CA1318591C - Composition containing heparin or derivative thereof - Google Patents

Abstract

ABSTRACT:

The invention relates to means for preventing or for delaying the rejection of allografts in humans and for the alleviation of and for treating autoimmune diseases in humans. The pharmaceutical compositions which are suitable to attain the above ends are based on heparin, on N-desulfated, N-acetylated heparin, O-desulfated, N-acetylated heparin or other suitable derivatives of heparin. The dosage which is effective is one which is substantially lower than that required for obtaining an anticoagulant effect in mammals.
It is generally of the order of about 0.5 per cent to about 5 per cent of the anti-coagulant effective dosage of such compounds.

Description

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FIELD OE THE INVENTION
There are provided pharmaceutical compositions adapted to prevent or diminish rejection of allografts, to prevent or alleviate autoimmune diseases comprising an effective dosage of heparin, or N-desulfated, N-acet:ylated heparin or O-desulfated, N-acetylated heparin, said dosage being of the order of from about 0.5 to 5 percent of a dosage which results in a perceptible anti-coagulant effect.
There may be also use other derivatives of, and modified forms of heparin, which result in the desired effect. Not all such derivatives are effective: for example, totally desulfated heparin is entirely without value for the intended purposes.
BACKGROUND OF THE INVENTION
Along with its vital role in protecting the individual against foreign invaders, the immune system may attack the individuals own tissues, thereby producing autoimmune diseases.
Another undesirable activity of the immune system is the rejection of critical transplanted organs. The ability of the immune system to produce autoimmune disease or reject allografts depends on the ability of lymphocytes, particularly activated T lymphocytes to enter the target organ or grafted tissue. Traffic to the target is by way of blood vessels and the activated T lymphocytes must be able to enter and exit through the vessel walls. Therefore, it is reasonable to suppose that the participation of T lymphocytes in autoimmune damage or graft rejection might be prevented by measures affecting their traffic.
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SUMMARY OE' THE INVENTION
In no previous studies were intact heparin or chemically modified heparins devoid of anti-coagulant activity shown to benefit autoimmune diseases or allograft rejection.
It was discovered recently by us that T lymphocytes expressed a heparanase enzyme that specifically attacked the glycsamino-glycan moiety of the extracellular matrix secreted by endothelial cells that line blood vessels (Naparstek, Y., Cohen, I.R., Fuks, Z.
and I, Vlodavsky. Activated T lymphocytes produce a matr.ix-degrad-ing heparan sulphate endoglycosidase. Nature 310:241 (1984)).
The presence of this enzyme was associated with the ability of autoimmune T lymphocytes to penetrate blood-vessel ~alls and to attack the brain in a model disease called experimental autoimmune encephalomyelitis.
Furthermore, it was found that the heparanase enzyme could be inhibited by heparin and some modified heparin molecules such as N-desulfated/ N-acetylated heparin but not by others such as totally desulfated heparin (Table 1).
We~ therefore tested whether heparin or modified heparins administered to experimental animals might be used to treat autoimmune diseases or to prevent graft rejection.
DETAILED DESCRIPTION
1. N-desulfated, N-acetylated modified heparin, or a low dose of intact heparin has no anti-coagulant effect in rats.
Table 2 shows that intact heparin at a dose of 2 mg per rat daily (10 mg/kg) caused an increase in the prothrombin time of recipient rats. In contrast, a dose of intact heparin of .~

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0.02 mg (0.1 mg/kg) or 2 mg of N-desulfated, N~acetylated heparir, (10 mg/kg) caused no anti-coagulant effect. Thus, -the potential dangers of hemorrhage attendant upon the administration of 10 mg/kg of intact heparin could be avoided by using intact heparin at a low dose (0.1 mg/kg) or a chemically modified heparin devoid of anti-coagulant activity.

2. Modified or low dose heparin inhibits skin allograft rejection. Figure 1 shows the survival of SJL/J skin grafts on (BALB/cxC57BL/6)Fl mice. The median survival time of the skin grafts on control mice treated with saline was 14 days while that on mice treated with 0.05 mg/kg daily of heparin was 26 days with maximal survival to 32 days. Figure 2 shows that treatment with 10 mg/kg daily of N-desulfated, N-acetylated heparin increased the median survival of the allogeneic skin grafts from 10 -to 20 days.
These results indicate that a low, sub-anti-coagulant dose of heparin or a modified, non-anti-coagulant heparin can significantly increase the survival time of allogeneic skin grafts on mice.

3. Modified or low dose heparin inhibits ability of anti-BP T lymphocytes to produce experimenta,l autoimmune encephalomyelitis (EAE).
EAE is an experimental autoimmune disease with some features reminiscent of multiple sclerosis in humans.The disease is caused by T lymphocytes immunized to the basic protein (BP) of the central nervous system myelin. To test the effect of heparins on the ability of T lymphocytes LO cause autoimmune disease, we used T lymphocytes sensiti~ed against BP, either as T cell ~31~9 ~

lines (Cohen, I.R. Experimental autoimmune encephalomyeli-tis:
Pa-thogenesis and prevention. In: Immunoregulatory Processes in Multiple Sclerosis and Experimen-taL Allergic Encephalomyelitis.
A.A. Vandenbark and J.C.M. Raus, eds. Elsevier Biomedical Res.
Amsterdam. 7:91-125 (1985)) or as populations of lymph node cells from BP immunized rats. Tab:Le 3 shows that a sub-anti-coagulant dose of intact heparin (0.1 mg/kg/day) or a dose of modified heparin (N-desulfated, N-acetylated) devoid of anti-coagulant activity (10 mg/kg/day) was able to inhibit markedly the severity of EAE produced by the anti-BP T
lymphocytes. Figure 3 shows graphically the inhibition of EAE
produced by treating rats with heparin (0.02 rng/rat/day;
0.1 mg/kg).

4. Modified or-low dose heparin inhibits adjuvant arthritis.
Adjuvant arthritis is an experimental disease inducible in some strains of rats by immunizing them to antigens of Mycobacterium tuberculosis (Pearson, C.M. Development of arthritis~ periarthritis and periostitis in rats given adjuvant.
Proc. Soc. Exp. Biol. Med. 91:91 (1956)). The disease is thought to be a model of rheumatoid arthritis in humans (Pearson, C.M.
Experimental models in rheumatoid disease. Arthritis Rheum. 7:80 (1964)). The arthritis appears to be caused by T lymphocytes that recognize an antigen of M. tuberculosis that is cross-reactive with cartilage (Cohen, I.R., J. Holoshitz, W. Van Eden, A. Frenkel. T lymphocytes illuminate pathogenesis and effect therapy of experimental arthritis. Arthritis Rheum. 29:841 (1985))~
Table 4 shows that sub-anti-coagulant doses of heparin ~ 3 ~

markedly inhlbited adjuvant arthritis. A dose of heparin of 0.001 mg daily had a marginal effect on arthritis. Doses of 0.005 and 0.01 mg wexe more effective while a dose of 0.02 mg was maximally effective in inhibiting arthritis. However, the higher dose of 0.04 mg had no inhibitory effect. Thus the dose-response characteristics of treatment were very sharp; doubling the most effective dose led to total loss of acti.vity. The sharpness Of the dose response curve makes the beneficial effect of heparin on autoimmunity and graft rejection easy to miss and probably accounts for the oversight of other investigators in making our observation. Modified heparins such as N-desulfated, N-acetylated heparin also showed a similarly sharp dose-response curve with a maximum effect at 0.02 mg per rat (0.1 mg/kg). A higher dose (0.04 mg) was ineffective (Figure 4).
Figure 5 illustrates that modified heparin (N-desulfated, N-acetylated) at a dose of 0.1 mg/kg/day given from day 21 to 51 produced early remission of established adjuvant arthritis. Thus, treatment was effective even when the arthritis was already clinically severe. Histologic examination of the joints showed severe signs of inflammation in the control rats and healthy ioints in the treated rats.
Table 5 tabulates the sources of commercially available heparin that were tested for their ability to produce long term inhibition (at day 60) of adjuvant arthritis subsequent to daily subcutaneous treatment for 5 days beginning on day 8 after induction of arthritis Heparin obtained from 3 of 4 sources were very effective while one source was only partially effective (Organon). Thus, a variety of sources can be used to obtain ~`~

active ma-terial.
Table 6 illustrates the various modified heparins that were tested for their ability to produce long term inhibition of adjuvant arthritis at day 60 as described above. Total desulfated and N-desulfated heparins were not effective in treating arthritis. However, N-desulfated, N-acetylated and O-desulEated, N-acetylated heparins were as effective as was native heparin.
As demonstrated in Table 2, the modified heparins had little anti-coagulant activity. Thus inhibition of undesirable immunological reactions can be achieved with various preparations of heparin devoid of the danger of anti-coagulant activity.
5. Modified heparin inhibits entry in-to, and exist from, blood vessels of activated T lymphocytes.
To test the effect of modified heparin on T lymphocyte traffic, we labelled the lymphocytes with Cr and measured the uptake of the labeled lymphocytes from a subcutaneous site and their persistence in the blood. We found that the labeled lymphocytes persisted in the site of in~ection into the tail of rats or mice for 5-6 days in treated animals (N-desulfated, N-acetylated heparin; 0.05 mg/kg) While the labeled lymphocytes migrated from the site of injection with 1-2 days in control animals.
Furthermore, treatment with the modified heparin led to persistence of labeled lymphocytes in the blood for 4-5 days, while the untreated rats or mice cleared the labeled lymphocytes from the blood in 1 day. Thus, treatment with modified heparin inhibited the ability of T lymphocytes to enter the blood vessels, '1,;,~; j~

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and once in the blood vessels, prevented the T lymphocytes from exiting. This can be attrlbuted to inhibition of the heparanase enzyme activity needed to penetrate the extracellular matri~ of the vessel wall. For this reason, the T lymphocytes were less able to cause atoimmune disease or graft rejection.
The results indicate that an inhibitor of heparanase, such as heparin or N-desulfated, N-acetylated heparin, can be used to prevent autoimmunity and allograft rejection. The effective dose of heparin of the order of 0.1 mg/kg, is about 1% or 16ss of that used to produce an anti-coagulant effect (10 mg/kg) and therefore prevention of undesirable immune reactions can be separated from anti-coagulation. N-desulfated, N-acetylated and O-desulfated, N-acetylated heparins are intrinsically devoid of anti-coagulant activity and can be used at a higher dose of the order of 10 mg/kg although lower doses (0.1 mg/kg) of these materials are more effective in preventing unwanted immune reactions. This dose is critical because, as shown inTable 4, 0.02 mg/rat (0.1 mg/kg) can be optimal in inhi.biting disease while a higher dose, 0.04 mg/rat (0.2 mg/kg~ can be ineffective. The same sensitivity of effect to dose was also observed with the modified heparins such as N-desulfated, N-acetylated.

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This treatment is novel and avoids -the generally toxic effects of immunosuppressive agen-tscurrently in use. We propose to patent the use of any heparin or modified heparin that inhibits T lymphocyte heparanase activity to treat autoimmune disease in humans, of the nervous system, joints, muscles, kidneys, liver, skin, digestive tract, liver, blood elements, endocrine organs or sex organs; or to prevent the rejection of allografts.

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Inhibition of DTH Type Skin Reactions -a. Assay sys-tem:
Mice were sensitized to 4 ethoxymethylene-2-phenyl oxazolone (oX) by painting their skins twice a-t 5 day in-tervals with abou-t 0.1 ml of 3% OX in a vehicle of 4:1 acetone:olive oil (by volume).
Their immunized draining lymph node cells (I-LNC) were then transferred (5x107) to recipient mice intravenously. The ability of the I-LNC to reach the site of antigen and produce a DTH
- reaction was assayed by challenging the recipien-t,mice 1 hr after I-LNC transfer with 0.02 ml of 0.5~ OX painted on -the ear. DTH
was ascertained by measuring the thickness of the ears 24 hrs later with an engineers micrometer.
The ability of the transferred I-LNC to reach the ears was tested by labeling the I-LNC before transfer with radioactive 51Cr (107 cells/ml incubated with 0.1 mCi 51Cr sodium chromate and washed) and measuring the amount cpm reaching the ears at the time of DTH.

b. Low dose heparin inhibits migration to site of DTH.
Figure 1 shows that a dose of 5 ,u.g. daily of heparin prevents the I-LNC both from reaching the ear (decreased cpm) and from producing a DTH reaction (decreased ear swelling). A
higher dose of heparin (20 ,u.g. daily) did not inhibit either I-LNC migration to the ears or DTH reactivity.

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c. Low dose heparin abrogates expression of heparanase in I-LNC
The above results indicated that treatment wi-th the low-dose ~5 ,u.g.) of hep~rin suppressed the ability of DTH mediating I-LNC
to enter blood vessels and to accumulate at the site of antigen deposit. To learn if these effects were associated with inhibi-tion of endogenous heparanase, we treated mice with high (25 ~.g.) or low (5 ,u.g.) doses of heparin, sensitized them to OX and tested their I-LNC for heparanase activity in vitro. Figure 2 shows that the I-LNC of mice treated with the low-dose (5 ~.g.) of heparin lacked heparanase activity. In contrast, the mice treated with the high (25 ~.g.) dose of heparin had heparanase activity that was similar to that of untreated control mice.
Thus, treatment with 5 ~.g. of heparin in vivo caused a substan-tial decrease of enzyme activity in sensitized lymphocytes.

d. Low-dose heparin does not abrogate an in situ DTH reaction If inhibition of heparanase and heparanase-dependent traffic is the major mechanism by which low dose heparin suppresses DTH, then one might be able to bypass the inhibition of DTH by bypassing the need for vascular traffic of sensitized T
lymphocytes. Accordingly, we treated recipient mice with an inhibitory dose (5 ~.g.) of heparin, and then injected the donor I-LNC directly into the ears, rather than intravenously Table 1 illustrates that putting the sensitized lymphocytes in situ bypassed the inhibitory effect on the DTH of 5 ,u.g. of heparin. Thus low dose heparin treatment appeared to inhibit DTH only when the DTH-mediat lymphocytes had to make their way to the site of the antigen by way of the circulation.

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Conclusions:
1. A low dose of heparin inhibits DTH reaction, as it does graft rejection and autoimmune diseases ln e~perimental animals.
2. These effects are associated with a decrease in T lymphocyte heparanase and T lymphocyte migration to the site of the antigen.
2. Low dose heparin inhibits DTH reactions in humans a. Healthy volunteers 6 young adult mèdical students were tested at g8 hrs for their spontaneous skin DTH reactions to tuberculin, tetanus toxoid, mumps antigen, or diphtheria toxoid and then treated with a single daily subcutaneous injection-of 300-500 un of heparin. Two weeks later during continued heparin treatment, their DTH reactions were again measured and the positive reactions in each of the 6 were found to be markedly reduced.
The heparin treatments were discontinued and 2 weeks later the DTH reactions were observed to return to their initial state of reactivity. Repeated administration of heparin again caused a marked decrement of DTH reactivity and stopping the treatment led to recovery of the original reactivity.
b. Patients suffering from multiple sclerosis In preparation fbr a clinical trial of low dose heparin in multiple sclerosis, 12 patients were treated with daily doses of 300-1000 units of heparin, and similar to the healthy volunteers~ all 12 had a decrease in DTH reactivity.
c. Low dose heparin induces improvement in rheumatoid arthritis.

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As DTH reactions to self antigens are involved in autoimmune diseases, we have begun to test low dose heparin (about 300-500 units daily) in patients with rheumatoid arthritis. Three patients with severe arthritis were treated for 1 month and all 3 were improved; they felt better subjectively and they had a decrease in their clinical disabili.ty and arthritis as assessed by their physicians.

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LEGENDS TO FIGURES:
FIGURE 1. Treatment wi-th heparin (005 mg/kg) prevents rejection of skin allografts. Mice of hybrid strain (BALB/c x C57BL/6)Fl were grafted with skin from allogeneic SJl/J mice. The mice (20 per group) were -treated daily with subcutaneous injections of saline (squares) or with heparin ("Leo", 0.05 mg/kg; diamonds) and scored for skin graft survival.
Median survival for the control group was 10 days while that for the heparin treated group was 24 days.

FIGURE 2. Treatment with N-desulfated, N-acetylated heparin (10 mg/~g) pre~ents rejection of skin allografts~ Mice were grafted as described in the legend to Figure 1 and treated daily with saline (squares) or N-desulfated, N-acetylated heparin (10 mg/kg; diamonds). The median survival of the skin allograft in the control groups was 10 days while that in the treated group was 20 days.

FIGURE 3. Treatment with heparin (0.1 mg/kg) inhibits EAE
produced by autoimmune T lymphocytes. Beginning 1 day before inoculation with the T lymphocytes, the rats were injected daily with 0.02 mg of heparin subcutaneously (0.1 mg/kg; squares).
Control rats were injected with saline (diamonds). EAE clinical score was estimated as tail weakness -25; paralysis of hind limbs -50; paralysis of all 4 limbs -75; moribund state -100.

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FI~URE 4. Treatment of adjuvant arthritis using modified heparin (N-desulfated, N-acetylated) at various doses. Rats were immunized to induce adjuvant arthritis as described in the legend to Table 4. On day 9 the rats were inoculated subcutaneously once daily with N-desulfated, N-acetylated heparin at doses of 0 mg ( ), 0.00:L mg (-), 0.02 my (x) or 0.04 mg ( ). The dose of 0.02 my caused a significant inhibition of arthritis.

FIGURE 5. Treatment with N-desulfated, N-acetylated heparin (0.1 mg/kg) induces remission of established adjuvant arthritis (~A). Twenty Lewis rats were inoculated with Mycobacteria tuberculosis to induce AA as described (3,11).
Clinical arthritis was scored on a scale of 0 (no arthritis) to 100 (marked swelling, tenderness and redness of all 4 paws).
On day 21, when all of the rats were suffering from marked arthritis, 10 were inoculated subcutaneously with saline (diamonds) and 10 were treated with N-desulfated, N-acetylated heparin (0.1 mg/kg) until day 51.

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_igure 6 Left:
Low dose heparin inhibits adoptively transferred DTH.
I-LNC were obtained from OX sensitized mice (groups A-C) and naive LNC from unsensitized mice (group D). The LNC were transferred intravenously to naive recipients that were treated (groups B-C) or unt~eated (groups A,D) with heparin (5 ,u.g. or 20 ,u.g.) injected 18hrs and 1 hr prior to cell transfer and 20 hrs after cell transfer. DTH ear swelling was elicited by OX at the time of cell transfer and measured 24 hrs later.

Right:
Heparin inhibits cell migration to DTE~ challenge site The experiment was done as described in Figure 1 (left), except that I-LNC were radiolabeled with 51Cr prior to cell transfer into naive recipient mice. The accumulation of I-LNC
in the OX challenged ears is indicated as the Cr (cpm/ear).

Figure 7 Heparin inhibits heparanase in vivo.
Mice were immunized with OX on days 0 and 5. Some of the mice were treated with heparin (5 ,u.g or 25 ,u.g per injection) 18 hrs before and 2, 10 and 20 hrs after the day 5 immunization with OX. On day 6, the I-LNC were removed and tested for heparanase activity by incubation for 48 hrs with labeled ECM.
Heparan sulfate degradation products are shown for I-LNC from the following groups of mice: No heparin - 0; 25 u.g heparan -X; 5 y.g heparin - 0. The LNC of control mice not immunized to OX had no heparanase activity (not shown).

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TABLE 1.
Inhibition of heparanase activity Test Inhibition of inhibitor degrada-tion of heparan sulfate by heparanase Heparin: intact yes Heparin: totally no desulfated Heparin:
N-desulfated yes N-acetylated Heparanase activity was induced into the extracellular medium bathing activated T lymphocytes and tested by incubating the medium with extracellular matrix whose heparan sulfate was labeled with 35S as described (Naparstek, Y., Cohen, I.R., Fuks, Z. and I. Vlodavsky. Activited T lymphocytes produce a matrix-degrading heparan sulphate endoglycosidase. Nature 310:241 tl984)). Inhibition of heparanase activity was tested by adding various concentrations of heparin or modified heparins to the reaction mixture and measuring the effect on degradation of the labeled heparin-sulfate as described (Naparstek, Y., Cohen, I.R., Fuks, Z. and I. Vlodavsky.

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~ ctivated T lymphocytes produce a matrix-deerading heparan qulphate endoglycoiida~e. Nature 310:241 (1~8ll)). Totally desulfated heparin and N-de.sulfated, N-acety!ated heparin was prepared as described. (Ayotte, L., A.S. Perlin. NMR spectroscopic observations related to the f~lction of sulfa~e group~ in heparin. Calcium binding vs. biolo~ical activity. Carbohydrate ~es. 145:267 (1y~6))~

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TABLE 2.

Effect on prothrombin time o~ heparins Injectc~ Dose Prothrom~in ~nti-coagulation ~aterial (mg) time (min) ~ . .. . _ _ . .
None 0 19 - Heparin 20 25 yes Heparin 0.2 17 no - Heparin:
N-desulfated N-acetylated 20 19 no Lewis rat:;, 10 weeks old weighing 250 ~n, were injected subcutaneously with the indicated dose of heparin once daily for 2 days. The prothrol~bin time was then tested as described in the "Pathromtin Kit - oTX8" (Hoechst-Behring, Marburg, FRG).

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'r~BLE 3.
Inhibition of ex~r~nel1tal aut()ilnlllune enccphal~nyelitis (EAE) by treatment with a sub-ant-coagulant dose of intact heparin or with modlfied heparin (N-desulfated, N-acetylated).

_ _ _ _ _ _ _ Ageht ~ose Mediation ~ D~y of Duration Clinical (mg/kg) of EAE incidence onset (days) score _ A. None - T cell100 5.2 4.2 2.4 line Modified Heparin 10 S0 6.4 1.8 0,8 B. None - T cell100 5.0 5.5 3.0 line Heparin 0.1 20 6.5 3.6 1.0 C. None - Pr~ned ~0 4.5 5.8 2.3 lymph node Modified - - ~
Heparin 10 D. None - Primed100 4.0 5.3 3.0 lymph node Heparin 0.1 75 6.3 4.0 1.5 ._ 13 3L`~5~

EAE was produced by inoculating Lewis rats with a T cell line of anti-BP T lym~1ocytes (10G cells) wi~h anti-BP primed lymph node cell~ (107 cells) intravet1ously (Col~en, l,~ xper~nental auto~nune encephalanyelitis: Pathogenesis and prevention, In: I~nunoregulatory Processes itl Multiple Sclerosis and Experitnental Allergic Encephalomyelitis, A,A, Vandenbark and ~'.E,M, ~aus, eds, Elsevier Biomedical Res, Amsterdam, 7:91-125 (1985)) One day before inoculation and daily for 10 days, the rats received either saLine or the heparins, The rats were observed for development of paralysis graded 1 for tail weakness; 2 for paralysis of hind limbs; 3 for paralysis of hind and forelimbs; and 4 for moribund state -- ~0 -~ 3 ~

TABLE 4.
Treatment of adjuvant arthritis by sub-anti-coagulan-t doses of heparin.

Heparin _ Adjuvant Arthritis dose Duration Maximum (mg) (days) clinical score 0 ~20 10 0.04 ~20 10 0.02 8 2 0.01 15 5 0.005 16 4 0.001 20 6 Rats were immunized with M. tuberculo is (1 mg) in oil to induce adjuvant arthritis (Pearson, C.M. Development of arthritis, periarthritis and periostitis in rats given adjuvant.
Proc. Soc. Exp. Biol. Med~ 91:91 (1956)). On day 9 the rats were incubated subcutaneously once daily for 5 days with various doses of heparin and scored for the development of arthritis on a scale of 0-16 as described (Holoshitz, Y., Y. Naparstek, A. Ben-Nun, I.R. Cohen. Lines of lymphocytes mediate or vaccinate against autoimmune arthritis. Science 219:56 (1983)).

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TABLE 5.

Sources of Heparin -tested for inhibition of adjuvan-t arthritis.

Heparin Company Arthritis Inhibition of Score Arthritis (day 60) __ Leo Leo Pharmaceutical Ballerp, Denmark 0 yes Sigma Sigma Chemical Co.
(bovine lung) St. Louis, MI, USA 0 yes BDH BDH Chemicals Poole, England 0 yes Thromboliquine Organon Teknika, Boxtel, Holland 2.5 partial Untreated --- 5 Adjuvant arthritis was induced in Lewis rats and the rats were treated with the indicated sources of heparin as described in the legend to Table 4. The mean arthritis score determined on day 60 was used to assay the efficacy of heparin treatment.

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TABLE 6.

Modified heparin3 tcsted for inhibition Or adjuvant arthritis.

_ Heparin ~rthritis score Inhibition of arthriti~

(d~y Go) None 6 Intact O yes N-desulfated O yes N-acetylated O-desulfated O yes N-acetylated Total ~1~sull`ated 5 no , N-desulfated 5 no Adjuvant arthritis w~s induced in Lewis rats and the rats were treated with modified heparins as described in the legends to Tables 4 and 5. The heparins were modified as described (Ayotte, L., A.S. Perlin. N;~R spectroscopic observations related to the function of sulfate groups in heparin. Calcium binding vs. biological activity. Carbohydrate Res. 145:267 (1986)).

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TABLE 7. This ~a~le illus~rales l~ cp~rin do~s not block 1ransfer of DTH when l-LNC are directly injected into the sitc of antige~
challenge Recipients of I-LNC sensitized to OX

~_________________________________________._______________________~___~ .
Heparin OX
treatment ChalLen~e Ear swelling (xlO~4inch) (5 u.g) +__________________________________________________.._________________~
' No Yes 22 ~ 3.4 : ' Yes Yes 20.4 + 1.6 No No 9 ~ 1.0 Yes No ~ + 1.0 +________________________.___________________________________________~
I-LNC were obtained froln BALB/c ~nice sensitized to OX ~ days earlier.
The I-LNC were centrifuged, resuspended in RP.~I medi~n and injected intradermally (3x10~ cells/20 ~./1) into the dorsal surface of ttle ears of naive recipient mice. m e ears were challenged with OX immediately after cell transfer. The maenitude of ~rH ear swelling was determined 24 h later.

Claims (12)

1. A pharmaceutical composition for delaying or for preventing the rejection of allografts, for alleviating and for treating autoimmune diseases, which composition contains as active ingredient of from about 0.5 per cent to about 5 per cent of the dosage of heparin, of N-desulfated, N-acetylated heparin or O-desulfated, N-acetylated heparin which is required to obtain an anticoagulant effect.

2. A composition according to claim 1, where the dosage is of 0.01 to 0.02 mg/kg of patient weight of heparin per day.

3. A composition according to claim 1 where the dosage is of from 0.01 to 0.02 mg/kg/day N-desulfated, N-acetylated heparin or O-desulfated, N-acetylated heparin.

4. A pharmaceutical anti-arthritically effective composition comprising about 0.02 mg/kg/day heparin.

5. Use of heparin, N-desulfated, N-acetylated heparin or O-desulfated, N-acetylated heparin in pharmaceutical compositions effective against autoimmune diseases, for the alleviation of such diseases and for the treatment of autoimmune diseases in mammals, including humans, and for preventing or delaying allograft rejection, which compositions contain as active ingredient of from 0.5 per cent to 5 per cent of the dosage of heparin, that is required to obtain an anticoagulant effect.

6. The use according to claim 5, wherein the dosage is of 0.01 to 0.02 mg/kg/day heparin.

7. The use according to claims 5, wherein the dosage is of 0.01 to 0.02 mg/kg/day N-desulfated, N-acetylated heparin or O-desulfated, N-acetylated heparin.

8. The use according to claim 5, wherein the dosage of 0.02 mg/kg/day heparin is effective against arthritis.

9. Use of heparin N-desulfated, N-acetylated heparin or O-desulfated, N-acetylated heparin for the preparation of pharmaceutical compositions effective against autoimmune diesases, for the alleviation of such diseases and for the treatment of autoimmune diseases in mammals, including humans, and for preventing or delaying allograft rejection, which compositions contain as active ingredient of from 0.5 per cent to 5 per cent of the dosage of heparin, that is required to obtain an anticoagulant effect.

10. The use according to claim 9, wherein the dosage is of 0.01 to 0.02 mg/kg/day heparin.

11. The use according to claim 9, wherein the dosage is of 0.01 to 0.02 mg/kg/day N-desulfated, N-acetylated heparin or O-desulfated, N-acetylated heparin.

12. The use according to claim 9, wherein the dosage of 0.02 mg/kg/day heparin is effective against arthritis.