Biological effects of cyclosporin A: A new antilymphocytic agent

The fungus metabolite cyclosporin A is a small peptide acting as a novel antilymphocytic agent. It strongly depressed appearance of both direct and indirect plaque-forming cells and produced a clear dose-dependent inhibition of haemagglutinin formation in mice upon oral administration. Skin graft rejection in mice and graft-versus-host disease in mice and rats were considerably delayed by cyclosporin A which also prevented the occurrence of paralysis in rats with experimental allergic encephalomyelitis. This compound was not only highly effective in preventing development of Freund's adjuvant arthritis, but in addition improved the symptoms in rats with established arthritis, although it is inactive in acute inflammation. This new agent contrasts with other immunosuppressives and cytostatic drugs in its weak myelotoxicity. Experimental evidence suggests that cyclosporin A, rather than being cytostatic or lympholytic, affects an early stage of mitogenic triggering of the immunocompetent lymphoid cell.Research Institute, Wander, Bern, a Sandoz Research Unit.     

Effects of the new anti-lymphocytic peptide cyclosporin A in animals.

The fungus metabolite cyclosporin A is a small cyclic peptide acting as a novel antilymphocytic agent. It is effective following either parenteral or oral administration in mice, rats and guinea-pigs. The suppressive effect after short and prolonged treatment on plaque-forming cells, the inhibition of the secondary humoral response and the reversibility of its effect on haemagglutinin formation is demonstrated. Cyclosporin A inhibits delayed hypersensitivity skin reaction to oxazolone (primary and secondary responses) in mice and to tuberculin in guinea-pigs. Its failure to suppress antibody synthesis to lipopolysaccharide antigens in nude mice suggests a selective effect on T cells. High doses of the compound affect the haemopoietic tissues very weakly as shown by the bone marrow and stem cell numbers in mice, which finding markedly contrasts with most other immunosuppressive and cytostatic drugs.     

Enzyme and combination therapy with cyclosporin A in the rat developing adjuvant arthritis

Recent knowledge of the pathophysiology of rheumatoid arthritis and the mechanism of drug effects have enabled the use of new drugs and drug combinations in rheumatoid arthritis therapy. This study investigates the efficacy of both enzyme therapy and combined therapy with cyclosporin in rats with adjuvant arthritis. Rats with adjuvant-induced arthritis were administered either cyclosporin A (2.5 or 5.0 mg/kg/day per os), a mixture of enzymes (Phlogenzym (PHL); 45 mg/kg twice daily intrarectally), or a combination of 2.5 mg cyclosporin A and 90 mg PHL for a period of 40 days from the adjuvant application. Levels of serum albumin, changes in hind paw swelling and bone erosions were measured in rats as variables of inflammation and arthritis-associated destructive changes. Treatment with 5 mg of cyclosporin A, as well as with the combination therapy with cyclosporin A plus PHL, significantly inhibited both the inflammation and destructive arthritis-associated changes. However, 2.5 mg of cyclosporin A and PHL alone inhibited these disease markers, although to a lesser extent and at a later stage of arthritis development. The results show the inhibitory effect of enzyme therapy on rat adjuvant arthritis, as well as the efficacy of a low dose of cyclosporin A given in combination with enzyme therapy, which may be useful in the treatment of rheumatoid arthritis.     

Effects of gold sodium thiomalate,cyclosporin A,cyclophosphamide, and placebo on collagen-induced arthritis in rats

The prophylactic and therapeutic effects of gold sodium thiomalate, cyclosporin A, cyclophosphamide, and placebo on collagen-induced arthritis (CIA) were evaluated in DA rats. Prophylactic treatment with cyclosporin A and cyclophosphamide suppressed the arthritis incidence, clinical inflammation, destructive bone changes, and development of anti-collagen antibody in DA rats subsequently injected with porcine type-II collagen. Therapeutic treatment with cyclosporin A and cyclophosphamide had a definite suppression on established CIA when started 21 days after the initial collagen injection, but the suppression was less marked than that of prophylactic treatment. Gold had no impact on CIA in DA rats when administered either prophylactically or therapeutically.     

Immunomodulation of rat antigen-induced arthritis by leflunomide alone and in combination with cyclosporin A

The effects of the new immunomodulating isoxazol derivative leflunomide, in comparison with cyclosporin A, on established antigen-induced arthritis in rats as well as serum antibody levels were determined. When treatment with leflunomide, at concentrations from 2.5 to 10 mg/kg/d, was started on day 3 of arthritis, the acute and chronic phases of arthritis were effectively inhibited. This was demonstrated by decreased joint swelling and reduced histopathological arthritis score at the end of experiment (day 26). Furthermore, the treatment resulted in a significantly reduced level of serum antibodies to the matrix components collagen type I, type II and proteoglycans. Neither leflunomide nor cyclosporin A, at doses of 1 mg/kg/d, had an effect on the severity of arthritis and antibody levels. However, when both drugs were used together, at these non-effective doses, the histopathological score of chronic arthritis was significantly reduced. The results of our experiments demonstrate that leflunomide has a strong suppressive effect on both acute and chronic phases of antigen-induced arthritis and formation of autoantibodies in rats. Furthermore, orally administered doses of leflunomide were as effective as doses of cyclosporin A given intraperitoneally. The combination of sub-effective doses of leflunomide and cyclosporin A resulted in significant inhibition of chronic arthritis.accepted by M. J. Parnham     

The effect of FK506 and cyclosporin A on antigen-induced arthritis.

FK506 and cyclosporin A inhibited the development of antigen-induced arthritis in the rat and rabbit. FK506 was five times more potent than cyclosporin A in the rat and approximately 20 times more potent in the rabbit. FK506 was effective in both species if administered either from the day of intra-articular administration of antigen or when the arthritis was established. In the rabbit, arthritis returned when administration of FK506 was stopped. FK506 (10 mg/kg/day) caused renal damage which was not observed at a dose of 2.5 mg/kg/day. Both of these doses were equally effective at inhibiting the arthritis. The conclusion from these studies is that FK506 is a more effective anti-arthritic agent than cyclosporin A and that a pronounced therapeutic effect can be achieved at non-toxic doses of the drug.     

Comparative study of effects of cyclosporins A and G on collagen arthritis in mice

The effects of the immunosuppressive agents cyclosporin G (CsG) and cyclosporin A (CsA) on collagen arthritis were compared in mice. When administered subcutaneously daily on days 0–13 after immunization with type II collagen, CsG and CsA were both capable of suppressing the development of collagen arthritis in mice as well as the immunological response to native type II collagen in a dose-dependent manner. Histopathologically, no marked inflammatory lesions were observed in diarthroidal joints from mice treated with 100 mg/kg per day of CsA or 800 mg/kg per day of CsG. However, an analysis of dose response showed CsG to be 8 times less potent than CsA in inhibiting the development of arthritis.     

Comparative studies of the effects of FK506 and cyclosporin A on passively transferred collagen-induced arthritis in rats.

We investigated the effect of a novel immunosuppressive agent, FK506, in comparison with cyclosporin A (CsA) on the development of passive arthritis induced by anti-type II collagen (CII) antisera in rats. FK506 pretreatment shortly before serum transfer markedly suppressed the incidence and the severity of passive arthritis, while CsA pretreatment had no observable effects on this disease when used in doses sufficient to suppress the development of active arthritis induced by CII immunization. In an additional study, we examined whether these agents affect antibody-mediated tolerance induction. CII-specific immunological tolerance was induced by serum transfer, but was unaffected by either FK506 or CsA pretreatment in our regimen. While its precise mechanism of the immunosuppressive activity remains to be elucidated, FK506 can act on the antibody-mediated effector phase of arthritis and may offer new insights into the possible role of potential therapeutic utility in human autoimmune diseases.     

Effects of deoxyspergualin on collagen arthritis in mice

We have studied the effect of the immunosuppressive agent deoxyspergualin (DSG) on collagen arthritis in mice. DSG, when given prophylactically, was capable of suppressing the development of collagen arthritis in mice as well as the immunological response to native type II collagen in a dose-dependent manner. Further, treatment of DSG, started 7 days after the primary immunization, also resulted in the inhibition of development of arthritis and immunity to collagen. The treatment of DSG, started after a booster injection, did not suppress the development of arthritis, despite suppression of antibody production in collagen. These findings suggest the possibility that a threshold level of anti-type II collagen antibodies may exist which must be exceeded before arthritis develops. Its therapeutic use in mice did not affect the clinical course of arthritis or the immune response to collagen, which is similar to the results obtained with cyclosporin.     

Leflunomide (HWA 486), a novel immunomodulating compound for the treatment of autoimmune disorders and reactions leading to transplantation rejection

Leflunomide has been shown to be very effective in preventing and curing several autoimmune animal diseases. Further, this agent is as effective as cyclosporin A in preventing the rejection of skin and kidney transplants in rats. Preliminary results from patients suffering from severe cases of rheumatoid arthritis demonstrated that clinical and immunological parameters could be improved with leflunomide therapy. Mode of action studies revealed that this substance antagonizes the proliferation inducing activity of several cytokines and is cytostatic for certain cell types. In this light, we could show that tyrosine phosphorylation of the RR-SRC peptide substrate and the autophosphorylation of the epidermal growth factor (EGF) receptor were, dose dependently, inhibited by leflunomide. EGF activates the intrinsic tyrosine kinase of its receptor, which stimulates the phosphorylation of a variety of peptides, the amino acid residue in all cases is tyrosine. These results indicate that much of leflunomide's activity could be due to the inhibition of tyrosine-kinase(s), which is an important general mechanism for the proliferation of various cell types. Thus, leflunomide, which is effective against autoimmune diseases and reactions leading to graft rejection, would seem to have a mode of action separating it from known immunosuppressive drugs.     

Serum transfer of collagen arthritis to cyclosporin-treated, type II collagen-tolerant rats

Collagen arthritis has been passively transferred with a serum concentrate from immunized donors to immunologically naive recipients as well as cyclosporin-treated, type II collagen-tolerant rats. These findings point to an important role for anticollagen antibody and appear to rule out a role for cellular immunity to type II collagen in the initiation of this disease. The passively transferred arthritis was a transient lesion in the majority of naive recipients and in the cyclosporin-treated, type II collagen-tolerant rats as well when a serum concentrate was transferred after the cessation of cyclosporin treatment. When cyclosporin-treated, type II collagen-tolerant rats received transfer concentrate while cyclosporin was administered continuously, arthritis was significantly enhanced, and lasted as long as cyclosporin was administered and in the majority of rats up to 2 weeks after the cessation of cyclosporin treatment. These results, together with a rapid clearance of anticollagen antibody from the serum, suggest that anticollagen antibody is not the sole regulatory factor and that a cellular suppressor system, sensitive to cyclosporin, might participate in the regulation of this disease process.     

Effect of FTY720, a novel immunosuppressant,on adjuvant-induced arthritis in rats

OBJECTIVE AND DESIGN: Anti-arthritic effect of FTY720, a novel immunosuppressant, was compared with those of immunosuppressants cyclosporin A and tacrolimus in adjuvant-induced arthritis in rats. MATERIAL: Male LEW rats. TREATMENT: FTY720 (0.03-0.3 mg/kg), cyclosporin A (1-10 mg/kg) or tacrolimus (0.3-3 mg/kg) were orally administered to rats for 21 days beginning on the day (day 0) of adjuvant inoculation. In addition, the anti-arthritic effect of FTY720 (0.3 mg/kg) and cyclosporin A (10 mg/kg) were evaluated by administration to animals for 5 consecutive days (days 2-6, 6-10, and 10-14). METHODS: Adjuvant-induced arthritis was produced by intradermal injection of 0.5 mg heat-killed Mycobacterium tuberculosis. Hindpaw edema was measured plethysmographically. The day of arthritis onset was determined macroscopically. Bone degradation was determined by radiography. Peripheral blood leukocytes were classified microscopically. RESULTS: All test compounds inhibited the incidence of arthritis, hindpaw edema and bone destruction. In addition, FTY720 but not cyclosporin A or tacrolimus markedly decreased the number of peripheral blood lymphocytes. FTY720 treatment on days 6 to 10 inhibited the bone destruction and hindpaw edema. CONCLUSION: These results suggest that the anti-arthritic effect of FTY720 in this adjuvant-induced arthritic model was more potent than those of cyclosporin A and tacrolimus. FTY720 administered on days 6 to 10 showed the inhibitory effect on the bone destruction and hindpaw edema. FTY720 may be effective in the treatment of rheumatoid arthritis.     

Collagen arthritis in rats: the importance of humoral immunity in the initiation of the disease and perpetuation of the disease by suppressor T cells

Arthritis could be passively transferred with a serum concentrate from collagen arthritic rats to nude rats and cyclosporin-treated, type II collagen-tolerant rats. These findings suggest that collagen arthritis could be inducible by humoral immunity alone in the absence of cellular immunity to type II collagen or functional T cells. In addition, passive arthritis induced by anticollagen antibody is a mild, transient disease from which the animals normally recover and the rats that have recovered from passive arthritis are resistant to develop a second phase of arthritis following a second administration of anticollagen antibody or the subsequent challenge with type II collagen. However, when a serum concentrate was transferred while cyclosporin was administered continuously, transferred arthritis in cyclosporin-treated, type II collagen-tolerant rats lasted as long as cyclosporin treatment and arthritis was significantly enhanced compared to those of naive recipients. Further, enhancement and prolongation of passively transferred arthritis in nude rats was observed. Furthermore, treatment with cyclophosphamide reversed acquired resistance to collagen arthritis subsequent to recovery from passive arthritis. These findings suggest that suppressor T cells might, at least in part, affect the clinical course of collagen arthritis and reverse acquired resistance to arthritis.     

Antischistosomal effects of cyclosporin A

Administration of cyclosporin A, a new selective immunosuppressive agent, to mice infected withSchistosoma mansoni resulted in a significant reduction in the number of mature and immature male and, to a greater extent, female worms. With lower, subeffective, doses a reduction in hemoglobinase activity and protein content of female schistosomes is produced. Evidence available so far suggests that the antischistosomal effects of cyclosporin A are mediated through a stimulation of host mechanisms directed against the parasite.Supported by grants from NIH (#GM 16492-12) and the Agency for International Development (#AID/ta-C-1312).     

Cyclosporin A (Sandimmun®) modulates the Ca2+ uptake of mitogen-stimulated lymphocytes

Incubation of murine spleen cells, or enriched T-cell populations, with the T-cell-dependent polyclonal mitogens Con-A or PHA resulted in a dose-dependent increase in⁴⁵Ca²⁺ uptake. This effect was observed after a delay of about 30 to 60 min, reached a maximum after 2–4 h and lasted up to 6 h. Similarly, the calcium ionophore A23187 caused an increased Ca²⁺ uptake, although this was faster, occurring within a few minutes, reaching a maximum at 15 min and lasting for about 2–4 h. No change in Ca²⁺ uptake was observed using a specific B-cell mitogen (lipopolysaccharide) or B-cell preparations. Nifedipine, a calcium channel-blocking agent, inhibited activation of lymphocytes in a primary immune response (mixed lymphocyte reaction and formation of plaque forming cellsin vitro) but was unable to interfere with proliferating cell lines or a secondary immune response.Incubation of Con-A-activated lymphocytes with the immunosuppressive agent CS-A caused an additional increase in calcium uptake, whereas no change in calcium uptake was observed when resting lymphocytes or B-cells activated by LPS were incubated with cyclosporin.A similar potentiation of Con-A-induced calcium uptake was seen with hydrocortisone, but not with cytostatic agents or anti-lymphocyte serum. Submaximal calcium uptake induced by the ionophore A23187 was potentiated by CS-A and hydrocortisone as well as by cytostatic agents and ALS.     

The effects of immunomodulatory thymic and splenic peptides and cyclosporin A on antigen-induced arthritis in the rat

Long-term treatment with natural and synthetic thymic and splenic peptides as well as cyclosporin A inhibited the development of antigen-induced arthritis in rats. This was demonstrated by decreased joint swelling and reduced degree of macroscopically and histologically evaluated severity of synovitis. The drug treatment also decreased serum levels of antibodies against the specific antigen methylated bovine serum albumin (mBSA) and against cartilage proteoglycans and collagens type I and II.

The conclusion from these studies is that the treatment with immunomodulatory thymic and splenic peptides and with the T-cell-directed immunosuppressive drug cyclosporin A inhibits the specific immune response against mBSA and/or the development of autoimmunity against cartilage constituents. The decreased immune reactivity in the joint may reduces the severity of chronic joint inflammation.

     

Influence of cyclosporin A on cytokine levels in synovial fluid and serum of rats with antigen-induced arthritis

The effects of the T-cell-directed immunosuppressant cyclosporin A (CsA) on the developmnnt of antigen-induced arthritis (AIA) in rats as well as on cytokine levels in synovial fluid and serum were determined. The treatment with CsA effectively inhibited the chronic phase of arthritis as demonstrated by decreased joint swelling and reduced histological arthritic score. In animals with AIA the level of IL-6 in the synovial fluid and serum is increased, showing good correlation with the severity of the disease. The CsA treatment reduced IL-6 levels to normal. IL-1, IL-2 and TNF- do not seem to be directly involved in the pathogenic mechanisms of chronic arthritis.     

Risks and Benefits of Low-Dosage Cyclosporin in Rheumatoid Arthritis

The effects of cyclosporin on the activity of rheumatoid arthritis have mainly been investigated in patients with active, refractory, long-standing disease. The data obtained in these trials suggest that cyclosporin is not only a symptomatic treatment for rheumatoid arthritis but can also be considered a disease-modifying antirheumatic drug (DMARD), since it seems to be capable of slowing the progression of cartilage and bone damage due to rheumatoid arthritis. The trials conducted so far have led to a better understanding of cyclosporin toxicity and, therefore, to better monitoring of patients in order to avoid it. The reasons for studying the role of cyclosporin in patients with early, active and potentially severe rheumatoid arthritis are the poor prognosis of the disease despite the use of the presently available DMARDs, and the hypothesis that the drug is more efficacious and better tolerated in early rheumatoid arthritis. A new classification of antirheumatic drugs proposes that disease-controlling antirheumatic therapies decrease inflammatory synovitis and prevent structural joint damage or significantly reduce its rate of progression. However, few existing drugs meet these criteria. The 12-month results of a disease-controlling antirheumatic therapy clinical trial with a blinded radiological end-point, named GRISAR (Gruppo Reumatologi Italiani Studio Artrite Reumatoide) comparing cyclosporin with conventional DMARDs in patients with early rheumatoid arthritis provide strong evidence that cyclosporin offers better control of ongoing joint damage than do conventional DMARDs.     

Effects of indomethacin,cyclosporin, cyclophosphamide,and placebo on collagen-induced arthritis of mice

The long term effects of indomethacin, cyclosporin, cyclophosphamide, and placebo on collagen-induced arthritis in mice were tested under two different treatment protocols. A prophylactic experiment examined the effects of the daily drug administration for 180 days beginning one day before the first collagen injection. Under this dosage schedule, cyclophosphamide and cyclosporin decreased the severity of arthritis, while indomethacin did not. A therapeutic protocol examined the effects of these same drugs when daily administration was delayed until the animals had active disease at 78 days after the first collagen injection. Under this protocol, all three drugs reduced the progression of disease. In both protocols, the most significant suppression of arthritis was seen in animals receiving cyclophosphamide which was associated with a decrease in anti-collagen antibody levels. Collagen-induced arthritis in mice should be further investigated as a model to study the long term effects of slow-acting anti-rheumatic drugs.Supported in part by a grant from Pennwalt Pharmaceutical Company, Rochester, NY, the Veterans Administration Medical Research program, Nora Eccles Treadwell Foundation, and grants from the National Institutes of Health AR-02255 and AM30763.     

The effect of some immunomodulating agents on the sensitization phase of experimental contact allergic reactions

In an experimental model which allows comparison of the macroscopic changes with the differentiated dermal inflammatory cell infiltrate of the allergic contact reaction to oxazolone in guinea pigs, the effects of single doses of the cytostatic agents cyclophosphamide, methotrexate and azathioprine and the anti-lymphocyte agent cyclosporin A administered prior to sensitization were studied. All the agents tested increased the degree of erythema and edema of reactions compared to controls. The dermal inflammatory mononuclear cell response showed little significant increase and would not seem to explain the enhancement of reactions. The number of basophils in reactions was not significantly different from controls, and there is no evidence for early arrival, degranulation and destruction of basophils prior to our first routine assessment at 24 h. The number of mast cells in the reactions was also unchanged. A role for the latter two cells involving degranulation without changes in the number of cells could provide a possible explanation for the augmentation of the vascular response. A mechanism for the effect of cyclophosphamide other than T-suppressor lymphocyte susceptibility based on selective cytostatic effects on precursor cells may need to be considered.