t with a m o n o c l o n a l a n t i b o d y to CD18 (the c o m m o n /32 integrin subunit) not only m o d i f i e d the initial acute

arthritis but also ameliorated the chronic arthritis which develops in a rabbit chronic antigen-induced arthritis model. Thus, like other pathologies involving the emigration and participation o f the cellular c o m p o n e n t s o f the i m m u n e system, R A may also be a m e n a b l e to intervention at the level o f the adhesion molecule.


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