Note: Descriptions are shown in the official language in which they were submitted.
CA 02388974 2002-04-25
Medicament for inducing tolerance
Description
The invention relates to a medicament for inducing
tolerance, for immunomodulation or/and for inhibiting
inflammation, comprising inhibitors of 11-j3-hydroxy-
steroid dehydrogenase (11(3-HSD), where appropriate in
combination with an antigen.
The immune system is distinguished by the property of
being able to differentiate between hazardous, disease-
promoting endogenous and/or exogenous and nonhazardous
antigens.
The state referred to as tolerance in this connection
is one distinguished by systemic "passivity" or else
"ignorance" of the immune system in relation to a
specific antigen. It is immaterial in this connection
whether this antigen is endogenous (self-antigens) or
exogenous. Collapse of tolerance leads, if endogenous
antigens continuously maintain an immune defense, to
autoimmune diseases. Overreactions to environmental
antigens which are not disease-promoting per se are
embraced by the term allergies. In the area of trans-
plantation medicine, when there are unwanted immune
responses there is said to be a rejection response
against the transplant or, in the case of a defense
response of the transplanted material against the
recipient, graft versus host disease (GVHD). The term
tolerance includes in particular desensitization, so
that exogenous substances are tolerated, and mechanisms
leading to a reduction in immune responses to
endogenous substances.
The nature of the immunological tolerance depends on
the type of antigen and on the form and dose in which
it is presented to the immune system.
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It is assumed that tolerance can be based on at least
three different mechanisms. Firstly, dangerous, poten-
tially autoreactive T cells which react in the thymus
with antigens present therein are negatively selected,
i.e. they are killed (deletion). Another mechanism is
clonal anergy. This entails T cells which recognize an
antigen on an antigen-presenting cell but not if
costimulatory signals are present simultaneously being
deactivated and being no longer able to elicit an
immune response against this antigen later either. The
third component of tolerance is assumed to be
regulatory T cells, called suppressor T cells, which
can have immunomodulatory effects on immunological
processes which have already been initiated.
In the case of an immune response in the elimination
for example of a virus or else in nonspecific inflam-
mations, regulatory T cells contribute to restoring an
immunological balance and to terminating the immune
response. A deficiency of self-regulation of
unexplained cause or prevention thereof for example by
medicaments may lead to a pathological state such as
autoimmunity and allergies, or to an unwanted immune
response in the area of transplantation medicine.
Autoimmune diseases represent a situation in which
tolerance has wholly or partly collapsed. In humans,
they generally have a chronic degenerative course.
Remission, spontaneous or under immunosuppressive
therapy, has to date been observed only rarely. The
longer the disease process lasts, the more difficult it
appears to be to break the vicious circle of chronic
degenerative inflammation. Mechanisms such as so-called
epitope spreading appear to play an important part in
this (Craft and Fatenejad 1997; Moudgil 1998; Vaneden,
Vanderzee et al. 1988).
Oral tolerance is based on the fact that antigens taken
by mouth usually elicit no immune responses and
CA 02388974 2002-04-25
moreover prevent the same antigen being followed by an
immune response when it gets into the body at a later
time via a route which normally produces immune
responses . This has led to vigorous attempts to modify
or to restore through presentation (in particular by
mucosal administration) of a suitable specific antigen
or of another antigen which brings about an immune
response similar to that of the disease-relevant
antigen (Liblau, Tisch et al. 1977; Weiner 1997; Bonnin
and Albani 1998; Strobel and Mowat 1998).
Tolerance induction is becoming increasingly important
in medicine, especially in the control of autoimmune
diseases, also in desensitization against environmental
antigens such as, for example, in the treatment of hay
fever and other allergies and of asthma (Tsitoura et
al. 1999; Tsitoura et al. 2000).
Very recent findings also ascribe importance to the
induction of tolerance in the area of conventional
vaccination (McSorley et al. 1999).
Modern immunosuppressive therapies still display
considerable side effects, however, and are inadequate
if the onset of the disease has already taken place,
i.e. they produce no cure.
According to the current state of knowledge, the
success of tolerance induction or immunomodulation
depends both on the mode of presentation of the antigen
and on the immunological milieu of the tissue/organism
in which the desired immune response is to proceed.
Various theories have been suggested about the immuno
logical processes leading to tolerance induction
in vivo, and some of them are very controversial.
It has to date been assumed that tolerance, and in
particular orally induced tolerance, is connected with
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certain cytokine patterns. These cytokine patterns in
turn suggest various T-helper cell populations. A
distinction is made between Thl, Th2 and, recently
also, Th3 cells. Thus, it is generally assumed that
autoimmunity is dominated mainly by Thl cells and
tolerance more by Th2 cells. However, it has been
possible to show that immunological tolerance in animal
models can be maintained even in the absence of
functional Thl and Th2 cells.
Some specialists assume that with high doses of a
mucosally administered antigen there is selective
elimination of autoreactive T cells which recognize the
particular antigen (clonal elimination) (Gutgemann,
Fahrer et al. 1998). On use of low antigen doses, this
theory starts from the induction of regulatory T cells
which actively suppress the pathological immune
response (bystander suppression). These likewise
antigen-specific T cells are assigned to the Th2 or
else Th3 type or to T cell classes which have not been
characterized in detail (Weiner 1997; Mason and Powrie
1998; Seddon and Mason 1999). The pathological situa-
tion is moreover frequently defined in such a way that
it is generally caused by Thl T cells. A further theory
postulates a regulation which has not to date been
characterized in detail and which depends on direct
contact between individual T cells as possible
mechanism a bystander suppression (Tsitoura, DeKruyff
et al. 1999). Other hypotheses again postulate
influences, which differ in each case, of different
cytokines or mechanisms independent of cytokines for
tolerance induction (Segal and Shevach 1998; Lundin,
Karlsson et al. 1999; Rizzo, Morawetz et al. 1999;
Seddon and Mason 1999).
Within the theory based on counter-regulation between
Thl and Th2/3 cytokines, it appears that the cytokine
milieu in which the immune responses proceed has
crucial importance in the induction of active bystander
i
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suppression; i.e. the proliferation mainly of
presumably protective Th2/3 T cells can take place only
in a suitable milieu (Liblau, Tisch et al. 1997; Weiner
1997; Strobel and Mowat 1998).
"Milieu" means in this connection in immunology the
combination of various purely immunological factors
which determine the direction of an antigen-induced
immune response. Hormones and other factors influencing
the immune system are, however, not included in this
classically immunological concept.
The aim in developing novel substances and methods is
to optimize this milieu. The intentions in this connec-
tion are firstly to allow the desired immune response
to proceed reliably and in a targeted manner, and
secondly to minimize the required amount of antigen.
In the current state of the art, therapeutic induction
of tolerance in advanced stages is a problem, for
example in cases of rheumatoid arthritis, which has not
yet been mastered. Thus, this new therapeutic approach
appears to be successful only if the duration of the
disease is less than 2 years. Thereafter mechanisms
such as epitope spreading and also nonspecific
inflammatory reactions presumably make oral tolerance
induction difficult to make difficult (Albani, UCSD,
personal communication).
W098/21951 (Haas et al.) discloses general methods for
oral tolerance induction using an antigen with a
derivatized amino acid as adjuvant.
In US patent No. 5,935,577 of Weiner et al. it was
attempted to improve tolerance induction by administer-
ing a bystander antigen in combination with
methotrexate. One aim was to reduce the amount of
methotrexate which is associated with serious side
effects because of its toxicity. However, the current
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view is that it is desirable to be able to dispense
entirely with substances such as methotrexate because
methotrexate has a cumulative toxicity, i.e. liver
damage occurs above a certain amount.
Attempts have also been made to modify the immuno-
logical milieu inter alia in the direction of a
presumably protective milieu which favors the
proliferation of regulatory Th-2i3 by means of various
auxiliary substances such as, for example, cytokines
and antibodies against cytokines (patent
No. W095/27500, W098/16248). W095/27500 discloses a
method for oral tolerance induction using a bystander
antigen together with cytokines, in particular IL-4,
which directs the immune system toward a response which
is dominated more by T-helper cells of type 2 (Th2).
W098/16248 uses inhibitors of IL-12 for oral tolerance
induction.
However, there have been only a very few studies
attempting to control via endogenous mechanisms the
immunological processes necessary for targeted
tolerance induction.
However, because of the complex functions of cytokines
in immunoregulation and thus possible uncontrollable
side effects, there are great problems in manipulating
cytokines as a possible way of optimizing immune
responses and thus for clinical use in the induction of
tolerance. It has emerged that interventions into the
cytokine network are frequently accompanied by
hazardous side effects and incalculable risks. Thus,
the FDA has recently drawn attention to the fact that
there have already been 10 deaths associated with
administration of TNF antibodies for rheumatoid therapy
because an inflammatory reaction associated with a
disorder other than the basic rheumatoid disorder was
no longer controllable and thus had a fatal cutcome.
Similar worries relate also to the use of IL-12
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neutralizing antibodies and with IL-4 because of known
side effects.
A further aspect associated with current therapeutic
methods is the fact that oral tolerance induction in
some cases encompasses therapy cycles in which the
antigen must be taken every day for several weeks. It
is to be assumed at present that these cycles must be
repeated more than once because the tolerance status in
some circumstances requires continuous presence of the
antigen, at least until a complete cure has occurred.
It is not possible to deduce from the data published to
date whether a complete cure is possible.
Strategies to date for tolerance induction are usually
based on administration of supraphysiological amounts
both of the antigen and of the adjuvants. The
disadvantage associated with the use of high
concentrations of the agents employed are high costs
and, in some cases, serious side effects, as, for
example, with methotrexate.
A further disadvantage of the studies of tolerance
induction carried out and published to date is that
there is use mostly of animal models in which induction
of tolerance is shown by absence of initiation in the
treated animals of the disease occurring in control
animals. However, agents able to intervene also in the
preexisting disease are of much greater importance.
Glucocorticoids are known for their antiinflammatory
and immunosuppressant properties (Cupps and Fauci 1982;
Chrousos 1995; Marx 1995; Almawi, Beyhum et al. 1996,
Wilckens and Derijk, 1997). They are therefore
preferentially used for treating inflammatory disorders
and also for treating autoimmune diseases.
It has been known for some time that glucocorticoids
(GCs) are able to downregulate the production of
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certain cytokines. The antiinflammatory effect of
glucocorticoids is based not only on their ability to
inhibit interleukin-2 (IL-2) and interferon-y (IFN-y)
but also to downregulate IL-1, tumor necrosis factor a
(TNF-a) and IL-6. The GC concentrations in tissues and
plasma is in turn regulated by a plurality of mecha
nisms, namely by cytokines, GC receptor expression and
by certain enzymes, e.g. 11-~-hydroxysteroid
dehydrogenase (11-a-HSD); Hult, Jornvall et al. 1998;
Krozowski 1999; Stewart and Krozowski 1999).
Whereas wide-ranging knowledge about the part played
for example by cytokines has accumulated during
research into tolerance induction, little or nothing is
known about the influence of steroid hormones in
general and of glucocorticoids in particular. It is of
interest, however, that it has been demonstrated that
estrogens classified as immunosuppressant in their
effect on the immune system (Jansson and Holmdahl 1998)
prevent the induction of tolerance (Mowat, Lamont et
al. 1988). This is particularly remarkable inasmuch as
estrogens per se promote a TH2 immune response which is
said in some systems to assist oral tolerance (Weiner
1997). Combination of inactive or suboptimal concentra-
tions, administered alone in each case, of estrogens
together with glucocorticoids brings about synergistic
immunosuppression (Carlsten, Verdrengh et al. 1996). It
is concluded from this in general that glucocorticoids
also inhibit tolerance induction.
No published data exist on the effect of glucocorti-
coids on tolerance induction. However, it was found in
a preclinical phase I study at the University of San
Diego in California that a glucocorticoid therapy
conflicts with an attempted therapy to induce oral
tolerance. This is also reflected by the fact that
patients treated with glucocorticoids are generally
excluded from clinical studies on tolerance induction
in autoimmune diseases. Administration even of low
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steroid concentrations blocks active induction of
tolerance (S. Albani, University of California, San
Diego and H. Weiner, Harvard University, Boston,
personal communications, (Weiner 1997; Bonnin and
Albani 1998)).
It is true that under certain conditions there may be a
shift, under the influence of glucocorticoids, in the
cytokine pattern in the direction of a Th2-like
cytokine profile. However, the latter is inadequate as
therapy of an autoimmune process also within the
framework of measures to induce tolerance. Both
administration of Th2 cytokines and transfer of Th2
cells is unable to completely prevent an autoimmune
reaction (Mason and Powrie 1998; Segal and Shevach
1998 ) . In addition, it has been demonstrated in a very
recent study that inhibition of 11-(3-HSD leads, just
like glucocorticoid injection, to killing of immature
T cells in the thymus (Gruber, Sgonc et al. 1994;
Horigome, Horigome et al. 1999). It is known that
glucocorticoids have a similar effect on peripheral
T cells and on undifferentiated, naive and immature
T cells (Cupps and Fauci 1982). Induction of tolerance
on the other hand promotes inter alia the activation
and proliferation of these immature T cells, whereas
tolerance apparently cannot be induced in memory cells
(Chung, Chang et al. 1999). It is additionally assumed
that glucocorticoids inhibit antigen presentation
(Piemonti, Monti et al. 1999; Piemonti, Monti et al.
1999)), which additionally conflicts with assistance of
tolerance induction by definition.
Thus, immunological therapeutic methods currently fail
because of the deficient possibility of controlling the
immune response and also because of the costs because
very large amounts both of protein and peptide antigen
and DNA must be used in order to induce relevant
clinical effects in humans. Whereas considerable
advances have been achieved in the identification of
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possible antigens, at present no methods suitable for
use in humans and suitable for long-term use are
available. Immunological approaches appear at present
not to be very promising because of uncontrollable side
effects.
The present invention was therefore based on the object
of improving and/or optimizing tolerance induction
through administration of particular antigens in combi
nation with a novel adjuvant.
This object is achieved by a novel medicament compris-
ing as active ingredient inhibitors of 11-(3-hydroxy-
steroid dehydrogenase in combination with one or mare
antigens and the use of the medicament for inducing
tolerance.
11-~i-Hydroxysteroid dehydrogenase (11-(3-HSD) is an
enzyme which is responsible for interconversion of
biologically active forms of glucocorticoids into and
out of their inactive forms. It has been found,
surprisingly, that inhibitors of enzymes which regulate
cortisol metabolism, in particular inhibitors of
11-(3-HSD, are able in combination with an antigen to
induce tolerance. A particularly advantageous effect is
achieved in this connection in combination with at
least one antigen.
This is unexpected inasmuch as it is generally known
that an increase in the cortisol level in the plasma
(e.g. on administration of glucocorticoids) has an
immunosuppressant effect. However, a distinction must
be made between the cortisol concentration in blood
plasma, which has systemic effects, and an increase in
the cortisol level which is possibly confined only to
very particular cells or tissues. The mechanisms
applying on inhibition of 11-~3-HSD are apparently
different from those resulting from systemic cortisol
administration. It is evident that inhibition of
a~
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11-~i-HSD causes immunostimulation progressing to
tolerance induction, possibly through activation of
suppressor T cells.
11-~i-HSD is a member of the short-chain
dehydrogenases/reductases (SDR). Members of the SDR
family typically comprise about 250 amino acids and
have an N-terminal coenzyme binding pattern (typically
GXXXGXG) and an active binding site with the sequence
YXXXK. The SDR family is highly divergent with a
typical identity agreement of from 15 to 30~ between
the individual members. The enzymes of the SDR family
encompass a wide range of specific substrates,
including steroids, alcohols and aromatic compounds
(Jornvall et al., 1999; Oppermann et al., 1996).
11-(3-Hydroxy-steroid dehydrogenase is the key enzyme in
the extrahepatic conversion of 11-(3-hydroxysteroids
such as, for example, cortisol and prenisone into their
inactive metabolites. 11-(3-hydroxysteroid dehydrogenase
is a bidirectional enzyme which displays a reductase
or/and a dehydrogenase activity depending on the
environment and the isoform. The reductase activity of
11-(3-hydroxysteroid dehydrogenase converts an
11-ketosteroid, for example the inactive cortisone,
into an 11-~3-hydroxysteroid, for example the active
cortisol. The dehydrogenase activity converts the
11-(3-hydroxysteroid into the 11-ketosteroid.
Various isoforms of 11-(3-HSD exist. For example,
11-(3-HSD of type 1 is a bidirectional enzyme which acts
mainly as reductase in vivo. 11-(3-HSD of type 2, by
contrast, is a unidirectional enzyme in vivo and acts
exclusively as an NAD-dependent dehydrogenase. The
11-(3-hydroxysteroid dehydrogenase inhibitor used herein
is preferably an inhibitor of the reductase activity
and particularly preferably an inhibitor of 11-~i-HSD-1.
The medicament of the invention comprises as active
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ingredient a combination of two or more substances
which may be present both as mixture or formulation and
separately, e.g. as kit.
On the one hand, the medicament comprises one or more
antigens which serve to induce a specific immunological
tolerance response.
The substances suitable as antigens of this type are
all those which elicit unwanted immune responses, such
as, for example, those associated with autoimmune
diseases, e.g. rheumatoid arthritis including juvenile
forms, lupus erythemadotes, multiple sclerosis,
uveitis, diabetes of type I (autoimmune diabetes), and
those associated with allergies or/and asthma.
However, it is also possible to use as antigens
substances which bring about an infection, in which
case the medicament of the invention is able to induce
a tolerance which can diminish or eliminate
disadvantageous or pathological responses to the infec-
tious pathogen. Examples of infectious pathogens are,
for example, bacteria, viruses or other microorganisms.
The medicament of the invention may include specific
epitopes or antigens of such infectious pathogens, but
it is also possible to use the whole microorganism or
parts thereof.
Classical vaccination normally entails intravenous
administration of a pathogen to the patient, eliciting
a protective immunity. However, it has now been found
that vaccine protection can be achieved not only in a
conventional way but also through tolerance induction
through mucosal administration of antigens (McSorley,
1999). The medicament combination of the invention
further enhances such an effect, so that a combination
of 11-(3-HSD inhibitor and antigen can advantageously be
employed as tolerogenic oral vaccine.
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It has surprisingly been found that induction of
tolerance has an advantageous effect on a subsequent
infection not only in cases of autoimmune diseases but
also in cases of infectious diseases. Thus, it was
possible to establish that it was possible to resolve
infections through induction of tolerance. It is
presumed that a vaccine protection through tolerance
induction is based on the following mechanism of
action: many infectious pathogens comprise a plurality
of antigens, and evidently some of these antigens
produce an overreaction in the host. This overreaction
may bring about, for example, conversion of T cells
into Th2 cells, although Th2 cells are unable to
control the invading pathogen. In the case of an
infectious disease, such antigens bring about an
overreaction of the host's immune system in a harmful
manner, while it is unable at the same time to control
the infectious pathogen effectively. It is possible by
inducing tolerance, for example by mucosal
administration of the antigen and simultaneous
administration of an 11-(3-HSD inhibitor, to eliminate
the disadvantageous effect of the antigens producing an
overreaction, so that the remainder of the immune
system can perform its protective function. In
tolerance induction to protect from or cure infectious
diseases, it is normally preferred to promote a
Thl-like response and eliminate a Th2-like response.
Accordingly, it is advantageous to generate a tolerance
for antigens which elicit a Th2-like response.
It is possible in principle, by suitable choice of the
antigen in accordance with the instructions given
herein, to provide vaccination protection through
tolerance induction for virtually all infectious
diseases. The antigen chosen is preferably one which is
associated with a bacterial or viral infection, for
example influenza, leishmania, fungal infections,
cytomegaly, pneumonia, streptococci B, chlamydias,
helicobacter, hepatitis C, herpes, human papilloma
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virus, mycobacteria tuberculosis and others.
The antigen employed according to the invention may
moreover preferably be a natural or synthetic protein,
protein constituent or peptide, in particular also a
so-called altered peptide ligand (APL), but carbo-
hydrates including polysaccharides and lipopoly-
saccharides are likewise suitable, as well as antigens
from biological resources. The latter comprise antigens
which play a part in the development and/or cure of
autoimmune diseases, allergies, or wanted and unwanted
immune response within the framework of transplantation
medicine. Tolerance induction brought about according
to the invention may thus be advantageous in trans
plantations too.
The antigen may moreover be one against which an immune
response is produced directly. However, it may also be
a so-called bystander antigen which elicits an
(auto)immune response to related or adjacent epitopes
on a protein.
Altered peptide ligands (APLs) are peptides which
essentially correspond to an antigen against which an
immune response is induced. However, individual amino
acids [lacuna] APLs have been exchanged. It has been
found that little or no additional adjuvant is neces-
sary on administration of APLs within the framework of
oral tolerance induction.
In the area of allergy prevention it is possible in
principle to use all nontoxic environmental antigens as
antigens suitable according to the invention.
Preference is given to benzylpenicilloyl, insulin,
ovalbumin, lactalbumin, but also various pollens, food
antigens, house dust mites and constituents thereof,
excreta thereof and the like.
Further suitable antigens comprise endogenous and other
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heat shock proteins (Prakken .et al. (1997); prakken et
al. (1998)), thyroglobulin, cellular constituents of
the uvea, of the skin, of various epithelial tissues,
of the thyroid, of the basement membrane, of the
muscles, of the myelin sheaths, of the nerve cells, of
the thymus, red blood corpuscles, further blood
constituents and cells, proteolipid, myelin basic
protein (MBP), myelin-oligodendrocyte glycoprotein
(MOG), or other constituents of normal or diseased body
tissue.
Preference is further given to a combination which
comprises a vaccine as disclosed by Rock et al. (2000),
in particular a bacterial vaccine.
The antigen may be present in the medicament of the
invention also as nucleic acid or oligonucleotide. It
is moreover possible on the one hand for the nucleic
acid or the oligonucleotide itself to represent the
antigen. On the other hand, the nucleic acid may code
for a particular peptide antigen. It can be given by
various routes of administration, e.g. by injection,
intravenously, intramuscularly, subcutaneously or with
the aid of a gene gun.
Antigen presentation can be effected preferably also by
dendritic cells. Dendritic cells are the most important
cells in the immune system for the presentation of
endogenous and foreign antigens. They are therefore
very potent stimulators of an immune response.
Dendritic cells can be cultured for example from
monocytes or bone marrow cells in the manner known in
the prior art (D. Rea et al. (2000); E. Dejong et al.
(1999); M. Mathiszak et al. (2000)). Dendritic cells
can be employed for antigen-specific immunotherapy
(Fairchild et al. (2000); Reid et al. (2000);
Kapsenberg et al. (1998)). For this purpose, they are
either pulsed in vitro with the required antigen, i.e.
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incubated with the antigen until they have taken it up
and express it on the surface. However, dendritic cells
can also be stimulated by gene transfer to express the
particular antigen, thus producing it themselves. It is
additionally possible to stimulate proliferation
ex vivo of cells already loaded with antigen and then
reinject them into the body. Antigen presentation takes
place by means of dendritic cells according to the
invention particularly preferably by maturing dendritic
cells in vitro with addition of glucocorticoids, for
example of cortisone, to the medium to give a
tolerance-inducing phenotype and then reinfusing them.
These dendritic cells can then be combined with an
11-(3-HSD inhibitor in order to assist the effect
further. It is surprisingly possible by treating
dendritic cells with glucocorticoids, for example with
cortisone, to increase the tolerance in addition to the
increase normally occurring in the immune response on
antigen presentation by dendritic cells.
This is presumably attributable to the fact that the
combination according to the invention of an 11-(3-HSD
inhibitor with the antigen-presenting dendritic cells
creates in the lymph node a milieu in which the
dendritic cells are more stable.
Besides genetic manipulation of dendritic cells to
express required antigens, it is also possible to
isolate dendritic cells with the required antigen from
patients suffering from the corresponding disease.
Artificial antigen-presenting cells or/and artificial
dendritic cells are preferably employed for antigen
presentation (Falcioni et al. (1999); Latouche et al.
(2000); Wu et al. (2000)).
In another preferred embodiment, antigen presentation
is effected by T cells. Regulatory or/and antigen-
specific T cells can be generated for example in vitro
s,
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(Ramirez et al. (2000); Shevach (2000)). In this case,
the antigen is presented by T cells or/and it is
possible for antigen-specific T cells to be influenced
by regulatory T cells.
11-(3-Hydroxysteroid dehydrogenase inhibitors which are
suitable in principle are all those disclosed to date
but also, where appropriate, as yet unidentified
inhibitors.
The inhibitors preferably inhibit all isoforms of
11-(3-HSD, but especially 11-(3-HSD-1, also preferably in
addition other isoforms, as well as tissue-specific
isoforms. An isoform which has not yet been unam-
biguously identified and which is likewise preferably
inhibited is 11-(3-HSD-3.
The inhibitors are preferably selected from endogenous
and exogenous inhibitors. Examples of endogenous inhi-
bitors are the following: substrates for 11-(3-HSD such
as, for example, 11-OH-progesterone, 3-alpha,5-beta-
tetrahydroprogesterone, 3-alpha,5-beta-tetrahydro-
11-deoxycorticosterone, 11-epicortisol, chenodeoxy-
cholic acid, cholic acid. Examples of exogenous inhibi-
tors are glycyrrhetinic acid (3(3-hydroxy-11-oxoolean-
12-en-30-oic acid) and derivatives thereof such as, for
example, glycyrrhizin, glycyrrhizic acid and carben-
oxolone; furosemide and derivatives thereof; flavonoids
and derivatives thereof such as, for example
naringenin; triterpinoids (e. g. CHAPS), ketoconazole,
saiboku-to, gossypol, metyrapone, 11-epiprednisolone.
Other suitable inhibitors are steroid-like such as, for
example, dexamethasone, budesonide, deflazacort and
stanozolol. There are suspected to be unidentified
derivatives among the ACTH-dependent inhibitors.
Particular preference is given to glycyrrhetinic acid
and derivatives, in particular glycyrrhizic acid,
glycyrrhizin, glycyrrhitic acid and derivatives
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thereof, and, in particular carbenoxolone.
Glycyrrhetinic acid (GA) is an extract from liquorice
(Glycyrrhiza glabra) and inhibits the metabolism of
endogenous glucocorticoids through blockade of the
enzyme 11-~i-hydroxysteroid dehydrogenase.
Carbenoxolone is a GA derivative which has a higher
affinity for 11-(3-HSD (Hult, Jornvall et al. 1998).
Carbenoxolone has, just like GA, been investigated in a
large number of studies on the pharmacokinetics,
toxicity and possible dosage regimens and mode of
administration in humans and is already approved for
human use, and possible side effects are not to be
expected or are nonhazardous and reversible (Ulick,
Wang et al. 1993; Bernardi, D'Intino et al. 1994;
Krahenbuhl, Hasler et al. 1994; Schambelan 1994).
It is also possible to employ antibodies against
11-(3-HSD or fragments thereof as inhibitors of
11-(3-HSD. Such antibodies against 11-(3-HSD can be
produced in a manner known to the skilled worker, e.g.
as monoclonal or polyclonal antibodies. The term
inhibitor of 11-(3-HSD as used herein additionally
encompasses substances which regulate the transcription
of 11-~i-HSD.
The amount of 11-(3-HSD available in the body can be
controlled by using such transcription regulators.
Suitable transcription regulators for 11-~3-HSD are
described for example in Williams et al. (2000) and
include in particular members of the C/EBP family.
Preference is further given to choosing the antigen
or/and the 11-(3-HSD modulator, in particular inhibitor,
from low molecular weight substances which preferably
have a molecular weight of <_ 500 Da, more preferably
<_ 250 Da.
CA 02388974 2002-04-25
- 19 -
Said inhibitors of 11-(3-HSD, in particular
glycyrrhetinic acid and its derivatives, are nontoxic
even in higher doses and have no serious side effects.
The therapeutic use of GA or similar inhibitors of
11-~i-HSD is possible ad hoc. Combination of an
inhibitor/method for inhibition of 11-~3-HSD with an
antigen thus represents a novel therapeutic approach to
curing various immunological disorders.
Possible dosages of the inhibitors, in particular of GA
and derivatives, for humans are up to 1 g per dose
unit.
The inhibitors are preferably administered in an amount
of at least 0.01, preferably at least 0.1 and particu-
larly preferably at least 1, mg/kg of body weight and
day and up to 100, preferably up to 50, particularly
preferably up to 10, mg/kg per day. An inhibitor of
11-~i-HSD is regarded as being a compound which inhibits
the in vivo 11-(3-HSD activity by at least 10%,
preferably at least 30%, more preferably at least 50%,
particularly preferably at least 70% and most prefer-
ably at least 80%. However, it may be advantageous to
use substances which inhibit the in vivo 11-~i-HSD
activity by at least 90%, preferably by at least 95%.
The inhibitors preferably have ICSO values as determined
in placental microsomes or MCF-7 cells of < 100 ~M,
preferably < 30 ~M, particularly preferably < 1 ~M.
Particularly potent inhibitors have ICSO values of
< 100 nM, preferably < 10 n~.M and particularly prefer-
ably < 1 nM.
The Ki values of the inhibitors employed according to
the invention are preferably < 1 200 ~,M, more pre-
ferably < 100 ~M and particularly preferably < 1 ~M.
Possible dosages of the inhibitors, in particular of GA
and derivatives, for humans are up to 1 g per dose
unit.
CA 02388974 2002-04-25
- 20 -
Apart from the use of said- inhibitors, it is also
possible and within the meaning of the invention to use
as inhibitor an antisense nucleic acid which hybridizes
with sequences which code for 11-(3-HSD. This may be
advantageous in particular if it is intended speci-
fically to inhibit an 11-~3-HSD in a particular tissue.
The ratio by weight of the inhibitor and antigen
constituents is preferably from 0.1:99.9 to 99.9:0.1,
particular preferably 90:10 to 10:90.
The medicament of the invention may be in the form of a
mixture or formulation of the two components antigens)
and inhibitor. However, the two constituents are
preferably administered not as a formulation but
separately. It is additionally possible for the medica-
ment or the two active ingredient components to
comprise pharmaceutically acceptable excipients and/or
additives (e. g. suitable solvents or diluents) and/or
adjuvants. These can easily be established by the
person skilled in the art.
There was additionally a need to provide compositions
with which immune responses, in particular autoimmune
diseases, can be beneficially influenced. The present
invention therefore relates further to the use of an
11-(3-hydroxysteroid dehydrogenase for obtaining a
medicament for tolerance induction, inhibition of
inflammation or/and immunomodulation. It has sur-
prisingly been found that it is possible to influence
beneficially immune diseases and in particular
autoimmune diseases by influencing the 11-~i-HSD as
representative of the SDR family. It is additionally
possible for allergies, transplant rejection and GVHD
to be treated therapeutically or/and prophylactically
by use of 11-(3-HSD. Immunomodulatory substances which
can be employed are, in particular, modulators such as,
for example, inhibitors or promoters of 11-(3-HSD in
order to bring about tolerance induction, inhibition of
CA 02388974 2002-04-25
- 21 -
inflammation or/and immunomodulation, in particular to
control autoimmune diseases, allergies, transplant
rejection or/and GVHD. Suitable immunomodulatory sub-
stances which can be employed are known inhibitors of
11-(3-HSD, or substances which interact with 11-(3-HSD
and, for example, are found by screening or computer-
assisted methods such as, for example, force field
calculations. It is additionally possible to use
substances which are known to be inhibitors of other
members of the SDR family and to test these substances
for their interaction with 11-(3-HSD by simple
experiments. In contrast to the previously described
immunosuppressant effect of 11-(3-HSD modulators, it has
surprisingly been found that the effects indicated
above can be obtained even on use of 11-(3-HSD modula-
tors alone, in particular of inhibitors.
The present invention further relates to the use of an
inhibitor of 11-(3-hydroxysteroid dehydrogenase for
producing a medicament for tolerance induction, inhibi-
tion of inflammation or/and immunomodulation. It has
surprisingly been found that a tolerance induction,
immunomodulation or/and inhibition of inflammation, not
an immunosuppressant effect, can be achieved through
use just of an 11-(3-HSD inhibitor. An inhibitor of
11-(3-HSD can be generally employed for inhibiting
inflammation in acute or/and chronic inflammatory
processes, including septic shock and sepsis. Advanta
geous effects have been observed both with infectious
and noninfectious inflammations.
The preferred use of the medicament of the invention,
in particular of the combination of inhibitor of
11-(3-HSD with one or more antigens, is for tolerance
induction, inhibition of inflammation or/and immuno-
modulation in a mammal, in particular a human. Areas of
application are for autoimmune diseases, e.g.
rheumatoid arthritis, including juvenile forms, lupus
erythematodes, multiple sclerosis, uveitis, diabetes of
CA 02388974 2002-04-25
- 22 -
type I, and for allergies and in transplantation
medicine, in particular transplant rejection and GVHD.
Tolerance induction can, however, also be employed in
immunization against pathogens of infection as stated
above.
The medicament can be administered by various routes.
It is possible to administer the inhibitors) of
11-~i-HSD, preferably together with the antigens) and,
where appropriate, additional adjuvants, it being
possible for said components to be in the form of a
formulation. The two active ingredient components and
the optionally additional excipients or adjuvants may,
however, also be given by various administration
routes. Possibilities on the one hand are mucosal
routes, e.g. intranasal, oral, sublingual or by
inhalation, but also others such as, for example,
intravenous, subcutaneous, intramuscular and intra-
peritoneal. In addition, the gene gun is suitable for
administering the nucleic acids.
Possibilities for administration suitable for presenta-
tion of the antigen are, in particular, the following:
the so-called mucosal, i.e. oral or nasal tolerance
induction and, recently, through administration of DNA
(Ragno, Colston et al. 1997; Lee, Corr et al. 1998;
Lobell, Weissert et al. 1999; McCluskie and Davis 1999;
McCluskie, Millan et al. 1999) which codes for the
relevant antigen and is injected into the organism to
be treated, e.g. a human.
Besides this it is also possible for antigen presenta
tion to be effected by means of dendritic cells or
T cells as described above.
The composition of the invention is preferably employed
for mucosal tolerance induction. In this connection,
mucosal means uptake through mucous membranes and
CA 02388974 2002-04-25
- 23 -
encompasses administration of an antigen inter alia by
oral intake or instillation into the nose, or inhala
tion and absorption via the lungs. However, administra
tion can also take place subcutaneously, intravenously
and/or intramuscularly.
Inhibitor and antigen can be administered together or
separately via different routes and also sequentially.
A particularly advantageous dosage form is represented
by a capsule which consists externally of an 11-(3-HSD
inhibitor, e.g. glycyrrhizic acid or glycyrrhetinic
acid, which envelopes an antigen inside.
The inhibitor can be administered by all known routes
and is for this purpose converted into the appropriate
form in each case, e.g. dissolved in a suitable solvent
for injection. It is also possible in this connection
to administer the inhibitor first, e.g. by intravenous,
intramuscular or subcutaneous injection, and then the
antigens) by mucosal routes. It may be advantageous to
give the inhibitor in more than one dose.
The invention is explained further by the appended
figures and the following examples.
Figure 1 shows the course of an experimentally induced
arthritis without (blue) or with (red) treatment by an
11-(3-HSD inhibitor. The inhibitor, in this case
glycyrrhetinic acid (GRA), was injected intradermally
at the base of the tail of rats in a dose of 2 mg in
200 ~tl of olive oil on days 0, 2 and 4 after injection
of complete Freund's adjuvant (CFA). CFA was adminis-
tered intradermally in a dose of 0.4 mg. The combina-
tion of inhibitor and antigen (present in CFA as
Mycobacterium t.) leads to a milder course of the dis-
order, that is to say has an immunomodulatory effect.
Figure 2 shows the enhancing effect of an 11-~i-HSD
CA 02388974 2002-04-25
- 24 -
inhibitor on nasal tolerance induction by means of a
peptide antigen. This entailed peptide 176 to 190
(Prakken et al. (1997)) being administered in 3% sodium
bicarbonate solution intranasally on days -15, -11, -7
and -3 before induction of the pathological episode.
GRA was given intranasally in a dose of 0.5 mg in 25 ~.l
per nostril, i.e. in total in an amount of 1 mg. The
red curve shows a control group. The blue curve shows
the course of the disorder during peptide treatment
(antigen) without addition of GRA. The yellow curve
shows the combination according to the invention of
peptide (antigen) and GRA, which were administered
intranasally.
Examples
Enhancement of tolerance induction in the model of
adjuvant-induced arthritis
This animal model, in which an arthritis-like auto-
immune disease can be induced, is known in the prior
art (Leech, Metz et al. 1998; Prakken, Wauben et al.
1998; Vaneden, Vanderzee et al. 1998):
This entails rats receiving an injection of a
suspension of oil and mycobacterium into the tail
(150 ~,l of a 10 mg/ml suspension with Mycobacterium
tuberculosis). After about 10-13 days, the animals
develop joint inflammations which reflect certain
characteristics of human rheumatoid arthritis. The
development of this type of arthritis is interpreted as
a result of a so-called cross-reaction of the immune
defenses both against antigens of the mycobacterium
(heat shock protein 60, hsp 60) and of the articular
cartilage (Vaneden, Vanderzee et al. 1998).
Example 1
Prevention of the development of the disorder:
The development and the progress of the disorder can be
i
CA 02388974 2002-04-25
- 25 -
beneficially influenced or prevented. To do this,
tolerance to particular antigens of mycobacterium is
induced in the rats before inducing the experimental
arthritis. This entails the antigen, e.g. hsp60 or a
so-called altered peptide ligand being presented to the
immune system as protein or peptides either orally or
nasally (Prakken, Wauben et al. 1998), or administered
as DNA (Ragno, Colston et al. 1997) before the sensiti-
zation with the oil/mycobacterium mixture takes place.
For this purpose, the APL for example is administered
on day -15, -10, -5 and on the day of sensitization
( 100 ~tg intranasally) .
The 11-(3-HSD inhibitor used in this example in each
case was glycyrrhetinic acid (GA) and a water-soluble
derivative thereof, namely carbenoxolone.
If the inhibitor is administered (e. g. 1-8 mg intra-
peritoneally in oil or intranasally in saline solution)
together with the antigen until the oil/peptide mixture
is injected, i.e. before the sensitization, the effect
of the latter is enhanced and the amount of antigen
necessary for inducing tolerance is reduced. There is
dose-dependent prevention or a diminution in the
incidence and the severity of the course of the rat's
disorder. It is additionally possible thereby to reduce
the amount of peptide necessary for tolerance
induction.
Example 2
Manipulation of the course of the disorder after
induction of autoimmune arthritis and after onset of
symptoms:
The induced autoimmune arthritis has a fulminant mono-
phasic course after the onset of the inflammatory
reaction 10 days after the injection of Freund's
adjuvant with mycobacterium or hsp60, with a pathology
peak around day 27; symptoms are no longer detectable
i
CA 02388974 2002-04-25
- 26 -
after 40 days.
The course of the inflammatory reaction can be
beneficially influenced with altered peptide ligands
(APLs), even after onset of the symptoms, if the treat-
ment takes place within 24-48 hours after appearance of
the first symptoms and is continued daily.
For this purpose, rats receive, simultaneously during
the nasal administration of the protein antigen (hsp60)
or of the APL, the inhibitor carbenoxolone by injection
or instillation through the nose (concentrations as in
example 1).
The increase in the from symptom incidence is in this
case influenced significantly more favorably than
without inhibition of 11-~i-HSD. Certain antigens such
as the pathogenic hsp60, which have no effect without
carbenoxolone, likewise lead in combination with
carbenoxolone to an improvement in the course of the
disorder. On use of carbenoxolone in combination with
APLs, an alleviation and shortening of the course of
the disorder can be achieved even with suboptimal
peptide concentrations.
The fact that inhibition of 11-~3-HSD using the peptide
(hsp60), which has not to date been suitable for thera-
peutic use, permits to attenuation of the course of the
disorder or reduces the amount of APL appears to be a
strong indicator that it may be possible even in late
stages to initiate successfully attempts at cure by
means of oral tolerance induction. Thus the data from
the animal experiment make an ad hoc attempt at cure
possible to assist tolerance induction in humans.
Example 3
Alleviation of experimentally induced arthritis by
treatment with an 11-(3-HSD inhibitor alone
i
CA 02388974 2002-04-25
- 27 -
An autoimmune arthritis was 'induced experimentally in
rats by administering Freund's complete adjuvant (CVA;
0.4 mg intradermally). The rats were divided into two
groups, and one group received intradermal injection of
2 mg of GRA (glycyrrhitic acid) in 200 ~.l of olive oil
at the base of the rat' s tail on days 0, 2 and 4 after
injection of CVA. The results for the two groups are
depicted in figure 1, which shows the course of the
experimentally induced arthritis without (blue) or with
treatment by the 11-(3-HSD inhibitor (red). As can
clearly be seen in figure 1, the combination of
inhibitor and antigen (present as Mycrobacterium t. in
CVA) leads to an alleviated course of the disorder.
Further experiments have shown that even the inhibitor
alone brings about a milder course of the disorder, or
weakens the disease-promoting effect of CVA and thus
has an immunomodulatory effect.
Example 4
Improved efficacy through mucosal tolerance induction
compared with systemic administration
It has been found that a distinctly improved efficacy
is to be observed on administration of an antigen plus
an 11-(3-HSD inhibitor (e. g. glycyrrhetinic acid)
intranasally compared with administration of the same
antigen alone or of the antigen plus 11-(3-HSD inhibitor
(e. g. glycyrrhizic acid) systemically (orally).
Figure 2 shows the enhancing effect of an 11-(3-HSD
inhibitor on nasal tolerance induction by means of a
peptide antigen. For this purpose, the peptide 176-190
was administered intranasally to all the experimental
groups on days -15, -11, -7 and -3 before induction of
a pathological episode of experimentally induced
arthritis (caused by CFA as described in example 3) in
3% sodium bicarbonate solution. GRA i.n. (intranasally)
was added in a dose of 0.5 mg in 25 ~1 per nostril,
i.e. in total a dose of 1 mg. The red curve shows a
i
CA 02388974 2002-04-25
- 28 -
control group with systemic administration of
glycyrrhizic acid in drinking water. The blue curve
shows the course of the disorder during peptide
treatment without addition of GRA and the yellow curve
shows the combination according to the invention of
peptide (antigen) and 11-(3-HSD inhibitor (e. g. GRA),
both of which were administered intranasally.
i
CA 02388974 2002-04-25
- 29 -
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