Note: Descriptions are shown in the official language in which they were submitted.
13~8~
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.
~i
-- 1 --
~ 3 ~
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
.~
~ 3 ~
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
-- 3 --
~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
-- 4 --
~ 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
- 5 -
~`~
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,
-- 6
'1,;,~; j~
~ 3 ~
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.
-- 7 --
~ 3~8~
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.
~ 3 ~
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.
~r
~ 3 ~
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.
-- 10 -
~ r
~ 3 ~
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.
-- 11 --
~ 3 ~
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.
- 12 -
~,~
~L 3 ~
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.
.1 .~ A.
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.
- 14 -
w
131~5~:~
_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).
- 15 -
131~
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.
.7~
~ 3 ~
~ 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))~
~ 3 ~
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).
- l~3 -
1 3 ~
'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
._
-- 19 -
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)).
~31~
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.
. . ~
, .
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)).
- 23 -
~ 3 ~
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.
- 24 -
