U.S. patent number 6,750,207 [Application Number 09/495,723] was granted by the patent office on 2004-06-15 for compositions for the regulation of cytokine activity.
This patent grant is currently assigned to Yeda Research and Development Co. Ltd.. Invention is credited to Liora Cahalon, Irun R. Cohen, Ofer Lider, Raanan Margalit, Oded Shoseyov.
United States Patent |
6,750,207 |
Cohen , et al. |
June 15, 2004 |
Compositions for the regulation of cytokine activity
Abstract
A pharmaceutical composition for the regulation of cytokine
activity in a subject. The composition features an effective amount
of an oligosaccharide to inhibit the cytokine activity, preferably
through inhibition of TNF-alpha activity. The oligosaccharide is
more preferably up to four saccharide units in length, and
optionally and more preferably, is N-sulfated with at least one
other sulfur moeity. The compositions are useful for the treatment
of various pathological conditions related to, caused by or
promoted through, cytokine activity.
Inventors: |
Cohen; Irun R. (Rehovot,
IL), Lider; Ofer (Rehovot, IL), Cahalon;
Liora (Givataim, IL), Shoseyov; Oded (Shimshon,
IL), Margalit; Raanan (Rehovot, IL) |
Assignee: |
Yeda Research and Development Co.
Ltd. (Rehovot, IL)
|
Family
ID: |
32475665 |
Appl.
No.: |
09/495,723 |
Filed: |
February 1, 2000 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
486127 |
Jun 7, 1995 |
6020323 |
|
|
|
436330 |
|
5861382 |
|
|
|
096739 |
Jul 23, 1993 |
|
|
|
|
974750 |
Oct 10, 1992 |
|
|
|
|
878188 |
May 1, 1992 |
|
|
|
|
Current U.S.
Class: |
514/53; 514/23;
514/54; 536/17.5; 536/21; 536/55.2 |
Current CPC
Class: |
A61K
31/70 (20130101); A61K 31/7016 (20130101); A61K
31/7024 (20130101); A61K 31/727 (20130101); C07H
11/00 (20130101) |
Current International
Class: |
A61K
31/726 (20060101); A61K 31/70 (20060101); A61K
31/727 (20060101); C07H 11/00 (20060101); A61K
031/715 (); A01N 043/04 (); C07H 015/00 () |
Field of
Search: |
;514/23,53,825,826,822,885,886,54 ;536/17.5,21,55.2 ;424/85.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0 114 589 |
|
Aug 1984 |
|
EP |
|
0 375 976 |
|
Jul 1990 |
|
EP |
|
0 394 971 |
|
Oct 1990 |
|
EP |
|
WO 88/05301 |
|
Jul 1988 |
|
WO |
|
WO 90/03791 |
|
Apr 1990 |
|
WO |
|
Other References
Asselot et al., "Heparin Fragments Regulate Collagen Phenotype And
Fibronectin In The Skin Of Genetically Diabetic Mice," Biochem.
Pharmacol. 38:895-899 (1989). .
Asselot-Chapel et al., "Biosyntheses Of Interstitial Collagens And
Fibronectin By Porcine Aorta Smooth Muscle Cells. Modulation By
Low-Molecular-Weight Heparin Fragments," Biochim. Biophys. Acta
993:240-244 (1989). .
Horvath et al., "Low Dose Heparin And Early Kidney Transplant
Function," Aust. N.Z. J. Med. 5:537-539 (1975). .
Kariya et al., "Preparation of Unsaturated Disaccharides by
Eliminative Cleavage of Heparin An Heparan Sulfate With
Heparitinases," Comp. Biochem. Physiol. 103B:473-479 (1992). .
Lider et al., "Inhibition Of T Lymphocyte Heparanase By Heparin
Prevents T Cell Migration And T Cell-Mediated Immunity," Eur. J.
Immunol. 20:493-499 (1990). .
Lider et al., "Suppression Of Experimental Autoimmune Disease And
Prolongation Of Allograft Survival By Treatment Of Animals With Low
Doses Of Heparins," J. Clin. Invest. 83:752-756 (1989). .
Naparstek et al., "Activated T Lymphocytes Produce A
Matrix-Degrading Heparan Sulphate Endoglycosidase," Nature
310:241-244 (1984). .
Psuja, "Affinity Of Binding Of Radiolabeled (.sup.125 l) Heparin
And Low Molecular Weight Heparin Fraction CY 222 To Endothelium In
Culture," Folio. Haematol. (Leipz) 114:429-436 (1987). .
Toivonen et al., "Rat Adjuvant Arthritis As A Model To Test
Potential Antirheumatic Agents," Meth. And Find. Exp. Clin.
Pharmacol. 4(6):359-363 (1982)..
|
Primary Examiner: Ponnaluri; Padmashri
Attorney, Agent or Firm: G. E. Ehrlich Ltd.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. Application Ser.
No. 08/486,127 filed Jun. 7, 1995, now U.S. Pat. No. 6,020,323,
which is a continuation of U.S. application Ser. No. 08/436,330
filed Jun. 29, 1995, the U.S. national phase of International
Application No. PCT/US93/10868 filed Nov. 9, 1993, U.S. Pat. No.
5,861,382, which is a continuation-in-part of U.S. application Ser.
No. 08/096,739 filed Jul. 23, 1993, (abandoned) which is a
continuation-in-part of U.S. application Ser. No. 07/974,750, filed
Nov. 10, 1992, (abandoned) which, in turn, is a
continuation-in-part of U.S. application Ser. No. 07/878,188, filed
May 1, 1992, (abandoned in favor of file wrapper continuation
application Ser. No. 08/384,203, filed Feb. 3, 1995, U.S. Pat. No.
5,474,987) the complete disclosures of which are incorporated by
reference herein.
Claims
What is claimed is:
1. A pharmaceutical composition for regulation of cytokine activity
in a subject comprising an amount of a substance effective to
inhibit or augment the activity of the cytokine in said subject,
said substance comprising a sulfated oligosaccharide or a
pharmaceutically acceptable salt thereof, wherein said
oligosaccharide consists of up to four saccharide units, wherein
said substance exhibits a non-zero "R" value as determined from an
in vivo bioassay that measures the relative experimental DTH
reaction of mice that have been treated with varying doses of said
substance ranging from 0 to about 2 micrograms per gram of mouse;
and a pharmaceutically acceptable carrier.
2. The composition of claim 1, wherein said oligosaccharide is
N-sulfated and wherein said oligosaccharide comprises at least one
other sulfate group.
3. The composition of claim 1, wherein said oligosaccharide
consists of from two to four saccharide units.
4. The composition of claim 1, being adapted for administration to
a subject in need thereof in low doses at weekly intervals.
5. The composition claim 1 wherein said oligosaccharide consists of
two saccharide units.
Description
1. FIELD OF THE INVENTION
The present invention relates to substances, their compositions,
and methods for the regulation of cytokine activity, for instance,
the up regulation or down regulation of Tumor Necrosis Factor alpha
(TNF-.alpha.) activity. In particular, substances and
pharmaceutically acceptable compositions are disclosed which, when
administered to a host in effective amounts, either inhibit or
augment the secretion of active TNF-.alpha. by host cells. It is
thought that the secretion of active cytokines, for example
TNF-.alpha., by the host's immune effector cells (e.g., the host's
activated macrophages) may be regulated by the methods of the
present invention.
The present invention also relates to methods for the prevention
and/or treatment of pathological processes or, conversely, the
initiation of a beneficial immune system-related response involving
the induction of cytokine production, secretion, and/or activity.
Selected compositions of the present invention comprise an
effective low dosage of a low molecular weight heparin (LMWH) to be
administered at intervals of up to between five to eight days.
Still other compositions include substances comprising carboxylated
and/or sulphate oligosaccharides in substantially purified form
obtained from a variety of primary sources including
chromatographic separation and purification of LMWHs, enzymatically
degraded heparin and enzymatically degraded extracellular matrix
(DECM).
Individually, the substances, compositions containing same, and
pharmaceutical compositions especially suited for parenteral, oral,
or topical administration, inhibit or augment TNF-.alpha. secretion
by resting T cells and/or macrophages in vitro in response to
activation by immune effector cell activators, including, but not
limited to, T cell-specific antigens, T cell mitogens, macrophage
activators, residual extracellular matrix (RECM), fibronectin,
laminin or the like. In vivo data, showing inhibition of
experimental delayed type hypersensitivity (DTH), are also
presented in further support of the in vitro results.
2. BACKGROUND OF THE INVENTION
2.1. Tumor Necrosis Factor Alpha
TNF-.alpha., a cytokine produced by monocytes (macrophages) and T
lymphocytes, is a key element in the cascade of factors that
produce the inflammatory response and has many pleiotropic effects
as a major orchestrator of disease states (Beutler, B. and Cerami,
A., Ann. Rev. Immunol. (1989) 7:625-655).
The biologic effects of TNF-.alpha. depend on its concentration and
site of production: at low concentrations, TNF-.alpha. may produce
desirable homeostatic and defense functions, but at high
concentrations, systemically or in certain tissues, TNF-.alpha. can
synergize with other cytokines, notably interleukin-1 (IL-1) to
aggravate many inflammatory responses.
The following activities have been shown to be induced by
TNF-.alpha. (together with IL-1); fever, slow-wave sleep,
hemodynamic shock, increased production of acute phase proteins,
decreased production of albumin, activation of vascular endothelial
cells, increased expression of major histocompatibility complex
(MHC) molecules, decreased lipoprotein lipase, decreased cytochrome
P450, decreased plasma zinc and iron, fibroblast proliferation,
increased synovial cell collagenase, increased cyclo-oxygenase
activity, activation of T cells and B cells, and induction of
secretion of the cytokines, TNF-.alpha. itself, IL-1, IL-6, and
IL-8. Indeed, studies have shown that the physiological effects of
these cytokines are interrelated (Philip, R: and Epstein, L. B.,
Nature (1986) 323(6083):86-89; Wallach., D. et al., J. Immunol.
(1988) 140(9):2994-2999).
How TNF-.alpha. exerts its effects is not known in detail, but many
of the effects are thought to be related to the ability of
TNF-.alpha. to stimulate cells to produce prostaglandins and
leukotrienes from arachidonic acid of the cell membrane.
TNF-.alpha., as a result of its pleiotropic effects, has been
implicated in a variety of pathologic states in many different
organs of the body. In blood vessels, TNF-.alpha. promotes
hemorrhagic shock, down regulates endothelial cell thrombomodulin
and enhances a procoagulant activity. It causes the adhesion of
white blood cells and probably of platelets to the walls of blood
vessels, and so, may promote processes leading to atherosclerosis,
as well as to vasculitis.
TNF-.alpha. activates blood cells and causes the adhesion of
neutrophils, eosinophils, monocytes/macrophages and T and B
lymphocytes. By inducing IL-6 and IL-8, TNF-.alpha. augments the
chemotaxis of inflammatory cells and their penetration into
tissues. Thus, TNF-.alpha. has a role in the tissue damage of
autoimmune diseases, allergies and graft rejection.
TNF-.alpha. has also been called cachectin because it modulates the
metabolic activities of adipocytes and contributes to the wasting
and cachexia accompanying cancer, chronic infections, chronic heart
failure, and chronic inflammation. TNF-.alpha. may also have a role
in anorexia nervosa by inhibiting appetite while enhancing wasting
of fatty tissue.
TNF-.alpha. has metabolic effects on skeletal and cardiac muscle.
It has also marked effects on the liver: it depresses albumin and
cytochrome P450 metabolism and increases production of fibrinogen,
l-acid glycoprotein and other acute phase proteins. It can also
cause necrosis of the bowel.
In the central nervous system, TNF-.alpha. crosses the blood-brain
barrier and induces fever, increased sleep and anorexia. Increased
TNF-.alpha. concentration is associated with multiple sclerosis. It
further causes adrenal hemorrhage and affects production of steroid
hormones, enhances collagenase and PGE-2 in the skin, and causes
the breakdown of bone and cartilage by activating osteoclasts.
In short, TNF-.alpha. is involved in the pathogenesis of many
undesirable inflammatory conditions in autoimmune diseases, graft
rejection, vasculitis and atherosclerosis. It may have roles in
heart failure and in the response to cancer. For these reasons,
ways have been sought to regulate the production, secretion, or
availability of active forms of TNF-.alpha. as a means to control a
variety of diseases.
The prime function of the immune system is to protect the
individual against infection by foreign invaders such as
microorganisms. It may, however, also attack the individual's own
tissues leading to pathologic states known as autoimmune diseases.
The aggressive reactions of an individual's immune system against
tissues from other individuals are the reasons behind the unwanted
rejections of transplanted organs. Hyper-reactivity of the system
against foreign substances causes allergy giving symptoms like
asthma, rhinitis and eczema.
The cells mastering these reactions are the lymphocytes, primarily
the activated T lymphocytes, and the pathologic inflammatory
response they direct depends on their ability to traffic through
blood vessel walls to and from their target tissue. Thus, reducing
the ability of lymphocytes to adhere to and penetrate through the
walls of blood vessels may prevent autoimmune attack, graft
rejection and allergy. This would represent a new therapeutic
principle likely to result in better efficacy and reduced adverse
reactions compared to the therapies used today.
Atherosclerosis and vasculitis are chronic and acute examples of
pathological vessel inflammation. Atherosclerosis involves
thickening and rigidity of the intima of the arteries leading to
coronary diseases, myocardial infarction, cerebral infarction and
peripheral vascular diseases, and represents a major cause of
morbidity and mortality in the Western world. Pathologically,
atherosclerosis develops slowly and chronically as a lesion caused
by fatty and calcareous deposits. The proliferation of fibrous
tissues leads ultimately to an acute condition producing sudden
occlusion of the lumen of the blood vessel.
TNF-.alpha. has been shown to facilitate and augment human
immunodeficiency virus (HIV) replication in vitro (Matsuyama, T. et
al., J. Virol. (1989) 63(6):2504-2509; Michihiko, S. et al., Lancet
(1989) 1(8648):1206-1207) and to stimulate HIV-1 gene expression,
thus, probably triggering the development of clinical AIDS in
individuals latently infected with HIV-1 (Okamoto, T. et al., AIDS
Res. Hum. Retroviruses (1989) 5(2):131-138).
Hence, TNF-.alpha., like the inflammatory response of which it is a
part, is a mixed blessing. Perhaps in understanding its physiologic
function, one may better understand the purpose of inflammation as
a whole and gain insight into the circumstances under which
"TNF-.alpha. deficiency" and "TNF-.alpha. excess" obtain. How best
to design a rational and specific therapeutic approach to diseases
that involve the production of this hormone may thus be closer at
hand.
2.2. Heparin
Heparin is a glycosaminoglycan, a polyanionic sulfated
polysaccharide, which is used clinically to prevent blood clotting
as an antithrombotic agent. In animal models, heparin has been
shown to reduce the ability of autoimmune T lymphocytes to reach
their target organ (Lider, O. et al., Eur. J. Immunol. (1990)
20:493-499). Heparin was also shown to suppress experimental
autoimmune diseases in rats and to prolong the allograft survival
in a model of skin transplantation in mice, when used in low doses
(5 .mu.g for mice and 20 .mu.g for rats) injected once a day
(Lider, O. et al., J. Clin. Invest. (1989) 83:752-756).
The mechanisms behind the observed effects are thought to involve
inhibition of release by T lymphocytes of enzyme(s) necessary for
penetration of the vessel wall, primarily the enzyme heparanase
that specifically attacks the glydosaminoglycan moiety of the
sub-endothelial extracellular matrix (ECM) that lines blood vessels
(Naparstek, Y. et al., Nature (1984) 310:241-243). Expression of
the heparanase enzyme is associated with the ability of autoimmune
T lymphocytes to penetrate blood vessel walls and to attack the
brain in the model disease experimental autoimmune
encephalomyelitis (EAE).
European Patent Application EP 0114589 (Folkman et al.) describes a
composition for inhibition of angiogenesis in mammals in which the
active agents consist essentially of (1) heparin or a heparin
fragment which is a hexasaccharide or larger and (2) cortisone or
hydrocortisone or the 11-.alpha. isomer of hydrocortisone.
According to the disclosure, heparin by itself or cortisone by
itself are ineffective; only the combination of both gives the
desired effects. Although there is no proof in the literature that
there is a connection between angiogenesis and autoimmune diseases,
the description on page 5 of the patent application connects
angiogenesis with psoriasis and with arthritis, indicating the use
of high doses of 25,000 units to 47,000 units of heparin per day
(i.e., about 160 to about 310 mg per day).
Horvath, J. E. et al., in Aust. N.Z.J. Med. (1975) 5(6):537-539,
describe the effect of subanticoagulant doses of subcutaneous
heparin on early renal allograft function. The daily dosage is high
(5000 U or about 33 mg) and the conclusion of the study is that
heparin in subanticoagulant doses has no effect on early graft
function or graft survival and that it may be associated with
increased hemorrhagic complications.
Toivanen, M. L. et al., Meth. and Find. Exp. Clin. Pharmacol.
(1982) 4(6):359-363, examined the effect of heparin in high dosage
(1000 U/rat or about 7 mg/rat) in the inhibition of adjuvant
arthritis in rats and found that heparin enhanced the severity of
the rat adjuvant arthritis.
PCT Patent: Application PCT/AU88/00017 published under No.
WO88/05301 (Parish et al.) describes sulphated polysaccharides that
block or inhibit endoglycosylase activity, such as heparanase
activity, for use as antimetastatic and anti-inflammatory agents.
Heparin and heparin derivatives, such as periodate oxidized,
reduced heparins, that had negligible anticoagulant activity, were
shown to have antimetastatic and anti-inflammatory activity when
used in dosages within, the range of 1.6-6.6 mg per rat daily,
administered by constant infusion (corresponding to 75-308 mg daily
for an adult human patient).
Heparin and heparan sulfate are closely related glycosaminoglycan
macromolecules. The degradation products of these polymeric
macromolecules, which are termed low molecular weight heparins
(LMWH), may have the same or greater pharmacologic effects on the
blood clotting system as the parent macromolecules. Furthermore,
because there is extensive but incomplete post-synthetic processing
of the polymer's basic disaccharide subunit, glucuronic acid and
N-acetyl glucosamine, the LMWH will be a heterogeneous mixture not
only of sizes but also of chemical compositions (See Goodman and
Gilman's The Pharmacological Basis of Therapeutics, 8th Ed.,
(Pergamon Press, New York, 1990) pp. 1313-1315. Methods to obtain
low molecular weight products from heparin, which are useful as
anticoagulants, are described in the art. These methods seek to
optimize the persistence in vivo or the extent of hemorrhagic side
effects of their products (See, for example, Alpino R. R., et al.,
U.S. Pat. No. 5,010,063; Choay, J., et al., U.S. Pat. No.
4,990,502; Lopez, L. L., et al., U.S. Pat. No. 4,981,955). Others
teach the use of affinity chromatographic methods to obtain low
molecular weight products (See, for example, Rosenberg, R. D., et
al., U.S. Pat. No. 4,539,398 and Jordan, R. E., et al., U.S. Pat.
No. 4,446,314).
Psuja, P., as reported in Folio Haematol. (Leipz), (1987)
114:429-436, studied the effect of the heterogeneity of heparins on
their interactions with cell surfaces. Psuja reported that there
are moderate affinity receptors for LMWH (D.sub.d =5.6 .mu.M) found
on cultured endothelial cells, but he determined that the upper
limit of the fraction of LMWH bound to these receptors was less
than 1% of total LMWH.
Other workers have demonstrated effects of LMWH on the metabolism
of a variety of cultured cell types. Asselot-Chapel, C., et al., in
Biochem. Pharmacol. (1989) 38:895-899 and Biochem. Biophys. Acta,
(1989) 993:240-244, report that LMWH cause cultured smooth muscle
cells to decrease the ratio of type III to type I collagen and
fibronectin synthesis. Rappaport, R. in U.S. Pat. No. 4,889,808,
teaches that LMWH can cause human diploid pulmonary fibroblasts,
cultured in the absence of serum, to respond to LMWH by increased
secretion of tissue plasminogen activator and related proteins.
Effects of LMWH on complex multicellular systems have been
reported. The work of Folkman et al. and Lider et al., in EPO
Application 0114589 and J. Clin. Invest. (1989) 83:752:756, have
been noted above. In addition, Diferrante, N., in published
International Application WO 90/03791, teaches the use of LMWH to
inhibit the reproduction of HIV in cultures of C8166 transformed
human lymphocytes (ALL). However, none of the prior art experiments
that have studied the effects of LMWH on cellular metabolism has
considered that the heterogeneity of LMWH may produce antagonistic
effects. Furthermore, none has shown or suggested a regulatory
effect on cytokine activity based on the use of substantially pure
oligosaccharide substances.
3. SUMMARY OF THE INVENTION
In the present invention, substances are disclosed which are
capable of regulating cytokine activity in a mammalian subject and
which are comprised of a carboxylated and/or sulfated
oligosaccharide in a substantially purified form. In particular,
the substance exhibits a consistent: (a) inhibitory "R" value of
about 200,000%.times.(.mu.g/gm).sup.-1 or more as determined from
an in vivo bioassay that measures the relative inhibition of
experimental DTH reactions in mice that have been treated with
varying dosages of said substance ranging from 0 to about 2
.mu.g/gm mouse; or (b) augmentative "R" value of about
0.03%.times.(pg/ml).sup.-1 or more as determined from an in vitro
bioassay that measures the relative activity of TNF-.alpha. that is
secreted by activated human CD4.sup.+ T cells in the presence of
varying concentrations of said substance from 0 to about
1.times.10.sup.7 pg/ml. Preferred substances exhibit in vivo
inhibitory "R" values selected from the group consisting of
300,000, 400,000, 500,000 and 600,000%.times.(.mu.g/gm).sup.-1 or
more.
Furthermore, the substances of the present invention having an
inhibitory effect on the secretion of active TNF-.alpha. may, in
addition, exhibit a consistent inhibitory "R" value of at least
about 0.4%.times.(pg/ml).sup.-1 as determined from an in vitro
bioassay that measures the relative activity of TNF-.alpha. that is
secreted by activated human CD4+T cells in the presence of varying
concentrations of said substance from 0 to about 1.times.10.sup.7
pg/ml.
In one embodiment of the present invention, the carbohydrate or
oligosaccharide has a molecular weight of no more than about 3000
daltons, preferably lying in the range of about 400 to about 2000
daltons, most preferably between about 400 and about 1100 daltons.
Generally, substances of the present invention which inhibit
TNF-.alpha. activity, as determined by biological assays (described
more fully, below) comprise molecules of various sugar units of
which the basic unit of activity is associated with a disaccharide.
However, larger oligosaccharide chains of up to about 10 sugar
units, containing the basic disaccharide unit of activity can also
function to inhibit TNF-.alpha. activity. On the other hand, the
substances of the present invention, which act to augment the
observed activity of TNF-.alpha., are generally of two types: (i)
relatively higher molecular weight aggregates of low molecular
weight molecules that, in a non-aggregated state, show inhibitory
activity; and (ii) disaccharide or monosaccharide subunits that
have lost sulfate groups (i.e., have experienced at least some
desulfation).
When purified these substances or the compositions that contain
them are substantially free of other substances that exert the
opposite or antagonistic effect. Thus, a substance exhibiting
inhibitory activity ("down" regulation) in a substantially purified
form would be substantially free not only of other substances, in
general, but of other substances that exhibit augmentation or
retard the inhibitory activity of the "down" regulator. The
situation would, of course, be reversed in the case of an
augmentative substance (i.e., "up" regulators), in which the
substance would be substantially free of other substances,
particularly those that "down" regulate or antagonize
augmentation.
The phrase "regulatory effect" includes both the up regulation or
down regulation of any process affecting the availability or
resulting activity in vivo or in vitro of cytokines, in general,
including IL-1, IL-6, IL-8 and, in particular, TNF-.alpha.. Thus,
compositions of the-present invention may exert a regulatory effect
on the host production of TNF-.alpha., on the host secretion of
TNF-.alpha., on the extracellular availability of TNF-.alpha., or
on the active forms of TNF-.alpha. in a host. For instance, but not
wishing to be limited by theory, the instant invention may act to
elicit the secretion of a substance, such as a protein, which may
bind to TNF-.alpha., change its conformation, and, consequently,
affect its biological activity. It is also possible that the
compositions of the present invention may, in penetrating activated
T cells or macrophages, bind to particular oligonucleotide
sequences and, thus, affect transcriptional or translational
processes that ultimately alter protein synthesis. The compositions
may also work through binding to cell surface receptors.
To simplify the following discussion, reference will be made, among
others, to the "secretion of active TNF-.alpha." or the regulation
of the "activity of TNF-.alpha." with the understanding that a much
broader meaning is to be attached to these phrases which
encompasses the actual mechanism that is responsible for or the
actual manner by which the observed augmentation or inhibition of
TNF-.alpha. activity is effected by the substances and compositions
of the present invention.
The substances of the present invention comprise a carboxylated
and/or sulfated oligosaccharide moiety that may be obtained from
natural sources, including living organisms. For example, active
substances have been isolated and purified from low molecular
weight heparin (LMWH) fractions, as well as extracellular matrices
that have been degraded by the action of an enzyme, e.g.,
heparanase derived from animals (mammals) or microorganisms
(bacteria). Yet another source of active substances is
enzyme-treated heparin (e.g., endoglycosylase-degraded
heparin).
Hence, the term "substantially purified form" means that specific
steps have been taken to remove non-active components, or
components that have an opposing effect, from the oligosaccharide
substances and to isolate the active moiety or moieties from
mixtures or supernatants, such as those obtained from enzymatic
degradation. Specifically, the substances claimed in the present
invention are obtained from a rigorous chromatographic process, in
which low pressure size-exclusion gel chromatography (i.e.,
chromatography on Sephadex columns) is but an initial step in the
purification scheme. Subsequent to the low pressure separation,
high pressure liquid chromatographic (HPLC) techniques are used to
isolate individual component oligbsaccharides. Preferably, these
steps have resulted in the purification of the individual active
substances to substantial homogeneity.
Such a preferred purification step may include, for example,
passing mixtures containing the active substance (e.g., fractions
obtained from low pressure gel chromatography) through gel
permeation HPLC or strong anion exchange (SAX) HPLC columns. Thus,
substances comprising oligosaccharides selected from the group
consisting of di-, tri-, tetra-, penta-, or hexasaccharides,
preferably disaccharides, have been observed and isolated. The
oligosaccharides of the present invention are carboxylated and/or
sulfated and are, therefore, negatively charged. Particular
embodiments of the invention preferentially include disaccharides
having three negatively charged groups. Those that exhibit a
specific inhibitory activity possess a molecular weight ranging
from about 400 to about 2000, preferably, about 400 to about
1100.
The present invention also provides a bioassay for quantifying the
effect of a test substance on the secretion of active TNF-.alpha..
The bioassay comprises the steps of preincubating human CD4.sup.+ T
cells in a medium with varying concentrations of a test substance,
adding a constant amount of an activator effective to elicit the
secretion of TNF-.alpha. by the T cells in the absence of said test
substance, collecting the medium after a sufficient period of time,
and subsequently testing the activity of the TNF-.alpha. in the
medium. Preferably, the human CD4.sup.+ T cells are obtained from
peripheral blood mononuclear leukocytes (PBL). Suitable immune
effector cell activators include, but are not limited to, T
cell-specific antigens, mitogens, macrophage activators, residual
extracellular matrix (RECM, defined in Section 4, below), laminin,
fibronectin, and the like.
The present invention relies on the specific regulatory activity of
particular substances as determined by in vitro and in vivo
bioassays described in greater detail, below. Briefly, the
substances useful in the present invention display a regulatory
(either inhibitory or augmentative) activity relating to the
induction of the secretion of active TNF-.alpha. which is dose
dependent. That is, a plot of the percent inhibition or
augmentation versus the dose (e.g., pg/ml of substance) gives rise
to a bell-shaped curve from which a maximum percent inhibition
(Inh.sub.max) or augmentation (Aug.sub.max) is readily apparent.
Thus, for every point on such a plot, a "ratio" between the percent
inhibition or augmentation and the concentration or dose can be
calculated. In the present case, a "specific regulatory activity"
or "R" value can be obtained from the ratio of the maximum percent
inhibition or augmentation (i.e., Inh.sub.max or Aug.sub.max) and
the concentration or dose of test substance which gave rise to such
maximum percent regulatory value. Furthermore, an "R" value can be
obtained for each bioassay. Hence, an "R" value can be associated
from an in vitro mouse spleen cell assay, an ex vivo mouse spleen
assay, an in vitro human PBL assay, and an in vivo assay based on
experimental DTH reaction. If no effect is observed, an "R" value
of zero is assigned.
Another object of the present invention is a method of regulating
cytokine activity in a mammalian subject comprising administering
to said subject an amount of a substance effective to inhibit or
augment the activity of a cytokine in said subject, said substance
comprising a carboxylated and/or sulfated oligosaccharide in a
substantially purified form and said substance exhibiting a
consistent: (a) non-zero inhibitory "R" value as determined from
(i) an in vitro bioassay that measures the relative activity of
TNF-.alpha. that is secreted by activated human CD4.sup.+ T cells
in the presence of varying concentrations of said substance from 0
to about 1.times.10.sup.7 pg/ml, and/or (ii) an in vivo bioassay
that measures the relative inhibition of experimental DTH reaction
in mice that have been treated with varying dosages of said
substance ranging from 0 to about 2 .mu.g/gm mouse; or (b) non-zero
augmentative "R" value as determined from an in vitro bioassay that
measures the relative activity of TNF-.alpha. that is secreted by
activated human CD4.sup.+ T cells in the presence of varying
concentrations of said substance from 0 to about 1.times.10.sup.7
pg/ml.
Yet another object of the present invention is a method of using
the active substance for the preparation of a pharmaceutical
preparation useful for the treatment of the host, which method
comprises combining the substance with a pharmaceutically
acceptable carrier to provide a unit dose, preferably of low
dosage, having an effective amount of the substance. The
pharmaceutical preparation may also comprise a stabilizing agent,
for example, protamine, in an amount sufficient to preserve a
significant, if not substantial, proportion of the initial activity
of the substance over an extended period, e.g., about 100 percent
over about 3 days. At storage temperatures below room temperature,
e.g., about -10 to about 10.degree. C., preferably 4.degree. C.,
more of the initial activity is preserved, for up to about 4
months.
Because the pharmaceutical compositions of the present invention
are contemplated for administration into humans, the pharmaceutical
compositions are preferably sterile. Sterilization is accomplished
by any means well known to those having ordinary skill in the art,
including use of sterile ingredients, heat sterilization or passage
of the composition through a sterile filter.
It should also be evident that a primary object of the present
invention is to provide a method of treating a host, such as a
mammalian subject, suffering from a medical condition the severity
of which can be affected by the activity of a cytokine in the host
comprising administering to such host an active substance
comprising the oligosaccharides of the instant invention in
substantially purified form or the pharmaceutical compositions that
can be prepared from same. Depending on the medical condition of
the particular host, substances or compositions can be administered
which either reduce the availability or activity of TNF-.alpha. or,
conversely, enhance TNF-.alpha. induction or amplify its activity.
Such compositions or pharmaceutical preparations may be
administered at low dosage levels and at intervals of up to about
5-8 days, preferably, once a week. Pharmaceutical compositions
containing oligosaccharide (e.g., mono-, di-, tri-, or
tetrasaccharides, preferably, comprising a disaccharide) substances
for parenteral, oral, or topical administration may be administered
daily according to convenience and effectiveness and at dosages
that would be readily determined by routine experimentation by one
of ordinary skill.
The present invention is also related to pharmaceutical
preparations for the prevention and/or treatment of pathological
processes involving the induction of active TNF-.alpha. secretion
comprising a pharmaceutically acceptable carrier and a low
molecular weight heparin (LMWH) present in a low effective dose for
administration at intervals of up to about 5-8 days and which LMWH
is capable of inhibiting in vitro secretion of active TNF-.alpha.
by resting T cells and/or macrophages in response to T
cell-specific antigens, mitogens, macrophage activators, residual
extracellular matrix (RECM), laminin, fibronectin, and the
like.
In a particular embodiment of the present invention the LMWH of the
pharmaceutical preparation has an average molecular weight of from
about 3,000 to about 6,000 and, furthermore, may be administered
every fifth or seventh day.
It is also an objective of the present invention to provide a
pharmaceutical preparation to be administered at intervals of up to
about 5-8 days for the prevention and/or treatment of pathological
processes involving the induction of active TNF-.alpha. secretion
comprising a pharmaceutically acceptable carrier and a low
molecular weight heparin (LMWH) present in a low effective
dose.
Active substances and compositions of the present invention are
capable of inhibiting experimental delayed type hypersensitivity
(DTH) reactions to an applied antigen as evidenced by a reduction
in the induration observed after the application of the antigen to
the skin up to about five to seven days after the administration of
the substance or pharmaceutical composition of same relative to the
induration observed after the application of the antigen to the
skin in the absence of or after recovery from the administration of
the substance or pharmaceutical composition of same. Examples of
the applied antigen include, but are not limited to, tetanus,
myelin basic protein, purified protein derivative, oxazolone, and
the like.
Furthermore, it is an objective of the present invention to provide
compositions or pharmaceutical preparations that may be
administered in any manner as dictated by the particular
application at hand including, but not limited to, enteral
administration (including oral or rectal) or parenteral
administration (including topical or inhalation with the aid of
aerosols). In preferred embodiments, the pharmaceutical
compositions of the present invention are administered orally,
subcutaneously, intramuscularly, intraperitoneally or
intravenously.
Thus, the present invention is useful, for example, in delaying or
preventing allograft rejection and treating or preventing a variety
of pathological processes such as those related to autoimmune
diseases, allergy, inflammatory diseases (in particular,
inflammatory bowel disease), or acquired immunodeficiency syndrome
(AIDS). The present invention also finds utility in the treatment
of diabetes type I, periodontal disease, skin diseases, liver
diseases, uveitis, rheumatic diseases (in particular, rheumatoid
arthritis), atherosclerosis, vasculitis, or multiple sclerosis.
Moreover, the present invention is useful, in the treatment of
tumors, viral infections and bacterial infections by administering
a substance of the invention so as to augment the'secretion of
active TNF-.alpha.. Examples of tumor treatment include, but are
not limited to, the treatment of breast, colon and prostate cancers
as well as lymphomas and other basal cell carcinomas. Bacterial
infection treatments include, but are not limited to, the treatment
of diphtheria, streptococcus, pneumonia, gonorrhea, leprosy, and
tuberculosis. Similarly, examples of viral infections which can be
treated by the invention include, but are not limited to, the
treatment of influenza, hepatitis, gastroenteritis, mononucleosis,
bronchiolitis, and meningitis.
In particular pharmaceutical compositions of the present invention,
low effective doses of the prescribed LMWH active substance are
present. Typically, the pharmaceutical composition contains a
single low dose unit of less than 5 mg of LMWH active substance,
preferably from about 0.3 to about 3 mg, and most preferably
contains a single low dose unit of from 1 to 1.5 mg.
The present invention also contemplates broadly a method of using a
low molecular weight heparin (LMWH) which is capable of inhibiting
in vitro secretion of active TNF-.alpha. by resting T cells and/or
macrophages in response to immune effector cell activators for the
preparation of a pharmaceutical preparation to be administered at
intervals of up to about 5-8 days for the prevention and/or
treatment of pathological processes involving induction of
TNF-.alpha. secretion which method comprises combining a low
effective dose of the LMWH with a pharmaceutically acceptable
carrier.
Yet another object of the present invention relates to methods for
providing sources of active substances according to the present
invention which comprise fractionating low molecular weight
heparins, enzymatically degrading intact heparin (DH), or
enzymatically degrading extracellular matrix (DECM).
A still further object of the present invention is to provide a
method of treating a subject or host suffering from a pathological
process involving induction of active TNF-.alpha. secretion
comprising administering to such subject or host a pharmaceutical
composition, as described above, at intervals of up to about 5-8
days, preferably once a week. As further described above,
pharmaceutical compositions comprising active oligosaccharide may
also be administered daily or up to weekly intervals.
The present invention also provides a pharmaceutical composition
for the inhibition of the production of active TNF-.alpha.
comprising a disaccharide of the formula (I) or its
pharmaceutically acceptable salt ##STR1##
in which X.sub.1 is hydrogen or sulfate; X.sub.2 is hydrogen or
sulfate; and X.sub.3 is sulfate or acetyl, provided that if X.sub.3
is sulfate, then at least one of X.sub.1 or X.sub.2 is sulfate and
if X.sub.3 is acetyl, then both X.sub.1 and X.sub.2 are sulfates;
and a pharmaceutically acceptable carrier. In particular, the
pharmaceutical composition may comprise a disaccharide which is
2-O-sulfate-4-deoxy-4-en-iduronic
acid-(.alpha.-1,4)-2-deoxy-2-N--
Yet another aspect of the present invention relates to a
pharmaceutical composition for augmenting the production of active
TNF-.alpha. comprising a non-sulfated N-acetylated
4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable
salt thereof and a pharmaceutically acceptable carrier.
Also contemplated by the present invention is a method of
inhibiting the production of an active cytokine in a subject
comprising administering to the subject, for example, a mammal,
such as a human patient, an effective amount of a disaccharide of
the formula (I) or its pharmaceutically acceptable salt
##STR2##
in which X.sub.1 is hydrogen or sulfate; X.sub.2 is hydrogen or
sulfate; and X.sub.3 is sulfate or acetyl, provided that if X.sub.3
is sulfate, then at least one of X.sub.1 or X.sub.2 is sulfate and
if X.sub.3 is acetyl, then both X.sub.1 and X.sub.2 are sulfates.
Another method relates to augmenting the production of an active
cytokine in a subject comprising administering to the subject an
effective amount of a disaccharide which is 4-deoxy-4-en-iduronic
acid-(.alpha.-1,4)-2-deoxy-2-N-acetylglucosamine or a
pharmaceutically acceptable salt thereof. Consistent with the
objectives of the present invention, such methods include the daily
or, preferably, weekly sulfateglucosamine, 4-deoxy-4-en-iduronic
acid-(.alpha.-1,4)-2-deoxy-2-N-sulfate-6-O-sulfateglucosamine,
2-O-sulfate-4-deoxy-4-en-iduronic
acid-(.alpha.-1,4)-2-deoxy-2-N-sulfate-6-O-sulfateglucosamine, or
2-O-sulfate-4-deoxy-4-en-iduronic
acid-(.alpha.-1,4)-2-deoxy-2-N-acetyl-6-O-sulfateglucosamine.
The present invention also contemplates a pharmaceutical
composition for augmenting the production of active TNF-.alpha.
comprising 4-deoxy-4-en-iduronic
acid-(.alpha.-1,4)-2-deoxy-2-N-acetylglucosamine or a
pharmaceutically acceptable salt thereof and a pharmaceutically
acceptable carrier. Such pharmaceutical compositions may, of
course, be adapted for various routes of administration including,
but not limited to, parenteral administration, oral administration,
or topical administration.
Furthermore, a pharmaceutical composition is provided for the
inhibition of the production of active TNF-.alpha. comprising a
compound which is an N-sulfated or N-acetylated
4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable
salt thereof. Such compound, if N-sulfated, has at least one other
sulfate group and, if N-acetylated, has at least two sulfate
groups. It should be noted that because of the unsaturation (i.e.,
the double bond at C-4 to C-5) at the "uronic" acid portion of
certain of the disaccharides of interest, there is no
stereochemistry associated with the C-6 carboxyl group that is
essentially in the plane of the six-membered ring. Hence, when the
double bond at C-4 to C-5 is present, an iduronic acid is the same
as a glucuronic acid. Consequently, the term "uronic" acid is meant
to encompass either a glucuronic or an iduronic acid. Likewise, a
"urono" group can mean either an idurono or glucurono group.
administration of the respective compounds or their
pharmaceutically acceptable salts.
The above-mentioned methods may also be utilized for inhibiting or
augmenting the production of an active cytokine in a subject
comprising administering to the subject an effective amount of the
pharmaceutical composition of the present invention.
The present invention also contemplates a method of using a
compound which is an N-sulfated or N-acetylated
4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable
salt thereof, the compound if N-sulfated having at least one other
sulfate group and the compound if N-acetylated having at least two
sulfate groups for the preparation of a pharmaceutical composition
for the prevention or treatment of a medical condition caused by or
related to the inappropriate production of TNF-.alpha..
Also contemplated is a method of using a compound which is a
non-sulfated N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a
pharmaceutically acceptable salt thereof for the preparation of a
pharmaceutical composition for the treatment of a medical condition
responsive to an increased production of TNF-.alpha..
Likewise, methods of preventing or treating a medical condition
caused by or related to the inappropriate production of an active
cytokine in a subject are also provided comprising administering to
the subject an effective amount of a compound which is an
N-sulfated or N-acetylated 4-deoxy-4-en-glucoronoglucosamine or a
pharmaceutically acceptable salt thereof, the compound if
N-sulfated having at least on other sulfate group and the compound
if N-acetylated having at least two sulfate groups. Methods of
treating a medical condition responsive to an increased production
of an active cytokine in a subject area also provided which
comprise administering to the subject an effective amount of a
compound which is a non-sulfated N-acetylated
4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable
salt thereof. Such treatments are particularly useful in cases
involving an autoimmune disease, a neoplastic condition or some
form of infection, including those induced by viral, bacterial or
fungal agents.
Other objects of the present invention concern methods of
protecting a subject from the harmful effects of exposure to
radiation comprising administering to the subject an effective
amount of a compound which is an N-sulfated or N-acetylated
4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable
salt thereof, the compound if N-sulfated having at least one other
sulfate group and the compound if N-acetylated have at least two
sulfate groups. Typically, the compounds of the present invention
are administered to the subject prior to radiation exposure. Most
advantageously, the radioprotective properties of the disclosed
compounds may be exploited during radiation therapy.
Further, methods of suppressing allograft rejection in a subject
are contemplated comprising administering to the subject an
effective amount of a compound which is an N-sulfated or
N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a
pharmaceutically acceptable salt thereof, the compound if
N-sulfated having at least one other sulfate group and the compound
if N-acetylated have at least two sulfate groups. The allograft
may, of course, include an organ transplant, including, but not
limited to, heart, liver, kidney or bone marrow transplants. The
disclosed methods may also apply to skin grafts.
Yet another object relates to a method of suppressing the
expression of an adhesion molecule in a subject comprising
administering to the subject an effective amount of a compound
which is an N-sulfated or N-acetylated
4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable
salt thereof, the compound if N-sulfated having at least one other
sulfate group and the compound if N-acetylated have at least two
sulfate groups. Examples of such adhesion molecules include, but
are not limited to, ICAM-1 or ELAM-1.
Also disclosed is an in vitro bioassay for quantifying the effect
of a test substance on the secretion of active TNF-.alpha.
comprising preincubating human CD4.sup.+ T cells in a medium with
varying concentrations of a test substance, adding a constant
amount of an activator effective to elicit the secretion of
TNF-.alpha. by the T cells in the absence of the test substance,
collecting the medium after a sufficient period of time, and
subsequently testing the activity of the TNF-.alpha. in the
medium.
Further objects of the present invention will become apparent to
those skilled in the art upon further review of the following
disclosure, including the detailed descriptions of specific
embodiments of the invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the adjuvant arthritis (AA) scores obtained from
groups of rats which were treated with weekly administrations of
Fragmin at various doses relative to a control group that received
only phosphate buffered saline (PBS).
FIG. 2 illustrates the AA scores obtained from groups of rats which
received a constant 20 microgram dose of Fragmin under various
dosage regimens, including single treatment, daily treatment, five
day intervals, and weekly.
FIG. 3 compares the effectiveness of weekly administration of
Fragmin versus Heparin and control (PBS).
FIG. 4 illustrates the results of daily administration of Fragmin,
Heparin or PBS.
FIG. 5 illustrates the AA scores obtained from groups of mice that
were treated either weekly or daily with various low molecular
weight heparins including Fraxiparin, Fraxiparine, and Lovenox.
FIG. 6 plots the percentage of survival rate of rats that had
undergone allogeneic heart transplants and had also received either
weekly administration of Fragmin or PBS.
FIG. 7 presents bar graphs illustrating the blood glucose levels of
two groups of NOD mice, one group receiving Fragmin and the other
receiving only PBS.
FIG. 8 illustrates the results of a DTH experiment involving a
human volunteer.
FIG. 9 illustrates the "bell-shaped" dose to response curve
exhibited by active Fragmin.
FIG. 10 illustrates the loss of inhibitory activity displayed by
inactivated Fragmin.
FIG. 11 shows the absorption at 206 nanometers of various fractions
obtained from the gel filtration of inactivated Fragmin, including
fractions F2, F8, F10 and F15.
FIGS. 12A, 12B and 12C illustrate the effects of active Fragmin,
fraction F15, and fraction F10. respectively, at various doses on
the sensitivity of mice to the DTH reaction.
FIG. 14 illustrates the absorption at 206 nanometers versus
fraction number for a number of fractions obtained from the
Sepharose 4B column separation of Fragmin and heparanase-degraded
ECM.
FIGS. 13 and 15 compare the elution profiles of fractions obtained
from the Sepharose 4B column separation of Fragmin and
heparanase-degraded ECM.
FIG. 16 shows that an oligosaccharide product (fraction 5 from FIG.
13) demonstrated a similar bell-shaped dose/response curve in its
ability to inhibit the secretion of active TNF-.alpha..
FIG. 17 shows that the areas of greatest anti-TNF-.alpha. effect
lie in the subfraction between about 5.65 and about 5.8.
FIGS. 18A and 18B illustrate the chromatogram obtained from the
HPLC separation of Fragmin and heparanase-degraded ECM,
respectively.
FIG. 19 illustrates the absorption at 206 nanometers of two
fractions, F5 and F8, obtained from the Sepharose 4B column
separation of heparanase-degraded ECM.
FIGS. 20A and 20B, on the other hand, illustrate the absorption at
206 and 232 nanometers, respectively, of a peak obtained from the
HPLC separation of fraction F5.
FIGS. 21A and 21B illustrate the uv absorption of additional HPLC
fractions obtained from fraction F5.
FIG. 22 illustrates the uv absorption of fractions F7 and F8
obtained from the Sepharose 4B column separation of
heparanase-degraded ECM.
FIG. 23 illustrates the substantially pure peak obtained from the
SAX-HPLC chromatography of combined fractions F7 and F8.
FIG. 24 illustrates another peak labeled "A23/4" obtained from
desalted preparations of the peak labeled "1" from FIG. 23.
FIGS. 25A, 25B and 25C illustrate the chromatograms that are
obtained from the SAX-HPLC column separation of disaccharide
standards obtained from Sigma labeled H-0895, H-1020 and H-9267,
respectively.
FIG. 26 illustrates the Sepharose 4B column separation of a mixture
obtained from the heparanase (MM 5) treatment of Heparin, yielding
fractions F7 and F8.
FIG. 27 illustrates the absorption at 206 nanometers of various
fractions obtained from the Sepharose 4B chromatography of PC3
heparanase alone and Heparin+PC3.
FIGS. 28A and 28B illustrate additional fractions obtained from the
HPLC separation of fraction F7 from FIG. 26.
FIGS. 29A and B illustrates fraction F90 obtained from the HPLC
separation of Fragmin.
FIG. 30, on the other hand, illustrates the chromatogram that is
obtained from a SAX-HPLC separation of an aged sample of A23/4.
FIG. 31 illustrates the proton NMR spectrum of a 20 microgram
sample of an ECM-derived disaccharide obtained from HPLC
chromatography, as shown in FIG. 23.
FIG. 32 illustrates a two-dimensional COSY spectrum of the sample
of FIG. 31.
FIG. 33 illustrates an expanded portion of the NMR spectrum of FIG.
31, showing the signal for the anomeric proton.
FIGS. 34 and 35 illustrate the FTIR spectra obtained from two
separate samples, one indicating the presence of a sulfated
compound (FIG. 34) and the other indicating the presence of a
partially desulfated analog (FIG. 35).
FIG. 36A illustrates the mass spectrum of a methylated derivative
of the sample obtained from FIG. 23 in a solvent matrix comprised
of DTT:thioglycerol (1:1).
FIG. 36B illustrates the mass spectrum of the solvent matrix
only.
FIGS. 37A and 37B illustrate the mass spectrum of the same sample
in a different solvent matrix comprised of methylnitrobenzyl
alcohol, FIG. 37A being the mass spectrum of the sample plus the
solvent matrix and FIG. 37B being the mass spectrum of the solvent
matrix only.
FIG. 38A illustrates the results of experiments comparing the
effectiveness of disaccharide 9392 and 1020 to improve the AA
scores of female Lewis rats suffering from experimentally induced
adjuvant arthiritis.
FIG. 38B illustrates the effect of disaccharide 0895 on the AA
scores of rats suffering from experimentally induced AA relative to
control (PBS).
FIG. 38C illustrates the effects of glucosamine treatment in the
improvement of the AA score of Lewis rats under various dosages of
glucosamine.
FIG. 38D, similarly, shows the effect of galactosamine at different
dosages on the AA score of Lewis rats.
FIGS. 38E and 38F illustrate the results of further experiments
carried out with disaccharide 9392 in which disaccharide is
administered either weekly or daily beginning at day zero (i.e.,
start of induction of AA) or at day 12 (i.e., when the rat is
already suffering from AA).
FIGS. 38G and 38H illustrate the results of a separate comparative
set of experiments that were carried out on groups of Lewis rats to
determine the effectiveness of disaccharide 9392 administered
weekly compared with the effectiveness of a known anti-inflammatory
agent, dexamethasone phosphate, on the suppression of
experimentally induced adjuvant arthritis.
FIG. 39A illustrates the effectiveness of subcutaneously injected
disaccharide 1020 against liposaccharide (LPS) induced inflammation
of rat corneas.
FIG. 39B presents the results of experiments relating to the
radioprotective effects of glucosamine at various dosages relative
to control (PBS).
FIG. 39C presents the results of similar irradiation experiments
involving the administration of disaccharide 9392 at various
dosages relative to control (PBS).
FIGS. 40A and 40B illustrate the results of experiments that
illustrate the ability of selected substances of the present
invention to suppress allograft rejection. The results presented in
FIG. 40A show that a 3 nanogram dose of disaccharide 9392 by
subcutaneous injection one day before grafting and weekly
thereafter, delayed the level of skin graft rejection at 50% by 5
days. However, a 300 nanogram dose of the same disaccharide failed
to produce a significant difference at 50% rejection relative to
control (PBS).
FIG. 41A illustrates the incidence of IDDM in groups of female NOD
mice which had been separately treated with either disaccharide
9392, glucosamine or saline.
FIG. 41B presents the mortality rate of female NOD mice that,
again, had been treated separately with the disaccharide 9392
glucosamine, or saline. It should be noted that in both FIGS. 41A
and 41B, the female NOD mice were approximately 31/2 months old,
meaning that the mice as a group already endured a 20% incidence of
IDDM.
FIG. 42 presents the respiratory distress (RD) score of six
immunized rats challenged with aerosolized antigen with (B1, B2 and
B3) and without (A1, A2 and A3) treatment by substance H-9392. See
text for details.
FIG. 43 represents the structure of
4-O-(2-deoxy-6-O-sulfo-2-sulfoamino-.alpha.-D-glucopyranosyl)-(2-O-sulfo-.
beta.-D-glucopyranoside)uronic acid, prepared by the synthetic
scheme of Example 6.23.
4. DETAILED DESCRIPTION OF THE INVENTION
In one aspect of the present invention, it was found that treatment
with low molecular weight heparins (LMWHs) inhibited the ability of
T cells and macrophages to secrete active TNF-.alpha.. In another
aspect of the present invention, other substances, comprising
carboxylated and/or sulfated oligosaccharides in substantially
purified form, are described which collectively represent a means
for regulating the biological activity of cytokines, such as
TNF-.alpha., in a host. For simplicity, the term "substance(s)" or
"active substance(s)" will be used to denote LMWHs, as used in the
method of treatment disclosed herein, as well as the substances
comprised of carboxylated and/or sulfated oligosaccharides that
have been isolated herein in substantially pure form: unless
otherwise noted.
One functional expression of this effect can be seen in the
inhibition in mice and humans of the delayed type hypersensitivity
(DTH) reaction, a T cell dependent inflammatory reaction that may
also be triggered by cells involving macrophages and other
inflammatory cells. Treatment with the active substances at doses
affecting active TNF-.alpha. production also was able to inhibit a
model of autoimmune arthritis called adjuvant arthritis (AA).
Active substance treatment also prolonged the survival of
allogeneic heart transplants in rats and abrogated insulin
dependent diabetes mellitus (IDDM) in NOD mice. Moreover, similar
treatment prevented the induction of active TNF-.alpha. production
by T cells and macrophages in response to the stimulus of damaged
or residual subendothelial extracellular matrix. This residual
extracellular matrix (RECM) that is responsible for signaling the
onset of TNF-.alpha. induction (and resulting inflammation) is to
be distinguished from the enzyme degraded extracellular matrix
(DECM), selected components of which have been isolated herein and
have been shown to either shut down TNF-.alpha. activity or amplify
it.
Since TNF-.alpha. at the site of vascular injury probably has a
role in the process of atherosclerosis, inhibition of TNF-.alpha.
activity at the site of damaged subendothelial ECM will ameliorate
the pathogenic process of atherosclerosis. A most surprising aspect
of treatment with the LMWH active substances is that such treatment
is most effective when administered at low doses at weekly
intervals. High doses of the LMWH active substances or doses of the
LMWH active substances given daily are not effective in inhibiting
TNF-.alpha. secretion or immune reactions.
Low molecular weight heparins, produced by fractionation or
controlled depolymerization of heparins, show improved
antithrombotic performance but also different pharmacokinetic
properties as compared to heparin: the half-life is doubled and the
bioavailability is higher with respect to their anticoagulant
effect after subcutaneous injection (Bratt, G. et al., Thrombosis
and Haemostasis (1985) 53:208; Bone, B. et al., Thrombosis Research
(1987) 46:845).
According to the present invention it has now been found that the
LMWH active substances administered at subanticoagulant doses at
several day intervals are effective in the prevention and/or
treatment of pathological processes involving induction of active
TNF-.alpha.. Moreover, it has now been found that discrete
substances, comprising an oligosaccharide of from 1-10 sugar units,
preferably 2-4 sugar units, can be identified which can either
inhibit or augment the activity of TNF-.alpha.. These discrete
substances can be obtained, for example, from the tissue of a
living organism, for instance, from the soluble degradation
products of substrate extracellular matrix.
4.1. Sources of Active Substances
The LMWHs to be used according to the invention are derived from
LMWHs with an average molecular weight of 3000-6000, such as, for
example the LMWHs disclosed in European Patent EP 0014184. Some
LMWHs are commercially available under different trade names, e.g.
FRAGMIN.RTM., FRAXIPARINE.RTM., FRAXIPARINE.RTM.,
LOVENOX.RTM./CLEXANE.RTM..
LMWHs can be produced in several different ways: enrichment by
fractionation by ethanol and/or molecular sieving, e.g., gel
filtration or membrane filtration of the LMWH present in standard
heparin and controlled chemical (by nitrous acid,
.beta.-elimination or periodate oxidation) or enzymatic (by
heparinases) depolymerization. The conditions for depolymerization
can be carefully controlled to yield products of desired molecular
weights. Nitrous acid depolymerization is commonly used. Also
employed is depolymerization of the benzylic ester of heparin by
.beta.-elimination, which yields the same type of fragments as
enzymatic depolymerization using heparinases. LMWH with low
anticoagulant activity and retaining basic chemical structure can
be prepared by depolymerization using periodate-oxidation or by
removing the antithrombin-binding fraction of LMWH, prepared by
other methods, using immobilized antithrombin for adsorption.
FRAGMIN.RTM. is a low molecular weight heparin with average
molecular weight within the range of 4000-6000 dalton, produced by
controlled nitrous acid depolymerization of sodium heparin from
porcine intestinal mucosa. It is manufactured by Kabi Pharmacia,
Sweden, under the name FRAGMIN.RTM., for use as an antithrombotic
agent as saline solutions for injection in single dose syringes of
2500 IU/0.2 ml and 5000 IU/0.2 ml, corresponding to about 16 mg and
32 mg, respectively.
FRAXIPARIN.RTM., and FRAXIPARINE.RTM. are LMWHs with average
molecular weight of approximately 4500 dalton, produced by
fractionation or controlled nitrous acid depolymerization,
respectively, of calcium heparin from porcine intestinal mucosa. It
is manufactured by Sanofi (Choay Laboratories) for use as an
antithrombotic agent in single doses comprising ca. 36 mg,
corresponding to 3075 IU/0.3 ml of water.
LOVENOX.RTM. (Enoxaparin/e), a LMWH fragment produced by
depolymerization of sodium heparin from porcine intestinal mucosa,
using .beta.-elimination, is manufactured by Pharmuka SF, France
and distributed by Rhone-Poulenc under the names CLEXANE.RTM. and
LOVENOX.RTM. for use as antithrombotic agent in single dose
syringes comprising 20 mg/0.2 ml and 40 mg/0.4 ml of water.
As shown in the present application, the novel properties of LMWHs
that have been discovered and are described herein are common to
all LMWHs regardless of the manufacturing process, the structural
differences (created by depolymerization or those dependent on
variation in the heparin used as raw material) or the anticoagulant
activity, provided that the LMWH employed is capable of inhibiting
active TNF-.alpha. secretion in vitro by resting T cells and/or
macrophages in response to activation by contact with T
cell-specific antigens, mitogens, macrophage activators, residual
ECM or its protein components, such as fibronectin, laminin, or the
like.
Another test useful for identifying the LMWHs that are effective
for the purpose of the present invention is the inhibition of
experimental delayed type hypersensitivity (DTH) skin reactions, a
T lymphocyte dependent reaction, to a variety of antigens (for
example, tetanus antigen, myelin basic protein (MBP), purified
protein derivative (PPD), and oxazolone). The LMWHs also inhibit T
cell adhesion to ECM and its protein components.
The LMWHs effective according to the invention are incorporated
into pharmaceutical compositions, for example, as water solutions,
possibly comprising sodium chloride, stabilizers and other suitable
non-active ingredients. The preferable way of administration is by
injection, subcutaneous or intravenous, but any other suitable mode
of administration is encompassed by the invention, including oral
administration.
According to the invention, the LMWH is to be administered at
intervals of up to about five to eight days, preferably once a
week. The other substances of the present invention, particularly
the lower molecular weight (below 2000) oligosaccharides, may be
administered in any convenient, effective manner (e.g., by
injection, orally, or topically) at dosage regimens that may
include daily or weekly administration.
4.2. Loss of Activity of LMWH Preparations Over Time. Influence of
Added Stabilizer
Time course studies conducted by the inventors demonstrate that
LMWH samples, such as FRAGMIN, lose their ability to inhibit the
activity of TNF-.alpha. within 72 h at ambient temperature and
within a few months at low temperature (e.g., 4.degree. C.).
Table XI, Section 6.1, below, indicates that about 53% of activity
of FRAGMIN.RTM. is lost after a day at ambient temperature. After
about two days, about 87% of activity is lost, and after about
three days, no activity is shown. As shown in Table XII, Section
6.2, below, experiments have shown that FRAGMIN.RTM. loses its
anti-DTH reactivity even at colder temperatures (4.degree. C.); the
process only requires more time. Conventional non-fractionated
heparins, in contrast, do not lose their classic anti-coagulant
activities at 4.degree. C.
In an effort to discover an agent capable of stabilizing or
preserving the cytokine inhibitory activity of the disclosed LMWH
preparations, the inventors turned to a well known heparin
additive. Protamine sulfate is known to neutralize the
anti-coagulant effects of heparinoid molecules and is used
clinically for that purpose (See, Goodman and Gilman's The
Pharmacological Basis of Therapeutics, Eighth Edition, Pergamon
Press, New York, 1990, p. 1317). It has been discovered, however,
according to the invention that added protamine sulfate does not
neutralize the inhibition of TNF-.alpha.-dependent activity by
LMWH; in fact, protamine sulfate actually stabilizes this activity
(See, Entries in Table XII, below, containing added protamine
sulfate).
In summary, one can conclude that (i) diluted LMWH solutions lose
activity quickly at 20.degree. C. and more slowly at 4.degree. C.
(it should be noted that the activity loss at 4.degree. C. is not a
feature of the standard anti-coagulant and anti-thrombotic
activities of heparin or LMWH); (ii) added protamine sulfate, the
classic neutralizer of the standard activities of heparins, does
not interfere with the novel activity of LMWHs against TNF-.alpha.
described in the present disclosure. Indeed, the inventors have
demonstrated that protamine sulfate actually preserves this novel
activity.
4.3. Fractionation of LMWH and Preparation of Degraded ECM or
Degraded Heparin, Discovery of Distinct Augmentative and Inhibitory
Activities for and Against TNF-.alpha. Activity
As already discussed above, Low Molecular Weight Heparin (Fragmin)
inhibits secretion of active TNF-a, Maximal inhibition or
Inh.sub.max (90%), was observed at a concentration of 1 pg/ml.
(See, FIG. 9). By contrast, inactivated Fragmin had no effect on
TNF-a production (See, FIG. 10). However, fractionation of the
inactivated material by low-pressure size-exclusion gel
chromatographic separation using a Sepharose 4B solid support (See,
FIG. 11 for a plot of the absorbance at 206 nm versus fraction
number) revealed active fractions of both inhibitory (F-15) and
augmentative (F8, F2) effects (See, FIG. 11 and Table XIII), The
inhibitory fraction of the inactivated Fragmin (F-15) also
inhibited the DTH reaction (See, FIGS. 12A; relating to active
Fragmins, 12B; relating to fraction F15, and 12C; relating to
fraction F10). Fraction F10 had no effect on TNF-aproduction or DTH
reactivity.
A SEPHAROSE 4B size-exclusion gel chromatographic separation was
also carried out on the degradation products obtained from
heparanase-treated ECM labeled with .sup.35 S-containing sulfate
groups. Several types of heparanase enzyme were used in the present
investigation. These enzymes include MM5 (Mammalian heparanase from
human placentas, obtained commercially from Rad-Chemicals, Weizmann
Industrial Park, Ness Ziona, Israel), PC3 (Bacterial
endoglycosidase, as described in Shoseiov, O. et al., Biochem.
Biophys. Res. Commun. (1990) 169:667-672), and an enzyme from a
bacterial source obtained from IBEX Technologies, Quebec, Canada. A
plot of the radioactivity (CPM) versus fraction number is presented
in FIG. 13. Another plot superimposing the elution profiles of
fractionated Fragmin and fractionated ECM-heparanase is shown in
FIG. 14. The conditions for the Sepharose 4B low-pressure
separation are listed in Table I, below.
TABLE I Sepharose 4B Chromatography Conditions Column: Sepharose 4B
(35 cm .times. 0.7 cm ID) Load: 1-1.5 ml Flow: 5 ml/hr Solvent: PBS
(pH = 7.4) Fraction: 0.2-0.5 ml/tube Detector Absorption Setting:
206 nm, 280 nm
The various fractions were assayed for their effect on TNF-.alpha.
production and these results are presented in Table XV, below.
Interestingly, fractions of similar elution properties from the two
sources (i.e., F-39 and F-42 from Fragmin and Heparanase-degraded
ECM) were found to have similar qualitative biological effects on
TNF-.alpha. production and/or activity.
FIGS. 15 and 13 illustrate one way of presenting the elution
profile, obtained on Sepharose 4B columns, of LMWH (Fragmin) and
.sup.35 S-sulfate labeled oligosaccharides of ECM, produced by
purified MM5 heparanase, respectively. It can be seen in FIG. 13
that the heparan sulfate of the ECM (the substrate of heparanase)
is degraded by the enzyme to produce heparan sulfate fragments with
elution properties comparable to fractionated LMWH.
FIG. 16 shows that an oligosaccharide product (Sepharose 4B
fraction #5, FIG. 13) obtained from the ECM+heparanase "soup"
(i.e., the mixture obtained from the heparanase degradation of
ECM), has a substantially similar dose/response characteristic as
LMHW in its effects on the secretion of active TNF-.alpha.: that
is, both display a bell-shaped dose/response curve and both exhibit
maximal inhibition of about 90% at a concentration of about 1 pg/ml
with less activity at either lower or higher concentrations. It is
advantageous, thus, that the administration of these active
substances includes dosages falling within an easily determined
"window" of physiological effect.
FIG. 17 shows that the anti-TNF-.alpha. effect of the
ECM-degradation products is highest in the area of a subfraction
(between about 5.65 and about 5.80) of the fragments under peak
number 5 of FIG. 13.
Thus, heparan sulfate can be acted upon by heparanase to generate
degradation products that, like LMWH, feed back on the T cells and
macrophages to shut off active TNF-.alpha. production and,
consequently, TNF-mediated inflammation.
It has also been discovered that low-molecular weight
oligosaccharide fragments, obtained from endoglycosylase treatment
of intact heparin, exhibit the desired regulatory effect over
TNF-.alpha. activity.
4.4. HPLC Separation of LMWH Fractions and Fragments Obtained from
DECK and DH
High performance liquid chromatography ("HPLC") techniques were
utilized to obtain better resolution of the fractions from the LMWH
(e.g., Fragmin), ECM-degradation, and heparin-degradation samples.
Initially, two types of HPLC conditions were used. Under the first
set of HPLC conditions, a ##STR3##
number of individual fractions were separated and isolated; their
ability to regulate the secretion of active TNF-.alpha. was then
examined. To the great surprise of the present inventors, it was
discovered that selected fractions can augment the activity of
TNF-.alpha. in the host while others inhibited TNF-.alpha.
activity. A second set of HPLC conditions was then utilized to
better separate the various components according to their molecular
weight.
In the first set of HPLC conditions, a TSK-GEL.RTM. G-Oligo-PW
column (30 cm.times.7.8 mm I.D.) equipped with a Guardcolumn Oligo
(4 cm.times.6 mm I.D.) was used. The conditions ("HPLC I") are
provided in Table II, below. A representative chromatogram for the
HPLC I separation of Fragmin and ECM+MM5 Heparanase is illustrated
in FIGS. 18A and 18B, respectively.
TABLE II HPLC I Chromatography Conditions Column: TSK-GEL
G-Oligo-PW 30 cm .times. 7.8 mm ID Guard Column: Guardcolumn Oligo
4 cm .times. 6.0 min ID Loop: 200 .mu.l Flow: 0.5 ml/min. Solvent:
0.2 M phosphate buffer (pH = 7.0) Fraction: 0.5 ml/tube Detector
Absorption 190 nm-400 nm Setting:
The second set of HPLC conditions ("HPLC II") are described in
Table III, below, and utilized conditions similar to those
described by Rice, K. G. et. al. in Analytical Biochem. (1985)
150:325-331. Hence, two columns connected in series were used: a
Toyo Soda TSK-Gel G3000SW (7.5 mm.times.50 cm) column connected to
a G2000SW (7.5 mm.times.50 cm) column. These columns, together with
a 7.5 mm.times.10 cm guard column attached to the inlet end of the
G2000 column, were obtained from Phenomenex. Further experimental
details are described in Sections 6.11, 6.14 and 6.15, below.
TABLE III HPLC II Chromatography Conditions Column: Toyo Soda
TSK-GEL G3000SW (50 cm .times. 7.5 mm ID) and a G2000SW (50 cm
.times. 7.5 mm ID) in series Guard Column: Guardcolumn (10 cm
.times. 7.5 mm ID) Loop: 20 or 100 .mu.l Flow: 1 ml/min. Solvent:
degassed 0.5 M NaCl Fraction: 0.5 ml/tube Detector Absorption 205
nm, 232 mn Setting:
Under these conditions, smaller substances are retained longer than
larger molecules.
In yet another set of HPLC conditions ("HPLC III"), the purity of
selected desalted HPLC fractions was examined with the aid of a
strong anion exchange (SAX) HPLC column. Such SAX HPLC columns are
known to separate similarly sized molecules according to the number
of negatively charged groups which are present in the molecules.
The greater the number of negatively charged groups in a substance,
the longer it is retained in the column. The HPLC III conditions
are outlined in Table IV, below.
TABLE IV HPLC III Chromatography Conditions Column: SAX-HPLC column
(25 cm .times. 4.6 mm ID, packed with Spherisorb, 5 .mu.m particle
size) Loop: 1 ml Flow: 1.5 ml/min. Solvent: linear gradient, below
Fraction: 1 ml/tube Detector Absorption 205 nm, 232 nm Setting:
Linear Gradient (See, Section 6.15, below)
It will also be apparent to one of ordinary skill in the art, after
considering the disclosure presented herein, that other HPLC
conditions can be contemplated and applied to the separation and
purification of the active substances of the present invention. In
particular, reverse-phase conditions can also be utilized to good
advantage. See, for example, Rice, K. G. et al., supra.
Again, without wishing to be limited by theory, it is suspected
that the activity of TNF-.alpha. is augmented by either increasing
the intracellular production of active TNF-.alpha., increasing the
amount of active TNF-.alpha. secreted by the host's immune effector
cells, or enhancing the activity of the cytokine through the action
of an agonist.
It also follows that the biological activity of TNF-.alpha. may be
inhibited by converse processes, including not only competition
offered by the active inhibitory substance for the receptors of
TNF-.alpha. (e.g., the inhibitory substance acting as or inducing
the production of another substance that acts as an antagonist of
TNF-.alpha.) but also the formation of a complex of TNF-.alpha. and
the inhibitory substance which is less active than free
TNF-.alpha.. Alternatively, it follows that a "souped-up" complex
between TNF-.alpha. and the augmentative substance may be
responsible for the observed increase in the activity of
TNF-.alpha..
4.5. Determination of Activity
The active substances of the present invention, both those able to
inhibit TNF-.alpha. activity and those able to augment TNF-.alpha.
activity, have been isolated and purified from mixtures containing
them. In some cases, these active substances have been purified to
substantial homogeneity by the powerful HPLC techniques described
herein.
As a further indication of the purity of these active substances,
the specific regulatory activities of the various substances were
determined.
Initially, however, a carbazole assay, performed in a manner
similar to that disclosed by Carney, S. L. in Proteoglycan
Analysis, A Practical Approach, Chaplin, M. F. and Kennedy, J. F.
(Eds.) IRL Press, Oxford, Washington, D.C. (1986) p. 129, was
utilized to determine the amount of oligosaccharide material
present (e.g., amount of sugar present) in a given test sample.
Picogram (pg) quantities of sugar can be quantified in this manner.
The assay is performed as described in Section 5, below.
Next, the apparent activity associated with that quantity of
substance is determined by one of the biological assays that are
described in great detail in Section 5, below, to provide a
dose/response profile. These bioassays may either be carried out in
vitro or under in vivo conditions.
It has, thus, been found-that the observed inhibition or
augmentation of TNF-.alpha. activity, expressed as a percentage of
the activity of TNF-.alpha. observed in the absence of the
substances of the present invention, depends on the concentration
or dose of such substance present in the test sample. The apparent
activity profile that results is approximately bell-shaped as
illustrated in FIGS. 9 and 16. The maximum value of percent
inhibition or augmentation observed for each substance is
designated Inh.sub.max or Aug.sub.max, as the case may be.
As described further, below, the bioassay used to establish the
"ideal" unit dose (i.e., the one that corresponds to Inh.sub.max or
Aug.sub.max) can be based on the in vitro or in vivo inhibition or
augmentation of the activity of TNF-.alpha. or DTH assay in
mice.
Alternatively, an in vitro assay based in human cells (described
further, below) may also be used. The specific regulatory activity
or "R" value is, as defined herein, the ratio of the Inh.sub.max or
Aug.sub.max and the "ideal" dose that gave rise to that maximum
percent inhibition or augmentation. For the in vitro assays, the
"R" values are typically expressed in units of
%.times.(pg/ml).sup.-1.
As stated above, the specific regulatory activity can also be
established under in vivo conditions by monitoring the inhibition
of experimental DTH reaction in mice or humans. It was found that
the ability of a particular dose of an inhibitory composition to
inhibit secretion of active TNF-.alpha. is positively correlated
with its ability to inhibit the delayed type hypersensitivity (DTH)
reaction, although the same composition may be more potent under
one assay versus another (i.e., between in vitro and in vivo
bioassays). Inhibitory or augmenting activity in this in vivo
cell-mediated inflammatory reaction is of great importance because
the DTH reaction is an expression of the processes involved in
autoimmune diseases, graft rejection, some types of blood vessel
inflammation and allergy. Thus, activity in this test is indicative
of utility in these types of diseases and possibly others, as
described further below.
Moreover, the new quantity, the specific regulatory activity, which
is defined as the ratio between the Inh.sub.max or Aug.sub.max
value and the amount or concentration of substance (the "ideal"
dose) which gave rise to that maximum percent value, can serve to
distinguish the novel active substances of the present invention
from those substances that may have been known, but unrecognized in
the art as possessing the cytokine regulatory activity disclosed
herein. This specific ratio is referred to herein as the "R" value,
for short. Hence, the novel substances or compositions of the
present invention can be described in terms of a minimum "R" value,
which can be calculated from the apparent activity versus dose
profile, and which "R" value will exceed the "R" value that can be
associated, by reference to the teachings of the present
disclosure, with known compositions.
4.6. Types of Disorders that May Benefit from the Present
Invention
The disorders that can be prevented or treated according to the
invention are all disorders linked to pathological processes
involving induction of active TNF-.alpha. secretion, including
atherosclerosis and vasculitis and pathological processes related
thereto; autoimmune diseases, e.g., rheumatoid arthritis, diabetes
type I (insulin-dependent diabetes mellitus or IDDM), multiple
sclerosis, lupus erythematosus, Graves disease; allergy; graft
rejection; acute and chronic inflammatory diseases, e.g. uveitis,
bowel inflammation; anorexia nervosa; hemorrhagic shock caused by
septicemia, and HIV infection in AIDS. In AIDS, the active
substances will suppress replication of HIV thereby preventing the
development of AIDS-related complex (ARC). Other disorders that may
benefit from a treatment designed to regulate cytokine activity
include, but are not limited to, psoriasis, pemphigus, asthma,
renal diseases, liver diseases, bone marrow failure, vitiligo,
alopecia, and myositis.
Further, augmentation of active TNF-.alpha. is useful in the
treatment of tumors, bacterial infections and viral infections.
Parenteral, oral or topical administration of the substances of the
present invention which augment the production of active
TNF-.alpha. in a pharmaceutically acceptable carrier may also help
combat skin cancer, such as basal cell cancer, squamous cell
cancer, or melanoma.
In the clinical application of the active substances of the present
invention, it should be kept in mind that the successful treatment
of certain types of disease consists, in large part, in the
restoration of homeostasis. To the endocrinologist, this implies
the judicious administration or antagonism of specific hormones.
For example, an insulin-dependent diabetic may be effectively
treated by insulin replacement therapy; a patient with. Graves'
disease may be helped by pharmacological measures that inhibit
thyroxine release. Only rarely can disease be alleviated by
administration of hormones that were never deficient to begin
with.
The use of cytokines, such as TNF-.alpha., as antineoplastic agents
provides one such instance. The rationale for administration of
immunomodulatory agents to cancer patients may be quite slender.
Many cytokines, like TNF-.alpha., exhibit toxicities that prove
dose-limiting long before a therapeutic goal is achieved. In such
an event, the augmentation of the activity of endogenously produced
TNF-.alpha. may provide an approach that is both novel and,
eventually, prove more effective than any previously contemplated
therapeutic regimen.
Clearly, our understanding of the role for TNF-.alpha. is still
evolving and, doubtless, new and useful uses of the hormone and the
substances able to regulate its activity will be uncovered. While
it goes without saying that all uses of the claimed compositions
and pharmaceutical preparations are within the scope of the present
invention, those uses that either alleviate the symptoms of
disease, prevent the onset of disease, or provide a cure for the
disease are especially contemplated.
4.7. Topical Applications of the Oligosaccharide Substances of the
Present Invention
The substances of the present invention also find use in topically
administered compositions, such as those preparations for the
treatment of edema or inflammation. Indeed, above and beyond a
purely therapeutic application, the substances of the present
invention may also find utility in supplementing the protective
action of cosmetic compositions, such as sunscreen or suntan
lotions. Few, if any, sunscreen preparations are fully effective in
blocking out all the harmful wavelengths (e.g., 290-320 nm) present
in the ultraviolet region of the electromagnetic spectrum. Hence,
overexposure to the sun often gives rise to an acute condition
known as solar erythema and prolonged, repeated exposure can, of
course, lead to leathery looking skin or, worse, skin cancer.
Thus, the incorporation of the active substances of the present
invention in cosmetic preparations is specifically contemplated
both for the purpose of preserving and protecting the skin, as well
as alleviating a medical condition, such as solar erythema. In
sunscreen or suntan preparations, it would be advantageous to
include an effective amount of the oligosaccharides of the present
invention along with conventional sunscreen agents. Generally, an
amount of active substance would be present to provide a dose of
about 1 .mu.g to about 100 mg per kilogram of subject, preferably
from about 0.01 mg to about 10 mg per kilogram of subject, and most
preferably about 0.1 mg to about 1 mg per kilogram of subject.
The cosmetic compositions, may contain conventional ingredients
known to those of ordinary skill in the art, such as those
described in Kirk-Othmer, Encyclopedia of Chemical Technology,
Third Edition (1979), Vol. 7, pp. 143-176. In sunscreen
preparations, the addition of the active substances of the present
invention increases the minimum erythemal dose (MED) and,
consequently, the sun protection factor (SPF). Specific
ingredients, including typical sunscreens, are listed in
Kirk-Othmer, supra, at pp. 153-154. In addition, topical
preparations and cosmetic formulations may be prepared as described
in U.S. Pat. Nos. 4,199,576, 4,136,165, and 4,248,861, the complete
disclosures of which are incorporated by reference herein. It
would, of course, be apparent to those of ordinary skill in the art
of cosmetology that the resulting compositions can be in many
forms, including, but not limited to, solutions, lotions, cremes,
pastes, emulsions, sprays, or aerosols.
4.8. Exemplary Dosage Regimens
It was thus established according to the invention that the lowest
dose of LMWH per kg causing inhibition of TNF-.alpha., production
or inhibition of DTH reactivity by at least 50% is considered to
constitute 12 mouse inhibitory units per kg (12 u/kg). Because of
the differences in surface area and metabolism between mice and
humans, humans should be treated with a lower dose of LMWH, and 12
u/kg in mice is established to correspond to 1 u/kg in humans. For
example, the dose of Fragmin.RTM. batch 38609 effective in
inhibiting both TNF-.alpha. secretion and DTH reactivity is 5 .mu.g
per mouse administered weekly. Since each mouse weighs about 25 g,
the dose of Fragmin.RTM. 38609 equivalent to 12 u/kg is 200
.mu.g/kg of mouse. The dose of 1 u/kg suitable for humans is
therefore 200 .mu.g/kg.div.12=16.67 .mu.g/kg. A human weighing
about 70 kg would then be treated by a dose of about 1.2 mg given
in a single dose subcutaneously once every 7 days. Since individual
humans vary biologically, the optimal dose may be different from
about 1.2 mg and will lie generally below 5 mg, particularly within
the range of 0.3 to 3 mg.
Hence a rough guide for conversion of the mice dosage regimen to
human dosage is the following:
Dose Human/kg=Dose Mouse/kg.div.10 or 12
The dose of LMWH that should be effective in rats can be derived
from the fact that the dose of LMWH per kg of rats is one-half the
dose per kg of mice, i.e. 6 u/kg. For example, if 12 u of
Fragmin.RTM. batch 38609 is 200 .mu.g/kg, then the 6 u dose
suitable for rats should be 100 .mu.g/kg or 20 .mu.g per 200 g rat,
administered once a week.
For most of the oligosaccharide substances of the present
invention, which have been isolated from LMWH, degraded heparin and
degraded ECM, the following is a way to predict the effective dose
of these oligosaccharide substances for treatment of humans from
the in vivo DTH bioassay.
FIG. 12B shows that an isolated fraction (F15) in vivo inhibits the
DTH in mice at a range of 10.1-5.0 .mu.g/mouse/week. Since our mice
weigh 25 gm, the in vivo dose is approximately (0.1.div.0.025 kg)
4-200 .mu.g/kg mouse/week (the equivalent of 0.01-10 pg/ml in
vitro).
To correct for the surface area difference between mice and humans,
we have to divide the mouse dose/kg by 12:
4-200 .mu.g/kg mouse.fwdarw.0.33-16.67 .mu.g/kg human. Thus, a 70
kg human should receive up to about 1.2 mg (about 1,200 .mu.g). To
be certain that we could cover the difference between people, we
might increase this dose to about 5 mg, an amount that is well
below any doses of heparinoids used for their effects on
coagulation or thrombosis. Hence, the dose for a 70 kg human, will
be about 5 mg or less, preferably about 3 mg or less, more
preferably about 1.5 mg or less, and most preferably about 1 mg or
less.
In fact for the highly purified materials of the present invention,
including those that have been obtained from HPLC chromatography,
the preferred dosages may be even less. For example, the
disaccharides, described in greater detail below, have been found
to exhibit inhibitory activity, when administered by injection, at
about 0.1 .mu.g to about 0.5 .mu.g per kilogram mouse. Hence, the
dosage for humans are estimated to be about 0.01 .mu.g to about
0.05 .mu.g per kilogram man or about 0.7 .mu.g to about 3.5 .mu.g
for a 70 kilogram man for the purified disaccharides. A general
range of dosage for a 70 kg man, then, may be about 0.1 .mu.g to
about 100 .mu.g, preferably about 1 .mu.g to about 10 .mu.g for the
disaccharides. The dosages may be somewhat higher for the known
disaccharide "markers," discussed further below.
The doses recited above may be given a number of times a day or at
daily, weekly or even greater intervals depending on the
responsiveness of the individual. For the LMWHs, however, the
dosage interval is preferably weekly, as stated previously.
The invention will now be illustrated by the following non-limiting
examples.
5. EXPERIMENTS USING LMWH (FRAGMIN) ONLY
5.1. Bioassay of Inhibition of Active TNF-.alpha. Secretion Using
Mouse Spleen Cells
Supernatants of spleen cells cultured in the presence or absence of
LMWH, or spleen cells obtained from mice treated or untreated with
LMWH in vivo are analyzed for their ability to secrete active
TNF-.alpha.. The TNF-.alpha. bioassay is based on the cytotoxic
effect of TNF-.alpha. on cycloheximide (CHI)-sensitized cells and
its quantitation by the neutral red uptake assay as described by
Wallach D., J. Immunol. (1984) 132:2464-2469. Briefly, the killing
of CHI-sensitized HeLa cells by TNF-.alpha. present in the
supernatants of the cells is measured, the concentration of
TNF-.alpha. in the supernatants being determined by comparison to
titration curves of TNF-.alpha. exogenously added. Cell viability
is determined by incubation with neutral red for two hours, washing
away excess dye, extracting the neutral red that was taken up by
the cells with Sorenson's citrate buffer-ethanol mixture, and
quantitating it calorimetrically at 570 nm with a Microelisa
Autoreader.
Cells from mice treated with LMWH are obtained as follows: female
mice of the BALB/c strain (25 grams, 2 months old), at least 5 mice
per group, are injected subcutaneously with various doses of LMWH,
usually in the range of 0.5 to 20 .mu.g per mouse. Five days later
the mice are killed by cervical dislocation, the spleens are
removed and suspensions of spleen cells, depleted of red blood
cells, are assayed for the production of TNF-.alpha. in response to
induction by residual extracellular matrix (RECM), Concanavalin A
(Con A) or lipopolysaccharide (LPS).
5.2. In Vivo Bioassay of Inhibition of Experimental DTH
Reactivity
Groups of inbred BALB/c (Jackson Laboratories, Bar Harbor, Me.) or
of outbred CD1 (Weizmann Institute Animal Breeding Center, Rehovot,
Israel) mice are sensitized on the shaved abdominal skin with 100
.mu.l of 2% oxazolone (OX) in acetone/olive oil (4/1, v/v) applied
topically. DTH sensitivity is elicited 5 days later as follows:
mice are challenged with 20 .mu.l of 0.5% OX (10 .mu.l administered
topically to each side of the ear) in acetone/olive oil. A constant
area of the ear is measured immediately before challenge and 24 and
48 h later with a Mitutoyo engineer s micrometer. The individual
measuring ear swelling is blinded to the identity of the groups of
mice. The increment (.DELTA.) of ear swelling is expressed as the
mean in units of 10.sup.-2 mm or 10.sup.-4 inch (+SE) depending on
the micrometer that is used. Percent inhibition is calculated as
follows: ##EQU1##
Mice are treated with LMWH as in Example 5.1, injected the day
before primary sensitization to OX. On the fifth day after
sensitization to OX, the mice are challenged to induce a DTH
reaction, as described above.
The positive control is the DTH reaction elicited in immunized mice
in the absence of treatment with LMWH. The negative control is the
background swelling produced by the antigen in naive
(non-immunized) mice.
5.3. Induction of TNF-.alpha. Secretion by T Cells and Macrophages
In Vitro
Microtiter plates were prepared as follows: fibronectin (FN) or
laminin (LN) (Sigma) were added to flat bottom 96-well plates
(Costar) at a concentration of 1 .mu.g/50 .mu.l PBS per well and
removed after 16 h. Remaining binding sites were blocked with
BSA/PBS (10 mg/ml) which was added to the wells for 2 h and washed
out.
ECM-coated wells were prepared as follows: bovine corneal
endothelial cells were cultured in flat bottom 96-well plates. The
confluent layers of endothelial cells were dissolved and the ECM
was left intact free of cellular debris (Gospodarowicz, D. et al.,
J. Biol. Chem. (1978) 253:3736). Disrupted or residual ECM (RECM)
was prepared by gently scratching the ECM three times with a 27G
syringe needle and the exposed sites were subsequently coated with
BSA/PBS. Resting cloned rat CD4.sup.+ T cells, designated Kl, which
recognize myelin basic protein (MBP), were propagated and
maintained in culture and were added to the wells, 10.sup.5 cells
per well with or without 3.times.10.sup.5 syngeneic splenic
macrophages, in 100 .mu.l per well RPNI 1640 (Gibco) supplemented
with 1% BSA and antibiotics.
The splenic macrophages were purified by removing the T and B cells
using specific monoclonal antibodies (mAb). Anti-murine TNF-.alpha.
mAb was obtained from Genzyme (Cambridge, Mass.), and was diluted
300-fold. A 10 .mu.l aliquot of this diluted solution was added to
each well. MBP (100 .mu.g/ml), Con A (2.5 .mu.g/ml), LPS (1
.mu.g/ml), FN (5 .mu.g/ml), and LN (5 .mu.g/ml) were added to the
wells where indicated.
The plates were incubated at 37.degree. C. in a humidified
incubator for 3 h. Subsequently, the contents of the wells (4 wells
per experimental group) were collected, centrifuged, and the media
were assayed for active TNF-.alpha. secretion as in the example
described in Section 5.1: That is, supernatants of cultured
macrophages and lymphocytes were added to cultures of HeLa cells,
which are sensitive to killing by TNF-.alpha., and death of these
cells in the presence of the test media was calibrated in
comparison to titration curves of exogenous added TNF-.alpha.. Cell
death is examined by the release of neutral red dye from the
preincubated HeLa cells. The results shown here represent data
obtained from a total of six experiments that produced essentially
similar results.
Table V shows that T cells and macrophages cultured together can be
induced to secrete TNF-.alpha. by contact with specific antigen MBP
(group 4), the mitogen Con A (group 6) or LPS (group 8). However,
in the absence of antigenic or mitogenic stimulus, the secretion of
TNF-.alpha. was also induced by residual extracellular matrix
(RECM; group 10) or by the ECM components, fibronectin (FN; group
12) or laminin (LN group 14). Intact ECM was a weak inducer of
TNF-.alpha. (group 16).
TABLE V TNF-.alpha. secretion by T cells and macrophages is induced
by specific antigen MBP, Con A, LPS, RECM, or ECM components. K1
cells cultured together with Secreted TNF-.alpha. (yes) or without
TNF-.alpha. Group inducer (no) macrophages (pg/ml) 1 none no 50 2
yes 65 3 MBP antigen no 30 4 yes 950 5 Con A no 120 6 yes 1300 7
LPS no 50 8 yes 1500 9 RECM no 30 10 yes 900 11 FN no 20 12 yes 650
13 LN no 50 14 yes 500 15 ECM no 30 16 yes 120
5.4. Regulation of TNF-.alpha. Secretion by LMWHs
T cell and accessory cell cultures were prepared as described in
Section 5.3. LMWH was added to the wells at the beginning of the
cell culture. The levels of TNF-.alpha. were examined after 3 h of
incubation.
Table VI shows that the presence of LMWH (Fragmin.RTM. batch 38609)
in vitro inhibited active TNF-.alpha. secretion induced by specific
antigen (MBP; group 4), mitogens (Con A and LPS; groups 6 and 8),
RECM or ECM components (groups 10, 12 and 14). Since TNF-.alpha.
secretion induced by RECM is likely to be involved in
atherosclerosis, inhibition of TNF-.alpha. by LMWH will be
beneficial in atherosclerosis.
TABLE VI Induction of TNF-.alpha. secretion induced in vitro is
inhibited by LMWH (Fragmin .RTM. batch 38609). Secretion of
TNF-.alpha. by cultures of T cells and TNF-.alpha. LMWH macrophages
Group Inducer (1 .mu.g/ml) (pg/ml) 1 none none 65 2 yes 30 3 MPB
antigen none 950 4 yes 60 5 Con A none 1300 6 yes 80 7 LPS none
1500 8 yes 80 9 RECM none 900 10 yes 90 11 FN none 650 12 yes 90 13
LN none 500 14 none 70
5.5. Ex Vivo Experiments with LMWH-Treated BALB/c Mice
To examine the effect of LMWH administered to mice in vivo on the
secretion of TNF-.alpha. by spleen cells in vitro, the following
experiment was conducted. BALB/c mice, 5 per group, were treated
with various doses of LMWH (Fragmin.RTM. batch 38609) diluted in
saline, injected subcutaneously. After one week, the animals were
killed and their spleen cells, devoid of red blood cells, were
examined for their ability to secrete TNF-.alpha. in response to
control wells without RECM (A) or to wells coated with RECM (B).
Measuring the levels of TNF-.alpha. secretion was done as described
in Section 5.1. Table VII shows the results which indicate that an
injection of 5 .mu.g of LMWH given once, 7 days earlier, inhibited
TNF-.alpha. secretion induced by RECM. Higher or lower doses of
LMWH were less effective. Thus, an optimal dose of LMWH
administered in vivo a week earlier was effective.
TABLE VII Ex vitro inhibition of T cell mediated TNF-.alpha.
secretion in response to residual ECM. In vitro TNF-.alpha.
secretion LMWH (pg/ml) by spleen cells treatment of cultured on:
BALB/c mice B. Residual ECM (weekly) A. None (% Inhibition) 1 None
30 400 -- 2 0.5 .mu.g 50 380 (5) 3 1 .mu.g 25 90 (78) 4 5 .mu.g 25
60 (85) 5 10 .mu.g 30 140 (65) 6 20 .mu.g 40 320 (20)
Table VIII shows that a 5 .mu.g dose in vivo of the LMWH
Fragmin.RTM. batch 38609 was also effective in inhibiting
TNF-.alpha. secretion induced by LPS. BALB/c (4 mice per
experimental group) mice were treated with the indicated amounts of
LMWH diluted in saline and injected subcutaneously. After one week,
the mice were injected intraperitoneally with 10 mg LPS, killed 4
hours later and their spleen cells, devoid of red blood cells, were
subsequently cultured in RECM coated wells for 3 hours in a
humidified incubator. The levels of TNF-.alpha. secreted in
response to the RECM was measured in the supernatants of the
cultures. The results are given in Table VIII.
TABLE VIII Treatment of mice with LMWH inhibits LPS mediated
secretion of active TNF-.alpha. by macrophages. In vitro
TNF-.alpha. LMWH secretion by treatment macrophages (pg/ml) of mice
(.mu.g) in response to LPS % Inhibition 0 690 -- 0.1 500 28 1 350
50 5 120 82 20 550 20
5.6. Experiments Using a Variety of LMWH Sources
To examine the effect of different LMWHs on the inhibition of
secretion of active TNF-.alpha. and on DTH responses, mice were
treated with the indicated LMWH administered subcutaneously in
different concentrations. After one week, some of the mice were
killed and the induction of secretion of active TNF-.alpha. in
response to Con A activation in vitro was measured (Table IX). The
remaining mice were examined for their ability to respond to the
antigen oxazolone (Table X). The results are expressed in the
Tables as percent inhibition compared to the responses of the LMWH
untreated mice.
Two conclusions can be made by inspecting the results shown in
Table IX and Table X:
1. Different batches of LMWH, each calibrated for by similar
antithrombotic effect (Factor X assay) have different optimal doses
for inhibition of secretion of active TNF-.alpha.. Moreover, there
are preparations of LMWH, such as Clexane.RTM. batch 4096, which
have no inhibitory effect on secretion of active TNF-.alpha., at
any of the doses tried. Therefore, it may be concluded that the
antithrombotic effect of a LMWH preparation is not related to the
potential of the LMWH preparation for inhibition of secretion of
active TNF-.alpha.. The two different bioassays are mediated by
different factors present in the preparations.
2. The ability of a particular dose of LMWH to inhibit secretion of
active TNF-.alpha. is positively correlated with its ability to
inhibit DTH reaction, and the dose of a LMWH preparation optimally
effective in inhibiting secretion of active TNF-.alpha. is also
optimally effective in inhibiting the DTH reaction.
TABLE IX Weekly Treatment of Mice with Different LMWHs Inhibits DTH
Sensitivity of Mice. DTH Inhibition Batch of Dose Response of DTH
"R" value LMWH (.mu.g/gm mouse) (10.sup.-2 mm) (%) % .times.
(pg/gm).sup.-1 Fragmin Batch 38609 None 25 (+) Control -- 2 (-)
Control -- 0.02 21 12 -- 0.04 23 10 -- 0.2 6 73 (max) 365 0.4 6 20
-- 2 0 0 -- Batch 45389 None 28 (+) Control -- 2 (-) Control --
0.004 26 6 -- 0.04 4 89 (max) 2225 0.2 24 13 -- 0.4 26 6 -- 2 29 0
-- Clexane Batch 2088 None 22 (+) Control -- 2 (-) Control -- 0.004
17 23 -- 0.04 3 87 (max) 2175 0.2 13 41 -- 0.4 23 0 -- Batch 2066
None 23 (+) Control -- 2 (-) Control -- 0.004 20 13 -- 0.04 8 65 --
0.2 7 70 (max) 350 0.4 7 70 -- Batch 4096 None 24 (+) Control -- 2
(-) Control -- 0.04 27 No effect 0 0.2 26 No effect 0 0.4 24 No
effect 0
TABLE X Weekly Treatment of Mice with Different LMWHs Inhibits Ex
Vivo Secretion of Active TNF Using Mouse Spleen Cell Bioassay. Con
A-Induced Batch of Dose (.mu.g/ TNF secretion Inhibition "R" value
LMWH gm mouse) (pg/ml) (%) % .times. (pg/gm).sup.-1 Fragmin Batch
38609 None 450 Control -- 0.02 425 5 -- 0.04 400 12 -- 0.2 68 85
(max) 425 0.4 350 22 -- 2 435 8 -- Batch 45389 None 320 Control --
0.004 280 13 -- 0.04 70 78 (max) 1950 0.2 260 18 -- 0.4 290 10 -- 2
310 4 -- Clexane Batch 2088 None 400 Control -- 0.004 360 10 --
0.04 64 84 (max) 2100 0.2 152 38 -- 0.4 380 4 -- Batch 2066 None
350 Control -- 0.004 338 6 -- 0.04 185 54 -- 0.2 192 57 (max) 285
0.4 186 55 -- Batch 4096 None 320 Control -- 0.04 335 No effect 0
0.2 325 No effect 0 0.4 330 No effect 0
5.7. Treatment of Adjuvant Arthritis (AA) in Rats with Low Doses of
LMWHs
Adjuvant arthritis is an experimental disease inducible in some
strains of rats by immunizing them to antigens of Mycobacterium
tuberculosis (Pearson, C. M., Proc. Soc. Exp. Biol. Med. (1956)
91:91). This experimental disease is considered to be a model of
human rheumatoid arthritis (Pearson, C. M., Arthritis Rheum. (1964)
7:80). The arthritis appears to be caused by T lymphocytes that
recognize an antigen of M. tuberculosis that is cross-reactive with
structures in the joint tissues (Cohen, I. R., et al., Arthritis
Rheum. (1985) 28:841).
Lewis rats were immunized with M. tuberculosis (1 mg) in oil to
induce adjuvant arthritis (Pearson, C. M., Proc. Soc. Exp. Biol.
Med. (1956) 91:91). Five days later the rats were inoculated
subcutaneously as indicated with the doses of LMWH and/or heparin
and scored for the development of arthritis on a scale of 0-16 as
described (Holoshitz, J., et al., Science (1983) 219:56). All the
experiments were performed with Fragmin.RTM., Batch 38609.
In order to study the dose response to Fragmin.RTM. (FIG. 1) rats
immunized to induce AA were injected subcutaneously weekly,
starting on the 5th day after injection with 0.5 .mu.g
(.smallcircle.), 1 .mu.g (.diamond-solid.), 2 .mu.g
(.circle-solid.), 10 .mu.g (.diamond.), 15 .mu.g (.DELTA.) 20 .mu.g
(.box-solid.); 30 .mu.g (.tangle-soliddn.), 40 .mu.g (X) and PBS
control (.rect-hollow.). The 20 .mu.g dose was maximally effective
in inhibiting arthritis.
The effect of the 20 .mu.g dose of Fragmin.RTM. on the course of AA
is shown in FIG. 2: PBS control (.quadrature.); single treatment on
5th day (.tangle-soliddn.); daily (.circle-solid.); every 5th day
(.smallcircle.); weekly (.box-solid.). It is shown that Fragmin
administration both at 5 day intervals and at 7 day intervals
inhibits arthritis.
FIG. 3 shows the effect of weekly administration of Fragmin.RTM.
(batch 38609) as compared to standard heparin on AA. Lewis rats
were immunized to induce AA. Beginning on day 5, the rats were
inoculated subcutaneously at weekly intervals with a 20 .mu.g dose
of Fragmin.RTM. (.circle-solid.), heparin (.smallcircle.) or
phosphate buffered saline (PBS) control (.quadrature.). The results
show a dramatic difference in potency between Fragmin.RTM. and
heparin: Fragmin.RTM. completely inhibited arthritis, while heparin
had no inhibitory effect.
No inhibitory effect on AA was found with daily administration of a
20 .mu.g dose of LMWH, although surprisingly the inhibitory effect
of heparin was stronger than that of Fragmin.RTM. in daily
administration, as shown in (FIG. 4: Fragmin.RTM. (batch
38609)(.circle-solid.), heparin (.smallcircle.), PBS control
(.quadrature.)).
A similar inhibitory effect was observed with several other LMWHs
administered to Lewis rats immunized to induce AA. FIG. 5 shows the
results of the injection of a 20 .mu.g dose of Fraxiparin.RTM.
(daily (.quadrature.); weekly (.box-solid.)); Fraxiparine.RTM.
(daily (.DELTA.); weekly (.tangle-soliddn.)),
Lovenox.RTM./Clexane.RTM. (daily (.circle-solid.); weekly
(.smallcircle.)), and PBS control (X). All the three LMWHs of
different types and sources showed a marked inhibition of
arthritis, when administered weekly, but not daily.
5.8. Treatment with LMWH Prevents Rejection of Allografts
Wistar rats were subjected to allogeneic BN heart transplant (Ono,
K. and Linsay, E. S., J. Thorac. Cardiovasc. Surg. (1969)
45:225-229). From the day before transplantation, the rats were
injected subcutaneously at 7 day intervals with 20 .mu.g of
Fragmin.RTM. or PBS control (FIG. 6, .circle-solid. and
.smallcircle., respectively) and scored for survival. The day of
rejection was determined as the day the transplanted heart stopped
beating, assayed by palpation of the abdomen. FIG. 6 shows that the
rats treated with the weekly dose of LMWH had a markedly increased
survival of the heart allografts.
5.9. Biological Effect of LMWH on Insulin Dependent Diabetes
Mellitus (IDDM) of NOD Mice
Mice of the NOD strain spontaneously develop a form of type I
insulin dependent diabetes mellitus (IDDM) that is the accepted
model for human IDDM (Castano, L. and Eisenbarth, G. S., Annu. Rev.
Immunol. (1990) 8:647-679). The disease begins at 4-5 weeks of age
by the appearance of inflammation of the pancreatic islets,
insulitis. The insulitis progressively damages the
insulin-producing beta cells which are sensitive to damage by
TNF-.alpha.. At about 4-5 months of age, a sufficient number of
beta cells are destroyed so that diabetes becomes overt.
To test whether treatment with LMWH could affect the IDDM process,
a group of 10 female NOD mice was treated with weekly subcutaneous
injections of 5 .mu.g per mouse of Fragmin.RTM. (batch 38669), the
dose determined to represent 12 mouse units per kg. A group of 10
control mice were treated with injections of saline. At the age of
5 months all the mice were bled to determine the development of
IDDM using a standard procedure (Elias, D. et al., Proc. Natl.
Acad. Sci. U.S.A. (199.0) 87:1576-1580). FIG. 7 shows that the
control mice ("none") had abnormal blood glucose (400 mg/dl). In
contrast the mice treated with LMWH had a normal blood glucose (100
mg/dl). Thus treatment with LMWH can indeed cure the IDDM
process.
5.10. LMWH Treatment of Allergy
In many allergic patients, intradermal challenge with specific
antigen or anti-IgE induces an immediate wheal and flare reaction
which is followed, 4-8 h later, by a period of persistent swelling
and leukocyte infiltration termed the late phase cutaneous
reaction. Late phase reactions (LPR).sup.2 were initially described
in the skin (Solley, G. O. et al., J. Clin. Invest. (1976)
58:408-420). However, it is now clear that late consequences of
IgE-dependent reactions, notably including infiltration of the
reaction sites with blood-borne leukocytes, also occur in the
respiratory tract and other anatomical locations (Lemanski, R. F.
and Kaliner, M., in Allergy: Principles and Practice, Vol. 1
(1988), Middeton, Jr., E. et al. (Eds.), pp. 224-246). Indeed, it
has been argued cogently that many of the clinically significant
consequences of IgE-dependent reactions, in both the skin and the
respiratory system, reflect the actions of the leukocytes recruited
to these sites during the LPR rather than the direct effects of the
mediators released at early intervals after antigen provocation
(Kay, A. B. J. Allergy Clin. Immunol. (1991) 87:893-910).
It has recently been widely recognized that chronic allergic
diseases such as asthma and atopic dermatitis are a result of an
underlying inflammatory process which includes the infiltration and
activation mainly of eosinophils and T cells (Kay, A. B. J. Allergy
Clin. Immunol. (1991) 87:893-910).
Several lines of evidence support the hypothesis that the leukocyte
infiltration associated with LPRs occurs as a result of mast cell
degranulation. In both man and experimental animals, agents that
induce cutaneous mast cell degranulation by either IgE-dependent of
certain other mechanisms can also promote infiltration of the
reaction sites with leukocytes (Solley, G. O. et al., J. Clin.
Invest. (1976) 58:408-420; Lemanski, R. F. and Kaliner, M., in
Allergy: Principles and Practice, Vol. 1 (1988), Middeton, Jr., E.
et al. (Eds.), pp. 224-246; Kay, A. B. J. Allergy Clin. Immunol.
(1991) 87:893-910). A review of the mediators that can be
elaborated by activated mast cells reveals many that might
contribute to leukocyte infiltration in LPRs, including lipid
mediators such as LTB.sub.4, LTC.sub.4, LTD.sub.4, PGD.sub.2, and
PAF (platelet activating factor), as well as several peptide or
proteinaceous chemotactic factors (Holgate, S. T. et al., in
Allergy: Principles and Practice, Vol. 1 (1988), Middleton, Jr. E.
et al. (Eds.), pp. 135-178). The latter agents range in size from
tetrapeptide "eosinophil chemotactic factors of anaphylaxis" to
very high molecular weight "neutrophil chemotactic factors".
Even more candidate mast cell associated mediators of leukocyte
infiltration recently have been identified, including cytokines
similar or identical to INF-.alpha., IL-1.alpha., and four members
of the MIP-1 gene family of small secreted peptides (Gordon, J. R.
et al, Immunol. Today (1990) 11:458-464). Four of these cytokines
(TNF-.alpha., IL-1.alpha., MIP-1.alpha. and MIP-1.beta.) have been
demonstrated to have the ability to promote leukocyte
infiltration.
More recently, (Wershil, B. K. et al., in J. Clin. Invest. (1991)
87:446-453, by using mast cell deficient mice have demonstrated
that the recruitment of leukocytes during IgE dependent LPR is mast
cell dependent and that this inhibition was partially blocked by
local administration of anti TNF-.alpha. antiserum. It is widely
accepted today that the inhibition of the cellular
infiltration/activation associated with IgE dependent LPR is a
crucial therapeutic approach in alleviating various allergic
diseases (Barnes, P. J. N. Eng. J. Med. (1989) 321:1517-1527).
To the surprise of the present applicants, it was found that LMWH
significantly inhibited the leukocyte infiltration during IgE
dependent cutaneous LPR in mice undergoing passive cutaneous
anaphylaxis (PCA).
Mice received an i.d., injection (into the ears) of monoclonal IgE
anti DNP Ab (.about.20 ng). A day later, the mice were i.v.
injected with DNP.sub.30-40 -HSA in saline. Ear swelling was
determined by measurement of ear thickness with a micrometer before
and at various intervals after the challenge with the DNP-HSA. In
all experiments, tissues from sites of PCA reactions were obtained
after sacrifice by cervical dislocation and were processed for
Giemsa-stained sections. LMWH was given once by s.c. injections (5
.mu.g/mouse) on Day-2.
Results
Swelling developed rapidly at sites of PCA reactions (.DELTA. of
35.times.10.sup.-4 inch at 15 min.) but not at control sites (ears
injected with diluent alone). Swelling of PCA sites diminished
markedly between 2 and 4 hours after i.v. antigen challenge.
PCA and control sites were assessed histologically at 6-8 hours
after the i.v. antigen challenge. The majority of mast cells at PCA
sites exhibited extensive or moderate degranulation. By contrast
<5% of mast cells at control sites exhibited marked
degranulation. There was a significant neutrophil infiltration only
in PCA sites at 6 hour post antigen challenge. This infiltration
was markedly reduced (by 60%) in mice which had been pretreated
with LMWH two days earlier. There was no effect of this drug on the
magnitude of mast cell degranulation. There was no effect of the
drug on the total and differential count of leukocytes in the
peripheral blood of these animals. It can be concluded that LMW
heparin inhibited the cellular infiltration associated with the IgE
dependent late cutaneous reaction. Additionally, the applicants
also anticipate that the administration of LMWH will exhibit a
beneficial effect on cutaneous LPR in animals with active cutaneous
anaphylaxis (specific IgE production will be induced with DNP-HSA
Alum). Similar therapeutic effects on pulmonary allergic
inflammation are also anticipated (Tarayre, J. P. et al. Int. J.
Immunopharmacol. (1992) 14(5):847-855.
5.11. LMWH Treatment of Human DTH
FIG. 8 shows an experiment in which a 40 year old male volunteer
weighing 85 kg was tested for DTH reactivity to tetanus antigen
(Merieux skin test applicator). About 18 mm of induration was
measured at 24 and 48 hours. The volunteer was then treated with a
subcutaneous injection of Fragmin.RTM. (batch 38609) 3 mg. Five
days later the volunteer was again tested for his DTH response to
tetanus and the induration was inhibited to about 5 mm. The
volunteer was tested again for DTH 3 weeks later ("Recovery") and
the test showed positive reactivity (23 mm of induration at 24 and
48 hours). The volunteer was then treated with Fragmin.RTM. as
before and the DTH reactivity was measured again 7 days later ("7
days post"). Again the DTH was inhibited to about 5 mm of
induration. Recovery of DTH again was found 3 weeks later. Thus,
LMWH at a dose of less than 5 mg can inhibit DTH in humans at
treatment intervals of 5 and 7 days.
6. EXPERIMENTS USING LMWH (FRAGMIN) AND OTHER ACTIVE SUBSTANCES
6.1. Stability Studies of LMWH (Fragmin) TNF-.alpha. Inhibitory
Activity
Fragmin batch 38609 was diluted in normal saline to a concentration
of 5 .mu.g/0.1 ml. Some of the vials were mixed with an equal
amount of protamine sulfate (5 .mu.g) and the vials were stored at
room temperature (21.degree. C.) for 0 to 72 hours (Table XI) or
were stored at 4.degree. C. for 1 to 4 months (Table XII). The
Fragmin with or without protamine sulfate was then used in vivo to
inhibit the DTH T cell reaction in BALB/c mice as described above.
For the present experiments, the positive control DTH was
17.5.+-.1.2.times.10.sup.-2 mm (0% inhibition) and the fully
inhibited DTH was 2.6.+-.0.5.times.10.sup.-2 mm (100%
inhibition).
The results of the incubation of LMWH at 20.degree. C. are listed
in the Table XI. It is evident from Table XI that LMWH loses its
inhibitory activity against TNF-.alpha.-dependent, T cell mediated
DTH reaction upon incubation at ambient temperature over 72 h. In
contrast, Heparin and LMWH lose their anti-coagulant activity at
ambient temperature only slowly.
TABLE XI Stability of Inhibitory Activity of Fragmin (Batch 38609,
5 .mu.g/0.1 ml), Without Protamine Sulfate, at 20.degree. C.
Against DTH-Reaction. No. Hours DTH Reaction % anti-DTH Reactivity
None 17.5 .+-. 1.2 Control* 0 2.6 .+-. 0.5 100** 24 9.6 .+-. 1 47
48 15.7 .+-. 1.6 13 72 17 .+-. 0.8 0 *no inhibition **full
inhibition
6.2. Loss of Anti-DTH Reactivity at Low Temperature and Stabilizing
Effect of Added Protamine
Table XII shows that Fragmin loses its ability to inhibit the DTH
reactivity of mouse T cells in dilute solution within 4 months at
4.degree. C. The addition of an equal concentration of protamine
sulfate does not interfere with inhibition of the DTH reaction, but
actually preserves this activity intact after 4 months at 4.degree.
C. Again, this result is contrary to the normal role of protamine
sulfate, when added to heparin or Fragmin, in which the protamine
sulfate neutralizes the anti-coagulant effects of the heparinoid
substances.
TABLE XII Loss of Anti-DTH Activity Over Time at Low Temperature.
Stabilizing Effect of Added Protamine. Fragmin Months at Protamine
DTH % Anti-DTH (38609) 4.degree. C. Sulfate (10.sup.-2 mm) Activity
none none none 15 .+-. 1 control* yes 1 none 2.8 .+-. 0.5 100** yes
1 yes 3 .+-. 0.4 100 yes 2 none 4 .+-. 0.8 82 yes 2 yes 2.4 .+-. 1
100 yes 3 none 9.6 .+-. 0.8 55 yes 3 yes 3 .+-. 0.5 100 yes 4 none
14.8 .+-. 1.4 0 yes 4 yes 3 .+-. 0.4 100 yes 0 yes 3 .+-. 0.5 100
*no inhibition **full inhibition
6.3. Preparation of ECM-Coated Plates
ECM-coated wells were prepared as follows. Freshly dissected bovine
eyes were obtained from a slaughter house within a few hours after
slaughter. The eyes were dissected in a hood to remove the cornea.
The cornea were then scratched or scraped with a scalpel to obtain
the corneal endothelial cells. These cells were cultured on tissue
culture plates with approximately 5 ml of media comprising DMEM
supplemented with 10% fetal calf serum, 5% calf serum and
antibiotics, such as 1% streptomycin or 1% neostatin, together with
1% glutamine as a stabilizer. The cells settled to the bottom of
the plates after approximately 2 days of seeding, were fed with
fresh media every four days, and incubated at 37.degree. C. in 5%
CO.sub.2 humidified incubators. If desired, some fibroblast growth
factor may also be added to the media, although the addition of FGF
is not crucial. When the cells were confluent (approximately 2
weeks later), the supernatant was aspirated off, and the cells were
then trypsinized with 1-2 mls of trypsin.
Eighty percent of these primary cells (the fate of the remaining
20% of the primary cells is described immediately below) were taken
and divided onto 5 flat-bottomed 96-well plates. The cells were
cultured in DMEM supplemented with 4% dextran T-40, 10% fetal calf
serum and 5% calf serum. After about 7 days of incubation at
37.degree. C. in a 10% CO.sub.2 humidified incubator, the resulting
confluent layers of endothelial cells were lysed. The lysing
buffer, comprising 0.025 M NH.sub.4 OH containing 0.25% Triton X in
PBS, was allowed to remain over the cells for 10 minutes and then
decanted. The contents of the plates were then washed three times
with PBS chilled to 4.degree. C. The preceding procedure left the
ECM intact, firmly attached to the entire area of the well. The
resulting ECM was also free of nuclei and cellular debris. The
ECM-coated plates can be stored at 4.degree. C. for at least three
months.
The remaining 20% of the primary cells were left on a single plate
and cultured in approximately 5 ml of media comprising DMEM
supplemented with 10% fetal calf serum, 5% calf serum and
antibiotics as described above. This secondary crop of cells was
allowed to become confluent and was treated with trypsin as
described above. Again, the trypsinized cells were divided, 80%
being cultured in 5 plates in the growth media containing 4%
Dextran T-40, and 20% being cultured in a single plate as before.
It is possible to perform this 80/20 division yet one more time
from this single plate.
6.4. Degradation of Sulfated Proteoglycans
.sup.35 (S)O.sub.4 -labelled ECM was incubated with 5 .mu.l of MM5
heparanase (4 u/ml) in 1 ml PBS and 100 .mu.l 8.2 M
phosphate-citrate buffer (pH 6.2) for 48 hrs. at 37.degree. C. The
medium was then collected, centrifuged at 1,000 g for 5 min.
(optional) and analyzed by gel filtration on Sepharose 4B columns.
Two ml fractions were eluted with PBS at a flow rate of 5 ml/hr and
were counted for radioactivity using Bio-Fluor Scintillation
fluid.
This .sup.35 (S)O.sub.4 -labelling experiment showed that the ECM
was actually being degraded, that the resulting degradation
products were successfully being released, and, furthermore, were
being properly filtered through the Sepharose 4B columns.
Subsequent experiments related to the degradation of sulfated
proteoglycans were carried out on non-labeled ECM, with the
degradation products being monitored by their absorption at 2.06 or
232 nm, instead.
Enzyme degradation experiments were carried out as above and, in
addition, the degradation products (DECM) were purified further by
loading the degraded proteoglycans that were eluted from the
Sepharose columns onto HPLC columns. HPLC analysis of the Sepharose
column fractions was carried out in a manner such as that described
in Section 6.11 et seq. Detection of the degradation products was
achieved by monitoring their absorption at 206 nm.
Additional enzyme degradation experiments were carried out with
similar results using PC3 enzyme and heparanase obtained from
IBEX.
6.5. Purification of Human CD4.sup.+ T Cells
CD4.sup.+ T cells were obtained from peripheral blood mononuclear
leukocytes obtained from healthy human donors as follows. The
mononuclear cells were isolated on a Ficoll gradient, washed in
RPMI supplemented with 10% FCS and antibiotics in petri dishes and
incubated at 37.degree. C. in a 10% CO.sub.2 humidified atmosphere.
After 1 h, the non adherent cells were removed and incubated on
nylon-wool columns (Fenwall, Ill.) for 45-60 min at 37.degree. C.
in a 10% CO.sub.2 humidified atmosphere. Non adherent cells were
eluted and washed. CD4.sup.+ T cells were negatively selected by
exposure of the eluted cells to a mixture of the following
monoclonal antibodies (mAb): anti-CD8, CD19, and CD14 conjugated to
magnetic-beads (Advanced Magnetics, Cambridge, Mass.). Unbound
cells were recovered and their phenotypes were examined. The
resultant purified cells were predominantly (>90%) CD3.sup.+
CD4.sup.+ as determined by FACScan analysis.
6.6. Bioassay of TNF-.alpha. Activity Using Human CD4.sup.+ T Cells
Derived from PBLs
Two hundred fifty thousand human CD4.sup.+ T cells were
preincubated with 150 .mu.l of ECM degradation products at various
concentrations for 1.5 h at 37.degree. C., under a 7% CO.sub.2
atmosphere. Then 100 .mu.l of PHA (Wellcome Co., England, 1
.mu.g/ml) were added for 3 h incubation, in flat-bottomed 96-well
plates (Costar). Subsequently, the contents of the wells (3-6 wells
per experimental group) were collected, centrifuged, and the media
were assayed for TNF-.alpha. secretion as previously described in
Section 5.1. Briefly, supernatants of cultured lymphocytes were
added to cultures of mouse fibrosarcoma cell clones (BALB/c.CL7).
BALB/c.CL7 cells are sensitive to killing by TNF-.alpha. in the
presence of actinomycin D (0.75 .mu.g/ml). Nophar, Y. et al. J.
Immunol. (1988) 140(10):3456-3460. The death of these cells, in the
presence of the test media, was calibrated in comparison to
titration curves of added exogenous TNF. Cell viability is
determined by incubation with MTT tetrazolium (Sigma, Cat. No.
M2128) for two hours, extracting the dye that was taken up by the
cells with isopropanol-HCl mixture and quantitating it
calorimetrically (at 570 nm) with a Microelisa Autoreade.
TNF-.alpha. typing was done by examining the neutralizing effect of
anti-murine TNF-.alpha. mAb (diluted 1/400; Genzyme, Mass.).
6.7. Degradation of Heparin
One milligram of heparin (Sigma) in 1 ml of PBS and 100 .mu.l 25 M
phosphate-citrate buffer (pH 6.2) was incubated with 20 .mu.l MM5
(5 u/ml) for 48 hrs. at 37.degree. C. The products of the reaction
were then analyzed by gel filtration on Sepharose 4B columns. Two
ml fractions were eluted with PBS at a flow rate of 5 ml/hr. To
further characterize the degradation products, the peaks eluted
from the Sepharose column were subjected to HPLC separation using
Toyo Soda-Gel G3000SW and G2000SW HPLC columns, as described in
Section 6.11 et seq.
Additional experiments were carried out using 20 .mu.l of PC3. The
PC3 enzymatic reaction was carried out with 1 mg of heparin under
the same conditions as described above for the MM5 except that the
reaction was incubated for 24 hrs instead of 48 hrs. The products
were then analyzed by gel filtration on Sepharose 4B columns (FIGS.
29A and 29B). The in vitro bioassay results are shown in Table XIX
below.
6.8. Elicitation of DTH Response in Mice and Examining Inhibitory
Effects
BALB/c mice (at least 5 mice per group) were sensitized on the
shaved abdomen with 3% 4-ethoxymethylene-2-phenyl oxazolone (OX;
BDH Chemicals, GB) in acetone/olive oil applied topically. DTH
sensitivity was elicited 5 days later as follows. Mice were
challenged with 0.5% OX in acetone/olive oil. The ear was measured
immediately before challenge and 24 h later with Mitutoyo
engineer's micrometer (Japan). The individual measuring the
swelling of the ear was blinded to the identity of the groups of
mice. To interfere with DTH response, the low molecular weight
immuno-regulatory fractions, diluted in PBS, were administrated
subcutaneously into the back of the treated mice at the indicated
time schedules and concentrations. Treated mice were inspected
during and after (>2 months) the treatment and no major
side-effects were observed clinically.
6.9. Separation of LMWH (Fragmin) on Size-Exclusion Gel
Chromatography Column (Sepharose 4B)
Fragmin (Batch 38.609) and inactive Fragmin were fractionated by
gel filtration on Sepharose 4B (Pharmacia) columns. Fractions of
0.5 ml were eluted with PBS at a flow rate of 5 ml/hr, and
monitored for absorbance at 206 nm. (No absorbance was detected at
280 nm). A plot of the fraction number versus absorption at 206 nm
appears on FIG. 11. The results of the bioassays for selected
fractions are presented in Tables XIII and XIV, below.
TABLE XIII Effect of Whole Fragmin, Sepharose 4B Fractions of
Fragmin, and an HPLC Fraction of a Sepharose 4B Fraction on the
Secretion of Active TNF Using Human PBL Bioassay. Bioassay of Test
TNF Activity "R" value Material conc. (pg/ml) (%) % .times.
(pg/ml).sup.-1 Active 1 Inh.sub.max (90%) 90 Frag/whole Inactivated
a No effect 0 Frag/whole Inactivated 5 Inh.sub.max (50%) 50
Frag/Seph.4B-F15 Inactivated a No effect 0 Frag/Seph.4B-F10
Inactivated 1000 Aug.sub.max (60%) 0.06 Frag/Seph.4B-F8 Inactivated
1000 Aug.sub.max (30%) 0.03 Frag/Seph.4B-F2 Frag/HPLC-F90 b No
effect 0 a At a conc range of 1 .mu.g/ml - 0.001 pg/ml. b At a conc
range of 1 .mu.g/ml - 0.01 pg/ml.
TABLE XIV Effect of Whole Fragmin and Sepharose 4B Fractions of
Fragmin on DTH Sensitivity of Mice. Inhibition Test Dose of DTH "R"
value Material (.mu.g/gm mouse) (>50%) % .times.
(.mu.g/gm).sup.-1 Active 0.2 50 250 Frag/whole Inactivated
0.2-0.004 No effect 0 Frag/whole Inactivated 0.004 50 12,500
Frag/Seph.4B-F15 Inactivated 0.2-0.004 No effect 0
Frag/Seph.4B-F10
6.10. Additional Experiments Involving the Fractionation of Fragmin
and Heparanase-degraded ECM
Fragmin and heparanase-degraded ECM were fractionated by gel
filtration on Sepharose 4B columns. Fractions of 0.2 ml were eluted
with PBS at a flow rate of 5 ml/hr, and monitored for absorbance at
206 nm. (No absorbance was detected at 280 nm). A plot of the
fraction number versus absorption at 206 n.m. appears on FIG. 14.
The results of the bioassays for selected fractions are presented
in Table XV below.
TABLE XV Effect of Sepharose 4B Fractions of Fragmin and DECM on
the Secretion of Active TNF Using Human PBL Bioassay. Bioassay of
conc TNF Activity "R" value Test Fraction (pg/ml) (%) % .times.
(pg/ml).sup.-1 Frag/Seph.4B-F39 100 Inh.sub.max (60%) 0.6
DEMC/Seph.4B-F39 10,000 Inh.sub.max (85%) 0.0085 Frag/Seph.4B-F42 a
No effect 0 DECM/Seph.4B-F42 a No effect 0 Frag/Seph.4B-F32 10
Aug.sub.max (55%) 5.5 DECM/Seph.4B-F46 10 Aug.sub.max (20%) 2 a At
a conc range of 1 .mu.g/ml - 0.001 pg/ml.
6.11. Separation of Active Substances from Fragmin Using High
Performance Liquid Chromatography
Two experiments were performed utilizing two sets of high
performance liquid chromatography conditions. The initial type of
column used was a 30 cm.times.7.8 mm I.D. TSK-Gel.RTM. G-Oligo-PW
column with a 4 cm.times.6 mm I.D. guard column. The column was
eluted with 0.2 M phosphate buffer, pH 7.0, at a flow of 0.5
ml/min. The fractions collected were each 0.5 mls in volume.
The second type of HPLC used was Toyo Soda TSK-Gel G3000SW (7.5
mm.times.50 cm) and G2000SW (7.5 mm.times.50 cm) columns (in
series) with a 7.5 mm.times.10 cm guard column from Phenomenex. The
column was eluted at 1 ml/min. with carefully degassed 0.5 M NaCl.
Fractions were collected at 0.5 ml per fraction. The detector was
set at 232 nm with 0.02 AUFS and retention times measured to
.+-.0.1 sec. The void and total volumes were measured by blue
dextran and sodium azide. The collections were also subjected to
detection at 206 nm under the same conditions as the 232 nm
setting.
A plot of the Fragmin HPLC fraction number versus absorption at 206
nm appears on FIG. 18A. The results of the bioassays for selected
fractions are presented in Table XVI, below. It is evident from the
results presented that certain substances are able to inhibit the
activity of TNF-.alpha. while others are able to augment its
activity.
TABLE XVI Effect of Whole and HPLC Fractions of Fragmin on the
Secretion of Active TNF Using Human PBL Bioassay. Bioassay of
Fragmin TNF Activity "R" value Fraction conc (pg/ml) (%) % .times.
(pg/ml).sup.-1 Whole.sup.a 10 Inh.sub.max (40%) 4 HPLC-F1 100
Aug.sub.max (60%) 0.6 HPLC-F3 10 Inh.sub.max (70%) 7 HPLC-F16 10
Inh.sub.max (100%) 10 HPLC-F22 10 Inh.sub.max (100%) 10 HPLC-F26
100 Inh.sub.max (50%) 0.5 HPLC-F30 100 Inh.sub.max (70%) 0.7
HPLC-F47 100 Inh.sub.max (55%) 0.55 .sup.a This whole Fragmin
sample had been aged at 4.degree. C. for 90 days.
6.12. Separation of Active Substances from ECM Using High
Performance Liquid Chromatography
Separation of active substances from ECM by high performance liquid
chromatography was carried out as discussed in Section 6.11.
A plot of the ECM HPLC fraction number versus absorption at 206 nm
appears on FIG. 18B. The results of the bioassays for selected
fractions are presented in Table XVII, below. It is evident from
the results presented that certain substances isolated from
heparanase-mediated degradation of ECM are able to inhibit the
activity of TNF-.alpha. while others are able to augment its
activity. The results also show that certain fractions obtained
from the HPLC separation exhibit no effect on the activity of
TNF.
TABLE XVII Effect of DECM HPLC Fractions on the Secretion of Active
TNF Using Human PBL Bioassay. Bioassay of DECM TNF Activity "R"
value Fraction conc (pg/ml) (%) % .times. (pg/ml).sup.-1 HPLC-F1 a
No Effect 0 HPLC-F5 a No Effect 0 HPLC-F10 10 Inh.sub.max (60%) 6
HPLC-F14 10 Inh.sub.max (70%) 7 HPLC-F17 a No Effect 0 HPLC-F25
1000 Aug.sub.max (40%) 0.04 HPLC-F33 100 Aug.sub.max (40%) 0.4
HPLC-F37 10 Aug.sub.max (100%) 10 HPLC-F39 100,000 Inh.sub.max
(60%) 0.0006 HPLC-F42 a No Effect 0 HPLC-F46 1000 Aug.sub.max (30%)
0.03 HPLC-F49 1000 Aug.sub.max (30%) 0.03 HPLC-F61 a No Effect 0 a
At a conc range of 1 .mu.g/ml - 0.001 pg/ml.
6.13. Carbazole Quantitative Sugar Assay
Briefly, 1500 .mu.l of borate sulphuric acid reagent is cooled on
an ice bath. The test solution (250 .mu.l containing 20 .mu.g of
uronic acid/ml) is then carefully layered onto the surface of the
boric acid reagent and allowed to diffuse for 10 minutes. The
solutions are then thoroughly mixed, put in a boiling bath for 10
minutes, and then cooled in an ice bath. Chilled carbazole (50
.mu.l) is then added to the mixture, vortexed, and put in a boiling
bath for 15 minutes. The solution is then cooled and the absorbance
is read at 525 nm. The results are compared with calibrated
solutions.
6.14. Isolation of a Disaccharide from ECM Degradation Products
A disaccharide substance was isolated by HPLC from bovine corneal
endothelial ECM that had been subjected to mammalian heparanase
(MM5).
More specifically:
An ECM-coated plate was incubated with 20 .mu.l of mammalian
heparanase (0.5 mg/ml) in 1 ml PBS buffer (that was preadjusted to
pH 6.2 by citric acid) for 48 hours at 37.degree. C. The medium was
then collected and applied on a Sepharose-4B column (35
cm.times.0.7 cm I.D.). The mobile phase was PBS buffer at a flow
rate of 5 ml/hr. Fractions of 2.2 ml were collected and monitored
at 206 nm (FIG. 19). A sample (100 ul) from Sepharose 4B fraction
no. 5 (8.8-11 ml elution volume) were injected into an HPLC column
(Toyo Soda TSK-Gel G3000SW (7.5 mm.times.50 cm) and G2000SW (7.5
mm.times.50 cm), in series with 7.5 mm.times.10 cm guard from
Phenomenex). The mobile phase was 0.5 M NaCl at a flow rate of 1
ml/min. One ml fractions were collected and monitored at 206 and
232 nm (FIGS. 20A and 20B). Peak no 1. (P1) was freeze dried in a
25 ml flask. The sample contains, ca. 20 .mu.g of oligosaccharide
(determined by carbazole assay) in 60 mg NaCl.
The sample has an elution profile that is similar to a disaccharide
standard or molecular weight "marker" obtained commercially from
the depolymerization of heparin (Sigma).
The Inh.sub.max value of the substance, based on similarly obtained
samples (See, for example peak F73 in FIG. 21A (elution time of
about 44 minutes) and entry F5/HPLC-F73 in Table XVIII, below), was
estimated at 87% in an in vitro TNF-.alpha. inhibition assay using
human PBLs, at a concentration of about 10 pg/ml. The bioassay was
conducted as previously described, above. This sample may be
purified further using a SAX-HPLC column, as described below.
6.15. Isolation of a Disaccharide from ECM Degradation Products
Including SAX-HPLC Chromatography
An ECM-coated plate was incubated with 20 .mu.l of mammalian
heparanase (0.5 mg/ml) in 1 ml PBS buffer (which had been
preadjusted to pH 6.2 by citric acid) for 48 hours at 37.degree. C.
The medium was then collected and applied on a Sepharose-4B column
(0.7.times.35 cm). The mobile phase was PBS buffer at a flow rate
of 5 ml/hr. Fractions of 1.6 ml were collected and monitored at 206
nm. (FIG. 22). Fractions nos. 7-8 were combined and freeze-dried;
the powder was resuspended in 1/10 of the initial volume. Samples
(100 .mu.l) were injected into an HPLC column (Toyo Soda TSWK-Gel
G3000SW 7.5 mm.times.50 cm and G2000SW 7.5 mm.times.50 cm, in
series with 7.5 mm.times.10 cm guard from Phenomenex), as before.
The mobile phase was 0.5 M NaCl at a flow rate of 1 ml/min. One ml
fractions were collected and monitored at 206 and 232 nm (FIG. 23).
The peak labeled "1" was collected from ten identical runs. The
substantially homogeneous fractions were combined and freeze
dried.
The material was resuspended in 2 ml double deionized water and
desalted on a Sephadex G-10 column (26.times.150 mm) eluted at 1.6
ml/min with double deionized (DD) water. One ml fractions were
collected and monitored at 232 nm and for conductivity (to
determine NaCl content) Desalted fractions were combined, freeze
dried and resuspended in 1 ml DD H.sub.2 O. A 1 ml sample, prepared
by combining 100 .mu.l of the resuspended solution and 900 .mu.l of
0.2 M NaCl at pH 3.5, was injected into an analytical SAX-HPLC
column (4.6.times.250 mm, packed with Spherisorb, 5-.mu.m particle
size). The flow rate was 1.5 ml/min and an NaCl linear gradient
program was employed as follows:
Time/Buffer (min) A (%) B (%) C (%) 0 100 0 0 2 100 0 0 35 38 62 0
40 38 62 0 45 0 100 0 47 0 100 0 50 0 0 100 55 0 0 100 58 100 0 0
60 100 0 0 A = 0.2 M NaCl, pH 3.5 B = 1.5 M NaCl, pH 3.5 C =
H.sub.2 O
The column eluent was monitored at 232 nm (FIG. 24) and peak A23/4
was collected and tested for TNF inhibition. The Inh.sub.max for
this SAX-HPLC fraction was found to be 60% at a concentration of
0.1 pg/ml, giving an "R" value of 600%.times.(pg/ml).sup.-1.
Heparin disaccharide standards with different levels of sulfation
were injected into the SAX-HPLC column under identical conditions.
The elution profiles of these standards are presented in FIGS.
25A-C. As can be seen from these Figures, the disaccharide
standards gave different retention times, with an unsulfated
disaccharide (Sigma Product No. H-0895) eluting fastest (FIG. 25A),
a disulfated disaccharide (Sigma Product No. H-1020) eluting at
less than 20 minutes (FIG. 25B), and a trisulfated disaccharide
(Sigma Product No. 9267) eluting last (FIG. 25C). The trisulfated
disaccharide standard H-9267 provided a retention time that was
very similar to that obtained for peak A23/4 (i.e., 23.07 min. vs
and 23.10 min., respectively).
6.16. Results of In Vitro Human PBL Bioassays for Various
Substances
The results of in vitro bioassays using human PBLs for various
active substances and starting "mixtures" are presented in Table
XVIII for the products obtained from the degradation of ECM,
including a peak "P1" from FIGS. 20A and 20B. Whereas, P1 provided
an "R" value of 10%.times.(pg/ml).sup.-1, the starting DECM "soup"
gave an "R" value of 0.000053%.times.(pg/ml).sup.-1.
TABLE XVIII Effect of ECM + MM5 Heparanase (DECM "Soup"), Sepharose
4B Fractions of "Soup", and HPLC Fractions of Sepharose 4B
Fractions on the Secretion of Active TNF Using Human PBL Bioassay.
Bioassay of Test TNF Activity "R" value Material conc (pg/ml) (%) %
.times. (pg/ml).sup.-1 DECM "Soup" 1 .times. 10.sup.6 Inh.sub.max
(53%) 5.3 .times. 10.sup.-5 Seph.4B-F5 100 Inh.sub.max (50%) 0.5
Seph.4B-F6 100 Inh.sub.max (60%) 0.6 Seph.4B-F7,8 100 Inh.sub.max
(81%) 0.8 F5/HPLC-F73 10 Inh.sub.max (87%) 8.7 F5/HPLC-F65 10
Inh.sub.max (78%) 7.8 F5/HPLC-F22 10 Inh.sub.max (33%) 3.3
F6/HPLC-F86 10 Inh.sub.max (43%) 4.3 P1 10 Inh.sub.max (100%)
10.0
6.17. Isolation of Oligosaccharides from Heparin Degradation
Products
In a manner similar to that described, above, for the degradation
of ECM, intact heparin was treated with heparanase enzymes obtained
from various sources, designated herein as MM5 and PC3 (See,
Shoseyov, O. et al., Biochem., Biophys. RES. COMM. (1990)
169:667-672, for the preparation of PC3 enzyme). Some of the
starting degradation mixtures were separated on Sepharose 4B (See,
FIG. 26 for Heparin+MM5 Sepharose 4B fractions F7 and F8, and FIG.
27 for Sepharose 4B chromatography of Heparin+PC3 and PC3 alone).
Still other fractions were further separated by the HPLC II methods
described above (See, FIGS. 28A and 28B for fractions F7/HPLC-F86,
-F84, and -F90). "Intact" heparin and Fragmin were also subjected
to direct HPLC II conditions and selected fractions were likewise
isolated (HPLC-F90 from Fragmin is presented in FIGS. 29A and B.
The results of in vitro bioassays using human PBLs for various
active substances and starting "mixtures" are presented in Table
XIX, including the products obtained from the degradation of
heparin.
TABLE XIX Effect of Intact Heparin, Heparin + MM5 or PC3 "SOUPS",
and Selected Sepharose 4B and HPLC Fractions of Same on the
Secretion of Active TNF Using Human PBL Bioassay. Bioassay of Test
TNF Activity "R" value Material conc (pg/ml) (%) % .times.
(pg/ml).sup.-1 Intact Heparin a No effect 0 Hep/HPLC-F90 a No
effect 0 Additional Heparin Fractions F7/HPLC-F86 0.1 Inh.sub.max
(26%) 260 F8/HPLC-F84 a No effect 0 F8/HPLC-F90 a No effect 0
Hep/PC3 "Soup" 0.1-10 .times. 10.sup.6 No effect 0 Seph.4B-F9 100
Inh.sub.max (50%) 0.5 Seph.4B-F8 100 Inh.sub.max (40%) 0.4 PC3 only
a No effect 0 Seph.4B-F8 a No effect 0 Seph.4B-F9 a No effect 0 a
At a conc range of 1 .mu.g/ml - 0.01 pg/ml.
6.18. Results of In Vivo DTH Reactivity of Mice Treated with
Various Substances
A variety of substances were tested under in vivo bioassay
conditions and found to inhibit the experimental DTH sensitivity of
mice to different extents depending on their state of purification.
The mice were treated with an active substance, as described in
Sections 5.1 and 5.2, above. Generally, the substances that have
been purified to substantial homogeneity by high pressure liquid
chromatography provide "R" values in the tens of thousands. The
results for one group of experiments are presented in Table XX.
TABLE XX Weekly Treatment of Mice with Various Substances and Their
Effect on the DTH Sensitivity of Mice. DTH Inhibition Test Dose
Response of DTH "R" value Material (.mu.g/gm mouse) (10.sup.-2 mm)
(%) % .times. (.mu.g/gm).sup.-1 None -- 17.2 .+-. 2 0 -- (-)
Control 2 -- -- 0.5 M NaCl -- 16.5 .+-. 1.5 5 -- Intact Heparin a
-- No effect 0 Fragmin Batch 38609 0.2 3 .+-. 1 85 425 DECM MM5
"Soup" a -- No effect -- Seph.4B-F6 0.032 16.5 .+-. 5 5 -- 0.016 16
.+-. 2 10 -- 0.0032 14 .+-. 2 20 -- 0.0006 7.1 .+-. 1 65 (max)
110,000 F6/HPLC-F9 0.032 11 .+-. 2 40 -- 0.01 17 .+-. 2 0 -- 0.002
6 .+-. 0.5 70 -- 0.0006 6 .+-. 1.2 70 (max) 120,000 F6/HPLC-F11
0.02 17 .+-. 3 0 -- 0.01 13 .+-. 1 25 -- 0.001 9.5 .+-. 2.5 55 --
0.0006 2.8 .+-. 0.5 90 (max) 150,000 F6/HPLC-F12 0.02 18 .+-. 2 0
-- 0.01 15 .+-. 2 15 -- 0.001 8.2 .+-. 1.5 60 -- 0.0006 4 .+-. 1 80
(max) 130,000 a At a dosage range of 0.04-0.0004 .mu.g/gm
mouse.
As noted in Table XX, intact heparin and the starting ECM+MM5
"soup" exhibited no in vivo effect. In the latter case, no effect
is obtained most likely because of the counterbalancing effects of
inhibitory and augmentative components. A fresh sample of Fragmin
(Batch 38609) exhibited a modest "R" value, comparable to th at
obtained in earlier experiments (See, first entry, Table IX). A
Sepharose 4B fraction manifested a slightly lower "R" value than
the corresponding fractions obtained under HPLC II conditions.
The results from another set of experiments, listed in Table XXI,
confirmed the absence of any effect from the starting ECM+MM5
"soup". Notably, an HPLC II fraction, no. F5/HPLC-L22, showing very
high specific regulatory activity when injected subcutaneously into
mice ("R" value=454,545%.times.(.mu.g/gm).sup.-1), also
demonstrated oral activity albeit at a higher dose ("R")
value+5,000%.times.(.mu.g/gm).sup.-1). It is also apparent from
Table XXI that active substances isolated from the ECM have greater
in vivo specific regulatory activity than those obtained from
Fragmin. Hence, the apparent desulfation reduces the specific
inhibitory activity of the active substances of the present
invention. In fact, under in vitro bioassay conditions,
augmentative "R" values are obtained from such "desulfated"
disaccharides.
Further experiments have also demonstrated that galactosamine, a
monosaccharide or simple sugar having no sulfate groups, is capable
of acting as an antagonist of the inhibitory activity of the
sulfated oligosaccharides of the present invention. Thus, it is
also possible that the desulfated oligosaccharides act as direct
augmentative components or as antagonists of the specific
inhibitory activity of the carboxylated and/or sulfated
oligosaccharides. The observations of the present investigators are
also consistent with a mechanism by which certain substances (e.g.,
a trisulfated disaccharide) behave as agonists of an as yet
unidentified natural inhibitor of active TNF-.alpha. secretion.
TABLE XXI In Vivo DTH Reactivity Data for Mice Treated
Subcutaneously With Various Substances Dose DTH Test (.mu.g/gm
Response Inh.sub.max "R" value Material mouse) (10.sup.-2 mm) (%) %
.times. (.mu.g/gm).sup.-1 ECM + MM5 a 20.4 .+-. 0.7 No effect 0
"Soup" F5/HPLC-L22 0.008.sup.b 13.2 .+-. 1.3 40 .+-. 12% 5,000
F5/HPLC-L22 0.000132 9.7 .+-. 1.3 60 .+-. 17% 454,545 FRAGMIN
FR/HPLC-2 0.00048 8.5 .+-. 1.3 70 .+-. 20% 145,833 a At a dose
range of 0.04-0.0004 .mu.g/gm mouse. .sup.b Administered orally.
Positive control group had DTH response of 20.0 .+-. 1.1 and the
negative control group had a DTH response of 2.0 .+-. 1.0.
6.19. Comparative In Vivo Activity of SAX-HPLC Fractions Versus
Known Disaccharide Markers
Two disaccharide compounds were tested under in vivo conditions to
determine their ability inhibit the relative DTH reactivity of
mice. As shown in Table XXII, the two compounds (H-1020 and H-9267)
exhibited moderate activity having "R" values between
140,000-160,000%.times.(.mu.g/gm).sup.-1 when injected
subcutaneously into mice. The compound, H-1020, was also tested
orally and found to have modest activity ("R"
value=531%.times.(.mu.g/gm).sup.-1). The H-1020 compound is an
O,N-di-sulfate, whereas the H-9267 marker is an O,O,N-tri-sulfate.
Their structures are depicted, below. ##STR4##
As presented in Table XXII, the SAX-HPLC fraction L22/SAX-A23/4
(FIG. 24) provided further improvement over the already high
specific regulatory activity of F5/HPLC-L22, giving an "R" value of
630,303%.times.(.mu.g/gm).sup.-1 compared with an "R" value of
454,545%.times.(.mu.g/gm).sup.-1 for F5/HPLC-L22 (Table XXI). It
was discovered, however, that this disaccharide substance, with a
retention time through the SAX-HPLC column which is almost
identical to the retention time of H-9267, loses its sulfate groups
at pH .about.3.5 over a few days at room temperature. Thus,
reanalysis of an aged sample through a SAX-HPLC column revealed
that the original peak at 23.10 min. had given way to three major
peaks, designated 2039/1, 203912, and 2039/3 in FIG. 30, all having
retention times shorter than A23/4. Peak 2039/3, having a retention
time similar to the H-1020 marker, is likely to have lost an
N-sulfate group. Peaks 2039/1 and 2039/2 likely correspond to
monosulfated or fully desulfated disaccharides. (Their retention
times are comparable to a disaccharides marker, H-0895, an
N-acetylglycosaminoglycan having no sulfate groups.)
These "desulfated" substances were each collected and tested under
the in vivo DTH bioassay conditions and found, surprisingly, to
have only moderate or no inhibitory activity. (See, Table XXII).
Indeed, under the in vitro human PBL assay, all three peaks
manifested augmentation of active TNF-.alpha. secretion. These in
vitro results are given, immediately below:
SAX-HPLC conc Aug.sub.max "R" value Peak (pg/ml) (%) % .times.
(pg/ml).sup.-1 20391/1 1 5 5 2039/2 1 35 35 2039/3 1 42 42
TABLE XXII Additional In Vivo Results Using a Variety of
Disaccharides Administered Subcutaneously Dose DTH "R" Value Test
(pg/gm Response Inh.sub.max % .times. Material mouse) (10.sup.-2
mm) (%) (.mu.g/gm).sup.-1 PBS -- 18.6 .+-. 0.7 -- -- (Pos. Control)
Naive -- 1.4 .+-. 0.2 -- -- (Neg. Control) SAX-HPLC Fractions A23/4
0.000132 3 .+-. 1 83 630,303 2039/3 0.0005 3.1 .+-. 1.1 83 166,000
2039/1 0.000132 18.5 .+-. 1.1 No effect 0 "Markers" H-1020 0.0003
4.7 .+-. 0.7 73 146,000 H-1020 0.128.sup.a 5.9 .+-. 0.9 68 531
H-9267 0.0005 3.5 .+-. 1 80 160,000 .sup.a Administered orally.
6.19.1. Further Results of the Ability of Selected Disaccharides to
Regulate the In Vivo Production of Active TNF-.alpha..
Additional experiments were performed in which selected
disaccharide molecules, commercially available from Sigma Chemical
Co. and identified herein by their respective Sigma Catalog Nos.,
were tested for their the ability to inhibit or augment the
experimental DTH reaction in mice and, thus, offer an indication of
their ability to regulate the production by these mammals of active
TNF-.alpha..
In particular, CD1 mice (available from the Weizmann Institute
Animal Breeding Center, Rehovot, Israel), 4-12 mice per group, were
treated as described in Section 5.2 or 6.8.
The results of the various experiments are summarized in Table
XXIIIA, below. As can be seen, four of the eleven disaccharides
tested exhibited an inhibitory effect on the swelling of the ears
of the mice in response to the administered oxazolone. The
inhibition of the T cell-mediated inflammatory response is thus
seen as an indication that the disaccharides exhibiting a non-zero
"R" value can down regulate the production of active TNF-.alpha..
From the results listed in the Table, the "R" values range from a
relatively modest 65,000%.times.(.mu.g/gm).sup.-1 to over about
1,500,000%.times.(.mu.g/gm).sup.-1. It should be pointed out that a
high "R" value is not necessarily the most desirable characteristic
of the active compounds of interest. In particular, the dose
"window" within which a particular compound exhibits physiological
effects should be as broad as possible so that there is less
likelihood that the dose administered will fall outside the
effective dose range. As indicated in the footnotes of Table
XXIIIA. the molecule H-9392 appears to have the broadest dose
window, 0.000132-0.004 .mu.g/gm, of the compounds tested.
Equally evident from the results is the surprising ability of one
of the eleven disaccharides tested to augment the swelling caused
by the experimental DTH T cell reaction. Compound H-0895, which has
no sulfate groups, displays a tremendous effect on the degree of
swelling at the very low dose of about 1.2 picograms disaccharide
per gram mouse. The resulting "R" value of about
76,700,000%.times.(.mu.g/gm).sup.-1 is presently unrivaled.
TABLE XXIIIA Additional In Vivo Results Using a Variety of
Disaccharide Markers Administered Subcutaneously. Test Dose DTH
Inh.sub.max "R" value material (.mu.g/gm) (10.sup.-2 mm) (%) %
.times. (pg/gm).sup.-1 H-9392.sup.a 0.0012 4.2 .+-. 0.7 78 6.5
.times. 10.sup.4 (17.8 .+-. 0.9) H-1020.sup.b 0.0004 6.7 .+-. 1.1
66 1.65 .times. 10.sup.5 (19.6 .+-. 1.2) H-9267.sup.c 0.0004 7.2
.+-. 1 64 1.60 .times. 10.sup.5 (19.6 .+-. 1.2) H-9517.sup.d
0.00004 7.6 .+-. 1 62 1.55 .times. 10.sup.6 (20.4 .+-. 0.7)
H-0895.sup.e 0.0000012 37.3 .+-. 0.7 +92 7.67 .times. 10.sup.7
(18.9 .+-. 0.7) H-9017.sup.f N.E. 0 H-8642.sup.g N.E. 0
H-9142.sup.h N.E. 0 H-8767.sup.i N.E. 0 H-8892.sup.j N.E. 0
H-1145.sup.k N.E. 0
The structures of four inhibitory disaccharide compounds, H-9392,
H-9517, H-1020 and H-9267, are presented below. ##STR5##
Of the eleven disaccharides tested six failed to exhibit any
consistent effects and, thus, may be classified as "neutral." The
structures of these neutral compounds are presented below.
##STR6##
Of the eleven, one disaccharide augmented the effects of the
experimental DTH reaction. This compound, H-0895, has the structure
presented below. ##STR7##
It is thus possible to propose a generic formula that embodies the
structural characteristics of the inhibitory compounds. This
generic formula (H-GENUS) is shown below: ##STR8##
in which, X.sub.1 is H or SO.sup.3 ; X.sub.2 is H or SO.sup.3 ; and
X.sub.3 is SO.sup.3 or COCH.sub.3, provided that if X.sub.3 is
SO.sup.3, then at least one of X.sub.1 or X.sub.2 is SO.sup.3 and
if X.sub.3 is COCH.sub.3, then both X.sub.1 and X.sub.2 are
SO.sup.3. In terms of a compound having a fairly broad window of
effective dosages, X.sub.1 is preferably SO.sup.3, X.sub.2 is
preferably H, and X.sub.3 is preferably SO.sup.3 (i.e., H-9392 has
the broadest window of effective inhibitory dosages).
One may also observe from the results presented above that, in
terms of inhibition, the preferred substituent at the glycosamine
nitrogen is sulfate. With a sulfate at the 2-N position of
glucosamine (X.sub.3), inhibitory activity is observed with the
presence of just one other sulfate either at the 2-position of the
iduronic acid residue or the 6-position of the glycosamine. The
presence of two additional sulfates at both hydroxyl positions
would also work, but the absence of any additional sulfate (as in
H-1145) produces a "neutral" compound.
By contrast, the introduction of an acetyl group at the 2-N
position of glucosamine requires the presence of two additional
sulfates, one each for 2-position of the iduronic acid (X.sub.1)
and the 6-position of the glucosamine (X.sub.2). The absence of any
substituent at the 2-N position of glucosamine, giving rise to a
positively charged ammonium group, effectively cancels any
inhibitory effect, as evidenced by the fact that all of the test
compounds, H-9017, H-8892; and H-9142, were "neutral." The presence
of one or two sulfate groups at the 2-position of the iduronic acid
or the 6-position of the glucosamine had no apparent effect.
Finally, the presence of an acetyl group at X.sub.3 combined with
the absence of sulfate groups at X.sub.1 and X.sub.2 give rise to
an augmenting regulatory activity (H-0895).
One should note the strong correlation between the negative charges
present in the disaccharide and its ability to inhibit the
production of TNF-.alpha.. The presence of the positively charged
ammonium substituent gives rise to "neutrality," whereas the
charge-neutral compound H-0895 augments the production of active
TNF-.alpha..
TABLE XXIIIB Empirical Rules Gleaned From the Results of In Vivo
DTH Studies Involving Commercially Available Disaccharides.
Identity of X.sub.# in H-GENUS Observed X.sub.3 X.sub.2 X.sub.1
Activity SO.sup.3 SO.sup.3 SO.sup.3 Inhibition SO.sup.3 SO.sup.3 H
Inhibition SO.sup.3 H SO.sup.3 Inhibition SO.sup.3 H H Neutral
COCH.sub.3 SO.sup.3 SO.sup.3 Inhibition COCH.sub.3 SO.sup.3 H
Neutral COCH.sub.3 H SO.sup.3 Neutral COCH.sub.3 H H Augmentor
H.sup.2+ SO.sup.3 SO.sup.3 Neutral H.sup.2+ SO.sup.3 H Neutral
H.sup.2+ H SO.sup.3 Neutral
6.19.2. Results of the Ability of Selected Monosaccharides to
Regulate the In Vivo Production of Active TNF-.alpha..
Additional experiments were performed in which selected
monosaccharides commercially available from Sigma, were tested for
their ability to regulate the in vivo production of active
TNF-.alpha.. Using substantially the same procedure described in
the preceding Section, CD1 mice, 6 to a group, were inoculated and
treated with a variety of control and test substances to determine
the effect, if any, of the subcutaneously injected substances on
the experimental DTH reaction of the test animals. The results of
these experiments are presented in the Table below.
TABLE XXIIIC Additional In Vivo Results Using a Variety of
Monosaccharides Administered Subcutaneously Test Dose DTH
Inh.sub.max "R" value material (.mu.g/gm) (10.sup.-2 mm) (%) %
.times. (.mu.g/gm).sup.-1 GlcN.sup.a 0.000012 5 .+-. 0.7 75 6.25
.times. 10.sup.6 (19.7 .+-. 1.2) 0.4 2.3 .+-. 0.9 88 2.2 .times.
10.sup.2 (19.7 .+-. 1.2) GlcN-2S.sup.b N.E. 0 GlcN-3S.sup.c N.E. 0
GlcN-6S.sup.d N.E. 0 GlcN-2,3S.sup.c 0.0012 6.3 .+-. 1 68 5.67
.times. 10.sup.4 (19.7 .+-. 1.2) GlcN-2,6S.sup.f 1.2 7.7 .+-. 1 61
5.08 .times. 10.sup. (19.7 .+-. 1.2) NAc-GlcN.sup.g N.E. 0
GalN.sup.h 0.00004 8.9 .+-. 0.6 50 1.25 .times. 10.sup.6 (18.9 .+-.
0.7) 0.12 6.5 .+-. 0.7 67 5.58 .times. 10.sup.2 (18.9 .+-. 0.7)
The stereochemistry of the hydroxyl at the 4-position of the
monosaccharide determines whether the sugar is glucosamine
(.alpha.-face) or galactosamine (.beta.-face), as indicated, below.
##STR9##
It thus appears that N-acetylation or the presence of one sulfate
in the monosaccharide interferes with the ability of glucosamine to
inhibit the DTH reaction in mice.
6.19.3. Treatment of Adjuvant Arthritis (AA) in Rats with Selected
Monosaccharides and Disaccharides.
AA was induced in female Lewis rats, 6-8 weeks old, as described in
Section 5.7, above. Groups of rats, 5-10 rats per group, were
treated with a test substance by subcutaneous injection 1 day
before induction of the experimental arthritis and subjected to
repeat treatments weekly thereafter. The effects, if any, were
scored as described in Section 5.7.
Of the three disaccharides tested, H-9392 showed the most
pronounced effect in lowering the AA score relative to control
groups of rats which received only saline (0.1 ml). As illustrated
in FIG. 38A, H-9392, administered at 120 ng/rat or 0.6 ng/gm rat,
suppressed the destructive inflammation of AA almost completely (up
to about 90%) 24days after induction and H-1020 inhibited the
development of AA by about 30% relative to control at day 24. In
contrast, the augmentor, H-0895, showed an increased level of AA
development within about 2 weeks of induction at 2 dosage levels:
0.1 and 0.4 ng/rat. This effect, however, faded rapidly after that
until, at day 24, the AA score was not substantially different from
control levels. (See, FIG. 38B.)
On the other hand, certain monosaccharides were also found to
exhibit in vivo inhibitory effects in this rat model. As shown in
FIG. 38C, glucosamine treatment at three dosage levels inhibits the
development of AA by about 60-80% of control levels. Galactosamine
also exhibits inhibitory effects but to a much lesser degree than
that shown by glucosamine. (See, FIG. 38D.)
Most interestingly, further experiments carried out with H-9392, in
which the disaccharide was administered either weekly or daily
beginning at day 0 (start of induction of AA) or at day 12 (the rat
is already suffering from AA), showed positive suppression of the
severity of AA in all cases. The results of these experiments are
presented in FIGS. 38E (weekly) and 38F (daily). As indicated in
FIG. 38E, weekly administration of H-9392 beginning at day 12, that
is, even after the rat is already afflicted with AA, is at least as
effective as weekly treatment at the start of induction (day 0)
relative to control. FIG. 38F shows that while daily treatment of
afflicted rats was not as effective as daily treatment beginning at
the start of induction, the daily treatment of rats with
established arthritis was still highly effective at lowering the
score of the AA relative to the control group.
These results show dramatically that the substances of the present
invention are effective not only in preventing the development of
severe arthritis but are also effective in treating established
arthritis. Furthermore, the present work also demonstrates that
while the LMWHs described previously show inhibitory
characteristics only when administered weekly, the disaccharides of
the present invention are able to manifest useful inhibitory
activity when administered weekly or daily.
As a further illustration of the superiority of the compounds of
the present invention in the treatment of experimentally-induced
AA, a separate, comparative set of experiments was carried out in
which groups of Lewis rats (5 rats per group) were treated by
subcutaneous injection with either dexamethasone phosphate
(purchased from Sigma, a known antiinflammatory agent) or the
disaccharide 9392. Treatments were begun 12 days after induction of
the adjuvant arthritis (AA) disease and consisted of two treatment
regimens: the first involving daily injection of the known
antiinflammatory agent; the second involving weekly injection of
the known antiinflammatory agent or the disaccharide. In all cases,
100 .mu.g of the known antiinflammatory agent in 0.1 ml of
phosphate buffer solution was administered to each rat, while 120
ng of the disaccharide, also in 0.1 ml of phosphate buffer
solution, was administered per rat. As a control, a group of rats
was injected with 0.1 ml of phosphate buffer solution only. For the
daily dose regimen of known antiinflammatory agents, treatments
were ended after day 17 post-induction and for the weekly dosage
regimen of known antiinflammatory agent or disaccharide, the
treatments were ended after day 26 post-induction.
The results of the above experiments are illustrated in FIGS. 38G
and 38H. Upon examination of FIG. 38G, one sees that the weekly
administration of the disaccharide 9392 compares well with the
daily administration of dexamethasone phosphate during about the
first week of treatment. Note, however, that after treatment has
ended (after day 26), the group of rats that received daily
dexamethasone phosphate suffered a relapse while the disaccharide
group continued to improve. At 30 days post-induction of AA, the
disaccharide group fared better than the dexamethasone phosphate
group.
What is more, comparing the effectiveness of weekly dexamethasone
phosphate vs. weekly disaccharide 9392, as illustrated in FIG. 38H,
one sees that at 30 days post-induction of the AA, weekly
administration of dexamethasone phosphate resulted in only a
moderate reduction in the severity of the AA score. In contrast,
the weekly administration of disaccharide 9392 at 30 days
post-induction of the AA gave rise to an almost complete
suppression of the experimentally-induced adjuvant arthritis.
Again, of particular note, after weekly treatment was ended for the
dexamethasone phosphate, the rat suffered a relapse of the adjuvant
arthritis. As noted previously, the rats treated with the
disaccharide 9392, however, continued to improve even after
administration of the disaccharide had ceased.
Thus, the weekly administration of the disaccharide 9392 is
manifestly superior over the daily or weekly administration of
dexamethasone phosphate over the long term. Whereas the rats
treated with dexamethasone phosphate, either daily or weekly,
suffered a relapse of the disease after treatment was ended, the
rats treated with the disaccharide continued to exhibit improved AA
scores, reflecting a continued post-treatment inhibition of the
disease.
6.19.4. Results of Experiments Relating to the Lipopolysaccharide
(LPS)-Induced Inflammation of the Rat Cornea.
LPS-induced inflammation of the cornea is TNF dependent, as shown
by the work of Vanderhagen, C. and co-workers in the Netherlands,
"Kinetics of Intraocular TNF and IL-6 in Endotoxin-Induced Uveitis
in the Rat," submitted for publication. Using a 30-gauge needle,
LPS (5 ng) was injected into the cornea of Lewis rats. After one
day, separate groups of rats, 2 to a group (or 4 eyes to a group),
were then injected subcutaneously with phosphate-buffered saline
(0.05 ml) or H-1020 (at a dose of either 50 ng/rat or 200 ng/rat).
The effects, if any, were scored as follows:
edema 0-3 points neovascularization 0-3 points redness 1/0 points
swelling 1/0 points hemorrhage 1/0 points miosis 1/0 points
synaechi 1/0 points (iris adherent to lens or cornea) hypopyon 1/0
points (pus or blood in anterior chamber) hazy cornea 1/0
points
As can be seen from the graphical representation of the results
(FIG. 39A), the 50 ng/rat dose was effective to suppress the
effects of the local LPS-induced inflammation relative to control.
Interestingly, a dose of 200 ng/rat failed to provide a significant
effect.
6.19.5. Results of Experiments Relating to the Lipopolysaccharide
(LPS)-Induced Uveitis in Rats.
Uveitis is the inflammation of the anterior chamber of the eye in
response to LPS given systemically. Like the inflammation produced
in the preceding Section by the local administration of LPS,
uveitis is TNF-dependent. In the present experiment, groups of
Lewis rats, 8 to 10 weeks old, 8 eyes per group, were treated at
Day 1 with either H-9392 (at a dose of 32 ng/rat or 500 ng/rat) or
saline (0.1 ml). At Day 2, a 2 mg/ml solution of LPS (50 .mu.l) was
injected into each foot pad; each rat received a total of 200 .mu.g
LPS. At Day 3, each eye was tapped and the concentration of total
protein was measured as a quantitative assay of the degree of
inflammation. The results of these experiments are provided
immediately below.
Median Protein Rats Treatment (mg/ml) No LPS -- 0.36 LPS Saline
18.4 LPS 32 ng H-9392 5.2 LPS 500 ng H-9392 4.8
Hence, a single administration of H-9392, at either dosage, was
effective to suppress the inflammation produced by the systemic
administration of LPS in the rat by over about 70% relative to
control.
6.19.6. Results of Experiments Relating to the Radioprotective
Effects of Selected Substances.
Groups of female BALB/c mice, 8 weeks old, 5-10 mice per group,
were injected subcutaneously with either saline (0.1 ml, control),
H-9392 (30 ng/mouse), or glucosamine (10,000 ng/mouse) 1 day before
irradiation, and weekly thereafter until termination of the
experiment on day 30. All the mice were irradiated to a dose of 700
rads using a .sup.60 Co gamma radiation source.
The mortality within the different groups of mice was then scored
over a 30 day period. The results showed that the mice that
received only saline suffered a 100% mortality rate by day 30,
whereas the mice that had been pretreated with H-9392, showed only
a 40% death rate in the same period. The group of mice which had
been injected with glucosamine also fared better than the control
group, showing a 20% mortality rate in the same period.
Thus, pretreatment with the test substances of the present
invention, allowed the pretreated mice to survive a radiation
regimen that would have ordinarily resulted in a 100% mortality
rate for the group within 30 days.
In a separate experiment, groups of BALB/c mice (5 mice to a group)
were likewise exposed to 750 rads of gamma-rays, except that these
groups of mice were treated with test substances (saline at 0.1 ml
per mouse; H-9392 at 0.3, 3, 30 and 300 ng/mouse; and glucosamine
at 1, 10 and 100 .mu.g/mouse) on the day before irradiation, on the
sixth day after irradiation, and once again on the thirteenth day
after irradiation only. All treatment ceased after the third and
last administration. The results of this experiment are illustrated
in FIGS. 39B (saline and glucosamine) and 39C (saline and H-9392)
and indicate that while all the animals in the control group had
died by the twenty-second day after irradiation, only one mouse in
the 30 ng H-9392 group had died by the thirtieth day after
irradiation. The 300 ng H-9392 and the 1 mg glucosamine treatment
regimens showed moderate activity in suppressing mortality after
irradiation.
These results thus indicate a possible utility of the substances of
interest in cancer therapy in which the toxicity associated with
radiation treatment may be reduced dramatically by
proadministration of the instant compounds. This approach may,
perhaps, allow an increase in the dosage of the radiation to higher
more effective levels without observing toxic side effects. As
illustrated by the experiments described above, the compounds of
the present invention are highly effective at certain very low
dosages even when treatment is limited to three-administrations of
disaccharide.
6.19.7. The Ability of Selected Substances to Suppress Allograft
Rejection.
The effect of H-9392 was also tested in skin graft rejection
experiments in mice. In particular, skin grafts from C57BL/6 donor
mice (H-2.sup.b) were applied to recipient BALB/c mice (H-2.sup.d)
according to the method of Baharav, E. et al. J. Immunol. Methods
(1986) 90:143-144. The number of days to rejection was measured by
the sloughing of the graft. The number of days to rejection was
determined for a control group (injected with saline only, 0.1 ml),
and test groups that received 3 ng or 300 ng of H-9392 by
subcutaneous injection one day before grafting and weekly
thereafter. The results, which are graphically represented in FIGS.
40A and 40B, reveal that the 3 ng/mouse dose delayed the level of
skin graft rejection at 50% by 5 days! However, the same compound,
administered at 300 ng/mouse failed to produce a significant
difference at 50% rejection relative to control. These results are
very significant given that rejection of a fully allogeneic skin
graft is recognized to be one of the most powerful immune responses
known.
6.19.8. The Ability of Selected Substances to Suppress the
Development of IDDM in NOD Mice.
It is well known that NOD mice serve as a faithful model of human
diabetes Type I. Indeed, all female NOD mice in our colony develop
diabetes spontaneously within about 4-5 months of age. Because Type
I diabetes or insulin-dependent diabetes mellitus (IDDM) is
recognized as an autoimmune disease that may be precipitated by
autoreactive T cells, selected compounds of the present invention
were tested for their ability to regulate this T cell-mediated
autoimmune reaction.
Hence, groups of female NOD mice, 6-12 to a group, were treated by
subcutaneous injection with saline (0.1 ml), H-9392 (30 ng/mouse),
or glucosamine (10,000 ng/mouse). All the mice were about 3.5
months old, as shown in FIG. 41A, meaning that the mice as a group
already endured a 20% incidence of IDDM. The incidence of IDDM can
be monitored by the level of glucose in the blood of the mice.
Non-diabetic mice exhibit a mean glucose level in the blood of
about 140.+-.10 mg/ml. A mouse is considered a diabetic if its
blood glucose level is equal to or exceeds 200 mg/ml (i.e., is
greater than about three times the standard deviation of the
"normal" level). For greater convenience, glucose urine levels were
measured using the Clinstix.TM. dipstick (Ames). This test provides
scores of 0 to +3, with a score of +2 or greater on two separate
occasions taken as a positive indication of diabetes.
It was thus very surprising to discover that both the H-9392 and
glucosamine inhibited the onset of diabetes in NOD mice such that
at 4.5 months, when all the control mice were considered diabetic,
only about 65% of the glucosamine-treated mice had become diabetic,
while among the H-9392-treated mice, less than 50% were afflicted
with the disease.
Put another way, FIG. 41B shows that by age 5 months, all the
control mice had died from their diabetic condition. In contrast,
only about half of the mice treated with 10,000 ng of glucosamine
had died within the same time frame. Quite strikingly, none of the
mice treated with 30 ng of H-9392 died within the same time frame;
that is, the plot for the H-9392 results is coincident with the
x-axis.
6.19.9. Effect of Selected Disaccharides on TNF-.alpha.-induced
Expression of Adhesion Molecules, ICAM-1 and ELAM-1, by Endothelial
Cells (EC).
The adhesion molecules, such as ICAM-1 and ELAM-1, are critical in
the recognition and subsequent "rolling" (i.e., adherence to
endothelium and migration through the endothpelium) of leukocytes
involved in the inflammatory response. In response to active
TNF-.alpha., endothelial cells (EC) express ICAM-1 and ELAM-1.
Thus, TNF-.alpha. can augment inflammation by up regulating the
signals for leukocyte adherence and migration. To determine the
effect of the disaccharides of the present invention on the
TNF-.alpha.-induced expression by EC of ICAM-1 and ELAM-1, the
following experiment was carried out.
Freshly isolated human umbilical vein EC were grown in M-199 (Gibco
Laboratories) supplemented with 10% FCS, 8% human serum,
antibiotics and 50 .mu.g per ml endothelial cell growth factor
(EC-GM; Sigma, St. Louis, Mo.). The EC were seeded by adding 0.1 ml
of the EC-GM medium (3.5.times.10.sup.5 cells per ml.) to
flat-bottom 96-well plates (Nunk Roskilde, Denmark).
Confluent monolayer cultures were washed and incubated with
selected disaccharide compounds at various concentrations in 50
.mu.l of M-199 at 37.degree. C. for 1 h. The compounds were then
washed away, and the cultures were incubated over-night with
preformed TNF-.alpha., 200 IU per ml, in EC-GM. The cells were then
washed three times at 37.degree. C. with Hank's solution containing
1% FCS (Hank's 1%), and fixed with 2% glutaraldehyde in PBS. The
cells were then washed three times with Hank's 1%, blocked with
2.5% BSA in PBS, and rewashed twice with Hank's 1%. Anti-ICAM-1 and
ELAM-1 mAb (Genzyme, Cambridge Mass.; diluted 1/1000 in PBS) were
incubated with the cells for 1 h at 22.degree. C. and, then, washed
off three times with Hank's 1%. Peroxidase-conjugated goat
anti-mouse Ab sigma, diluted 1/1000) was incubated with cells for 1
h and, subsequently, washed off. After adding the
o-Phenylenediamine (OPD) prepared by dissolving an OPD
hydrochloride tablet in water (OPD is a substrate for peroxidase
and can be obtained from Sigma, Cat. No. p9187), the absorbance was
detected in an ELISA reader at 492 nm. Samples were assayed in
triplicate, and the average of at least three different assays were
calculated.
TABLE XXIIID Effect of Disaccharides on the Expression of Adhesion
Molecules by Endothelial Cells in Response to Preformed
TNF-.alpha.. Inhibitory Recombinant human TNF.alpha.- TNF.alpha.
treatment compound induced adhesion of EC [pg/ml] molecules (O.D
492)*: None None 0.12 .+-. 0.01 0.18 .+-. 0.01 Yes None 1.2 .+-.
0.1 2.2 .+-. 0.2 Yes 9392 [50] 0.6 .+-. 0.1 1.0 .+-. 0.04 Yes 9392
[100] 0.9 .+-. 0.07 1.4 .+-. 0.03 Yes 1020 [50] 0.7 .+-. 0.05 1.2
.+-. 0.1 Yes 1020 [100] 0.9 .+-. 0.03 1.5 .+-. 0.07 *Expression of
ELAM-1 and ICAM-1 detected by ELISA binding of specific monoclonal
antibodies
The experiments described above demonstrate that pretreatment of EC
with disaccharide compounds 9392 and 1020 endows the EC with
significant resistance to preformed TNF-.alpha.. Hence, the up
regulation of EC expression of adhesion molecules induced by
TNF-.alpha. was inhibited by up to 50%. These results mean that the
compounds of the invention can influence the target cells of
TNF-.alpha. (e.g., EC), as well as the cells that produce
TNF-.alpha. (e.g., T cells, macrophages) to inhibit not only the
production of active TNF-.alpha. but also the propensity of the
target cells to respond to TNF-.alpha. (i.e., regulation of
peripheral reception of the cytokine). Certain disease states,
then, may benefit from the administration of the substances of the
present invention by imparting on the target cells of TNF-.alpha. a
type of resistance against the cell-induced inflammatory response
initiated by the activated. T-cells and macrophages.
6.19.10. The Ability of Substance H-9392 to Suppress the Signs of
Experimental Allergic Asthma in Rats
Experimental allergic bronchial asthma is an immediate type
hypersensitivity reaction in rats which have been immunized and
then rechallenged by inhalation of the priming antigen in a
aerosolized solution. (Edelman, et al., Am. Rev. Rest. Dis. (1988)
137:1033-37). The etiology and pathophysiology of this experimental
disease closely parallel the naturally occurring human
counterpart.
To assess the ability of substance H-9392 to prevent a bronchial
asthma attack, 6 male Brown Norway rats were primed to ovalbumin
(OVA) by subcutaneous injection of 1 mg of OVA in suspension with
200 mg AlOH/ml 0.9% saline solution accompanied by intraperitoneal
injection of 1 ml solution containing 6.times.10.sup.6 heat killed
Bordetella pertussis bacteria (Pasteur Merieux, S. V.), on day
zero. Subsequent challenges consisted of a 5 minute period of
inhalation of OVA (1 mg/ml solution) aerosolized in a Devilbiss
Nebulizer operated at an airflow of 6 L/min. Respiratory Distress
(RD) responses were scored as Grade 0, no signs of distress; Grade
1, tachypnea; Grade 2, moderate labored breathing; Grade 3 severe
labored breathing with mouth open; Grade 4, loss of consciousness
and muscular tone. Sixteen days following primary immunization all
animals were exposed to an initial aerosolized challenge to
establish the positive control. The animals were coded so that the
observer was ignorant of the subject animal's history. All rats
were sensitive to OVA and asthma was uniformly induced in all
animals.
On day thirty, the animals were divided into 2 groups and given
either saline (control Group A) or 30 ng of substance H-9392, s.c.,
(Group B) and challenged on day 35 as above. The results are
presented in FIG. 42. As shown in FIG. 42, the animals which
received saline only on day 30 experienced Grade 3 or 4 respiratory
distress. The animals treated with substance H-9392 displayed mere
tachypnea. The results presented show that administration of
substance H-9392 (5 days prior to secondary challenge), to an
animal with established hypersensitivity, blocks an asthmatic
attack.
6.20. Results of In Vitro Human PBL Bioassay on Commercially
Available Heparin Derived Oligosaccharides
Commercially available samples of heparin derived disaccharides and
polysaccharides ranging in molecular weight from 1,800 and 18,000
were tested for biological activity. The results are presented in
Table XXIII. As is evident from the data obtained, all the samples
tested, with one exception, gave either no effect or inconsistent
effects. As explained in the table footnote, a test result for a
particular entry was designated inconsistent if the same
qualitative result was not obtained for all three bioassay runs.
(Each entry in all the Tables included in this disclosure, which
present results of bioassays, was the product of at least three
tests. In the human PBL bioassay, each run used blood obtained from
different individuals.)
TABLE XXIII Effect of Commercially Available Heparin Disaccharides
on the Secretion of Active TNF Using Human PBL Bioassay. Bioassay
of Test TNF Activity "R" value Material.sup.a conc (pg/ml) (%) %
.times. (pg/ml).sup.-1 H 9517 .sup.b .sup. Inconsistent.sup.c -- H
8642 .sup.b Inconsistent -- H 8767 .sup.b Inconsistent -- H 0895
.sup.b Inconsistent -- H 8892 .sup.b No effect 0 H 9017 .sup.b No
effect 0 H 9142 .sup.b Inconsistent -- H 9267 .sup.b Inconsistent
-- H 1020 1 25% to 35% 25 to 35 H 9392 .sup.b Inconsistent -- MW
1,800.sup.d .sup.c Inconsistent -- MW 2,400 .sup.c Inconsistent --
MW 3,000 .sup.c Inconsistent -- MW 3,600 .sup.c Inconsistent -- MW
4,200 .sup.c Inconsistent -- MW 4,800 .sup.c Inconsistent -- MW
5,400 .sup.c No effect 0 MW 6,000 .sup.c No effect 0 MW 9,000
.sup.c No effect 0 MW 16,000 .sup.c Inconsistent -- MW 18,000
.sup.c Inconsistent -- .sup.a Product Number found in Sigma
Chemical Co. Product Catalog (1992). .sup.b At a conc range of 1
.mu.g/ml - 0.01 pg/ml. .sup.c Three bioassays using PBLs obtained
from three separate individuals were performed on each test
material. A test result for any given test material was designated
"inconsistent" if the same result (i.e., inhibition, augmentation,
or no effect) was not obtained for all three bioassays. .sup.d
Molecular weight designations for various Heparin Oligosaccharide
Fragments, as described in Serbio Product Catalog (Dec. 26, 1991).
.sup.c At a conc range of 1 .mu.g/ml - 0.01 pg/ml.
6.21. Results of In Vitro Human PBL Bioassay Versus Assay Kit Based
on mAb
A comparison of the activity data obtained from the in vitro
bioassay based on human PBLs described herein and the results of a
conventional monoclonal antibody-based assay kit shows that much
more protein is present in the media being tested than what is
being detected by the human PBL bioassay as being "active" TNF. The
results are presented in Table XXIV. For example, the difference
between the amounts of protein produced by T cells in "Control"
levels (ca. 274 pg/ml) and when Fragmin HPLC-F16 is present (ca.
200 pg/ml) is not nearly as dramatic as the difference in activity
detected by the human PBL assay for the same samples (a 100 percent
change in activity). Hence, one may conclude from these results
that although the activated immune effector cell may secrete
significant amounts of TNF protein, even in the presence of the
active substances of the present invention, only a small proportion
of the secreted protein is active enough to kill TNF-sensitive
cells. This conclusion supports the notion that TNF is produced in
both active and inactive forms.
TABLE XXIV Comparison of TNF Activity Detected by Human PBL
Bioassay Versus Amount of Protein Detected by mAb Immunoassay Kit.
Bioassay of mAb Assay Kit Test TNF Activity TNF Material (% killed)
(pg/ml) None 45 Control 273.9 Control Fragmin HPLC-F1 72
Aug.sub.max (60%) 284.5 Aug.sub.max (3.8%) HPLC-F3 13.5 Inh.sub.max
(70%) 224 Inh.sub.max (17.9%) HPLC-F16 0 Inh.sub.max (100%) 199
Inh.sub.max (27%) DECM HPLC-F10 18 Inh.sub.max (60%) 225
Inh.sub.max (17.5%) HPLC-F14 13.5 Inh.sub.max (70%) 232 Inh.sub.max
(15%) HPLC-F25 63 Aug.sub.max (40%) 292 Aug.sub.max (6.5%) HPLC-F37
90 Aug.sub.max (100%) 301 Aug.sub.max (10%)
6.22. Results of Preliminary Investigation of the Structural
Characteristics of ECM-Derived Disaccharide
A 20 .mu.g sample of an ECM-derived disaccharide was obtained after
HPLC II chromatography (FIG. 23) and desalting, as previously
described. Subsequent purification, under SAX-HPLC conditions,
showed this sample to be greater than 90% pure (FIG. 24, peak A23/4
at 23.10 min.). However, the proton NMR spectrum of this sample
(FIG. 31) shows that a contaminant having a repetitive aliphatic
--CH.sub.2 -- moiety is present. It is important to note,
nevertheless, that the SAX-HPLC purification; tested positively for
inhibition under both in vitro and in vivo bioassay conditions.
The proton NMR spectrum was recorded in D.sub.2 O at 500 MHz at
23.degree. C. A 2-dimensional COSY spectrum was also recorded. The
final matrix size was 512.times.512, N-COSY with presaturation,
1536 scans (FIG. 32). Typical sugar signals are evident between
3-5.5 ppm in both the one dimensional and 2-D spectra. A doublet
signal for the anomeric proton is found at 5.39 ppm having a
coupling factor of 3 Hertz (FIG. 33), consistent with the presence
of a glucosamine sugar unit in an alpha configuration.
The chemical shift of the anomeric proton, together with what is
believed to be the beta-glucuronide specificity of placental
heparanase, leads to the tentative conclusion that the disaccharide
has a glucosamine at the non-reducing end, in an alpha
configuration, attached 1.fwdarw.4 to a glucuronic acid residue at
the reducing end. Furthermore, the absence of a signal at 6 ppm
indicates that the disaccharide is saturated (i.e., there is no
double bond at C.sub.4 -C.sub.5 of the glucosamine residue). Thus,
the heparanase is not an eliminase but apparently a hydrolase.
FTIR spectra were recorded on a Mattson Galaxy 6020, equipped with
a transmission film detector; DTGS at resolution of 2 cm.sup.-1 ;
128 scans using a ZnSe window. FIGS. 34-35 indicate the presence of
a sulfated compound and a partially desulfated analog,
respectively. FIG. 34, in particular, exhibits absorptions at
3500-3000 (characteristic of carboxyl and hydroxyl groups), 1594
(carbonyl), 1394, 1121, 1107, 1074, 1005, 993, 936, and 852
cm.sup.-1, at least some of which, particularly the last, are
associated with sulfate.
The mass spectrum of a methyl derivative, believed to have lost
some sulfate, was obtained on a Jeol JMS HX/110A FAB. Xe beam was
used at E=6 kV, emission current=10 mA, acceleration voltage was 10
kV. First, the methyl derivative was prepared by treating a sample
of the oligosaccharide with diazomethane in acidic media. The
methylated product was next extracted with ethyl acetate. A
characteristic ion was observed above the background having M/Z
(M+H.sup.+)=531. Thus, it is believed that the data are consistent
with a molecular weight, for a methylated derivative, of about 530.
In order to verify the results, two different matrices were used;
DTT:thioglycerol (1:1) and methylnitrobenzyl alcohol (FIGS. 36A-B
and 37A-B, respectively). The "A" spectra correspond to
sample+matrix, whereas the "B" spectra relate to the specific
matrix only.
Based on such mass spectral data, a tentative chemical formula for
the methylated (partially desulfated) derivative can be proposed:
C.sub.13 H.sub.23 NO.sub.17 S.sub.2, MW=529.47.
4-O-(2-deoxy-6-O-sulfo-2-sulfoamino-.alpha.-D-glucopyranosyl)-(2-O-sulfo-.b
eta.-D-glucopyranoside)uronic acid is synthesized according to the
following protocol.
6.23. Synthesis of
4-O-(2-deoxy-6-O-sulfo-2-sulfoamino-.alpha.-D-glucopyranosyl)-(2-O-sulfo-.
beta.-D-glucopyranoside)uronic acid
6.23.1. Preparation of
6-O-Acetyl-2-azido-3,5-di-O-benzyl-2-deoxy-.beta.-D-glucopyranosyl
chloride
2-O-Tosyl-1,6:3,4-dianhydro-.beta.-D-galactopyranose [2] is
prepared from 1,6-anhydro-.mu.-D-glucopyranose [1], according to
Cerny et al. (1961) Coll. Chech. Chem. Cos. 26:2547.
2-O-Tosyl-1,6-anhydro-.beta.-D-glucopyranose [3] is prepared from
2-O-Tosyl-1,6:3,4-dianhydro-.beta.-D-galactopyranose [2] according
to Cerny et al. (1965), Coll. Chech. Chem. Soc. 30:1151.
1,6:2,3-dianhydro-.beta.-D-mannopyranose [4] is prepared from
2-O-Tosyl-1,6-anhydro-.beta.-D-glucopyranose [3] according to
Stanek and Cerny (1972) SYNTHESIS p. 698.
6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-.beta.-D-glucopyranosyl
chloride [A] is prepared from
1,6:2,3-dianhydro-.beta.-D-mannopyranose [4] according to Paulsen
and Slenzel (1978) Chem. Ber. 111:2334.
6.23.2. Preparation of Methyl-(benzyl-2-O-acetyl-3-O-benzyl-.beta.
and .alpha.-L-glucopyranosid)-uronate
1,2:5,6-Di-O-isopropylidene-.alpha.-D-glucofuranose [6] is prepared
from D-glucose [5] according to Stevens (1978), Methods Carbohydr.
Chem. 6:124.
3-O-Benzyl-1,2-O-isopropylidene-.alpha.-D-glucofuranose [7] is
prepared from 1,2:5,6-Di-O-isopropylidene-.alpha.-D-glucofuranose
[6] according to Whistler and Lake (1972), Methods Carbohydr. Chem.
6:286.
Methyl-(benzyl-2-O-acetyl-3-O-benzyl-.beta. and
.alpha.-L-glucopyranosid)-uronate is prepared from
3-O-Benzyl-1,2-O-isopropylidene-D-glucofuranose [7] according to
Jacquinet et al. (1984), Carbohydr. Res. 130:221.
6.23.3. Condensation of Products of 6.23.1 and 6.23.2
Coupling of the products of Section 6.23.1 and 6.23.2 above is
conducted according to Jacquinet et al. (1988), Carbohydr. Res.
174:253. O-deacetylation, hydrolysis, O-Sulfation, reduction, and
debenzylation is conducted according to Jacquinet et al. (1984)
(supra) to give
4-O-(2-deox-6-O-sulfo-2-sulfoamino-.alpha.-D-glucopyranosyl)-(2-O-sulfo-.b
eta.-D-glucopyranoside)uronic acid, which is further purified by
SAX-HPLC according to Rice et al. (1985), Anal. Biochem. 150:325.
The structure of this product is given in FIG. 43 and it is
believed it has the same biological activity as other compounds
described herein.
It should be apparent to those skilled in the art that other
compositions and methods not specifically disclosed in the instant
specification are, nevertheless, contemplated thereby. Such other
compositions and methods are considered to be within the scope and
spirit of the present invention. Hence, the invention should not be
limited by the description of the specific embodiments disclosed
herein but only by the following claims.
The disclosure of all references cited herein are incorporated
herein in their entirety by reference.
* * * * *