U.S. patent application number 12/383849 was filed with the patent office on 2010-01-07 for compositions for mucosal and oral administration comprising hcg fragments.
Invention is credited to Robbert Benner, Nisar A. Khan.
Application Number | 20100004172 12/383849 |
Document ID | / |
Family ID | 36461674 |
Filed Date | 2010-01-07 |
United States Patent
Application |
20100004172 |
Kind Code |
A1 |
Khan; Nisar A. ; et
al. |
January 7, 2010 |
Compositions for mucosal and oral administration comprising hcg
fragments
Abstract
The invention relates to the field of immunology, more
specifically to the field of immune-mediated disorders such as
allergies, auto-immune disease, transplantation-related disease and
other inflammatory diseases. The invention in particular relates to
the systemic treatment of inflammatory disease by oral or mucosal
administration of a pharmaceutical composition with a
gene-regulatory peptide. The invention provides a pharmaceutical
composition in a form for mucosal application for the treatment of
a subject suffering from disease, the pharmaceutical composition
comprising a pharmacologically effective amount of a
gene-regulatory peptide or a functional analogue thereof together
with a pharmaceutically acceptable diluent.
Inventors: |
Khan; Nisar A.; (Rotterdam,
NL) ; Benner; Robbert; (Barendrecht, NL) |
Correspondence
Address: |
TRASKBRITT, P.C.
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
36461674 |
Appl. No.: |
12/383849 |
Filed: |
March 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11243438 |
Oct 4, 2005 |
7517529 |
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12383849 |
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PCT/EP2004/003747 |
Apr 8, 2004 |
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11243438 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61P 3/10 20180101; A61K
38/1709 20130101; A61P 29/00 20180101; A61P 37/06 20180101; A61K
9/0019 20130101; A61P 37/00 20180101; A61P 19/02 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 29/00 20060101 A61P029/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 8, 2003 |
EP |
03076021.9 |
Apr 8, 2003 |
EP |
03076022.7 |
Apr 8, 2003 |
EP |
03076023.5 |
Apr 8, 2003 |
EP |
03076024.3 |
Apr 8, 2003 |
EP |
03076025.0 |
Apr 8, 2003 |
EP |
03076026.8 |
Apr 8, 2003 |
EP |
03076027.6 |
Apr 8, 2003 |
EP |
03076028.4 |
Apr 8, 2003 |
EP |
03076029.2 |
Apr 8, 2003 |
EP |
03076030.0 |
Apr 30, 2003 |
CN |
03131227.6 |
Claims
1. A pharmaceutical composition in a form for mucosal application
for the treatment of a subject suffering from a disease, said
pharmaceutical composition comprising: a pharmacologically
effective amount of a gene-regulatory peptide or a functional
analogue thereof together with a pharmaceutically acceptable
diluent.
2. The pharmaceutical composition of claim 1, wherein said form for
mucosal application is selected from the group of sprays, liquids,
and gels.
3. The pharmaceutical composition of claim 1, wherein said
pharmaceutical composition is in a form for oral
administration.
4. The pharmaceutical composition of claim 3, wherein said form for
oral administration is selected from the group consisting of
capsules, tablets, liquids, oral suspensions, emulsions, and
powders.
5. The pharmaceutical composition of claim 1, wherein said
gene-regulatory peptide or functional analogue modulates
translocation and/or activity of a gene transcription factor.
6. The pharmaceutical composition of claim 5, wherein said gene
transcription factor comprises an NF.kappa.B/Rel protein.
7. The pharmaceutical composition of claim 6, wherein translocation
and/or activity of said NF.kappa.B/Rel protein is inhibited.
8. The pharmaceutical composition of claim 1, wherein said
gene-regulatory peptide or functional analogue regulates expression
of a gene encoding an inflammatory mediator
9. The pharmaceutical composition of claim 8, wherein said
inflammatory mediator comprises a cytokine selected from the group
of TNF-.alpha., TGF-.beta., interferon .gamma., IL-1.beta., IL-4,
IL-5, IL6, IL-10, IL-12, IL-23 and IL-40.
10-20. (canceled)
21. The pharmaceutical composition of claim 3, wherein the
gene-regulatory peptide modulates translocation and/or activity of
a gene transcription factor.
22. The pharmaceutical composition of claim 21, wherein the gene
transcription factor comprises an NF.kappa.B/Rel protein.
23. The pharmaceutical composition of claim 22, wherein
translocation and/or activity of the NF.kappa.B/Rel protein is
inhibited.
24. The pharmaceutical composition of claim 4, wherein the
gene-regulatory peptide modulates translocation and/or activity of
a gene transcription factor.
25. The pharmaceutical composition of claim 24, wherein the gene
transcription factor comprises an NF.kappa.B/Rel protein.
26. The pharmaceutical composition of claim 25, wherein
translocation and/or activity of the NF.kappa.B/Rel protein is
inhibited.
27. The pharmaceutical composition of claim 2, wherein said
gene-regulatory peptide or functional analogue regulates expression
of a gene encoding an inflammatory mediator
28. The pharmaceutical composition of claim 27, wherein the
inflammatory mediator comprises a cytokine selected from the group
of TNF-.alpha., TGF-.beta., interferon .gamma., IL-1.beta., IL-4,
IL-5, IL6, IL-10, IL-12, IL-23 and IL-40.
29. The pharmaceutical composition of claim 4, wherein said
gene-regulatory peptide or functional analogue regulates expression
of a gene encoding an inflammatory mediator
30. The pharmaceutical composition of claim 29, wherein the
inflammatory mediator comprises a cytokine selected from the group
of TNF-.alpha., TGF-.beta., interferon .gamma., IL-1.beta., IL-4,
IL-5, IL6, IL-10, IL-12, IL-23 and IL-40.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S. patent
application Ser. No. 11/243,438, filed Oct. 4, 2005, U.S. Pat. No.
7,517,529 (Apr. 14, 2009), which is a continuation of PCT
International Patent Appl'n No. PCT/EP2004/003747, filed on Apr. 8,
2004, designating the United States of America, and published, in
English, as PCT International Publication No. WO 2004/093897 on
Nov. 4, 2004, which International Application itself claims
priority to EP 03076028.4, filed Apr. 8, 2003 with the European
Patent Office (EPO), EP 03076029.2, filed Apr. 8, 2003 with the
EPO, of EP 03076027.6, filed Apr. 8, 2003, with the EPO, of EP
03076026.8, filed Apr. 8, 2003 with the EPO, of EP 03076022.7 filed
Apr. 8, 2003 with the EPO, of U.S. Ser. No. 10/409,671, filed Apr.
8, 2003, of EP 03076021.9, filed Apr. 8, 2003 with the EPO, of EP
03076025.0, filed Apr. 8, 2003 with the EPO, of EP 03076024.3,
filed Apr. 8, 2003 with the EPO, of EP 03076030.0, filed Apr. 8,
2003 with the EPO, of EP 03076023.5, filed Apr. 8, 2003 with the
EPO, and of CN 03131227.6, filed Apr. 30, 2003 with the Chinese
Patent Office, the contents of the entirety of each of which are
incorporated by this reference.
TECHNICAL FIELD
[0002] The invention relates generally to biotechnology and to the
field of immunology, more specifically to the field of
immune-mediated disorders such as allergies, auto-immune disease,
transplantation-related disease and other inflammatory diseases.
The invention in particular relates to the treatment of
inflammatory disease by administration of a pharmaceutical
composition with a gene-regulatory peptide.
BACKGROUND
[0003] The immune system produces cytokines and other humoral and
cellular factors to respond with an inflammation to protect the
host when threatened by noxious agents, microbial invasion, or
injury. In most cases, this complex defense network successfully
restores normal homeostasis, but at other times the immunological
or inflammatory mediators may actually prove deleterious to the
host. Some examples of immune disease and immune system-mediated
injury have been extensively investigated including anaphylactic
shock, autoimmune disease, and immune complex disorders.
[0004] Recent advances in humoral and cellular immunology,
molecular biology and pathology have influenced current thinking
about auto-immunity being a component of immune-mediated
inflammatory disease. These advances have increased our
understanding of the basic aspects of antibody, B-cell, and T-cell
diversity, the generation of innate (effected by monocytes,
macrophages, granulocytes, natural killer cells, mast cells,
.gamma..delta. T-cells, complement, acute phase proteins, and such)
and adaptive (T- and B-cells and antibodies) or cellular and
humoral immune responses and their interdependence, the mechanisms
of (self)-tolerance induction and the means by which immunological
reactivity develops against auto-antigenic constituents.
[0005] Since 1900, the central dogma of immunology has been that
the immune system does not normally react to self. However, it has
recently become apparent that auto-immune responses are not as rare
as once thought and that not all auto-immune responses are harmful;
some responses play a distinct role in mediating the immune
response in general. For example, certain forms of auto-immune
response such as recognition of cell surface antigens encoded by
the major histocompatibility complex (MHC) and of anti-idiotypic
responses against self idiotypes are important, indeed essential,
for the diversification and normal functioning of the intact immune
system.
[0006] Apparently, an intricate system of checks and balances is
maintained between various subsets of cells (i.e., T-cells) of the
immune system, thereby providing the individual with an immune
system capable of coping with foreign invaders. In that sense,
auto-immunity plays a regulating role in the immune system.
[0007] However, it is now also recognized that an abnormal
auto-immune response is sometimes a primary cause and at other
times a secondary contributor to many human and animal diseases.
Types of auto-immune disease frequently overlap, and more than one
auto-immune disorder tends to occur in the same individual,
especially in those with auto-immune endocrinopathies. Auto-immune
syndromes may be mediated with lymphoid hyperplasia, malignant
lymphocytic or plasma cell proliferation and immunodeficiency
disorders such as hypogammaglobulinaemia, selective Ig deficiencies
and complement component deficiencies.
[0008] Auto-immune diseases, such as systemic lupus erythematosus,
diabetes, rheumatoid arthritis, post-partum thyroid dysfunction,
auto-immune thrombocytopenia, to name a few, are characterized by
auto-immune inflammatory responses, for example, directed against
widely distributed self-antigenic determinants, or directed against
organ- or tissue-specific antigens. Such disease may follow
abnormal immune responses against only one antigenic target, or
against many self antigens. In many instances, it is not clear
whether auto-immune responses are directed against unmodified
self-antigens or self-antigens that have been modified (or
resemble) any of numerous agents such as viruses, bacterial
antigens and haptenic groups.
[0009] There is as yet no established unifying concept to explain
the origin and pathogenesis of the various auto-immune disorders.
Studies in experimental animals support the notion that auto-immune
diseases may result from a wide spectrum of genetic and
immunological abnormalities which differ from one individual to
another and may express themselves early or late in life depending
on the presence or absence of many superimposed exogenous (viruses,
bacteria) or endogenous (hormones, cytokines, abnormal genes)
accelerating factors. However, one common aspect of all these
various diseases comes to the eye; all share an, at times mostly
systemic, inflammatory response.
[0010] It is evident that similar checks and balances that keep
primary auto-immune diseases at bay are also compromised in other
immune-mediated disorders, such as allergy (asthma), acute
inflammatory diseases such as sepsis or septic shock, chronic
inflammatory disease (i.e., rheumatic disease, Sjogrens syndrome,
multiple sclerosis), transplantation-related inflammatory responses
(graft-versus-host-disease, post-transfusion thrombocytopenia), and
many others wherein the responsible antigens (at least initially)
may not be self-antigens but wherein the inflammatory response is
in principle not wanted and detrimental to the individual.
[0011] As a particular example of an acute systemic inflammatory
response, the sepsis/SIRS concept is here discussed. Sepsis is a
syndrome in which immune mediators, induced by, for example,
microbial invasion, injury or through other factors, induce an
acute state of inflammation which leads to abnormal homeostasis,
organ damage and eventually to lethal shock. Sepsis refers to a
systemic response to serious infection. Patients with sepsis
usually manifest fever, tachycardia, tachypnea, leukocytosis, and a
localized site of infection. Microbiologic cultures from blood or
the infection site are frequently, though not invariably, positive.
When this syndrome results in hypotension or multiple organ system
failure (MOSF), the condition is called sepsis or septic shock.
Initially, micro-organisms proliferate at a nidus of infection. The
organisms may invade the bloodstream, resulting in positive blood
cultures, or might grow locally and release a variety of substances
into the bloodstream. Such substances, when of pathogenic nature
are grouped into two basic categories: endotoxins and exotoxins.
Endotoxins typically consist of structural components of the
micro-organisms, such as teichoic acid antigens from staphylococci
or endotoxins from gram-negative organisms like LPS). Exotoxins
(e.g., toxic shock syndrome toxin-1, or staphylococcal enterotoxin
A, B or C) are synthesized and directly released by the
micro-organisms. As suggested by their name, both of these types of
bacterial toxins have pathogenic effects, stimulating the release
of a large number of endogenous host-derived immunological
mediators from plasma protein precursors or cells
(monocytes/macrophages, endothelial cells, neutrophils, T-cells,
and others). Sepsis/SIRS is an acute systemic inflammatory response
to a variety of noxious insults (particularly insults of an
infectious origin such as a bacterial infection, but also
non-infectious insults are well known and often seen). The systemic
inflammatory response seen with sepsis/SIRS is caused by
immunological processes that are activated by a variety of
immunological mediators such as cytokines, chemokines, nitric
oxide, and other immune-mediating chemicals of the body. These
immunological mediators are generally seen to cause the
life-threatening systemic disease seen with sepsis/SIRS. These
immunological mediators are, on the one hand, required locally, for
example, as effective antibacterial response, but are, in contrast,
potentially toxic when secreted into the circulation. When secreted
into the circulation, these mediators can cause, in an upward
spiral of cause and effect, the further systemic release of these
mediators, in the end leading to severe disease, such as multiple
organ failure and death. Crucial inflammatory mediators are tumor
necrosis factor-.alpha. (TNF-.alpha.), tissue growth factor-.beta.
(TGF-.beta.), interferon .gamma., interleukins (IL-1, IL-4, IL-5,
IL-6, IL-10, IL-12, IL-23, IL-40, and many others), nitric oxide
(NO), arachidonic acid metabolites and prostaglandins 1 and 2 (PGE1
and PGE2), and others.
[0012] In essence, sepsis, or septicemia, relates to the presence
in the blood of pathogenic microorganisms or their toxins in
combination with a systemic inflammatory disease associated with
such presence. Central in the development of sepsis in a subject is
an infection of a subject with a microorganism which gives origin
to the systemic release of immunological mediators by its presence
in the blood of an affected subject or by the presence of its
toxins in the blood of the subject. Only when the presence gives
rise to a disease that pertains to or affects the body as a whole,
a systemic disease, one speaks of sepsis.
[0013] The field of sepsis is thus limited to those conditions that
are characterized by the presence of microorganisms or their toxins
in the blood of a subject and simultaneously to (respectively) the
subject's systemic response(s) to the microorganism or to a
subject's systemic response(s) toxins. Sepsis herein includes
severe sepsis and septic shock, whereby severe sepsis relates to
sepsis accompanied with organ dysfunction and septic shock relates
to sepsis accompanied with hypotension or perfusion abnormalities
or both. SIRS relates to the type of severe systemic disease seen
in cases of sepsis but also relates to systemic inflammatory
disease wherein pathogenic microorganisms or their toxins are not
present in the blood.
[0014] Central in the development of SIRS in a subject is the
presence and effects of immunological mediators that give rise to a
disease that pertains to or affects the body as a whole, a systemic
disease. This systemic immunological response can be caused by a
variety of clinical insults, such as trauma, burns and
pancreatitis. Also, burn patients with or without inhalation injury
commonly exhibit a clinical picture produced by systemic
inflammation. The phrase "systemic" inflammatory response syndrome
(SIRS) has been introduced to designate the signs and symptoms of
patients suffering from such a condition. SIRS has a continuum of
severity ranging from the presence of tachycardia, tachypnea, fever
and leukocytosis, to refractory hypotension and, in its most severe
form, shock and multiple organ system dysfunction. In thermally
injured patients, the most common causes of SIRS are the burn
itself. Sepsis, SIRS with the presence of infection or bacteremia,
is also a common occurrence. Pathological alterations of metabolic,
cardiovascular, gastrointestinal, and coagulation systems occur as
a result of the hyperactive immune system. Both cellular and
humoral mechanisms are involved in these disease processes and have
been extensively studied in various burn and sepsis models. The
phrase systemic inflammatory response syndrome (SIRS) was
recommended by the American College of Chest Physicians/Society for
Critical Care Medicine (ACCP/SCCM) consensus conference in 1992 to
describe a systemic inflammatory process, independent of its cause.
The proposal was based on clinical and experimental results
indicating that a variety of conditions, both infectious and
noninfectious (i.e., burns, ischemia-reperfusion injury, multiple
trauma, pancreatitis), induce a similar host response. Two or more
of the following conditions must be fulfilled for the diagnosis of
SIRS to be made:
[0015] Body temperature >38.degree. C. or <36.degree. C.;
[0016] Heart rate >90 beats/minute;
[0017] Respiratory rate >20/minute or Paco.sub.2<32 mmHg;
[0018] Leukocyte count >12.000/.mu.l, <4000/.mu.L, or >10%
immature (band) forms.
[0019] All of these pathophysiologic changes must occur as an acute
alteration from baseline in the absence of other known causes for
them such as chemotherapy-induced neutropenia and leukopenia.
[0020] As a particular representative of a subacute or chronic
systemic inflammatory response, the clinical symptoms seen with an
auto-immune inflammatory disease such as diabetes are here
discussed. The non-obese diabetic (NOD) mouse is a model for
auto-immune disease, in this case insulin-dependent diabetes
mellitus (IDDM) which main clinical feature is elevated blood
glucose levels (hyperglycemia). The elevated blood glucose level is
caused by auto-immune inflammatory destruction of insulin-producing
.beta.-cells in the islets of Langerhans of the pancreas. This is
accompanied by a massive cellular infiltration surrounding and
penetrating the islets (insulitis) composed of a heterogeneous
mixture of CD4+ and CD8+ T-lymphocytes, B-lymphocytes, macrophages
and dendritic cells. Also in subacute and chronic inflammation,
crucial inflammatory mediators are tumor necrosis factor-.alpha.
(TNF-.alpha.), tissue growth factor-.beta. (TGF-.beta., interferon
.gamma., interleukins (IL-1, IL-4, IL-5, IL-6, IL-10, IL-12, IL-23,
IL-40), nitric oxide (NO), arachidonic acid metabolites and
prostaglandins 1 and 2 (PGE1 and PGE2), and others.
[0021] The NOD mouse represents a model in which a primary
inflammatory response mediated by inflammatory mediators and
directed against .beta.-cells is the primary event in the
development of IDDM. When the NOD mouse is not yet diabetic, an
inflammation invariably directed at the .beta.-cells develops.
Diabetogenesis is mediated through a multifactorial interaction
between a unique MHC class II gene and multiple, unlinked, genetic
loci, as in the human disease. Moreover, the NOD mouse demonstrates
beautifully the critical interaction between heredity and
environment, and between primary and secondary inflammatory
responses, its clinical manifestation, for example, depending on
various external conditions, most importantly of the microorganism
load of the environment in which the NOD mouse is housed. During
the diabetic phase, the inflammatory responses in the mice (and
humans suffering from established diabetes) are much more diverse,
due to the vascular damage caused by the high glucose levels tissue
damage results throughout the body, again inflammatory mediators
get released and secondary inflammations flourish, resulting in
inflammation throughout whole body, however, with much more serious
consequences to the patient than the earlier phase at first sight
seems to cause.
[0022] As for auto-immunity demonstrable in NOD mice, most
antigen-specific antibodies and T-cell responses which are measured
directed against various antigens were detected as self-antigens in
diabetic patients. Understanding the role these auto-antigens play
in NOD diabetes allows to further distinguish between an initial
inflammatory response directed at pathogenic auto-antigens leading
to the diabetic phase per se and the secondary inflammatory
responses that are observed as an epiphenomenon.
[0023] In general, T-lymphocytes play a pivotal role in initiating
the immune-mediated disease process. CD4+ T-cells can be separated
into at least two major subsets Th1 and Th2. Activated Th1 cells
secrete IFN-.gamma. and TNF-.alpha., while Th2 cells produce IL-4,
IL-5 and IL-10. Th1 cells are critically involved in the generation
of effective cellular immunity, whereas Th2 cells are instrumental
in the generation of humoral and mucosal immunity and allergy,
including the activation of eosinophils and mast cells and the
production of IgE. A number of studies have now correlated diabetes
in mice and human with Th1 phenotype development. On the other
hand, Th2 T-cells are shown to be relatively innocuous. Some have
even speculated that Th2 T-cells in fact, may be protective. It was
shown that the ability of CD4+ T-cells to transfer diabetes to
naive recipients resided not with the antigen specificity
recognized by the TCR per se, but with the phenotypic nature of the
T-cell response. Strongly polarized Th1 T-cells transferred disease
into NOD neonatal mice, while Th2 T-cells did not, despite being
activated and bearing the same TCR as the diabetogenic Th1 T-cell
population. Moreover, upon co-transfer, Th2 T-cells could not
ameliorate the Th1-induced diabetes, even when Th2 cells were
co-transferred in ten-fold excess.
[0024] In summary, the crucial pathophysiologic event that
precipitates acute as well as systemic inflammation is tissue
damage after which inflammatory mediators, in particular cytokines,
are released that initiate the inflammatory process. This can occur
as a result of the direct injury to tissues from mechanical or
thermal trauma as well as cellular injury induced by immunological
or inflammatory mediators such as seen after, for example,
ischemia-reperfusion injury or during a microbial infection of the
tissue. Cellular injury results in the acute release of
proinflammatory cytokines. If injury is severe, such as in
extensive tissue damage, a profound release of cytokines occurs,
resulting in the induction of a systemic inflammatory reaction. The
ability of the host to adapt (acutely or chronically) to this
systemic inflammatory response is dependent on the magnitude of the
response, the duration of the response, and the adaptive capacity
of the host.
[0025] The current invention relates to the body's innate way of
modulation of important physiological processes and builds on
insights reported in WO99/59617, WO01/72831 and PCT/NL02/00639.
[0026] In these earlier applications, small gene-regulatory
peptides are described that are present naturally in pregnant women
and are derived from proteolytic breakdown of placental
gonadotropins such as human chorionic gonadotropin (hCG) produced
during pregnancy. These peptides (in their active state often only
at about 4 to 6 amino acids long) were shown to have unsurpassed
immunological activity that they exert by regulating expression of
genes encoding inflammatory mediators such as cytokines.
Surprisingly, it was found that breakdown of hCG provides a cascade
of peptides that help maintain a pregnant woman's immunological
homeostasis. These peptides are nature's own substances that
balance the immune system to assure that the mother stays
immunologically sound while her fetus does not get prematurely
rejected during pregnancy but instead is safely carried through its
time of birth.
[0027] Where it was generally thought that the smallest breakdown
products of proteins have no specific biological function on their
own (except to serve as antigen for the immune system), it now
emerges that the body in fact routinely utilizes the normal process
of proteolytic breakdown of the proteins it produces to generate
important gene-regulatory compounds, short peptides that control
the expression of the body's own genes. Apparently the body uses a
gene-control system ruled by small broken down products of the
exact proteins that are encoded by its own genes.
[0028] It is long known that during pregnancy the maternal system
introduces a status of temporary immuno-modulation which results in
suppression of maternal rejection responses directed against the
fetus. Paradoxically, during pregnancy, often the mother's
resistance to infection is increased and she is found to be better
protected against the clinical symptoms of various auto-immune
diseases such as rheumatism and multiple sclerosis. The protection
of the fetus can thus not be interpreted only as a result of immune
suppression. Each of the above three applications have provided
insights by which the immunological balance between protection of
the mother and protection of the fetus can be understood.
[0029] It was shown that certain short breakdown products of hCG
(i.e., short peptides which can easily be synthesized, if needed
modified, and used as pharmaceutical composition) exert a major
regulatory activity on pro- or anti-inflammatory cytokine cascades
that are governed by a family of crucial transcription factors, the
NF.kappa.B family which stands central in regulating the expression
of genes that shape the body's immune response.
[0030] Most of the hCG produced during pregnancy is produced by
cells of the placenta, the exact organ where cells and tissues of
mother and child most intensely meet and where immuno-modulation is
most needed to fight off rejection. Being produced locally, the
gene-regulatory peptides which are broken down from hCG in the
placenta immediately balance the pro- or anti-inflammatory cytokine
cascades found in the no-man's land between mother and child. Being
produced by the typical placental cell, the trophoblast, the
peptides traverse extracellular space; enter cells of the immune
system and exert their immuno-modulatory activity by modulating
NF.kappa.B-mediated expression of cytokine genes, thereby keeping
the immunological responses in the placenta at bay.
SUMMARY OF THE INVENTION
[0031] It is herein postulated that the beneficial effects seen on
the occurrence and severity of auto-immune disease in the pregnant
woman result from an overspill of the hCG-derived peptides into the
body as a whole; however, these effects must not be overestimated,
as it is easily understood that the further away from the placenta,
the less immuno-modulatory activity aimed at preventing rejection
of the fetus will be seen, if only because of a dilution of the
placenta-produced peptides throughout the body as a whole. However,
the immuno-modulatory and gene-regulatory activity of the peptides
should by no means only be thought to occur during pregnancy and in
the placenta; men and women alike produce hCG, for example, in
their pituitaries, and nature certainly utilizes the
gene-regulatory activities of peptides in a larger whole.
[0032] Consequently, a novel therapeutic inroad is provided, using
the pharmaceutical potential of gene-regulatory peptides and
derivatives thereof. Indeed, evidence of specific up- or
down-regulation of NF.kappa.B driven pro- or anti-inflammatory
cytokine cascades that are each, and in concert, directing the
body's immune response was found in silico in gene-arrays by
expression profiling studies, in vitro after treatment of immune
cells and in vivo in experimental animals treated with
gene-regulatory peptides. Also, considering that NF.kappa.B is a
primary effector of disease (A. S. Baldwin, J. Clin. Invest., 2001,
107:3-6), using the hCG-derived gene-regulatory peptides offer
significant potential for the treatment of a variety of human and
animal diseases, thereby tapping the pharmaceutical potential of
the exact substances that help balance the mother's immune system
such that her pregnancy is safely maintained.
BRIEF DESCRIPTION OF DRAWING
[0033] FIG. 1. Mucosal treatment with gene-regulatory peptides
4+5+6 (experiment 3, LQGV+GVLPALPQ+VLPALP (SEQ ID NOS:1, 23 and 4,
respectively)) significantly increased the percentage of immature
MP20 high positive bone marrow cells.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The invention provides the treatment of a subject suffering
or believed to be suffering from disease by mucosal, preferably
oral administration of a pharmaceutical composition comprising a
pharmacologically effective amount of a gene-regulatory peptide or
functional analogue thereof together with a pharmaceutically
acceptable diluent to the subject. A particularly useful
pharmaceutically acceptable diluent is sterile water or an isotonic
salt solution such as 0.9% saline or phosphate buffered salt (PBS).
In a preferred embodiment, the invention provides the treatment of
a subject suffering or believed to be suffering from disease by
mucosal, preferably oral administration of a pharmaceutical
composition comprising a pharmacologically effective amount of two
or more gene-regulatory peptides or functional analogues thereof
together with a pharmaceutically acceptable diluent to the subject.
The administration dose of the gene-regulatory peptide may be
varied over a fairly broad range. The concentrations of an active
molecule which can be administered would be limited by efficacy at
the lower end and the solubility of the compound at the upper end.
The optimal dose or doses for a particular patient should and can
be determined by taking into consideration relevant factors such as
the condition, weight and age of the patient, and other
considerations of the physician or medical specialist involved.
[0035] The invention thus provides use of a regulatory peptide
pharmaceutical composition for mucosal, preferably oral application
to a subject for generating a systemic modulation of the expression
of a gene in a cell throughout the body of the subject. Useful
examples of such a gene-regulatory peptide can be selected from the
group of oligopeptides LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID
NO:2), LQGA (SEQ ID NO:3), VLPALPQVVC (SEQ ID NO:21), VLPALP (SEQ
ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ
ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ
ID NO:10), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12), VLPALPQ
(SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID NO:15),
GVLPALP (SEQ ID NO:16), GVLPALPQ (SEQ ID NO:23), LQGVLPALPQVVC (SEQ
ID NO:17), VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:24),
RPRCRPINATLAVEK (amino acids 1-15 of SEQ ID NO:25),
EGCPVCITVNTTICAGYCPT (amino acids 16-35 of SEQ ID NO:25),
SKAPPPSLPSPSRLPGPS (SEQ ID NO:26), SIRLPGCPRGVNPVVS (SEQ ID NO:27),
LPGCPRGVNPVVS (SEQ ID NO:18), LPGC (SEQ ID NO:19), MTRV (SEQ ID
NO:20), MTR, VVC, QVVC (SEQ ID NO:29) and functional analogues or
derivatives thereof. Functional analogues can, for example, be
found in urinary fractions derived from pregnant women or in
commercial preparations of hCG; at least in those commercial
preparations that contain substantial amounts of breakdown products
of hCG and have gene-regulatory activity, however, a disadvantage
of using such urinary fractions or even commercial hCG preparations
lies in the fact that they may or not may contain (sufficient)
quantities of the active compound, synthetic peptides are thus
preferred.
[0036] A preferred size of a gene-regulatory peptide for inclusion
in a pharmaceutical composition according to the invention is at
most 15 amino acids, although much smaller molecules have been
shown to be particularly effective. Surprisingly, the invention
provides here the insight that gene expression can be modulated or
regulated systemically by small peptides by applying them locally
to the mucosae. Oral treatment is preferred, but mucosal treatment
other than oral treatment is herein also provided. Preferred
peptides are breakdown products of larger polypeptides such as
chorionic gonadotrophin (CG) and growth hormones or growth factors
such as fibroblast growth factor, EGF, VEGF, RNA 3' terminal
phosphate cyclase and CAP18, or synthetic versions thereof.
Preferred for oral treatment are peptides smaller then five amino
acids, i.e., three or four amino acids long. In principle, such
regulating peptide sequences can be derived from any protein of
polypeptide molecule produced by prokaryotic and/or eukaryotic
cells, and the invention provides the insight that breakdown
products of polypeptides, preferably oligopeptides at about the
sizes as provided herein that are naturally involved as
gene-regulatory peptide in modulation of gene expression can be
applied via the mucosa to generate a systemic effect. In
particular, a (synthetic) gene-regulatory peptide is provided
obtainable or derivable from .beta.-human chorionic gonadotrophin
(.beta.-hCG), preferably from nicked .beta.-HCG. It was thought
before that breakdown products of .beta. hCG were involved in
immuno-modulation via regulation of gene expression (WO99/59671,
WO01/72831, PCT/NL02/00639) or in the treatment of wasting syndrome
(WO97/49721) but a relationship with systemic modulation of gene
expression, in particular via local application at or through the
mucosa was not forwarded in these publications. Of course, such a
gene-regulatory peptide, or functional equivalent or derivative
thereof, is likely obtainable or derivable from other proteins that
are subject to breakdown or proteolysis and that are close to a
gene-regulatory cascade. Preferably, the peptide signaling molecule
is obtained from a peptide having at least 10 amino acids such as a
peptide having an amino acid sequence MTRVLQGVLPALPQVVC (SEQ ID
NO:30), SIRLPGCPRGVNPVVS (SEQ ID NO:27),
VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:24),
RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT (SEQ ID NO:25),
CALCRRSTTDCGGPKDHPLTC (SEQ ID NO:31), SKAPPPSLPSPSRLPGPS (SEQ ID
NO:26), CRRSTTDCGGPKDHPLTC (SEQ ID NO:32),
TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO:33).
EXAMPLES
[0037] Not wishing to be bound by theory, it is postulated herein
that an unexpected mode of gene regulation with far reaching
consequences for the oral or mucosal treatment of disease has been
uncovered. Polypeptides, such as endogenous CG, EGF, etc., but also
polypeptides of pathogens such as viral, bacterial or protozoal
polypeptides, are subject to breakdown into distinct oligopeptides,
for example, by intracellular proteolysis. Distinct proteolytic
enzymes are widely available in the cell, for example, in
eukaryotes in the lysosomal or proteasomal system. Some of the
resulting breakdown products are oligopeptides of 3 to 15,
preferably 4 to 9, most preferably 4 to 6, amino acids long that
are surprisingly not without any function or effect to the cell,
but as demonstrated herein may be involved, possibly via a feedback
mechanism in the case of breakdown of endogenous polypeptides, as
signaling molecules in the regulation of gene expression, as
demonstrated herein by the regulation of the activity or
translocation of a gene transcription factor such as NF.kappa.B by,
for example, peptides LQGV (SEQ ID NO:1), VLPALPQVVC (SEQ ID
NO:21), LQGVLPALPQ (SEQ ID NO:17), LQG, GVLPALPQ (SEQ ID NO:23),
VLPALP (SEQ ID NO:4), VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID
NO:16), VVC, MTRV (SEQ ID NO:20), and MTR. Synthetic versions of
these peptides as described above, and functional analogues or
derivatives of these breakdown products, are herein provided to
modulate gene expression in a cell and be used in methods to
rectify errors in gene expression or the mucosal or oral treatment
of systemic disease. Oligopeptides such as LQG, AQG, LQGV (SEQ ID
NO:1), AQGV, LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4), ALPALP (SEQ
ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7), VLPAAPQ (SEQ
ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10), VLAALP (SEQ
ID NO:11), VLPALA (SEQ ID NO:12), VLPALPQ (SEQ ID NO:13), VLAALPQ
(SEQ ID NO:14), VLPALPA (SEQ ID NO:15), GVLPALP (SEQ ID NO:16),
GVLPALPQ (SEQ ID NO:23), LQGVLPALPQVVC (SEQ ID NO:17),
SIRLPGCPRGVNPVVS (SEQ ID NO:27), SKAPPPSLPSPSRLPGPS (SEQ ID NO:26),
LPGCPRGVNPVVS (SEQ ID NO:18), LPGC (SEQ ID NO:19), MTRV (SEQ ID
NO:20), MTR, VVC, or functional analogues or trimer or tetramer
derivatives (including breakdown products) of the longer sequences
thereof, are particularly effective. In particular, preferred for
oral administration are LQG, QVV, PALP (SEQ ID NO:34), AQG, LAG,
LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), and LAGV (SEQ ID
NO:10).
[0038] In a preferred embodiment, the invention provides the
treatment of a subject suffering or believed to be suffering from
inflammatory disease by mucosal, preferably oral administration of
a pharmaceutical composition comprising a pharmacologically
effective amount of a gene-regulatory peptide capable of regulating
expression of genes encoding inflammatory mediators such as
cytokines. Useful gene-regulatory peptides for inclusion in a
pharmaceutical application for mucosal administration for the
treatment of disease, in particular inflammatory disease, are those
peptides that are present naturally in pregnant women and are
derived from proteolytic breakdown of placental gonadotropins such
as human chorionic gonadotropin (hCG) produced during pregnancy,
however, synthetic variants and modifications of these peptides
that have functional equivalent or analogue activity can be
synthesized and tested for their activity easily by the person
skilled in the art, using, for example, animal experiments, such as
experiments with NOD mice as explained herein. In another
embodiment, the invention provides a pharmaceutical composition for
mucosal application comprising a gene-regulatory peptide or
functional analogue thereof, and use of a gene-regulatory peptide
or functional analogue thereof for the production of a
pharmaceutical composition for mucosal application. Such a
composition is most useful to apply to a mucosal surface area, the
inner buccal surfaces and surfaces of the tongue, the surfaces of
the (upper and lower) intestinal tract, the mucosal surfaces of the
nose and (upper and lower) respiratory tract, and thereby,
considering that in general the mucosal surfaces are permeable for
most gene-regulatory peptides that are smaller than nine, but
preferably smaller than seven amino acids, such as three or four
amino acids long, often affects more than only the area to which it
is applied, and is most useful to treat the body systemically,
i.e., as a whole, as well.
[0039] The inventors have now unearthed an insight in the biology
and physiology of the nature of regulatory factors in gene
regulation in cellular organisms that allows for an unexpected fast
progress in the identification and development of an artificial or
synthetic compound acting as a gene regulator and its use as new
chemical entity for the production of a pharmaceutical composition
for mucosal application or its use in the treatment of inflammatory
disease via the mucosal application of a gene-regulatory peptide.
The insight is herein provided that many of small peptides that are
derivable by proteolytic breakdown of endogenous proteins of an
organism, or that are derivable by proteolytic breakdown of
proteins of a pathogen, i.e., during the presence of the pathogen
in a host organism, that exert an often very specific
gene-regulatory activity on cells of the organism can actually
exert this activity even on a systemic level when administered via
mucosal uptake, such as by oral use, rectal application, nasal
spray, upper airway aerosol application, and so on. In a particular
embodiment, the present invention has major value for investigators
in furthering the quality and quantity of knowledge regarding the
systemic mechanisms controlling NF.kappa.B-initiated gene
expression and resulting inflammatory responses in a subject by
treatment of a subject with a pharmaceutical composition for
mucosal application, for example, by oral use.
[0040] This insight was gained in a two fold way. In one
experiment, designed to test the influence of mucosal uptake of
gene-regulatory peptides, it was shown that
NF.kappa.B-down-regulating peptides, instilled once daily in the
buccal sac of non-diabetic NOD mice had an unexpected beneficial
influence on the overall development of diabetes in these mice. The
incidence of the development of the insulitis, the primary
inflammation in the pathogenesis of diabetes, was severely reduced.
In another experiment, already diabetic NOD mice were given
drinking water with or without NF.kappa.B-down-regulating peptides,
and also a beneficial effect was observed, the clinical
consequences of the typical secondary and systemic inflammation
caused by the vascular damage were remarkably less severe, whereby
the drinking water therapy contributed to a much better physical
appearance of the treated versus the untreated group. Similar
results were seen in mice treated mucosally or orally with a
pharmaceutical composition comprising a functional analogue to a
gene-regulatory peptide, a human chorionic gonadotropin (hCG)
produced during pregnancy and proteolytic breakdown products
thereof, however, batch wise differences in dose and effect were
observed, likely reflecting batch wise differences in concentration
of the regulatory peptides involved.
[0041] With these insights the invention provides among others a
screening method for identifying or obtaining a gene-regulatory
peptide suitable for mucosal or oral application comprising a
peptide or functional derivative or analogue thereof capable of
modulating expression of a gene in a cell, be it in vitro or in
vivo in an experimentally diseased animal such as a monkey or a
small laboratory animal such as a rat or mouse, comprising
providing the animal via a mucosal route with at least one peptide
or derivative or analogue thereof and determining the clinical
response of the animal to the treatment or the expression of one or
more genes in an animal or the activity and/or nuclear
translocation of a gene transcription factor. It is in particular
useful when the peptide is 3 to 15 amino acids long, more
preferably, wherein the peptide is 3 to 9 amino acids long, most
preferred wherein the peptide is 3 or 4 to 6 amino acids long.
[0042] Functional derivative or analogue herein relates to the
gene-regulatory effect or activity as, for example, can be measured
by measuring the peptide's or its analogue's or derivative's effect
on gene expression or on nuclear translocation of a relevant
transcription factor, such as NF.kappa.B in an NF.kappa.B assay, or
AP-1 in an AP-1 assay, or by another method as is available in the
art. Fragments can be somewhat (i.e., one or two amino acids)
smaller or larger on one or both sides, while still providing
functional activity.
[0043] A screening method according to the invention is also
provided wherein the method further comprises determining whether
the gene transcription factor regulates the transcription of a
cytokine as, for example, measured by detecting cytokine transcript
levels or the actual presence as such in the treated cell or
animal, or wherein the gene transcription factor comprises an
NF.kappa.B/Rel protein, or by determining relative up-regulation
and/or down-regulation of at least one gene of interest expressed
in the animal or of a multitude of genes expressed in the animal,
as easily can be done with gene chip technology.
[0044] Of course, the invention aims at providing pharmaceutical
compositions for mucosal application, such as oral use, that act as
a signaling molecule useful in modulating expression of a gene in a
cell and are identifiable or obtainable by employing a screening
method according to the invention as provided herein. Useful
signaling molecules are already provided herein as modulators of
NF.kappa.B/Rel protein-mediated gene-expression, as detailed
further on. The invention also provides use of a signaling molecule
as thus provided for the production of a pharmaceutical composition
for the modulation of gene expression, for example, by inhibiting
NF.kappa.B/Rel protein activation, or its use for the production of
a pharmaceutical composition for the treatment of a primate or
domestic animal.
[0045] That small peptides, and even breakdown products, can have
biological activity, is already known. Proteolytic breakdown
products of endogenous or pathogen-derived proteins are, for
example, routinely generated by the proteasome system and presented
in the context of class I or II major histocompatibility complex
(MHC). Also, it has been recognized that classically known
neuropeptides (also known as peptide neurotransmitters) or small
peptide hormones, such as antidiuretic hormone, oxytocin,
thyrotropin-releasing hormone, gonadotropin-releasing hormone,
somatostatins gastrin, cholecystokinin, substance-P, enkephalins,
neurotensin, angiotensins, and derivatives or equivalents thereof
have distinct biological activity which is, in general, mediated by
cell-surface receptor interaction. Furthermore, it is now known
that certain small and arginine- or lysine- or proline-rich
peptides, i.e., having more than 50% of arginine, or 50% of lysine
or 50% of proline, or having more than 50% arginine and lysine, or
more than 50% arginine and proline, or more than 50% lysine and
proline, or more than 50% arginine and lysine and proline residues,
have distinct membrane-permeation properties that may result in
biological activity. The gene-regulatory peptides as used herein
are other than the classically known neuropeptides or peptide
hormones, and other than the above identified arginine- or lysine-
or proline-rich peptides.
[0046] The present invention relates to small peptides suitable for
mucosal application to treat disease systemically, in that the
mucosal application has a systemic effect on a disease or condition
in a subject treated via mucosal application with such a small
peptide. Mucosal use and systemic effect of the gene-regulatory
peptides is surprising. It is preferred that the peptides of the
invention are small. A most preferred size is 4 to 6 amino acids,
peptides of 2 to 3 amino acids are also very well feasible, a size
of 7 to 15 amino acids is also feasible but becomes less practical
for mucosal application and peptides from 10 to 15 amino acids or
larger are preferably broken down to smaller, functionally more
active, fragments.
[0047] As said, the invention provides the insight that small
peptides that are derivable or obtainable by proteolytic breakdown
of endogenous proteins of an organism, or that are derivable or
obtainable by proteolytic breakdown of proteins of a pathogen,
i.e., during the presence of the pathogen in a host organism, can
exert an often very specific and systemic gene-regulatory activity
on cells throughout the body of the organism, even after they have
been applied only to a mucosal surface of the organism. This
insight produces an immediate incentive for systematic approaches
to practice or execute a method as provided herein to identify a
signaling molecule, by obtaining information about the capacity or
tendency of a small (oligo)peptide, or a modification or derivative
thereof, (herein jointly called lead peptide) to systemically
regulate expression of a gene after mucosal application and
provides an incentive to try and test the chances of intradermal,
transdermal, or hypodermal application.
[0048] The gene-regulatory peptide can be administered and
introduced in-vivo preferably via any mucosal route, and possibly
via passage through the skin. The peptide, or its modification or
derivative, can be administered as the entity as such or as a
pharmaceutically acceptable acid- or base-addition salt, formed by
reaction with an inorganic acid (such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid); or with an organic acid (such
as formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid); or by reaction with an inorganic
base (such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide); or with an organic base (such as mono-, di-, trialkyl
and aryl amines and substituted ethanolamines). A selected peptide
and any of the derived entities may also be conjugated to DMSO,
translocating peptides, sugars, lipids, other polypeptides, nucleic
acids and PNA; and function in-situ as a conjugate or be released
locally after reaching a targeted tissue or organ.
[0049] The invention also provides a pharmaceutical composition for
the treatment of a subject suffering from a disease or disorder,
the pharmaceutical composition comprising a pharmacologically
effective amount of a gene-regulatory peptide together with a
pharmaceutically acceptable diluent. In particular, the invention
provides a pharmaceutical composition for mucosal application
comprising a gene-regulatory peptide or functional analogue
thereof, and use of a gene-regulatory peptide or functional
analogue thereof for the production of a pharmaceutical composition
for mucosal application. In a preferred embodiment, the invention
provides a pharmaceutical composition for mucosal application
comprising two or more gene-regulatory peptides or functional
analogues thereof, and use of two or more gene-regulatory peptides
or functional analogues thereof for the production of a
pharmaceutical composition for mucosal application.
[0050] In one embodiment, it is preferred that the pharmaceutical
composition is in a form suitable for mucosal administration. In a
much preferred embodiment, the form for mucosal administration is
selected from the group consisting of sprays, liquids and gels,
preferably with a watery base. In a much preferred embodiment, the
invention provides a pharmaceutical composition for the treatment
of a subject suffering from a disease or disorder, the
pharmaceutical composition comprising a pharmacologically effective
amount of a gene-regulatory peptide together with a
pharmaceutically acceptable diluent wherein the pharmaceutical
composition is in a form suitable for oral administration. It is
preferred that the form for oral administration is selected from
the group consisting of capsules, tablets, liquids, oral
suspensions, emulsions and powders.
[0051] Although the gene-regulatory peptide may be prepared by
other methods known for the preparation of analogous compounds
(e.g., by use of a solid phase synthesis), a method of making the
gene-regulatory peptide is described in the detailed description
herein. During the process of preparation, solvents such as
N,N-dimethylformamide (DMF), 1-butanol, 2-butanol, ethanol,
methanol, ethyl acetate, methylene chloride, hexane, diethyl ether,
water, acetic acid, and others may be used. Catalysts containing
palladium or molybdenum may also be used in the preparation of the
gene-regulatory peptide.
[0052] However made, the gene-regulatory peptide forms
pharmacologically acceptable salts from pharmacologically
acceptable organic and inorganic acids such as hydrochloric,
hydrobromic, fumaric, phosphoric, ascorbic, tartaric, citric,
lactic, maleic, palmitic, and other well-known acids. Especially
preferred are the hydrochloric and acetic acid salts. The acid
addition salts are obtained by reacting the gene-regulatory peptide
with the acid.
[0053] Methods of crystallizing compounds are described in Chase et
al., Remington's Pharmaceutical Sciences (16th ed., Mack Publishing
Co., Easton. PA, U.S.A., 1980) ("Remington's"), at page 1535.
[0054] A crystalline gene-regulatory peptide can be used to make
numerous dosage forms such as powders for insufflations, powders
for reconstitution, tablet triturates (e.g., dispensing tablets and
hypodermic tablets), other tablets, and so forth.
[0055] The pharmaceutical compositions containing the crystalline
gene-regulatory peptide are preferably dispensed in unit dosage
forms, such as tablets, capsules, pills, powders, granules,
suppositories, sterile parenteral solutions or suspensions and
non-parenteral solutions or suspensions, containing suitable
quantities of the pharmaceutically acceptable salt of the
gene-regulatory peptide.
[0056] Methods and compositions for making such dosage forms are
well-known to those skilled in the art. For example, methods of
making powders and their compositions are described at pages 1535
through 1552 of Remington's. Insufflations are described at page
1552, and insufflators are described at 1792. Methods and
compositions for making tablets and pills, containing active
ingredients, are described in Remington's, at pages 1553 through
1584. Methods of coating pharmaceutical dosage forms and making
prolonged release pharmaceuticals are described at pages 1585-1613
of Remington's. The contents of these pages are hereby incorporated
by this reference.
[0057] The crystalline gene-regulatory peptide may also be
incorporated into devices intended for implantation into a patient.
Such devices, polymers intended for use therein, and methods of
making both are described in U.S. Pat. Nos. 3,773,919, 4,767,628,
and 4,675,189. For example, a sufficient quantity of the
crystalline gene-regulatory peptide could be incorporated into a
PLAGA implant to allow for the release of gene-regulatory peptide
(e.g., 5 mg per day for one month) into the patient's body.
[0058] One advantage with pharmaceutical compositions containing
the crystalline versus the amorphous product, is that the
pharmaceutical composition containing the crystalline salt product,
having twice the bioavailability of the amorphous product, may need
only contain half the absolute amount of the active ingredient on
certain mucosa thus decreasing the amount of ingredient needed to
be insufflated or otherwise administered and decreasing the
ultimate cost of the composition. Such mucosa would include the
nasal and the buccal mucosa.
[0059] Although the pharmaceutical compositions containing the
crystalline gene-regulatory peptide may be formulated with
adjuvants such as solubilizers, they need not be. The ability to
use solely the crystalline gene-regulatory peptide (i.e., the
crystalline acid addition salt of the gene-regulatory peptide) in a
pharmaceutical composition to be applied to, for example, a nasal
mucosa has advantages. For one thing, certain adjuvants are not
suitable for chronic administration. However, long term
administration may be necessary for the particular person ingesting
the gene-regulatory peptide. Another advantage is that the
adjuvants necessarily take up a portion of the pharmaceutical
composition, which portion may be better suited for the
gene-regulatory peptide in order to decrease mucosal
discomfort.
[0060] However if it is desired, suitable solubilizers, buffers,
swelling agents, etc. may be used in such formulations. Buffering
agents are preferably those which keep the gene-regulatory peptide
in its unionized form.
[0061] The dosage of the crystalline acid addition
salt/gene-regulatory peptide administered will generally be
dependent upon the kind of disorder to be treated, the type of
patient involved, his age, health, weight, kind of concurrent
treatment, if any, and length and frequency of treatment.
[0062] The dosage forms will be administered over varying
durations. To treat a disorder, the compounds are administered to a
patient for a length of time sufficient to alleviate the symptoms
associated with the disorders that the patient is suffering from.
This time will vary, but periods of time exceeding two months are
especially preferred. After the symptoms have been alleviated, the
compound may then be discontinued to determine whether it is still
required by the particular patient.
[0063] To prevent the occurrence of inflammatory disease, and thus
alleviate the need for treatment, the compounds are administered to
a person believed to be susceptible to suffering from inflammatory
disease some time in the future (e.g., patients undergoing
treatment with cytotoxic drugs such as vincristine; diabetics;
alcoholics; etc.) for so long as he or she is believed susceptible.
The length of such prophylactic administration of the compounds
will of course vary, but again, periods of time exceeding two
months are preferred. If the reason for the supposed susceptibility
to an inflammatory disease has ceased to exist, the compound may
then be discontinued. If, however, the reason for the disorder has
not ceased to exist (e.g., in the case of diabetes) the compound
may be needed to be administered for the person's lifetime.
[0064] Illustratively, dosage levels of the administered active
ingredients can be (intranasally): between 0.55 mg and 270 mg per
day. In human therapy, daily doses of between 8 mg and 120 mg,
administered orally, will preferably be used.
[0065] The invention provides a pharmaceutical composition for oral
application comprising a gene-regulatory peptide or functional
analogue thereof, and use of a gene-regulatory peptide or
functional analogue thereof for the production of a pharmaceutical
composition for oral application. In a preferred embodiment, the
invention provides a pharmaceutical composition for oral
application comprising two or more gene-regulatory peptides or
functional analogues thereof, and use of two or more
gene-regulatory peptides or functional analogues thereof for the
production of a pharmaceutical composition for oral
application.
[0066] The gene-regulatory peptide(s) is (are) incorporated into
dosage units for oral administration. The term "dosage unit"
generally refers to physically discrete units suitable as unitary
dosages for humans or animals, each containing a predetermined
quantity of active material (e.g., gene-regulatory peptide)
calculated to produce the desired effect. Methods and compositions
for making such dosage units are well-known to those skilled in the
art. For example, methods and compositions for making tablets and
pills, containing active ingredients, are described in the standard
reference, Chase et al., Remington's Pharmaceutical Sciences (16th
ed., Mack Publishing Co., Easton, Pa., U.S.A., 1980)
("Remington's"), at pages 1553 to 1584. Methods of making powders,
and their composition are described at pages 1535 to 1552 of the
reference. Methods of coating pharmaceutical dosage forms are
described at pages 1585 to 1593 of Remington's.
[0067] For making dosage units, e.g., tablets, the use of
conventional additives, e.g., fillers, colorants, polymeric binders
and the like is contemplated. In general any pharmaceutically
acceptable additive which does not interfere with the function of
the active compounds can be used in the one or more of the
compositions.
[0068] Suitable carriers with which the compositions can be
administered include lactose, starch, cellulose derivatives and the
like used in suitable amounts. Lactose is a preferred carrier.
Mixtures of carriers can also be used.
[0069] A process of manufacturing the pharmaceutical composition
for oral use involves mixing predetermined quantities of peptide
with predetermined quantities carrier and converting the mixture
into the first dosage units (e.g., by filling capsules or molding
tablets with the mixture and any desired excipients)
[0070] A preferred process of manufacturing the gene-regulatory
product according to the invention involves incorporating the
desired dosages of gene-regulatory peptide into a tablet by known
techniques. Tablets or other dosage units containing different
amounts and types of gene-regulatory peptides may be of different
colors, and kept in different portions of, for example, a blister
pack.
[0071] In another embodiment, the invention provides a
pharmaceutical composition for rectal application, such as a
suppository comprising a gene-regulatory peptide or functional
analogue thereof, and use of a gene-regulatory peptide or
functional analogue thereof for the production of a pharmaceutical
composition for rectal application.
[0072] In another embodiment, the invention provides a
pharmaceutical composition for sub- or transdermal application
comprising a gene-regulatory peptide or functional analogue
thereof, and use of a gene-regulatory peptide or functional
analogue thereof for the production of a pharmaceutical composition
for sub- or transdermal application.
[0073] A pharmaceutical composition for mucosal application or
application via the skin as provided herein is particularly useful
for the modulation of gene expression by inhibiting NF.kappa.B/Rel
protein-mediated cytokine activation.
[0074] NF.kappa.B/Rel proteins are a group of structurally related
and evolutionarily conserved proteins (Rel). Well known are c-Rel,
RelA (p65), RelB, NF.kappa.B1 (p50 and its precursor p105), and
NF.kappa.B2 (p52 and its precursor p100). Most NF.kappa.B dimers
are activators of transcription; p50/p50 and p52/p52 homodimers
repress the transcription of their target genes. All NF.kappa.B/Rel
proteins share a highly conserved NH2-terminal Rel homology domain
(RHD). RHD is responsible for DNA binding, dimerization, and
association with inhibitory proteins known as I.kappa.BS. In
resting cells, NF.kappa.B/Rel dimers are bound to I.kappa.Bs and
retained in an inactive form in the cytoplasm. I.kappa.Bs are
members of a multigene family (I.kappa.B.alpha.; I.kappa.B.beta.,
I.kappa.B.gamma., I.kappa.Bepsilon, Bcl-3, and the precursor
Rel-proteins, p100 and p105. Presence of multiple copies of ankyrin
repeats interact with NF.kappa.B via the RHD (protein-protein
interaction. Upon appropriate stimulation, I.kappa.B is
phosphorylated by I.kappa.B Kinase (IKKs), polyubiquitinated by
ubiquitin ligase complex, and degraded by the 26S proteosome.
NF.kappa.B is released and translocates into nucleus to initiate
gene expression.
[0075] NF.kappa.B regulation of gene expression includes innate
immune responses: such as regulated by cytokines IL-1, IL-2, IL-6,
IL-12, TNF-.alpha., LT-.alpha., LT-.beta., GM-CSF; expression of
adhesion molecules (ICAM, VCAM, endothelial leukocyte adhesion
molecule [ELAM]), acute phase proteins (SAA), inducible enzymes
(iNOS and COX-2) and antimicrobial peptides (.beta.-defensins). For
adaptive immunity, MHC proteins IL-2, IL-12 and IFN-.alpha. are
regulated by NF.kappa.B. Regulation of overall immune response
includes the regulation of genes critical for regulation of
apoptosis (c-IAP-1 and c-IAP-2, Fas Ligand, c-myc, p53 and cyclin
D1).
[0076] Considering that NF.kappa.B and related transcription
factors are cardinal pro-inflammatory transcription factors, and
considering that the invention provides a gene-regulatory peptide
and functional analogue or derivative suitable for mucosal
application that is capable of systemically inhibiting NF.kappa.B
and likely also other pro-inflammatory transcription factors,
herein also called NF.kappa.B inhibitors, the invention provides a
method and pharmaceutical composition for systemically modulating
NF.kappa.B activated gene expression, in particular for inhibiting
the expression and thus inhibiting a central pro-inflammatory
pathway. In a preferred embodiment, the gene-regulatory peptide is
administered orally, to exert its activity systemically, beyond the
mucosal surface to which it is applied.
[0077] The consequence of this potency to inhibit this
pro-inflammatory pathway systemically via a mucosal, such as an
oral application, is wide and far-reaching.
[0078] For one, a novel therapeutic inroad is provided using the
pharmaceutical potential of gene-regulatory peptides and
derivatives applied mucosally or orally for generating a systemic
response directed at modulating NF.kappa.B-mediated disease.
Earlier, we presented evidence of specific up- or down-regulation
of NF.kappa.B driven pro- or anti-inflammatory cytokine cascades
that are each, and in concert, directing the body's immune response
was found in silico in gene-arrays by expression profiling studies,
in vitro after treatment of immune cells and in vivo in
experimental animals treated with gene-regulatory peptides. Also,
considering that NF.kappa.B is a primary effector of disease, using
the hCG-derived gene-regulatory peptides via an oral or otherwise
mucosal application offers significant potential for the treatment
of a variety of human and animal diseases, thereby tapping the
systemic pharmaceutical potential of the exact substances that help
balance the mother's immune system such that her pregnancy is
safely maintained by applying a gene-regulatory peptide mucosally,
preferably orally.
[0079] Examples of NF.kappa.B-modulated disease are foremost found
among the earlier discussed inflammatory conditions.
[0080] Conditions that can be treated orally with a pharmaceutical
composition as provided herein preferably include subacute or
chronic inflammatory disease, such as diabetes type I or II,
rheumatic disease, Sjogrens syndrome, multiple sclerosis),
transplantation-related immune responses such as
graft-versus-host-disease, post-transfusion thrombocytopenia,
sub-acute and chronic transplant rejection, pre-eclampsia,
rheumatoid arthritis, inflammatory bowel disease, the inflammatory
component of neurological or psychiatric disorders,
atherosclerosis, asthma, allergy and chronic auto-immune disease.
Especially the oral treatment of systemic autoimmune disease will
be very helpful in the treatment of patients with chronic,
immune-mediated inflammation, as is the case in autoimmune disease.
A non-limiting list of thus treatable autoimmune diseases includes:
Hashimoto's thyroditis, primary mysxoedema thyrotoxicosis,
pernicious anemia, autoimmune atrophic gastritis, Addison's
disease, premature menopause, insulin-dependent diabetes mellitus,
stiff-man syndrome, Goodpasture's syndrome, myasthenia gravis, male
infertility, pemphigus vulgaris, pemphigoid, sympathetic
ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune
hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic
leucopenia, primary biliary cirrhosis, active chronic hepatitis,
cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome,
rheumatoid arthritis, dermatomyositis, polymyositis, scleroderma,
mixed connective tissue disease, discoid lupus erythematosus, and
systemic lupus erythematosus.
[0081] The invention thus also relates to the treatment of the
inflammatory component of neurological disorders or so called
neuroimmune disorders such as schizophrenia, manic depression and
other bipolar disorders, post-partum psychosis and autism. The
invention provides a method for modulating a neurological disorder
in a subject comprising providing the subject with a
gene-regulatory peptide or functional analogue thereof. The
invention also provides use of an NF.kappa.B down-regulating
peptide or functional analogue thereof for the production of a
pharmaceutical composition for the treatment of a neurological
disorder.
[0082] The invention provides a method for modulating a
neurological disorder in a subject comprising providing the subject
orally with a gene-regulatory peptide or functional analogue
thereof, in particular wherein the regulatory peptide
down-regulates translocation and/or activity of a gene
transcription factor, such as an NF.kappa.B/Rel protein. Preferred
peptides for modulating a neurological disorder by oral treatment
are LQG, QVV, PALP (SEQ ID NO:34), AQG, LAG, LQGV (SEQ ID NO:1),
AQGV (SEQ ID NO:2), or LAGV (SEQ ID NO:10). The peptides are also
useful for the production of a pharmaceutical composition for the
treatment of a neurological disorder, especially wherein the
peptide or analogue is selected from the group of peptides
analogues having NF.kappa.B down-regulating activity in
LPS-stimulated RAW264.7 cells or in LPS-unstimulated RAW264.7
cells.
[0083] The invention also relates to the oral treatment of multiple
sclerosis, and in particular to the treatment of the inflammatory
injury seen in the progressive stages in the disease such as seen
with the recurrent upsurges of acute disease, classically known as
relapses or exacerbations herein identified as relapsing/remitting
disease seen in multiple sclerosis. The invention provides a method
for modulating relapsing/remitting disease as seen with multiple
sclerosis in a subject comprising providing the subject orally with
a gene-regulatory peptide or functional analogue thereof.
[0084] The invention in particular provides a method for modulating
relapsing/remitting disease as seen in multiple sclerosis in a
subject comprising orally providing the subject with a
gene-regulatory peptide or functional analogue thereof, in
particular wherein the gene-regulatory peptide down-regulates
translocation and/or activity of a gene transcription factor,
preferably wherein the gene transcription factor comprises an
NF.kappa.B/Rel protein of which translocation and/or activity is
inhibited. It is preferred to orally administer such a peptide,
preferably selected from the group of LQG, QVV, PALP (SEQ ID
NO:34), AQG, LAG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ
ID NO:10), when the subject is presenting clinical signs of
exacerbations.
[0085] The invention also provides use of such an NF.kappa.B
down-regulating peptide or functional analogue thereof for the
production of a pharmaceutical composition for the treatment of
relapsing/remitting disease as seen with multiple sclerosis.
[0086] The invention, thus, also relates to the treatment of
diabetes. The invention provides a method for modulating diabetes
in a subject comprising providing the subject orally with a
gene-regulatory peptide or functional analogue thereof. The
invention also provides use of an NF.kappa.B down-regulating
peptide or functional analogue thereof for the production of a
pharmaceutical composition for the oral treatment of diabetes.
[0087] The invention provides a method for modulating diabetes in a
subject comprising providing the subject orally with a
gene-regulatory peptide or functional analogue thereof, in
particular wherein the regulatory peptide down-regulates
translocation and/or activity of a gene transcription factor, such
as an NF.kappa.B/Rel protein. Preferred peptides for modulating
diabetes by oral treatment are LQG, QVV, PALP (SEQ ID NO:34), AQG,
LAG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), or LAGV (SEQ ID
NO:10). The peptides are also useful for the production of a
pharmaceutical composition for the treatment of diabetes by oral
administration, especially wherein the peptide or analogue is
selected from the group of peptides analogues having NF.kappa.B
down-regulating activity in LPS-stimulated RAW264.7 cells or in
LPS-unstimulated RAW264.7 cells.
[0088] The invention also provides a method of treatment of a
menopausal condition or a post-menopausal condition, such as
osteoporosis, comprising oral or mucosal treatment with a
gene-regulatory peptide according to the invention allowing
systemic modulation and inhibition of osteoclast differentiation
and inhibiting TNF-.alpha.-induced apoptosis of osteoblasts,
thereby limiting the dissolve of bone structures, otherwise so
prominent in post-menopausal women that have no longer a natural
source of hCG and thus lack the modulatory effect of the signal
molecules that are derived of hCG as shown herein. The invention
thus also provides a method of mucosal or oral treatment of a bone
disease, such as osteoporosis (which is often, but not exclusively,
seen with post-menopausal women). Furthermore, NO and TNF-.alpha.
modulators as provided herein inhibit the inflammatory response and
bone loss in periodontitis. Furthermore, considering that there is
a correlation between TNF-.alpha. activity and severity of clinical
manifestations in ankylosing spondylitis, the invention provides
the treatment of spondylitis by use of a gene-regulatory peptide as
provided herein. Preferred peptides for modulating a menopausal,
post-menopausal or osteoporosis condition by oral treatment are
QVV, PALP (SEQ ID NO:34), AQG, LAG, LQGV (SEQ ID NO:1), AQGV (SEQ
ID NO:2), or LAGV (SEQ ID NO:10).
[0089] The invention also relates to the oral or mucosal treatment
of an ischemic event such as a stroke or myocardial infarction.
[0090] An ischemic event refers to an event in which the blood
supply to a tissue is obstructed. Due to this obstruction, the
endothelial tissue lining the affected blood vessels becomes
"sticky" and begins to attract circulating white blood cells. The
white cells bound to the endothelium eventually migrate into the
affected tissue, causing significant tissue destruction. Although
neither acute myocardial infarction nor stroke is directly caused
by inflammation, much of the underlying pathology and the damage
that occurs after an acute ischemic event are caused by acute
inflammatory responses during reperfusion, the restoration of blood
flow to the affected organ. Early restitution of blood flow to
ischemic tissues is essential to halt the progression of cellular
injury associated with decrease of oxygen supply and nutrient
delivery. This fact provides the basis for the traditional view
that minimizing ischemic time is the only important intervention
for diminishing the extent of ischemic injury. However, it is now
well recognized that reperfusion of ischemic tissues initiates a
complex series of reactions that can paradoxically injure tissues.
Although several mechanisms have been proposed to explain the
pathogenesis of ischemia--reperfusion injury, most attention has
focused on a role for reactive oxygen and nitrogen metabolites and
inflammatory leukocytes. In addition to the local tissue injury,
distant organs can also be affected, particularly if the intensity
of the inflammatory reaction in postischemic tissue (e.g.,
intestine) is great. The remote effects of ischemia--reperfusion
injury are most frequently observed in the lung and (cardio- or
cerebro-) vascular system, and can result in the development of the
systemic inflammatory response syndrome (SIRS) and multiple organ
dysfunction syndrome (MODS), both of which account for 30 to 40% of
the mortality in tertiary referral intensive care units (ICUs).
[0091] The invention provides a method for modulating or treating
such an ischemic event in a subject comprising providing the
subject with orally or mucosally with a gene-regulatory peptide or
functional analogue thereof, in particular wherein the peptide
down-regulates translocation and/or activity of a gene
transcription factor, preferably wherein the gene transcription
factor comprises an NF.kappa.B/Rel protein and wherein
translocation and/or activity of the NF.kappa.B/Rel protein is
inhibited. For mucosal or oral application, it is preferred that
the peptide is selected from the group of peptides having
NF.kappa.B down-regulating activity in LPS-stimulated RAW264.7
cells, especially when the subject is at risk to experience
reperfusion injury occurring after the ischemic event.
[0092] For achieving a rapid clinical intervention by oral or
mucosal administration it is preferred that the peptide is selected
from the group of peptides having NF.kappa.B down-regulating
activity in LPS-unstimulated RAW264.7 cells, then the subject may
also be provided with a thrombolytic agent, such as when the
thrombolytic agent comprises tissue plasminogen activity.
[0093] Furthermore, the invention provides use of a gene-regulatory
peptide, preferably comprising an NF.kappa.B down-regulating
peptide or functional analogue thereof, for the production of a
pharmaceutical composition for the oral or mucosal treatment of
reperfusion injury occurring after an ischemic event in a subject.
The most preferred peptide for treating such a reperfusion injury
orally is AQGV (SEQ ID NO:2).
[0094] The invention furthermore relates to the oral or mucosal
treatment of immunosuppressive effects such as those seen after
trauma or major surgery. In the United States, posttraumatic sepsis
is responsible for 60% of all late deaths after trauma. The
susceptibility of trauma patients to sepsis seems to be caused at
least in part by a profound suppression of cellular immunity often
found after trauma, burns and hemorrhage. The relationship between
the nervous and the immune system following trauma or other
life-threatening events is poorly understood and under
investigation. Recent reviews have highlighted the complex nature
of the tremendous surge of hormone and catecholamine output from
the pituitary-adrenal axis following trauma, which may be mediated
through the spinal cord along afferent neurons from the site of
tissue destruction. Also, often a generalized depression of the
immune system exists. The invention provides a method for oral or
mucosal treatment of an immunosuppressive state in a subject
comprising providing the subject via oral or mucosal application
with a gene-regulatory peptide or functional analogue thereof. Such
treatment is particularly useful when the subject has experienced
trauma or major surgery likely resulting in an immunosuppressive
state. It is preferred that the peptide or analogue up-regulates
translocation and/or activity of a gene transcription factor such
as an NF.kappa.B/Rel protein AP-1 protein. In a much preferred
embodiment, the peptide is selected from the group of peptides
having NF.kappa.B up-regulating activity in LPS-unstimulated
RAW264.7 cells; such treatment is also very useful when the subject
is at risk to experience a counter anti-inflammatory response
syndrome, especially when the peptide is selected from the group of
peptides having NF.kappa.B up-regulating activity in LPS-stimulated
RAW264.7 cells. Further therapy may include providing the subject
with an agent directed against disseminated intravascular
coagulation, such as when the agent comprises Activated Protein C
activity. Also, the invention provides use of a gene-regulatory
peptide, in particular an NF.kappa.B up-regulating peptide or
functional analogue thereof for the production of a pharmaceutical
composition for the oral or mucosal treatment of an
immunosuppressive state or a counter anti-inflammatory response
syndrome in a subject.
[0095] The invention also relates to the field of (veterinary)
medicine and to the oral or mucosal treatment of subjects (be it
man or animal) that suffer from iatrogenic disease, i.e.,
experience problems or complications resulting from a medical
treatment. Iatrogenic events that result from activities of, for
example, physicians or surgeons are commonplace in modern medicine
and can often not be avoided. Various adverse conditions can occur
due to malpractice or neglect, such as wrongly selecting or
executing a therapy, misplacing or forgetting to remove surgical
utensils during surgery, and the like. However, most therapeutic or
surgical interventions, even those well selected and properly
executed, may, even beyond their beneficial effects, cause adverse
and often inflammatory conditions in a patient. Furthermore, also
tried and tested therapies in infectious disease, such as
treatments with antibiotics or antivirals, have their iatrogenic
side-effects, often related to the lysis or destruction of the very
micro-organism they are designed to be used against, and the
release of microbe membrane fragments and/or toxins which induces
additional pro-inflammatory cytokine release. Whatever the cause
may be, most iatrogenic events, herein defined as a disorder or
disease resulting from a treatment of a human or animal subject
with a pharmaceutical composition or by a medical or surgical
procedure, result in the damage, destruction or lysis of cells or
tissue of the subject, resulting in additional pro-inflammatory
cytokine release.
[0096] The invention provides a method for treating an iatrogenic
event in a subject comprising providing the subject orally or
mucosally with a gene-regulatory peptide or functional analogue
thereof, particularly when the peptide modulates translocation
and/or activity of a gene transcription factor such as an
NF.kappa.B/Rel protein or causes inhibition of an NF.kappa.B/Rel
protein-mediated cytokine gene expression. It is very useful to
treat a subject orally or mucosally when the iatrogenic event
comprises destruction or lysis of a cell or tissue of the subject
or of a pathogen hosted by the subject, for example, when the lysis
is due to treatment of the subject with a pharmaceutical
composition, such as a pharmaceutical composition that is selected
from the group of antigens, vaccines, antibodies, anticoagulants,
antibiotics, antitoxins, antibacterial agents, antiparasitic
agents, antiprotozootic agents, antifungal agents, antiviral
agents, cytolytic agents, cytostatic agents, thrombolytic agents.
Such treatment is also useful when the lysis is due to treatment of
the subject with a virus, such as a lytic phage. The invention also
provides use of a signaling molecule comprising an NF.kappa.B
down-regulating peptide or functional analogue thereof for the
production of a pharmaceutical composition for the oral or mucosal
treatment of a pro-inflammatory cytokine response occurring after
an iatrogenic event in a subject.
[0097] Other examples of disease or disorders that can be treated
mucosally or orally with a pharmaceutical composition as provided
herein include acute inflammatory disease, such as (hyper)acute
transplant rejection, sepsis/SIRS, for example, after burn injury
and acute autoimmune disease.
[0098] In particular, the invention provides oral or mucosal
treatment of an acute systemic disease such as sepsis/SIRS.
Sepsis/SIRS is an acute systemic inflammatory response to a variety
of noxious insults (particularly insults of an infectious origin
such as a bacterial infection, but also non-infectious insults are
well known and often seen). The systemic inflammatory response seen
with sepsis/SIRS is caused by immunological processes that are
activated by a variety of immunological mediators such as
cytokines, chemokines, nitric oxide, and other immune-mediating
chemicals of the body. These immunological mediators are generally
seen to cause the life-threatening systemic disease seen with
sepsis/SIRS.
[0099] The invention provides a method for treating sepsis/SIRS in
a subject comprising providing the subject orally or mucosally with
a gene-regulatory peptide or functional analogue thereof,
particularly when the peptide modulates translocation and/or
activity of a gene transcription factor such as an NF.kappa.B/Rel
protein or causes inhibition of an NF.kappa.B/Rel protein-mediated
cytokine gene expression. It is very useful to treat a subject
orally or mucosally when the sepsis/SIRS finds its basis in the
ongoing destruction or lysis of a cell or tissue of the subject or
of a pathogen hosted by the subject. The invention also provides
use of a gene-regulatory peptide, in particular of an NF.kappa.B
down-regulating peptide or functional analogue thereof for the
production of a pharmaceutical composition for the treatment of a
systemic inflammatory response syndrome or sepsis of a subject.
[0100] The gene-regulatory activity of a gene-regulatory peptide,
in particular of an NF.kappa.B-regulating peptide such as selected
from the group of peptides LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ
ID NO:2), LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4), ALPALP (SEQ ID
NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7), VLPAAPQ (SEQ ID
NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10), VLAALP (SEQ ID
NO:11), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12), VLPALPQ (SEQ
ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID NO:15), GVLPALP
(SEQ ID NO:16), LQGVLPALPQVVC (SEQ ID NO:17), LPGCPRGVNPVVS (SEQ ID
NO:18), LPGC (SEQ ID NO:19), MTRV (SEQ ID NO:20), MTR, VVC is
manifested in the following way. Classically, many genes are
regulated not by a signaling molecule that enters the cells but by
molecules that bind to specific receptors on the surface of cells.
Interaction between cell-surface receptors and their ligands can be
followed by a cascade of intracellular events including variations
in the intracellular levels of so-called second messengers
(diacylglycerol, Ca.sup.2+, cyclic nucleotides). The second
messengers in turn lead to changes in protein phosphorylation
through the action of cyclic AMP, cyclic GMP, calcium-activated
protein kinases, or protein kinase C, which is activated by
diaglycerol. Many of these classic responses to binding of ligands
to cell-surface receptors are cytoplasmatic and do not involve
immediate gene activation in the nucleus. Some receptor-ligand
interactions, however, are known to cause prompt nuclear
transcriptional activation of a specific and limited set of genes.
However, progress has been slow in determining exactly how such
activation is achieved. In a few cases, the transcriptional
proteins that respond to cell-surface signals have been
characterized.
[0101] One of the clearest examples of activation of a pre-existing
inactive transcription factor following a cell-surface interaction
is the nuclear factor (NF).kappa.B, which was originally detected
because it stimulates the transcription of genes encoding
immunoglobulin light chains of the .kappa. class in B-lymphocytes.
The binding site for NK.kappa.B in the K gene is well defined (see,
for example, P. A. Baeuerle and D. Baltimore, 1988, Science
242:540), providing an assay for the presence of the active factor.
This factor exists in the cytoplasm of lymphocytes complexed with
an inhibitor. Treatment of the isolated complex in vitro with mild
denaturing conditions dissociates the complex, thus freeing
NK.kappa.B to bind to its DNA site. Release of active NF.kappa.B in
cells is now known to occur after a variety of stimuli including
treating cells with bacterial lipopolysaccharide (LPS) and
extracellular polypeptides as well as chemical molecules (e.g.,
phobol esters) that stimulate intracellular phosphokinases. Thus a
phosphorylation event triggered by many possible stimuli may
account for NF.kappa.B conversion to the active state. The active
factor is then translocated to the cell nucleus to stimulate
transcription only of genes with a binding site for active
NF.kappa.B. We have found that a variety of short peptides as
indicated above exert a modulatory activity on NF.kappa.B
activity.
[0102] Considering that the inflammatory response involves the
sequential release of mediators and the recruitment of circulating
leukocytes, which become activated at the inflammatory site and
release further mediators (Nat. Med. 7:1294; 2001), we provided
using NF.kappa.B-regulating peptide in the field of medicine, e.g.,
by providing pharmaceutical compositions and methods for use in the
medicine. Considering that NF.kappa.B is thought by many to be a
primary effector of disease (A. S. Baldwin, J. Clin. Invest., 2001,
107:3-6), numerous efforts are underway to develop safe inhibitors
of NF.kappa.B to be used in treatment of both chronic and acute
disease situations.
[0103] For example, concomittantly or separately with a method for
perfusing a transplant with a perfusing fluid, the invention
herewith provides treating the recipient of the transplant with a
pharmaceutical composition for oral or mucosal use comprising at
least one gene-regulatory peptide, preferably an NF.kappa.B
down-regulating peptides as provided herein; ischemic or
post-implantation damage due to activation of NF.kappa.B in the
transplant and/or the recipient can then be greatly diminished,
allowing a longer survival and use of the transplants. It is now
provided that the use also allows reducing the risk on chronic
transplant rejection, allowing increasing transplant survival. The
invention provides a method for avoiding acute and in particular
chronic rejection of a transplant and increasing transplant
survival in a recipient of the transplant comprising providing the
recipient orally or mucosally with a gene-regulatory peptide or
functional analogue thereof, herein also called a signaling
molecule. It is preferred that the peptide is 3 to 15 amino acids
long, more preferably, that the peptide is 3 to 9 amino acids long,
it is most preferred that the peptide is 4 to 6 amino acids long.
It is in particular preferred that the signaling molecule is
capable of inhibiting NF.kappa.B/Rel protein activity.
[0104] Functional analogue herein relates to the signaling
molecular effect or activity as, for example, can be measured by
measuring nuclear translocation of a relevant transcription factor,
such as NF.kappa.B in an NF.kappa.B assay, or AP-1 in an AP-1
assay, or by another method as provided herein. Fragments can be
somewhat (i.e., one or two amino acids) smaller or larger on one or
both sides, while still providing functional activity. In one
embodiment of the invention, the peptide used as a signaling
molecule or gene-regulatory peptide is a chemically modified
peptide. A peptide modification includes phosphorylation (e.g., on
a Tyr, Ser or Thr residue), N-terminal acetylation, C-terminal
amidation, C-terminal hydrazide, C-terminal methyl ester, fatty
acid attachment, sulfonation (tyrosine), N-terminal dansylation,
N-terminal succinylation, tripalmitoyl-S-Glyceryl Cysteine (PAM3
Cys-OH) as well as farnesylation of a Cys residue. Systematic
chemical modification of a peptide can, for example, be performed
in the process of peptide optimalization.
[0105] Synthetic peptides can be obtained using various procedures
known in the art. These include solid phase peptide synthesis
(SPPS) and solution phase organic synthesis (SPOS) technologies.
SPPS is a quick and easy approach to synthesize peptides and small
proteins. The C-terminal amino acid is typically attached to a
cross-linked polystyrene resin via an acid labile bond with a
linker molecule. This resin is insoluble in the solvents used for
synthesis, making it relatively simple and fast to wash away excess
reagents and by-products. The peptide, or its functional analogue,
modification or derivative, can be administered as the entity as
such or as a pharmaceutically acceptable acid- or base-addition
salt, formed by reaction with an inorganic acid (such as
hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid,
thiocyanic acid, sulfuric acid, and phosphoric acid); or with an
organic acid (such as formic acid, acetic acid, propionic acid,
glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic
acid, succinic acid, maleic acid, and fumaric acid); or by reaction
with an inorganic base (such as sodium hydroxide, ammonium
hydroxide, potassium hydroxide); or with an organic base (such as
mono-, di-, trialkyl and aryl amines and substituted
ethanolamines). A selected peptide and any of the derived entities
may also be conjugated to sugars, lipids, other polypeptides,
nucleic acids and PNA; and function in-situ as a conjugate or be
released locally after reaching a targeted tissue or organ.
[0106] In response to a variety of pathophysiological and
developmental signals, the NF.kappa.B/Rel family of transcription
factors are activated and form different types of hetero- and
homodimers among themselves to regulate the expression of target
genes containing KB-specific binding sites. NF.kappa.B
transcription factors are hetero- or homodimers of a family of
related proteins characterized by the Rel homology domain. They
form two subfamilies, those containing activation domains
(p65-RELA, RELB, and c-REL) and those lacking activation domains
(p50, p52). The prototypical NF.kappa.B is a heterodimer of p65
(RELA) and p50 (NF.kappa.B1). Among the activated NF.kappa.B
dimers, p50-p65 heterodimers are known to be involved in enhancing
the transcription of target genes and p50-p50 homodimers in
transcriptional repression. However, p65-p65 homodimers are known
for both transcriptional activation and repressive activity against
target genes. KB DNA-binding sites with varied affinities to
different NFB dimers have been discovered in the promoters of
several eukaryotic genes and the balance between activated
NF.kappa.B homo- and heterodimers ultimately determines the nature
and level of gene expression within the cell. The term
"NF.kappa.B-regulating peptide" as used herein refers to a peptide
or functional analogue or a modification or derivative thereof
capable of modulating the activation of members of the
NF.kappa.B/Rel family of transcription factors. Examples of such
peptides that are particularly useful in a method or composition
according to the invention are selected from the group of
VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQGV (SEQ ID
NO:1), AQGV (SEQ ID NO:2), GVLPALPQ (SEQ ID NO:23), VLPALP (SEQ ID
NO:4), VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16), VVC, MTRV
(SEQ ID NO:20), and MTR. Modulation of the activation of NF.kappa.B
can lead to enhanced transcription of target genes. Also, it can
lead to transcriptional repression of target genes. NF.kappa.B
activation can be regulated at multiple levels. For example, the
dynamic shuttling of the inactive NF.kappa.B dimers between the
cytoplasm and nucleus by I.kappa.B proteins and its termination by
phosphorylation and proteasomal degradation, direct
phosphorylation, acetylation of NF.kappa.B factors, and dynamic
reorganization of NF.kappa.B subunits among the activated
NF.kappa.B dimers have all been identified as key regulatory steps
in NF.kappa.B activation and, consequently, in NF.kappa.B-mediated
transcription processes. Thus, an NF.kappa.B-regulating peptide is
capable of modulating the transcription of genes that are under the
control of NF.kappa.B/Rel family of transcription factors.
Modulating comprises the up-regulation or the down-regulation of
transcription.
[0107] The term "pharmaceutical composition" as used herein is
intended to cover both the active regulatory peptide or analogue
alone or a composition containing the regulatory peptide or
analogue together with a pharmaceutically acceptable carrier,
diluent or excipient. Acceptable diluents of a peptide are, for
example, physiological salt solutions or phosphate buffered salt
solutions. It is in particular useful to provide a pharmaceutical
composition wherein the gene transcription factor comprises an
NF.kappa.B/Rel protein. For example, to counter
ischemia-reperfusion damage of a transplant, for example, derived
from a brain dead donor or, to prevent ischemia-reperfusion damage
during cold storage and transport of a transplant, it is herein
recommended to provide a pharmaceutical composition by which
translocation and/or activity of the NF.kappa.B/Rel protein is
inhibited. Such a composition can be a transplant preservation or
perfusion fluid as described herein, comprising a gene-regulatory
peptide or functional analogue thereof. It is useful to select the
peptide from the group of peptides LQG, AQG, LQGV (SEQ ID NO:1),
AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4),
ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7),
VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10),
VLAALP (SEQ ID NO:11), VLAALP (SEQ ID NO:1), VLPALA (SEQ ID NO:12),
VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID
NO:15), GVLPALP (SEQ ID NO:16), LQGVLPALPQVVC (SEQ ID NO:17),
LPGCPRGVNPVVS (SEQ ID NO:18), LPGC (SEQ ID NO:19), MTRV (SEQ ID
NO:20), MTR, VVC, or functional analogues thereof, but other
gene-regulatory peptides can also be selected. As described above,
under certain circumstances it is preferred that the pharmaceutical
composition is hypertonic. It may also be useful to add to the
perfusion fluid an anticoagulant, such as heparin, or in conditions
where disseminated intravascular coagulation (DIC) of the
transplant is expected (such as with cadaveric donors) to add
(recombinant) Activated Protein C to a perfusion fluid as herein
provided. Where the Activated Protein C resolves the diffuse
coagulation leading to ischemia, the NF.kappa.B-regulating peptide
in the perfusion fluid helps reduce reperfusion damage. In most
circumstances, the treatment with the preservation or perfusion
fluid comprises providing the transplant with the signaling
molecule after the transplant has been taken out of the donor. It
is in particular useful to further treat the recipient with one of
the above mentioned classically known pharmaceutical compositions
for further reducing the risk of transplant rejection, especially
in those cases wherein the HLA-type of the transplant mismatches
with the HLA-type of the recipient.
[0108] The invention also provides a transplant preservation fluid
or a transplant perfusion fluid comprising, as a signaling
molecule, a peptide or functional analogue capable of modulating
translocation and/or activity of a gene transcription factor.
[0109] In a specific embodiment, such a fluid also comprises
(recombinant) Activated Protein C, especially when the gene
transcription factor comprises an NF.kappa.B/Rel protein, or the
AP-1 protein. The peptides added to such a fluid, such as LQG, AQG,
LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP
(SEQ ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ
(SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV
(SEQ ID NO:10), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12),
VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID
NO:15), GVLPALP (SEQ ID NO:16),
VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:24),
RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT (SEQ ID NO:25),
SKAPPPSLPSPSRLPGPS (SEQ ID NO:26), LQGVLPALPQVVC (SEQ ID NO:17),
SIRLPGCPRGVNPVVS (SEQ ID NO:27), LPGCPRGVNPVVS (SEQ ID NO:18), LPGC
(SEQ ID NO:19), MTRV (SEQ ID NO:20), MTR, and VVC and others are,
for example, prepared by solid-phase synthesis detailed
description.
[0110] In response to a variety of pathophysiological and
developmental signals, the NF.kappa.B/Rel family of transcription
factors is activated and forms different types of hetero- and
homodimers among themselves to regulate the expression of target
genes containing KB-specific binding sites. NF.kappa.B
transcription factors are hetero- or homodimers of a family of
related proteins characterized by the Rel homology domain. They
form two subfamilies, those containing activation domains
(p65-RELA, RELB, and c-REL) and those lacking activation domains
(p50, p52). The prototypical NF.kappa.B is a heterodimer of p65
(RELA) and p50 (NF.kappa.B1). Among the activated NF.kappa.B
dimers, p50-p65 heterodimers are known to be involved in enhancing
the transcription of target genes and p50-p50 homodimers in
transcriptional repression. However, p65-p65 homodimers are known
for both transcriptional activation and repressive activity against
target genes. KB DNA-binding sites with varied affinities to
different NFB dimers have been discovered in the promoters of
several eukaryotic genes and the balance between activated
NF.kappa.B homo- and heterodimers ultimately determines the nature
and level of gene expression within the cell. The term
"NF.kappa.B-regulating peptide" as used herein refers to a peptide
or a modification or derivative thereof capable of modulating the
activation of members of the NF.kappa.B/Rel family of transcription
factors. Activation of NF.kappa.B can be gene-regulatory to
enhanced transcription of target genes. Also, it can be
gene-regulatory to transcriptional repression of target genes.
NF.kappa.B activation can be regulated at multiple levels. For
example, the dynamic shuttling of the inactive NF.kappa.B dimers
between the cytoplasm and nucleus by I.kappa.B proteins and its
termination by phosphorylation and proteasomal degradation, direct
phosphorylation, acetylation of NF.kappa.B factors, and dynamic
reorganization of NF.kappa.B subunits among the activated
NF.kappa.B dimers have all been identified as key regulatory steps
in NF.kappa.B activation and, consequently, in NF.kappa.B-mediated
transcription processes. Thus, an NF.kappa.B-regulating peptide is
capable of modulating the transcription of genes that are under the
control of NF.kappa.B/Rel family of transcription factors.
Modulating comprises the up-regulation or the down-regulation of
transcription. In a preferred embodiment, a peptide according to
the invention, or a functional derivative or analogue thereof, is
used for the production of a pharmaceutical composition. Examples
of useful NF.kappa.B down-regulating peptides to be included in
such a pharmaceutical composition are VLPALPQVVC (SEQ ID NO:21),
LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ
ID NO:23), VLPALP (SEQ ID NO:4), VVC, MTR and circular
LQGVLPALPQVVC (SEQ ID NO:17). More gene-regulating peptides and
functional analogues can be found in a (bio)assay, such as an
NF.kappa.B translocation assay as provided herein. Most prominent
among NF.kappa.B down-regulating peptides are VLPALPQVVC (SEQ ID
NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1), and
VLPALP (SEQ ID NO:4). These are also capable of reducing production
of NO by a cell. It is herein also provided to use a composition
that comprises at least two oligopeptides or functional analogues
thereof, each capable of down-regulation of NF.kappa.B, and thereby
reducing production of NO and/or TNF-.alpha. by a cell, in
particular wherein the at least two oligopeptides are selected from
the group LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), and VLPALP (SEQ
ID NO:4). Useful NF.kappa.B up-regulating peptides are VLPALPQ (SEQ
ID NO:13), GVLPALP (SEQ ID NO:16) and MTRV (SEQ ID NO:20). As
indicated, more gene-regulatory peptides may be found with an
appropriate (bio)assay. A gene-regulatory peptide as used herein is
preferably short. Preferably, such a peptide is 3 to 15 amino acids
long, and capable of modulating the expression of a gene, such as a
cytokine, in a cell. In a preferred embodiment, a peptide is a
signaling molecule that is capable of traversing the plasma
membrane of a cell or, in other words, a peptide that is
membrane-permeable. More preferably, wherein the lead peptide is 3
to 9 amino acids long, most preferred wherein the lead peptide is 4
to 6 amino acids long.
[0111] Functional derivative or analogue herein relates to the
signaling molecular effect or activity as, for example, can be
measured by measuring nuclear translocation of a relevant
transcription factor, such as NF.kappa.B in an NF.kappa.B assay, or
AP-1 in an AP-1 assay, or by another method as provided herein.
Fragments can be somewhat (i.e., one or two amino acids) smaller or
larger on one or both sides, while still providing functional
activity. Such a bioassay comprises an assay for obtaining
information about the capacity or tendency of a peptide, or a
modification thereof, to regulate expression of a gene. A scan
with, for example, a 15-mer, or a 12-mer, or a 9-mer, or a 8-mer,
or a 7-mer, or a 6-mer, or a 5-mer, or a 4-mer or a 3-mer peptides
can yield valuable information on the linear stretch of amino acids
that form an interaction site and allows identification of
gene-regulatory peptides that have the capacity or tendency to
regulate gene expression. Gene-regulatory peptides can be modified
to modulate their capacity or tendency to regulate gene expression,
which can be easily assayed in an in vitro bioassay such as a
reporter assay. For example, some amino acid at some position can
be replaced with another amino acid of similar or different
properties. Alanine (Ala)-replacement scanning, involving a
systematic replacement of each amino acid by an Ala residue, is a
suitable approach to modify the amino acid composition of a
gene-regulatory peptide when in a search for a signaling molecule
capable of modulating gene expression. Of course, such replacement
scanning or mapping can be undertaken with amino acids other than
Ala as well, for example, with D-amino acids. In one embodiment, a
peptide derived from a naturally occurring polypeptide is
identified as being capable of modulating gene expression of a gene
in a cell. Subsequently, various synthetic Ala-mutants of this
gene-regulatory peptide are produced. These Ala-mutants are
screened for their enhanced or improved capacity to regulate
expression of a gene compared to gene-regulatory polypeptide.
[0112] Furthermore, a gene-regulatory peptide, or a modification or
analogue thereof, can be chemically synthesized using D- and/or
L-stereoisomers. For example, a gene-regulatory peptide that is a
retro-inverso of an oligopeptide of natural origin is produced. The
concept of polypeptide retro-inversion (assemblage of a natural
L-amino acid-containing parent sequence in reverse order using
D-amino acids) has been applied successfully to synthetic peptides.
Retro-inverso modification of peptide bonds has evolved into a
widely used peptidomimetic approach for the design of novel
bioactive molecules which has been applied to many families of
biologically active peptide. The sequence, amino acid composition
and length of a peptide will influence whether correct assembly and
purification are feasible. These factors also determine the
solubility of the final product. The purity of a crude peptide
typically decreases as the length increases. The yield of peptide
for sequences less than 15 residues is usually satisfactory, and
such peptides can typically be made without difficulty. The overall
amino acid composition of a peptide is an important design
variable. A peptide's solubility is strongly influenced by
composition. Peptides with a high content of hydrophobic residues,
such as Leu, Val, Ile, Met, Phe and Trp, will either have limited
solubility in aqueous solution or be completely insoluble. Under
these conditions, it can be difficult to use the peptide in
experiments, and it may be difficult to purify the peptide if
necessary. To achieve a good solubility, it is advisable to keep
the hydrophobic amino acid content below 50% and to make sure that
there is at least one charged residue for every five amino acids.
At physiological pH Asp, Glu, Lys, and Arg all have charged side
chains. A single conservative replacement, such as replacing Ala
with Gly, or adding a set of polar residues to the N- or
C-terminus, may also improve solubility. Peptides containing
multiple Cys, Met, or Trp residues can also be difficult to obtain
in high purity partly because these residues are susceptible to
oxidation and/or side reactions. If possible, one should choose
sequences to minimize these residues. Alternatively, conservative
replacements can be made for some residues. For instance,
Norleucine can be used as a replacement for Met, and Ser is
sometimes used as a less reactive replacement for Cys. If a number
of sequential or overlapping peptides from a protein sequence are
to be made, making a change in the starting point of each peptide
may create a better balance between hydrophilic and hydrophobic
residues. A change in the number of Cys, Met, and Trp residues
contained in individual peptides may produce a similar effect. In
another embodiment of the invention, a gene-regulatory peptide
capable of modulating gene expression is a chemically modified
peptide. A peptide modification includes phosphorylation (e.g., on
a Tyr, Ser or Thr residue), N-terminal acetylation, C-terminal
amidation, C-terminal hydrazide, C-terminal methyl ester, fatty
acid attachment, sulfonation (tyrosine), N-terminal dansylation,
N-terminal succinylation, tripalmitoyl-S-Glyceryl Cysteine (PAM3
Cys-OH) as well as farnesylation of a Cys residue. Systematic
chemical modification of a gene-regulatory peptide can, for
example, be performed in the process of gene-regulatory peptide
optimalization.
[0113] Synthetic peptides can be obtained using various procedures
known in the art. These include solid phase peptide synthesis
(SPPS) and solution phase organic synthesis (SPOS) technologies.
SPPS is a quick and easy approach to synthesize peptides and small
proteins. The C-terminal amino acid is typically attached to a
cross-linked polystyrene resin via an acid labile bond with a
linker molecule. This resin is insoluble in the solvents used for
synthesis, making it relatively simple and fast to wash away excess
reagents and by-products.
[0114] The peptides as mentioned in this document such as LQG, AQG,
LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP
(SEQ ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ
(SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV
(SEQ ID NO:10), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12),
VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID
NO:15), GVLPALP (SEQ ID NO:16),
VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:24),
RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT (SEQ ID NO:25),
SKAPPPSLPSPSRLPGPS (SEQ ID NO:26), LQGVLPALPQVVC (SEQ ID NO:17),
SIRLPGCPRGVNPVVS (SEQ ID NO:27), LPGCPRGVNPVVS (SEQ ID NO:18), LPGC
(SEQ ID NO:19), MTRV (SEQ ID NO:20), MTR, and VVC were prepared by
solid-phase synthesis using the fluorenylmethoxycarbonyl
(Fmoc)/tert-butyl-based methodology with 2-chlorotrityl chloride
resin as the solid support. The side-chain of glutamine was
protected with a trityl function. The peptides were synthesized
manually. Each coupling consists of the following steps: (i)
removal of the .alpha.-amino Fmoc-protection by piperidine in
dimethylformamide (DMF), (ii) coupling of the Fmoc amino acid (3
eq) with diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole
(HOBt) in DMF/N-methylformamide (NMP), and (iii) capping of the
remaining amino functions with acetic
anhydride/diisopropylethylamine (DIEA) in DMF/NMP. Upon completion
of the synthesis, the peptide resin was treated with a mixture of
trifluoroacetic acid (TFA)/H.sub.2O/triisopropylsilane (TIS)
95:2.5:2.5. After 30 minutes, TIS was added until decolorization.
The solution was evaporated in vacuo and the peptide precipitated
with diethylether. The crude peptides were dissolved in water (50
to 100 mg/ml) and purified by reverse-phase high-performance liquid
chromatography (RP-HPLC). HPLC conditions were: P column: Vydac
TP21810C18 (10.times.250 mm); elution system: gradient system of
0.1% TFA in water v/v (A) and 0.1% TFA in acetonitrile (ACN) v/v
(B); flow rate 6 ml/minute; absorbance was detected from 190 to 370
nm. There were different gradient systems used. For example, for
peptides LQG and LQGV (SEQ ID NO: 1): ten minutes 100% A followed
by linear gradient 0 to 10% B in 50 minutes. For example, for
peptides VLPALP (SEQ ID NO:4) and VLPALPQ (SEQ ID NO:13): five
minutes 5% B followed by linear gradient 1% B/minute. The collected
fractions were concentrated to about 5 ml by rotation film
evaporation under reduced pressure at 40.degree. C. The remaining
TFA was exchanged against acetate by eluting two times over a
column with anion exchange resin (Merck II) in acetate form. The
elute was concentrated and lyophilized in 28 hours. Peptides later
were prepared for use by dissolving them in PBS.
[0115] RAW 264.7 macrophages, obtained from American Type Culture
Collection (Manassas, Va.), were cultured at 37.degree. C. in 5%
CO.sub.2 using DMEM containing 10% FBS and antibiotics (100 U/ml of
penicillin, and 100 .mu.g/ml streptomycin). Cells
(1.times.10.sup.6/ml) were incubated with peptide (10 .mu.g/ml) in
a volume of 2 ml. After eight hours of cultures cells were washed
and prepared for nuclear extracts.
[0116] Nuclear extracts and EMSA were prepared according to
Schreiber et al. Methods (Schrieber et al. 1989, Nucleic Acids
Research 17). Briefly, nuclear extracts from peptide-stimulated or
nonstimulated macrophages were prepared by cell lysis followed by
nuclear lysis. Cells were then suspended in 400 .mu.l of buffer (10
mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA,
1 mM DTT, 0.5 mM PMSF and protease inhibitors), vigorously vortexed
for 15 seconds, left standing at 4.degree. C. for 15 minutes, and
centrifuged at 15,000 rpm for two minutes. The pelleted nuclei were
resuspended in buffer (20 mM HEPES (pH 7.9), 10% glycerol, 400 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease
inhibitors) for 30 minutes on ice, then the lysates were
centrifuged at 15,000 rpm for two minutes. The supernatants
containing the solubilized nuclear proteins were stored at
-70.degree. C. until used for the Electrophoretic Mobility Shift
Assays (EMSA).
[0117] Electrophoretic mobility shift assays were performed by
incubating nuclear extracts prepared from control (RAW 264.7) and
peptide-treated RAW 264.7 cells with a 32P-labeled double-stranded
probe (5' AGCTCAGAGGGGGACTTTCCGAGAG 3' (SEQ ID NO:28)) synthesized
to represent the NF.kappa.B-binding sequence. Shortly, the probe
was end-labeled with T4 polynucleotide kinase according to the
manufacturer's instructions (Promega, Madison, Wis.). The annealed
probe was incubated with nuclear extract as follows: in EMSA,
binding reaction mixtures (20 .mu.l) contained 0.25 .mu.g of
poly(dI-dC) (Amersham Pharmacia Biotech) and 20,000 rpm of
32P-labeled DNA probe in binding buffer consisting of 5 mM EDTA,
20% Ficoll, 5 mM DTT, 300 mM KCl and 50 mM HEPES. The binding
reaction was started by the addition of cell extracts (10 .mu.g)
and was continued for 30 minutes at room temperature. The
DNA-protein complex was resolved from free oligonucleotide by
electrophoresis in a 6% polyacrylamide gel. The gels were dried and
exposed to x-ray films.
[0118] The transcription factor NF.kappa.B participates in the
transcriptional regulation of a variety of genes. Nuclear protein
extracts were prepared from LPS and peptide-treated RAW264.7 cells
or from LPS-treated RAW264.7 cells. In order to determine whether
the peptide modulates the translocation of NF.kappa.B into the
nucleus, on these extracts EMSA was performed. Here we see that
indeed peptides are able to modulate the translocation of
NF.kappa.B since the amount of labeled oligonucleotide for
NF.kappa.B is reduced. In this experiment peptides that show the
modulation of translocation of NF.kappa.B are: VLPALPQVVC (SEQ ID
NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1),
GVLPALPQ (SEQ ID NO:23), VLPALP (SEQ ID NO:4), VLPALPQ (SEQ ID
NO:13), GVLPALP (SEQ ID NO:16), VVC, MTRV (SEQ ID NO:20), MTR.
[0119] RAW 264.7 mouse macrophages were cultured in DMEM,
containing 10% or 2% FBS, penicillin, streptomycin and glutamine,
at 37.degree. C., 5% CO.sub.2. Cells were seeded in a 12-wells
plate (3.times.10.sup.6 cells/ml) in a total volume of 1 ml for two
hours and then stimulated with LPS (E. coli 026:B6; Difco
Laboratories, Detroit, Mich., USA) and/or gene-regulatory peptide
(1 microgr/ml). After 30 minutes of incubation, plates were
centrifuged and cells were collected for nuclear extracts. Nuclear
extracts and EMSA were prepared according to Schreiber et al. Cells
were collected in a tube and centrifuged for five minutes at 2000
rpm (rounds per minute) at 4.degree. C. (Universal 30 RF, Hettich
Zentrifuges). The pellet was washed with ice-cold Tris buffered
saline (TBS pH 7.4) and resuspended in 400 .mu.l of a hypotonic
buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA,
1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail (Complete.TM.
Mini, Roche) and left on ice for 15 minutes. Twenty-five
microliters 10% NP-40 was added and the sample was centrifuged (two
minutes, 4000 rpm, 4.degree. C.). The supernatant (cytoplasmic
fraction) was collected and stored at -70.degree. C. The pellet,
which contains the nuclei, was washed with 50 .mu.l buffer A and
resuspended in 50 .mu.l buffer C (20 mM HEPES pH 7.9, 400 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor
cocktail and 10% glycerol). The samples were left to shake at
4.degree. C. for at least 60 minutes. Finally the samples were
centrifuged and the supernatant (nucleic fraction) was stored at
-70.degree. C.
[0120] Bradford reagent (Sigma) was used to determine the final
protein concentration in the extracts. For electrophoretic mobility
shift assays an oligonucleotide representing NF.kappa.B-binding
sequence (5'-AGC TCA GAG GGG GAC TTT CCG AGA G-3' (SEQ ID NO:28))
was synthesized. One hundred pico mol sense and antisense oligo
were annealed and labeled with .gamma.-32P-dATP using T4
polynucleotide kinase according to the manufacturer's instructions
(Promega, Madison, Wis.). Nuclear extract (5 to 7.5 .mu.g) was
incubated for 30 minutes with 75000 cpm probe in binding reaction
mixture (20 microliters) containing 0.5 .mu.g poly dI-dC (Amersham
Pharmacia Biotech) and binding buffer BSB (25 mM MgCl.sub.2, 5 mM
CaCl.sub.2, 5 mM DTT and 20% Ficoll) at room temperature. The
DNA-protein complex was resolved from free oligonucleotide by
electrophoresis in a 4 to 6% polyacrylamide gel (150 V, two to four
hours). The gel was then dried and exposed to x-ray film. The
transcription factor NF.kappa.B participates in the transcriptional
regulation of a variety of genes. Nuclear protein extracts were
prepared from either LPS (1 mg/ml), peptide (1 mg/ml) or LPS in
combination with peptide-treated and untreated RAW264.7 cells. In
order to determine whether the peptides modulate the translocation
of NF.kappa.B into the nucleus, on these extracts EMSA was
performed. Peptides are able to modulate the basal as well as
LPS-induced levels of NF.kappa.B. In this experiment peptides that
show the inhibition of LPS-induced translocation of NF.kappa.B are:
VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV
(SEQ ID NO:1), GVLPALPQ (SEQ ID NO:23), VLPALP (SEQ ID NO:4), VVC,
MTR and circular LQGVLPALPQVVC (SEQ ID NO:17). Peptides that in
this experiment promote LPS-induced translocation of NF.kappa.B
are: VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16) and MTRV (SEQ
ID NO:20). Basal levels of NF.kappa.B in the nucleus was decreased
by VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG and
LQGV (SEQ ID NO:1) while basal levels of NF.kappa.B in the nucleus
was increased by GVLPALPQ (SEQ ID NO:23), VLPALPQ (SEQ ID NO:13),
GVLPALP (SEQ ID NO:16), VVC, MTRV (SEQ ID NO:20), MTR and
LQGVLPALPQVVC (SEQ ID NO:21). In other experiments, QVVC also
showed the modulation of translocation of NF.kappa.B into nucleus
(data not shown).
Further Modes of Identification of Gene-Regulatory Peptides by
NF.kappa.B Analysis
[0121] Cells: Cells will be cultured in appropriate culture medium
at 37.degree. C., 5% CO.sub.2. Cells will be seeded in a 12-well
plate (usually 1.times.10.sup.6 cells/ml) in a total volume of 1 ml
for two hours and then stimulated with regulatory peptide in the
presence or absence of additional stimuli such as LPS. After 30
minutes of incubation, plates will be centrifuged and cells are
collected for cytosolic or nuclear extracts.
[0122] Nuclear Extracts: Nuclear extracts and EMSA could be
prepared according to Schreiber et al., Method (Schreiber et al.
1989, Nucleic Acids Research 17). Cells are collected in a tube and
centrifuged for five minutes at 2000 rpm (rounds per minute) at
4.degree. C. (Universal 30 RF, Hettich Zentrifuges). The pellet is
washed with ice-cold Tris buffered saline (TBS pH 7.4) and
resuspended in 400 .mu.l of a hypotonic buffer A (10 mM HEPES pH
7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and
protease inhibitor cocktail (Complete.TM. Mini, Roche) and left on
ice for 15 minutes. Twenty-five microliters 10% NP-40 is added and
the sample is centrifuged (two minutes, 4000 rpm, 4.degree. C.).
The supernatant (cytoplasmic fraction) was collected and stored at
-70.degree. C. for analysis. The pellet, which contains the nuclei,
is washed with 50 .mu.l buffer A and resuspended in 50 .mu.l buffer
C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,
0.5 mM PMSF and protease inhibitor cocktail and 10% glycerol). The
samples are left to shake at 4.degree. C. for at least 60 minutes.
Finally the samples are centrifuged and the supernatant (nucleic
fraction) is stored at -70.degree. C. for analysis.
[0123] Bradford reagent (Sigma) could be used to determine the
final protein concentration in the extracts.
[0124] EMSA: For Electrophoretic mobility shift assays, an
oligonucleotide representing NF.kappa.B-binding sequence such as
(5'-AGC TCA GAG GGG GAC TTT CCG AGA G-3' (SEQ ID NO:28)) are
synthesized. One hundred pico mol sense and antisense oligo are
annealed and labeled with .gamma.-.sup.32P-dATP using T4
polynucleotide kinase according to the manufacturer's instructions
(Promega, Madison, Wis.). Cytosolic extract or nuclear extract
(5-7.5 .mu.g) from cells treated with regulatory peptide or from
untreated cells is incubated for 30 minutes with 75,000 cpm probe
in binding reaction mixture (20 .mu.l) containing 0.5 .mu.g poly
dI-dC (Amersham Pharmacia Biotech) and binding buffer BSB (25 mM
MgCl.sub.2, 5 mM CaCl.sub.2, 5 mM DTT and 20% Ficoll) at room
temperature. Or cytosolic and nuclear extract from untreated cells
or from cells treated with stimuli could also be incubated with
probe in binding reaction mixture and binding buffer. The
DNA-protein complexes are resolved from free oligonucleotide by
electrophoresis in a 4 to 6% polyacrylamide gel (150 V, two to four
hours). The gel is then dried and exposed to x-ray film. Peptides
can be biotinylated and incubated with cells. Cells are then washed
with phosphate-buffered saline, harvested in the absence or
presence of certain stimulus (LPS, PHA, TPA, anti-CD3, VEGF,
TSST-1, VIP or know drugs etc.). After culturing, cells are lysed
and cells lysates (whole lysate, cytosolic fraction or nuclear
fraction) containing 200 micrograms of protein are incubated with
50 microliters Neutr-Avidin-plus beads for one hour at 4.degree. C.
with constant shaking. Beads are washed five times with lysis
buffer by centrifugation at 6000 rpm for one minute. Proteins are
eluted by incubating the beads in 0.05 N NaoH for one minute at
room temperature to hydrolyze the protein-peptide linkage and
analyzed by SDS-polyacrylamide gel electrophoresis followed by
immunoprecipitated with agarose-conjugated anti-NF.kappa.B subunits
antibody or immunoprecipitated with antibody against the to be
studied target. After hydrolyzing the protein-peptide linkage, the
sample could be analyzed on HPLS and mass-spectrometry. Purified
NF.kappa.B subunits or cell lysate interaction with biotinylated
regulatory peptide can be analyzed on biosensor technology.
Peptides can be labeled with FITC and incubated with cells in the
absence or presence of different stimulus. After culturing, cells
can be analyzed with fluorescent microscopy, confocal microscopy,
flow cytometry (cell membrane staining and/or intracellular
staining) or cells lysates are made and analyzed on HPLC and
mass-spectrometry. NF.kappa.B-transfected (reporter gene assay)
cells and gene array technology can be used to determine the
regulatory effects of peptides.
[0125] HPLC and mass-spectrometry analysis: Purified NF.kappa.B
subunit or cytosolic/nuclear extract is incubated in the absence or
presence of (regulatory) peptide is diluted (2:1) with 8 N
guanidinium chloride and 0.1% trifluoroacetic acid, injected into a
reverse-phase HPLC column (Vydac C18) equilibrated with solvent A
(0.1% trifluoroacetic acid), and eluted with a gradient of 0 to
100% eluant B (90% acetonitrile in solvent A). Factions containing
NF.kappa.B subunit are pooled and concentrated. Fractions are then
dissolved in appropriate volume and could be analyzed on
mass-spectrometry.
Example
[0126] The invention in particular relates to the, preferably oral,
treatment of neurological disorders or so called neuroimmune
disorders such as schizophrenia, manic depression and other bipolar
disorders, post-partum psychosis, autism, chronic fatigue syndrome
(CFS), fibromyalgia, Alzheimer's, mood disorders and certain forms
of stress. Although there are major differences in etiology and
mechanisms of pathogenesis of each of these syndromes and or
diseases, there are in fact common inflammatory and
immunomodulatory pathways that are shared within the pathogenesis
of neurological disorders.
[0127] Evidence of immune abnormalities in patients suffering from
psychological disease clearly shows the implication of the immune
system in pathogenesis. Neuroimmune disorders have become
recognized as common pathogenetic factors in the development of
psycho- or neuropathologies. The neurochemical and immunologic
findings indicate multiple pathways of the pathogenesis; herein, we
discuss the role of inflammatory disease in neurological disorders.
For example, chronic fatigue syndrome is a condition that affects
women in disproportionate numbers, and that is often exacerbated in
the premenstrual period and following physical exertion. The signs
and symptoms, which include fatigue, myalgia, and low-grade fever,
are similar to those experienced by patients infused with cytokines
such as interleukin-1. In general, during the development of a
neuroimmune disorder, the TNF-.alpha. family and other
pro-inflammatory cytokines are highly elevated in cerebrospinal
fluid (CSF), demonstrative of foci of inflammation in the brain
leading to an array of destructive and degenerative responses
directed at diverse areas in the CNS. Major mood disorders are
leading causes of disability from early adolescence onward and
leading sources of disease burden, surpassing cardiovascular
diseases, dementia, lung cancer and diabetes. As said, there is a
major role for inflammatory cytokines and immune cells in the
pathophysiology of mood disorders, it was recently also found that
T-cells and monocytes function at a higher, pro-inflammatory level
in patients with bipolar disorder. Successful therapy of these
destructive and degenerative disorders that affect the adult human
central nervous system (CNS) will require the ability both to
reduce the rate and extent of tissue injury, and to restore or
replace destroyed tissue. Neuroimaging studies have shown that
functional organization occurs spontaneously in the adult human
brain in response to tissue insults. The extent of this
compensatory mechanism may be limited, necessitating development of
active methods of intervention. Replacement of a single
neurotransmitter, neurohormone or trophic factor may suffice if the
injury is limited or effected as a suppression or altered pathway
within the CNS through proinflammatory regulators. The hippocampus
is a source for mitotically active neuronal progenitor cells which
can hypothetically replace neurons and myelinating cells. It is the
control of these cells and the health and activity of other cells
which offers new insight and hope of treating heretofore chronic
CNS disease. It is areas such as cells in the adult human dentate
gyrus which may be part of the key to controlling immunomodulation
and growth support of the brain and its diverse functions which
span from memory and cognition to its endocrine and immunologic
activities. As with all complex traits, a neurological disorder
results from an interplay between as yet unidentified environmental
factors and susceptibility genes. Together, these factors trigger a
cascade of events, involving engagement of the immune system, acute
inflammatory injury of the central nervous system, notably axons
and glia, recovery of function and structural repair,
post-inflammatory gliosis, and neurodegeneration. The sequential
involvement of these processes underlies the clinical course
characterized by episodes with recovery interchanged with episodes
leaving persistent deficits, episodes which we generally call
psychological disorders.
[0128] For a more detailed example, although there are several
forms of autism (which often present themselves already at birth)
which may have clear genetic etiologies, the most common forms
however occur long after normal births and are associated with
proinflammatory cytokine dysregulation. According to recent
epidemiological surveys, autistic spectrum disorders have become
recognized as common childhood psychopathologies. These
life-lasting conditions demonstrate a strong genetic determinant
consistent with a polygenic mode of inheritance for which several
autism susceptibility regions have been identified. Parallel
evidence of immune abnormalities in autistic patients argues for an
implication of the immune system in pathogenesis. This introduction
summarizes advances in the molecular genetics of autism, as well as
recently emerging concerns addressing the disease incidence and
triggering factors. The neurochemical and immunologic findings are
analyzed in the context of a neuroimmune hypothesis for specific
neurological disorders. For example, pregnancy and the post partum
period are important modulators of the immune system and the immune
suppression in pregnancy is followed by an immune activation in the
puerperium. In another example, autism is influenced by specific
food allergies or even the early use of vaccines which may cause
changes in the regulation of innate or acquired immunity and set up
neuroendocrine dysfunction. Also, neurological disorders are often
associated with autoimmune disorders in the patients' relatives. A.
M. Comi et al. (J. Child Neurol. 1999 June; 14(6):388-94) evaluated
the frequency of autoimmune disorders, as well as various prenatal
and postnatal events in autism, and surveyed the families of 61
autistic patients and 46 healthy controls using questionnaires. The
mean number of autoimmune disorders was greater in families with
autism; 46% had two or more members with autoimmune disorders. As
the number of family members with autoimmune disorders increased
from one to three, the risk of autism was greater, with an odds
ratio that increased from 1.9 to 5.5, respectively. In mothers and
first-degree relatives of autistic children, there were more
autoimmune disorders (16% and 21%) as compared to controls (2% and
4%), with odds ratios of 8.8 and 6.0, respectively. The most common
autoimmune disorders in both groups were type 1 diabetes, adult
rheumatoid arthritis, hypothyroidism, and systemic lupus
erythematosus. Forty-six percent of the autism group reported
having relatives with rheumatoid diseases, as compared to 26% of
the controls. Prenatal maternal urinary tract, upper respiratory,
and vaginal infections; asphyxia; prematurity, and seizures were
more common in the autistic group, although the differences were
not significant. Thirty-nine percent of the controls, but only 11%
of the autistic group, reported allergies. The increased number of
autoimmune disorders shows that in autism, immune dysfunction
interacts with various environmental factors to play a role in
autism pathogenesis. According to S. B. Edelson and D. S. Cantor
(Toxicol. Ind. Health 1998 July-August; 14(4):553-63) the advances
in medical technology during the last four decades have provided
evidence for an underlying neurological basis for autism. The
etiology for the variations of neurofunctional anomalies found in
the neurological disorder spectrum behaviors appears inconclusive
as of this date but growing evidence supports the proposal that
chronic exposure to toxic agents, i.e., xenobiotic agents,
resulting in a inflammatory reaction directed towards a developing
central nervous system may be the best model for defining the
physiological and behavioral data found in these populations. Also,
an examination of 18 autistic children in blood analyses that were
available showed that 16 of these children showed evidence of
levels of toxic chemicals exceeding adult maximum tolerance. In the
two cases where toxic chemical levels were not found, there was
abnormal D-glucaric acid findings suggesting abnormal xenobiotic
influences on liver detoxification processes. A proposed mechanism
for the interaction of xenobiotic toxins with immune system
dysfunction and continuous and/or progressive endogenous toxicity
is presented as it relates to the development of behaviors found in
the autistic spectrum. H. Jyonouchi et al. (J. Neuroimmunol. 2001
Nov. 1; 120(1-2):170-9) determined innate and adaptive immune
responses in children with developmental regression and autism
spectrum disorders (ASD, N=71), developmentally normal siblings
(N=23), and controls (N=17), and found a clear relationship between
proinflammatory and regulatory cytokine production associated with
innate and adaptive immune responses in children with autism
spectrum disorders and developmental regression. With
lipopolysaccharide (LPS), a stimulant for innate immunity,
peripheral blood mononuclear cells (PBMCs) from 59/71 (83.1%) ASD
patients produced >2 SD above the control mean (CM) values of
TNF-.alpha., IL-1.beta., and/or IL-6 produced by control PBMCs. ASD
PBMCs produced higher levels of proinflammatory/counter-regulatory
cytokines without stimuli than controls. With stimulants of
phytohemagglutinin (PHA), tetanus, IL-12p70, and IL-18, PBMCs from
47.9% to 60% of ASD patients produced >2 SD above the CM values
of TNF-.alpha. depending on stimulants. These results indicate
excessive innate immune responses as a result of NF.kappa.B-induced
cytokine expression in a number of ASD children that is most
evident in TNF-.alpha. production. Furthermore, according to S.
Messahel et al. (Neurosci. Lett. 1998 Jan. 23; 241(1):17-20) the
pterins, neopterin and biopterin, occur naturally in body fluids
including urine. It is well established that increased neopterin
levels are associated with activation of the cellular immune system
and that reduced biopterins are essential for neurotransmitter
synthesis. It has been also been suggested that some autistic
children may be suffering from an autoimmune disorder. To
investigate this further, the above authors performed high
performance liquid chromatography analyses of urinary pterins in a
group of pre-school autistic children, their siblings and
age-matched control children. Both urinary neopterin and biopterin
were raised in the autistic children compared to controls and the
siblings showed intermediate values.
[0129] As yet another example, the chronic fatigue syndrome (CFS)
is a clinically defined condition characterized by severe disabling
fatigue and a combination of symptoms that prominently features
self-reported impairments in concentration and short-term memory,
sleep disturbances, and musculoskeletal pain. Heretofore, the
diagnosis of the chronic fatigue syndrome could only be made after
other medical and psychiatric causes of chronic fatiguing illness
were excluded. No pathognomonic signs or clear diagnostic tests for
this condition have yet been validated. Thus far, no definitive
treatment exists. Recent longitudinal studies suggest that some
persons affected by the chronic fatigue syndrome improve with time
but that most remain functionally impaired for several years. CFS
is characterized by debilitating fatigue that is not attributable
to known clinical conditions, that has lasted for >6 months,
that has reduced the activity level of a previously healthy person
by >50%, and that has been accompanied by flu-like symptoms
(e.g., pharyngitis, adenopathy, low grade fever, myalgia,
arthralgia, headache) and neuropsychological manifestations (e.g.,
difficulty concentrating, exercise intolerance, and sleep
disturbances). CFS is frequently of sudden onset. There have been
considerable advances in our understanding of the mediators of CFS,
with several careful studies of immunologic function, activation,
and cytokine dysregulation. An increasing number of independent
groups have reported abnormalities of both T- and B-cell lymphocyte
and NK cell function, with one group correlating levels of NK cell
function to disease severity. It was suggested that the illness be
named chronic immune activation syndrome given the abnormally
elevated markers of T-cell activation measured on T-cells and
cytotoxic T-cells.
[0130] Over the last decade, investigators have demonstrated that
individuals with CFS have significantly increased proportions of
activated CD8+ T-cells, decreased natural killer cell (NK)
cytotoxic and lymphoproliferative activities, elevated serum levels
of tumor necrosis factor (TNF)-.alpha. and .beta., and detectable
TNF-.beta., interleukin (IL)-1.beta. and IL-6 mRNA in peripheral
blood mononuclear cells (PBMC). CFS patients, as a group, also have
significantly higher levels, as compared to controls, of soluble
TNF receptor type I (sTNF-RI), sIL-6R and .beta.2-microglobulin
(.beta.2-m), but not of IL-1 receptor antagonist (IL-1Ra).
Correlative and population distribution studies that included
lymphoid phenotypic distributions and function as well as soluble
immune mediator expression levels revealed the existence of at
least two mainly nonoverlapping categories among CFS patients with
either: (1) dysregulated TNF-.alpha./.beta. expression in
association with changes in the serum levels of IL-1.alpha., IL-4,
sIL-2R, and IL-1Ra, PBMC-associated expression of IL-1.beta., IL-6,
and TNF-.beta. mRNA, and T-cell activation; or (2) interrelated and
dysregulated expression of sTNF-R1, sIL-6R, and
.beta.2-microglobulin and significantly decreased
lymphoproliferative and NK cell cytotoxic activities. Furthermore,
allostasis--the ability to achieve stability through change--is
critical to survival, and many psychological disorders are
manifestations of the fact that such stability is not present.
Through allostasis, the autonomic nervous system, the
hypothalamic-pituitary-adrenal (HPA) axis, and the cardiovascular,
metabolic, and immune systems protect the body by responding to
internal and external stress. The price of this accommodation to
stress can be allostatic load, which is the wear and tear that
results from chronic overactivity or underactivity of allostatic
systems.
[0131] The core of the body's response to a challenge is twofold,
turning on an allostatic response that initiates a complex adaptive
pathway, and then shutting off this response when the threat is
past. The most common allostatic responses involve the sympathetic
nervous systems and the HPA axis. For these systems, activation
releases catecholamines from nerves and the adrenal medulla and
leads to the secretion of corticotropin from the pituitary. The
corticotropin, in turn, mediates the release of cortisol from the
adrenal cortex. Inactivation returns the systems to base-line
levels of cortisol and catecholamine secretion, which normally
happens when the danger is past. However, if the inactivation is
inefficient, there is overexposure to stress hormones. Over weeks,
months, or years, exposure to increased secretion of stress
hormones results in a so-called allostatic load and its
immunopathophysiologic consequences. It has been shown that
allostatic load over a lifetime may cause the allostatic systems to
wear out or become exhausted. Frailty in old age is generally seen
as a consequence of a worn-out allostatic system. A vulnerable link
in the regulation of the HPA axis and cognition is the hippocampal
region. Wear and tear on this region of the brain leads to
dysregulation of the HPA axis and cognitive impairment. Indeed,
some, but not all, of the aging people have impairment of episodic,
declarative, and spatial memory and hyperactivity of the HPA axis,
all of which can be traced to inflammatory hippocampal damage.
Recent data show that similar events occur at a younger age in
humans with unexplained mood disorders. In one type of allostatic
load inadequate responses by some allostatic systems trigger
compensatory increases in others. When one system does not respond
adequately to a stressful stimulus, the activity of other systems
increases, because the underactive system is not providing the
usual counter-regulation. For example, if cortisol secretion does
not increase in response to stress, secretion of inflammatory
cytokines (which are counter-regulated by cortisol) increases. The
negative consequences of an enhanced inflammatory response are, for
example, that the affected subjects are very susceptible to
autoimmune and inflammatory disturbances, aggravated often by a
genetically determined hyporesponsiveness of the HPA axis.
[0132] Also, the months following childbirth are a time when some
women are susceptible to serious mood disorders. The illnesses can
be resistant to conventional psychiatric treatment methods. Cases
of postpartum depression or puerperal psychosis often occur in
women with a past history of major depression or bipolar disorder.
There has been considerable debate as to whether postpartum
psychosis is a discrete diagnostic entity or whether it represents
a rapidly evolving psychosis, that is a manifestation of an
underlying bipolar (or manic-depressive) disorder. To date,
existing psychiatric research supports the latter view.
[0133] The invention provides a method for the treatment of a
subject believed to be suffering from a neurological disorder, with
a specific aim of reducing the frequency, and limit the lasting
effects of the psychological manifestations of neuroimmune disease,
and in particular the treatment of the inflammatory component of
neurological or mood disorders to relieve symptoms that arise from
the release of additional pro-inflammatory cytokines, in particular
during disease progression, to prevent disability arising from
disease progression, and to promote CNS tissue repair. The
invention provides a pharmaceutical composition, in particular for
oral administration, for the treatment of a neurological disorder
occurring in a subject, for example, in a primate, and a method for
the treatment of the disease associated with additional
pro-inflammatory cytokine release, for example, in a primate
comprising subjecting the subject to a signaling molecule according
to the invention, preferably to a mixture of such signaling
molecules. The invention aims at countering the involvement of
cell-mediated immunity in the etiology of neurological disease, and
treating the inflammatory component of neurological disorders by
targeting the central role of NF.kappa.B-induced cytokine
expression. As a consequence of (likely CNS-based) NF.kappa.B
expression, toxic inflammatory mediators are released, sustaining
breakdown of the blood-brain barrier and leading to injury of axons
and glia. Nitric oxide might act directly on normal or
hypomyelinated axons, transiently blocking conduction and
reversibly increasing deficits arising from already compromised
pathways. As acute inflammation resolves, pathways are released
from nitric oxide-induced physiological conduction block. Symptoms
also improve as surviving functional pathways are reorganized at
the cellular and systems level. Together, these mechanisms account
for remission early in the disease. But tissue vulnerability is
easily exposed. When compounded by high axonal firing frequency,
nitric oxide causes structural (and hence irreversible) changes to
axons. Cytokines and growth-promoting factors released by reactive
astrocytes and microglia as part of the acute inflammatory process
promote endogenous remyelination. But, over time, astrocyte
reactivity seals the lesion and gliosis causes a physical barrier
to further remyelination, reducing the capacity to accommodate
cumulative deficits, and marking transition to the stage of
persistent deficit. Since permanent disability can be caused by
incomplete recovery from the inflammation, the invention provides a
method for modulating a neurological disorder in a subject believed
to be in need thereof comprising providing the subject with a
signaling molecule comprising a short, gene-regulatory peptide or
functional analogue thereof, wherein the signaling molecule is
administered in an amount sufficient to modulate the exacerbating
event. The signal molecule is preferably a short peptide,
preferably at most 30 amino acids long, or a functional analogue or
derivative thereof. In a much preferred embodiment, the peptide is
an oligopeptide of from about 3 to about 15 amino acids long,
preferably 4 to 12, more preferably 4 to 9, most preferably 4 to 6
amino acids long, or a functional analogue or derivative thereof.
For oral treatment, it is preferably 3 to 6, even more preferably 3
to 5, most preferably 3 or 4 amino acids long. Most preferred for
oral treatment is a peptide selected from the group of peptides
LQG, QVV, PALP (SEQ ID NO:34), AQG, LAG, LQGV (SEQ ID NO:1), AQGV
(SEQ ID NO:2), or LAGV (SEQ ID NO:10). Of course, such signaling
molecule can be longer, for example, by extending it (N- and/or
C-terminally), with more amino acids or other side groups, which
can, for example, be (enzymatically) cleaved off when the molecule
enters the place of final destination, however, by virtue of its
small size of smaller than 15, preferably smaller than nine amino
acids, even more preferably smaller then six amino acids, a peptide
or functional analogue according to the invention thereof readily
be taken up by the intestinal mucosae after oral administration and
readily be crossing the blood brain barrier. Furthermore such a
small peptide as provided herein is very stable and has a
pharmaceutical half life greater than four hours. Herewith, the
invention also provides a method of, preferably oral, treatment of
mood disorders such as cases of postpartum depression or puerperal
psychosis and a use of a signal molecule according to the invention
for the preparation of a pharmaceutical composition for the
treatment of cases of postpartum depression or puerperal psychosis,
in particular by at least partly restoring or mimicking the
anti-inflammatory activity of the gene-regulatory peptides LQGV
(SEQ ID NO:1), AQGV (SEQ ID NO:2), and VLPALP (SEQ ID NO:4) and
their functional analogues. In particular a method is provided
wherein the signaling molecule modulates translocation and/or
activity of a gene transcription factor. It is particularly useful
when the gene transcription factor comprises an NF.kappa.B/Rel
protein or an AP-1 protein. Many of the neurological disorder
events as mentioned above involve increased expression of
inflammatory cytokines due to activation of NF.kappa.B and AP-1,
and in a preferred embodiment the invention provides a method
wherein translocation and/or activity of the NF.kappa.B/Rel protein
or AP-1 protein is inhibited. In this way, the destruction of brain
tissues like the myelin lining of nerves or plaque formation which
disrupts--the brain which have been found to be significantly based
on autoimmune or proinflammatory destruction caused by a
dysregulated release of cytokines and chemokines is inhibited by an
oral treatment according to the invention. In one embodiment, the
peptide is selected from the group of synthetic peptides LQG, AQG,
LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP
(SEQ ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ
(SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV
(SEQ ID NO:10), VLAALP (SEQ ID NO:1), VLPALA (SEQ ID NO:12),
VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID
NO:15), GVLPALP (SEQ ID NO:16), LQGVLPALPQVVC (SEQ ID NO:17),
LPGCPRGVNPVVS (SEQ ID NO:18), LPGC (SEQ ID NO:19), MTRV (SEQ ID
NO:20), MTR, VVC, which is used for the preparation of a
pharmaceutical composition. As said, additional expression of
inflammatory cytokines is often due to activation of NF.kappa.B and
AP-1. Inflammatory cytokines can be expressed by endothelium,
perivascular cells and adherent or transmigrating leukocytes, all
inducing numerous pro-inflammatory and procoagulant effects.
Together these effects predispose to inflammation, thrombosis and
hemorrhage. Of clinical and medical interest and value, the present
invention provides the opportunity to selectively control
NF.kappa.B-dependent gene expression in tissues and organs in a
living subject, preferably in a primate, allowing up-regulating
essentially anti-inflammatory responses such as IL-10, and
down-regulating essentially pro-inflammatory responses such as
mediated by TNF-.alpha., nitric oxide (NO), IL-5, IL-6 and
IL-1.beta..
[0134] The invention thus provides use of an NF.kappa.B-regulating
peptide or derivative thereof for the production of a
pharmaceutical composition for the treatment of neurological
disorders, preferably in a primate, and provides a method of
treatment of neurological disorders, notably in a primate. It is
preferred when the treatment comprises administering to the subject
a pharmaceutical composition comprising an NF.kappa.B
down-regulating peptide or functional analogue thereof. Examples of
useful NF.kappa.B down-regulating peptides are VLPALPQVVC (SEQ ID
NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1),
GVLPALPQ (SEQ ID NO:23), VLPALP (SEQ ID NO:4), VVC, MTR and
circular LQGVLPALPQVVC. More down-regulating peptides and
functional analogues can be found using the methods as provided
herein. Most prominent among NF.kappa.B down-regulating peptides
are VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV
(SEQ ID NO:1), and VLPALP (SEQ ID NO:4). These are also capable of
reducing production of NO by a cell. It is herein also provided to
use a composition that comprises at least two oligopeptides or
functional analogues thereof, each capable of down-regulation
NF.kappa.B, and thereby reducing production of NO and/or
TNF-.alpha. by a cell, in particular wherein the at least two
oligopeptides are selected from the group LQGV (SEQ ID NO:1), AQGV
(SEQ ID NO:2), and VLPALP (SEQ ID NO:4), for the treatment of
recurring disease seen with neurological disorders. In a preferred
embodiment, a peptide according to the invention, or a functional
derivative or analogue thereof is used for the production of a
pharmaceutical composition for the oral treatment of neurological
disorders. NF.kappa.B-regulating peptide can be given alone or
concomitantly to other treatments, the peptide (or analogue)
concentration preferably being from about 1 to about 1000 mg/l, but
the peptide can also be given on its own, for example, in a bolus
injection or oral preparation. In acute cases, doses of 1 to 5
mg/kg bodyweight, for example, every eight hours in a bolus
injection or per infusionem until the patient stabilizes, are
recommended, however, maintenance dosages afterwards are preferably
administered orally. For example, in cases where large adverse
response are expected or diagnosed, it is preferred to monitor
cytokine profiles, such as TNF-.alpha., IL-6 or IL-10 levels, in
the plasma of the treated patient, and to stop treatment according
to the invention when these levels are normal. In a preferred
embodiment it is herein provided to provide the patient
experiencing a severe and acute bipolar disorder with a bolus
injection of NF.kappa.B down-regulating peptide such as AQGV (SEQ
ID NO:2), LQGV (SEQ ID NO:1) or VLPALP (SEQ ID NO:4) at 2 mg/kg and
continue the infusion with an NF.kappa.B-down-regulating peptide
such as AQGV (SEQ ID NO:2), LQGV (SEQ ID NO:1) or VLPALP (SEQ ID
NO:4) or a functional analogue thereof at a dose of 1 mg/kg
bodyweight for every eight hours. Dosages may be increased or
decreased, for example, depending on the outcome of monitoring the
cytokine profile in the plasma or CSF of the patient. As said,
disease progression is dramatically mediated by cytokines and
chemokines. For example, the TNF-.alpha. family is then highly
elevated in CSF. The down-regulation or T-cell regulation of these
cytokines and chemokines can prevent T-cell and dendritic cells
from reaching the CNS and then further down-regulate the
proinflammatory response which produces pathology of the brain and
spinal cord. This model of migration of cells to the CNS and then
the release of proinflammatory cytokines and chemokines is seen in
the following and can be treated by a peptide according to the
invention through NF.kappa.B regulation, the development of
T-regulator cells, and the intervention of expression early or
pregenes such as C-jun or C-erg. For the pathologist, neurological
disorders often present as a disorder of the central nervous
system, manifesting as acute focal inflammatory demyelination and
axonal loss with limited remyelination. Thus, the primary nature of
inflammation is undisputed and is central for treatments that
modulate the immune system. There are, however, several aspects
that limit the therapeutic efficacy of strategies directed against
the inflammatory component of the disease. Currently, immune
suppression with corticosteroids is unable to specifically stop the
inflammatory regimes. Also, the inflammatory forms of neurological
disorder, such as described above with autism, which are now
epidemic in United States and European studies responds well in
part to the use of a NF.kappa.B-down-regulating peptides according
to the invention.
[0135] It is herein also provided to use a composition that
comprises at least two oligopeptides or functional analogues
thereof, each capable of down-regulation NF.kappa.B, and thereby
reducing production of NO and/or TNF-.alpha. by a cell, in
particular wherein the at least two oligopeptides are selected from
the group LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), and VLPALP (SEQ
ID NO:4). Useful NF.kappa.B up-regulating peptides are VLPALPQ (SEQ
ID NO:13), GVLPALP (SEQ ID NO:16) and MTRV (SEQ ID NO:20). As
indicated, more gene-regulatory peptides may be found with an
appropriate (bio)assay. A gene-regulatory peptide as used herein is
preferably short. Preferably, such a peptide is 3 to 15 amino acids
long, more preferably, wherein the lead peptide is 3 to 9 amino
acids long, most preferred wherein the lead peptide is 4 to 6 amino
acids long, and capable of modulating the expression of a gene,
such as a cytokine, in a cell. In a preferred embodiment, a peptide
is a signaling molecule that is capable of traversing the plasma
membrane of a cell or, in other words, a peptide that is
membrane-permeable.
Example
[0136] This invention in particular relates to the, preferably
oral, treatment of multiple sclerosis, and in particular to the,
preferably oral, treatment of the inflammatory injury seen in the
progressive stages in the disease such as seen with the recurrent
upsurges of acute disease, classically known as relapses or
exacerbations, herein identified as relapsing/remitting disease
seen with multiple sclerosis (MS).
[0137] Multiple sclerosis (MS) is the prototype inflammatory
autoimmune disorder of the central nervous system and, with a
lifetime risk of one in 400, potentially the most common cause of
neurological disability in young adults. In experimental animals,
an experimental autoimmune/allergic encephalomyelitis (EAE) can be
induced in which MS is studied. Exacerbations in EAE and MS both
are dramatically mediated by cytokines and chemokines. During an
exacerbation, the TNF-.alpha. family and other pro-inflammatory
cytokines is highly elevated in CSF. As with all complex traits,
the disorder results from an interplay between as yet unidentified
environmental factors and susceptibility genes. Together, these
factors trigger a cascade of events, involving engagement of the
immune system, acute inflammatory injury of axons and glia,
recovery of function and structural repair, post-inflammatory
gliosis, and neurodegeneration. The sequential involvement of these
processes underlies the clinical course characterized by episodes
with recovery, episodes leaving persistent deficits, and secondary
progression. Despite limited success in each of these categories,
everyone touched by multiple sclerosis looks for a better dividend
from applying an improved understanding of the pathogenesis to
clinical management.
[0138] Now, multiple sclerosis is recognized throughout the world,
with around 2 to 5 million affected individuals. For the
pathologist, multiple sclerosis is a disorder of the central
nervous system (CNS), manifesting as acute focal inflammatory
demyelination and axonal loss with limited remyelination,
culminating in the chronic multifocal sclerotic plaques from which
the disease gets its name. Demyelination in MS develops by a T-cell
driven inflammatory process. Thus, the primary nature of
inflammation is undisputed and will remain central for treatments
that modulate the immune system. There are, however, several
aspects that limit the therapeutic efficacy of strategies directed
exclusively against the inflammatory component of the disease.
Currently, immune suppression is unable to stop the inflammatory
regimes using interferon .beta. or co-polymer I, these treatments
may decrease, but do not abolish, inflammation. Furthermore, it is
currently not possible to intervene more specifically in the
inflammatory process because neither the trigger of inflammation
(virus-induced versus autoimmunity) nor the specific target antigen
in the CNS of affected patients is known.
[0139] For the patient, multiple sclerosis entails an apparently
infinite variety of symptoms but with certain recurring themes and
an unpredictable course. For the neurologist, multiple sclerosis is
a disorder of young adults diagnosed on the basis of clinical and
paraclinical evidence for a least two demyelinating lesions,
affecting different sites within the brain of spinal cord,
separated in time. For the clinical scientist, multiple sclerosis
is the prototype inflammatory autoimmune disease of the central
nervous system in which knowledge gained across a range of basic
and clinical neuroscience disciplines has already allowed rational
strategies for treatment. For all these groups, multiple sclerosis
remains a difficult disease for which solutions seem attainable yet
remain elusive.
[0140] The oligodendrocyte, a principal target of immune attack in
multiple sclerosis, synthesizes and maintains the myelin sheath of
up to 40 neighboring nerve axons in the central nervous system.
Compact myelin consists of a condensed membrane, spiraled around
axons to form the insulating segmented sheath needed for a
saltatory axonal conduction: voltage-gated sodium channels cluster
at the unmyelinated nodes of Ranvier, between myelin segments, from
where the action potential is propagated and spreads passively down
the myelinated nerve segment to trigger another action potential at
the next node. The consequences of demyelination for saltatory
conduction explain many clinical and laboratory features of
multiple sclerosis. Partially demyelinated axons conduct impulses
at reduced velocity--explaining the characteristic delays in
conduction of evoked potentials. Demyelinated axons can discharge
spontaneously and show increased mechanical sensitivity--accounting
for the flashes of light on eye movement (phosphenes) and
electrical sensation running down the spine or limbs on neck
flexion (Lhermitte's symptom and sign). Partially demyelinated
axons, whose safety factor for conduction is compromised, cannot
sustain the fall in membrane capacitance induced by a rise in
temperature, and conduction fails--leading to the characteristic
appearance of symptoms and signs after exercise or a hot bath
(Uhthoff's phenomenon). Ephaptic transmission (cross-talk) can
arise between neighboring demyelinated axons, resulting in
paroxysmal symptoms--trigeminal neuralgia, ataxia, and dysarthria,
or painful tetanic posturing of the limbs, lasting one or two
minutes and often triggered by touch or movement. Individuals with
multiple sclerosis characteristically tire during physical and
cognitive tasks, and take longer to recover: although poorly
understood, and probably multifactorial, fatigue in multiple
sclerosis can be very disabling, even in isolation.
[0141] Multiple sclerosis affects twice as many women as it does
men; this unexplained bias is similar to that seen in many other
putative autoimmune diseases. The disease has an incidence of about
seven per 100,000 every year, prevalence of around 120 per 100,000,
and lifetime risk of one in 400.80% of patients present with
relapsing/remitting disease and, typically, the illness passes
through phases of relapse with full recovery, relapse with
persistent deficit, and secondary progression. In about a quarter
of patients, multiple sclerosis never affects activities of daily
living; conversely, up to 15% become severely disabled within a
short time. Episodes happen at random intervals, but initially
average about one per year, decreasing steadily thereafter. In 20%
of patients, the disease is progressive from onset, hence termed
primary progressive--affecting the spinal cord and, less
frequently, the optic nerve, cerebrum, or cerebellum. Disease onset
is usually in the third or fourth decade, but 2% of patients with
multiple sclerosis present before age 10 years, and 5% before age
16 years. In children, the distinction from acute disseminated
encephalomyelitis (ADEM) can often only be established by observing
the subsequent natural history. Overall, life expectancy is at
least 25 years from disease onset with most patients dying from
unrelated causes.
[0142] Healthy individuals harbor autoreactive myelin T-cells,
presumed to normally be kept in check by regulatory T-cells. One
hypothesis to explain the breakdown of immune regulation in these
autoimmune diseases is molecular mimicry, which suggests that
peptide (the environmental factor), presented in the groove of
specific HLA/MHC class II molecules (one component of inherited
risk), is immunologically indistinguishable from self-antigen and,
hence, an appropriate response to infection generates inappropriate
inflammation against some component of the oligodendrocyte-myelin
unit. In common with all organ-specific autoimmune diseases, this
systemic defect results not in a sustained autoimmune attack on the
entire target organ, but, rather, in inflammatory lesions that are
temporally and spatially segregated.
[0143] Failure of regulation leads to proliferation, activation,
and entry into the circulation of autoreactive T-cells; they
express adhesion molecules and induce reciprocal changes in
endothelia, allowing access across the blood-brain barrier into the
central nervous system. There, activated T-cells re-encounter
antigen and activate microglia (the CNS macrophage); these, in
turn, express class II molecules, re-present antigen to T-cells,
and set up a proinflammatory loop, which provides an infiltrate
rich in activated T-cells and microglia with some neutrophils.
[0144] Toxic inflammatory mediators are released, sustaining
breakdown of the blood-brain barrier and leading to injury of axons
and glia. Nitric oxide might act directly on normal or
hypomyelinated axons, transiently blocking conduction and
reversibly increasing deficits arising from already compromised
pathways. As acute inflammation resolves, pathways are released
from nitric oxide-induced physiological conduction block. Symptoms
also improve as surviving functional pathways are reorganized at
the cellular and systems level. Together, these mechanisms account
for remission early in the disease. But tissue vulnerability is
easily exposed. When compounded by high axonal firing frequency,
nitric oxide cause structural (and hence irreversible) changes to
axons. Axonal transection in acute inflammatory plaques is shown
histologically and radiologically through reduction in the neuronal
spectroscopic marker, N-acetyl aspartate (NAA). These transected
axons undergo Wallerian degeneration during the subsequent 18
months, but this action does not seem to extend the lesion or shape
the clinical deficit.
[0145] Cytokines and growth-promoting factors released by reactive
astrocytes and microglia as part of the acute inflammatory process
promote endogenous remyelinaction. But, over time, astrocyte
reactivity seals the lesion and gliosis causes a physical barrier
to further remyelination, reducing the capacity to accommodate
cumulative deficits, and marking transition to the stage of
persistent deficit.
[0146] Since permanent disability can be caused by incomplete
recovery from disease episodes, relapse frequency is bound to
correlate with accumulation of disability during the
relapsing-remitting phase of multiple sclerosis. Type-1 interferons
were first used in multiple sclerosis for their anti-viral action,
in view of the propensity of viral infections to trigger relapses.
In fact, their mechanism of action is immunological and complex: we
prefer the evidence for functional antagonism of proinflammatory
cytokines and down-regulation of class II MHC antigen expression;
but other modes of action--including effects on the blood brain
barrier (BBB)--can equally well be argued.
[0147] Only in trials of the two interferon .beta.-1a preparations,
not interferon .beta.-1b, was this change in relapse rate also
accompanied by reduction in the accumulation of disability. But
this reduction could be accounted for by a fall in the accumulation
of relapse-related deficits, rather than an effect on secondary
progression.
[0148] Three other agents reduce relapse frequency, and the
accumulation of disability, in relapsing-remitting multiple
sclerosis; each has similar efficacy to the .beta.-interferons and
acceptable adverse effects profiles. Glatiramer acetate (Copaxone,
Teva), a mixture of synthetic polypeptides was noted
serendipitously to suppress experimental autoimmune/allergic
encephalomyelitis, perhaps by inhibiting the binding of myelin
basic protein (MBP) to the T-cell receptor or by altering the
phenotype of myelin-autoreactive T-cells. The drug is licensed for
the treatment of relapsing-remitting multiple sclerosis in the USA
and in Europe on the basis of results from a trial of 251 patients,
in which the annual relapse rate was reduced by 25% in the treated
group.
[0149] Azathioprine inhibits lymphocyte proliferation by inhibiting
purine synthesis, and probably has similar efficacy to the .beta.
interferons, although the trial data were obtained in a less
rigorous manner and reported more candidly.
[0150] Mitoxantrone inhibits DNA repair and synthesis in dividing
and non-dividing cells through inhibition of DNA topoisomerase II;
it is potentially much more toxic than the .beta. interferons, but
has a USA license for the treatment of aggressive relapsing
disease, including patients with high relapse frequency in the
progressive phase.
[0151] In view of the fact that the ability to suppress relapses
and limit their consequences is partial, no informed analyst could
reasonably conclude that (despite their achievements) the
.beta.-interferons are a definitive therapy in multiple sclerosis.
The pharmaceutical industry has responded by sponsoring studies
with combinations of established drugs (such as .beta. interferon
and cyclophosphamide) without compelling evidence for synergistic
benefit to date, together with a significant investment in novel
immunotherapeutic strategies. Interferon .beta.-1b and .beta.-1a
and glatiramer acetate are widely prescribed in North America to
patients with relapsing MS. However, these drugs have significant
limitations, including cost (US $11,000 per year), inconvenience
(parenteral administration), frequency of adverse effects
(especially "flu-like" symptoms for several hours in many patients
after each injection of interferon) and a relatively modest overall
impact on disease course (for example, reductions in relapse rate
of less than 35%). Furthermore the therapeutic effect of interferon
.beta. more than one year after onset of treatment in
relapsing-remitting MS is unclear. The National Multiple Sclerosis
Society has issued a proactive directive recommending the use of
these medications by all patients with clinically significant,
relapsing MS. Other therapies directed against MS include the
treatment of the MS patient with a (monoclonal) antibody directed
against a cytokine, such as TNF-.alpha., IL-6 or IL-12, However,
although few would disagree that using these cytokine-blocking
agents such as anti-TNF-.alpha. therapy may be an important
therapeutic addition in the treatment of patients with MS, adverse
effects related to single cytokine neutralizing therapies have
emerged. Also, for unknown reasons, single cytokine blocking
proteins may cause the formation of anti-dsDNA antibodies, and
after repeated treatment the cumulative ANA incidence can be as
high as 50%. Nonetheless, anti-TNF-.alpha. antibody therapy is
associated with lupus-like symptoms. Also, demyelinizing disease
and aplastic anaemia have been reported in a small number of thus
treated patients. A major problem of repeated administration of
chimeric therapeutic antibodies is immunogenicity, and up to 60% of
antibody-treated patients develop human antichimeric antibodies
(HACAs) which are related to infusion reactions and reduce
therapeutic efficacy.
[0152] The invention provides a method for the treatment of a, in
particular human, subject believed to be suffering of multiple
sclerosis, with a specific aim to reduce the frequency, and limit
the lasting effects, of relapses or exacerbations, to relieve
symptoms that arise from the release of additional pro-inflammatory
cytokines during the relapse, to prevent disability arising from
disease progression after the relapse, and promote tissue repair
after the relapse. The invention provides a pharmaceutical
composition for the oral treatment during relapses in case of
relapsing/remitting multiple sclerosis occurring in a subject, in
particular in a human, and a method for the oral treatment during
the relapses of the exacerbations associated with additional
pro-inflammatory cytokine release, for example, in a primate
suffering from MS or EAE comprising subjecting the subject to a
signaling molecule according to the invention, preferably to a
mixture of such signaling molecules. Most preferred for oral
treatment during relapses is a peptide, or a mixture of peptides
selected from the group of peptides LQG, QVV, PALP (SEQ ID NO:34),
AQG, LAG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), or LAGV (SEQ ID
NO:10).
[0153] Furthermore, the invention provides a method for the
prevention of the development of multiple sclerosis in a subject
believed to be in need thereof, in particular for the treatment of
a human being after a sign of neurological failure, such as
neuritis optica has been observed but MS has not developed, and use
of a signaling molecule according to the invention for the
production of a pharmaceutical composition for the prevention of
multiple sclerosis, for treatment of relapsing/remitting multiple
sclerosis occurring in a subject, in particular in a human being,
and a method for the treatment of the exacerbations associated with
additional pro-inflammatory cytokine release, in particular in a
human being.
[0154] Administration of such a signaling molecule or mixture
preferably occurs systemically, e.g., by intravenous,
intramuscular, intraperitoneal or subcutaneous administration and
leads to a dampening of the effect of the additionally released
pro-inflammatory cytokines during the exacerbation phase. In severe
cases, intrathecal administration may be considered. However, a
most preferred treatment comprises mucosal administration,
preferably oral.
[0155] In a preferred embodiment, the invention provides a method
for modulating relapsing/remitting disease of MS in a subject
believed to be in need thereof comprising providing the subject
with a signaling molecule comprising a short, gene-regulatory
peptide or functional analogue thereof, wherein the signaling
molecule is administered orally in an amount sufficient to modulate
the iatrogenic event. The signal molecule is preferably a short
peptide, preferably of at most 30 amino acids long, or a functional
analogue or derivative thereof. In a much preferred embodiment, the
peptide is an oligopeptide of from about 3 to about 15 amino acids
long, preferably 4 to 12, more preferably 4 to 9, most preferably 3
to 4 to 6 amino acids long, or a functional analogue or derivative
thereof. Of course, such a signaling molecule can be longer, for
example, by extending it (N- and/or C-terminally), with more amino
acids or other side groups, which can, for example, be
(enzymatically) cleaved off when the molecule enters the place of
final destination. In particular a method is provided wherein the
signaling molecule modulates translocation and/or activity of a
gene transcription factor. It is particularly useful when the gene
transcription factor comprises an NF.kappa.B/Rel protein or an AP-1
protein. Many of the relapsing/remitting events as mentioned above
induce increased expression of inflammatory cytokines due to
activation of NF.kappa.B and AP-1, and in a preferred embodiment
the invention provides a method wherein translocation and/or
activity of the NF.kappa.B/Rel protein or AP-1 protein is
inhibited. In one embodiment, the peptide is selected from the
group of peptides LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2),
LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4), ALPALP (SEQ ID NO:5),
VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8),
VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10), VLAALP (SEQ ID NO:1),
VLPALA (SEQ ID NO:12), VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID
NO:14), VLPALPA (SEQ ID NO:15), GVLPALP (SEQ ID NO:16),
LQGVLPALPQVVC (SEQ ID NO:17), LPGCPRGVNPVVS (SEQ ID NO:18), LPGC
(SEQ ID NO:19), MTRV (SEQ ID NO:20), MTR, VVC. Most preferred for
oral treatment is a peptide selected from the group of peptides
LQG, QVV, PALP (SEQ ID NO:34), AQG, LAG, LQGV (SEQ ID NO:1), AQGV
(SEQ ID NO:2), or LAGV (SEQ ID NO:10). As said, additional
expression of inflammatory cytokines is often due to activation of
NF.kappa.B and AP-1. Inflammatory cytokines can be expressed by
endothelium (for example, by trauma), perivascular cells and
adherent or transmigrating leukocytes, inducing numerous
pro-inflammatory and procoagulant effects. Together these effects
predispose to inflammation, thrombosis and hemorrhage. Of clinical
and medical interest and value, the present invention provides the
opportunity to selectively control NF.kappa.B-dependent gene
expression in tissues and organs in a living subject, preferably in
a primate, allowing up-regulating essentially anti-inflammatory
responses such as IL-10, and down-regulating essentially
pro-inflammatory responses such as mediated by TNF-.alpha., nitric
oxide (NO), IL-5, IL-6. IL-12 and IL-1.beta..
[0156] In comparison with single cytokine therapy, such as the use
of anti-TNF-.alpha., anti IL-5, anti-IL-6, anti-IL-12, anti-IL-23,
anti-IL-12p40, anti-IL23p40 or anti-IL-1.beta. antibodies, using an
NF.kappa.B down-regulating peptide or functional analogue thereof
according to the invention has the major advantage that a major
network of pro-inflammatory cytokines is down-regulated.
[0157] The invention thus provides use of an NF.kappa.B-regulating
peptide or derivative thereof for the production of a
pharmaceutical composition for the treatment of relapsing/remitting
disease seen with MS, preferably in a primate, and provides a
method of treatment of relapsing/remitting disease seen with MS,
notably in a primate. It is preferred that treatment comprises
administering to the subject a pharmaceutical composition
comprising an NF.kappa.B down-regulating peptide or functional
analogue thereof. Examples of useful NF.kappa.B down-regulating
peptides are VLPALPQVVC (SEQ ID NO:21), LQGVLPALPQ (SEQ ID NO:22),
LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:23), VLPALP (SEQ ID
NO:4), VVC, MTR and circular LQGVLPALPQVVC (SEQ ID NO:17). More
down-regulating peptides and functional analogues can be found
using the methods as provided herein. Most prominent among
NF.kappa.B down-regulating peptides are VLPALPQVVC (SEQ ID NO:21),
LQGVLPALPQ (SEQ ID NO:22), LQG, LQGV (SEQ ID NO:1), and VLPALP (SEQ
ID NO:4). These are also capable of reducing production of NO by a
cell.
[0158] In one embodiment, the invention provides a method of
treating a subject suffering from a relapsing/remitting disease
seen during relapses with MS with a method and signaling molecule
according to the invention concomitantly, or at least timely, with
a treatment with a single cytokine blocking protein, such as an
anti-TNF-.alpha., anti IL-5, anti-IL-6, anti-IL-12, anti-IL-23,
anti-IL-12p40, anti-IL23p40 or anti-IL-1.beta. antibody or
functional analogue thereof. It is herein also provided to use a
signaling molecule according to the invention for the production of
a pharmaceutical composition for the treatment of a subject
believed to be suffering from MS and receiving treatment with an
anti-TNF-.alpha., anti IL-5, anti-IL-6, anti-IL-12, anti-IL-23,
anti-IL-12p40, anti-IL23p40 or anti-IL-10 antibody. It is herein
also provided to use a composition that comprises at least two
oligopeptides or functional analogues thereof, each capable of
down-regulation NF.kappa.B, and thereby reducing production of NO
and/or TNF-.alpha. by a cell, in particular wherein at least two
oligopeptides are selected from the group LQGV (SEQ ID NO:1), AQGV
(SEQ ID NO:2), and VLPALP (SEQ ID NO:4), for the treatment of
relapsing/remitting disease seen with MS. In response to a variety
of signals received by the body in the course of the
relapsing-remitting disease seen with MS, the NF.kappa.B/Rel family
of transcription factors are activated and form different types of
hetero- and homodimers among themselves to regulate the expression
of target genes containing KB-specific binding sites. NF.kappa.B
transcription factors are hetero- or homodimers of a family of
related proteins characterized by the Rel homology domain. They
form two subfamilies, those containing activation domains
(p65-RELA, RELB, and c-REL) and those lacking activation domains
(p50, p52). The prototypical NF.kappa.B is a heterodimer of p65
(RELA) and p50 (NF.kappa.B1). Among the activated NF.kappa.B
dimers, p50-p65 heterodimers are known to be involved in enhancing
the transcription of target genes and p50-p50 homodimers in
transcriptional repression. However, p65-p65 homodimers are known
for both transcriptional activation and repressive activity against
target genes. KB DNA-binding sites with varied affinities to
different NFB dimers have been discovered in the promoters of
several eukaryotic genes and the balance between activated
NF.kappa.B homo- and heterodimers ultimately determines the nature
and level of gene expression within the cell. The term
"NF.kappa.B-regulating peptide" as used herein refers to a peptide
or a modification or derivative thereof capable of modulating the
activation of members of the NF.kappa.B/Rel family of transcription
factors. Activation of NF.kappa.B can lead to enhanced
transcription of target genes. Also, it can lead to transcriptional
repression of target genes. Modulating comprises the up-regulation
or the down-regulation of transcription. In a preferred embodiment,
a peptide according to the invention, or a functional derivative or
analogue thereof is used for the production of a pharmaceutical
composition for oral use for the treatment of relapsing/remitting
disease seen with MS. NF.kappa.B-regulating peptide can be given
concomitantly to other MS treatments, the peptide (or analogue)
concentration preferably being from about 1 to about 1000 mg/l, but
the peptide can also been given on its own, for example, in a bolus
injection. Doses of 1 to 5 mg/kg bodyweight, for example, every
eight hours in a bolus injection or per infusionem until the
patient stabilizes, are recommended initially, however, the
potential of oral treatment allows a rapid transition to oral
administration thereafter. For example, in cases where large
adverse response are expected or diagnosed, it is preferred to
monitor cytokine profiles, such as TNF-.alpha., IL-6 or IL-10
levels, in the plasma of the treated patient, and to stop treatment
according to the invention when these levels are normal. In a
preferred embodiment it is herein provided to give a patient
experiencing a severe and acute exacerbation (relapse) with a bolus
injection of NF.kappa.B down-regulating peptide such as AQGV (SEQ
ID NO:2), LQGV (SEQ ID NO:1) or VLPALP (SEQ ID NO:4) at 2 mg/kg and
continue the infusion with an NF.kappa.B-down-regulating peptide
such as AQGV (SEQ ID NO:2), LQGV (SEQ ID NO:1) or VLPALP (SEQ ID
NO:4) or a functional analogue thereof at a dose of 1 mg/kg
bodyweight for every eight hours. The oral treatment commences,
using dosages of 0.01 to 10 mg/kg bodyweight, and preferably 0.1 to
1 mg/kg bodyweight until the relapse has stabilized. Dosages may be
increased or decreased, for example, depending on the outcome of
monitoring the cytokine profile in the plasma or CSF of the
patient. Of course, when the relapse seems of a milder nature, oral
treatment is the first choice to begin with. As said, exacerbations
and disease progression in experimental autoimmune/allergic
encephalomyelitis (EAE) and MS both are dramatically mediated by
cytokines and chemokines. During an exacerbation of MS, the
TNF-.alpha. family is highly elevated in CSF and plasma. IL-12
activity is often also high. The down-regulation or T-cell
regulation of these cytokines and chemokines can prevent T-cell and
dendritic cells from reaching the CNS and then further
down-regulate the proinflammatory response which produces
demyelination of the brain and spinal cord. This model of migration
of cells to the CNS and then the release of proinflammatory
cytokines and chemokines is seen particularly in the course of
relapsing/remitting disease and can be treated by a peptide
according to the invention through NF.kappa.B regulation, the
development of T-regulator cells, and the intervention of
expression of early or pregenes such as C-jun or C-erg. The
treatment protocols as given herein can also be used for other
diseases that resemble or include exacerbations of multiple
sclerosis and its variants, additional pro-inflammatory cytokine
release in EAE and other infectious and/or immune based
meningoencephalopathies, such as seen with measles, i.e., SSPE,
mumps, infections with hemorrhagic viruses, Progressive Multifocal
Encephalopathy or a papillomavirus (JC virus) disease, Bacterial
Endocarditis inducing immune encephalopathy, malaria with cerebral
encephalopathy, angiostrongyliasis and other parasitic
encephalitis, Lyme Disease, Herpes 1-8 disease including the mono
like viruses such as EBV, CMV, and HHV6, rickettsial disease, i.e.,
Typhus, Rocky Mountain Spotted Fever, and Q fever, Chlamydia
disease, i.e., Trachoma, NSU, Chlamydial Pneumonia, mycoplasma
arthritis and encephalitis, HIV-1 and 2 encephalitis and dementia,
Arbovirus disease, Togavirus disease and other lentivirus or Bunya
virus or Flavivirus disease. Other forms of infectious and/or
inflammatory meningo-encephalomyelites are acute bacterial
infections, sprirochetal infections (neurolues, lyme
neuroborreliosis, tuberculosis, viral infections (enteroviruses,
mumps, herpes simplex type 2, togaviruses (arbovirus), HIV type 1
and 2, HTLV-1 infections), fungal infections (Cryptococcus
neoformans, Coccidiodes immitis, Blastomyces dermatitidis,
Paracoccidioides brasiliensis, sporothrix schenkii, Histoplasma
capsulatum, Pseudallescheria boydii and the dermatiaceous fungi,
mostly opportunistic infections such as candida and aspergillus
species and zygomycetes), protozoan infections (cerebral malaria,
toxoplasmosis, trypanosoma species, naegleria species and
helminths), neurosarcoidosis, Creutzfeldt-Jacob disease, and
neurological complications following vaccination.
Mucosal and Oral Administration
Experiment 1:
[0159] Material and methods: Female NOD mice were bred and
maintained in a pathogen-free facility at Lucky Farm, Balkbrug, The
Netherlands. All mice were given free access to food and water.
[0160] Twenty-one to 22-week-old diabetic female NOD mice (n=5)
were given four weeks long free access to either water containing 4
IU per ml hCG (pregnyl; batch number 235863) or mixture of
gene-regulatory peptides LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID
NO:23) and VLPALP (SEQ ID NO:4) (each 1 microgram per milliliter).
Control mice were given plain water only. During these four weeks
of treatment mice were daily observed for their drinking behavior,
urination, and the look of the fur.
[0161] Results: During four weeks of treatment, mice without
treatment drank much water because on daily bases their drinking
bottle had to be refilled and they had percolated fur, which is a
normal sign of heavily diabetic mice. Mice with treated water with
hCG or a mixture of gene-regulatory peptides drank a normal amount
of water after four days of starting the experiment and thereafter
during the test period their fur was normal.
[0162] Conclusion: This experiment shows that due to oral treatment
with a commercial hCG preparation as well as due to oral treatment
with gene-regulatory peptides mice that were already diabetic had
less severe diabetic symptoms compared to the control group.
Therefore, the oral treatment of hCG preparation or gene-regulatory
peptides do have visual therapeutic effects and crude hCG
preparation and gene-regulatory peptides are able to be taken up in
the mouth or through the digestive tract.
Experiment 2:
[0163] Material and methods: Female NOD mice were bred and
maintained in a pathogen-free facility at Lucky Farm, Balkbrug, The
Netherlands. All mice were given free access to food and water.
[0164] Twelve- to 14-week-old non-diabetic female NOD mice (n=14 to
16) were given seven weeks long free access to either water
containing 4 IU per ml hCG from four different batches (pregnyl;
batch number 235863, 248455, 293703 and 313692) or plain water
only. At the end of the treatment, mice were assessed for diabetes
by determining the presence of glucose in urine. Mice were
considered diabetic after two consecutive glucose measurements in
urine.
[0165] Results: Five of the sixteen mice treated with plain water
were diabetic at the end of the experiment, while two of the
sixteen mice treated with batch 235863, three of the sixteen mice
treated with batch 248455, one of the fourteen mice treated with
batch 293703 and two of the fourteen mice treated with 313692 were
diabetic.
[0166] Conclusion: This experiment shows that the oral treatment of
NOD mice with a commercial hCG preparation reduce the incidence of
diabetes during the treatment period. In addition, this experiment
also shows the therapeutic effect of Pregnyl varies among different
batches.
Experiment 3:
[0167] Material and methods: Female NOD mice were bred and
maintained in a pathogen-free facility at Lucky Farm, Balkbrug, The
Netherlands. All mice were given free access to food and water.
[0168] Eleven- to 13-week-old non-diabetic female NOD mice (n=9)
were given five weeks long three times a week one drop (50
microliters) of water containing gene-regulatory peptides LQGV (SEQ
ID NO:1), GVLPALPQ (SEQ ID NO:23) and VLPALP (SEQ ID NO:4) (each 1
microgram per milliliter) or drops of plain water. Drops were
instilled in the mouth in the buccal sac. After the treatment, mice
were left alive for another fifteen weeks. At the age 31 to 33
weeks, mice were assessed for diabetes by determining the presence
of glucose in urine. Mice were considered diabetic after two
consecutive glucose measurements in urine. The mice were then
sacrificed and the percentage of MP12/20 high positive bone marrow
cells was determined by FACS analyses.
[0169] Result: Eight of the nine mice treated with plain water drop
were diabetic at the age of 31 to 33 weeks, while five of the nine
mice treated with water containing gene-regulatory peptides were
diabetic at the age of 31 to 33 weeks.
[0170] Conclusion: This experiment shows that the mucosal treatment
of NOD mice with gene-regulatory peptides reduces the incidence of
diabetes.
Example 4
[0171] Material and Methods: Female NOD mice were bred and
maintained in a pathogen-free facility at Lucky Farm, Balkbrug, the
Netherlands. All mice were given free access to food and water.
[0172] Thirteen- to 14-week-old non-diabetic female NOD mice (n=10)
were given five weeks long daily one drop (50 microliters) of PBS
containing gene-regulatory peptides LQG, LQGV (SEQ ID NO:1), VLPALP
(SEQ ID NO:4), VVC and MTRV (SEQ ID NO:20) each 20 mcg or one drop
PBS only. Drops were instilled in the mouth in the buccal sac.
After the oral treatment, mice were left alive for another 20
weeks. Every week mice were assessed for diabetes by determining
the presence of glucose in urine. Mice were considered diabetic
after two consecutive glucose measurements in urine.
[0173] Results: Nine weeks after end of the treatment, all ten mice
treated orally with PBS were diabetic, while only four of the ten
mice treated orally with a mixture of gene-regulatory peptides
(LQG, LQGV (SEQ ID NO:1), VLPALP (SEQ ID NO:4), VVC and MTRV (SEQ
ID NO:20)) were diabetic. However, anti-diabetic effect of
gene-regulatory peptides seemed to be weakened over a period of
time since twenty weeks after end of the treatment eight of the ten
mice became diabetic.
[0174] Conclusion. This experiment shows that the oral treatment of
NOD mice with a mixture of gene-regulatory peptides delayed the
incidence of diabetes.
TABLE-US-00001 Diabetes incidence Age Group A Group B (weeks)
(mixture of peptides) (PBS treatment only) 13 to 14 0% 0% 14 to 15
20% 0% 15 to 16 20% 10% 16 to 17 40% 20% 17 to 18 40% 30% 18 to 19
40% 40% 19 to 20 40% 100% 20 to 21 40% 100% 21 to 22 40% 100% 22 to
23 40% 100% 23 to 24 40% 100% 24 to 25 40% 100% 25 to 26 40% 100%
26 to 27 40% 100% 27 to 28 80% 100% 28 to 29 80% 100% 29 to 30 80%
100% 30 to 31 80% 100% 31 to 32 80% 100% 32 to 33 80% 100% 33 to 34
80% 100% 34 to 35 80% 100% 35 to 36 80% 100% 36 to 37 80% 100% 37
to 38 80% 100%
[0175] Common to both type 1 and type 2 diabetes is the development
of inflammatory and vascular complications that result from high
glucose levels and, over time, portend significant morbidity and
early mortality in affected subjects. Although multiple studies
have suggested a direct role for adverse effects of glucose itself
in modulating cellular properties, both in the extra- and
intracellular milieu, recent observations also suggest an emerging
role for the products of nonenzymatic glycoxidation of proteins
and/or lipids--the advanced glycation end products (AGEs)--in the
pathogenesis of diabetic complications.
[0176] A number of epidemiological studies have suggested that
elevated levels of circulating insulin contribute independently to
cardiovascular risk. Other factors, such as hyperlipidemia and
intermittently elevated levels of blood glucose, are tightly linked
to syndromes characterized by elevated levels of insulin such as
metabolic syndrome or syndrome x. Activation of NF.kappa.B in the
pathogenesis of atherosclerosis, ischemia-reperfusion injury, and
diabetes play in this respect a special role. For example, target
genes of NF.kappa.B, such as tumor necrosis factor-.alpha.
(TNF-.alpha.) and vascular cell adhesion molecule-1, have long been
speculated to participate in the earliest stages of atherogenesis.
Indeed, RelA/p65, one of the components of NF.kappa.B, has been
identified within the nuclei of vascular smooth muscle cells
(VSMCs) and mononuclear phagocytes in human atheromata.
[0177] One of the consequences of hyperglycemia in both type 1 and
type 2 is the generation of advanced glycation end products (AGEs).
Interaction of these products of nonenzymatic glycation/oxidation
of proteins, with their key signal transduction receptor RAGE
(receptor for AGE), results in activation of NF.kappa.B in
endothelial cells, mononuclear phagocytes, and VSMCs, by processes
that involve, at least in part, generation of reactive oxygen
intermediates and activation of p21.sup.ras and ERK1/2 kinases.
Recently, a specific AGE, carboxy(methyl lysine) adducts of
proteins, has been shown to bind RAGE and mediate cellular
activation, both in vitro and in vivo. Evidence definitively
linking RAGE to these ligand-mediated effects was demonstrated by
blockade of AGE-mediated activation of NF.kappa.B in the presence
of blocking antibodies to RAGE, soluble RAGE (sRAGE; the
extracellular ligand-binding domain), or transient transfection
into wild-type RAGE-bearing cells of a construct in which solely
the cytosolic domain of the receptor was deleted. In the latter
case, a dominant-negative effect resulted, as AGE-stimulated
activation of NF.kappa.B was significantly suppressed. Furthermore,
a novel property of insulin is its ability to activate prenyl
transferases, farnesyl transferases, and geranylgeranyl
transferases I and II. Because these molecules possess the capacity
to post-translationally modify Ras, Rho, and Rab proteins, their
activation links them to signal transduction pathways. Incubation
of VSMCs with insulin (largely at physiologically relevant doses)
increased availability of geranylgeranylated Rho-A, thereby
invoking an established mechanism to link increased levels of
insulin to activation of NF.kappa.B. In VSMCs, insulin, and AGEs,
hyperglycemia or angiotensin II synergized to enhance NF.kappa.B
activation to even greater degrees than that observed by any of
these mediators alone. It is also known that insulin primes the
vasculature for enhanced activation on contact with these
traditional mediators; the vascular microenvironment in type 2
diabetes or syndromes of insulin resistance is enriched in factors
that appear to lead to a common pathway, activation of NF.kappa.B.
In the above experiments, the oral treatment of pre-diabetic NOD
mice with gene-regulatory peptides reduced the incidence of
diabetes showing an inhibitory effect of the treatment on
pancreatic inflammation, .beta.-cell destruction and on autoimmune
process. Furthermore, when the treatment was started at late stage
in diabetes (in already diabetic mice), we observed the inhibition
of inflammatory effects of prolonged hyperglycemia and reduction of
clinical symptoms of inflammation which was observed by their
changes in drinking behavior, reduction in urination, and the look
of the fur. The ongoing impairment of glucose tolerance and/or
prolonged hyperglycemia which with time if uncontrolled in patients
results in serious diabetic complications such as kidney
failure/damage, impaired blood macro- and microcirculation,
retinopathy, neuropathy, nephropathy and accelerated
arteriosclerosis was thus countered by mucosal treatment with
gene-regulatory peptides directed at down-regulation of NF.kappa.B.
NF.kappa.B is a target to prevent or suppress the
vascular-perturbing properties of a range of injurious molecules
linked to diabetes and insulin resistance, from oxidized
lipoproteins, to AGEs, to high levels of glucose or insulin.
NF.kappa.B is a pleiotropic transcription factor. In the context of
both type 1 and 2 diabetes (hyperinsulinemia and hyperglycemia), a
range of environmental stimuli, such as AGEs, hyperglycemia, and
angiotensin II triggers signal transduction pathways leading to
NF.kappa.B activation. Prominently included among these signaling
mechanisms is a role for Ras, Rho-A, cdc42, and Rac1. Nuclear
translocation of NF.kappa.B leads to activation of a range of genes
involved in host and cellular defense responses. In certain
settings, activation of NF.kappa.B may lead to "good" inflammation,
manifested by resolution and regeneration, or "bad" inflammation,
causing tissue destruction. However, it is likely that separation
of good from bad inflammation triggered by NF.kappa.B will be
difficult because mechanisms underlying both operate in tandem,
delicately balanced, under many conditions. Our gene-regulatory
peptides have regulatory effects on, for example, NF.kappa.B in
this respect and play an important therapeutic role.
FURTHER REFERENCES
[0178] WO99/59617; WO01/72831; WO97/49721; WO01/10907; WO01/11048
EP 1 138 692; US 2002/0064501; Khan et al., Human Immunology
63:1-7, 2002; Christman et al., Intens. C are Med. 24:1131-1138,
1998; Tak et al., J. Clin. Invest. 107:7-11, 2001; EP 1 300418;
U.S. Pat. No. 5,851,997; U.S. Pat. No. 6,319,504 B1; Blackwell et
al., Am. J. Respir. Cell Mol. Biol. 17:3-9, 1997; WO01/10907; U.S.
Pat. No. 6,319,504; U.S. Pat. No. 6,489,296; WO02/085117;
WO98/35691; DE 3715662; Patil et al., Acta Neurochirurgica
87:76-78, 1987; Slater et al., Transplantation 23:104-104, 1977;
Blackwell et al., Am. J. Respir. Cell Mol. Biol. 17:3-9, 1997;
WO99/59617; Tan et al., Acta Physiol. Sinica. 55:58-64, 2003; US
2002/0041871; DE 19953339; Jyonouchi Harumi et al., J. Neuroim.
120:170-179, 2001; Khan et al., Human Immunology 62:1315-1323,
2001; Roehrig et al., Zentralblatt Bakt 289:89-99, 1999; Tovey et
al., J. Interferon Cytokine Res. 19:911-921, 1999; Kanungo et al.,
J. Adv. Zool. 20:1-5, 1999; Khan et al., Human Immunology 63:1-7,
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et al., J. Exp. Med. 160:1672-1685, 1984; U.S. Pat. No. 4,977,244.
Sequence CWU 1
1
3414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Leu Gln Gly Val 124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Ala
Gln Gly Val 134PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 3Leu Gln Gly Ala 146PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Val
Leu Pro Ala Leu Pro 1 556PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Ala Leu Pro Ala Leu Pro 1
566PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Val Ala Pro Ala Leu Pro 1 577PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Ala
Leu Pro Ala Leu Pro Gln 1 587PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Val Leu Pro Ala Ala Pro Gln
1 597PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Val Leu Pro Ala Leu Ala Gln 1 5104PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Leu
Ala Gly Val 1116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 11Val Leu Ala Ala Leu Pro 1
5126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Val Leu Pro Ala Leu Ala 1 5137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Val
Leu Pro Ala Leu Pro Gln 1 5147PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 14Val Leu Ala Ala Leu Pro Gln
1 5157PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Val Leu Pro Ala Leu Pro Ala 1 5167PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Gly
Val Leu Pro Ala Leu Pro 1 51713PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 17Leu Gln Gly Val Leu Pro Ala
Leu Pro Gln Val Val Cys 1 5 101813PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 18Leu Pro Gly Cys Pro Arg
Gly Val Asn Pro Val Val Ser 1 5 10194PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Leu
Pro Gly Cys 1204PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 20Met Thr Arg Val 12110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Val
Leu Pro Ala Leu Pro Gln Val Val Cys 1 5 102210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Leu
Gln Gly Val Leu Pro Ala Leu Pro Gln 1 5 10238PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Gly
Val Leu Pro Ala Leu Pro Gln 1 52438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Val
Val Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu Pro 1 5 10
15Gly Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu
20 25 30Ser Cys Gln Cys Ala Leu 352535PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Arg
Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu Ala Val Glu Lys Glu 1 5 10
15Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr Ile Cys Ala Gly Tyr
20 25 30Cys Pro Thr 352618PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 26Ser Lys Ala Pro Pro Pro Ser
Leu Pro Ser Pro Ser Arg Leu Pro Gly 1 5 10 15Pro
Ser2716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn
Pro Val Val Ser 1 5 10 152825DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 28agctcagagg gggactttcc gagag
25294PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Gln Val Val Cys 13017PRTartificialSynthetic
peptide 30Met Thr Arg Val Leu Gln Gly Val Leu Pro Ala Leu Pro Gln
Val Val1 5 10 15Cys3121PRTArtificialSynthetic peptide 31Cys Ala Leu
Cys Arg Arg Ser Thr Thr Asp Cys Gly Gly Pro Lys Asp1 5 10 15His Pro
Leu Thr Cys 203218PRTArtificialSynthetic peptide 32Cys Arg Arg Ser
Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu1 5 10 15Thr
Cys3337PRTArtificialSynthetic peptide 33Thr Cys Asp Asp Pro Arg Phe
Gln Asp Ser Ser Ser Ser Lys Ala Pro1 5 10 15Pro Pro Ser Leu Pro Ser
Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 20 25 30Pro Ile Leu Pro Gln
35344PRTArtificialSynthetic peptide 34Pro Ala Leu Pro1
* * * * *