U.S. patent application number 14/880892 was filed with the patent office on 2016-04-07 for yersinia outer protein m (yopm) in the treatment of psoriasis.
The applicant listed for this patent is Westfaelische Wilhelms-Universitaet Muenster. Invention is credited to Christian Rueter, Alexander Schmidt.
Application Number | 20160095898 14/880892 |
Document ID | / |
Family ID | 47913915 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160095898 |
Kind Code |
A1 |
Rueter; Christian ; et
al. |
April 7, 2016 |
YERSINIA OUTER PROTEIN M (YOPM) IN THE TREATMENT OF PSORIASIS
Abstract
The present invention relates to the use of Yersinia outer
protein M (YopM) in the prevention and/or treatment of psoriasis by
cutaneous, intradermal or subcutaneous administration. In
particular, the present invention relates to the use of YopM in the
treatment of plaque psoriasis, guttate psoriasis, inverse
psoriasis, pustular psoriasis, erythrodermic psoriasis, arthritis
psoriasis.
Inventors: |
Rueter; Christian;
(Muenster, DE) ; Schmidt; Alexander; (Havixbeck,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Westfaelische Wilhelms-Universitaet Muenster |
Muenster |
|
DE |
|
|
Family ID: |
47913915 |
Appl. No.: |
14/880892 |
Filed: |
October 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14344395 |
Aug 1, 2014 |
9155779 |
|
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PCT/EP2012/053209 |
Feb 24, 2012 |
|
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14880892 |
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61539693 |
Sep 27, 2011 |
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Current U.S.
Class: |
514/18.6 |
Current CPC
Class: |
A61P 17/06 20180101;
A61K 38/164 20130101 |
International
Class: |
A61K 38/16 20060101
A61K038/16 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 23, 2011 |
EP |
11007785.6 |
Claims
1. A method for the prevention of psoriasis in a human subject,
comprising a step of administering a pharmaceutical composition
comprising Yersinia outer protein M (YopM) to a human subject
cutaneously, wherein administering prevents the psoriasis and
wherein YopM does not enter the lymph node.
2. The method according to claim 1 wherein the psoriasis is
selected from the group consisting of plaque psoriasis, guttate
psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic
psoriasis, and arthritis psoriasis.
3. The method according to claim 1 wherein the YopM is from a
species selected from the group consisting of Yersinia
enterocolitica, Yersinia pseudotuberculosis or Yersinia pestis.
4. The method according to claim 1, wherein the Yersinia outer
protein M (YopM) is provided in a cream, lotion, gel or
ointment.
5. The method according to claim 4, wherein the cream is a
semi-solid emulsion.
6. The method according to claim 5, wherein the cream is an oil-in
water or water-in oil cream.
7. The method according to claim 1, wherein the composition further
comprises one or more of carriers, excipients, or diluents.
8. The method according to claim 1, wherein the composition further
comprises one or more of cetearyl alcohol, paraffinum liquidum,
glycerol, dimethicone, sodium cetearyl sulfate, phenoxyethanol,
methylparaben, ethylparaben, propylparaben, or sodium
hydroxide.
9. The method according to claim 1, wherein the step of
administering provides YopM in an amount in the range of 1
.mu.g/cm.sup.2 to 20 mg/cm.sup.2 (YopM/cutaneous area).
10. The method according to claim 9, wherein the amount is in the
range of 1 .mu.g/cm.sup.2 to 20 .mu.g/cm.sup.2.
11. The method according to claim 10, wherein the amount is 2
.mu.g/cm.sup.2.
12. The method according to claim 1, wherein the step of
administering is performed every 12 hours, or every 24 hours.
Description
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present non-provisional application is a continuation of
U.S. patent application Ser. No. 14/344,395, filed on Aug. 1, 2014,
which claims benefit from International Application No.
PCT/EP2012/053209, which was filed on Feb. 24, 2012, which in turn
claims priority to U.S. Provisional Patent Application No.
61/539,693, filed on Sep. 27, 2011; and European Patent Application
11007785.6 filed on Sep. 23, 2011; wherein the entireties of said
patent applications are incorporated herein by reference.
2. FIELD OF THE INVENTION
[0002] The present invention relates to the use of Yersinia outer
protein M (YopM) in the prevention and/or treatment of psoriasis by
cutaneous, intradermal or subcutaneous administration.
3. BACKGROUND ART
[0003] The YopM protein consists of two amino terminal helices
followed by variable numbers of an approximately 20 amino acid
leucine-rich repeat (LRR) motif (12-20 LRR among different Yersinia
strains), forming a horseshoe-shaped protein. The LRR, which have
been implicated in protein-protein interactions, make up most of
YopM (FIG. 1).
[0004] Besides protein-protein interactions with serum proteins
such as .alpha.-thrombin and .alpha.1-antitrypsin (Hines et al.,
2000; Heusipp et al., 2006) and a scaffolding function of YopM with
two cytoplasmic kinases, RSK1 and PRK2 (McDonald et al., 2003) the
molecular function of YopM during infection is only poorly
understood.
[0005] The fact that yopM mutants of Y. enterocolitica and Y.
pestis cannot establish a systemic infection in infected mice
(Trulzsch et al., 2004; Kerschen et al., 2004) indicates that YopM
is important for full virulence and resistance to innate immunity
during infection (Leung et al., 1990). Interestingly, Kerschen et
al. showed that YopM of Y. pestis caused a global depletion of
natural killer (NK) cells (Kerschen et al., 2004). In association
with this NK cell depletion, the spleen/liver tissues of infected
mice and in the same manner the macrophages/NK cells isolated from
those tissues showed a decreased expression of several
proinflammatory cytokines including TNF-.alpha., IFN-.gamma.,
IL-1.beta., IL-12, IL-15, IL-15R.alpha. and IL-18.
[0006] How YopM interferes with the signalling pathways of the
proinflammatory cytokines is just speculative and has not been
investigated. Specifically, it remains elusive how YopM as a
locally delivered, bacterial host-cell-contact dependently
translocated protein can have a global effect on the host response
(Kerschen et al., 2004).
[0007] According to a model of Y. enterocolitica infection, YopM is
translocated through the T3SS into the host cell cytoplasm.
However, other studies also suggest an additional extracellular
role of YopM, like binding to the acute-phase protein
.alpha.1-antitrypsin, and binding of YopM to serum protein
.alpha.-thrombin which ist supported by a humoral immune response
to YopM after infection of mice (Benner et al., 1999, Heusipp et
al., 2006; Huck et al. 1998). Furthermore, an apolar secretion (7%)
of YopM during in vitro infection has been described by Cheng and
Schneewind (Cheng & Schneewind, 2000).
[0008] Patent Application having publication number WO2009/115531
reports an additional delivery function of YopM which is
independent from the direct delivery of the effector protein by
T3SS into the cytoplasm of host cells. In particular, WO2009/115531
discloses Yersinia outer protein M (YopM), a YopM fragment or a
YopM variant which are capable of auto penetrating the cell
membrane and integrating into the cell cytosol without the
requirement of additional factors. The use of this function for
delivering a cargo molecule across the membrane to the cytosol of a
cell is described therein. WO2009/115531 further discloses that
once YopM is integrated into the cell cytosol, YopM is capable of
effectively downregulating cytokines, in particularly
pro-inflammatory cytokines.
[0009] Psoriasis is one of the most common immune-mediated chronic
inflammatory skin disorders affecting approximately 2-3% of the
population. It is considered as an incurable, life-long skin
condition that affects all races, age groups and both sexes. The
pathogenesis of psoriasis is complex and not clearly understood.
Ongoing research indicates that immune T-cells appear to play a
dominant role for the development of this disease. These cells
release TNF-.alpha., IFN-.alpha. and other pro-inflammatory
cytokines that lead to vasodilation, leukocyte migration, and
activation of keratinocytes. This further activates dendritic cells
which in turn leads to inflammation.
[0010] Psoriasis appears in a variety of forms with distinct
characteristics. Typically, an individual has only one type of
psoriasis at a time.
[0011] Plaque psoriasis (psoriasis vulgaris) is the most prevalent
form of the disease. About 80 percent of those who have psoriasis
have this type. It is characterized by raised, inflamed, red
lesions covered by a silvery white scale. It is typically found on
the elbows, knees, scalp and lower back.
[0012] Guttate psoriasis is a form of psoriasis that often starts
in childhood or young adulthood. This form of psoriasis appears as
small, red, individual spots on the skin. Guttate lesions usually
appear on the trunk and limbs. These spots are not usually as thick
as plaque lesions.
[0013] Guttate psoriasis often comes on quite suddenly. A variety
of conditions can bring on an attack of guttate psoriasis,
including upper respiratory infections, streptococcal throat
infections (strep throat), tonsillitis, stress and injury to the
skin.
[0014] Inverse psoriasis is found in the armpits, groin, under the
breasts, and in other skin folds around the genitals and the
buttocks. This type of psoriasis appears as bright-red lesions that
are smooth and shiny. Inverse psoriasis is subject to irritation
from rubbing and sweating because of its location in skin folds and
tender areas. It can be more troublesome in overweight people and
those with deep skin folds.
[0015] Primarily seen in adults, pustular psoriasis is
characterized by white blisters of noninfectious pus (consisting of
white blood cells) surrounded by red skin. It may be localized to
certain areas of the body, such as the hands and feet, or covering
most of the body. It begins with the reddening of the skin followed
by formation of pustules and scaling.
[0016] Erythrodermic psoriasis is a particularly inflammatory form
of psoriasis that affects most of the body surface. The reddening
and shedding of the skin are often accompanied by severe itching
and pain, heart rate increase, and fluctuating body
temperature.
[0017] Erythrodermic psoriasis causes protein and fluid loss that
can lead to severe illness. The condition may also bring on
infection, pneumonia and congestive heart failure.
[0018] Several drugs have been used in the treatment of psoriasis.
The four most commonly prescribed systemic medications to treat
psoriasis are Methotrexate, Ciclosporin, Acitretin and
Hydroxycarbamide. Methotrexate, Ciclosporin and Hydroxycarbamide
all work by suppressing the immune system, whereas Acitretin is a
Vitamin A derived treatment.
[0019] A common disadvantage (besides their side effects) of all
drugs currently available for treating this primarily cutaneous
disease is that they are only effective when given systemically but
are largely ineffective when applied topically because of poor
absorption.
[0020] Systemic medications are prescription medications that
affect the entire body. The systemic treatments that are available
to treat psoriasis and psoriatic arthritis are associated with
significant short- and long-term side effects. The benefits of
psoriasis clearance or improvement must be balanced against the
risk of these side effects.
[0021] There is therefore the need of an effective therapeutic
agent useful in the prevention and/or treatment of psoriasis having
a good patient compliance and reduced side effects.
[0022] It must be noted that as used herein, the singular forms
"a", "an", and "the", include plural references unless the context
clearly indicates otherwise. Thus, for example, reference to "a
reagent" includes one or more of such different reagents and
reference to "the method" includes reference to equivalent steps
and methods known to those of ordinary skill in the art that could
be modified or substituted for the methods described herein. Unless
otherwise indicated, the term "at least" preceding a series of
elements is to be understood to refer to every element in the
series. At least one includes for example, one, two, three, four,
or five or even more. Those skilled in the art will recognize, or
be able to ascertain using no more than routine experimentation,
many equivalents to the specific embodiments of the invention
described herein. Such equivalents are intended to be encompassed
by the present invention. Throughout this specification and the
claims which follow, unless the context requires otherwise, the
word "comprise", and variations such as "comprises" and
"comprising", will be understood to imply the inclusion of a stated
integer or step or group of integers or steps but not the exclusion
of any other integer or step or group of integer or step.
[0023] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, etc.), whether supra
or infra, are hereby incorporated by reference in their entirety.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
4. DESCRIPTION OF THE INVENTION
[0024] The present inventors have surprisingly found that Yersinia
outer protein M (YopM) when administered via cutaneous, intradermal
or subcutaneous route prevents and/or treats psoriasis. Even more
surprisingly, it has been found by the present inventors that the
cutaneous, intradermal, or subcutaneous routes of administration
provide an improved method of prevention and/or treatment of
psoriasis when compared to the intra-peritoneal route.
Interestingly, in contrast to sub-cutaneous treatment,
epicutaneously applied YopM did not enter systemic distribution,
mainly remained in the epidermis, dermis, and subcutis, and
couldn't be found in local lymph nodes. As sub-cutaneous injected
YopM was also found in local lymph nodes, an epicutaneous treatment
with YopM is clearly advantageous and favoured to avoid unwanted
side effects. The cutaneous administration route is therefore the
preferred administration route of the present invention.
[0025] It has been found that Yersinia outer protein M (YopM) when
administered via cutaneous, intradermal or subcutaneous route is
able to treat psoriasis in a patient affected by this disease. A
"patient" thereby includes mammals like for example mouse, rat,
cat, dog, apes and humans, humans being preferred.
[0026] In particular, the Yersinia outer protein M (YopM) is able
to treat plaque psoriasis, guttate psoriasis, inverse psoriasis,
pustular psoriasis, erythrodermic psoriasis, arthritis
psoriasis.
[0027] In one aspect of the invention, Yersinia outer protein M
(YopM) is selected from YopM of the species Yersinia
enterocolitica, Yersinia pseudotuberculosis or Yersinia pestis.
[0028] YopM is administered in a dose effective to prevent and/or
treat psoriasis. The dose for cutaneous administration ranges from
1 .mu.g/cm.sup.2 to 20 mg/cm.sup.2, preferably 1 .mu.g/cm.sup.2 to
20 .mu.g/cm.sup.2, more preferably about 2 .mu.g/cm.sup.2.
[0029] The term "Yersinia outer protein M (YopM)" according to the
invention relates to a Yersinia outer protein M. The term includes
the Yersinia outer protein M full-length amino acid sequence, YopM
fragments and YopM variants thereof. The term includes a Yersinia
outer protein M as described in Boland A, et al. EMBO J. 1996 Oct.
1; 15 (19):5191-201; Cornelis GR. J. Bacterid. 1998 November; 180
(21):5495-504; Skrzypek, E., Cowan, C. and Straley, S. C. (1998)
Mol. Microbiol. 30: 1051-1065; McDonald, C, Vacratis, P. O.,
Bliska, J. B. and Dixon, J. E. (2003) J. Biol. Chem 278:
18514-18523; Skrzypek E, Myers-Morales T, Whiteheart S W, Straley S
C. Infect. Immun. 2003 February; 71 (2):937-47; Kerschen, E. J.,
Cohen, D. A., Kaplan, A. M. and Stranley, S. C. (2004) Infect.
Immun. 72: 4589-4602; Heusipp, G., Spekker, K., Brast, S., Falker,
S. and Schmidt, M. A. (2006) Microbiol. 152: 1327-1335; Kloss E,
Barrick D. Protein Sci. 2009 September; 18 (9):1948-60; McCoy et
al. Infect Immun. 2010 June; 78 (6):2584-98; McPhee J B et al.
Infect Immun. 2010 August; 78 (8):3529-39; Ruter C et al. J Cell
Sci. 2010 Jul. 1; 123(Pt 13):2190-8; Hentschke Met al. PLoS One.
2010 Oct. 5; 5 (10). pii: e13165 and Vieux E F et al. Biophys Chem.
2011 November; 159 (1):152-61; or derivable from any biological
database known to the person skilled in the art, e.g. from the
Genbank database.
[0030] In a preferred embodiment, Yersinia outer protein M (YopM)
is a Yersinia outer protein M of a Yersina strain naturally
comprising a YopM encoding virulence plasmid. The term "YopM
encoding virulence plasmid" relates to plasmid pYV or pCD1 as
described to be present, for instance, in Yersinia enterocolitica,
Yersina pseudotuberculosis and Yersinia pestis (Cornelis et al.,
Microbiol. Mol. Biol. Rev. 62:1315-1352 (1998)).
[0031] In a further preferred embodiment, the Yersinia outer
protein M (YopM) is a Yersinia outer protein M selected from the
species Yersinia enterolitica, Yersinia pseudotuberculosis and
Yersinia pestis. More preferably, Yersinia outer protein M (YopM)
is a Yersinia outer protein M selected from Yersinia enterolitica
8081 v, serotype O:8.
[0032] In a further embodiment of the invention, a YopM fragment is
used in the prevention and treatment of psoriasis. In the context
of the present invention, the term "YopM fragment" means that the
fragment is a therapeutically active fragment of the YopM full
length protein. The term "therapeutically active" means that the
fragment has the above mentioned therapeutically activities of YopM
full length amino acid sequence protein, i.e. a fragment of the
invention has the capability of preventing and/or treating
psoriasis. Hence a "YopM fragment" according to the present
invention is a YopM fragment that retains the property of the full
length YopM protein to preventing and/or treating psoriasis. In
particular, a YopM fragment of the invention is able to treating
psoriasis in the mouse model used in Example paragraph 5.2 below.
In a particular embodiment, the fragment retains or comprises or
consists of the "immunomodulatory domain" of the YopM full length
protein or the fragment retains or comprises or consists of a part
of the "immunomodulatory domain" of the YopM full length protein.
An "immunomodulatory domain of YopM" comprises at least one
leucine-rich repeat (LRR) of YopM, i.e. one, two, three, four,
five, six, seven or eight LRRs up to 20 LRRs. In an embodiment, the
YopM fragment may also retain the property of YopM full length
protein to allow translocation into the cytosol and/or to and/or
into the nucleus of a target cell. Translocation of a YopM fragment
into the cytosol and/or nucleus of the target cell can be permitted
if the fragment of YopM retains the domain or part of the domain of
the YopM full length protein which permits translocation.
Alternatively, the translocation function can be performed by a
"cell penetrating peptide" (CCP) which is heterologous to Yersinia.
Preferably, the YopM fragment of the invention is able to
distribute in the cells of the epidermis and/or dermis and/or
hypodermis, and/or is able to distribute in the cells present in
the epidermis and/or dermis and/or hypodermis. In particular, the
YopM fragment of the invention is able to distribute in at least
one kind amongst the cells of the epidermis and/or dermis and/or
hypodermis, and/or is able to distribute in at least one kind
amongst the cells present in the epidermis and/or dermis and/or
hypodermis, wherein the term "distribute" means that the YopM
fragment of the invention is able to enter in the cytosol of a cell
and optionally to reach and/or optionally to enter the nucleus of
said cell. The cells of the epidermis, dermis and/or hypodermis
and/or the target cells for the prevention and/or treatment of
psoriasis are for examples melanocytes, Langerhans' cells, Merkel's
cells, keratinocytes, T helper cells, T suppressor cells, T killer
cells, T memory cells, B cells, macrophages, mast cells or any
other cells of the local immune system. Hence, preferably the YopM
fragment of the invention is able to distribute in at least one of
the target cells of the epidermis and/or dermis and/or hypodermis,
and/or is able to distribute in at least one of the target cells
present in the epidermis and/or dermis and/or hypodermis to prevent
and/or treat psoriasis. In addition, the YopM fragment of the
invention is able to down-regulate at least one cytokine. A list of
cytokines is disclosed below. In particular, the fragment of the
invention is able to down-regulating TNF.alpha.. The "YopM
fragment" according to the invention is a fragment of the
corresponding YopM full length protein of at least 100 aminoacids,
more preferably is of at least 150 aminoacids, more preferable is
of at least 150-300 aminoacids.
[0033] In a further embodiment of the invention, a YopM variant is
used in the prevention and/or treatment of psoriasis. In the
context of the present invention the term "YopM variant" includes
that the variant is a therapeutically active full length YopM
protein variant or a fragment thereof, preferably the term means
that the variant has the therapeutic activities of full length YopM
protein or fragment thereof. It is envisaged that a variant of the
invention has the capability of preventing and/or treating
psoriasis. Hence a "YopM variant" according to the present
invention is a YopM variant that retains the property of the full
length YopM protein of preventing and/or treating psoriasis. In a
particular embodiment, this variant retains or comprises or
consists of the "immunomodulatory domain" of YopM full length
protein or the variant retains or comprises or consists of part of
the "immune-modulatory domain" of the YopM full length protein. An
"immunomodulatory domain of YopM" comprises at least one
leucine-rich repeat (LRR) of YopM, i.e. one, two, three, four,
five, six, seven or eight LRRs up to 20 LRRs. The addition of
further LRRs is also envisaged. Variants and fragments of said
immunomodulatory domain are likewise envisaged. In a particular
embodiment, the YopM variant may also retain the property of full
length YopM protein to allow translocation into the cytosol and/or
nucleus of a target cell. Translocation of a YopM variant into the
cytosol of the target cell can be permitted if the variant of YopM
retains the property of the protein which permits translocation.
Alternatively, the translocation function can be performed by a
"cell penetrating peptide" (CCP) which is heterologous to Yersinia.
Such variants may include deletions, insertions, inversions,
repeats, and substitutions selected according to general rules
known in the art so as to have little effect on activity. For
example, guidance concerning how to make phenotypically silent
amino acid substitutions is provided in Bowie et al., (Science 247:
1306-1310 (1990)), wherein the authors indicate that there are two
main strategies for studying the tolerance of an amino acid
sequence to change. Preferably, the YopM variant of the invention
is able to distribute in the cells of the epidermis and/or dermis
and/or hypodermis, and/or is able to distribute in the cell present
in the epidermis and/or dermis and/or hypodermis. In particular,
the YopM variant of the invention is able to distribute in at least
one kind amongst the cells of the epidermis and/or dermis and/or
hypodermis, and/or is able to distribute in the cells present in
the epidermis and/or dermis and or hypodermis, wherein the term
"distribute" means that the YopM variant of the invention is able
to enter in the cytosol of a cell and optionally to reach and/or
optionally to enter the nucleus of said cell. The cells of the
epidermis, dermis and/or hypodermis are for examples melanocyte,
Langerhans' cell, Merkel's cell, keratinocytes, T helper cells, T
suppressor cells, T killer cells, T memory cells, B cells,
macrophages, mast cells or any other cells of the local immune
system. Hence, the YopM variant of the invention is able to
distribute in at least one of the target cells of the epidermis
and/or dermis and/or hypodermis, and/or is able to distribute in at
least one of the target cell present in the epidermis and/or dermis
and/or hypodermis to prevent and/or treat psoriasis. In addition,
the YopM variant of the invention is able to down-regulate at least
one cytokine. A list of cytokines is disclosed below. In
particular, the variant of the invention is able to down-regulate
TNF.alpha.. The term "YopM variant" includes the "YopM variant" as
defined in patent application WO2009/115531 which is incorporated
herein by reference.
[0034] The invention also encompasses YopM polypeptides having a
lower degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the YopM
protein, fragment and variant as described herein above.
[0035] Thus, in a further embodiment the full length YopM, YopM
fragment, or YopM variant as described herein, is capable of
autopenetrating the cell membrane and of integrating into the cell
cytosol without the requirement of additional factors and is
capable of downregulating cytokines i.e. the compounds of the
invention comprise in this embodiment the immunomodulatory
domain(s) of YopM, particularly at least one leucine-rich repeat
(LRR), i.e. one, two, three, four, five, six, seven or eight LRRs
up to 20 LRRs. The addition of further LRRs is also envisaged.
[0036] The term "cytokines" relates to soluble proteins and
peptides that act as humoral regulators, which, either under normal
or pathological conditions, modulate the functional activities of
individual cells and tissues and also mediate interactions between
cells directly and regulate processes taking place in the
extracellular environment. The term encompasses type 1 cytokines
produced by Th1 T-helper cells, type 2 cytokines produced by Th2
T-helper cells, interleukins, chemokines or interferons, e.g. IL-1
ra (antagonist), CNTF, LIF, OSM, Epo, G-CSF, GH, PRL, IP10, I309,
IFN-alpha, IFN-beta, IFN-gamma, IL2, IL3, IL4, IL5, IL6, IL7, IL8,
IL9, IL10, IL11, IL12 (p35+p40), IL13, IL14, IL15, IL16, IL17 A-F,
IL18, IL19, IL20, IL21, IL22, IL23 (p19+p40), IL24, IL25, IL26,
IL27 (p28-EBI3), IL28A, IL28B, IL29, IL30, IL31, IL32, IL33, IL35
(p35-EBI3), LT-alpha, LT-beta, LIGHT, TWEAK, APRIL, BAFF, TL1A,
GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1 BBL, TRAIL, RANK,
GM-CSF, M-CSF, SCF, IL1-alpha, IL1-beta, aFGF, bFGF, int-2, KGF,
EGF, TGF-alpha, TGF-beta, TNF-alpha, TNF-beta, betacellulin, SCDGF,
amphiregulin or HB-EGF, as is known to the person skilled in the
art and can be derived, for example, from Tato, CM. & Cua, D.
J. (Cell 132: 900; Cell 132: 500, Cell 132: 324, (2008)) or from
Cytokines & Cells Online Pathfinder Encyclopaedia.
"Pro-inflammatory cytokines" are also contemplated. The term
"proinflammatory cytokine" means an immunoregulatory cytokines that
favours inflammation. Typically, pro-inflammatory cytokines
comprise IL-1 -alpha, IL-1-beta, IL-6, and TNF-alpha. These
pro-inflammatory cytokines are largely responsible for early
responses. Other pro-inflammatory mediators include LIF, IFN-gamma,
IFN-alpha, OSM, CNTF, TGF-beta, GM-CSF, TWEAK, IL-11, IL-12, IL-15,
IL-17, IL-18, IL-19, IL-20, IL-8, IL-16, IL-22, IL-23, IL-31, and
IL-32 (Tato, C M. & Cua, D J. Cell 132:900; Cell 132:500, Cell
132, 324 (2008)). These pro-inflammatory cytokines may act as
endogenous pyrogens (IL-1, IL-6, TNF-alpha), up-regulate the
synthesis of secondary mediators and pro-inflammatory cytokines by
both macrophages and mesenchymal cells (including fibroblasts,
epithelial and endothelial cells), stimulate the production of
acute phase proteins, or attract inflammatory cells. Preferably,
the term "pro-inflammatory cytokine" relates to TNF-alpha, IL-15,
IFN-gamma, IFN-alpha, IL-1-beta, IL-8, IL-16 and IL-22.
[0037] The term "downregulates" or "downregulating" means that the
mRNA levels of an expressed gene, e.g. of a cytokine gene, and/or
the protein levels expressed by such mRNAs is reduced in the
presence of YopM as described herein. The downregulation of mRNA
and/or protein expressed by that mRNA in the context of the YopM of
the invention can be tested and determined by methods known to a
person skilled in the art.
[0038] In the context of the invention the term "cutaneous
administration" sometimes also referred to as "epicutaneous
administration" (e.c.) means the application of suitable drug
dosage forms directly onto the skin for either local (topical) or
systemic effects. According to the present invention, cutaneous
administration is preferred. Cutaneous topical administration is
likewise preferred. The cutaneous administration is preferably
repeated in constant intervals such as for example every 12 hours,
i.e. after 12 hours, 24 hours, 36 hours, 48 hours, 60 hours etc. In
a preferred embodiment the cutaneous administration is repeated
every 24 hours (i.e. daily administration).
[0039] Any pharmaceutical composition suitable for cutaneous
administration is encompassed by the present invention. Suitable
pharmaceutical compositions are e.g., cream, lotion, gel, paste,
ointment, patch, spray, solution. Creams are semi-solid emulsions,
that is mixtures of oil and water. They are divided into two types:
oil-in-water (O/W) creams which are composed of small droplets of
oil dispersed in a continuous phase, and water-in-oil (W/O) creams
which are composed of small droplets of water dispersed in a
continuous oily phase--both types are specifically included into
the scope of the present invention. Creams, lotions, gels and
ointments are preferred. The aforementioned pharmaceutical
compositions comprise in a preferred embodiment cetearyl alcohol,
paraffinum liquidum, glycerol, dimethicone, sodium cetearyl
sulfate, phenoxyethanol, methylparaben, ethylparaben,
propylparaben, and sodium hydroxide.
[0040] Any pharmaceutical composition suitable for sub-cutaneous
administration is encompassed by the present invention. Suitable
pharmaceutical compositions are e.g. solutions for subcutaneous
injection. The solution may be administered for example in a
pre-filled, multi-dose pen that delivers the effective dose of the
YopM of the invention.
[0041] Any pharmaceutical composition suitable for intradermal
administration is encompassed by the present invention.
Pharmaceutical compositions suitable for intradermal administration
are known in the art.
[0042] Further non-limiting examples of dosage forms include
dispersions, liquid dosage forms suitable for cutaneous or
intradermal or subcutaneous administration such as suspensions
(e.g. aqueous or non-aqueous liquid suspensions, oil-in-water
emulsions or a water-in-oil liquid emulsion), solutions, liquid
dosage forms suitable for intradermal or subcutaneous
administration to a patient.
[0043] In a further embodiment of the invention, the active
ingredient of the invention may be delivered by a vehicle such as
vesicles, biopolymers, liposomes, nanoparticles, microspheres,
micelles, or virus incorporated. Biodegradable needles can also be
used for the delivery of the YopM active ingredient via intradermal
or subcutaneous administration.
[0044] The pharmaceutical composition can be used in the
preparation of individual, single unit dosage forms. Pharmaceutical
compositions and dosage forms of the invention can further comprise
one or more carriers, excipients or diluents.
[0045] The composition, shape and type of dosage forms of the
invention will typically vary depending on their use. For example,
a dosage form used in the acute treatment of psoriasis disease may
contain larger amounts of the active ingredients than a dosage form
used in the chronic treatment of the same disease.
[0046] The pharmaceutical formulations can also be delayed release
or controlled release formulations. YopM of the invention can be
administered by delayed or controlled release means or by delivery
devices that are well known to those of ordinary skill in the
art.
[0047] This disclosure may best be understood in conjunction with
the accompanying figures, incorporated herein by references.
Furthermore, a better understanding of the present invention and of
its many advantages can be derived from the following examples,
which are given by way of illustration and are not intended as
limiting the disclosure.
5. FIGURES
[0048] FIG. 1: discloses the domain organization and functional
regions of YopM. Type-III secretion (S) and translocation (T)
signal required by the T3SS [N-terminal residues aa 34-40 (S) &
aa 40-100 (T), (Ghosh 2004)]. NLS, nuclear localization signal.
NLS-I: three N-terminal LRRs and NLS-II: 32 C-terminal residues of
YopM. Interaction with serum proteins .alpha.-thrombin and
.alpha.1-antitrypsin seems to depend on conserved residues in LRR
domains and not on specific domains of the LRR-containing region of
the protein. The figure describes information gained from analyses
of YopM from all three pathogenic Yersinia species.
[0049] FIG. 2: refers to the purification of recombinant YopM. FIG.
2a) is the SDS-PAGE analysis following isolation and purification
of YopM. Proteins were separated on a 12.5% SDS polyacrylamide gel
and subsequently silver stained. lane M: "See Blue Plus2" protein
ladder, RE: crude extract, W: wash I, lanes E2-5: elution fractions
2-5. FIG. 2b) shows the LPS content in YopM elution fractions by
Limulus amebocyte lysate Toxin sensor assay.
[0050] FIG. 3: shows the effect of YopM on an imiquimod
(IMQ)-induced psoriasis mouse model. Daily topical application of
imiquimod (5% Aldara Creme Sachets b-f), a TLR7/8 ligand and potent
immune activator, on mouse back skin (Balb/c) induces and
exacerbates psoriasis FIG. 3b). FIG. 3c) refers to the results
obtained on mice after a i.p. 100 .mu.g of YopM in PBS every 48 h,
FIG. 3e) shows the results obtained on mice treated with an
YopM-containing cream (mice balmed with 200 mg cream containing
approx. 30 .mu.g of YopM); FIG. 3f) shows the results obtained on
mice treated with YopM by subcutaneous injection (s.c. 25 .mu.g in
PBS every 48 h). FIG. 3a) is the negative control (untreated mice)
and FIG. 4d) is the placebo control (200 mg cream without
YopM).
[0051] FIG. 4: refers to the histological analyses of psoriatic and
non-psoriatic epidermis of back-skin tissue sections (5 .mu.m)
stained using a PAN Cytokeratin Antibody (green) to determine the
beneficial effects of YopM on epidermal proliferation, and abnormal
differentiation. The nuclei were stained with Draq5 (blue). FIG.
4a) refers to the negative control; FIG. 4b) to the IMQ-positive
control; FIG. 4c) to the YopM intraperitoneally injected; FIG. 4d)
to the placebo, FIG. 4e) to the YopM balmed, and FIG. 4f) to the
YopM subcutaneously injected. Pictures were taken with confocal
laser scanning microscope. Magnification 40.times..
[0052] FIG. 5: shows the histological analyses of psoriatic and non
psoriatic epidermis of back-skin tissue sections (5 .mu.m) stained
using CD4+ (green) and IL17 (red) antibodies to determine the
amount of epidermal infiltrates consisting of CD4+ T cells induced
by IMQ, the epidermis of back-skin tissue sections (5 .mu.m) was
stained using CD4+ (green) and IL17 (red) antibodies. The nuclei
were stained with Draq5 (blue). FIG. 5a) refers to negative
control; FIG. 5b) to the IMQ-positive control; FIG. 5c) refers to
the YopM intra-peritoneally injected; FIG. 5d) refers to the YopM
balmed. Pictures were taken with confocal laser scanning
microscope. Magnification 40.times..
[0053] FIG. 6: Distribution of YopM in the Epidermis and Dermis
after 48 h of application. YopM specific immunostaining (green) of
skin tissue samples. YopM balmed: Epidermis (a), Dermis (b); YopM
i.p. injected: Epidermis (c), Dermis (d); YopM s.c. injected:
Epidermis (e), Dermis (f); negative control: Epidermis (g), Dermis
(h). Actin was stained with Texas Red Phalloidin (red). DNA was
stained with Draq5 (blue). Pictures were taken with confocal laser
scanning microscope. Magnification 40.times..
[0054] FIG. 7: Distribution of YopM in the epidermis and dermis
after 48 h of application. YopM specific immunostaining (green) of
skin tissue samples. untreated IMQ-skin: epidermis (a), dermis (d),
subcutis (g), and local lymph nodes (j). YopM applied
epicutaneously: epidermis (b), dermis (e), subcutis (h), and local
lymph nodes (k).YopM subcutaneously injected: epidermis (c), dermis
(f), subcutis (i), and local lymph nodes (I). Actin was stained
with Texas Red Phalloidin (red). DNA was stained with Draq5 (blue).
Pictures were taken with a confocal laser scanning microscope.
Magnification 40.times..
[0055] FIG. 8: Effect of YopM on papillomatosis. IMQ treatment
induces regular and symmetrical extensions of rete ridges,
separated by elongated dermal papillae (papillomatosis). H&E
staining of the back skin of mice after different treatment
regimes. A line indicates regular and symmetrical extensions of
rete ridges and the elongated dermal papillae. (a) healthy skin;
(b) untreated IMQ-skin; (c) placebo control; (d) YopM applied
epicutaneously; (e) 12.5 pg YopM subcutaneously injected; (f) (e)
100 .mu.g YopM subcutaneously injected. Pictures were taken with a
light microscope. Magnification 40.times..
6. EXAMPLES
[0056] To investigate whether YopM might be functional as an
immune-modulator for the treatment of psoriasis, the murine
imiquimod (IMQ)-induced psoriasis model has been utilized. In this
model, daily topical application of imiquimod (IMQ), a TLR7/8
ligand and potent immune activator, on shaven mouse back skin
induces and exacerbates a psoriasis-like condition (Van der Fits L,
et al. (2009) Imiquimod-induced psoriasis-like skin inflammation in
mice is mediated via the IL-23/IL-17 axis. J. Immunol. 182:
5836-5845). Therefore, this model might serve for the analysis of
pathogenic mechanisms in psoriasis-like dermatitis. Nestle and
Nickoloff (Nestle, F. O., and B. J. Nickoloff. (2006) Animal models
of psoriasis: a brief update. J. Eur. Acad. Dermatol. Venereol.
20(Suppl. 2): 24-27) defined several criteria for an ideal
psoriasis model: 1) epidermal changes based on keratinocyte
hyperproliferation and altered differentiation; 2) papillomatosis;
3) presence of inflammatory cells including T cells, DC, and
neutrophils; 4) a functional role for T cells; 5) and altered
vascularity. As the Imiquimod-induced psoriasis like skin model
fulfils these criteria, it might serve for the analysis of
pathogenic mechanisms in psoriasis-like dermatitis (Van der Fits L,
et al. (2009) Imiquimod-induced psoriasis-like skin inflammation in
mice is mediated via the IL-23/IL-17 axis. J. Immunol. 182:
5836-5845). For this, we applied YopM either epicutaneously (e.c.)
as a component of a specific cream (e.c.) or by subcutaneous (s.c.)
injection (every 48 h) over a period of two weeks to groups of mice
suffering from Imiquimod-induced psoriasis. Furthermore, an
additional group of psoriasis-mice was treated with YopM by
intraperitoneally injection (i.p.).
1.1 Expression and Purification of Recombinant YopM in E. Coli
BL21
[0057] For the expression of recombinant YopM (rYopM), the
pET24b(+) expression vector which also provides the coding sequence
for a C-terminal 6.times. His-tag has been chosen. Protein
purification was performed by nickel-nitrilotriacetic acid (Ni-NTA)
metal-affinity chromatography under native conditions. After
purification, fractions of highest purity were pooled and dialyzed
in PBS or H.sub.2O bidest. The protein solution was applied to a
dialysis tube (pore size: 6-10 kDa) and dialyzed overnight in 2 l
PBS or H.sub.2O bidest. at 4.degree. C. with gentle stirring. After
dialysis, the protein was concentrated using centrifugal filters at
500.times.g and 4.degree. C. The expressed and purified recombinant
proteins were quantified using the bicinchoninic acid (BCA) assay
(Smith et al., 1985). Successful purification was confirmed by
SDS-PAGE analysis. Proteins were separated on a 12.5% SDS
polyacrylamide gel and subsequently silver stained (exemplified in
FIG. 1a). Furthermore, the LPS content in elution fractions of
recombinant YopM was determined by Limulus amebocyte lysate Toxin
sensor assay (exemplified in FIG. 1b, approx. 0.33 EU/mg).
1.2 Effects of YopM on an Imiquimod (IMQ)-Induced Murine Psoriasis
Model
[0058] The impact of rYopM applied topically (balmed and s.c.) and
i.p. on inflammation linked to psoriasis-like conditions induced by
imiquimod (FIG. 2) was investigated.
[0059] Daily epicutaneous (e.c.), topical application of imiquimod
(IMQ) (5% Aldara Creme Sachets), a TLR7/8 ligand and potent immune
activator, on mouse back skin (Balb/c) induces and exacerbates
psoriasis. After induction of psoriatic skin by IMQ, injections
(i.p. and s.c.) of YopM (in PBS) into IMQ-treated Balb/c mice were
performed every 48 h. Furthermore, YopM (in H.sub.2O bidest.) was
emulsified in a cream (150 .mu.g YopM per 1 g cream). Said cream
contained cetearyl alcohol, paraffinum liquidum, glycerol,
dimethicone,sodium cetearyl sulfate, phenoxyethanol, methylparaben,
ethylparaben, propylparaben,and sodium hydroxide. Mice were balmed
with 200 mg YopM-containing cream (approx. 30 .mu.g of YopM) every
48 h.
[0060] In order to determine the beneficial effects of YopM on
epidermal proliferation, abnormal differentiation, epidermal
infiltrates consisting of CD4+ T cells, and papillomatosis induced
by IMQ, we visually inspected the development of the psoriasis-like
skin conditions. Pictures of the back skin of mice were taken after
a period of two weeks (FIG. 3).
[0061] Further, immunohistochemistry staining on cryosections of
biopsy taken from back skin of treated mice and employing
antibodies against YopM (FIG. 6) has been performed. In particular,
the distribution of YopM in the epidermis and dermis after 48 h of
application has been determined. YopM balmed: Epidermis (a), Dermis
(b); YopM i.p. injected: Epidermis (c), Dermis (d); YopM s.c.
injected: Epidermis (e), Dermis (f); negative control: Epidermis
(g), Dermis (h). Actin was stained with Texas Red Phalloidin (red).
DNA was stained with Draq5 (blue).
[0062] As further histological analyses of psoriatic and non
psoriatic epidermis of back-skin tissue skin tissues was performed
using a PAN Cytokeratin Antibody (green) in order to identify
keratinocytes and to determine the beneficial effects of YopM on
epidermal proliferation, and abnormal differentiation (FIG. 4). The
epidermis of back-skin tissue sections (5 .mu.m) was stained using
a PAN Cytokeratin Antibody (green). The nuclei were stained with
Draq5 (blue). FIG. 4a) is the negative control; FIG. 4b) is the
IMQ-positive control; FIG. 4c) is the YopM intraperitoneally
injected; FIG. 4d) refers to the placebo, FIG. 4e) is YopM balmed,
and FIG. 4f) refers to the YopM subcutaneously injected.
[0063] A further additional histological analysis of psoriatic and
non psoriatic epidermis of back-skin tissue sections of the treated
mice was performed in order to determine the amount of epidermal
infiltrates consisting of CD4+ T cells induced by IMQ (FIG. 5). The
epidermis of back-skin tissue sections (5 .mu.m) was stained using
CD4+ (green) and IL17 (red) antibodies. The nuclei were stained
with Draq5 (blue). FIG. 5a) is the negative control; FIG. 5b)
refers to IMQ-positive control; FIG. 5c) refers to the YopM
intraperitoneally injected and FIG. 5d) to the YopM balmed.
[0064] The results confirm the "self-delivering" abilities of YopM
across the cutaneous barrier when applied epicutaneously (e.c.) as
a cream (FIG. 6a), b)), and already indicated a remarkable
dampening of overt inflammatory reactions (FIG. 3e), 4e), 5d)).
Repression of disease symptoms was also found in the group of mice
subcutaneously injected with YopM (FIGS. 3f), 4f), 6e) and 6f)),
whereas no significant phenotypic changes were observed in
intraperitoneally injected mice (FIGS.; 3c), 4c), 5c), 6c) and
6d)). Interestingly, in contrast to s.c. treated mice,
epicutaneously applied YopM did not enter systemic distribution,
mainly remained in the epidermis, dermis, and subcutis, and
couldn't be found in local lymph nodes. As s.c. injected YopM was
also found in local lymph nodes, an epicutaneous treatment with
YopM is clearly advantageous and favoured to avoid unwanted side
effects.
[0065] These results clearly demonstrate a superior effect of the
YopM-containing formulation or direct application of rYopM vs.
control in cutaneous (e.c. and s.c.) vs. an i.p. application in
reducing psoriasis-like conditions in a murine psoriasis model.
Subsequent to cutaneous YopM application in the murine psoriasis
model we observed nearly no detrimental epidermal changes based on
keratinocyte hyperproliferation, lack of papillomatosis, and
depletion of inflammatory T cells.
* * * * *