WO2013041247A1 - Protéine m externe de yersinia (yopm) dans le traitement du psoriasis induit par imiquimod - Google Patents

Protéine m externe de yersinia (yopm) dans le traitement du psoriasis induit par imiquimod Download PDF

Info

Publication number
WO2013041247A1
WO2013041247A1 PCT/EP2012/053224 EP2012053224W WO2013041247A1 WO 2013041247 A1 WO2013041247 A1 WO 2013041247A1 EP 2012053224 W EP2012053224 W EP 2012053224W WO 2013041247 A1 WO2013041247 A1 WO 2013041247A1
Authority
WO
WIPO (PCT)
Prior art keywords
yopm
imiquimod
psoriasis
treatment
yersinia
Prior art date
Application number
PCT/EP2012/053224
Other languages
English (en)
Inventor
Christian Rueter
Alexander Schmidt
Original Assignee
Westfällsche Wilhelms-Universitaet Muenster
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Westfällsche Wilhelms-Universitaet Muenster filed Critical Westfällsche Wilhelms-Universitaet Muenster
Publication of WO2013041247A1 publication Critical patent/WO2013041247A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics

Definitions

  • Yersinia outer protein M (YopM) in the treatment of psoriasis induced by imiquimod
  • 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.
  • YopM Yersinia outer protein M
  • 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.
  • LRR leucine-rich repeat
  • YopM 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.
  • YopM is translocated through the T3SS into the host cell cytoplasm.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • AK clinically typical, nonhyperkeratotic actinic keratosis
  • sBCC primary superficial basal cell carcinoma
  • IMQ topical application of imiquimod
  • IMQ can induce and exacerbate psoriasis, a chronic inflammatory skin disorder in model mouse and in human patients.
  • 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.
  • 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.
  • Yersinia outer protein M 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.
  • 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.
  • AK actinic keratosis
  • sBCC superficial basal cell carcinoma
  • common warts common warts
  • external genital warts external genital warts
  • external perianal warts external genital warts
  • MC molluscum contagiosum
  • skin cancers vulvar intraepithelial neoplasia
  • vaginal intraepithelial neoplasia vaginal intraepithelial neoplasia.
  • 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.
  • patient refers to an animal, preferably mammals and more preferably to humans.
  • imiquimod including its structure is disclosed herein elsewhere.
  • 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,
  • R 2 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
  • 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,
  • 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.
  • 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 ⁇ g/cm 2 to 20mg/cm 2 , preferably 1 ⁇ g/cm 2 to 2C ⁇ g/cm 2 , more preferably about 2 ⁇ g/cm 2 .
  • Yersinia outer protein M (YopM) is selected from YopM of the species Yersinia enterocolitica, Yersinia pseudotuberculosis or Yersinia pestis.
  • Yersinia outer protein M (YopM)
  • YopM 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.
  • Yersinia outer protein M 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)).
  • the Yersinia outer protein M 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.
  • YopM as used herein also includes “YopM fragment” and/or "YopM variant” (both explained herein elsewhere.
  • a YopM fragment means that the fragment is a therapeutically active fragment of the YopM full length protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • LRR leucine-rich repeat
  • 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.
  • the translocation function can be performed by a "cell penetrating peptide" (CCP) which is heterologous to Yersinia.
  • 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.
  • 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.
  • 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.
  • a "YopM variant” 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.
  • 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.
  • LRR leucine-rich repeat
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • LRR leucine-rich repeat
  • 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.
  • 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.
  • compositions suitable for cutaneous administration are 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.
  • YopM 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.
  • 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.
  • 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.
  • 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.
  • compositions and dosage forms of the invention can further comprise one or more carriers, excipients or diluents.
  • composition, shape and type of dosage forms of the invention will typically vary depending on their use.
  • the pharmaceutical formulations can also be a delayed release or controlled release formulation.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition comprising YopM is in the form of cream, lotion, gel, paste, ointment, patches, spray or solution.
  • 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.
  • 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.
  • IMQ imiquimod
  • Fig. 3c refers to the results obtained on mice after a i.p. 100 ⁇ 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 ⁇ g of YopM); Fig.
  • FIG. 3f shows the results obtained on mice treated with YopM by subcutaneous injection (s.c. 25 ⁇ 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).
  • Figure 4: refers to the histological analyses of psoriatic and non-psoriatic epidermis of back- skin tissue sections (5 ⁇ ) 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;
  • Figure 5 shows the histological analyses of psoriatic and non psoriatic epidermis of back- skin tissue sections (5 ⁇ ) 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 ⁇ ) 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.
  • FIG. 6 Distribution of YopM in the Epidermis and Dermis after 48 h of application.
  • FIG. 7 Distribution of YopM in the epidermis and dermis after 48 h of application.
  • 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 ⁇ g YopM subcutaneously injected; (f) (e) 100 ⁇ g YopM subcutaneously injected. Pictures were taken with a light microscope. Magnification 40X.
  • 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.
  • IMQ murine imiquimod
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • YopM recombinant YopM
  • 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 H 2 0 bidest.. The protein solution was applied to a dialysis tube (pore size: 6-10 kDa) and dialyzed overnight in 2 I PBS or H 2 0 bidest. at 4°C with gentle stirring.
  • Ni-NTA nickel-nitrilotriacetic acid
  • 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).
  • 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 ⁇ g of YopM) every 48 h.
  • 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).
  • Fig. 4 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).
  • 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
  • Fig. 4f) refers to the YopM subcutaneously injected.
  • FIG. 5 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 ⁇ ) 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.
  • YopM did not enter systemic distribution, mainly remained in the epidermis, dermis, and subcutis, and could ' t be found in local lymph nodes.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Dermatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne l'utilisation de la protéine M externe de Yersinia (YopM) dans la prévention et/ou le traitement par une administration cutanée, intradermique ou sous-cutanée du psoriasis induit par imiquimod ou des sels pharmaceutiquement acceptables de celui-ci ou dérivés d'imiquimod. L'invention concerne en outre des compositions pharmaceutiques comprenant YopM et imiquimod, et une trousse comprenant une composition pharmaceutique contenant YopM et une composition pharmaceutique contenant imiquimod.
PCT/EP2012/053224 2011-09-23 2012-02-24 Protéine m externe de yersinia (yopm) dans le traitement du psoriasis induit par imiquimod WO2013041247A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP11007786.4 2011-09-23
EP11007786 2011-09-23
US201161539693P 2011-09-27 2011-09-27
US61/539,693 2011-09-27

Publications (1)

Publication Number Publication Date
WO2013041247A1 true WO2013041247A1 (fr) 2013-03-28

Family

ID=47913916

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/053224 WO2013041247A1 (fr) 2011-09-23 2012-02-24 Protéine m externe de yersinia (yopm) dans le traitement du psoriasis induit par imiquimod

Country Status (1)

Country Link
WO (1) WO2013041247A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4689338A (en) 1983-11-18 1987-08-25 Riker Laboratories, Inc. 1H-Imidazo[4,5-c]quinolin-4-amines and antiviral use
WO2006109303A2 (fr) * 2005-04-11 2006-10-19 Yeda Research And Development Co.Ltd. Proteines chimeres et leur preparation, et compositions pharmaceutiques contenant lesdites proteines
WO2009115531A2 (fr) 2008-03-17 2009-09-24 Universitätsklinikum Münster Protéine yopm utilisée comme véhicule pour les molécules cargo et comme agent thérapeutique biologique pour l’immunomodulation de réactions inflammatoires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4689338A (en) 1983-11-18 1987-08-25 Riker Laboratories, Inc. 1H-Imidazo[4,5-c]quinolin-4-amines and antiviral use
WO2006109303A2 (fr) * 2005-04-11 2006-10-19 Yeda Research And Development Co.Ltd. Proteines chimeres et leur preparation, et compositions pharmaceutiques contenant lesdites proteines
WO2009115531A2 (fr) 2008-03-17 2009-09-24 Universitätsklinikum Münster Protéine yopm utilisée comme véhicule pour les molécules cargo et comme agent thérapeutique biologique pour l’immunomodulation de réactions inflammatoires

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
BOLAND A ET AL., EMBO J., vol. 15, no. 19, 1 October 1996 (1996-10-01), pages 5191 - 201
BOWIE ET AL., SCIENCE, vol. 247, 1990, pages 1306 - 1310
CORNELIS ET AL., MICROBIOL. MOL. BIOL. REV., vol. 62, 1998, pages 1315 - 1352
CORNELIS GR., J. BACTERID., vol. 180, no. 21, November 1998 (1998-11-01), pages 5495 - 504
CROMME C ET AL: "ANTI-INFLAMMATORY EFFECTS OF YOPM IN RHEUMATOID ARTHRITIS FIBROBLAST-LIKE SYNOVIOCYTES", ANNALS OF THE RHEUMATIC DISEASES, vol. 70, no. Suppl. 2, March 2011 (2011-03-01), & 31ST EUROPEAN WORKSHOP FOR RHEUMATOLOGY RESEARCH; AMSTERDAM, NETHERLANDS; MARCH 03 -06, 2011, XP009155682, ISSN: 0003-4967 *
HENTSCHKE M ET AL., PLOS ONE., vol. 5, no. 10, 5 October 2010 (2010-10-05), pages E13165
HEUSIPP, G.; SPEKKER, K.; BRAST, S.; FALKER, S.; SCHMIDT, M.A., MICROBIOL., vol. 152, 2006, pages 1327 - 1335
INTERNATIONAL JOURNAL OF DERMATOLOGY, vol. 45, 2006, pages 1464 - 1465
KERSCHEN EDWARD J ET AL: "The plague virulence protein, YopM, targets innate immunity by suppressing proinflammatory cytokine and IL-15 production and depleting NK cells", FASEB JOURNAL, FED. OF AMERICAN SOC. FOR EXPERIMENTAL BIOLOGY, US, vol. 18, no. 4-5, 1 January 2004 (2004-01-01), XP009127970, ISSN: 0892-6638 *
KERSCHEN, E.J.; COHEN, D.A.; KAPLAN, A.M.; STRANLEY, S.C., INFECT. IMMUN., vol. 72, 2004, pages 4589 - 4602
KLOSS E; BARRICK D., PROTEIN SCI., vol. 18, no. 9, September 2009 (2009-09-01), pages 1948 - 60
M. ALEXANDER SCHMIDT: "Sekretionsmechanismen Gram-negativer Pathogene", 9 January 2012 (2012-01-09), XP002667668, Retrieved from the Internet <URL:http://campus.uni-muenster.de/uploads/media/09012012_Netzfolien.pdf> [retrieved on 20120120] *
MCCOY ET AL., INFECT IMMUN., vol. 78, no. 6, June 2010 (2010-06-01), pages 2584 - 98
MCDONALD, C; VACRATIS, P.O.; BLISKA, J. B.; DIXON, J. E., J.BIOL.CHEM, vol. 278, 2003, pages 18514 - 18523
MCPHEE JB ET AL., INFECT IMMUN., vol. 78, no. 8, August 2010 (2010-08-01), pages 3529 - 39
NESTLE, F. O.; B. J. NICKOLOFF.: "Animal models of psoriasis: a brief update", J. EUR. ACAD.DERMATOL. VENEREOL., vol. 20, no. 2, 2006, pages 24 - 27
PATEL U ET AL., BR J DERMATOL., vol. 164, no. 3, March 2011 (2011-03-01), pages 670 - 2
POCEVA M ET AL: "YopM as a bacteria-derived anti-inflammatory cell-penetrating peptide (CPP) in a murine Psoriasis model", IJMM INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, vol. 301, no. Suppl. 47, 26 August 2011 (2011-08-26), & 63RD ANNUAL MEETING OF THE GERMAN-SOCIETY-FOR-HYGIENE-AND-MICROBIOLO GY; ESSEN, GERMANY; SEPTEMBER 25 -28, 2011, pages 26, XP009155685, ISSN: 1438-4221 *
RUTER C ET AL., J CELL SCI., vol. 123, no. 13, 1 July 2010 (2010-07-01), pages 2190 - 8
RUTER C ET AL: "A newly identified bacterial cell-penetrating peptide that reduces the transcription of pro-inflammatory cytokines", JOURNAL OF CELL SCIENCE 20100701 COMPANY OF BIOLOGISTS LTD GBR LNKD- DOI:10.1242/JCS.063016, vol. 123, no. 13, 1 July 2010 (2010-07-01), pages 2190 - 2198, XP009155676, ISSN: 0021-9533 *
SCHAMERT J ET AL: "YopM from Yersinia enterocolitica penetrates host cell membranes and down-regulates transcription of pro-inflammatory cytokines", IJMM INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, vol. 299, no. Suppl. 46, September 2009 (2009-09-01), & 61ST CONFERENCE OF THE DEUTSCHEN-GESELLSCHAFT-FUR-HYGIENE-UND-MIKROBI OLOGIE; GOTTINGEN, GERMANY; SEPTEMBER 20 -23, 2009, pages 33, XP009155674, ISSN: 1438-4221 *
SKRZYPEK E; MYERS-MORALES T; WHITEHEART SW; STRALEY SC, INFECT. IMMUN., vol. 71, no. 2, February 2003 (2003-02-01), pages 937 - 47
SKRZYPEK, E.; COWAN, C.; STRALEY, S.C., MOL. MICROBIOL., vol. 30, 1998, pages 1051 - 1065
VAN DER FITS L ET AL.: "Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis", J. IMMUNOL., vol. 182, 2009, pages 5836 - 5845
VAN DER FITZ ET AL., J IMMUNOL., vol. 182, no. 9, 1 May 2009 (2009-05-01), pages 5836 - 45
VIEUX EF ET AL., BIOPHYS CHEM., vol. 159, no. 1, November 2011 (2011-11-01), pages 152 - 61
WAGSTAFF ANTONA J ET AL: "Topical imiquimod: a review of its use in the management of anogenital warts, actinic keratoses, basal cell carcinoma and other skin lesions", DRUGS, ADIS INTERNATIONAL LTD, NZ, vol. 67, no. 15, 1 January 2007 (2007-01-01), pages 2187 - 2210, XP009158526, ISSN: 0012-6667 *

Similar Documents

Publication Publication Date Title
EP1553962B1 (fr) Chimiotherapie combinee comprenant la chlorotoxine
JP6625071B2 (ja) 慢性疼痛用のナトリウムチャネルを標的とする非麻薬性ｃｒｍｐ２ペプチド
EP3322430B1 (fr) Peptides anti-inflammatoires et leurs utilisations
ES2371671T3 (es) Uso de ácido fenilbutírico o sales del mismo para tratar el prurito.
US20220370560A1 (en) Ptd-smad7 therapeutics
JPWO2006009114A1 (ja) Ｉｌ−１８レセプターアンタゴニスト、および当該アンタゴニストを含む薬学的組成物
KR20190101990A (ko) 펩티드 wkdeagkplvk를 포함하는 조성물
CN105705160B (zh) Il-22二聚体在制备用于治疗胰腺炎的药物中的用途
JPWO2017164409A1 (ja) 疾患の要因となる生体内タンパク質を標的とするコンジュゲートワクチン
KR102551575B1 (ko) 글루코스 운반을 촉진하는데 사용하기 위한 펩티드
JP2023126760A (ja) 抗炎症剤
WO2013041247A1 (fr) Protéine m externe de yersinia (yopm) dans le traitement du psoriasis induit par imiquimod
US20190365859A1 (en) Prophylactic or therapeutic agent for inflammatory skin disease
Quagliata et al. Therapeutic applications of thymosin peptides: a patent landscape 2018-present
AU2018202086B2 (en) Yersinia outer protein m (yopm) in the treatment of psoriasis
WO2020149282A1 (fr) Utilisation de l&#39;omiganan dans le traitement d&#39;une rosacée
US20190175601A1 (en) Composition for inhibiting or alleviating itching including riboflavin
TW202345888A (zh) 治療顱腦損傷的方法
WO2023196916A1 (fr) Procédés de traitement de l&#39;hidradénite suppurée

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12710179

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12710179

Country of ref document: EP

Kind code of ref document: A1