WO2013041247A1

WO2013041247A1 - Yersinia outer protein m (yopm) in the treatment of psoriasis induced by imiquimod

Info

Publication number: WO2013041247A1
Application number: PCT/EP2012/053224
Authority: WO
Inventors: Christian Rueter; Alexander Schmidt
Original assignee: Westfällsche Wilhelms-Universitaet Muenster
Priority date: 2011-09-23
Filing date: 2012-02-24
Publication date: 2013-03-28

Abstract

The present invention relates to the use of Yersinia outer protein M (YopM) in the prevention and/or treatment by cutaneous, intradermalor subcutaneous administration,of psoriasis induced by imiquimod or pharmaceutically acceptable saltsthereof or imiquimod derivatives. It further relates to pharmaceutical compositionscomprising YopM and imiquimod and a kit comprising apharmaceutical compositioncontaining YopM and a pharmaceutical composition containing imiquimod.

Description

Yersinia outer protein M (YopM) in the treatment of psoriasis induced by imiquimod

1. Field of the invention

The present invention relates to the use of Yersinia outer protein M (YopM) in the prevention and/or treatment by cutaneous, intradermal or subcutaneous administration, of psoriasis induced by imiquimod or pharmaceutically acceptable salt thereof or imiquimod derivatives.

2. Background art

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 ).

Besides protein-protein interactions with serum proteins such as a-thrombin and a1 - 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.

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/N K cells isolated from those tissues showed a decreased expression of several proinflammatory cytokines including TNF-a, IFN-γ, I L- 1 β , IL-12, IL-15, IL-15Ra and IL-18. 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 dependency translocated protein can have a global effect on the host response (Kerschen et ai, 2004).

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 a1 -antitrypsin, and binding of YopM to serum protein a-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).

Patent Application having publication number WO2009/1 15531 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. WO2009/1 15531 further discloses that once YopM is integrated into the cell cytosol, YopM is capable of effectively downregulating cytokines, in particularly pro-inflammatory cytokines.

The compound 1-isobutyl-1 H-imidazo[4,5-c]-quinolin-4-amine, known as imiquimod (also IMQ) has the following formula

Imiquimod is commercially marketed in the U.S. under the brand name Aldara(R) and is disclosed in U.S. Pat. No. 4,689,338 (which is incorporated herein by reference). It is described therein as an antiviral agent and as an interferon inducer. Imiquimod is known to be a TLR7/8 ligand and potent immune activator. Currently, the FDA has approved a 5% imiquimod cream, commercially available under the brand name Aldara(R), to treat certain dermal and mucosal associated conditions, such as (1) the topical treatment of clinically typical, nonhyperkeratotic actinic keratosis (AK) on the face or scalp in immunocompetent adults, (2) topical treatment of biopsy-confirmed, primary superficial basal cell carcinoma (sBCC) in immunocompetent adults, and (3) the topical treatment of external genital and perianal warts/condyloma acuminate in patients 12 years or older. It is known that topical application of imiquimod (IMQ), can induce and exacerbate psoriasis, a chronic inflammatory skin disorder in model mouse and in human patients. Van der Fitz et al. (J Immunol. 2009 May 1 ;182 (9): 5836-45) hypothesized that IMQ-induced dermatitis in mice can serve as a model for the analysis of pathogenic mechanisms in psoriasis-like dermatitis and assessed its IL-23/IL-17 axis dependency. Daily application of IMQ on mouse back skin induced inflamed scaly skin lesions resembling plaque type psoriasis. These lesions showed increased epidermal proliferation, abnormal differentiation, epidermal accumulation of neutrophils in microabcesses, neoangiogenesis, and infiltrates consisting of CD4(+) T cells, CD1 1 c(+) dendritic cells, and plasmacytoid dendritic cells. IMQ induced epidermal expression of IL-23, IL-17A, and IL-17F, as well as an increase in splenic Th17 cells. IMQ- induced dermatitis was partially dependent on the presence of T cells, whereas disease development was almost completely blocked in mice deficient for IL-23 or the IL-17 receptor, demonstrating a pivotal role of the IL-23/I L-17 axis. In conclusion, the sole application of the innate TLR7/8 ligand IMQ rapidly induces a dermatitis closely resembling human psoriasis, critically dependent on the IL-23/I L-17 axis. Cases of localized psoriasis at the sites of application of topical imiquimod, have been also for human patient, e.g. for patient with pre-existing psoriasis that had been stable for 14 years was treated with imiquimod 5% cream daily for 6 weeks to three superficial basal cell carcinomas. International Journal of Dermatology 2006 45, 1464-1465 (PateJJJ et al. Br J Dermatol. 2011 Mar; 164(3):670-2) reports a case where a topical treatment with Imiquimod 5% cream was prescribed once daily 3 times/week for 6 weeks for the actinic keratosis. After the second week of treatment the patient developed moderate psoriasis from changes at the sites of application of the imiquimod cream. A 4-day rest period from application of the drug was advised, after which the treatment was restarted. After the fourth week, the patient was referred to showing a severe psoriatic reaction of the scalp and widespread small erythemato-scaling plaques over the trunk.face and limbs.

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. 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.

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. There is therefore the need of an effective therapeutic agent useful in the prevention and/or treatment of psoriasis induced by imiquimod and derivatives thereof having a good patient compliance and limited side effects.

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.

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.

3. Description of the invention

The present inventors have surprisingly found that Yersinia outer protein M (YopM) when administered via cutaneous, intradermal or subcutaneous route is able to treat psoriasis induced by imiquimod in a patient affected by psoriasis induced by this drug (see the appended examples). Cutaneous administration of YopM (sometimes also referred to as epicutaneous administration) is thereby preferred. Thus, the present inventors recognized that YopM is surprisingly able to treat, ameliorate and/or mitigate unwanted side effects, such as psoriasis, nonspecific inflammation and dermatitis, caused by an imiquimod therapy. The aforementioned side effects are well known in the art and accompany the typical imiquimod therapy. Imiquimod is frequently used for the treatment of actinic keratosis (AK), superficial basal cell carcinoma (sBCC), common warts, external genital warts, external perianal warts, molluscum contagiosum (MC), skin cancers, vulvar intraepithelial neoplasia, and vaginal intraepithelial neoplasia. Diseases for which imiquimod or a pharmaceutical salt thereof or a derivative thereof is prescribed include specifically, although not being limited thereto (1) the topical treatment of clinically typical, nonhyperkeratotic actinic keratosis (AK) on the face or scalp in immunocompetent adults, (2) topical treatment of biopsy-confirmed, primary superficial basal cell carcinoma (sBCC) in immunocompetent adults, and (3) the topical treatment of external genital and perianal warts (condyloma acuminate) , preferably in patients 12 years or older, as well as (4) skin cancers (basal cell carcinoma, Bowen's disease, superficial squamous cell carcinoma, some superficial malignant melanomas, and actinic keratosis). It has also been tested for treatment of molluscum contagiosum, vulvar intraepithelial neoplasia, common warts that have proven difficult to treat, and vaginal intraepithelial neoplasia.

Based on the present invention, it is now possible to treat, ameliorate and/or mitigate unwanted side effects, such as psoriasis, nonspecific inflammation and dermatitis, caused by an imiquimod therapy. Many imiquimod side effects have been reported, ranging from local reactions such as redness, skin peeling, flaking, swelling, crusting, itching/burning, to systemic effects, such as fever, muscle aches, psoriasis, nonspecific inflammation and dermatitis, to name some. The skilled person, for example a medical doctor is however able to detect further imiquimod-induced side effects. Side effects which become manifest on the skin of an imiquimod treated patient are preferred side effects for the treatment with YopM in accordance with the present invention. Such side effects include inter alia psoriasis and dermatitis. Psoriasis is an even more preferred imiquimod induced side effect of the present invention.

The patient in accordance with the present invention is either (1) a patient who is going to be treated with imiquimod; (2) is already treated with imiquimod but has not yet the first symptoms of an imiquimod induced side effect; (3) is already treated with imiquimod and has already the first symptoms of an imiquimod induced side effect; or (4) has already been treated with imiquimod and still suffers from side effects caused by that imiquimod therapy. The term "patient" refers to an animal, preferably mammals and more preferably to humans.

"Imiquimod" including its structure is disclosed herein elsewhere. When used herein, the term "imiquimod" also includes its pharmaceutical salts and derivatives thereof. These are described herein elsewhere. The term "imiquimod derivatives" includes the compounds of formula (I) as disclosed in US patent US 4,689,338:

wherein is selected from the group consisting of alkyl of one to about ten carbon atoms, hydroxylalkyl of one to about six carbon atoms, acyloxyalkyl wherein the acyloxy moiety is alkanoyloxy of two to about four carbon atoms or benzoyloxy, and the alkyl moiety contains one to about six carbon atoms, benzyl, (phenyl)ethyl and phenyl, said benzyl, (phenyl)ethyl or phenyl substituent being optionally substituted on the benzene ring by one or two moieties independently selected from the group consisting of alkyl of one to about four carbon atoms, alkoxy of one to about four carbon atoms and halogen, with the proviso that if said benzene ring is substituted by two of said moieties, then said moieties together contain no more than 6 carbon atoms;

R₂ is selected from the group consisting of hydrogen, alkyl of one to about eight carbon atoms, benzyl, (phenyl)ethyl and phenyl, the benzyl, (phenyl)ethyl or phenyl substituent being optionally substituted on the benzene ring by one or two moieties independently selected from the group consisting of alkyl of one to about four carbon atoms, alkoxy of one to about four carbon atoms and halogen, with the proviso that when the benzene ring is substituted by two of said moieties, then the moieties together contain no more than 6 carbon atoms; and each R is independently selected from the group consisting of alkoxy of one to about four carbon atoms, halogen and alkyl of one to about four carbon atoms, and n is an integer from 1 to 2, with the proviso that if n is 2, then said R groups together contain no more than 6 carbon atoms; or a pharmaceutically acceptable acid addition salt thereof.

Both imiquimod and the imiquimod derivatives as defined by above formula (1) may exist in the form of pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salt thereof means those salts that are safe and effective for topical (cutaneous, intradermal and subcutaneous administration) use in mammals and that possess the desired biological activity. Pharmaceutically acceptable salts include salts of acidic or basic groups present in imiquimod and derivatives thereof. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1 , 1'-methylene-bis-(2-hydroxy-3- naphthoate)) salts. Imiquimod and derivatives thereof can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.

YopM can be administrated previously or concomitantly to an imiquimod therapy, i.e. YopM is used to prevent the onset of imiquimod induced side effects. Needless to say that the YopM therapy must be closely followed in order to safeguard that the imiquimod therapy still exerts it's therapeutically effect. Thus, YopM can be used to prevent the appearance of unwanted imiquimod-induced side effects such as psoriasis, nonspecific inflammation and dermatitis. Alternatively, YopM may be administered as soon as the first symptoms of psoriasis (or other imiquimod induced side effects) begin to appear as a side effect due to the imiquimod therapy. In this setting, YopM is used to ameliorate unwanted imiquimod-induced side effects such as psoriasis and/or nonspecific inflammation and dermatitis, in the course of the imiquimod-therapy.

Alternatively, YopM may be administered as imiquimod post treatment, i.e. as a follow up in the treatment of the psoriasis or other imiquimod induced side effects, i.e. YopM is administered once the treatment with imiquimod has been completed (provided that the patient still suffers from imiquimod induced side effects). YopM can thus be administered subsequently as imiquimod post treatment, if psoriasis (and/or other imiquimod induced side effects) are not completely remitted. This "follow up" treatment is particularly preferred.

YopM is administered in a dose effective to prevent and/or treat psoriasis or other side effects induced by imiquimod. The dose for cutaneous administration ranges for example from 1 μg/cm² to 20mg/cm², preferably 1 μg/cm² to 2C^g/cm², more preferably about 2μg/cm². 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.

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, fragments and 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 ; Cornells GR. J. Bacterid. 1998 Nov; 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 SW, Straley SC. Infect. Immun. 2003 Feb; 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 Sep; 18(9): 1948-60; McCoy et al. Infect Immun. 2010 Jun;78(6):2584-98; McPhee JB et al. Infect Immun. 2010 Aug;78(8):3529-39; Ruter C et al. J Cell Sci. 2010 Jul 1 ;123(Pt 13):2190-8; Hentschke M et al. PLoS One. 2010 Oct 5;5(10). pii: e13165 and Vieux EF et al.Biophys Chem. 201 1 Nov; 159(1): 152-61 ; or derivable from any biological database known to the person skilled in the art, e.g. from the Genbank database.

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 (Cornells et al., Microbiol. Mol. Biol. Rev. 62: 1315-1352 (1998)).

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 0:8.

The term "YopM" as used herein also includes "YopM fragment" and/or "YopM variant" (both explained herein elsewhere. Such a 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 ameliorating and/or treating imiquimod induced side effects. 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, ameliorating and/or treating imiquimod induce side effects. In particular, a YopM fragment of the invention is able to treat an imiquimod induces side effect, such as psoriasis as shown 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. 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.

In a further embodiment of the invention, a YopM variant is used in the prevention and/or treatment of psoriasis or other side effects induced by imiquimod or pharmaceutically active salt thereof or derivative thereof. 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 (or other side effects) induced by imiquimod. 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 (or other side effects) induced by imiquimod. 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 term "YopM variant" further includes the "YopM variant" as defined in patent application WO2009/115531 which is incorporated herein by reference. 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. 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.

In the context of the invention the term "cutaneous administration" of YopM sometimes also referred to as "epicutaneous administration" of YopM means the application of suitable drug dosage forms of YopM directly onto the skin for either local (topical) or systemic effects. According to the present invention, cutaneous administration of YopM or pharmaceutical compositions comprising YopM 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.

The route of administration of imiquimod is based on the recommendation of the physical doctor and/or the supplier of imiquimod. Imiquimod is normally applied cutaneously, or transdermally.

Any pharmaceutical composition suitable for cutaneous administration is encompassed by the present invention (including pharmaceutical compositions comprising YopM and/or imiquimod). Pharmaceutical compositions for cutaneous administration are e.g., in the form of a cream, lotion, gel, paste, ointment, patches, spray, or 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 of YopM comprise in a preferred embodiment cetearyl alcohol, paraffinum liquidum, glycerol, dimethicone, sodium cetearyl sulfate, phenoxyethanol, methylparaben, ethylparaben, propylparaben, and sodium hydroxide.

Any pharmaceutical composition suitable for sub-cutaneous administration is also encompassed by the present invention. 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 and/or imiquimod described herein.

Any pharmaceutical composition suitable for intradermal administration is likewise encompassed by the present invention. Pharmaceutical compositions suitable for intradermal administration are known in the art. Further non-limiting examples of dosage forms include dispersions, liquid dosage forms suitable for cutaneous, 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 for subcutaneous administration to a patient.

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. Needles can also be used for the delivery of the YopM active ingredient or imiquimod via intradermal or subcutaneous administration.

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.

The composition, shape and type of dosage forms of the invention will typically vary depending on their use.

The pharmaceutical formulations (comprising either YopM and/o imiquimod) can also be a delayed release or controlled release formulation. In dependence of the kind of treatment, YopM and imiquimod or pharmaceutical acceptable salts thereof or derivatives thereof may be formulated together in the same dosage form for the contemporaneous administration of the two drugs. Alternatively, YopM and imiquimod or pharmaceutical acceptable salts thereof or derivatives thereof may be formulated in two distinct dosage forms for the previous, contemporaneous or subsequent administration of YopM with respect to imiquimod or pharmaceutical acceptable salts thereof or derivatives thereof. Preferably, the dosage form is a cream.

Imiquimod suitable dosage form and pharmaceutical composition are already known in the art. An additional aspect the present invention refers to a kit comprising a pharmaceutical composition comprising YopM and a pharmaceutical composition comprising imiquimod or a pharmaceutically acceptable salt thereof or a derivative thereof. In particular, the pharmaceutical composition comprising YopM is in the form of cream, lotion, gel, paste, ointment, patches, spray or solution. Additionally, the pharmaceutical composition comprising imiquimod or a pharmaceutically acceptable salt thereof or a derivative thereof is in form of cream, lotion, gel, paste, ointment, patch, spray and solution.

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.

Figures

Figure 1 : discloses the domain organization and functional regions of YopM. Type-Ill 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 localisation signal. NLS-I: three N-terminal LRRs and NLS-II: 32 C-terminal residues of YopM. Interaction with serum proteins a- thrombin and a1 -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.

Figure 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.

Figure 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 μg 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 μg of YopM); Fig. 3f) shows the results obtained on mice treated with YopM by subcutaneous injection (s.c. 25 μg 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). Figure 4: refers to the histological analyses of psoriatic and non-psoriatic epidermis of back- skin tissue sections (5μηι) 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 40X.

Figure 5: shows the histological analyses of psoriatic and non psoriatic epidermis of back- skin tissue sections (5μηι) 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 (5μηι) 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 40X.

Figure 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 withTexas Red Phalloidin (red). DNA was stained with Draq5 (blue). Pictures were taken with confocal laser scanning microscope. Magnification 40X.

Figure 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 withTexas Red Phalloidin (red). DNA was stained with Draq5 (blue). Pictures were taken with a confocal laser scanning microscope. Magnification 40X.

Figure 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 μg YopM subcutaneously injected; (f) (e) 100 μg YopM subcutaneously injected. Pictures were taken with a light microscope. Magnification 40X.

4. Examples

To investigate whether YopM might be functional as an immune-modulator for the treatment of psoriasis induced by imiquimod, 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 psoriasislike 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.).The effect of YopM has been tested in different administration routes. YopM has been applied either topically as a component of a specific cream (balmed) or by subcutaneously (s.c.) injection or (every 48 h) by intraperitoneally injection (i.p.) over a period of two weeks to groups of mice suffering from imiquimod-induced psoriasis.

4.1 Expression and purification of recombinant YopM in E. Coli BL21

For the expression of recombinant YopM (rYopM), the pET24b(+) expression vector which also provides the coding sequence for a C-terminal 6 x 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 H₂0 bidest.. The protein solution was applied to a dialysis tube (pore size: 6-10 kDa) and dialyzed overnight in 2 I PBS or H₂0 bidest. at 4°C with gentle stirring. After dialysis, the protein was concentrated using centrifugal filters at 500 x g and 4°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. 1 a). Furthermore, the LPS content in elution fractions of recombinant YopM was determined by Limulus amebocyte lysate Toxin sensor assay (exemplified in Fig. 1 b, approx. 0.33 EU/mg).

4.2 Effects of YopM on an imiquimod (IMQ)- induced murine psoriasis model

The impact of rYopM applied topically (balmed and s.c.) and i.p. on inflammation linked to psoriasis-like conditions induced by imiquimod (Figure 2) was investigated.

Daily 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 H₂0 bidest.) was emulsified in a cream (150 μg 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 μg of YopM) every 48 h.

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).

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). 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 μηι) 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.

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 μηι) 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.

The results confirm the "self-delivering" abilities of YopM across the cutaneous barrier when applied epicutaneously 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 (Fig: 3f), 4f), 6e) and 6f)), whereas no significant phenotypic changes were observed in intraperitoneally injected mice (Fig.; 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.

These results clearly demonstrate the ability of YopM to treat psoriasis induced by imiquimod. 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 imiquimod induced psoriasis model is further shown. 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.

Claims

1. A Yersinia outer protein M (YopM) for use in the prevention, amelioration and/or treatment of imiquimod-induced side effects in a human patient.

2. The YopM of claim 1 , wherein said YopM is administered by the cutaneous, intradermal or sub-cutaneous route.

3. The YopM of claims 1 or 2, wherein said imiquimod-induced side effects become manifest on the skin of the patient.

4. The YopM of claims 1 , 2 or 3, wherein said imiquimod-induced side effects are psoriasis or dermatitis.

5. The Yersinia outer protein M (YopM) according to any one of the preceding claims, wherein the YopM is selected from the YopM of the species Yersinia enterocolitica, Yersinia pseudotuberculosis or Yersinia pestis.

6. A pharmaceutical composition comprising imiquimod, YopM and optionally a pharmaceutical acceptable excipient.

7. The pharmaceutical composition of claim 6 which is formulated as a cream, a lotion, a gel, a paste, an ointment, patch, spray or a solution.

8. A kit comprising a pharmaceutical composition comprising YopM and a pharmaceutical composition comprising imiquimod.

9. A kit comprising (1) a pharmaceutical composition comprising imiquimod for use in the treatment of actinic keratosis (AK), superficial basal cell carcinoma (sBCC), common warts, external genital warts, external perianal warts, molluscum contagiosum (MC), skin cancers, vulvar intraepithelial neoplasia, and vaginal intraepithelial neoplasia, and (b) a pharmaceutical composition comprising YopM.

10. The kit of claim 8 or 9, wherein said YopM is for use in the treatment of imiquimod- induced side effects.