WO2015022326A1

WO2015022326A1 - Peptides as active agents for treating primary graft dysfunction

Info

Publication number: WO2015022326A1
Application number: PCT/EP2014/067248
Authority: WO
Inventors: Peter Petzelbauer; Sonja Reingruber
Original assignee: Xiber Science Gmbh
Priority date: 2013-08-12
Filing date: 2014-08-12
Publication date: 2015-02-19

Abstract

The present invention relates to peptides for use in treating and/or preventing primary graft dysfunction (PGD). The present invention further relates to polynucleotides for use in treating and/or preventing PGD which encode these peptides. Another aspect of the invention relates to pharmaceutical compositions for use in treating and/or preventing PGD comprising the above mentioned peptides and/or polynucleotides and further comprising a pharmaceutically acceptable carrier and/or diluent.

Description

Peptides as active agents for treating primary graft dysfunction

Transplantation represents the primary method of care for patients with end-stage organ failure. While the number of individuals on the waiting list to receive an organ donation has increased exponentially the demand has not been met due to difficulties with organ procurement and immunological rejection responses.

Those individuals who are lucky enough to undergo organ transplantation are still faced with many challenges, and one of the major challenges is primary graft dysfunction (PGD). PGD is an ischemia-reperfusion injury occurring in the early (i.e. within the first 72 h) period following transplantation (see, e.g. Oto, Am J Transpl, 2008; 8: 2132-9). The cause is the change from hypoxia to normal oxygen tension as given in any normal perfused tissue. It is most often described in the transplanted lung, liver or kidney, but is relevant in all organs and tissues exposed to ischemia followed by reconnection to the blood circulation. An association between PGD among lung, kidney and heart recipients from the same multiorgan donor has been described (Oto, Am J Transpl, 2008; 8: 2132-9). PGD has to be distinguished from graft versus host disease which occurs, when bone marrow cells from an allogenic donor are transferred into an irradiated host and consists of an immune response of the donor cells against host cells. Patients with PGD have markedly worse 90-day post-operative mortality and 3-year survival (Arcasoy, J Heart Lung Transplant, 2005; 24: 1483-1488). The incidence of PGD after lung, kidney and heart transplantation is estimated as 20%, 24% and 20%, respectively, and it is considered to be a significant cause of morbidity and mortality after solid-organ transplantation.

WO 201 1/157819 A2 describes that Rho GTPases are useful in treating or preventing diseases that are related to a loss of endothelial or epithelial barrier function and US 2004/0053250 A1 suggests using arginine-rich protein (ARP)-like nucleic acids and polypeptides for treating autoimmune disorders. However, none of these documents provides for medicaments for the treatment of PGD.

Thus, there is a need for medicaments for the therapeutic intervention of PGD. Thus, the technical problem underlying the present invention is the provision of means and methods for the medical intervention of primary graft dysfunction, in particular of lung, kidney or heart PGD.

This technical problem has been solved by the embodiments provided herein and as described in the appended illustrative examples and in the claims.

The present invention provides for peptides to be used in treating and/or preventing primary graft dysfunction (PGD) comprising or consisting of the amino acid sequence

GXi RPX₂X₃X4X₅GGX₆ (SEQ ID NO: 1 ) wherein

Xi is an amino acid selected from the group consisting of R and A;

X2 is either omitted or an amino acid selected from the group consisting of L and V;

X3 is either omitted or an amino acid sequence consisting of PPP;

X is either omitted or an amino acid sequence consisting of GG; X₅ represents two amino acids selected from the group consisting of A, I and S; and

Xe is either omitted or an amino acid sequence consisting of 1 to 5 amino acids; provided that if X₃ is omitted, than X is not omitted. It is also provided in context of the invention that if X₄ is omitted, than X₃ is not omitted. Thus, said peptide to be used in treating and/or preventing PGD must have at least one of X3 and X₄. Or, in other words, the peptide to be used in treating and/or preventing PGD must contain PPP (i.e. X3 is PPP) and/or GG (i.e. X₄ is GG). An example for a peptide wherein X₃ is omitted and wherein X₄ is present is the peptide of the amino acid sequence GRRPLGGISGG (SEQ ID NO: 3). An example for a peptide wherein X₃ is present and wherein X₄ is omitted is the peptide GRRPLPPPISGG (SEQ ID NO: 8).

The illustrative appended Examples demonstrate that ex vivo flushing of the lungs to be transplanted prior to transplantation suppresses the proliferation of sensibilized T cells targeting the MHC molecules of the donor lungs. Moreover, after transplantation of allogenic grafts which have been treated with the inventive peptides of SEQ ID NO: 1 , no alloreactivity could be detected. In addition, as illustrated in the appended examples, the peptides of the invention surprisingly inhibit the activation of macrophages. In particular, the herein described peptides of the form GX1 RPX2X3X4X₅GGX6 (SEQ ID NO: 1 ) have surprisingly been demonstrated to reduce uptake of bacteria by macrophages and to suppress prolonged extracellular acidification of macrophages after LPS treatment. It is noted that macrophages have repeatedly been shown to be involved in PGD (see, e.g., Suzuki, Semin Respir Crit Care Med; 2013; 34: 305-319). Accordingly, the means and methods described and provided herein are particularly useful in treating and/or preventing PGD. For example, the peptides comprising or consisting of GX1 RPX2X3X4X₅GGX6 (SEQ ID NO: 1 ) may be used in treating recipients of (a) lung(s), (a) kidney(s) and/or a heart. In addition or alternatively, these peptides may be used in treating (a) donor lung(s), (a) donor kidney(s) and/or a donor heart prior to transplantation.

US 2004/0053250 A1 discloses arginine-rich protein (ARP)-like polypeptides for suppressing immune function and treating autoimmune disorders. In contrast, as described in detail herein below, the efficacy of the peptides provided herein does not rely on immunosuppression. Rather, the herein described peptides reduce organ damage during the grafting procedure resulting in a diminished immune response of the host. Moreover, the appended illustrative examples surprisingly show that, to be useful in the treatment of PGD, a peptide requires a flexible center (e.g. a central PPP or GG, preferably GG) and N-terminal from this flexible center, the peptide has to have the amino acid sequence GXi RP, wherein Xi is R or A. Accordingly, the peptides provided herein are considerably different form the ARP-like polypeptides disclosed in US 2004/0053250 A1 .

As mentioned, the herein described peptides comprise or consist of the amino acid sequence GXi RPX2X3X4X₅GGX6 (SEQ ID NO: 1 ). To be active, a flexible center (e.g. wherein X₃ is PPP and/or wherein X₄ is GG) is required in said peptide to be used in the medical intervention of primary graft dysfunction (PGD). Preferably, said flexible center consists of GG (i.e. X₄ is GG).

In addition to peptides of SEQ ID NO: 1 , the invention also relates to peptides of SEQ ID NO: 45 for use in treating and/or preventing PGD. It is noted that in context of this aspect of the invention, if X is GG, than X₃ can be omitted or can also be any amino acid or a sequence of 1 to 5 amino acids. In context of the invention and as commonly known, said "any amino acid" may be alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophane (Trp; W), tyrosine (Tyr; Y), valine (Val; V), selenocysteine (Sec; U) or pyrrolysine (Pyl; O). Also non-natural are envisaged under the term "any amino acid".

Accordingly, if the peptide of the invention comprises the flexible center GG (i.e. X₄ is GG), than X₃ can be omitted or can consist of an amino acid sequence of 1 to 5 amino acids. In other words, the invention relates to a peptide for use in treating primary graft dysfunction comprising or consisting of the amino acid sequence

GX1 RPX2X3X4 XsGGXe (SEQ ID NO: 45) wherein

Xi is an amino acid selected from the group consisting of R and A;

X3 is either omitted or an amino acid sequence consisting of 1 to 5 amino acids; X is either omitted or an amino acid sequence consisting of GG;

X₅ represents two amino acids selected from the group consisting of A, I and S; and

Xe is either omitted or an amino acid sequence consisting of 1 to 5 amino acids; provided that if X₃ is omitted, that X is an amino acid sequence consisting of GG; and provided that if X is omitted, than X₃ is an amino acid sequence consisting of PPP. Thus, said peptide to be used in treating and/or preventing PGD must have at least one of PPP (i.e. wherein X₃ is PPP) and GG (i.e. wherein X is GG). Or, in other words, the peptide to be used in treating and/or preventing PGD must contain PPP (i.e. X3 is PPP) and/or GG (i.e. X₄ is GG). An example for a peptide wherein X₃ is omitted and wherein X₄ is GG is the peptide of the amino acid sequence GRRPLGGISGG (SEQ ID NO: 3). An example for a peptide wherein X₃ is PPP and wherein X₄ is omitted is the peptide GRRPLPPPISGG (SEQ ID NO: 8).

The cumulative graft failure rate is 50% at 10 years (Parang, 2009, Can.J. Cardiol., 25:e57-e62). The reason is that organ transplantation faces several problems including organ take, extra-corporal organ preservation, successful grafting resulting in perfusion, cell survival and cell function. The first step (i.e. organ take, extracorporal organ preservation and grafting) does not require immunosuppression. Immunosuppression is usually necessary in the second step, because even if the first step is successfully performed, the host normally initiates an immune response, ultimately resulting in the destruction of the grafted organ. Of note, if a syngeneic graft is used, e.g. in situations of vein grafts in patients with coronary or carotic artery occlusion, no immunosuppression is required. Still, the outcome of such grafts (graft patency) depends on a successful step one.

The peptides described and provided herein protect the organ during "step one", i.e. they reduce organ damage during the grafting procedure. As an indirect effect, the host is less exposed to foreign major histocompatibility complex (MHC) molecules from the grafted organ resulting in delayed or diminished sensitizing of the host^'s immune system (not immunosuppression) and a delayed or diminished immune response.

Thus, one aspect of the present invention is the use of (a) peptide(s) described herein (e.g. peptides of SEQ ID NO: 1 ) to protect an organ to be grafted during the procedure of organ-take, extra corporal storage and grafting. As shown in the appended illustrative examples, using the peptides of the invention to protect an organ to be grafted has the unexpected advantage that the organ function is increased and the exposure of donor MHC to host antigen presenting cells is reduced as compared to a grafting procedure wherein the organ to be grafted is not treated with the peptides of the invention. It is noted that the efficacy of the peptides described herein does not rely on an immunosuppression but on a reduced sensitization. The Examples document that the peptides as described herein prevent prolonged extracellular acidification of macrophages (see appended Example 12 and Figure 6) and reduce bacterial uptake of macrophages (see appended Example 13 and Figure 7). Without being bound by theory, this has the advantage that the side-effects of suppressed T cell function through the use of e.g. cyclosporine can be avoided and/or reduced by using the peptides of the invention. Accordingly, increased susceptibility to infections and decreased cancer immunosurveillanceis are avoided or are at least reduced.

The appended illustrative examples demonstrate that the peptides provided herein are capable of inhibiting Rho GTPases. This has been exemplarily demonstrated for RhoA. Furthermore, the inventors of the present invention found that the efficacy of the herein described peptides in the treatment of PGD is dependent on the ability of the peptides to inhibit Rho GTPases. WO 201 1/157819 A2 describes that Rho GTPases are useful in treating or preventing diseases that are related to a loss of endothelial and/or epithelial barrier function, such as burns, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator induced lung injury (VILI), systemic inflammatory response syndrome (SIRS), acute kidney injury (AKI), sepsis, multiorgan dysfunction syndrome (MODS) or edema. However, the finding that Rho GTPase-inhibiting peptides are able to prevent PGD is surprising since, as discussed in more detail below, Rho GTPases are involved in a sophisticated network and play a role in the regulation of several different cell functions such as the organization of the cytoskeleton, cell migration, cell cycle progression, cell proliferation, cell differentiation, gene expression, cell survival, apoptosis, vascular and endothelial permeability as well as tight junction dissociation and assembly (Nature, 2006, 440(7087):1069-1072; Curr Drug Targets, 2010, 1 1 (9): 1043-1058; Cardiovasc Res 2010, 87(2): 243-253; Thromb Haemost 2010, 103(1 ): 40-55; Am J Physiol Lung Cell Mol Physiol, 2005, 288(2): L294-L306; Am J Pathol, 2010, 177(2): 512-524; Physiology (Bethesda), 2010, 25(1 ): 16-26; Mol Biol Cell, 2007, 18: 3429- 3439 and Nat Cell Biol, 201 1 , 13(2): 159-166). PGD is a disease or disorder associated an innate immune response, whereas e.g., graft versus host disease is a response to the adaptive immunity. Moreover, diseases related to a loss of endothelial and/or epithelial barrier function are considerably different from PGD. In particular, diseases such as ALI, ARDS and SIRS are mainly pathogen-induced, i.e. the inadequate response of the host to a foreign stimulus. In contrast thereto, PGD is an intrinsic disease which is unrelated to MHC-mismatch. It is noted that PGD may even occur when syngeneic grafts are used. In addition, in context of the present invention it has been shown that the herein provided peptides act distinct from epithelial and and endothelial cells. For example, the illustrative examples demonstrate that the inventive peptides inhibit stress induced responses of macrophages. More specifically, the appended examples show that the peptides of the invention prevent prolonged extracellular acidification of macrophages (Example 12 and Figure 6) and reduce bacterial uptake of macrophages (Example 13 and Figure 7).

Moreover, as demonstrated in the appended illustrative examples, the peptides provided herein are advantageous over conventional Rho GTPase-inhibitors. In particular, conventional Rho GTPase- and Rho-kinase-inhibitors act systemically, i.e. they influence Rho-GTPases and Rho-kinases also in healthy cells/tissue and, thus, may imply severe side effects such as toxic myopathy (for statins) and may cause hypotension (Fasudil) (Surg. Neurol, 2007, 68(2):126-131 ). In contrast thereto, the inventive peptides provided herein can act locally (e.g. if administered directly into the organ to be grafted) and are well tolerable. Moreover, statins have to be supplied as a prophylactic treatment, i.e. prehospital treatment is required (Crit Care Med, 201 1 , 39(6): 1343-1350). This also applies for Fasudil where pretreatment was required in animal models (Life Sci, 201 1 , 88(1 -2): 104-109). In contrast, as demonstrated in the appended illustrative examples, the peptides provided herein can be flushed extra-corporal through the donor organ vessels to be effective in the treatment of PGD. Accordingly, the treatment of PGD with the inventive peptides does not require pre-transplantation treatment of the recipient. However, it is noted that the time point of administration of the inventive peptides is not restricted to the transplantation procedure. Rather, the invention relates to (1 ) administration of the inventive peptides to the donor and/or the recipient prior to the transplantation procedure in order to prevent PGD; (2) administration of the inventive peptides to the donor organ/tissue and/or the recipient during the transplantation procedure in order to prevent PGD; and (3) administration of the inventive peptides to the donor after the transplantation procedure in order to prevent or treat PGD. A further advantage of the herein provided peptides is that, in addition to be RhoA inhibitors, the peptides improve stress induced metabolic responses as illustrated and exemplified in Figure 6.

As explained above, the invention relates to peptides which comprise or consist of the amino acid sequence of GXi RPX2X3X4X₅GGX6 (SEQ ID NO: 1 ) for use in treating and/or preventing PGD. In one embodiment, the invention relates to a peptide for use in treating primary graft dysfunction consisting of the amino acid sequence of SEQ ID NO: 1 . The peptide for use in treating and/or preventing PGD may be of the form G-R(A)-R-P-L(V)-P-P-P-I(A)-S(A)-G-G. In a preferred embodiment, the peptide for use in treating and/or preventing PGD is of the form G- R(A)-R-P-L(V)-G-G-I(A)-S(A)-G-G.

The peptides for use in treating and/or preventing PGD preferably comprise or consist of not more than 50 amino acids, not more than 40 amino acids, not more than 30 amino acids, not more that 20 amino acids or not more that 19 amino acids, more preferably not more than 14 amino acids, most preferably not more than 12 amino acids, not more than 1 1 amino acids or not more than 9 amino acids. In one embodiment, the peptide for use in treating PGD comprises or consists of 1 1 amino acids. In another embodiment, the peptide for use in treating PGD comprises or consists of 12 amino acids. Thus, the peptide for use in treating PGD may consist of not more than 19, 14, 12, 1 1 or 9 amino acids. In a prioritized embodiment, the peptide for use in treating and/or preventing PGD consists of 1 1 amino acids.

As used herein, the term "amino acid" refers to any amino acid known in the art and comprises proteinogenic as well as non-proteinogenic amino acids as known in the art. Proteinogenic amino acids comprise alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophane (Trp; W), tyrosine (Tyr; Y), valine (Val; V), selenocysteine (Sec; U) and pyrrolysine (Pyl; O). Non-limiting examples for non-proteinogenic amino acids are hydroxyproline, selenomethionine, carnitine, gamma-aminobutyric acid (GABA), lanthionine, dehydroalanine, ornitine, or citrulline. As the skilled person is readily aware of, it is possible that in some cases also non-proteinogenic amino acids may be part of proteins. Amino acids are abbreviated herein by the one-letter code or the three-letter code as commonly used in the art and as also set forth hereinabove.

The peptides provided by the present invention are derived from cingulin, a known GEF-H1 inhibitor (NY Acad Sci, 2009, 1 165: 88-98; Dev Cell, 2005, 8: 777-786; Dev Cell, 2007, 12: 699-712). Furthermore, the peptides described and provided herein show sequence similarity with the VE-cadherin binding and RhoA-inhibiting protein Ββι_{5- 2} (PLoS ONE, 2009, 4(4): e5391 ). The activity of Ββι_{5- 2} has repeatedly been described as being strongly dependent on the first four amino acids (Int J Cancer, 2009, 125: 577-584). Particularly Hisi₆ (2^nd position of Ββι_{5- 2}) and Argi₇ (3^rd position of Ββ15-42) of Ββι_5-42 have been described as being critical (Biochemistry, 2002, 41 : 4107-41 16) for functionality of Ββι_5-42- In contrast thereto, the peptides described and provided by the present invention do not have a His at the 2^nd position but are nevertheless shown to have strong RhoA-inhibitory effects. Also, the herein described peptides target endothelial as well as epithelial cells, the latter lacking VE-cadherin. Moreover, it has surprisingly been found in the present invention that peptides described and provided herein are significantly more effective than Ββι_{5- 2} as exemplarily demonstrated using an animal model for acute lung injury (ALI; see, e.g., Example 6). In addition, the appended illustrative Examples demonstrate that the inventive peptides are more useful in treating PGD than Ββ_15-42. In particular, the appended illustrative Examples show that the peptides of the invention inhibit the activation of macrophages, wherein Ββι_{5- 2} does not have this capability. More specifically, the peptides comprising or consisting of GX1 RPX2X3X4X5GGX6 (SEQ ID NO: 1 ) reduce the uptake of bacteria by macrophages and suppress prolonged extracellular acidification of macrophages after LPS treatment, wherein Ββι_{5- 2} fails to have this immunomodulatory activity.

The peptides for use in treating and/or preventing PGD as described and provided herein may further comprise X₇ at the C-terminus. As defined herein X₇ may be a moiety which is suitable to delay primary renal filtration, to prolong serum half life and/or to protect against proteolytic degradation (particularly peptidases) of the peptides provided herein. Such moieties are known in the art. Non-limiting examples for such moieties are NH₂, albumin, polyethyleneglycol, dextrane, ferritine, hydroxyethyl-starch and the Fc-moiety of an antibody. X₇ may also be or comprise an amino acid stretch that is capable of prolonging serum half-life as described in, e.g., WO 08/155134 or amino acid sequences as described in WO 07/103515 or in Nat Biotechnol, 2009, 27: 1 186-1 190.

In the following, non-limiting examples for the variables Xi to Xe of the general sequence of the peptides described and provided herein are described. The peptides for use in treating and/or preventing PGD of the present invention may comprise one of the following specific examples of the variables or a combination of two, three or more thereof. Xi is for example an R. It is preferred that X₂ is an L or a V, for example an L. X₃ may be omitted or PPP, for example omitted. X₄ may be GG. X₅ may be IS or AS, for example IS. Xe may be omitted or may be an additional amino acid or amino acid stretch. In one embodiment of the invention, X₆ is omitted. If Xe is not omitted, said 1 to 5 amino acids of Xe may be selected from any amino acid as described herein. Furthermore, in certain circumstances, Xe may comprise or consist of more than 5 amino acids. It is also envisaged that X₆ may comprise a longer amino acid stretch, even an amino acid stretch which is 7, 9, 1 1 , 13 or 15 amino acids or larger than 15 amino acids. Such a stretch may also comprise an amino acid stretch that prolongs serum half-life as described for X₇ above, like the "PAS" sequences provided in WO 08/155134, or the peptide sequences provided in WO 07/103515 or in Nat Biotechnol, 2009, 27: 1 186-1 190. However, as mentioned, Xe may also be omitted.

As described herein, the invention relates to peptides for use in treating and/or preventing PGD comprising or consisting of the amino acid sequence of SEQ ID NO: 1 , provided that if X₃ is omitted, than X is not omitted and provided that if X is omitted, than X₃ is not omitted. Accordingly, if at all, either X or X₃ can be omitted within the peptide for use in treating and/or preventing PGD provided herein.

One aspect of the invention relates to the herein described peptides, wherein Xi is R, X₂ is L, X₃ is omitted, X₅ is IS and Xe is omitted, or wherein Xi is R, X₂ is V, X₃ is omitted, X₅ is IS and Xe is omitted. In one embodiment, the peptide of the present invention comprises or consists of the amino acid sequence GRRPLX₄ISGG (SEQ ID NO: 2), e.g., GRRPLGGISGG (SEQ ID NO: 3) or GRRPLISGG (SEQ ID NO: 4). In another embodiment, the peptide of the present invention comprises or consists of the amino acid sequence GRRPVX₄ISGG (SEQ ID NO: 5), e.g., GRRPVGGISGG (SEQ ID NO: 6) or GRRPVISGG (SEQ ID NO: 7). In one embodiment, the peptide of the invention comprises or consists of the amino acid sequence GRRPVGGAAGG (SEQ ID NO: 1 1 ), GRRPLPPPISGG (SEQ ID NO: 8) or GRRPVGGISGG (SEQ ID NO: 6). In a particular embodiment, the peptide of the present invention comprises or consists of the amino acid sequence GRRPLGGISGG (SEQ ID NO: 3).

The present invention relates to the following non-limiting specific examples for the peptides described and provided herein. In particular, the inventive peptides may comprise or consist of an amino acid sequence selected from the group consisting of:

GRRPLGGISGG (SEQ ID NO: 3);

GRRPVGGAAGG (SEQ ID NO: 1 1 ); GRRPLPPPISGG (SEQ ID NO: 8);

GRRPVGGISGG (SEQ ID NO: 6);

GRRPVPPPISGG (SEQ ID NO: 9);

GRRPLGGAAGG (SEQ ID NO: 10);

GRRPLPPPAAGG (SEQ ID NO: 12);

GRRPVPPPAAGG (SEQ ID NO: 13);

GRRPLGGASGG (SEQ ID NO: 14);

GRRPVGGASGG (SEQ ID NO: 15);

GRRPLPPPASGG (SEQ ID NO: 16);

GRRPVPPPASGG (SEQ ID NO: 17);

GRRPLGGIAGG (SEQ ID NO: 18);

GRRPVGGIAGG (SEQ ID NO: 19);

GRRPLPPPIAGG (SEQ ID NO: 20);

GRRPVPPPIAGG (SEQ ID NO: 21 );

GARPLGGISGG (SEQ ID NO: 22);

GARPVGGISGG (SEQ ID NO: 23);

GARPLPPPISGG (SEQ ID NO: 24);

GARPVPPPISGG (SEQ ID NO: 25);

GARPLGGAAGG (SEQ ID NO: 26);

GARPVGGAAGG (SEQ ID NO: 27);

GARPLPPPAAGG (SEQ ID NO: 28)

GARPVPPPAAGG (SEQ ID NO: 29);

GARPLGGASGG (SEQ ID NO: 30);

GARPVGGASGG (SEQ ID NO: 31 );

GARPLPPPASGG (SEQ ID NO: 32);

GARPVPPPASGG (SEQ ID NO: 33);

GARPLGGIAGG (SEQ ID NO: 34);

GARPVGGIAGG (SEQ ID NO: 35);

GARPLPPPIAGG (SEQ ID NO: 36); or

GARPVPPPIAGG (SEQ ID NO: 37).

In one embodiment of the invention, the peptide for use in treating PGD comprises any one of the sequences of SEQ ID NO: 3, SEQ ID NO: 1 1 , SEQ ID NO: 8 or SEQ ID NO: 6. For example, the peptide described herein may comprise SEQ ID NO: 3. In context of the invention, "treating" also refers to "preventing". Accordingly, a peptide comprising any one of the sequences of SEQ ID NO: 3, SEQ ID NO: 1 1 , SEQ ID NO: 8 or SEQ ID NO: 6 may also be used to prevent PGD.

In a specific embodiment, the peptide for use in treating and/or preventing PGD consists of any one of the following sequences:

GRRPLGGISGG (SEQ ID NO: 3);

GRRPVGGAAGG (SEQ ID NO: 1 1 );

GRRPLPPPISGG (SEQ ID NO: 8);

GRRPVGGISGG (SEQ ID NO: 6);

GRRPVPPPISGG (SEQ ID NO: 9);

GRRPLGGAAGG (SEQ ID NO: 10);

GRRPLPPPAAGG (SEQ ID NO: 12);

GRRPVPPPAAGG (SEQ ID NO: 13);

GRRPLGGASGG (SEQ ID NO: 14);

GRRPVGGASGG (SEQ ID NO: 15);

GRRPLPPPASGG (SEQ ID NO: 16);

GRRPVPPPASGG (SEQ ID NO: 17);

GRRPLGGIAGG (SEQ ID NO: 18);

GRRPVGGIAGG (SEQ ID NO: 19);

GRRPLPPPIAGG (SEQ ID NO: 20);

GRRPVPPPIAGG (SEQ ID NO: 21 );

GARPLGGISGG (SEQ ID NO: 22);

GARPVGGISGG (SEQ ID NO: 23);

GARPLPPPISGG (SEQ ID NO: 24);

GARPVPPPISGG (SEQ ID NO: 25);

GARPLGGAAGG (SEQ ID NO: 26);

GARPVGGAAGG (SEQ ID NO: 27);

GARPLPPPAAGG (SEQ ID NO:28)

GARPVPPPAAGG (SEQ ID NO: 29);

GARPLGGASGG (SEQ ID NO: 30);

GARPVGGASGG (SEQ ID NO: 31 ); GARPLPPPASGG (SEQ ID NO: 32);

GARPVPPPASGG (SEQ ID NO: 33);

GARPLGGIAGG (SEQ ID NO: 34);

GARPVGGIAGG (SEQ ID NO: 35);

GARPLPPPIAGG (SEQ ID NO: 36); or

GARPVPPPIAGG (SEQ ID NO: 37).

In one embodiment of the invention, the peptide for use in treating PGD consists of any one of the sequences of SEQ ID NO: 3, SEQ ID NO: 1 1 , SEQ ID NO: 8 or SEQ ID NO: 6. For example, the peptide provided herein may consist of SEQ ID NO: 3. As described herein, a peptide consisting of any one of the sequences of SEQ ID NO: 3, SEQ ID NO: 1 1 , SEQ ID NO: 8 or SEQ ID NO: 6 may also be used to prevent PGD.

As already mentioned, in accordance with the present invention, a peptide comprising or consisting of any one of the above particular sequences may further comprise a moiety X₇ at the C-terminus as defined herein.

Methods for synthesizing peptides are know in the art and comprise, e.g., standard FMOC-synthesis as described in the literature (e.g., solid phase peptide synthesis - "A practical approach" by E. Atherton, R.C. Sheppard, Oxford University press 1989) or liquid phase synthesis, where the peptides are assembled using a mixed strategy by BOC-chemistry and fragment condensation as described in the literature (E. Wunsch, "Synthese von Peptiden" in "Methoden der organischen Chemie" (Houben-Weyl), 15 Ausg. 4, Teil 1 und 2 Thieme, Stuttgart, 1974). Another method for synthesizing the peptide(s) is the generation of transgenic cells which express the peptide(s) of the invention. After expression of a desired peptide by a transgenic cell, said peptide may be purified.

The present invention also relates to a polynucleotide for use in treating and/or preventing PGD, wherein the polynucleotide encodes the peptide described herein. These polynucleotides may be nucleic acid analogues such as, e.g., DNA molecules, RNA molecules, oligonucleotides thiophosphates, substituted ribo- oligonucleotides, LNA molecules, PNA molecules, GNA (glycol nucleic acid) molecules, TNA (threose nucleic acid) molecules, or morpholino polynucleotides. Furthermore, the term "polynucleotide" is to be construed equivalently with the term "nucleic acid molecule" in context of the present invention and may inter alia refer to DNA, RNA, PNA or LNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 5,525,71 1 , US 4,71 1 ,955, US 5,792,608 or EP 302175 for examples of modifications). Nucleic acid residues comprised by the polynucleotides described and provided herein may be naturally occurring nucleic acid residues or artificially produced nucleic acid residues. Examples for nucleic acid residues are adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), xanthine (X), and hypoxanthine (HX). As understood by the person of skill in the art, thymine (T) and uracil (U) may be used interchangeably depending on the respective type of polynucleotide. For example, as the skilled person is aware of, a thymine (T) as part of a DNA corresponds to an uracil (U) as part of the corresponding transcribed mRNA. The polynucleotides described and provided herein may be single- or double-stranded, linear or circular, natural or synthetic. It is prioritized in context of the invention that the peptides are linear.

The present invention further relates to pharmaceutical compositions for use in treating PGD comprising the peptide described herein and/or the polynucleotide as described and provided herein. In context of the present invention, said "pharmaceutical composition(s)" are medicaments. Such pharmaceutical compositions may be administered to a subject in need of medical intervention of PGD. Thus, the invention also relates to a method of treating and/or preventing primary graft dysfunction by administering an effective dose of the peptide as provided herein, the polynucleotide as provided herein, or the pharmaceutical composition as provided herein to a subject in need of such treatment. In context of the present invention, a subject may be a mammal, e.g., a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine species, horse, camel, primate or a human being. It is prioritized that the subject is a human being.

As explained herein above and below, the present invention relates to peptides, polynucleotides and methods for use in treating and/or preventing primary graft dysfunction. In one embodiment of the invention the graft is a syngeneic, an allogeneic or a xenogeneic graft. "Syngenic" or "syngeneic" means genetically identical, or sufficiently identical and immunologically compatible as to allow for transplantation. For instance, a syngeneic graft may be a graft transplanted from an identical twin. A syngeneic graft is also known as an "isograft". A "xenogeneic" graft is derived from a different species as the recipient. In context of the invention, the graft may also be an autologous graft, which is collected from the same patient on whom it will be used. An "allogeneic" graft (which is also called allograft, allogeneic transplant or homograft) is a graft which is transplanted from a donor who is of the same species as the recipient but genetically not identical with the recipient. An immune response against an allograft or xenograft is termed rejection. Most human tissue and organ transplants are allografts. Therefore, in context of the invention, it is prioritized that the graft is an allogeneic graft. Orthotopic transplantation means that a tissue or organ transplant is grafted into its normal place in the body. For example, during orthotopic lung transplantation, the previous lung is removed and the transplant is placed at that location (i.e. the location of the previous lung) in the body.

In context of the present invention, the graft may be cells, organs and/or tissues. It is prioritized that the graft is an organ or a tissue. In one particular aspect of the invention the graft is an organ. For example, the graft may be at least one organ selected from the group consisting of lung, kidney, heart, liver, pancreas and intestine. The intestine may be small intestine. In one aspect of the invention, the peptides of the invention may be used to treat and/or prevent PGD in recipients of lung, kidney, heart or liver. In a particular aspect of the invention, the herein provided peptides are used in the treatment and/or prevention of PGD, wherein the graft is lung or kidney. The graft may also be limbs, such as arms, legs, hands or feet. In addition, the graft can also be the uterus. In another aspect of the present invention, the graft is at least one tissue selected from the group consisting of a vein, an artery, skin, a valve, bone, eye tissue, amnion, connective tissue and a sinew. The eye tissue may be, e.g., corneal tissue. In addition, the graft may also be cranial tissue. It is also envisaged that a patient be treated with the peptide/polynucleotide/pharmaceutical composition as described and provided herein, wherein said patient receives more than one organ and/or tissue, such as two lungs or two kidneys. In addition, the peptides described herein may be used during blood transfusion to inhibit sensitization of the receiver^'s immune system.

One embodiment of the present invention relates to the peptide as provided herein, the polynucleotide as provided herein, the pharmaceutical composition as provided herein, or the method of treating PGD as described herein, wherein the peptide, the polynucleotide or the pharmaceutical composition is co-administered with at least one other active agent. Said other active agent may be at least one active agent selected from the group consisting of corticosteroids, cyclosporine, tacrolimus, sirolimus, methotrexate, azathiopine, mercatopurine, antibiotics, polyclonal antibodies, monoclonal antibodies, interferon, opioids, TNF binding proteins, mycophenolate, FTY720 and any other immunosuppressive drug.

In one embodiment of the invention the peptides of the invention are to be administered to the subject prior to the transplantation of the graft. In another embodiment, the transplantation of the graft and the administration of the inventive peptides are performed simultaneously. In a further embodiment the inventive peptides are to be administered after the graft has been transplanted into the subject. Accordingly, the herein provided inventive peptides may be administered in order to prevent PGD and/or in order to treat an already manifested PGD.

The present invention also relates to a method for the preparation of a pharmaceutical composition for use in treating primary graft dysfunction, comprising the following steps:

(a) contacting the peptide provided herein and/or the polynucleotide provided herein with a liquid carrier or a solid carrier;

(b) optionally, adjusting the pH and/or the osmolarity of the product obtained in step (a);

(c) optionally, sterilizing the product obtained in step (a) or (b); and

(d) formulating and/or packaging the product obtained in step (a), (b) or (c) as a finished medical product. The resulting pharmaceutical composition may be in the form of a liquid solution, an erodible implant, a pill, a tablet, a capsule, a thin film, a powder, a solid crystal or liposomes. In the herein described method for the preparation of a pharmaceutical composition, the carrier may be a carrier selected from the group consisting of water, saline (e.g. physiological saline), Ringer's solution, dextrose solution, a fixed oil, ethyl oleate, liposomes and an organ preservation solution. The organ preservation solution may be, e.g., Perfadex^® (Perfadex, or a corresponding preservation solution may comprise or consist of 5% dextran 40 (Mw 40.000), Na⁺ 138 mmol, K+ 6 mmol, Mg ²⁺ 0.8 mmol, CI^" 142 mmol, SO ₄ ^2" 0.8 mmol, H₂PO 4-plus HPO4 2-0.8 mmol and glucose 5 mmol per 1000 ml). Other useful preservation solutions are known in the art.

In step (b) of the herein described preparation method, the pH may be adjusted to be the pH of blood (e.g. a pH of 7.35-7.45). In addition, in step (b) of the preparation method, the osmolarity of the product may be adjusted to be isotonic with blood. For example, the NaCI content may be adjusted to be isosmotic with blood. In accordance with text books, Na⁺⁺ in blood is in the range of about 135-144 mmol/l.

In step (c) of the above described method, the medical product is sterilized. Methods for sterilization are known in the art. For example, sterilization may be accomplished by, e.g., filtration through sterile filtration membranes (e.g., 0.2 micron membranes).

In step (d) of the herein described production method, formulating of the pharmaceutical product is conducted. For example, the product may be shaped into the desired formulation (e.g. into a pill, a tablet, a capsule, a thin film, a powder or a solid crystal). Finally, the pharmaceutical product may be packaged. For example, the pharmaceutical product may be placed into a container having a sterile access port, for example, an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. Alternatively, the pharmaceutical product may be packed in unit or multi-dose containers, for example, sealed ampoules or vials. The method for the preparation of a pharmaceutical composition as described herein results in the production of a finished medical product. Such a product is a product which is ready for administration, sale and distribution. In addition, the package of the pharmaceutical composition may comprise instructions regarding the use of the pharmaceutical composition.

The inventive peptides or the pharmaceutical compositions as described herein may be administered to a subject in need of medical intervention of PGD in an amount of about 1 ng/kg body weight to about 100 mg/kg body weight per day. For example, the pharmaceutical composition for use in treating and/or preventing PGD may be administered to the subject in an amount of about 1 pg/kg body weight to about 40 mg/kg body weight per day, or about 1 mg/kg body weight to about 30 mg/kg body weight per day, or about 1 mg/kg body weight to about 20 mg/kg body weight per day, or about 1 mg/kg body weight to about 15 mg/kg body weight per day, or about 1 mg/kg body weight to about 10 mg/kg body weight per day, about 10 mg/kg body weight to about 15 mg/kg body weight per day, or about 1 pg/kg body weight to about 1 mg/kg body weight per day.

As described herein, the inventive peptides or pharmaceutical compositions may be injected into the organ/tissue to be grafted (i.e. the organ/tissue to be grafted may be flushed with the inventive peptides or pharmaceutical compositions). The peptides or pharmaceutical compositions as described herein may be administered to the organ/tissue to be grafted in an amount of about 1 ng/kg organ/tissue weight to about 10.000 mg/kg organ/tissue weight. For example, the peptide or pharmaceutical composition for use in treating and/or preventing PGD may be administered to the organ/tissue to be grafted in an amount of about 1 pg/kg organ/tissue weight to about 100 mg/kg organ/tissue weight, in an amount of about 100 pg/kg organ/tissue weight to about 40 mg/kg organ/tissue weight, or about 1 mg/kg organ/tissue weight to about 30 mg/kg organ/tissue weight, or about 1 mg/kg organ/tissue weight to about 20 mg/kg organ/tissue weight, or about 1 mg/kg organ/tissue weight to about 15 mg/kg organ/tissue weight, or about 1 mg/kg organ/tissue weight to about 10 mg/kg organ/tissue weight, or about 10 mg/kg organ/tissue weight to about 15 mg/kg organ/tissue weight, or about 1 pg/kg organ/tissue weight to about 1 mg/kg organ/tissue weight.

The peptides or pharmaceutical compositions of the invention may be administered once in a single dose or daily until the desired therapeutic effect is achieved. In a prioritized aspect of the invention, the peptides or pharmaceutical compositions of the invention are administered several times to the organ to be grafted and/or to the recipient. For example, the inventive peptides or pharmaceutical compositions may be administered to the recipient of the graft at four time points (e.g. 20 min prior to reperfusion, during reperfusion, 20 min after reperfusion and 60 min after reperfusion). In addition or alternatively, the inventive peptides or pharmaceutical compositions may be injected into the organ/tissue to be grafted (i.e. the donor organ/tissue may be flushed with the inventive peptides or pharmaceutical compositions).

An example of an infusion-therapy with the inventive peptides described herein in the medical use of treatment and/or prevention of primary graft dysfunction is a dosage range of 0.01 -1 mg/kg/h for 1 day or for 3 days or for 7 days. Again, further dosages and administration schemes are envisages.

In context of the present invention, the pharmaceutical composition for use in treating PGD comprising the herein provided peptides and/or polynucleotides, may further comprise a pharmaceutically acceptable carrier, excipient and/or diluent. In addition, the pharmaceutical composition for use in treating PGD may further comprise vectors and/or host cells as described herein below. Accordingly, the present invention also relates to a pharmaceutical composition for use in treating PGD comprising peptides, polynucleotides, vectors and/or host cells as described and provided herein and, optionally, further comprising a pharmaceutically acceptable carrier, excipient and/or diluent. Generally, examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. As described above, the pharmaceutical compositions of the invention can be administered to the subject at a suitable dose, i.e. about 1 pg/kg body weight to about 40 mg/kg body weight per day, or about 1 mg/kg body weight to about 30 mg/kg body weight per day, or about 1 mg/kg body weight to about 20 mg/kg body weight per day, or about 1 mg/kg body weight to about 15 mg/kg body weight per day, or about 1 mg/kg body weight to about 10 mg/kg body weight per day, or about 10 mg/kg body weight to about 15 mg/kg body weight per day or about 1 pg/kg body weight to about 1 mg/kg body weight per day. Furthermore, also doses below or above of the exemplary ranges described hereinabove are envisioned, especially considering the aforementioned factors. The skilled person knows that the effective amount of pharmaceutical compositions administered to an individual will, inter alia, depend on the nature of the compound. For example, the dose may be further decreased or increased as subject to therapeutic discretion, in particular if concomitantly certain lipids are applied or if the peptide is subject to certain chemical modifications. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

The peptides of the present invention may also be used in combinations of two or more peptides provided herein. Accordingly, the pharmaceutical compositions of the present invention may comprise two or more peptides provided herein, optionally also in combination with other compounds described and provided herein. Moreover, the peptides of the present invention may be used in co-therapy in conjunction with vasoactive agents such as nitric oxide, prostacyclin, exogenous surfactants, anticoagulants, agents targeting tissue factor activity, agents with the potential to improve alveolar fluid clearance such as 2-agonists, agents inhibiting TNF actions, anti-IL-8 and anti-CD40L therapies (Curr Med Chem, 2008, 15(19): 191 1-1924), inhaled activated protein C (Crit. Care, 2010, 14(2): R70), immunosuppressants such as glucocorticosteroids or cyclosporine, antibiotics, HES solutions, colloids used for volume expansion (Emerg Med J, 2003, 20: 306-315), or agents targeting pathologic imbalance of the renin-angiotensin system.

In addition, the peptides, polynucleotides or pharmaceutical compositions provided herein may be used in co-therapy in conjunction with active agents which prevent ischemia-reperfusion injury and/or delayed graft function. Such active agents are, for example, disclosed in Lancet, 2004, 364(9447): 1814-1827. In particular, the herein described peptides, polynucleotides and pharmaceutical compositions may be used in co-therapy with preservation solutions (e.g. University of Wisconsin solution, histidine-tryptophan-ketoglutarate solution or celsior solution), agents which affect recipient fluid management (e.g. fluid expansion with colloid or crystalloids, mannitol or furosemide), vasodilatory agents (e.g. calcium-channel blockers, prostacyclin, atrial natriuretic peptide or selective or non-selective endothelin receptor antagonists), antioxidants (e.g. agents that result in heme- oxygenase-1 induction or overexpression in the graft, N-acetylcysteine, propionyl-L- carnitine or inhibitors of inducible NO synthase), anti-inflammatory agents (e.g. antagonists of platelet activating factor receptor, monoclonal antibodies to TNFa, inhibitors or antagonists of cytokines [e.g. interleukins 1 , 10, and 13, CXCL-8 or MCP-1], monoclonal antibodies to ICAM1 and leucocyte function-associated antigen 1 , soluble P-selectin glycoprotein ligands, immunosuppressants [e.g. CTLA4-lg fusion proteins or mycophenolate mofetil], complement inhibitors or statins) or growth factors (e.g. insulin-like growth factor).

Administration of the pharmaceutical compositions for use in treating and/or preventing PGD, the peptides for use in treating and/or preventing PGD or the polynucleotides for use in treating and/or preventing PGD may be effected by different ways, e.g., parenterally (e.g., intravenously, subcutaneous, transdermal^, intramuscularly or intraperitoneally), via inhalation (e.g., intrabronchially), as an erodible implant made of biodegradable polymers (e.g., polylactate or polyglycolate) or enterally (e.g., pill, tablet, [e.g. buccal, sublingual, orally or disintegrating], capsule, thin film, liquid solution suspension, powder, solid crystals or liquid), rectally (e.g., suppository, enema), transdermal^, topically, vaginally, epicutaneously or intranasally.

The pharmaceutical compositions for use in treating and/or preventing PGD comprising the inventive peptides, polynucleotides, vectors and/or host cells as described and provided herein may be administered locally or systemically. The pharmaceutical compositions, polynucleotides or peptides for use in treating and/or preventing PGD may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

As described herein above, the pharmaceutical compositions, polynucleotides and/or peptides of the invention may be administered by different ways, e.g., parenteral, subcutaneous, intraperitoneal, topical, intrabronchial, intrapulmonary, intranasal or by infusion or injection. In a particular aspect of the invention the herein described pharmaceutical compositions, peptides or polynucleotides are flushed extra-corporal through the donor organ vessels or injected into donor tissue; or are administered intravenously, subcutaneously or intramuscularly to the recipient.

Accordingly, one embodiment of the invention relates to the peptide for use in treating and/or preventing PGD, the polynucleotide for use in treating and/or preventing PGD, the pharmaceutical composition for use in treating and/or preventing PGD, or the method of treating and/or preventing PGD, wherein the peptide for use in treating and/or preventing PGD, the polynucleotide for use in treating and/or preventing PGD or the pharmaceutical composition for use in treating and/or preventing PGD is flushed extra-corporal through the donor organ vessels or injected into donor tissue.

Flushing an active agent (e.g. the peptide for use in treating and/or preventing PGD as described herein) extra-corporal through the donor organ vessels means that the transplanted organ is treated ex-vivo with said active agent (e.g. with the peptide for use in treating and/or preventing PGD). In particular, the organ to be transplanted is removed from the donor and afterwards the active agent (e.g. the peptide for use in treating and/or preventing PGD) is injected into the vessels of the organ ex vivo. As described herein, the active agent may also be the polynucleotide or the pharmaceutical composition provided herein. In addition or alternatively to ex vivo flushing, the peptides provided herein may be infused into cadaveric donors, i.e. intravascular into the full donor organism; afterwards the respective organ is taken for grafting.

As mentioned, another embodiment of the invention relates to the herein described peptide, polynucleotide, pharmaceutical composition, or method of treating and/or preventing PGD, wherein the peptide, the polynucleotide or the pharmaceutical composition is administered intravenously, subcutaneously or intramuscularly to the recipient.

The pharmaceutical composition described and provided herein may be also administered by (a) sustained release system(s). Suitable examples of sustained- release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP-A1 58481 ), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Biopolymers, 1983, 22: 547-556), poly (2- hydroxyethyl methacrylate) (J Biomed Mater Res, 1981 , 15: 167-277; Langer, Chem Tech, 1982, 12: 98-105), ethylene vinyl acetate (Langer, loc. cit.) or poly-D-(-)-3- hydroxybutyric acid (EP-A1 133988). Sustained release pharmaceutical compositions may also include liposomally entrapped compounds. Liposomes containing the pharmaceutical composition may be prepared by methods known in the art, such as described in DE 3218121 ; Proc Natl Acad Sci USA, 1985, 82: 3688- 3692; Proc Natl Acad Sci USA 77: 4030-4034, 1980; EP-A1 52322; EP-A1 36676; EP-A1 88046; EP-A1 143949; EP-A1 142641 ; Japanese Pat. Appl. 83-1 18008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP-A1 102324.

In context of the present invention, the formulations described herein may be prepared by contacting the components of the pharmaceutical composition for use in treating and/or preventing PGD uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product may be shaped into the desired formulation. The carrier may be a parenteral carrier, e.g., a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be useful herein, as well as liposomes as described herein. The carrier may suitably contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are preferably non-toxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

In context of the present invention, the components of the pharmaceutical composition to be used for therapeutic administration are preferably sterile. Sterility may readily be accomplished by, e.g., filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The components of the pharmaceutical composition ordinarily may be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As a non-limiting example of a lyophilized formulation, 10-ml vials may be filled with 5 ml of sterile-filtered 1 % (w/v) aqueous solution, and the resulting may be is lyophilized. The infusion solution may be prepared by reconstituting the lyophilized compound(s) using bacteriostatic Water-for-lnjection. As described herein, the polynucleotides for use in treating and/or preventing PGD described and provided herein may be cloned into a vector. Thus, the present invention also relates to a vector for use in treating and/or preventing PGD comprising the polynucleotide as described and provided herein. The term "vector" as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In a preferred embodiment, these vectors are suitable for the transformation of cells, like fungal cells, cells of microorganisms such as yeast or prokaryotic cells. In a particularly preferred embodiment, such vectors are suitable for stable transformation of bacterial cells, for example to express the polynucleotides for use in treating and/or preventing PGD of the present invention.

Accordingly, in one aspect of the invention, the vector for use in treating and/or preventing PGD as provided herein is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is preferably at least one restriction site or a polylinker which enables the insertion of a nucleic acid sequence/molecule desired to be expressed.

The expression vector may comprise a promoter suitable to be employed in context of this invention, for example for the expression of a polynucleotide as described hereinabove. The nucleic acid construct to be expressed may be inserted into that vector. The skilled person knows how such insertion can be put into practice.

Non-limiting examples for the vector into which a polynucleotide for use in treating PGD as described and provided herein may be cloned are adenoviral, adeno- associated viral (AAV), lentiviral, HIV-based lentiviral, or nonviral minicircle-vectors. Further examples of vectors suitable to comprise the polynucleotide of the present invention to form the vector described herein are known in the art and are, for example, other vectors for bacterial and eukaryotic expression systems. Furthermore, as described herein, the herein described polynucleotides and/or vectors may be transduced, transformed or transfected or otherwise introduced into a host cell. Thus, the present invention also relates to a host cell comprising the polynucleotide and/or the vector as described and provided herein. For example, the host cell may be a prokaryotic cell, for example, a bacterial cell. As a non-limiting example, the host cell may also be a mammalian cell or an insect cell. The host cell described herein is intended to be particularly useful for generating the peptides for use in treating and/or preventing PGD as described and provided herein. Generally, the host cell described herein may be a prokaryotic or eukaryotic cell, comprising the polynucleotide or the vector described and provided herein or a cell derived from such a cell and containing the nucleic acid construct or the vector. In one embodiment, the host cell is genetically modified with the polynucleotide or the vector described and provided herein in such a way that it contains the polynucleotide for use in treating and/or preventing PGD integrated into the genome. For example, such host cells described herein may be bacterial, yeast, insect, mammalian or fungus cells. In one particular aspect, the host cell may be capable to express or expresses a polynucleotide for use in treating and/or preventing PGD of the present invention. An overview of examples of different corresponding expression systems to be used for generating the host cell described herein is for instance contained in Methods in Enzymology 153, 1987, 385-516, in Bitter (Methods in Enzymology 153, 1987, 516-544), in Sawers (Applied Microbiology and Biotechnology 46, 1996, 1 -9), Billman-Jacobe (Current Opinion in Biotechnology 7, 1996, 500-4), Hockney (Trends in Biotechnology 12, 1994, 456- 463), and in Griffiths, (Methods in Molecular Biology 75, 1997, 427-440). The transformation or genetically engineering of the host cell with a polynucleotide or vector described and provided herein can be carried out by standard methods, as for instance described in Sambrook and Russell, 2001 , Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. In context of the present invention, the peptides, polynucleotides, vectors, host cells, compositions and pharmaceutical compositions described and provided herein may be used in treating or preventing PGD

For example, the present invention relates to a peptide comprising or consisting of the sequence GRRPLGGISGG (SEQ ID NO: 3) for use in treating or preventing PGD.

As described above, the present invention further relates to a method of treating or preventing PGD. Such a method particularly comprises the administration of an effective dose of (a) peptide(s), polynucleotide(s), vector(s), host cell(s), composition(s) and/or pharmaceutical composition(s) described and provided herein to a subject in need of the treatment or prevention of PGD. In one embodiment, the subject is human.

As mentioned above, primary graft dysfunction (PGD) is an ischemia-reperfusion injury occurring in the early period (i.e. within the first 72 h) following transplantation (see, e.g., Oto, Am J Transpl, 2008; 8: 2132-9). The cause of PGD is the change from hypoxia to normal (i.e. physiologic, normal physiological oxygen tension of about 92% to about 98%) oxygen tension. PGD is most often described in transplanted lung, liver, or kidney, but it can occur in all tissues and organs exposed to ischemia followed by reconnection to the blood circulation. Accordingly, PGD can be diagnosed by diagnosing ischemia in any grafted organ and/or tissue. For example, PGD can be diagnosed by diagnosing ischemia in a grafted heart, lung, kidney, liver, pancreas and/or in grafted intestine. In addition, PGD can be diagnosed by diagnosing ischemia in a grafted valve and/or in grafted skin, amnion, or connective tissue (in particular within the first 72 h after transplantation). In addition, also in a transplanted vein, artery or sinew or in transplanted bone or eye tissue, PGD may be diagnosed by diagnosing ischemia.

Regarding lung, kidney and heart transplants a more detailed definition of PGD exists which is disclosed in Oto, Am J Transpl, 2008; 8: 2132-9 and discussed herein in the following. For lung transplants, the PGD grading system of the International Society for Heart and Lung Transplantation which is based on the PaO₂ FiO₂ ratio and chest radiograph infiltrates can be used to diagnose PGD and the assessment may be performed at 6 h after transplantation (Christie, J Heart Lung Transplant 2005; 24: 1454-1459; oto, J Heart Lung Transplant 2007; 26: 431-436). PGD grade 3 representing a PaO₂ FiO₂ ratio < 200 with radiographic infiltrates, including the requirement of extracorporeal membrane oxygenation or inhaled nitric oxide beyond 48 h after transplantation, may be considered as PGD. However, in context of the invention, also PGD of grade 1 or 2 may be considered as PGD. When there are two single lung recipients from one donor, it may be defined as PGD if both recipients develop PGD (Oto, J Thorac Cardiovasc Surg 2006; 132: 1441-1446). However, in context of the invention, when there are two single lung recipients from one donor, it may also be defined as PGD if one recipient develops PGD. In addition, in context of the invention, if one recipient receives two lungs, it may be defined as being PGD if one or both lungs develop PGD.

For kidney transplants, PGD may be defined as no-immediate post-transplant graft function, as represented by no spontaneous fall in serum creatinine within the first 72 h, including a requirement for hemodialysis (Oto, Am J Transpl, 2008; 8: 2132-9 and Cecka, Clin Transplant 2004; 1-16; McDonald, Am J Transplant 2007; 7: 1201- 1208,). Also a severe loss of renal function as well as a steep rise in blood urea nitrogen (BUN) within 24-72h post-ischemia indicates PGD in kidney recipients.

For heart transplants, PGD can be defined as the requirement for a mechanical assist device (e.g. ventricular assist devices, extracorporeal membrane oxygenation and intraaortic balloon pumping) within 72 h after transplantation, excluding prophylactic use of an intraaortic balloon pump (Potapov, Transplantation 2001 ; 71 : 1394-1400; Lima, Circulation 2006; 1 14: 127-132;. Alternatively, a more detailed heart PGD definition may be used as described in Marasco, J Heart Lung Transplant 2005; 24: 2037-2042. It is known in the art that there is a significant correlation between PGD defined by the two definitions of PGD in heart recipients (Oto, Am J Transpl, 2008; 8: 2132-9). The peptides described and provided herein are capable of inhibiting activity of a Rho GTPase, e.g., RhoA. A method for measuring whether a given peptide is capable of inhibiting activity of a Rho GTPase is described in the following. Rho GTPase containing material (e.g. a cell lysate) may be contacted with the peptide to be tested and the Rho GTPase activity may be analysed and compared to a control sample which is not contacted with the peptide to be tested. Methods for assessing Rho GTPase activity are known in the art and as described and exemplified herein. Non-limiting examples for methods suitable for assessing Rho GTPase activity include determination of global Rho GTPase activity as described in J Biol Chem, 2004, 279: 7169-7179. Such an assay may be performed by using Rho substrates (e.g., Rhotekin) tagged with GST which are mixed into cell lysates, followed by a pull down using anti-GST antibodies. Detection may be carried by gel electrophoresis and Western blot using anti-Rho antibodies as known in the art. Another suitable method for assessing Rho GTPase activity is a G-LISA assay as described in Basic Res Cardiol, 2009, 104: 333-340. Still another way to assess Rho GTPase activity may be the determination of site spatio-temporal Rho activation within a given cell by using Rho GTPase activation biosensors such as GFP-effector sensors or unimolecular or bimolecular FRET sensors transfected or recombinantly expressed in a given cell. These biosensors allow spatio-temporal in vivo imaging of individual active Rho GTPases (J Cell Science, 2010, 123: 1841 - 1850). Also, commercial kits for assessing RhoGTPase activity are available such as, e.g., "RhoGEF Exchange Assay Biochem Kit" from Cytoskeleton, Inc. For example, a given peptide may be considered a peptide of the present invention (1 ) if it comprises or consists of a sequence as defined herein, and (2) if it decreases Rho GTPase (e.g., RhoA) activity of a test cell at least 1 .5-fold, at least 2-fold, at least 2.5-fold or at least 3-fold compared to the respective Rho GTPase activity (e.g., RhoA) of a reference cell (belonging to the same cell line) not treated with the peptide.

As mentioned, in context of the present invention it has been identified that Rho GTPase-inhibiting peptides are able to treat and/or prevent PGD. This is surprising since, as discussed in the following, Rho GTPases are involved in a sophisticated network and they play a role in several different diseases.

The fact that the herein described peptides are able to inhibit Rho GTPases does not imply that these peptides are useful in the treatment and prevention of PGD, since Rho GTPases are involved in several different regulative pathways. For example, Rho GTPases control many aspects of cell behaviour such as the organization of the cytoskeleton, cell migration, cell cycle progression, cell proliferation, cell differentiation, gene expression, cell survival and apoptosis (Nature, 2006, 440(7087):1069-1072; Curr Drug Targets, 2010, 1 1 (9): 1043-1058). A main aspect in their function is the control of permeability of vascular (Cardiovasc Res 2010, 87(2): 243-253; Thromb Haemost 2010, 103(1 ): 40-55; Am J Physiol Lung Cell Mol Physiol, 2005, 288(2): L294-L306) and epithelial surfaces (Am J Pathol, 2010, 177(2): 512-524; Physiology (Bethesda), 2010, 25(1 ): 16-26). Because of their central role in regulating permeability, the activation of Rho GTPases is decisive for many pathophysiological processes associated with a breakdown in epithelial and endothelial barrier function. Statins and bisphosphonates are substances which affect biosynthesis of isoprenoids and, thus, prevent lipid modification of Rho GTPases which are necessary for their activation as described below. Statins and bisphosphonates are tested in clinical studies as pharmaceuticals against cancer and cardiac diseases as well as for their capability to improve disturbed vascular and epithelial barrier function. For example, Fasudil is a Rho-kinase inhibitor which is used in vasospasms of brain arteries and lung hypertension. Furthermore, a VE-cadherin binding, fibrin-derived protein, Ββι_{5- 2}, has been described to stabilize endothelial barriers via inhibition of the Rho GTPase RhoA (PloS ONE, 2009, 4(4): e5391 ).

Because of their central role in cell biology, the activity of Rho GTPases is strictly controlled. Rho GTPases cycle between an inactive, GDP-bound, state and an active GTP-bound state. Rho GTPases can interact with their effector molecules and affect their functions only in the GTP-bound form. Most GTPases in active form are bound to the cell membrane. This membrane-targeting is mediated by C- terminal polybasis region and a post-translational isoprenylation of the Rho GTPases. The active state exists only for a limited time as due to the hydrolyse- activity of Rho GTPases, the bound GTP is quickly converted to GDP. The GDP- bound state is more stable, therefore the major part of cellular Rho GTPases is inactive. So-called guanidine dissociation inhibitors (GDIs) mask the membrane- targeting sequences of the Rho GTPases and stabilize the GDP-bound conformation. The activation of RhoGTPases is mediated by specific guanine- nucleotide-exchange factors (GEFs), which catalyze the exchange of GDP for GTP; see also herein below. GEFs enhance the rate of dissociation of GDP and stabilize the nucleotide-free form of Rho GTPases. Since GTP is present in the cell in high molecular excess, the binding of GTP is favored. GTP-binding evokes a conformational change of the Rho GTPase such that the GEFs dissociate. The balance between active and inactive Rho GTPAses is further regulated by another group of regulatory proteins, the GTPase-activating proteins (GAPs). GAPs increase the intrinsic Rho GTPAse hydrolyse activity of the Rho GTPases and, thus favour their inactivation (Trend Cell Biol, 2008, 18: 210-219). In their active form, Rho GTPases interact with high affinity with one of several downstream effectors. The active state is very transient; it is terminated by hydrolysis of GTP to GDP, a reaction that is stimulated by GAPs. In addition, guanine nucleotide dissociation factors stabilize the inactive form of Rho GTPases (Genes Dev. 1997; 1 1 : 2295- 2322; Biochem Soc Trans,2005; 33: 891 -895; Cell, 2004; 1 16: 167-179).

One possibility to control context-specific Rho GTPase activity is via guanine- nucleotide-exchange factors (GEFs). GEFs are upstream regulators of Rho GTPase activity. GEFs control Rho GTPase activity in a spatio-temporal- and partially context-specific manner (Nature Cell Biol, 201 1 , 13: 159-166). In other words, GEFs allow activation of Rho in a defined time and location within a given cell. They integrate and process multiple outer signals and are themselves strictly controlled. They act like interfaces linking incoming signals to certain Rho GTPase driven cell biologic responses (Trends Cell Biol 2008; 18:210-19). The regulatory features of GEFs are due to their multi-domain architecture. The first Rho GEF was isolated from lymphoma cells as transformed gene and was named Dbl (Nat Rev Mol Cell Biol, 2005, 6(2): 167-180). Meanwhile, the Dbl homology family comprises over 70 proteins. Most GEFs contain a highly conserved homology domain of about 200 amino acids which mediates the exchange of GDP and GTP in Rho GTPases. This domain is designated the DH-domain. The specificity of GEFs for a single or group of GTPases is conferred by this DH domain. The N-terminal domain is autoinhibitory, i.e. in the inactive state the N-terminus is phosphorylated and interacts with the DH-domain. Upon dephosphyorylation, the auto-inhibition is resolved (Trend Cell Biol, 2008, 18: 210-219; Protein Sci, 201 1 , 20: 107-1 1 1 ). Additionally, there is a second subfamily of GEFs comprising 1 1 members which do not carry a DH domain. Instead of a DH domain, they contain two homology regions, namely DHR1 and DHR2 (dock homology region 1 and 2) (Trends Cell Biol, 2008, 18: 210-219; J Cell Sci, 2005, 1 18: 4937-4946; Nat Rev Mol Cell Biol, 2005, 6(2): 167-180). GEFs bind the Rho GTPases via their homology domain(s) and, thus, assist exchange of GDP with GTP.

GEFs comprise a Pleckstrin-Domain (PH-Domain) close to the DH domain. The PH domain is involved in catalytic activity and mediation of protein-protein interaction. Together, the DH and the PH domain provide the minimal structure that is required for GTPase activation. The PH-domain is involved in the subcellular distribution of GEFs and in regulating activity (Genes Dev, 2002, 16: 1587-1609; Nat Rev Mol Cell Biol, 2005, 6(2): 167-180). For example, GEF-H1 is inactive when it is associated with microtubuli and tight junctions. In the active state, GEF-H1 relocates into the cytoplasm (Mol Biol Cell, 2008, 19(5): 2147-2153; Dev Cell, 2005, 8: 777-786).

GEF activity is controlled by intramolecular inhibition. The N-terminal domain of the GEFs functions as auto-inhibitor wherein intramolecular interaction is neutralized by phosphorylation. Thus, the target-GTPase can interact with the DH domain. Targeting of Rho GEFs at specific subcellular regions is also an important control mechanism of GEF activity. For example, inactive GEF-H1 is associated with microtubuli where it is bound to the inner membrane. In an active state, GEF-H1 dissociates and re-localizes in the cytoplasm (Trends in Cell Biol, 2008, 18: 210- 219).

GEF/RhoGTPases pathways regulate a number of central cell biologic processes such as organization of the cytoskeleton, gene expression, cell cycle progression and cell differentiation as well as apoptotic and non-apoptotic processes and cell motility, antigen presentation, and epithelial and endothelial permeability (Cardiovasc Res, 2010, 87(2): 243-253; Thromb Haemost, 2010, 103(1 ): 40-55; Am J Physiol Lung Cell Mol Physiol, 2005, 288(2): L294-L306; Am J Pathol, 2010, 177(2): 512-524; Physiology (Bethesda), 2010, 25(1 ): 16-26). Due to its central role in cell physiology, the dysregulation of GEF/ RhoGTPase pathways is a major component of pathophysiologic signal transduction in inflammatory diseases, endothelial and epithelial barrier dysfunction and cancer.

The GEF-H1/RhoA-pathway activates the cellular contractile apparatus consisting of actin and myosin and is required for junction dissociation (Mol Biol Cell, 2007, 18: 3429-3439). Conversely, the p1 14RhoGEF induced RhoA activation is required for tight junction assembly (Nat Cell Biol, 201 1 , 13(2): 159-166). A site and context- specific regulation of RhoA is decisive for maintenance of physiological barriers such as epithelial and endothelial layers. Endothelial and epithelial cells form continuous layers lining the inner lumen of blood vessels or the visceral cavities respectively. They form semi-permeable barriers and regulate the exchange of fluid and nutrients of neighboring tissues. A balanced RhoA activity is curial for physiologic epithelial and endothelial barrier function. Quiescent endothelial and epithelial cells show a basal RhoA activity, actin fibers and myosin bundles are restricted to the cell boarders to stabilize the tissue (Endothelial Biomedicine, Cambridge Press, 2007, 696-706; J Cell Biol, 1996, 133: 1403-1415). Stimulation with pro-inflammatory or pro-thrombotic agents results in activation of the GEF/RhoA pathway that in turn induces cyto-skeletal activation (J Cell Biol, 1996, 133: 1403-1415). Actin and myosin form contractile bundles that pervade the cells; as a result the cells constrict, neighboring cells loosen their contact. Opening of the cell-cell boarder is an important physiologic process e.g., in tissue proliferation and inflammatory processes to facilitate migration of inflammatory cells. But aberrant GEF/RhoA over-activation results in the breakdown of epithelial and /or endothelial barriers and is a major contributor to the patho-physiology of many serious diseases. (Adv Drug Deliv Rev, 2000, 41 : 329-40; Ann NY Acad Sci, 2008, 1 123: 134-45; Trends Cell Biol, 2008, 18: 210-219) For example, GEF-H1 inhibition prevents acute lung injury (ALI) caused by mechanical ventilation in a mouse model (Am J Physiol Lung Cell Mol Physiol, 2010, 298(6): L837-L848). For example, there are studies that suggest a contribution of myosin light chain kinase (MLCK) and Rho kinase to the development of systemic inflammatory response syndrome (SIRS) and capillary leak after burns. Both molecules, MLCK and Rho kinase, are downstream effectors of RhoA. It is shown that endothelial cells lose their barrier function upon re-incubation with plasma isolated from burned rats. The endothelial hyper- permeability can be reverted by treating the endothelial cells with a MLCK inhibitor (AM J Physiol Lung Cell Mol Physiol, 2004, 286: L841 -L847). Pharmacologic inhibition of MLCK after scald injury improves outcome in vivo (Shock, 2003, 20: 363-368). A knockout of MLCK-210 in mice reduces capillary leak and improves survival in a mouse model of burns (Shock, 2007, 28: 589-595). Inhibition of Rho kinase decreases the vascular leak after scald injury in vivo (Burns, 2003, 29(8): 820-827).

Accordingly, Rho GTPases are involved in a large and complicated network which is far from being unscrambled. However, as described above, in context of the present invention it has unexpectedly been identified that Rho GTPase-inhibiting peptides are useful in the treatment of PGD.

As used herein, the terms "treatment" and "treating" and the like also means "preventing" and "ameliorating" of a disease such as PGD. These terms are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term "treatment/treating" as used herein covers any treatment of a disease in a subject and includes: (a) preventing and ameliorating PGD from occurring in a subject which may be predisposed to the disease; (b) inhibiting PGD, e.g. arresting its development; or (c) relieving PGD, e.g. causing regression of PGD. In accordance with the present invention, the term "prevention" or "preventing" of an disease means the disease per se can be hindered of developing or to develop into an even worse situation. Accordingly, it is one aspect of the present invention that the herein described peptides can be employed in avoidance of PGD. In accordance with the present invention, the peptides as described herein may be employed before PGD develops.

As described herein, the peptides for use in treating PGD as described herein may also be employed in the amelioration and/or treatment of disorders wherein the diseased status has already developed, i.e. in the treatment of an existing PGD. Accordingly, the term "treatment/treating" as used herein also relates to medical intervention of an already manifested disorder, like the treatment of an already defined and manifested PGD. Thus, the present invention relates to the treatment or prevention of PGD by using the peptides as described herein, the polynucleotides as described herein or the pharmaceutical compositions as defined herein.

Herein, the term "peptide(s) for use in treating (and/or preventing) PGD" is used interchangeably with the terms "inventive peptide(s)", "peptide(s) of the (present) invention" "peptide(s) provided herein" "peptide(s) described herein" and "herein described peptide(s)" and relates to peptides for use in treating and/or preventing primary graft dysfunction (PGD) comprising or consisting of the amino acid sequence

GXi RPX₂X₃X4X₅GGX₆ (SEQ ID NO: 1 ) wherein

Xi is an amino acid selected from the group consisting of R and A;

X3 is either omitted or an amino acid sequence consisting of PPP;

X₄ is either omitted or an amino acid sequence consisting of GG;

Xe is either omitted or an amino acid sequence consisting of 1 to 5 amino acids; provided that if X₃ is omitted, than X₄ is not omitted.

As described above, the invention also encompasses polynucleotides encoding said peptides and pharmaceutical compositions comprising said peptides and/or said polynucleotides. Furthermore, herein, the term "treating" also encompasses "preventing". Therefore, the term "peptide/polynucleotide/pharmaceutical composition for use in treating PGD" also refers to a "peptide/polynucleotide/pharmaceutical composition for use in preventing PGD".

The Figures show:

Figure 1 : Ventilator-induced Lung Injury (VILI). Total cell counts, neutrophil counts, protein and IgM content served as surrogate parameter to asses barrier dysfunction. (^* p>0.05 ^**p>0.01 ; ^*** p>0.001 ). Abbreviations are: LPS: LPS inhalation; LVt: low volume tide ventilation; HVt: high volume tide ventilation, sc: scrambled peptide GGGGGSRRIPL (SEQ ID NO: 38); XIB1 -b: GRRPLGGISGG (SEQ ID NO: 3).

Figure 1 compares effects of LPS challenge followed by LVt or HVt on total cell counts, neutrophil counts, total protein content and IgM content within broncheoalveolar lavages in animals treated with sc or GRRPLGGISGG (SEQ ID NO: 3). In all groups, GRRPLGGISGG (SEQ ID NO: 3) reduces all parameters. Significance was obtained as indicated by asterices (^*p<0.5, ^***p<0.01 ).

Figure 2: Thrombin effects are antagonized by several different peptides of the present invention.

Figure 3: Cingulin-ZO-1 complexes following thrombin treatment.

Immunoprecipitation has been performed with an anti-ZO-1 antibody, western blot has been performed with an anti-cingulin antibody. Ββ_15- ₄₂/FX06 = GHRPLDKKREEAPSLRPAPPPISGGGYR (SEQ ID NO: 41 ).

Figure 4: The peptides of the present invention prevent PGD. Donor lungs have been flushed ex vivo with 1 mg GRRPLGGISGG (SEQ ID NO: 3) or GGGGGSRRIPL (random, SEQ ID NO: 38) in 20ml Perfadex. Then the recipient received four injections i.p., 4 mg/kg bodyweight each, at the following time points:

(T-20): 20min before reconnection of lung circulation

(T-0): at the time of reconnection

(T+20): 20min later than T-0

(T+60): 60min later than T-0.

In addition to the above, the rats received 25 mg urbason/rat i.p. as a single dose at time T=0.

The Figure shows the wet/dry ratio, 28 days after grafting the allogenic graft; n=8/group. Control lungs show PGD as reflected by persistent post-transplantation lung edema. Any symptoms of PGD are completely abrogated when animals were treated with GRRPLGGISGG (SEQ ID NO: 3).

Figure 5: The peptides of the present invention protect the transplanted organ during the grafting procedure. Control animals have sensibilized T cells targeting the MHC of the lung donor, whereas GRRPLGGISGG (SEQ ID NO: 3)-treated animals have no detectable allo-reactivity (back to baseline seen with naive cells). In particular, whereas lymphocytes of naive as well as GRRPLGGISGG (SEQ ID NO: 3)-treated rats to not proliferate in response to donor-derived dendritic cells, lymphocytes derived from control animals show alloreactivity (indicating reduced exposure of the recipient to donor tissue due to protection of the organ during the grafting/reperfusion period).

Figure 6: The peptides of the invention normalizes LPS-induced metabolic disturbance in response to LPS in macrophages. LPS treatment of macrophages induces a rapid and sustained extracellular acidification (Phase 1 , 0-30 min following LPS), which slowly returns to baseline (Phase 2, 30-180 min following LPS). Random peptides (GGGGGSRRIPL; SEQ ID NO: 38) as well as GHRPLDKKREEAPSLRPAPPPISGGGYR (B i_{5- 2}/FX06; SEQ ID NO: 41 ) do not change kinetics, whereas peptides GRRPVGGAAGG (SEQ ID NO: 1 1 ), GRRPLGGAAGG (SEQ ID NO: 10), GRRPLPPPISGG (SEQ ID NO: 8) and GRRPLGGISGG (SEQ ID NO: 3) induce a faster return to baseline (condition without LPS). The graphs show the mean rate of pH-change as extracellular acidification rate (ECAR) +/- standard deviation compared to control without LPS

Figure 7: The peptides of the invention reduce uptake of bacteria by macrophages. The peptides GRRPVGGAAGG (SEQ ID NO: 1 1 ) and GRRPLGGISGG (SEQ ID NO: 3) reduce uptake of Escherichia coli by macrophages as compared with random peptide and Ββι_5-42 ΡΧ06. (B i5_-42/FX06 = GHRPLDKKREEAPSLRPAPPPISGGGYR; SEQ ID NO: 41 )

Figure 8: Efficacy of the peptides of the invention in the treatment and prevention of PGD in kidney recipients. The Figure shows serum- creatinine (μηηοΙ/Ι). All groups showed an increase of s-creatinine after ischemia. Mice treaded with the peptides corresponding to SEQ ID NO: 1 as defined herein (i.e. GRRPLGGISGG (SEQ ID NO: 3) and GRRPLGGAAGG (SEQ ID NO: 10)) show significant less s-creatinine than mice treated with scrambled peptide.

Figure 9: 3-dimensional shape of the peptides GRRPGGISGG (SEQ ID NO: 42);

GRRPISGG (SEQ ID NO: 43) and GAAPGGISGG (SEQ ID NO: 44).

Figure 10: RhoA pull down. HUVEC grown to confluence were incubated with indicated peptides (20 g/ml each), thrombin (0.1 U/ml) or thrombin+peptides for 5 minutes or were left untreated. Active RhoA was pulled down. Bound proteins were separated on a 15% polyacrylamid gel and blotted on Nitrocellulose-Membrane. RhoA was detected with anti-RhoA antibody. Total RhoA contents were determined in western blots performed from the same lysates before pull downs were performed. The lines shown in this Figure represent the following samples:

Line 1 : untreated

Line 2: 0.1 U/ml thrombin

Line 3: 0.1 U/ml thrombin + GGGGGSRRIPL (SEQ ID NO: 38;

20Mg/ml)

Line 4: 0.1 U/ml thrombin +GRRPVPPPISGG (SEQ ID NO: 9;

20Mg/ml)

Line 5: 0.1 U/ml thrombin + GRRPLGGISGG (SEQ ID NO: 3;

20Mg/ml)

Line 6: 0.1 U/ml thrombin + GRRPLGGAAGG (SEQ ID NO: 10;

20Mg/ml)

Figure 11 : Intra-operative situs during bronchial suture anastomosis (A)

GRRPLGGISGG (SEQ ID NO: 3)-treated, no edema draining via the left main bronchus, (B) scrambled peptide-treated, severe fluid escaping through the left main bronchus.

Figure 12: Allo-grafted left lungs after reperfusion, homogenous pattern of reperfusion in GRRPLGGISGG (SEQ ID NO: 3)- treated animals (left), in-homogenous reperfusion in controls (right).

Figure 13: Oxygen saturation in % 120 minutes after opening of the vascular clips and reperfusion in grafted lungs.

The following examples illustrate the invention.

Example 1 : Peptides

For the following exemplary studies, peptides with the amino acid sequence GRRPGGASGG (SEQ ID NO: 39; also called XIB1 -a), GRRPLGGISGG (SEQ ID NO: 3; also called XIB1 -b), GRRPVGGAAGG (SEQ ID NO: 1 1 ), GRRPLPPPISGG (SEQ ID NO: 8), GRRPVGGISGG (SEQ ID NO: 6) were used as active agent. For control purposes, if not indicated otherwise, a random peptide with the amino acid sequence GGGGGLSRRIP (SEQ ID NO: 40) or solvent control (0,9% NaCI) were used. These peptide as well as all other peptides described herein were synthesized by standard FMOC-Synthesis as described in the literature (e.g., solid phase peptide synthesis - "A practical approach" by E. Atherton, R.C. Sheppard, Oxford University press 1989) or by liquid phase synthesis where the peptides are assembled using a mixed strategy by BOC-chemistry and fragment condensation as described in the literature (E. Wunsch, "Synthese von Peptiden" in "Methoden der organischen Chemie" (Houben-Weyl), 15 Ausg. 4, Teil 1 und 2 Thieme, Stuttgart, 1974).

Example 2: Inhibition of GEF-Activity

To measure the GEF-inhibitory effect, the following cell lines were used: Caco-2 (epithelial cells from adeno-carcinoma), ECV304 (epithelial cells from bladder carcinoma) and HpMec (endothelial cells, immortalized pulmonary micro-vascular cells). All cells were grown at standard conditions (37 °C, 5% CO2 and 95% relative humidity (rH)). Culture Medium used for Caco-2: DMEM + 1 mM sodium pyruvate + 20% FCS + 1 % Penicillin Streptomycin; for ECV 304: RPMI 1640 + 10% FCS + 1 % Penicillin Streptomycin; and for HpMec: IMDM + 25 mM Hepes + 10% Human Serum + 1 % Penicillin Streptomycin + 1 % L-Glutamine + ECGS/Heparin 2 ml. 4 h before the experiment, cells were starved by serum withdrawal. To induce GEF- activity, cells were stimulated with thrombin, lipopolysaccharide (LPS) or PMA for the indicated time in presence or absence of 50 g/ml GRRPLGGISGG (SEQ ID NO: 3). After stimulation, membrane fractions were prepared by using the commercial available „Compartemental Protein Extraction Kit" from Biochain Institutes. Membrane fractions were prepared according to the manufacturer instructions. GEF-activity in membrane lysates was determined by using„RhoGEF Exchange Assay Biochem Kit" from Cytoskeleton Inc. according to manufacturer instructions. GEF-activity was measured as fluorescence at using the Fluoroskan Ascent FL 2.6 from Thermo Electron Corporation. The excitation Filter wavelength was set at 355 nm and the emission filter wavelength at 460 nm. Table 1

Relative values compared to unstimulated control; (^*) p < 0.05 compared to tests w/o GRRPLGGISGG (SEQ ID NO: 3)

As can be taken from Table 1 , in ECV304 cells, thrombin and LPS stimulation resulted in an increase of GEF-activity compared to untreated control cells. ECV 304 cells stimulated with thrombin or LPS in the presence of GRRPLGGISGG (SEQ ID NO: 3) show a significant reduction of GEF-activity compared with treatment with thrombin or LPS alone.

In CaCo-2 cells, a 1 min PMA-stimulus resulted in an increase of GEF-activity by 2.5 fold. The magnitude of GEF-activation was significantly reduced when CaCo-2 cells were co-treated with PMA and GRRPLGGISGG (SEQ ID NO: 3).

In HepMec cells, thrombin induced a 3.5-fold increase in GEF-activity after 1 min and a 3-fold increase after 5 min of stimulation. Co-treatment of cells with GRRPLGGISGG (SEQ ID NO: 3) significantly reduced the magnitude of GEF- activity after 1 min and after 5 min. Treatment with GRRPLGGISGG (SEQ ID NO: 3) alone did not alter basic GEF-activity.

The results demonstrate that peptides of the present invention such as GRRPLGGISGG (SEQ ID NO: 3) reduce GEF-activity induced by different stimulating agents in epithelial and endothelial cells, but do not alter basic GEF- activity in unstimulated cells. This shows that the peptides of the present invention can be used in therapy, e.g., in the treatment or prevention of diseases or disorders associated with aberrant GTPase activity.

Example 3: Reduction of GTP-associated RhoA

To measure the GTP-associated active RhoA, the following cell lines were used: Caco-2, ECV304 and HpMec. All cells were grown at standard conditions (37 °C, 5% CO₂ and 95% rH). 4 h before the experiment, cells were starved by serum withdrawal. To induce GEF-activity, cells were stimulated with thrombin, LPS or PMA for the indicated time in presence or absence of 50 g/ml GRRPLGGISGG (SEQ ID NO: 3). After stimulation, membrane fractions were prepared by using the commercial available „Compartemental Protein Extraction Kit" from Biochain Institutes. Membrane fractions were prepared according to the manufacturer instructions. The membrane fraction was separated on 15% polyacrylamide gel according to standard procedures of gel electrophoresis. The gels were afterwards blotted on a nitrocellulose membrane according to standard procedures of western blotting. GTP-bound RhoA was detected using RhoA-GTP monoclonal antibody from NewEast Inc. in a dilution of 1 :5000. Protein bands were analyzed with the Dolphin-1 D Gel analysis system (Wealtec).

Table 2

GRRPLGGISGG 1 min

Thrombin 1 U/ml + 1 ,3^* 0,4

GRRPLGGISGG 5 min

As can be taken from Table 2, in ECV304 cells, thrombin and LPS stimulation resulted in an increase of RhoA-activity compared to untreated control cells. ECV 304 cells stimulated with thrombin or LPS in the presence of GRRPLGGISGG (SEQ ID NO: 3) show a significant reduction in RhoA-activity compared with treatment with thrombin or LPS alone.

In CaCo-2 cells, PMA-stimulus resulted in a 2.3 fold increase of RhoA-activity after 1 min of stimulation and in a 2-fold increase of RhoA-activity after 5 min of stimulation. The magnitude of RhoA-activation after 1 and after 5 min was significantly reduced when CaCo-2 cells were co-treated with PMA and GRRPLGGISGG (SEQ ID NO: 3).

In HepMec cells, thrombin induced a 3.8-fold increase in RhoA-activity after 1 min and a 3.2-fold increase after 5 min of stimulation. Co-treatment of cells with GRRPLGGISGG (SEQ ID NO: 3) significantly reduced the magnitude of RhoA- activity after 1 min and after 5 min. Treatment with GRRPLGGISGG (SEQ ID NO: 3) alone did not alter basic RhoA-activity.

These results demonstrate that peptides of the present invention such as GRRPLGGISGG (SEQ ID NO: 3) reduce RhoA-activity induced by different stimulating agents in epithelial and endothelial cells, but do not alter basic GEF- activity in unstimulated cells. RhoA-activity is controlled by GEF-activation as described above. The results demonstrate that GRRPLGGISGG (SEQ ID NO: 3) decreases RhoA-activity by inhibiting GEF-activityand, thus, is useful in the treatment and/or prevention of diseases or disorders which are associated with incrased Rho-GTPase- (in particular RhoA)-activity. Example 4: Phosphorylated myosin light chain (MLC) and actin stress fiber formation

To measure MLC phosphorylation and actin stress fiber formation, the following cell lines were used: Caco-2, ECV304 and HpMec. All cells were grown at standard conditions (37 °C, 5% CO₂ and 95% rH). Culture Medium used: for Caco-2: DMEM + 1 mM sodium pyruvate + 20% FCS + 1 % Penicillin Streptomycin; for ECV 304: RPMI 1640 + 10% FCS + 1 % Penicillin Streptomycin; and for HpMec: IMDM + 25 mM Hepes + 10% Human Serum + 1 % Penicillin Streptomycin + 1 % L-Glutamine + ECGS/Heparin 2 ml. 4 h before the experiment, cells were starved by serum withdrawal. To induce GEF-activity, cells were stimulated with thrombin, LPS or PMA for the indicated time in presence or absence of 50 g/ml GRRPLGGISGG (SEQ ID NO: 3). After stimulation, cells were fixed using 4% PFA. Phospho MLC was detected by using the„rabbit anti phosphor-myosin light chain antibody" from Chemicon in a concentration of 3 μΙ/ml in PBS (Gibco) supplemented with 0,1 % Triton X-100. As detection antibody, the Alexa 448 tagged „anti Rabbit IgG Antibody" from Invitrogen was used in a concentration of 0,5 μΙ/ml in PBS (Gibco) supplemented with 0,1 % Triton X-100. Aktin was detected using TRITC-labeled Phalloidin in a concentration of 0,5 μΙ/ml in PBS (Gibco) supplemented with 0,1 % Triton. Stained cells were analyzed by using a Zeiss Laser Scan microscope. Evaluation of the cyto-skeletal activation was performed by 2 independent observers that were blinded to the conditions. Evaluation criteria were set as follows: Actin: parallel actin bundles absent=0; distinct bundle formation=1 ; prominent parallel bundles=2; Phospho-MLC: present at cell poles=0; slight co-localization with actin bundles= 1 ; prominent co-localization with actin bundles=2

Table 3

Relative values compared to unstimulated control; (^*) p < 0.05 compared to tests w/o GRRPLGGISGG (SEQ ID NO: 3); (^*) p < 0.05 compared to tests w/o GRRPLGGISGG (SEQ ID NO: 3)

ECV 304 Zellen Mean SD

Control Peptide 1 min 0 0

Control Peptide 5 min 0 0

Peptid GRRPLGGISGG 1 1 0,2

min

Peptid GRRPLGGISGG 0 0

GRRPLGGISGG; 5 min

As can be taken from Table 3, in ECV304 cells, thrombin and LPS stimulation induced MLC phosphorylation and actin stress fiber formation. ECV 304 cells stimulated with thrombin or LPS in the presence of GRRPLGGISGG (SEQ ID NO: 3) show a significant reduction in MLC phosphorylation and actin stress fiber formation compared with treatment with thrombin or LPS alone.

In CaCo-2 cells, a PMA-stimulus induced an increase in MLC phosphorylation and actin stress fiber formation after 1 min and after 5 min of stimulation. The magnitude of Cytoskeletal activation after 1 and after 5 min was significantly reduced when CaCo-2 cells were co-treated with PMA and GRRPLGGISGG (SEQ ID NO: 3).

In HepMec cells, thrombin induced an increase in MLC phosphorylation and actin stress fiber formation after 1 min and after 5 min of stimulation. Co-treatment of cells with GRRPLGGISGG (SEQ ID NO: 3) significantly reduced the magnitude of cytoskeletal activation after 1 min and after 5 min. Treatment with GRRPLGGISGG (SEQ ID NO: 3) alone did not alter basic cytoskeletal activity.

The results demonstrate that peptides of the present invention such as GRRPLGGISGG (SEQ ID NO: 3) reduce MLC phosphorylation and actin stress fiber formation induced by different stimulating agents in epithelial and endothelial cells. MLC phosphorylation and actin stress fiber formation is controlled RhoA-activity as described above. The results demonstrate that peptides of the present invention such as GRRPLGGISGG (SEQ ID NO: 3) is decreasing MLC phosphorylation and actin stress fiber by inhibiting GEF-activity and subsequent RhoA-activity and, thus, is useful in the treatment and/or prevention of diseases or disorders which are associated with MLC phosphorylation and actin stress fiber formation.

Example 5: Endothelial and epithelial permeability

To measure permeability across endothelial and epithelial barriers, the following cell lines were used: Caco-2, ECV304 and HpMec. All cells were grown to confluence at standard conditions on a transwell system (Costar) with a pore size of 4 μιτι. At the start of the experiment, growth media were withdrawn and substituted with Hank's buffered salt solution. The upper chamber was supplemented with 2 mg/ml FITC- labeled dextran (Sigma Aldrich). Cells were stimulated as indicated in Table 4. 30 min-samples from the lower chambers were collected and determined for fluorescence (Flouroscan Ascent FL, Thermo Electron). 50 g/ml GRRPLGGISGG (SEQ ID NO: 3) was added where indicated. Table 4

As can be taken from Table 4, in ECV304 cells, thrombin and LPS stimulation increases barrier permeability. ECV 304 cells stimulated with thrombin or LPS in the presence of GRRPLGGISGG (SEQ ID NO: 3) show a significant reduction in barrier permeability compared with treatment with thrombin or LPS alone.

In CaCo-2 cells, PMA-stimulation induced an increase in barrier permeability. The barrier function was significantly improved when CaCo-2 cells were co-treated with PMA and GRRPLGGISGG (SEQ ID NO: 3).

In HepMec cells, thrombin induced an increase in barrier permeability. Treatment of the cells with GRRPLGGISGG (SEQ ID NO: 3) significantly reduced the thrombin induced barrier permeability. Treatment with GRRPLGGISGG (SEQ ID NO: 3) or control peptide alone did not alter barrier function. These results demonstrate that peptides of the present invention such as GRRPLGGISGG (SEQ ID NO: 3) reduce barrier permeability induced by different stimulating agents in epithelial and endothelial cells. Barrier permeability and barrier function is controlled by actin and myosin fibers. Activation of this cytoskeletal component results in cell contraction and cell rounding. Neighboring cells lose contact thereby increasing tissue permeability. Accordingly, the experiments demonstrate that peptides of the present invention such as GRRPLGGISGG (SEQ ID NO: 3) are decreasing barrier permeability induced by different agents in epithelial and endothelial cells.

Example 6: LPS induced Lung Injury

Male C57BI/6 Mice (Charles River, Germany) were kept at the animal facility of the Medical University of Vienna, feed with standard diet and water was provided ad libitum. All interventions were performed according to the guide lines of AAALAC (/Association for /Assessment and /Accreditation of Laboratory /Animal Care). All experiments were approved by the Ethic committee of the Medical University of Vienna. Mice were anesthetized with isoflouran and treated with 100 ng of LPS (E. coli 055: B5, Sigma Aldrich) intranasally. The peptide GRRPLGGISGG (SEQ ID NO: 3) was applied either intra-peritoneally (2 x 2 mg/kg) or via inhalation (2 x 4 mg/kg), first application was performed concomitantly with LPS administration, the second application was performed 1 h after the LPS inhalation.

Bronchoalveolar lavage

After 6 h, mice were anesthetized with Ketamine (Pfizer, Vienna, Austria) and sacrificed by bleeding out the vena cava inferior. The trachea was exposed through a midline incision and canulated with a sterile 20-gauge catheter (BD Venflon™, Becton Dickinson Infusion Therapy, Helsingborg, Sweden). Bilateral bronchoalveolar lavage fluid (BALF) was gained by instilling two 0.5 ml aliquots of sterile saline. Approximately 0.9-1 ml BALF was retrieved per mouse. Total cell numbers were counted from each sample using a hemo-cytometer (Turck chamber), BALF differential cell counts were done on cytospin preparations stained with Giemsa. For protein measurements, BALF was diluted 1 :2 in buffer containing 300 mM NaCI, 30 mM Tris, 2 mM MgCI₂, 2 mM CaCI₂, and Pepstatin A, Leupeptin and Aprotinin (all 20 ng/ml; pH 7.4). Protein levels in BALF were measured using the BCA protein according to the manufacturer's instructions (Pierce, Rockford, IL).

Table 5

Neutrophil counts and albumine content of the bronchio alveolar lavages serves as surrogate parameter for barrier dysfunction

n=20 per experimental group, intraperitoneal application of GRRPLGGISGG (SEQ ID NO: 3)

Values represent counts of neutrophils/ml (x 10³) in the BALF (mean +/ SD) 6 h after LPS administration. The difference between GRRPLGGISGG (SEQ ID NO: 3) and controls is significant (p<0.05)

The values represent the albumine content of BALFs (pg/ml; mean +/ SD) 6 h after LPS administration. The difference between GRRPLGGISGG (SEQ ID NO: 3) and controls is si nificant <0.05

n=20 per experimental group, intratracheal application of GRRPLGGISGG (SEQ ID NO: 3)

Values represent counts of neutrophils/ml (x 10³) in the BALF (mean +/ SD) 6 h after LPS administration. The difference between GRRPLGGISGG (SEQ ID NO: 3) and

As can be taken from Table 5, intranasal treatment of mice with LPS induced barrier dysfunction in the lung conveyed by increased neutrophil influx and albumin accumulation in the bronchio-alveolar space. Treatment of mice with GRRPLGGISGG (SEQ ID NO: 3) significantly improves barrier function, the mice showed less neutrophils and decrease of albumin in the BALF. The beneficial effect of GRRPLGGISGG (SEQ ID NO: 3) was equal in animal groups treated intraperitoneally and intratracheally.

Treatment of mice with control peptide did not alter the neutrophil counts and the albumin content of the BALF.

The LPS inhalation model is an accepted animal model to mimic ALI/ARDS as it resembles the human disease in regard to permeability changes in endothelial and epithelial cells and subsequent neutrophil and albumin accumulation in the bronchio alveolar space (Lung Cell Mol Physiol (2008), 295: L379-L399). The beneficial effect of GRRPLGGISGG (SEQ ID NO: 3) in the LPS-inhalation model demonstrates the usefulness of the peptides of the present invention to treat and/or prevent of diseases or disorders associated with a localized or systemic breakdown of epithelial or endothelial barrier functions. Specifically, the peptides of the present invention are useful in treating and/or preventing diseases and disorders such as, e.g., acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

Comparison of the peptides of the present invention with Ββ15-42

In addition, the same set-up as described above was used for comparing the impact of the peptide GRRPLGGISGG (SEQ ID NO: 3) compared to Ββ15-42 (PLoS ONE, 2009, 4(4): e5391 ), a peptide derived from fibrin having the sequence GHRPLDKKREEAPSLRPAPPPISGGGYR (SEQ ID NO: 41 ). Ββ15-42 was added in the same manner as the peptide GRRPLGGISGG (SEQ ID NO: 3). Again, protein content and cell count of BALF was measured.

Table 6

n=20 per experimental group, intraperitoneal application of GRRPLGGISGG (SEQ ID NO: 3) or Ββ15-42

LPS + NaCI LPS + Control-peptide LPS + LPS + Ββ15- GRRPLGGISGG 42

89+/-29 91 +/-33 27+/-26 33+/-22 n=20 per experimental group, intraperitoneal application of GRRPLGGISGG (SEQ ID NO: 3) or Ββ15-42

The values represent the albumine content of BALFs (pg/ml; mean +/ SD) 6 h after LPS administration. The difference between GRRPLGGISGG (SEQ ID NO: 3) and controls as well as between GRRPLGGISGG (SEQ ID NO: 3) and Ββ15-42 is significant (p<0.05)

n=20 per experimental group, intratracheal plus intraperitoneal application of GRRPLGGISGG (SEQ ID NO: 3) or Ββ15-42

Values represent counts of neutrophils/ml (x 10³) in the BALF (mean +/ SD) 6 h after LPS administration. The difference between GRRPLGGISGG (SEQ ID NO: 3) and controls as well as between GRRPLGGISGG (SEQ ID NO: 3) and Ββ15-42_ί5 significant (p<0.05)

Using the LPS-inhalation model, the effects of the peptide GRRPLGGISGG (SEQ ID NO: 3) and Ββ15-42 on neutrophil influx and albumin accumulation in the BALF were compared. Intraperitoneal treatment of mice with GRRPLGGISGG (SEQ ID NO: 3) or Ββ15-42 reduced neutrophil infiltration in the BALF in a comparable range. Yet, as surprisingly found herein, GRRPLGGISGG (SEQ ID NO: 3) was significantly more effective in reducing albumin content of the BALF as Ββ15-42. This clearly shows that the peptides of the present invention, e.g. GRRPLGGISGG (SEQ ID NO: 3), are more efficient than Ββ15-42. Example 7: Ventilator-induced Lung Injury (VILI)

Experiments were performed with healthy C57BL/6 (aged 8 - 10 weeks, with weights ranging from 19 - 25 g). All interventions were performed according to the guide lines of AAALAC. All experiments were approved by the Ethic committee of the University of Amsterdam.

Pre-challenge with LPS.

Mice were challenged with LPS (dosage: 50 g per mouse) (or saline), via intranasal injection 2 h before initiation of mechanical ventilation, to induce lung injury.

Administration of GRRPLGGISGG (SEQ ID NO: 3) or random peptide.

GRRPLGGISGG (SEQ ID NO: 3) or random peptide was administered i.v. 10 min before start of mechanical ventilation (dosage: 4 mg/kg loading dose, followed administration i.v. injections 1 mg/kg/h).

Instrumentation and anesthesia during mechanical ventilation.

Throughout the experiments rectal temperature was maintained between 36.5 - 37.5 °C using a warming path. Anesthesia was achieved with intra-peritoneal injection of a mix of ketamine, medetomidine, and atropine.

Mechanical ventilation strategies.

A Y-tube connector with 1 .0 mm outer diameter and 0.6 mm inner diameter was surgically inserted into the trachea under general anesthesia. Mice were placed in a supine position and connected to a ventilator. Mice were pressure-controlled ventilated with either an inspiratory pressure of 10 cm H₂O (resulting in V_T ~ 7.5 mL/kg; low V_T, LV_T) or an inspiratory pressure of 18 cm H₂O (resulting in V_T ~ 15 mL/kg; high V_T, HV_T). Positive end-expiratory pressure (PEEP) is set at 2 cm H₂O with both MV-strategies. The fraction of inspired oxygen was kept at 0.5 throughout the experiment. The inspiration to expiration ratio was kept at 1 :1 throughout the experiment. Fluid support strategies

Mice received intra-peritoneal boluses of normal saline 1 hour before start of MV, followed by boluses of normal saline via an intra-peritoneal catheter every 30 min.

Hemodynamic and ventilatory monitoring

Systolic blood pressure and heart rate were non-invasively monitored throughout the complete experiment. V_T was checked hourly with a pneumotach system.

Measurements

BALF was obtained by instilling 3 times 0.5 mL aliquots of saline by a 22-gauge Abbocath-T catheter (Abbott, Sligo, Ireland) into the trachea. Approximately, 1 .0 mL of BALF was retrieved per mouse and cell counts were determined using a hemacytometer (Beckman Coulter, Fullerton, CA). Subsequently, differential counts were done on cytospin preparations stained with a modified Giemsa stain, Diff— Quick (Dade Behring AG, Dudingen, Switzerland). Supernatant was stored at -80 °C.

Assays

Total protein levels in BALF are determined using a Bradford Protein Assay Kit (OZ Biosciences, Marseille, France) according to manufacturers' instructions with bovine serum albumin as standard. Mouse IgM was determined by ELISA by using anti- Mouse IgM sensitized 96-strip micro-well plates according to manufacturers' instructions (IMMUNO-TEK kit from ZeptoMetrix).

As a result, it was shown that GRRPLGGISGG (SEQ ID NO: 3) reduces lung inflammation which correlates with less lung damage and reduces pulmonary edema; cf. Figure 1 .

In this experiment, lung injury was induced by a pre-exposure of mice to LPS followed by mechanical ventilation with high or low tidal volume. Mice were either treated with GRRPLGGISGG (SEQ ID NO: 3) or scrambled peptide. Treatment of mice with GRRPLGGISGG (SEQ ID NO: 3) resulted in significant decrease of total cell count, neutrophil count, total protein content and IgM content in the BALF in the most aggressive experimental protocol (LPS-HTV). No improvement was observed by using scrambled peptide. Using the modest experimental protocol, GRRPLGGISGG (SEQ ID NO: 3) (LPS+LTV) significantly reduced total cell counts and neutrophil counts in the BALF compared to scrambled peptide. The modest treatment protocol did not cause a pronounced increase in total protein content and IgM content, thus no effect of GRRPLGGISGG (SEQ ID NO: 3) could be observed.

The present animal model resembles the clinical situation of patients that develop acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) as a sequel of pneumonia. The positive results obtained with GRRPLGGISGG (SEQ ID NO: 3) demonstrate the suitability of the peptides of the present invention for treating or preventing diseases or disorders. An example of diseases which may be treated with the peptides of the present invention are diseases or disorders associated with a localized or systemic breakdown of epithelial or endothelial barrier functions. Such diseases and disorders comprise burns, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator induced lung injury (VILI), systemic inflammatory response syndrome (SIRS), acute kidney injury (AKI), sepsis, multiorgan dysfunction syndrome (MODS), or edema.

Example 8: Thrombin effects are antagonized by various different peptides of the present invention

Methods

Human dermal microvascular endothelial cells (hDMEC) were isolated from human foreskin and cultured to passage 2 in endothelial medium (EGM-2) with supplements for microvascular cells (Lonza CC-3156 and CC-4147). Cells were split using Trypsin/EDTA (Gibco 25300) at 95% confluence and cells were seeded into chamber slides and cultured to confluence. Cells were incubated for 2 min with medium containing 1 U/ml Thrombin (Sigma T6884) or 1 U/ml Thrombin+20 g/ml of indicated peptide. After 2 min cells were fixed with 4% PFA (SAV LP), permeabilized with 1 % Triton (Bio-Rad 161 -0407) and stained with mouse-a-ZO-1 (BD 610966) and mouse lgG1 (Sigma M9035) controls. Secondary antibody used was Alexa488- conjugated goat-a-mouse F(ab)2-fragment (Invitrogen A1 1017). Hoechst dye was used to visualize nuclei. Cells were imaged by laser scan microscopy. Results and Discussion

The results are shown in Figure 2 and demonstrate that the effects of thrombin are antagonized by several different peptides of the present invention.

In particular, as can be seen from Figure 2, the addition of thrombin to microvascular endothelial cells induces rupture (arrows) of the continuous band of ZO-1 staining (green, nuclei are stained blue) in random peptide-treated cells as well as in Ββι_{5- 2} (FX06)-treated cells. However, thrombin-induced rupture of the continuous ZO-1 band is prevented by several different peptides of the invention; i.e. by peptides corresponding to SEQ ID NOs 3, 8 and 1 1 . Accordingly, this experiment demonstrates that a peptide falling under SEQ ID NO: 1 of the present invention is effective and may be used in therapy.

Example 9: Peptides inhibit the formation of actin-linked cingulin/ZO-1 complexes

Methods

Immunoprecipitation studies using anti-ZO-1 antibodies for precipitation and anti- cingulin antibodies for detection have been performed as follows.

Lysates

Human Pulmonary Microvascular Endothelial Cells (HPMEC) were cultured to confluence in TC75 flasks. The peptides indicated in Figure 3 were added, then 2U/ml Thrombin was added for 10 or 30 minutes as indicated. Then, RIPA buffer was added and cells were scrapped.

Immunoprecipitation

Protein G Sepharose Fast Flow (Sigma; P3296) was washed with Tris buffer, then, 1 g ZO-1 monoclonal mouse Antibody (BD-Bioscience; Cat#: 610966) or isotype control antibody was added. After washing, 750 g lysate was added to beads. After 12h incubation at 4°C, samples were centrifuged, supernatants removed and beads added to 50 μΙ of 2x reducing loading buffer and boiled at 96 °C for 5 minutes. Following centrifugation, supernatatants were run on SDS-PAGE. Primary antibodies used in western blot

ZO-1 mouse mAB (BD Biosciences; Cat#: 610966; use: 1 :550); CGN rabbit Ab (Sigma; HPAO 27657; use: 1 :800)

Secondary antibodies used in western blot

Goat anti rabbit HRP (Bio-Rad; Cat: 170-6515; use: 1 :50.000); goat anti mouse HRP (Bio-Rad; Cat: 170-6516; use: 1 :25.000)

Results and Discussion

After treatment of microvascular endothelial cells with thrombin, immunoprecipitation has been performed with an anti-ZO-1 antibody and western blot has been performed with an anti-cingulin antibody.

Figure 3 shows Cingulin-ZO-1 complexes following thrombin treatment. As can be seen from this Figure, thrombin increases the formation of actin-linked cingulin/ZO-1 complexes, which is inhibited by a peptide falling under the definition of SEQ ID NO: 1 {i.e. SEQ ID NO: 3, GRRPLGGISGG). In contrast, Ββι_5-42 (i.e. FX06 or GHRPLDKKREEAPSLRPAPPPISGGGYR; SEQ ID NO: 41 ) did not inhibit formation of actin-linked cingulin/ZO-1 complexes.

Example 10: Lung grafts

Methods

Donor: Fisher 344 rats; Recipient: Wistar rats Treatment protocol (total dose/rat 5mg)

1 . Ex vivo flushing of donor lungs with 1 mg GRRPLGGISGG (SEQ ID NO: 3) or GGGGGSRRIPL (SEQ ID NO: 38) in 20ml Perfadex.

2. Treatment of recipient rat, (Time T-0min corresponds to the time point when the grafted Fisher344 lung is reconnected to the circulation of Wistar rats). Four injections 1 mg each rat at times T-20min, T-0min, T+20min and T+60min. In addition, each rat received a single dose of 25mg urbason at T- Omin intraperitoneally (i.p). Surgical procedure

Donor operation

The abdomen was opened, the infrahepatic inferior vena cava was cannulated using a 27G needle and the donor was heparinized (5,000 I.U.). A 21 G needle was introduced into the pulmonary trunk and the lungs were perfused with 40 ml of cold preservation fluid of low potassium dextran glucose (Perfadex®) with 1 μΙ/ml of sodium bicarbonate via this cannula with the addition of peptide as indicated. The perfusion solution was drained via the left atrial appendage. The thoracic cavity was filled with mashed ice to induce cardiac arrest. The trachea was ligated while the lungs were fully inflated; and the heart and lungs were removed en-block. Then the broncho-vascular structures were dissected and cut to an appropriate length to allow secure anastomosis. The left pulmonary vein was dissected. The pulmonary artery was mobilized and cut. Both vessels were flushed with 500 IU of Heparin. Then the left main bronchus, both the trachea and right main bronchus were dissected, ligated and cut.

Implantation surgery

The lateral chest wall was opened via the 4^th intercostal space and the ribs retracted (Aesculap BV074R, 130mm). The left inferior pulmonary ligament was cut and the left native lung was mobilized outside the thoracic cavity. The distal pulmonary artery was ligated (7-0 silk suture, Catgut, Germany) and cut distal to the ligature. The superior segmental vein was double ligated using a 7-0 silk ligature and cut and the left main bronchus was clamped (Aesculap FE720 Miniclip) and the left lung was excised. The donor lung was introduced into the recipient thoracic cavity and constantly cooled. The bronchial anastomosis was initiated by two interrupted stabilization-sutures and completed with approximately 14 interrupted sutures (8-0 Prolene, BV 130-5). The bronchial anastomosis was checked for patency and the lung was re-inflated. Vascular anastomoses started with the pulmonary artery utilizing the cuff technique. The pulmonary artery was clamped with a microvascular hemostat clip and stabilized. The ligature was resected and the proximal end of the artery passed through an 18G polyethylene intravenous catheter. Heparin was topically applied and the vessel wall was everted over the cuff and fixed using a 7-0 silk suture ligature. The corresponding donor pulmonary artery was pulled over the complex of cuff and everted recipient vessel and secured by a 7-0 silk ligature. Finally, the pulmonary vein was anastomosed utilizing the same technique used for the artery. Once a good reperfusion was obtained, the thorax was closed utilizing 4- 0 Prolene, subcutis and skin closure was performed by 4-0 Vicryl, respectively.

Results

The results are presented in Figure 4. The numeric data as well as the p values corresponding to the results shown in Figure 4 are shown in Table 6.

Table 6: Numeric data of the results shown in Figure 4 and the corresponding p values.

GRRPL - GRRPL- control GGISGG control GGISGG

P=0,02069 P=0,00051

right lungs left lungs naive

Interpretation

Control lungs show PGD as reflected by persistent post-transplantation lung edema. In animals treated with GRRPLGGISGG (SEQ ID NO: 3) edema is reduced which is due to reduced alloreactivity. These data indicate that the peptides of the present invention, e.g. GRRPLGGISGG (SEQ ID NO: 3), reduce exposure (and sensitizing) of the recipient to donor tissue due to protection of the organ during the grafting/reperfusion period. These results demonstrate that the peptides provided herein are useful in the treatment of primary graft dysfunction, including but not limited to, PGD in lung, kidney and heart recipients. Example 11 : Mixed Lymphocyte Reaction (MLR)

Methods

Procedure of the Experiment

• 10 ml of isolation medium was added to a 10 cm petri dish with a nylon mesh (cell strainer F2350, BD Falcon) set in place. Freshly isolated spleens from donor animal, recipient animals and na^'fve animals were placed into the strainer. Using the plunger of a sterile tuberculin syringe spleens were pressed through the mesh and centrifuged at 275 g for 10 min at room temperature. To remove red blood cells a Red Blood Cell Lysis Buffer was used. Serial dilutions were prepared to achieve the following cell numbers / ml of the assay: 0.5x10⁶, 1x10⁶, 2x10⁶ and 4x10⁶.

• Donor spleens are those animals from which lung was taken.

• Recipient spleens were those who had received the allogenic lung implant.

• Na^'fve spleens were those which were never exposed to allogenic lung grafts.

• Spleen cells from donors were irradiated with 10gray and added to spleen cells of the recipient and incubate at 37°C, 95% H₂O, 5 % CO₂ for 3 days. Then, cells were pulsed with 3-H thymidine (I mCi/ml) diluted 1 :20. To 200 μΙ of Mixed Lymphocyte Reaction 20 μΙ of diluted 3-H Thymidine was added and incubated for another 24 hrs. Then plates were harvested and H3 thymidine content of cells quantified.

Mixed Lymphocyte Reaction

effector cells: spleens of 10 transplanted Wistar rats (7 untreated, 3 treated) target cells: irradiated spleens of Fisher rats

Results

The results are presented in Figure 5. The p values of the values shown in Figure 5 are shown in Table 7.

Table 7: p values of the values shown in Figure 5.

two-sided t test, Error bars represent Standard Deviation Interpretation

Control animals have sensibilized T cells targeting the major histocompatibility complex (MHC) of the lung donor, whereas GRRPLGGISGG (SEQ ID NO: 3)- treated animals have no detectable alloreactivity (back to baseline seen with naive cells) based on organ protection during the grafting procedure. Accordingly, these results indicate that the peptides for use in treating and/or preventing PGD as provided herein reduce organ damage during the grafting procedure. As an indirect effect, the host is less exposed to foreign MHC molecules from the grafted organ resulting in delayed or diminished sensitizing (not immunosuppression) and a delayed or diminished immune response.

Thus, using the peptides of the invention to protect an organ to be grafted increases organ function and reduces exposure of donor MHC to host antigen presenting cells as compared to the organ function of an organ which is not treated with the peptides of the invention. Accordingly, the efficacy of the herein described peptides does not rely on an immunosuppression but a reduced sensitization.

In summary, these data demonstrate that the peptides provided herein are useful in the prevention and/or treatment of primary graft dysfunction. The inventive peptides may be used in the prevention and/or treatment of PGD in recipients of a lung, a kidney, a heart, a liver, a pancreas, intestine, a vein, an artery, skin, a valve, bone, eye tissue, amnion, connective tissue or a senew.

Example 12: Extracellular Acidification Rate (ECAR)

Methods

Mouse macrophage cell line Raw264.7 was cultured in DMEM. Cells were split using Trypsin/EDTA (Gibco 25300) at 95% confluence and cells were seeded into Seahorse 24-well plates and cultured to 95% confluence. Medium was changed to not buffered assay medium (Seahorse 102352) prior to measurements. LPS and peptides were diluted in assay medium and H₂O was accounted for in control wells. At indicated time points, treatment medium was released into the assay wells. The rate of pH-change and oxygen consumption were monitored by electrodes connected to the Seahorse device. Results

The results are presented in Figure 6. The graphs of Figure 6 show the mean rate of pH-change as extracellular acidification rate (ECAR) +/-standard deviation.

Interpretation

LPS induces a rapid and sustained extracellular acidification. Random peptides as well as GHRPLDKKREEAPSLRPAPPPISGGGYR (B 15-42/FX06; SEQ ID NO: 41 ) to not change the kinetics, whereas peptides GRRPVGGAAGG (SEQ ID NO: 1 1 ), GRRPLPPPISGG (SEQ ID NO: 8) and GRRPLGGISGG (SEQ ID NO: 3) induce a faster return to baseline (not-stressed condition). Accordingly, the peptides provided herein are capable of shortening the time period of stress induced metabolic disturbances as exemplified here in LPS-stressed macrophages, where acidification normalized significantly faster in the presence of peptides described herein. These data indicate that the peptides described herein are useful in the treatment or prevention of graft rejection, in particular of PGD. The peptides of the invention may be used to treat or prevent PGD in recipients of, e.g., lung, kidney, heart, liver, pancreas, intestine, a vein, an artery, skin, a valve, bone, eye tissue, amnion, connective tissue or a senew. For instance, the peptides described herein may be used to treat PGD in lung and kidney recipients.

Example 13: Phagocytosis Assay

Methods

Mouse macrophage cell line Raw264.7 was cultured in RPMI (Gibco 21875) + 10% FBS. Cells were split using Trypsin/EDTA (Gibco 25300) at 95% confluence and 500.000 cells were seeded into 12-well plates and cultured overnight.

Cells were washed twice with PBS (Lonza BE17-513F) and RPMI + 3% Serum containing therapeutic peptides was added to the cells. After 30 min pre-incubation heat-killed FITC-labelled Escherichia coli were added at a MOI of 100. Cells were incubated for time points indicated in Figure 7. Another plate was incubated at 4°C as negative control.

For analysis, cells were washed twice with ice-cold PBS. Subsequently 1 ml 50 g/ml proteinase K solution was added for 15min at room temperature. Again cells were washed twice with PBS, unless otherwise indicated steps were done at 4°C. Add 1 ml PBS containing 2mM EDTA to detach the cells and transfer the supernatant to FACS tubes for analysis. Wash with PBS before FACS-analysis.

Interpretation

GRRPVGGAAGG (SEQ ID NO: 1 1 ) and GRRPLGGISGG (SEQ ID NO: 3) reduce uptake of E. coli as compared with random peptide and Ββι_5-42 ΡΧ06 (see Figure 7). These results demonstrate that the peptides provided herein are useful in the treatment of primary graft dysfunction, including but not limited to, in lung, kidney and heart recipients.

Example 14: Treatment and/or Prevention of PGD after Kidney Transplantation

Introduction

Primary Graft Dysfunction (PGD) after kidney transplantation accounts for oliguria, increased immunogenicity, risk for acute rejection and decreased long-term survival of the graft. To mimic PGD a mice model of bilateral renal pedical clamping for 30min was introduced. This causes a disease pattern with severe loss of renal function and steep rise in creatinine and blood urea nitrogen (BUN) within 24-72h post-ischemia.

Aim of the Study

In this study the therapeutic peptides of the invention for the treatment and/or prevention of PGD after kidney transplantation were tested.

Study Design

Animal Model

12-15 week old male C57BI/6 mice (Charles River, Germany) were used for the study. They were anesthetized using isofluran via a nose mask and placed supine on a heating table to maintain a body temperature around 32 °C. Bilateral flank incisions was performed and both renal pedicals were clipped with a microaneurysm clip for 30 min.

Preparation of the Test Article The peptides GRRPLGGISGG (SEQ ID NO: 3), GRRPLGGAAGG (SEQ ID NO: 10) and the random peptide was dissolved in physiological saline at a concentration 30mg/ml and stored at -20°C.

For iv. injection peptides were used in a dose of 3mg/kg and was administered prior to ischemia and 5 min after reperfusion. The stock solutions were diluted 1 :50 to a working solution of 0.6mg/ml and 5μΙ per g bodyweight were injected.

Study Groups and Administration of the Test Article

The two different Peptides (GRRPLGGISGG (SEQ ID NO: 3) and GRRPLGGAAGG (SEQ ID NO: 10)) were tested versus random peptide in the bilateral IR-model. Route of administration was iv via retrobulbar injection. Renal function were studied in n=10 mice each group.

Table 8: Study groups.

Renal Function

Blood samples were obtained under light ether anaesthesia from the cavernous sinus with a capillary at different time points (baseline 2-4 days prior surgery= day 0, and at days 1 , 2 and 3 post surgery) and renal function (s-creatinine,) was measured on an Olympus analyser (AU400).

Statistics

Data are shown as mean ± standard error (SEM). Statistical significance was calculated by ANOVA with post hoc Scheffe test. Differences were considered significant at p<0.05. SPSS 16.0 software was used. Significance of survival was calculated by log rank test and Prism software was used. Results and Discussion

Serum-creatinine (umol/l)

All groups showed an increase of s-creatinine after ischemia. Mice treaded with GRRPLGGISGG (SEQ ID NO: 3) or GRRPLGGAAGG (SEQ ID NO: 10) show significant less s-creatinine than mice treated with scrambled peptide; see Figure 8. These results demonstrate that the peptides provided herein are useful in the treatment of organ transplant rejection, particularly primary graft dysfunction, including but not limited to, in kidney recipients.

It is noted that PGD is an ischemia-reperfusion injury occurring in the early period following transplantation which is most often described in the transplanted lung, liver, or kidney, but is relevant in all tissues exposed to ischemia followed by reconnection to the blood circulation. Thus, the data shown above indicate that the peptides provided herein can be used in the treatment and/or prevention of PGD of several transplanted organs and/or tissues including, but not limited to, heart, lung, kidney, liver, pancreas, intestine, a vein, an artery, skin, a valve, bone, eye tissue, amnion, connective tissue or a sinew.

Example 15: Functionality of the Inventive Peptides requires a flexible center

Methods

The peptides GRRPGGISGG (SEQ ID NO: 42); GRRPISGG (SEQ ID NO: 43) and GAAPGGISGG (SEQ ID NO: 44) have been analyzed for their 3-dimensional shape. In particular, the used technique was "Molecular Dynamics Simulation" as described, e.g., in Karplus, 2002, Nature Structural Biology 9: 646 - 652 and Hansson, 2002, Curr Opin Struct Biol 12: 190-196. The used software was "GROMACS" as described, e.g., in Pronk, 2013, Bioinformatics. 29(7): 845-54.

Results and Discussion

The peptides described herein (e.g. GRRPGGISGG; SEQ ID NO: 42) have a flexible center (i.e. X3 is PPP or X4 is GG, for example, X4 is GG) and N-terminal from this flexible center there is the amino acid sequence GX1 RP [Xi is R or A] and C-terminal from this flexible center there is the amino acid sequence GG. Peptides of this type have been demonstrated to be useful for the treatment of PGD; see, e.g., Examples 10 to 14, above. Such a peptide (i.e. the peptide of SEQ ID NO: 42) has a bent shape as illustrated in Figure 9 (see left peptide). The peptide of SEQ ID NO: 43, which does not have a flexible center (i.e. a central PPP or GG) has a stretched shape; see Figure 9, middle. In addition, the peptide of SEQ ID NO: 44, wherein, N-terminal to the flexible center, the amino acid sequence GXiRP [Xi is R or A] is lacking has no apparent shape; see Figure 9, right peptide.

These data indicate that to be useful in the treatment of PGD, a peptide requires a flexible center (i.e. a central PPP or GG, preferably GG) and N-terminal from this flexible center the amino acid sequence GXiRP [Xi is R or A].

Example 16: RhoA Pull Down.

Methods

HUVEC grown to confluence were incubated with indicated peptides (20 g/ml each), thrombin (0.1 U/ml) or thrombin+peptides for 5 minutes or were left untreated. The peptides used were:

Line 3: 0.1 U/ml thrombin + GGGGGSRRIPL (SEQ ID NO: 38) (20 pg/ml)

Line 4: 0.1 U/ml thrombin + GRRPVPPPISGG (SEQ ID NO: 9) (20 pg/ml)

Line 5: 0.1 U/ml thrombin + GRRPLGGISGG (SEQ ID NO: 3) (20 pg/ml)

Line 6: 0.1 U/ml thrombin + GRRPLGGAAGG (SEQ ID NO: 10) (20 pg/ml)

Active RhoA was pulled down by using Rho Assay Reagent (GST-coupled Rhotekin, Upstate) according to manufactures instructions. Bound proteins were separated on a 15% polyacrylamid gel and blotted on Nitrocellulose-Membrane (Bio-Rad). RhoA was detected with anti-RhoA antibody (clone55; Upstate). Total RhoA contents were determined in western blots performed from the same lysates before pull downs were performed.

Results and Discussion

The Results are shown in Figure 10. As evident from this Figure, all peptides corresponding to SEQ ID NO: 1 effectively inhibit RhoGTPases. In addition, Figure 10 shows that a peptide corresponding to SEQ ID NO: 3 is the best RhoGTPase inhibitor. This peptide has also been shown to be most active in the treatment and prevention of PGD; see, e.g., Examples 12 and 13. Thus, these data indicate that the efficacy of the herein described peptides in the treatment of PGD is dependent on the ability of the peptides to inhibit Rho GTPases

Example 17: Effects of the peptide GRRPLGGISGG (SEQ ID NO: 3) in an orthotopic rodent lung transplantation model 100 minutes after reperfusion of the lung graft.

1) Bronchial anastomosis and pulmonary edema

After flushing the donor lung with a colloid containing preservation solution (15ml of Perfadex®), the first dose of GRRPLGGISGG (SEQ ID NO: 3) or scrambled peptide (1 mg) was injected directly into the pulmonary arterial circulation of the donor lung. Thereafter, the donor lung is explanted and the left main bronchus is ligated to keep the donor lung inflated for as long as possible. After about 100min the ligated bronchus is cut open with microscissors to prepare both the donor bronchial stump and the recipient bronchial stump for suture anastomosis. This is the first time for drug effects to become clinically evident. In GRRPLGGISGG (SEQ ID NO: 3)-treated lungs almost no pulmonary edema fluid draining through the donor main brochus can be detected whereas in scrambled peptide treated animals, there is moderate to severe fluid escaping through the bronchial stump (see Figure 1 1 ).

The following peptide doses (1 mg each) were injected into recipient rats, at times T-20, 0, +20 and +60 min. whereas TO is the time point, when the grafted lung is connected to the circulation of the recipient.

2) Reperfusion

The reperfusion process of the allo-grafted left lung starts with opening of the vascular clips. In GRRPLGGISGG (SEQ ID NO: 3)-treated animals, lungs show a homogenous pattern of reperfusion, whereas scrambled peptide treated controls display a patchy pattern of reperfusion (see Figure 12).

3) Postoperative systemic arterial oxygen saturation (P02)

Rats tend to tolerate clamping of the left pulmonary artery and unilateral pneumectomy, respectively, very well without severe effects on systemic arterial oxygen tension and saturation. However, when the bronchial clamp is removed after transplantation, the effect of post-operative lung edema and fluid extravasation becomes evident. Once serous fluid is pushed through the pulmonary capillaries into alveoli the stage is set for fluid to quickly enter smaller bronchioles, larger bronchi and fluid finally drains into the contra-lateral airways, exceeding the rats' elaborate coping mechanisms and thereby decreasing systemic arterial oxygen tension. The mean systemic arterial oxygen tension of GRRPLGGISGG (SEQ ID NO: 3)-treated animals was 95.2+/-2% compared to the 84.7+/- 3% of scrambled peptide treated animals (p<0.001 ); see Figure 13. Both groups were ventilated with an FI02 of 0,7 both throughout the procedure and during the subsequent process of reperfusion.

Claims

1 . A peptide for use in treating and/or preventing primary graft dysfunction comprising or consisting of the amino acid sequence

GX1 RPX2X3X4 X₅GGX₆ (SEQ ID NO: 1 ) wherein

Xi is an amino acid selected from the group consisting of R and A;

X₂ is either omitted or an amino acid selected from the group consisting of L and V;

X3 is either omitted or an amino acid sequence consisting of PPP;

X is either omitted or an amino acid sequence consisting of GG;

Xe is either omitted or an amino acid sequence consisting of 1 to 5 amino acids;

provided that if X₃ is omitted, than X₄ is not omitted.

2. The peptide as defined in claim 1 for use in treating and/or preventing primary graft dysfunction which is capable of inhibiting activity of a Rho GTPase.

3. The peptide as defined in claim 1 or 2 for use in treating and/or preventing primary graft dysfunction, further comprising X₇ at the C-terminus of the sequence, wherein X₇ is a moiety selected from the group consisting of NH₂, albumin, polyethyleneglycol, dextrane, ferritine, hydroxyethyl-starch and Fc- moiety of an antibody.

4. The peptide as defined in any one of claims 1 to 3 for use in treating and/or preventing primary graft dysfunction, wherein Xi is R.

5. The peptide as defined in claim 1 for use in treating and/or preventing primary graft dysfunction, wherein X₂ is L.

6. The peptide as defined in any one of claims 1 to 5 for use in treating and/or preventing primary graft dysfunction, wherein X₃ is omitted.

7. The peptide as defined in any one of claims 1 to 6 for use in treating and/or preventing primary graft dysfunction, wherein X₄ is GG.

8. The peptide as defined in any one of claims 1 to 7 for use in treating and/or preventing primary graft dysfunction, wherein X₅ is IS or AS.

9. The peptide as defined in claim 8 for use in treating and/or preventing primary graft dysfunction, wherein X₅ is IS.

10. The peptide as defined in any one of claims 1 to 9 for use in treating and/or preventing primary graft dysfunction, wherein X₆ is omitted.

1 1 . The peptide as defined in any one of claims 1 to 10 for use in treating and/or preventing primary graft dysfunction, wherein Xi is R, X₂ is L, X₃ is omitted, X₅ is IS and Xe is omitted, or wherein Xi is R, X₂ is V, X₃ is omitted, X₅ is IS and Xe is omitted.

12. The peptide as defined in any one of claims 1 to 1 1 for use in treating and/or preventing primary graft dysfunction, wherein said peptide comprises or consists of an amino acid sequence selected from the group consisting of GRRPLGGISGG (SEQ ID NO: 3);

GRRPVGGAAGG (SEQ ID NO: 1 1 );

GRRPLPPPISGG (SEQ ID NO: 8);

GRRPVGGISGG (SEQ ID NO: 6);

GRRPVPPPISGG (SEQ ID NO: 9);

GRRPLGGAAGG (SEQ ID NO: 10); GRRPLPPPAAGG (SEQ ID NO: 12);

GRRPVPPPAAGG (SEQ ID NO: 13);

GRRPLGGASGG (SEQ ID NO: 14);

GRRPVGGASGG (SEQ ID NO: 15);

GRRPLPPPASGG (SEQ ID NO: 16);

GRRPVPPPASGG (SEQ ID NO: 17);

GRRPLGGIAGG (SEQ ID NO: 18);

GRRPVGGIAGG (SEQ ID NO: 19);

GRRPLPPPIAGG (SEQ ID NO: 20);

GRRPVPPPIAGG (SEQ ID NO: 21 );

GARPLGGISGG (SEQ ID NO: 22);

GARPVGGISGG (SEQ ID NO: 23);

GARPLPPPISGG (SEQ ID NO: 24);

GARPVPPPISGG (SEQ ID NO: 25);

GARPLGGAAGG (SEQ ID NO: 26);

GARPVGGAAGG (SEQ ID NO: 27);

GARPLPPPAAGG (SEQ ID NO: 28);

GARPVPPPAAGG (SEQ ID NO: 29);

GARPLGGASGG (SEQ ID NO: 30);

GARPVGGASGG (SEQ ID NO: 31 );

GARPLPPPASGG (SEQ ID NO: 32);

GARPVPPPASGG (SEQ ID NO: 33);

GARPLGGIAGG (SEQ ID NO: 34);

GARPVGGIAGG (SEQ ID NO: 35);

GARPLPPPIAGG (SEQ ID NO: 36); and

GARPVPPPIAGG (SEQ ID NO: 37).

13. The peptide as defined in any one of claims 1 to 12 for use in treating and/or preventing primary graft dysfunction, wherein the graft is a syngeneic, an allogeneic or a xenogeneic graft.

14. A polynucleotide for use in treating and/or preventing primary graft dysfunction, wherein the polynucleotide encodes the peptide of any one of claims 1 to 13.

15. A pharmaceutical composition comprising the peptide of any one of claims 1 to 13 and/or the polynucleotide of claim 14, for use in treating and/or preventing primary graft dysfunction, further comprising a pharmaceutically acceptable carrier and/or diluent.

16. The peptide of any one of claims 1 to 13, the polynucleotide of claim 14, or the pharmaceutical composition of claim 15, wherein the graft is an organ or a tissue.

17. The peptide of any one of claims 1 to 13 and 16, the polynucleotide of claim 14 or 16, or the pharmaceutical composition of claim 15 or 16, wherein the graft is at least one organ selected from the group consisting of lung, kidney, heart, liver, pancreas and intestine.

18. The peptide of any one of claims 1 to 13 and 16, the polynucleotide of claim 14 or 16, or the pharmaceutical composition of claim 15 or 16, wherein the graft is at least one tissue selected from the group consisting of a vein, an artery, skin, a valve, bone, eye tissue, amnion, connective tissue and a sinew.

19. Method of treating and/or preventing primary graft dysfunction by administering an effective dose of the peptide of any one of claims 1 to 13 and 16 to 18, the polynucleotide of any one of claims 14 and 16 to 18, or the pharmaceutical composition of any one of claims 15 to 18, to a subject in need of such treatment.

20. The peptide of any one of claims 1 to 13 and 16 to 18, the polynucleotide of any one of claims 14 and 16 to 18, the pharmaceutical composition of any one of claims 15 to 18, or the method of claim 19, wherein the peptide, the polynucleotide or the pharmaceutical composition is co-administered with at least one other active agent. The peptide of claim 20, the polynucleotide of claim 20, the pharmaceutical composition of claim 20, or the method of claim 20, wherein the other active agent is at least one active agent selected from the group consisting of corticosteroids, cyclosporine, tacrolimus, sirolimus, methotrexate, azathiopine, mercatopurine, antibiotics, polyclonal antibodies, monoclonal antibodies, interferon, opioids, TNF binding proteins, mycophenolate, FTY720 and any other immunosuppressive drug.

The peptide of any one of claims 1 to 13, 16 to 18, 20 and 21 , the polynucleotide of any one of claims 14, 16 to 18, 20 and 21 , the pharmaceutical composition of any one of claims 15 to 18, 20 and 21 , or the method of any one of claims 19 to 21 , wherein the peptide, the polynucleotide or the pharmaceutical composition is flushed extra-corporal through the donor organ vessels or injected into donor tissue.

The peptide of any one of claims 1 to 13, 16 to 18 and 20 to 22, the polynucleotide of any one of claims 14, 16 to 18 and 20 to 22, the pharmaceutical composition of any one of claims 15 to 18 and 20 to 22, or the method of any one of claims 19 to 22, wherein the peptide, the polynucleotide or the pharmaceutical composition is administered intravenously, subcutaneously or intramuscularly to the recipient.

The pharmaceutical composition of any one of claims 15 to 18 and 20 to 23, wherein the pharmaceutical composition is in the form of a liquid solution, an erodible implant, a pill, a tablet, a capsule, a thin film, a powder, a solid crystal or liposomes.

A method for the preparation of a pharmaceutical composition for use in treating and/or preventing primary graft dysfunction, comprising the following steps: (a) contacting the peptide of any one of claims 1 to 13, 16 to 18 and 20 to 23 and/or the polynucleotide of any one of claims 14, 16 to 18 and 20 to 23 with a liquid carrier or a solid carrier;

(c) optionally, sterilizing the product obtained in step (a) or (b); and

(d) formulating and/or packaging the product obtained in step (a), (b) or (c) as a finished medical product.

The method of claim 25, wherein the carrier is at least one carrier selected from the group consisting of water, saline, Ringer's solution, dextrose solution, a fixed oil, ethyl oleate, liposomes and an organ preservation solution.