The immunomodulator Linomide: role in treatment and prevention of autoimmune diabetes mellitus

International Immunopharmacology 1 Ž2001. 1131–1139 www.elsevier.comrlocaterintimp

Review

The immunomodulator Linomide: role in treatment and prevention of autoimmune diabetes mellitus David J. Gross a,) , Lola Weiss b, Israel Reibstein b, Gunnar Hedlund c , Eva Dahlen ´ c, d b Micha J. Rapoport , Shimon Slavin a

Department of Endocrinology and Metabolism, Hadassah UniÕersity Hospital, P.O. Box 12000, 91120, Jerusalem, Israel b Department of Bone Marrow Transplantation and Cancer Immunobiology Research Laboratory, Hebrew UniÕersity-Hadassah Medical Center, Jerusalem, Israel c Pharmacia and Upjohn, Lund Research Center, Lund, Sweden d Department of Internal Medicine A B B , Assaf Harofeh Medical Center, Zerifin, Israel Received 6 May 2000

Abstract Insulin-dependent diabetes mellitus ŽIDDM. is considered to be an autoimmune disorder characterized by destruction of the pancreatic b-cells by auto-reacting lymphocytes. An attractive therapeutic approach to this disease would be to abrogate the autoimmune process at an early stage, thus preserving a critical mass of pancreatic b-cells necessary for maintenance of normal glucose tolerance. Linomide Žquinoline-3-carboxamide, Roquinimex, LS 2616., is a novel, orally absorbed, immunomodulatory drug that has been shown to be effective in various models of autoimmunity without causing non-specific immunosuppression. In this review, we describe the efficacy of Linomide for ameliorating the autoimmune process and diabetes in the non-obese diabetic ŽNOD. model of IDDM when administered at early stages of the disease. We also show that advanced disease in the NOD mouse can be treated effectively by combining Linomide with therapeutic modalities designed to increase pancreatic b-cell mass. Subsequent clinical studies have shown that Linomide preserves b-cell function in individuals with new-onset IDDM. Based on these data, Linomide or derivatives thereof might be useful for treatment of human IDDM. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Linomide; Insulin-dependent diabetes mellitus; Pancreas

1. Introduction Insulin-dependent diabetes mellitus ŽIDDM. is characterized by immune-mediated destruction of the ) Corresponding author. Tel.: q972-2-6777-648; fax: q972-26437-940. E-mail address: [email protected] ŽD.J. Gross..

pancreatic b-cells by auto-reacting lymphocytes. This pathological process results in a progressive and relentless decrease in number of the b-cells, reaching a critical mass at which time the endocrine pancreas cannot meet the metabolic demand for insulin. Subsequently, hyperglycemia appears and the clinical syndrome of diabetes ensues. An obvious approach for treatment of IDDM would be to abro-

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gate the autoimmune process, thus preserving a critical mass of pancreatic b-cells necessary for maintenance of normal glucose tolerance. In patients with recent onset IDDM, trials were initially attempted with non-specific immunosuppressive agents. Studies with prednisone, prednisone combined with azothioprine, anti-thymocyte globulin and cyclosporin A resulted in a delay of insulin dependency or decreased insulin requirement in a minority of patients; however, discontinuation of such treatments resulted in loss of the metabolic benefit w1x. Thus, the modest effects of non-specific immunosuppression, and the need for continued treatment with these agents in order to attain a sustained beneficial effect are offset by considerable toxicity associated with chronic drug administration. An attractive therapeutic alternative to non-specific immunosuppression for treatment of IDDM would be an immunomodulatory approach, namely, reinduction of tolerance towards self antigens at an early stage of the disease, thus preventing further destruction of the pancreatic b-cell mass. Linomide Žquinoline-3-carboxamide, Roquinimex, LS 2616, Fig. 1., is a novel, orally absorbed, immunomodulatory drug. This compound was originally found to have a proinflammatory effect when screened with chemically similar compounds for non-steroid anti-inflammatory drug ŽNSAID.-like activity. Thus, in contrast to NSAID properties, Linomide was found to have a potentiating activity on delayed type hypersensitivity reactions w2x. Subsequently, the drug was found to increase lymphocyte proliferation in response to T-cell-dependent mitogens accompanied by an enhanced production of

Fig. 1. Molecular structure of linomide.

IL-2 w3x and to enhance natural killer ŽNK. cell activity both in mice and humans w4–6x. Finally, Linomide was shown to have a potent ameliorative effect in a variety of murine models of human autoimmune disease such as the systemic lupus erythematosus-like syndrome w7,8x, type II collagen-induced arthritis w9x and experimental autoimmune encephalomyelitis w10,11x. In all of these models of autoimmunity, the beneficial therapeutic effect of Linomide was achieved in the absence of general immunosuppression. In light of these properties and good tolerance of the drug in experimental animals and humans, we evaluated the potential of Linomide for treatment of IDDM.

2. The non-obese diabetic (NOD) mouse as a model for IDDM The NOD mouse spontaneously develops diabetes with many features in common to human IDDM. In both human IDDM and the NOD model, a polygenic background confers genetically determined susceptibility interacting with as yet poorly defined environmental factors. In this mouse model, an islet of Langerhans inflammatory lesion, termed insulitis, which initially involves the periphery of the islet Žperi-insulitis., develops at 4–5 weeks of age, preceding diabetes. At later stages of the disease, insulitis infiltrates the islet Žpan-insulitis. and is associated with b-cell destruction leading to diabetes at 10–40 weeks. The NOD islet infiltrate is similar to that observed in patients with recent onset IDDM. Substantial experimental evidence indicates that the basis for insulitis in the NOD mouse is a T lymphocytemediated autoimmune process. Although a variety of autoantibodies to islet proteins including insulin, glutamic acid decarboxylase ŽGAD., ICA512 and phogrin have been identified in both the mouse model and the human disease, the initiating antigenŽs. of the T-cell-mediated autoimmune process have not yet been elucidated Žfor review of the immunopathology of the NOD mouse, see Ref. w12x.. At variance with human IDDM, the NOD mouse displays sexual dimorphism with ) 80% of females developing the disease by 40 weeks in contrast to 10–20% in males.

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3. Linomide for chemoprevention of IDDM in the NOD mouse 3.1. Protection from diabetes We initially assessed the effect of Linomide on the occurrence of diabetes in female NOD mice prior to development of the disease. Groups of mice were treated with Linomide 0.5 mgrml in drinking water starting at 5 weeks of age. At approximately 15 weeks of age, untreated animals started to develop diabetes, reaching a cumulative incidence of 61% at 40 weeks. In sharp contrast, all mice in the Linomide-treated groups remained free of diabetes for the duration of the experiment. In order to obtain better assessment of metabolic control, intraperitoneal glucose tolerance tests ŽIPGTT. done at 16 and 40 weeks of age revealed normal glucose tolerance in 90% of the drug-treated animals, indistinguishable from that of normal control BALBrc mice w13x. Although an approximately 10% decrease in body weight was noted, the treated animals appeared well and histological examination of skin and internal organs revealed no evidence of drug toxicity w13x. The absence of drug toxicity is in keeping with previous studies in mice w7,8x. Furthermore, the NOD mice also develop two other autoimmune pathologies, thyroiditis and sialoadenitis, which Linomide effectively inhibited w14x. 3.2. Islet of Langerhans morphology Histological examination of the pancreas at 40 weeks of age revealed a paucity of islets, the surviving islets being afflicted with typical insulitis in all untreated mice. However, most drug-treated animals showed islets predominantly normal in size, number and morphology; occasional mild peri-insulitis was seen in a minority of islets. Further studies ŽEva Dahlen ´ et al., manuscript in preparation. showed that Linomide regulates and inhibits insulitis in late as well as in early phases of NOD IDDM and that infiltration by T lymphocytes, B lymphocytes and dendritic cells are effectively inhibited by Linomide. The density of macrophages though is not changed but their activity in situ probably is Žsee below..

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3.3. Effect of Linomide dosage and duration of treatment We chose to treat NOD mice with the dosage of 0.5 mgrml in drinking water since in preliminary experiments we observed that the mice refused water containing higher concentrations of the drug. Lower doses of Linomide Ž0.1 and 0.02 mgrml orally. resulted in similar protection from diabetes as determined by glucosuria at 0.5 mgrml; however, at the reduced dosage a higher frequency of glucose intolerance was seen, presumably resulting from the more severe insulitis and reduced pancreatic insulin content observed in these animals ŽGross et al., unpublished observations.. Interestingly, we found that a dose of 0.5 mgrml for limited periods of 5 or 15 weeks at the onset Ž5 weeks of age. resulted in protection from diabetes at 42 weeks, similar to that conferred by the same dose of drug given for the duration of the experiment w13x. 4. Linomide for therapy of established IDDM In IDDM patients at the initial presentation of clinical disease, the pancreatic b-cell mass is markedly reduced Žapproximated to be less than 10%. and inadequate for maintenance of normoglycemia. Therefore, the use of various immunosuppressive modalities, known to downregulate antiself reactivity for treatment of recent onset IDDM, proved to be of only marginal benefit w15x. Recently, on the basis of our pre-clinical studies in the NOD mouse, Coutant et al. conducted a double blind, placebo-controlled clinical study with low dose Linomide Ž2.5 mgrday. given orally in young patients with recent onset diabetes w16x. In keeping with previous clinical studies with immunosuppression in patients with recent onset IDDM, an improvement in b-cell function was noted only in patients with residual b-cell function at the onset of the study, and independence from insulin injections was not achieved w16x. In an emulation of the clinical situation in humans, we treated female NOD mice with Linomide starting at 16 weeks of age, at which stage the animals have advanced insulitis and glucose intolerance. Similar to the situation in human IDDM, only a partial beneficial effect was noted, and the mice, although protected from frank diabetes, re-

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mained glucose-intolerant at the end of the experiments. Accordingly, it has become apparent that in patients with recent onset IDDM, in addition to immunomodulation, augmentation of the pancreatic b-cell mass is required in order to reestablish normoglycemia. From a theoretical view point, in order to attain this objective two therapeutic options could be considered: Ža. islet transplantation and Žb. induction of endogenous b-cell replication andror neogenesis. 4.1. Linomide for protection of islet transplants to diabetic NOD mice Islet transplantation has been attempted for reversal of IDDM; however, re-occurrence of autoimmune insulitis due to the underlying autoimmune process has remained a major problem in both experimental animals and man w17,18x, thus, successful treatment of IDDM by islet transplantation still represents a major challenge yet to be accomplished. We designed experiments to explore the feasibility of protecting transplanted syngeneic islets in diabetic NOD mice from insulitis by administration of Linomide. Female NOD mice with advanced disease Žage 23–24 weeks. received renal subcapsular syngeneic islet transplantation combined with oral Linomide administration. The combined treatment resulted in prevention of diabetes and graft insulitis and in the Linomide group up to 40 weeks, and the extent of metabolic control imparted by the combination of transplantation with Linomide was superior to that of Linomide alone w19x. Thus, Linomide could be a potential immunoregulatory modality for protection from recurrent autoimmune destruction of the graft in patients undergoing pancreatic islet or whole organ transplantation. However, since Linomide has been found to abrogate the protective effect of cyclosporin A on allografts w20,21x, careful selection of means for graft protection from rejection compatible with Linomide treatment will be necessary w22x. 4.2. Treatment of diabetic NOD mice with Linomide combined with reg protein Until recently, pancreatic b-cells have been regarded to play a passive role in this pathogenesis of IDDM. It is becoming increasingly clear, however, that the appearance of clinical diabetes is dependent

on a balance between the destructive autoimmune process on one hand, and the capacity of the pancreas to expand the b-cell mass on the other. Thus, it was shown many years ago that in the pancreases of patients with new onset IDDM, neoformation of b-cells occurs w23–25x, an observation also documented in animal models of diabetes with reduced b-cell mass w26x, in transgenic mice in which g-interferon expression by the b-cells induces lymphocytic infiltration and b-cell damage w27x and in NOD mice w28,29x. Moreover, the fact that only a small minority of individuals with islet cell antibodies, an indicator of autoimmunity towards the b-cell, proceed to develop w30,31x indicates that other factors, namely b-cell function and capacity for proliferation andror neoformation, might play an important role in the development of the disease. The limited proliferative capacity of mature b-cells coupled with b-cell autoimmunity poses a major problem for treatment of IDDM, illustrated by the low success rates of various immunomodulatory approaches in patients with newly diagnosed disease w15x. In this setting, the unsatisfactory outcome can be accounted for by the lack of a critical b-cell mass necessary to maintain normoglycemia, despite successful abrogation of the autoimmune process. Reg protein, the gene product of a cDNA isolated from a regenerating rat islet library w32x, has been previously shown to induce expansion of b-cell mass in pancreatectomized rats w33x. Therefore, we decided to determine the combined effect of immunomodulation Žfor reinduction of b-cell tolerance. and reg protein Žfor expansion of b-cell mass. on advanced autoimmune diabetes, we treated 14-week-old female NOD mice with oral Linomide and intraperitoneal reg protein injections. In animals with less severe disease Žglucose tolerant., treatment with each agent alone resulted in amelioration of diabetes, as did treatment with reg alone in 5-week-old pre-diabetic mice. In animals with more severe disease Žglucose intolerant. only treatment with the combination of both agents, but not with each separately, resulted in amelioration of diabetes w34x. Interestingly, reg protein, which has previously shown to induce b-cell replication in islets w33x appears to also induce b-cell neogenesis in the NOD mouse model ŽFig. 2.. Thus, treatment with Linomide, aimed at abrogation of autoimmunity combined with expansion of b-cell mass could be a

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Fig. 2. Photomicrograph of a pancreas section with immunoperoxidase staining for insulin obtained from a female NOD mouse at the age of 40 weeks. Mice in this group were treated with linomide 0.5 mgrml in drinking water and Reg protein, 1 mgrkgrday i.p. daily = 6 daysrweek from 15 to 40 weeks of age. Note lack of insulitis and small clusters of insulin positive cells adjacent to a pancreatic duct, compatible with b-cell neogenesis.

potential therapeutic approach for treatment of IDDM.

5. Mechanism of Linomide-induced abrogation of autoimmunity in the NOD mouse In our studies in the NOD mouse, we found that Linomide treatment did not induce non-specific immunosuppression since comparable cell surface phenotype, in-vitro responses of mononuclear cells to Tand B-dependent mitogens and mixed lymphocyte reaction, cytotoxicity assays against NK-sensitive and NK-resistant target cells, humoral antibody responses as well as the in-vivo DTH responses, were observed in treated and control mice w13,35x. Moreover, in experiments in which lymph node-derived cells from female NOD mice treated for a prolonged period with Linomide were stimulated with Con A, an enhanced proliferative effect was noted w36x, indicating a immunostimulatory effect of the drug on the known impaired T-cell function in these animals w37x. Establishment of tolerance to self-antigens by Linomide, as confirmed by adoptive transfer experiments, in the presence of normal or even enhanced T-cell-dependent mitogenic responses, suggests that

Linomide may be effective for downregulation of autoreactive lymphocyte activity w13x. Since the primary antigenŽs. that initiate b-cell directed autoimmunity in the NOD mouse is unknown, demonstration of reinduction of tolerance towards such antigenŽs. is unfeasible at this time. However, in models in which autoimmunity is induced by known antigens, specific downregulation of immune responses towards the instigating antigens has been observed w11,38,39x. What could be the mechanism of Linomide-induced suppression of autoreactivity in the NOD mouse? 5.1. The effect of Linomide on NOD T-cell anergy Thymic T-cell anergy, as manifested by impaired thymocyte proliferative to antigen, is thought to impair the development of immunological self-tolerance. It has been previously shown that thymocytes and peripheral T-cells in young female NOD mice have a decreased response Žanergy. to TCR stimulation associated with decreased activation of the PKCrp21RasrMAP kinase pathway w40x. This thymic cell hyporesponsiveness is associated with a shift of the T-cell population from a Th2 towards a Th1 phenotype, reflected by a high IFNgrIL-4 in the

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islet T-cell infiltrates of diabetes prone, female NOD mice in contrast to the high IL-4rIFNg ratio in the diabetes-resistant males w41x. Treatment of young female NOD mice with IL-4, a Th2 cytokine, completely reverses the thymocyte unresponsiveness and confers protection from diabetes w42x. Similar to the effect of IL-4, treatment with Linomide corrects the T-cell-proliferative defect and is associated with the upregulation of the T-cell PKCrp21RasrMAP kinase pathway transduction defect w36x. 5.2. The effect of Linomide on NOD macrophage phenotype and cytokine production It has been shown that defects of macrophage differentiation and function, including reduced costimulatory potency w43x, are present in NOD mice and that correction of macrophage function with a variety of immunostimulants confers protection from diabetes, presumably by improved presentation of self-antigens to tolerogenic mechanisms w44x. Linomide has been found to have a profound immunostimulatory effect on macrophages of BALBrc mice w3x and was observed to increase the Mac-3rMac-1 expression ratio on NOD peritoneal cells, indicating a possible effect on maturation of macrophage phenotype w13x. Furthermore, Linomide showed distinct stimulatory effects on antigen presenting cells correlating to its capacity to inhibit EAE in SJL mice w45x. Linomide also modifies the NOD macrophage cytokine secretion profile, namely, reduction both of basal and LPS induced secretion by peritoneal macrophages of IL-1 b and TNF-a ŽWeiss et al., manuscript in preparation. cytokines, which are cytotoxic to pancreatic b-cells w46x. Measurement of NOD pancreatic mRNA levels for TNF-a revealed that this cytokine is inhibited both after short- Ž3–7 days. and long-term Linomide treatment Žas above.. The short-term treatment was performed to reveal potentially Linomide-induced cytokine modulation without changed inflammation density indicating that TNF-a was indeed modulated in situ. Furthermore, as discussed below, IL-12 p40 mRNA was also increased ŽEva Dahlen ´ et al., manuscript in preparation.. This phenomenon is in accordance with modulation of in situ macrophages activating a downregulatory circuit of signals and thus with the hypothesis underlining a suboptimal immune activation as the

etiology of certain autoimmune conditions. The downregulatory circuit interferes with the recruitment of new inflammatory cells and inhibits the pro-inflammatory cytokines. The Linomide-induced modulation of NOD macrophage cytokine profile is in accordance with results in other experimental systems showing that cells from the macrophage lineage are regulated by the drug in their capacity to produce cytokines and other effector molecules: TNF-a , IL-1 and IL-6 being inhibited by Linomide, GM-CSF unaffected and plasminogen activator type 2 stimulated w47,48x. 5.3. The effect of Linomide on NOD T-cell Th1 r Th2 ration The T-cell anergy repairing effect of Linomide could be mediated by IL-4 and other Th2 related cytokines, since Linomide treatment causes increased secretion by female NOD splenocytes of the Th2 related cytokines IL-4, IL-6 and IL-10 and decreased secretion of the Th1 related cytokines IL-2 and IFNg ŽWeiss et al., manuscript in preparation.. These observations indicate that Linomide-induced expression of the Th2 phenotype in the NOD mouse could downregulate autoreactive Th1-cells, thereby inducing tolerance to b-cell antigens. A similar effect of the drug on the Th1rTh2 ratio has been observed in animal models of experimental allergic encephalomyelitis ŽEAE. w49–51x; however, in a model of myesthenia gravis, a pathology where antibodies play a pivotal role, downregulation of both Th1 and Th2 was observed w39x. Thus, Linomide regulates the autoimmune response by causing a shift from a Th1 to a suppressive Th2 response or when acting in inhibition of Th2. 5.4. The effect of Linomide on NOD T-cell IL-12 production The skewing of the T-cell population in young female NOD mice towards a Th1 phenotype, and its subsequent correction by Linomide, might possibly be due to modulation of IL-12, a cytokine elaborated by macrophages, B-cells and dendritic cells that directs T-cells towards a Th1 phenotype w12x. The expression of IL-12 in the NOD pancreas correlates with the development of diabetes, and exogenous

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administration of the cytokine causes acceleration of the disease w52x. Biologically active IL-12 exists as a 70–75 kDa heterodimer comprising 40 Žp40. and 35 kDa Žp35. monomers bound by disulfide bonds. Interestingly, both p40 monomers and homodimers bind to the IL-12 receptor and inhibit IL-12 binding, and treatment with the homodimer protects b-cells and prevents diabetes in NOD mice w53x. Linomide treatment of female NOD mice induces downregulation of IL-12 secretion by splenocytes ŽWeiss et al., manuscript in preparation., as observed in other experimental models of autoimmunity w39,51x. Moreover, in addition to inhibition of IL-12 secretion, Linomide might also act by inhibition of IL-12 receptor activity. At the mRNA level, Linomide causes marked upregulation of NOD pancreatic IL-12 p40 transcripts without a concomitant increase of p35 transcripts ŽEva Dahlen ´ et al., manuscript in preparation.. This combination would be expected to lead to a reduction of the IL-12 heterodimer and enhancement of the IL-12 p40 homodimer formation, the overall result being reduction of IL-12 bioactivity, thus redirecting the Th1rTh2 balance towards a Th2 AtolerogenicB environment. In summary, Linomide modulates accessory and antigen presenting cells, e.g. macrophages, resulting in the inhibition of further infiltration of T-cells, B-cells or dendritic cells into the pancreatic islets. Linomide might also affect T-cells directly by correcting NOD specific T-cell anergy. The net result of these effects is the downregulation of the autoimmune responses pivotal for development of insulitis and diabetes in the NOD mouse.

6. Concluding remarks The immunomodulator Linomide is highly effective for prevention and treatment of IDDM in the NOD mouse model, apparently by downregulation of autoreactivity towards b-cell antigens. Use of the drug in this model in conjunction with islet grafts or reg protein indicates that the paradigm of b-cell expansion combined with immunoprotection could be a viable approach for treatment of patients with established IDDM. Linomide treatment of young patients with IDDM indicates a beneficial effect on b-cell function, with very low drug toxicity w16x.

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However, a higher than expected rate of cardiovascular events in older patients with multiple sclerosis treated with Linomide required premature termination of a large multicenter clinical trial w54,55x. The beneficial effects documented with Linomide in both experimental IDDM as well as in IDDM clinical trials emphasize the value of future drugs with Linomide-like activity.

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