Note: Descriptions are shown in the official language in which they were submitted.
2~6~3~
WO94/18988 PCT~4/00506
USE OF HEPARINS FOR THE TREATMENT OF INFLAMMATORY
OR IMMUNOLOGICAL DISEASES
.
Bac~ground of the invention
Field of the invention
The invention is related to a pharmaceutical composition
for Ihe trea~ment of inflammatory and/or imml~nological
diseases and a method for the treatment of said diseases.
These diseases can be multiple sclerosis, primary biliary
cirrhosis, rheumatism, lupus erythematosus (LE), post-
infarct-syndrome, Ghost versus Host reaction (GvH), auto-
agressive neuritides (german: Neuritiden), mi~raine,
hyper-IgE-syndro...e, Cr~hr,'s disease (german: Morbus
Crohn), systemic carcinoma diseases and the like.
Sys~emic carcinoma diseases can be melanoma, Burkitt
lymphom~, leukaemia, Non-Hotrhk; n lymph~mA and the like.
.
DescriPtion of the prior art
Heparin and heparan sulfate which are known as an-
ticoagulant substances found in the liver and lungs and
Whi ~h C~n ~1 C~ ~ p~ ~ ~_ti f~ ~ rc-.m..~
for the treatment and prophylaxis of thrombosis. The ab-
ove-mentioned pharmaceuticals are known at least since
1930. ~hey are especially used for the treatmen~ of
hyperlipidaemia, arteriosclerosis and are used during
biood transfusion and after operations. The interaction of
heparins with lymphocyte adhesion patterns was studied by
Ekre et al. (Ekre H.P., F~ellner B. and Hagermark O.
(1986) Inhibition of complement dependant experimental
inflammation in human skin by different heparin fractions.
Int. J. Tmmllnophramacol. 8 (3):277-286; in the following
WO94/18988 ~ 6 7 3 5 PCT~4/00506
.
-- 2 --
Ekre et al.1986) concerning the activity of heparin which
suppresses the ;mmllnoresponse in the human skin
permantently at concentrations between 2 and 5 i.U. per kg
and day with a dose limitation of 300 i.U. per day.
However, it is not known to use these pharmaceuticals for
treating inflammatory or lmmllnological diseases.
One of the most known adhesion molecule is the fibrinogen
in the tissue-pl~sm;nogen-activator-system (TPA). There
are also other important systems. These are e.g.
fibronectin and lactoferrin which exist in large amounts
if the patient has tumors and which are a sign for the
decreasing cellular contact inhibition. These proteins are
also detectable for chronic inflammations which do not
have a tumoric origin and are called acute-phase-proteins
(ge_man: ~utphaser.proteine). Our own results show that
the acute-phase-proteins are increased corresponding to
the C-reactive protein and are directly correlated to the
erythrocyte sedimentation rate.
Multiple sclerosis (MS) has become a major health problem
in industrialized countries. It affects between 50 and 100
individuals out of 100.000. A review of the literature
gave evidence that up to now there is no convincing clue
what the main cause of this disease could be (Reinherz
E.L., Kung P.C., Goldstein G. and Schlossmann S.F. (1979)
Separation of functional subsets of human T-cells by
monoclonal antibody. Proc.Nat.Acad.Sci.USA 76:4061; in the
following: Re;nherz et al. ;979). Until recently no causai
therapy was avalaible.
Other inflammatory and/or lmml~nological diseases are also
a major health problem not only in industrialized but also
in countries at the stage of economic take-off and also
~5673~
WO 94/18988 - PCT/EP94100506
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those of the third world. These diseases are e.g. primary
biliary cirrhosis, rheumatism, lupus erythematosus (LE),
post-infarct syndrome, ghost-versus-host reaction (GvH),
systemic carcinoma diseases and others.
Various pharmaceuticals are used to treat the above
mentioned diseases. However, there does not exist any
pharmaceutical which allows a healing of the patients
without any side effects. Moreover, the probability of a
compiete recovery of the patients is low. It is thus an
object of the invention to find a pharmaceutical and a
method of treatment to cure patients from the above
mentioned diseases with few or even no side effects at
all.
The high endothelial cells (XEC), oligodendrocytes and
Schwann cells of multiple sclerosis (MS) patients carry a
hypervariable activated cellular DNA, a so called inter-
cellular adhesion molecule cellular DNA (pICAMhec
cDNA) which encoaes a hypervariabie lC~vlhec. Actlvated
lymphocytes of the type Tal and T113 adhere strongly to
cells expressing this ICAMhec and from the studies of
Ford (Ford W.L. (1978) Possible clues to the mechanism
underlying selective migration of lymphocvtes from ~he
blood. Symp. Soc. Exp. Biol. 32:359; in the following:
Ford 1978) and Woodruff et al. (Woodruff J.J. and
Kuttr.er B.J. ~1980) Adherence of lymphocytes to ~E~ of
lymph nodes in vitro Excerpta Medica: Blood cells and
vessels wails: functional interactions (Cibal Foundation
Symposium) 243-263; in the following Woodruff et. al.1980)
we know that adherent lymphocytes migrate into the
surrounding tissue. A possible role of cell surface
molecules involved in this adhesive interaction has been
postulated already for some time by Stoolman et al.
2~7~5
WO94118988 PCT~4/00506
-- 4 --
(Stoolman L.M. and Rosen S.D. (1983) Possible role for
cell surface carbohydrate b; n~; ng molecules in lymphocyte
recirculation. J. Cell Biol.96:722; in the ~ollowing:
Stoolman et. al.1983). In a routinely performed adhesion
assay we observed that lymphocytes preincubated with MS
patient serum adhere on HEC.
However, it was not known which type of lymphocytes adhere
on HEC and secondly which molecules regulate this
adhesion. From the litera~ure pubiisnea by Reinherz et
al.1979, Bach et al. (Bach M.A., Tournier E. et al.(1980)
Deficit of suppressor T-cells in active multiple
sclerosis. Lancet 2:1221; in the following Bach et
al.1980), Traugott et al.(Traugott U., Re;nherz E.L. and
Raine C.S. (1983) Multiple Sclerosis: distribution of T-
cells s~sets wit~.in active relapsir.g and chronic pro-
gressive diseases. Ann. Neurol 14:445; in the following:
Traugott et al.1983) and Weiner et ai. (Weiner n.~ han
A.K. and Burks J. (1984) Tm~llnohistochemical analysis of
~he cellular infil~ra~e in multiple sclerosis lesions.
Neurology (NY) 34: Suppl 1:112; in the following Weiner et
al.1984) it is known that the adherent lymphocytes could
be Ta1 and T113 cells.
Objects and SummarY of the Invention
It is therefore an object of the invention to provide an
improved ~hAr~ceutical compositior, for the treatment of
inflammatory and immllnological diseases and a method of
treatment of said diseases.
In accordance with these objects the invention provides
pharmaceutical compositions comprising heparins,
heparinoids, proteoglycans or low-molecular-weight
heparins or mixtures therof to be used for the treatment
WO 94/18988 215 6 7 ~ 5 PCT/EP94100506
-- 5 --
of the above-mentioned diseases. The present invention
furthermore provides a method of treatment of such
diseases involving the use of said pharmaceutical
compositions.
The pharmaceutical compositions of the invention and the
method of treatment make it possible to cure patients from
the above-mentioned diseases with only few side effects or
even no side effects at ail.
The pharmaceuticals according to the invention generally
comprise heparins of various molecular weights.
Preferably, low-molecular-weight heparin is used.
However, heparins of high molecular weight, heparinoids,
heparan sulfate as well as proteoglycans can also be used.
The pharmaceutical can be applied directly to the mucosa
_r ~ ca., ~e ~jected subcutaneously or intravenous'y. It
should be noted that the pharmaceuticals should not be
applied to the eye because there a toxic rezct~cn could
occur. For a usage of the pharmaceutical nasally a spray
seems to be suitable so that the patient is able to treat
himself easily with the pharmaceutical. Swallowing pills
is another possibility for an easy treatment of the pati-
ent himself because the dosage can be controlled very
accurately. However, an injection is also suitable.
The dos2ges for the treat~..ent depends on the disease
itself and its strength and the pharmaceutical which is
used. However, it turned out that dosages of up to 40 i.U
per kg and day are suitable to effect MS-patients and also
not more than 40 i.U. per kg and day should be suitable to
- effect patients with the other mentioned diseases. It is
WO94/18988 21~ ~ 7 3 5 PCT~4/00506
.
- 6 -
most suitable to use dosages between 1 and 5 i.U. for all
mentioned diseases.
If the patient will be treated with one of the mentioned
pharmaceuticals it is important to mention that the inter-
national units are not always defined the same manner. One
should always calculate the correct e~uivalent molar
concentration to e.g. Fragmin-D. If e.g. heparin is used a
factor of ca. 30 has to be taken to receive the correct
amount of this pharmaceutical. If other heparinoids or
heparan sulfate are used the correct equivalent molar
concentration must also be calculated. The used dosage
depends on the concentration of the pharmaceutical in the
plasma (german: Plasmaspiegel) which should be more than
0,5 i.U./ml for LWHM's.
Most suitable for diseases like MS, rheumatism, primary
biliary cirrhosis, migraine, hyper-IgE-syndrome and neu-
ritides are pharmaceuticals as Fragmin and Fragmin-D.
It turned out that for diseases like GvH and systemic
carcinoma e.g. leukaemia or lymphoma Reviparin is
a preferred pharmaceutical.
If Fragmin is used a dosage between 1-40 i.U. antifactor
Xa per kg and day which means an amount of more than 0,5
i.U./ml in the plasma (german: Plasmaspiegel) should be
achieved.
The present invention is based on observations obtained in
in vitro studies on the influence of heparin on the
adhesive interaction between HEC cells and peripheral
blood lymphocyte subtypes.
SUB~TIME SHEET (RULE 26)
~ WO94/18988 21~ ~ 7 3 ~ PCT~4/00506
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Between 1985 and 1991 we took blood samples from 1216
patients and we conducted adhesion assays on cultivated
high endothelial cells (HEC). We observed reproducible
pathognomonic lymphocyte adhesion of two types of
peripheral blood lymphocytes, one bearing the T-cell-
specific activation antigen Ta1 and the other activation
antigen is the so called T113 lymphocytes to HECs.
Therefore, we started analyzing the Intercellular Adhesion
Molecule (ICAM) on HEC, oligodendrocytes, Schwann cells
and lymphocytes as well as the Lymphocyte Function Antigen
(LFA) on Ta1 and T113 lymphocytes of MS patients by means
of protein structure analysis. Furthermore, in situ
hybridization to the ICAM's and LFA genes in lymphocytes
and high endothelial cells of MS patients was carried out.
It was found that MS patients express a pathognomonic
hypervariable cellular DNA which encodes a hypervariable
ICAMhec on HEC, oligodendrocytes and Schwanncells in 95%
of the cases. The metabolized degradation products of
this ICAMhec product leads to the activation of T-cells,
i.e. the expression of Ta1 and T113 antigens on
lymphocytes. The molecular structure of this ICAMhec shows
a 75% homology to heparansulfateproteoglycane. Adding
heparin to Ta1- and T113-positive lymphocytes inhibited
the pathognomonic adhesion patterns of MS patients
lymphocytes. The evaluation of these in vitro studies led
to the concept of the pharmaceutical compositions and the
method of treatment of the present invention.
The results of extensive studies show that the effect of
inhibition of coagulation of the pharmaceutical is caused
by a blocking or modelling of adhesion molecules including
those expressed on thrombocytes. This effect prevents the
SUB~TITUTE SI~EET (RULE 26~
WO94/18988 ~ 6 7 ~ ~ PCT~4/00506
-- 8 --
thrombocytes or other blood cells from aggregating or ~rom
adhering to an injured vascular wall.
Moreover, the viscosity of the blood cells is altered by
medication with the pharmaceutical in such a manner that
they can increase their length up to 7 times over their
physiological length.
The results show that the pharmaceutical has a direct
effect on the LFA-receptors and the ICAM-system because it
has the function of a modulating receptor or ligand for
both systems. Fibronectin, lactoferrin, fibrinogen and the
whole TPA and others belong also to this system. Moreover,
the suppression of transplantation and GvH reactions can
be explained by the effect on the LFA and the ICAM.
The diseases to he treated can be mu'ti~la s~lerosis, GvH,
primary biliary cirrhosis, post-infarct-syndrome, LE,
rheumatism or systemic carcinoma. The dosage of this
pharmaceutical should be up to 40 i.U. (international
units) per kg and day.-However, a dosage between l and 5
i.U. per kg and day is preferred.
The NMR controlled reduction of sclerotic patches with
TM~ f~^ eY~ Q ~ n-n ~ C ~ gn~ ficant and thP nl mhe~
of negative side effects compared to other st~nA~rd drugs
such as dexamethason or azathioprine is negligible. The
only ob~erved ~ide effects were a diSt~nct sleepi~ess,
nausea, and ophth~ c migraine. The sleepiness persisted
usually during tne whoie treatment period. Therefore, most
patients received their ~edication in the evening. Nausea
and migraine disappeared within a fortnight. A combination
of Prostavasin with LWXM leads to an improved biological
availability. Thus, Prostavasin is used to improve local
blood flow. This is another new therapeutic regime with
21~3~
WO 94/18988 ^ PCT/EP94/00506
rather encouraging results. Prostavasin is a pro-
staglandin. The substance is Alprostadil. However, it has
to be proven which other possible indications for this
drug are.
We found that low-molecular-weight heparins in vitro as
well as in vivo have significant better antiflammatory and
antiactivation potentials in MS than the medication tested
in this study (p = ~,002; n = 1216j. There was iess posi-
tive ir,f~uer.ce by dex&,.ethasor. or a~ath~opr ne co~ ared to
heparin (LMWH).
Other objects and advantages of the present invention and
some more results of the extensive studies will become
apparent from the following detailed description of the
invention when considered in conjunction with the
accompanying drawings.
Brief Description of the Drawinqs
Fig. 1: The results of the analysis G peripheral blood
mo~onuclear cells with anti-Ta1, anti-T113 and
control ascites in a patient with progressive MS
and a healthy control are shown. The
flowcytometric analysis was performed with a
linear scale of fluorescence intensitiy.
Fig. 2: The Tmmlmoprecipitation of ICAM from 125J-labelled
cells was demonstrated by the use of the
monoclon~l antibody 84-H10.
a: Regulation of ICAM synthesis in HEC by
cytokines;
b: Regulation of ICAM synthesis in oligodendro-
cytes by cytokines;
21~673~
WO94/18988 ~ PCT~4/00506
-- 10 --
Fig. 3: One can observe the expression and structure of
the pICAMhec gene shown by method
a: RNA blot hybridization and
b: genomic DNA blot hybridization.
Fig. 4: Reduction of sclerotic patches and symptom
improvement after longterm treatment of MS-
patients with Fragmin-D, dexamethasone,
azathioprine or a control substance.
Fig. 5: Significant therapeutic improvement in 73 acute MS
cases after treatment with LMWH compared to
treatment with dexamethasone or azathioprine
(p = 0,002; n = 73).
Fig. 6: Nucleotide sequence of the cellular DNA clone
pICAMhec (a), and the associated protein se~uence
(b). The nucleotide numbering goes from left to
right, the aminoacid sequence from right to left.
Fig. 7: Typical alteration of the adhesion rate in a
case of MS. The last attack was 15 years ago.
Fig. 8: Typical picture of an acute MS attack. Upon serial
dilution more than 10.000 adherent lymphocytes
per HEC were observed in the adhesion assay.
HEC were cultivated according to v. Arnim (1987, German
Patent No. DE 3536955). 5x10 HEC per ml were cultivated
on PRIMARIA-multiwell plates (Becton & Dickinson, Heidel-
berg) with culture medium RPMI-1640 (Gibco, Karlsruhe)
cont~;n;ng 16% Hyclone defined Fetal Calf Serum, 200mM
L-glutamine, 20 ~g/ml Endothelial Cell Growth Supplement
(ECGS; Sigma), 20 ~g/ml Multiple Stimulating Activity
SUBStITUrE SHEET (RULE ~6)
2~5~
oWO 94/18988 - PCT/EP94/00506
(MSA; Sigma), 10 ,ug/ml Heparin (H-3125, Sigma) and 15
~g/ml Insulin (Sigma). After a 10 day cultivation period
the HEC-monolayer was confluent and therefore the cells
were fixed for 10 min at 4C in 0,2Mol L-lysine (Gibco,
Karlsruhe) and deepfrozen in liquid nitrogen.
..
The blood samples were collected by iv puncture. The
lymphocytes and the serum of each sample were extracted by
centrifugation in Lymphoprep-tubes (Becton & Dickinson,
Heidelberg). Thereafter ser-~m and lymphocytes were
deepfrozen in liquid nitrogen.
Anti-Tal immunoprecipates a single major 105 kd band.
Unlike antibodies to the interleukine-2 receptor, anti-Ta1
does not inhibit T cell proliferative responses to antigen
or interleukine containing medium. Anti-T113 reacts with a
uni~ue epitope that becomes expressed on T cells after
activation. The weight of the pICAMhec cDNA encoded prote-
in varies between 100 and 125 kd. Concerning also the 105
kd band immllnoprecipita~ed by ant -Tal and LFA-1 being a
glycoprotein of a weight also varying between 100 and 125
kd this leaves no other conclusion then ICAM being the
ligand of LFA-1-carrying Ta1-cells (Stoolman L.M. (1989)
Adhesion molecules controlling lymphocyte migration; Cell
56:907-910; in the following: Stoolman 1989). Taking all
this into account ICAM expressing cells play a major role
in the induction of the pathophysiologic response in MS.
Gesner et al. (Gesner B.M. and Ginsburg V. (1964) Effect
of glycosidases on the fate of transfused lymphocytes.
Proc. Natl. Acad. Sci. USA 52:750; in the following:
Gesner et al.1964) , Ford 1978, Curtis (Curtis A.S.G.
(1974) The specific control of cell positioning. Arch.
Biol. 32:105; in the following: Curtis 1974) and Aklyama
(Aklyama S.K. (1981) The structure of fibronectin and
W094/18988 21 5 ~ 7 3 ~ PCT~4/00506
- 12 -
its role in cellular adhesion. J. Supramolecular structure
and cellular Biochem. 16:345; in the following Aklyama
1981) postulated that cells involved in lymphocyte
adhesion and migration must (1) produce, release and
transport recognition signal; (2) receive signals;
(3) activate an effector system in the receiving cell;
and (4) induce a specific answer in the receiving cell
via the effector system. In case of the adhesion of
Ta1 and T113 cells on ICAMhec expressing cells.
Stoolman et al.1983 and Holzmann et al. (1989) have
demonstrated the specificity of adhesion, migration and
cellular interaction and its physiologic versus patho-
physiologic relevance. The LFA-1 is a member of the inte-
grin family of cell surface receptors (Stoolmann 1989).
The tripeptide motif Arg-Gly-Asp (RGD) is a common feature
of the ligands for this family and is required for ligand
receptor interaction.
But ICAM contains no RGD motifs bearing instead a single
RGE sequence at position 152 (see Fig.4). Nevertheless
there is a striking similarity between ICAMhec, NCAM and
between heparansulfatproteoglykane and LFA-1. This
similarity is particularly interesting as it brings
together surface mucopolysaccharids, lymphoid and neuronal
adhesion molecules.
According to Reinherz et al.1979, Bach et al.1980, Weiner
et al.1984 and Traugott et al.1983 multiple sclerosis (MS)
patients carry Ta1 and T113 lymphocytes. Traugotts
laboratory gave us Anti-Ta1 and Anti-T113. All
monoclonal antibodies were used in antibody excess at a
dilution of 1:500 except for anti-T3, which was used at a
dilution of 1:250. Cytofluorographic analysis of cell
populations were performed by means of indirect
SUB~TITUl E S~IE~T (RULE 26~
WO 94/18988 21~ ~ 7 ~ 5 PCT/EP94/00506
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immunofluorescence with fluoresceinisothiocyanate-
conjugated (FITC) goat antirat antibodies (Wellcome) on a
flow cytometer using a linear scale. Bac~ground
fluorescence activity was determined by control ascites
fluid obtained from rats immtlnlzed with a nonsecreting
hybridoma.
For the adhesion assays lymphocytes from healthy controls
(no known immttnologic, allergic or lnfectious disease)
were incubated in patient serum.. for 60m.in at 37C,
centrifuged with 300g, washed in phosphate buffered saline
(PBS), centrifuged once more and resuspended in RPMI-1640
cont~i n; ng lOmM magnesium. Thereafter the fixed HEC-mono-
layer was incubated with the resuspended lymphocytes for
60min at 37C. After 60min the incubated monolayer was
washed three times with PBS, stained with acridine orange
and observed under the microscope. The number of adherent
lymphocytes per HEC was documented by photography (Film
Agfachrome professional ISO 200).
Peripheral blood mononuclear cells of a patient with
progressive MS and of a healthy control individual were
analyzed with anti-Tal, anti-T113 and control ascites by
flowcvtometric analvsis with a linear scale of
fluorescence intensity (see Fig. 1). Tal lymphocytes in
both T4 and T8 subsets from a patient with MS. Subsets
were prepared by complement-mediated lysis of reciprocal
populations and stained with anti-T3, anti-T4, anti-T8,
anti-Tal and control ascites. Cytofluorographic analysis
using a log scale was then performed. The T8 subset shows
st~; n ' ng with anti-T3, anti-T8 and anti-Tal, and no
St~ n i ng with anti-T4. The reciprocal T4 subset also
stains with anti-Tal. Anti-T3 reacts with virtually all
peripheral blood T cells. Anti-Tal lmmnnoprecipitates a
WO94/18988 2 ~ ~ 6 7 ~ ~ PCT~4/00506
.
- 14 -
single major 105 kd band. Unlike antibodies to the inter-
leukine-2 receptor, anti-Ta1 does not inhibit T cell pro-
liferative responses to antigen or interleukine cont~; n; ng
medium. Anti-T113 reacts with a uni~ue epitope that be-
comes expressed on T cells after activation. The
percentage of activated antigen positive T cells was
calculated by dividing the number of activated antigen
positive T cells by the total number of T3-positive cells.
The average percentage of T3-positive mononuclear cells
was signi'icantly di''erer.t ir. the di~ferent sroups.
To ;mmllnoprecipitate the Inter Cellular Adhesion
Molecule (ICAM) on 125J-labelled HEC, oligodendrocytes,
and Schwann cells we used the commercially available
monoclonal antibody 84-H10 (Becton & Dickinson, Heidel-
berg). A transfection with plCAMhec was done according to
the protocols of Berger et al. (Berger S.L.
and K;mmel A.R. (1987) Guide to .-"olecular Cloning
techni~ues Academic Press Inc. Vol 152:1-812; in the
following: Berger et al.1987) and Glover (Glover D.M.
(1987) DNA cloning a practical approach. IRL Press Vol 1-
3; ed.: D.M. Glover; in the following: Glover 1987)
episomal DNA was extracted from washed HEC. The HEC.
oligodendrocytes, Schwann cells were set to 5x105 cells
per ml. Thereafter, the cells were incu~ated for 48h with
the following test reagents; 50ng/ml
12/0/tetradekanoylphorbol-13-acetate (TDA, Sigma), 100~/ml
gamma-interferon, 200~/ml TNF, 10~/ml interleukine-1-beta.
For the function analysis the XEC, oligodendrocytes and
Schwann cells were transfected with the control vector PE-
H3M. We then conducted a lymphocyte adhesion assay.
Therefore HEC, oligodendrocytes and Schwann-cells were
incubated for lh at 37C in PBS containing 12,5% defined
fetal calf serum (Hyclone) and l~g/ml of the st~ rd
~ WO94/l8988 2 1 5 6 7 3 5 PCT~W4/00506
antibody 84-H10. Once in each preparation the antibody was
washed of to avoid lymphocyte binding by the antibody
excess. In all the other cases the antibody excess was
necessary to avoid lymphocyte migration through the cells.
The imm-lnoprecipitation of ICAM from 125J-labelled cells
was demonstrated by the use of the monoclonal antibody 84-
H10 (Fig. 2)
a: Regulation of ICAM synthesis in HEC by cytokines;
gam.ma-interferon-induced without antibody (lane 1), with
antibody (lane 2); tumor necrosis factor (TNF)-induced
without antibody (lane 3) with antibody (lane 4)
b: Regulation of ICAM synthesis in oligodendrocytes by
cytokines; gam.ma-interferon-induced without antibody (lane
1), with antibody (lane 2); TNF-induced without antibody
(lane 3), with antibody (lane 4); interleukine-l~-induced
without antibody (lane 5), with antibody (lane 6).
In Fig. 3 one can observe the expression and structure of
tne pICAMhec gene shown by the method of
a: RNA blot hybridization. Total RNA (24 ~g) was denatured
in formaldehyde, electrophoresed, transfered to nylon
mem.~branes and hybridized with ICAM-l cDNA.
Lane 1 uninduced HEC; lane 2 TDA induced; lane 3 gamma IFN
induced; lane 4 IL-l~ induced; lane 5 TNF induced; lane 6
glomerular endothellum; lane 7 oligodendrocytes from an MS
patient; lane 8 Tal; lane 9 T113; lane 10 lymphokine
activated killer cells. The RNA blot analysis revealed a
3,2 kb and a 1,9 kb species in HEC stimulated with TDA,
gam.ma-interferon, TNF, and interleukine-l~. It was not
found in llnin~l)çed cells. The 1,9 kb band is blocked by
18S rRNA which is present as a cont~min~tion in poly-A
selected RNA and is therefore considered unspecific.
W094/18988 21~ ~ ~ 3 ~ PCT~4/00506
- 16 -
The results lead to the conclusion that the expression of
ICAM is regulated by inflammatory cytokines on the
transcription level.
b: genomic DNA blot hybridization (pICAMhec cDNA from
high endothelial cells from MS patients): lane 1: EcoRI;
lane 2: BamHI; lane 3: HindIII endonuclease from Hemophilus
influenza; lane 4: DraI endonuclease.
For the DNA-hybridization human chromosomai piacenta DNA
(24~ug) was digested with restriction enzymes, separated
electrophoretically and transfered to a nylon membrane.
Then it was hybridized with the cellular pICAMhec-cDNA
clone. For the RNA blot hybridization we used the protocol
from Berger et al.(1987) and Glover (1987); 24,ug
cellular RNA was denatured in formaldehyde, separated
electrophoretically and transferred to a nylone membrane.
Thereafter the RNA was hybridized with ceiluiar DNA of the
clone plCAMhec-cDNA. A cellular DNA library was
constructed by extracting RNA stimulated with
12/0/tetradecanoylphorbol-13-acetate (TDA; Sigma) from MS
patients' lymphocytes, HEC, oligodendrocytes and Schwann
cells. This library was transfected into HEC. HEC
expressina the pICAMhec were identified bv the antibodv
84-H10. From these cells the episomal DNA was extracted to
isolate the clone pICAMhec-cDNA. The se~uencing was
conducted via the dideoxy-chain-termination using a
combination of subclones and specific oligonucleotides and
the align program of the Protein Identification Ressours.
The T cells for the tests werde obtained by culture of
peripheral blood lymphoyctes with phytohemagglutinin for
48h.
WO94/18988 21~ 6 ~ 3 ~ PCT~W4/00506
- 17 -
Synthesis of a cDNA library:
RNase H from Escherichia coli is used to nick the RNA in
the hybrids, leaving only small RNA primers with free 3'-
OH groups attached to the cDNA. These 3'-OH groups can
subsequently be used by DNA polymerase I to synthesize
efficiently a second strand all along the length of the
first strand (original cDNA). Double stranded
oligonucleotides called BamHI-linkers are used to add
complementary ends to blunt-ended double helical DNA. The
linkers are first ligated to both ends of the given DNA.
Then the product is treated with BamHI-endonuclease.
HindIII endonuclease is from Hemophilus influenzae. The
rest of the synthesis of the cDNA library is in accordance
to Kimmel et al. (K~mm~l, A.R. and Berger, S.L.:
Preparation of cDNA and Generation of cDNA Libraries:
Overview, Methods in Enzymology, 1987, 152:307).
The here described transcript pICAMhec which encodes a
protein of a relative molecular weight of 110 kd was
discovered in T cells induced by MS. Therefore, we
established the homogeneity of the pICAMhec transcript and
the activated pICAMhec-cDNA. The blot-hybridization of the
pICAMhec transcript with the placental genomic DNA re-
vealed a pattern indicating a single copy gen. To
establish the pathognomic role of the pICAMhec encoded
protein HEC, oligodendrocytes and Schwann cells expressing
pICAMhec were studied according to their ability to bind
Ta1 and T113 cells. Within 60 min Ta1 and T113 cells
adhered strongly to HEC, oligodendrocytes and Schwann
cells expressing pICAMhec while the lymphocytes did not
adhere to cells with a related transfection.
The sequencing of the cellular DNA clone pICAMhec and the
associated protein sequence is shown in Fig. 6. The
SUBSTITUrE SHEET (RULE 26)
WO94/18988 ~1~ 6 7 3 ~ PCT~4/00506
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- 18 -
nucleotide numbering goes from left to right, the
aminoacid sequence from right to left. The RGD-motive at
position 152 is underlined, the potential N-dependent
point of glycosilation is marked by "-CHO-". The trans-
membrane ~om~; n is stained by "-TM-". The aminoacid
sequence is numbered from the projected separation point
of the signal peptide. R~m~lnlng cysteine units are
circled. The pICAMhec-cDNA clone is build out of 1846 i
185 nucleotides. The computer analysis of 40 different
pICAMhec clones from HEC, oligodendrocytes, and Schwann
cells compared to 40 clones isolated ~rom lymphocytes
showed that under physiologic conditions the intercellular
sequence variability is less than 10%. Under patho-
physiological conditions the intercellular sequence
variability is also less than 10% but the interpatient
variability is more than 25%. As ICAM belongs to the
~am~ly of the ;mmllnoglobulins we established another com-
puter analysis and found out that the pICAMhec-cDNA clone
expresses a constant region with 1600 i 160 nucleotides
and a hypervariable region with 246 + 25 nucleotides. The
constant region has a sequence variability of less than 5%
while the hypervariable region has a variability of more
than 60%.
The predicted protein sequence has the typical
characteristics of a tr~nsm~mhrane protein, which includes
a potential signal sequence, a possible separation point a
glycine-25 and asparagine-26, and a singular 25 unit
tr~n sm~mhrane dom~ ' n which terminates in a cytoplasmatic
~om~ i n of high potential. The extracellular ~m~ i n in-
cludes 7 potentially N-linked glycosylation points. At
least this could explain the weight difference between the
deglycosylated precursor (60kd) and the endproduct (100-
125kd). The specific use of these glycosylation points
WO94/18988 ~ 3 5 PCT~4/00506
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could also explain the heterogenic molecular mass of the
plCAMhec product i.e. ICAMhec. A search of a laboratory
database cont~inlng recently published surface proteins,
however, did reveal a surprising and significant
similarity between ICAMhec, the neuronal cell adhesion
molecule (NCAM). The optimal alignment score obtained,
using the National Biomedical Research Foundation (NBRF)
Align Programm is eight standard deviation above the mean
score obtained from 500 random permutations of the
sequence. Using a database of known immtlnoglobulin related
sequences it has been shown that ICAMhec may be divided
into five ~om~;n~ (28-112, 115-206, 217-310, 312-391, and
399-477) each of which shows significant similarity with
members of the immunoglobulin. For example ~om~;n I is
similar to CD3, whereas ~omA;n III and IV align to ~om~l nS
of myelin associated glycoprotein (Holzmann B., McIntyre
B.W. and Weissman I.L., Cell 1989 56:37-46) and a
cinoembryonic antigen (Stoolman 1989). All five ~om~;n~ of
ICAMhec align with NCAM.
Example 1:
Studies on MS patients were carried out with the
assumption that heparins might be potentive immuno-
suppressors in human at concentrations of 2-5 i.U. per kg
and day. Therefore, a placebo controlled doubleblind
crossover study with a Low-Molecular-Weight-Heparin (LMWH)
(Fragmin-D, Kabi, Erlangen) was performed. Fragmin-D was
chosen because the halflife time is 5 times longer than
that of other heparins and the dilution row revealed that
its an~; infl ammatory activity is 30 times higher than
standard heparins.
The placebo controlled double blind crossover study was
conducted according to the proposals of the EG-GCP-Note
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for Guidance. 1216 MS patien~s were divided into seven
randomized groups with a male female ratio of 1:1,7 and a
mean age of 29,7 $ 6,2 years.
Table 1: Population Characteristics
Group sexratio mean age aisease Group
dura~on S~Q
m/f years T 2S years T 2S n
Whole popula~1 / 1.9 31.1 _ 6.22 6.6 _ 1.32 1216
tion
T~H 1/ 1.7 23.4 ~ 4.68 6.8 _ 1.36 323
~EXA 1/ 2.1 33.2 6.64 7.4 _ 1.48 287
METHASON
1.9 29.? ~ 5.94 5.9~ 302
T~OP~tIN
PLACEBO 1 / 2.0 38.1 ' 7.62 6.3 ' 1,26 304
Acute cases1 / 1.7 31.1 ~ 6.22 6.6 = 1.32 73
out of all
groups
L~V~ 1 / 1.~ 23.4 _ 4.68 6.8 _ 1.36 26
D~Ya- 1/ 2.1 33.2 - 6.64 7.4 - 1.48 23
meth~on
Azathiop~in1/1.9 29.7 1 5.94 5.9 ~ 1.18 24
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The control group had a male female ratio of 1:2 and a
mean age of 33,2 + 7,4 years. Each group was treated over
six months by the same physician with 5 i.U. Fragmin per
kg and day with a maximum dose of 300 i.U. Fragmin-D plus
31,2 i.U. Alprostadil per day, dexamethason plus 31,2 i.U.
Alprostadil, azathioprine 2,5 mg/kg per day plus 31,2 i.U.
Alprostadil or placebo (NaCl 9,9%) plus 31,2 i.U.
Alprostadil. The patients received one subcutaneous injec-
tion once a day and three times a day the prostavasine
intranasal. The maximum dose of 300 i.U. per day for
LMWH's was chosen to ml n;m~ ze the risk of insufficient
;mmnnocompetent heparin treatment and to avoid high doses
of heparin in patients with designated risk factors for
bleeding and to reduce the rereptor desensitization effect
of heparin doses exceeding 5 i.U. per kg and day.
Each patient had a neurologic evaluation done once a week
and a Nuclear Magnetic Resonance scan (NMR-scan) once a
month. This neurologic ex~minAtion consisted of a
neurologic status (reflexes, visus, muscular tonus,
coordination and a dyn~mnmetric evaluation of the grasp
force (Dynacheque-test)). See table 4.
CoL,cern ns this status the cr~er a were: reduced (-1
point), ~lnch~nged (0 points) or improved (+1 point). These
data were evaluated in a variance analysis.
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Table 4: variance analysis o~ the variables: reflexes,
visus, muscular tonus, coordination and Dynacheque-test
during the di~ferent treatment periods. p stands ~or the
significance niveau.
Vanables ~M~VH DEXA AZAPL4CEBO
n2 323 n-287 n-302 n-304
refl~Yes F:1,8S6 F:2,364 F:6,473F:2,751
(R) p:O,OOO p:O,069 p:O,OS 1p:O,141
~sus F:47,99 F:20,69 ~:3,630~:47,08
(V) p:O,OOO p:O,701 p:O,865p:O,965
musn~ar t~ F:8,674 F:15,69 ~:0,029~:17,93
nus (~) p:O,OOO p:O,654 p:O,865p:O,913
coor~in~o~ F:3,681 F:91,65 ~:1,130~:11,27
(C) p:O,~12 p:Q,053 p:O,042p:O,756
Dyn~he~ue- F:74,23 F:1,266 ~:1.066~:0,399
test(D) p:O,OGO p:C,268 p:~,38 p:O.~.3
In the NMR-scans number and size of the plaques were
documented. After six months the crossover took place so
that within 24 months each patient received a six month
treatment course of Fragmin-D, dexamethason, azathioprine
and placebo. The physicians treating the patients and
evaluatin~ their neurolcsical status did not know which
therapy the patients received. The medication was prepared
as an injectable solution with a volume of l ml in ampules
carrying a code, the daily injected amount was lml. In
addition we received blood samples once a week conducting
adhesion assays. After the 24 month treatment period the
initial LMWH group received a further continuous treatment
for l8 months with Fragmin-D.
Results are snown in tabie 3.
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Table 3: NMR-scans of the central nervous system.
Gr~up Numl~er of Plaques Size of Plaques
MEAN 2S mm2 2S
WHOLEPOPULATIO~ 35.6 1 14.Z 65.9- 26.4
BEFORE TRE4.TMENT
n~ 1216
6 months tre~tmPnt 17.2 ' 6.9 31.4_ 13.2
with L~IWH
after2ysLMWH 4.3 ' 1.7 7.8 2.6
n- 323
DEXAMETH~ON 6 mor 26.7_ 10.7 49.4_ 19.8
n ~ 287
AZATHIOPRIN 6 mon 23.4_ 9.4 37.8_15.1
n~ 302
PLACEBO 6 mon 32.1 _ 12.8 59.4 _ ~,~
n - 304
Change of the number and the size of sclerotic plaques in
the different groups which are found in the NMR-scans are
shown. Comparing the dexamethason and azathioprine group
to the LMWH group the NMR controlled reduction of
sclerotic patches in the LMWH group (FRAGMIN-D) is
signi~icant (p=0,002; n=1216). Compared to other st~nA~r~
drugs such as Dexamethason and Azathioprin the number of
negative side effects is negligible. Figure 4 shows the
mean therapeutic results graphically.
There were 73 acute cases of MS amongst the group of 1216
patients of example l; see table 1 and also Fig. 5. These
patients had a breething failure and needed a mechanical
breething support on admission. They were r~n~omized into
three groups and treated either with a LMWH i.e. Fragmin-
D, dexamethason or azathioprine. The only improvement
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WO94/18988 PCT~4/00506
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criteria was the possibility to reduce or turn off the
mechanical breething support.
A signi~icant therapeutic improvement in the 73 acute
cases could be ~emonstrated after treatment with LMWH as
compared to treatment with dexamethason or azathioprine (p
= O,002; n = 73). Patients treated with Fragmin-~ showed
the highest amount of reduction of vitally impairing
symptoms.
Example 2:
Adhesion assays were performed with blood samples ~rom MS
patients treated with low-molecular-weight heparin,
dexamethason, azathioprine ana a controi substance.
Adhesion assays were carried out as described above. The
results are shown in table 2.
Table 2: Pathologic adhesion patter,. cf lymphocytes/cell.
Group HEC Oligo~1Pnrirocytes Schwann' ce31s
percent per 100 cells - 2S
L~rWH n ~ 323 1.2 + 0.2 0 0
DEXA 4S.6_ 19.2 52.8_ 21.2 34.7- 13.9
METHASON
n~ 287
AZAT~OPRIN 97.5 48.8 85,9_ 45.4 69.4, 27.8
n 5 302
PLACEBO 450,6 _ 246,2 2~4.6 - 117.5 237.1 + 118.6
n= 30~1
W~OLE 148.7_ 53.6 122.1- 61.1 96.4- 38.6
POPULATION n
- 1216
He~lthy 1.4_ 0.3 0 0
Control
n - 200
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WO 94/18988 PCT/EP94/00506
- Z5 -
To determine the anti-inflammatory and anti-activation
potential of heparins versus corticosteroids and
azathioprine we preincubated Ta1 and T113 cells isolated
from the MS patients for 60min at 37C with 0,004 i.U./ml
LMWH, with 0,0014 mg/ml dexamethason or with 0,0025mg/ml
azathioprine and performed an adhesion assay on pICAMhec
expressing HEC, oligodendrocytes and Schwann cells which
showed no pathologically increased adhesion in case of
LMWH-pretreatment while the pathologic adhesion patterns
were not positivly influenced by dexamethason or
azathioprine.
Refering to table 2 the in vitro adhesion assays showed
pathognomonic patterns of lymphocyte adhesion on in vitro
cultivated HEC. According to Woodruff et al. (1980) the
physiologic adhesion rate is one lymphocyte per high
endothelial cell. We observed similar rates. Putting
pathophysiologic conditions into account such as MS the
adhesion rate varies from 2 lymphocytes per HEC to 10.000
lymphocytes per HEC depending on the state of disease and
activity.
Fig. 7 shows the typical alteration of the adhesion rate
in a case of MS. The last attack is already 15 years ago.
Fig. 8 reveals the typical picture of an acute MS attack.
By dilution row we managed to observe more than 10.000
adherent lymphocytes per HEC.
The adhesion assay was routinely performed for all pati-
ents and we observed that the assay is a highly specific
and the most sensitive tool for diagnosis of MS compared
to the Nuclear Magnetic Resonance Scan which needs
sclerotic patches of at least 5mm diameter in vivo to come
SUBSTI~ SHEET (RULE 26)
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W094/18g88 PCT~4/00506
.
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up with a diagnosis. This is due to the fact that the
adhesion assay shows changes of the adhesion pattern
im~ediately even when the patient has not got any
symptoms.
The results o~ table 2 show that the adhesion is more
efficiently reduced with LMWH compared to dexamethason
and azathioprine. Moreover, the rate o~ adhesion upon
heparin treatment is similar to that o~ healthy control
individuals.
Example 3:
There are also other diseases that have been treated with
heparins, heparinoids, proteoglycans and LWHM in
doubleblind crossover studies. In these studies 174
patients with Alzheimer's disease, 174 with GvH, 174 with
leukaemia, 174 with lym~hom~, 174 with primary biliary
cirrhosis, 173 with rheuma~ism and 173 patien.s with
neuritides (german: Neuritiden) have been treated with the
above-mentioned pharmaceuticfi~, corticosterolds and
placebo.
It turned out that results similar to those obtained by
treatment of MS patients with LWXM have been received by
treatment of rheumatism, primary biliary cirrhosis (PBC),
neuritides i~ autoagressi~e (this means if it has an
immll~ological origin), GvH and systemic carcinoma e.g.
leukaemia and lymp~m~s with the same pharmaceuticals.
Most suitable ~or the treatment of rheumatism, PBC,
neuritides and of course MS are pharm.aceuticals as Fragmin
and Pragmin-D which are both LWHM's. Fragmin-D has 2.500
i.U./ml and Fragmin has 10.000 i.U./ml. Thus, Fragmin has
a higher yield than Fragmin-D and can be cheaper in use.
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WO94/18988 - 27 - PCT~4/00506
Fragmin-P is also suitable for the treatment of the above-
mentioned diseases.
Most suitable for the treatment of GvH and systemic
carcinoma e.g. leukaemia and lymphomas is Reviparin.
The bone marrow dif~usion of Reviparin is much better than
that of Fragmin or Fragmin-D. There is a higher
concentration of a factor 103 of this pharmaceutical in the
bone marrow which is a result of the higher diffusion
(also a factor of 103).
All other LWHM's which can be bought until now vary in
concentration up to 20% . Thus, the usage for treatment is
much more intensive in time because the correct
concentration has to be determined before it can be used
to treat the patients.
1~ turned out that Fragmin and Fragmin-D is also very
suitable to treat patients with migraine and hyper-IgE-
syndrome.
To treat Crohn's disease a special medication
(application) has to be used. In this case pills with a
galenic preparation that is soluble in the small intestine
is used. Thus, it is possible to treat the disease where
it occurs with very few side effects.
