Compositions for prevention of adhesions and other barrier applications

(56) References cited:

WO-A-2004/007532 WO-A-2006/116524

WO-A-2006/014570 WO-A-2007/142757

EP 2 581 097 Β1

Note: Within nine months ofthe publication ofthe mention ofthe grant ofthe European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall nőt be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).

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EP 2 581 097 Β1

Description

FIELD OF THE INVENTION [0001] The present invention is generally in the field offormulations for application to tissues for prevention ofadhesions and other barrier applications.

BACKGROUND OF THE INVENTION [0002] Adhesions may be present at birth (congenital) or may form after abdominal surgery or inflammation. Most adhesions typically form after surgery. Adhesions are more common after procedures on the colon, appendix, or uterus than after surgery on other organs, such as the stomach, gall bladder, or pancreas. The risk of developing adhesions increases with the passage of time after the surgery.

[0003] Abdominal adhesions are bands offibrous scar tissue that form on organs in the abdomen, causing the organs to stick to one another or to the wall of the abdomen. Intestinal adhesions are bands of fibrous tissue that connect the loops of the intestines to each other; the intestines to other abdominal organs; or the intestines to the abdominal wall. These bands can puli sections of the intestines out of piacé and may block the passage of food. In people living in developed countries, this scar tissue most commonly develops after abdominal surgery, in which organs are handled by the surgical team and are shifted temporarily from their normál positions. The scar tissue can alsó form in people who develop peritonitis, an infection that has spread to the membráné that covers the abdominal organs. Peritonitis commonly occurs after appendicitis or other abdominal infections. Another cause of adhesions is endometriosis, an inflammatory condition that affects somé women and may involve the abdomen and serious abdominal trauma, including cesarean sections.

[0004] Adhesions are a major cause of intestinal obstruction. Adhesions can cause partial or complete obstruction of the intestines. The symptoms exhibited due to the adhesions depend on the degree and the location ofthe obstruction. Symptoms include cramps, abdominal pain, vomiting, bloating, an inability to pass gas, and constipation. In a small number of people who have adhesions, however, the fibrous bands of scar tissue block the intestines either completely or partially. This blockage is called a bowel obstruction, and leads to death in about 5% of cases. Sometimes, an area of intestine that is affeeted by adhesions can become blocked then unblocked, causing symptoms to come and go. In about 10% of small-bowel obstructions, a portion ofthe bowel twists tightly around a bánd ofadhesions. This cuts off the normál blood supply to the twisted bowel, a disorder known as strangulation, causing that section of bowel to die. When this emergency happens, the person must undergo surgery immediately. The death rate is as high as 37% in people who develop strangulation.

[0005] Percutaneous epidural adhesiolysis and spinal endoscopic adhesiolysis are interventional pain management techniques used to treat patients with refractory low back pain due to epidural scarring. Standard epidural steroid injections are often ineffective, especially in patients with prior back surgery. Adhesions in the epidural space can prevent the fiow of medicine to the target area; lysis of these adhesions can improve the delivery of médication to the affeeted areas, potentially improving the therapeutic efficacy ofthe injected medications. Prevention ofsuch adhesions, however, would be more preferable.

[0006] Many different materials have been tried as a means of preventing adhesions. Most of these are hydrogels that are applied as Solutions at the time of surgery. Efficacy of these materials varies due to rapid degradation and/or failure to form a sufficiently thick barrier. Others materials work only in combination with anti-proliferative drugs. Nőne of these materials has been shown to be effective in a highly fluid environment, which usually is present during surgery due to bleeding and leakage of other bodily fluids.

[0007] U.S. Patent Nos. 5,670,483, 6,548,630, and 7,098,028 by Zhang et al. describe amphiphilic peptides having alternating hydrophobic and hydrophilic residues. Zhang alleges that the membranes are potentially useful in biomaterial applications such as slow-diffusion drug delivery systems, artificial skin, and separation matrices, and as experimental models for Alzheimer’s disease and serapie infection. However, Zhang does nőt disclose the use ofsuch materials for the prevention ofadhesions.

[0008] WO 2007/142757 and U.S.S.N. 11/411,745 describe compositions including peptides with alternating hydrophilic and hydrophobic monomers that allow them to self-assemble under physiological conditions are formulated for application to wounds.

[0009] WO 2006/116524 relates to compositions that include nanoscale structured materials or precursors thereof, which can include other substanoes such as vasoconstrictors.

[0010] WO 2006/014570 relates to amphiphilic peptide chains having alternating hydrophilic and hydrophobic amino acids, wherein the peptide contains at least 8 amino acids, are complementary and structurally compatible, and selfassemble intő a beta-sheet macroscopic scaffold wherein peptide at least about 75% of the chains have the same sequence.

EP 2 581 097 Β1 [0011] WO 2004/007532 relates to materials comprising ribbons, fibrils or fibres characterised in that each of the ribbons, fibrils or fibres have an antiparallel arrangement of peptides in a beta-sheet tape-like substructure.

[0012] However, these applications do nőt describe the use of such materials for the prevention of adhesions.

[0013] lt is therefore an object ofthe present invention to provide compositions for preventing or minimizing adhesions and for other barrier applications, which can be applied to tissues or cells which are bleeding or in the presence of fluids. [0014] lt is anotherobject ofthe present invention to provide such a composition that can be formulated as a bandage, spray, coating, or powder.

[0015] lt is a still further object ofthe present invention to provide a composition that can be used to prevent adhesions bút is sufficiently clear to allow a physician to see and work through the matériái.

BRIEF SUMMARY OF THE INVENTION [0016] The invention is as defined in the accompanying claims.

[0017] Compositions including peptides which self-assemble under physiological conditions are formulated for application to tissues for prevention of adhesions or other barrier applications, such as minimizing contamination or infection (e.g., from bacteria, fungi, viruses, or other pathogenic agents), limiting spread of metastasis following cancer surgery, or for delivery of a therapeutic, diagnostic or prophylactic agent in a confined area, after bleeding or fluid leakage has been substantially stopped.

[0018] In one embodiment, the composition comprises self-assembling peptides having a length within the rangé of 6 to 200 amino acids and comprise a sequence of amino acid residues conforming to one or more of Formulas l-IV:

<td> ((Xaa^neu-Xaa⁺)x(Xaa^neu-Xaa^_)y)_n</td><td> (I)</td>

<td> ((Xaa^neu-Xaa)x(Xaa^neu-Xaa⁺)y)_n</td><td> (II)</td>

<td> ((Xaa⁺-Xaa^neu)x(Xaa-Xaa^neu)y)_n</td><td> (III)</td>

<td> ((Xaa-Xaa^neu)x(Xaa⁺-Xaa^neu)y)_n</td><td> (IV)</td>

wherein Xaa^neu represents an amino acid residue having a neutrai charge; Xaa⁺ represents an amino acid residue having a positive charge; Xaa represents an amino acid residue having a negative charge; x and y are integers having a value of 1,2, 3, or 4, independently; and n is an integer having a value of 1-5, [0019] wherein the self-assembling peptides can form a self-assembled barrier structure, that inhibits or prevents passage of bodily fluid or bodily substances through the structure wherein the self-assembling peptides further comprise an amino acid sequence that interacts with the extracellular mátrix, wherein the amino acid sequence anchors the self assembling peptides to the extracellular mátrix, for use in a method of preventing adhesions following surgery or injury at a site in need thereof.

[0020] The concentration of the self-assembling peptides in any given formulation can vary and can be between approximately 0.1 % and 99%, inclusive, preferably between 0.1% and 10%. In one embodiment, the concentration of the self-assembling peptides (e.g., in a liquid formulation) can be approximately 0.1-3.0% (1-30 mg/ml) (e.g., 0.1-1.0%; 1.0-2.0%; 2.0-3.0% or 1.0-3.0%). The concentration of self-assembling peptides can be higher in stock Solutions and in solid (e.g., powdered) formulations. Solid preparations may have a concentration of self assembling peptides approaching 100% (e.g., the concentration of self-assembling peptides can be 95, 96, 97, 98, 99% or more (e.g., 99.99%) ofthe composition). Whether in liquid or solid form, the peptides can be brought to the desired concentration prior to use by addition of a pharmaceutically acceptable diluent (e.g. deionized water), fillers, or oil. The formulations may include a pharmaceutically acceptable carrier or therapeutic, prophylactic or diagnostic agents. These include, bút are nőt limited to, anti-inflammatories, vasoactive agents, anti-infectives, anesthetics, growth factors, and/or cells. Metals may be added as chelators or to further decrease adhesion.

[0021] The formulation can be administered as appropriate for treatment ofone or more disorders or conditions, such as those noted above. For example, theformulation may be applied after repair of an injury or during surgery ofthe lung, eye or dura, or following an epidural or spinal táp, to prevent or minimize formation of adhesions. The self-assembling peptides can alsó be used to prevent postlaminectomy adhesions (e.g., laminectomy-induced cauda equina adhesions) and post-spinal decompression adhesions (e.g., dural adhesions). The self-assembling peptides may alsó be used as an immuné system blockade or filter, particularly in the case of injury to an organ or tissue, to prevent red blood cell accumulation and/or platelet aggregation atthe site of injury. This has been demonstrated in wounds to the liver and brain. [0022] In somé embodiments, the self-assembling peptides may allow the passage ofselect materials while preventing the passage or introduction of other materials, such as bacteria, viruses, fungi, etc.

[0023] The formulation may be applied as a hydrogel, laminate including oil, or as a spray. In one embodiment, the

EP 2 581 097 Β1 formulation is provided as a dry or lyophilized powder which can be administered directly as a powder or a tablet, disc, or wafer which hydrates at the site of application, or suspended or dissolved in a liquid, most preferably aqueous, and applied as a spray, paint, injection or a hydrogel including a matéria! such as chitin, collagen, alginate, or synthetic polymer. In the preferred embodiment, the self-assembling peptides are provided in combination with an oil, the combination of which forms a laminate. In yet another embodiment, the formulation is provided in a bandage, foam or mátrix, in which the self-assembling peptides may be dispersed or absorbed. The formulation could alsó be in the form of sutures, tape, or adhesive. The liquid formulations may be provided in a syringe or pipette having a barrel containing a composition including self-assembling peptides and a means fór expelling the composition from an open tip ofthe syringe or pipette (e.g., a plunger or bulb). The syringe may consist of one or more compartments, so that mixing of the selfassembling peptides with one or more other agents occurs at the time of application. The compartments may alsó contain excipients such as a matéria! forming a hydrogel or adhesive in one compartment and the self-assembling peptides in the other compartment. In another embodiment, one compartment may contain lyophilized or particles of self-assembling peptides, and another compartment may contain solution to dissolve or hydrate the peptides, or mixed with other powders fór dry application. The liquid and powder compositions are stable, preferably fór a period greater than one year, more preferably greater than two years and most preferably greater than three years.

[0024] One or more ofthe compositions described herein can be assembled in kits, together with instructions fór use. Fór example, the kits can include a biocompatible composition including self-assembling peptides (or a concentrated solution or powdered formulation thereof, together with a diluent) and a vasoconstrictor, a coloring agent, or an analgesic or anesthetic agent and instructions fór their combination (if nőt already combined) and use (e.g., dilution and administration). The kits can further include one or more ofthe additional agents described herein. These agents can be present within the self-assembling composition or packaged separately, and they can include one or more types of biological cells, an antibiotic or other therapeutic, collagen, an anti-inflammatory agent, a growth factor, or a nutrient. The kit may alsó include one or more of a syringe (e.g., a barrel syringe or a bulb syringe), a needle, a pipette, gauze, sponges, cotton, swabs, a bandage, a disinfectant, surgical thread, scissors, a scalpel, a sterilé fluid, a spray canister, including those in which a liquid solution is sprayed through a simple hand pump, a sterilé Container, or disposable gloves. The kit may alsó include one or more additives to vary the assembly kinetics ofthe matéria! depending on the environment in which the self-assembling peptides are to be used.

DETAILED DESCRIPTION OF THE INVENTION

I. Formulations [0025] Adhesions, as used herein, generally refers tofibrous tissue and/or scar tissue attached to organ and/or tissue surfaces, capable of connecting, covering, or distorting organs and/or tissue. Adhesions can be caused by previous infections and/or surgery. Adhesions can occur in a variety of areas of the body including, bút nőt limited to, the pelvic area, abdomen, bowel, and reproductive organs, such as fallopian tubes or ovaries.

[0026] Biocompatible, as used herein, refers to compatibility with living tissue or a living system by nőt being toxic, injurious, or physiologically reactive and nőt causing immunological rejection.

[0027] Complementary means having the capability of forming ionic or hydrogen bonding interactions between hydrophilic residues from adjacent peptides in a structure. Each hydrophilic reside in a peptide either hydrogen bonds or ionically pairs with a hydrophilic residue on an adjacent peptide, or is exposed to solvent. Pairing may alsó involve van dér Waals forces.

[0028] Effective amount, in reference to an active agent such as a self-assembling peptide or biomolecule, pharmaceutical agent, etc. refers to the amount necessary to elicit a desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount ofan agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the natúré ofthe site to which the agent is delivered, the natúré ofthe conditions fór which the agent is administered, etc. Fór example, the effective amount of a composition fór treatment of diabetic retinopathy may be an amount sufficient to promote recovery to a greater extent than would occur in the absence of the composition.

[0029] Hemostasis refers to the cessation of bleeding.

[0030] Preventing refers to causing a condition, state, or disease, orsymptom or manifestation ofsuch, or worsening ofthe severity ofsuch, nőt to occur. Preventing includes reducing the risk that a condition, state, or disease, orsymptom or manifestation ofsuch, or worsening of the severity ofsuch, will occur.

[0031] Repair, as used in reference to the repair of tissue in various embodiments ofthe invention, may include any aspect of anatomical or functional restoration of the condition of the tissue prior to an injury, deterioration, or other damage. Fór example, it may include restoration of physical continuity between portions of tissue that were separated by injury, deterioration, or other damage. Preferably such restoration of physical continuity includes reposition or reconnection ofthe portions of tissue without appreciable separation by tissue ofa type that was nőt present prior to the injury,

EP 2 581 097 Β1 such as scar tissue. Repair may, bút need nőt, include growth or development of new tissue. Repair and Healing are used interchangeably herein.

[0032] Self-assembling, as used herein, refers to the assembly of molecules intő defined, stable, noncovalently bonded assemblies that are held together by intermolecular forces. The assembly may be spontaneous or induced.

II. Self-Assembling Materials

A. Self-assembling peptides [0033] The term peptide, as used herein includes polypeptide, oligopeptide, and protein, and refers to a chain of at least two α-amino acid residues linked together by covalent bonds (e.g., peptide bonds). Useful peptides can vary in length so long as they retain the ability to self-assemble to an extent useful for one or more ofthe purposes described herein. The number of amino acid residues in the peptide may rangé from about 6 to about 200 residues, preferably from about 6 to about 64 residues, more preferably from about 8 to about 36 residues, most preferably from about 8 to about 24 residues. The peptides can be at least six amino acids in length (e.g., eight or 10 amino acids), at least 12 amino acids in length (e.g., 12 or 14 amino acids), or at least 16 amino acids in length (e.g., 16, 18, 20, 22, or 24 amino acids). Peptides that are less than 100 amino acid residues long, more preferably less than approximately 50 amino acids in length, may assemble more readily. In one embodiment, the peptide has from about 8 to about 16 residues. In another embodiment, the peptide has from about 12 to about 20 residues. In yet another embodiment, the peptide has from about 16 to about 20 residues. Peptide may refer to an individual peptide or to a collection of peptides having the same or different sequences, any of which may contain naturally occurring α-amino acid residues, non-naturally occurring α-amino acid residues, and combinations thereof. α-Amino acid analogs are alsó known in the art and may alternatively be employed. In particuíar, D-a-amino acid residues may be used.

[0034] In addition, one or more of the amino acid residues in a self-assembling peptide can be altered or derivatized by the addition ofone or more Chemical entities including, bút nőt limited to, acyl groups, carbohydrate groups, carbohydrate chains, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, or a linker which allows for conjugation orfunctionalization ofthe peptide. For example, either or both ends of a given peptide can be modified. For example, the carboxyl and/or amino groups ofthe carboxyl- and amino-terminal residues, respectively can be protected or nőt protected. The charge at a terminus can alsó be modified. For example, a group or radical such as an acyl group (RCO-, where R is an organic group (e.g., an acetyl group (CH₃CO-)) can be present at the N-terminus of a peptide to neutralize an extra positive charge that may otherwise be present (e.g., a charge nőt resulting from the side chain of the N-terminal amino acid). Similarly, a group such as an amine group (RNH-, where R is an organic group (e.g., an amino group -NH₂)) can be used to neutralize an extra negative charge that may otherwise be present at the C-terminus (e.g., a charge nőt resulting from the side chain ofthe C-terminal amino acid residue). Where an amine is used, the Cterminus bears an amidé (-CONHR). The neutralization of charges on a terminus may facilitate self-assembly. One of ordinary skill in the art will be able to select other suitable groups.

[0035] Useful peptides can alsó be branched, in which case they will contain at least two amino acid polymers, each of which consists ofat least three amino acid residues joined by peptide bonds. The two amino acid polymers may be linked by a bond other than a peptide bond.

[0036] While the sequences of the peptides can vary, useful sequences include those that convey an amphiphilic natúré to the peptides (e.g., the peptides can contain approximately equal numbers of hydrophobic and hydrophilic amino acid residues), and the peptides can be complementary and structurally compatible. Complementary peptides have the ability to form ionic or hydrogen bonds between residues (e.g., hydrophilic residues) on adjacent peptides in a structure. For example, one or more hydrophilic residues in a peptide can either hydrogen bond or ionically pair with one or more hydrophilic residues on an adjacent peptide. Hydrophilic residues are those residues that typically contain a polar functional group or a functional group that is charged at physiological conditions. Exemplary functional groups include, bút are nőt limited to, carboxylic acid groups, amino groups, sulfate groups, hydroxy groups, halogén groups, nitro groups, phosphate groups, etc. Hydrophobic residues are those residues that contain non-polar functional groups. Exemplary functional groups include, bút are nőt limited to, alkyl groups, alkene groups, alkyne groups, and phenyl groups. [0037] In one embodiment, the hydrophilic residue has the formula -NH-CH(X)-COO-, wherein X has the formula (CH₂)_yZ, wherein y = 0-8, preferably 1-6, more preferably 1-4 and most preferably 1-3, and Z is a polar or charged functional group including, bút nőt limited to, a carboxylic acid group, an amino group, a sulfate group, a hydroxy group, a halogén group, a nitro group, a phosphate group, or a functional group containing a quaternary amine. The alkyl chain can be in a linear, branched, or cyclic arrangement. X may alsó contain one or more heteroatoms within the alkyl chain and/or X may be substituted with one or more additiönai substituents. In a preferred embodiment, Z is a carboxylic acid group or an amino group. In one embodiment, the hydrophobic residue has the formula -NH-CH(X)-COO-, wherein X has the formula (CH₂)_yZ, wherein y = 0-8, preferably 1-6, more preferably 1-4, and more preferably 1-3, and Z is a non5

EP 2 581 097 Β1 polar functional group including, bút nőt limited to, an alkyl group, an alkene group, an alkyne group, or a phenyl group. The alkyl, alkene, or alkyne chain can be in a linear, branched, or cyclic arrangement. X may alsó contain one or more heteroatoms within the alkyl chain and/or X may be substituted with one or more additional substituents. In a preferred embodiment, X is an alkyl group, such as a methyl group.

[0038] Where self-assembling peptides are used, it is thought that their side chains (or R groups) partition intő two faces, a polar face with positively and/or negatively charged ionic side chains (e.g., side chains containing -OH, -NH, -CO₂H, or -SH groups), and a nonpolar face with side chains that are considered neutral or uncharged at physiological pH (e.g., the side chain of an alanine residue or residues having other hydrophobic groups). The positively charged and negatively charged amino acid residues on the polar face of one peptide can form complementary ionic pairs with oppositely charged residues of another peptide. These peptides may therefore be called ionic, self-complementary peptides. Ifthe ionic residues alternate with one positively and one negatively charged residue on the polar face (-+-++-+), the peptides may be described as modulus I; ifthe ionic residues alternate with two positively and two negatively charged residues (-++-++) on the polar face, the peptides are described as modulus II; ifthe ionic residues alternate with three positively and three negatively charged residues (+++—+++—) on the polar face, the peptides are deseribe as modulus III; ifthe ionic residues alternate with four positively and four negatively charged residues (++++—++++— -) on the polar face, they are described as modulus IV. A peptide having four repeating units ofthe sequence EAKA (SEQ ID NO: 111) may be designated EAKA16-I (SEQ. ID NO. 410), and peptides having other sequences may be described by the same convention.

[0039] Unpaired residues can interact (e.g. form hydrogen bonds, etc,) with the solvent. Peptide-peptide interactions may alsó involve van dér Waals forces and/or forces that do nőt constitute covalent bonds. The peptides are structurally compatible when they are capable of maintaining a sufficiently constant intrapeptide distance to allow self-assembly and structure formation. The intrapeptide distance can vary. Intrapeptide distance, as used herein, refers to the average of a representative number of distances between adjacent amino acid residues. In one embodiment, the intrapeptide distance is less than about 4 angstroms, preferabíy less than about 3, more preferabíy less than about 2 angstroms, and most preferabíy less than about 1 angstrom. The intrapeptide distance may be larger than this, however. These distances can be calculated based on moleeular modeling or based on a simplified procedure described in U.S. Patent Number No. 5,670,483 to Zhang et al.

[0040] The structures described herein can be formed through self-assembly ofthe peptides described in U.S. Patent Nos. 5,670,483; 5,955,343; 6,548,630; and 6,800,481 to Zhang et al.; Holmes et al., Proc. Natl. Acad. Sci. USA, 97:6728-6733 (2000); Zhang et al., Proc. Natl. Acad. Sci. USA, 90:3334-3338 (1993); Zhang et al., Biomaterials, 16:1385-1393 (1995); Caplan etal., Biomaterials, 23:219-227 (2002); León et al., J. Biomater. Sci. Polym. Ed., 9:297-312 (1998); and Caplan et al., Biomacromolecules, 1:627-631 (2000).

[0041] Self-assembling peptides containing alternating hydrophobic and hydrophilic amino residues can be used. Examples of representative hydrophobic and hydrophilic peptides are listed in Table 1.

No.

1.

2.

3.

4.

5.

6.

7.

8. 9. 10 11 12

Table 1. Representative Self-Assembling Peptides Sequence (N -&gt; C) n-SGSGSGSGSGSGSGSG-c (SEQ ID NO: 2) n-SASASASASASASASA-c (SEQ ID NO: 3) n-SVSVSVSVSVSVSVSV-c (SEQ ID NO: 4) n-SLSLSLSLSLSLSLSL-c (SEQ ID NO: 5) n-SISISISISISISISI-c (SEQ ID NO: 6) n-SMSMSMSMSMSMSMSM-c (SEQ ID NO: 7) n-SFSFSFSFSFSFSFSF-c (SEQ ID NO: 8) n-SWSWSWSWSWSWSWSW-c (SEQ ID NO: 9) n-SPSPSPSPSPSPSPSP-c (SEQ ID NO: 10) n-TGTGTGTGTGTGTGTG-c (SEQ ID NO: 11) n-TATATATATATATATA-c (SEQ ID NO: 12) n-TVTVTVTVTVTVTVTV-c (SEQ ID NO: 13) n-TLTLTLTLTLTLTLTL-c (SEQ ID NO: 14) n-TITITITITITITITI-c (SEQ ID NO: 15) n-TMTMTMTMTMTMTMTM-c (SEQ ID NO: 16) n-TFTFTFTFTFTFTFTF-c (SEQ ID NO: 17) n-TWTWTWTWTWTWTWTW-c (SEQ ID NO: 18) n-TPTPTPTPTPTPTPTP-c (SEQ ID NO: 19)

EP 2 581 097 Β1 (continued)

No. Sequence (N -&gt; C)

TT n-CGCGCGCGCGCGCGCG-c (SEQ ID NO: 20)

20. n-CACACACACACACACA-c (SEQ ID NO: 21)

21. n-CVCVCVCVCVCVCVCV-c (SEQ ID NO: 22)

22. n-CLCLCLCLCLCLCLCL-c (SEQ ID NO: 23)

23. n-CICICICICICICICI-c (SEQ ID NO: 24)

24. n-CMCMCMCMCMCMCMCM-c (SEQ ID NO: 25)

25. n-CFCFCFCFCFCFCFCF-c (SEQ ID NO: 26)

26. n-CWCWCWCWCWCWCWC-c (SEQ ID NO: 27)

27. n-CPCPCPCPCPCPCPCP-c (SEQ ID NO: 28)

28. n-YGYGYGYGYGYGYGYG-c (SEQ ID NO: 29)

29. n-YAYAYAYAYAYAYAYA-c (SEQ ID NO: 30)

30. n-YVYVYVYVYVYVYVYV-c (SEQ ID NO: 31)

31. n-YLYLYLYLYLYLYLYL-c (SEQ ID NO: 32)

32. n-YIYIYIYIYIYIYIYI-c (SEQ ID NO: 33)

33. n-YMYMYMYMYMYMYMYM-c (SEQ ID NO: 34)

34. n-YFYFYFYFYFYFYFYF-c (SEQ ID NO: 35)

35. n-YWYWYWYWYWYWYWYW-c (SEQ ID NO: 36)

36. n-YPYPYPYPYPYPYPYP-c (SEQ ID NO: 37)

37. n-NGNGNGNGNGNGNGNG-c (SEQ ID NO: 38)

38. n-NANANANANANANANA-c (SEQ ID NO: 39)

39. n-NVNVNVNVNVNVNVNV-c (SEQ ID NO: 40)

40. n-NLNLNLNLNLNLNLNL-c (SEQ ID NO: 41)

41. n-NINININININININI-c (SEQ ID NO: 42)

42. n-NMNMNMNMNMNMNMNM-c (SEQ ID NO: 43)

43. n-NFNFNFNFNFNFNFNF-c (SEQ ID NO: 44)

44. n-NWNWNWNWNWNWNWNW-c (SEQ ID NO: 45)

45. n-NPNPNPNPNPNPNPNP-c (SEQ ID NO: 46)

46. n-QGQGQGQGQGQGQGQG-c (SEQ ID NO: 47)

47. n-QAQAQAQAQAQAQAQA-c (SEQ ID NO: 48)

48. n-QVQVQVQVQVQVQVQV-c (SEQ ID NO: 49)

49. n-QLQLQLQLQLQLQLQL-c (SEQ ID NO: 50)

50. n-QIQIQIQIQIQIQIQI-c (SEQ ID NO: 51)

51. n-QMQMQMQMQMQMQMQM-c (SEQ ID NO: 52)

52. n-QFQFQFQFQFQFQFQF-c (SEQ ID NO: 53)

53. n-QWQWQWQWQWQWQWQW-c (SEQ ID NO: 54)

54. n-QPQPQPQPQPQPQPQP-c (SEQ ID NO: 55)

55. n-AEAKAEAKAEAKAEAK-c (SEQ ID NO: 56)

56. n-RADARADARADARADA-c (SEQ ID NO: 1)

57. n-RAEARAEARAEARAEA-c (SEQ ID NO: 58)

58. n-KADAKADAKADAKADA-c (SEQ ID NO: 59) [0042] Other peptides or proteins can be used in combination or alternation with the disclosed self-assembling peptides or compositions. It will be appreciated that the additiönai peptides can include other self-assembling peptides or proteins. Alternatively, the peptide may be peptides that do nőt self-assemble. Representative additiönai peptides, proteins, or chemically modified variants thereof include, bút are nőt limited to the peptides provided in Table 2.

Table 2. Additiönai Peptides

1. Pmp-Y(Me)-l-T-N-C-P-Orn-Y-NH₂ (SEQ ID NO: 60)

2. Mpr-Y-F-Q-N-C-P-R (SEQ ID NO: 61)

3. C-Y-F-Q-N-C-P-R-G-NH₂ (SEQ ID NO: 62)

EP 2 581 097 Β1 (continued)

<td></td><td> 4.</td><td> C-Y-F-Q-N-C-P-R (SEQ ID NO: 63)</td>

<td></td><td> 5.</td><td> C-Y-lle-Q-N-C-P-R-G-NH₂ (SEQ ID NO: 64)</td>

<td> 5</td><td> 6.</td><td> Y-F-Q-N-Asu-P-R-G-NH₂ (SEQ ID NO: 65)</td>

<td></td><td> 7.</td><td> Y-lle-Q-N-Asu-P-R-G-NH₂ (SEQ ID NO: 66)</td>

<td></td><td> 8.</td><td> Mpr-D-PyridylAnine-F-Q-N-C-P-R-G-NH₂ (SEQ ID NO: 67)</td>

<td></td><td> 9.</td><td> Deamino-Pen-Y-F-V-N-C-P-DR-G-NH₂ (SEQ ID NO: 68)</td>

<td rowspan="2"> 10</td><td> 10.</td><td> Mpr-Y-F-Q-N-C-P-R-G-NH₂ (SEQ ID NO: 69)</td>

<td> 11.</td><td> Mpr-Y-F-Q-N-C-P-DR-G-NH₂ (SEQ ID NO: 70)</td>

<td></td><td> 12.</td><td> Mpr-Y-F-Q-N-C-P-K (SEQ ID NO: 71)</td>

<td></td><td> 13.</td><td> C-Y-F-Q-N-C-P-K-G-NH₂ (SEQ ID NO: 72)</td>

<td></td><td> 14.</td><td> C-Y-F-Q-N-C-P-K (SEQ ID NO: 73)</td>

<td> 15</td><td> 15.</td><td> Mpr-Y-F-V-N-C-P-DR-G-NH₂ (SEQ ID NO: 74)</td>

<td></td><td> 16.</td><td> C-F-lle-Q-N-C-P-Orn-G-NH₂ (SEQ ID NO: 75)</td>

<td></td><td> 17.</td><td> Pmp-DY(OEt)-F-V-N-C-P-Cit-G-NH₂ (SEQ ID NO: 76)</td>

<td></td><td> 18.</td><td> Pmp-Y(OEt)-F-V-N-C-P-R-G-NH₂ (SEQ ID NO: 77)</td>

<td></td><td> 19.</td><td> Pmp-Y(Me)-F-Q-N-C-P-R-G-NH₂ (SEQ ID NO: 78)</td>

<td> 20</td><td> 20.</td><td> Pmp-Y(Me)-l-Q-N-C-P-Orn-G-NH₂ (SEQ ID NO: 79)</td>

<td></td><td> 21.</td><td> G-DR-G-D-S-P (SEQ ID NO: 80)</td>

<td></td><td> 22.</td><td> G-DR-G-D-S-P-A-S-S-K (SEQ ID NO: 81)</td>

<td></td><td> 23.</td><td> G-P-R</td>

<td> 25</td><td> 24.</td><td> G-Pen-G-R-G-D-S-P-C-A (SEQ ID NO: 82)</td>

<td></td><td> 25.</td><td> GRADSP (SEQ ID NO: 83)</td>

<td></td><td> 26.</td><td> GRGD-DS-P (SEQ ID NO: 84)</td>

<td></td><td> 27.</td><td> GRGDNP (SEQ ID NO: 85)</td>

<td></td><td> 28.</td><td> GRGDS (SEQ ID NO: 86)</td>

<td> 30</td><td> 29.</td><td> GRGDSP (SEQ ID NO: 87)</td>

<td></td><td> 30.</td><td> GRGDSPC (SEQ ID NO: 88)</td>

<td></td><td> 31.</td><td> GRGDSPK (SEQ ID NO: 89)</td>

<td></td><td> 32.</td><td> GRGDTP (SEQ ID NO: 90)</td>

<td> 35</td><td> 33.</td><td> GRGES (SEQ ID NO: 91)</td>

<td></td><td> 34.</td><td> GRGESP (SEQ ID NO: 92)</td>

<td></td><td> 35.</td><td> GRGETP (SEQ ID NO: 93)</td>

<td></td><td> 36.</td><td> KGDS (SEQ ID NO: 94)</td>

<td></td><td> 37.</td><td> GAVSTA(SEQ ID NO: 95)</td>

<td> 40</td><td> 38.</td><td> WTVPTA (SEQ ID NO: 96)</td>

<td></td><td> 39.</td><td> TDVNGDGRHDL (SEQ ID NO: 97)</td>

<td></td><td> 40.</td><td> REDV (SEQ ID NO: 98)</td>

<td></td><td> 41.</td><td> RGDC (SEQ ID NO: 99)</td>

<td rowspan="2"> 45</td><td> 42.</td><td> RGDS (SEQ ID NO: 100)</td>

<td> 43.</td><td> RGDSPASSKP (SEQ ID NO: 101)</td>

<td></td><td> 44.</td><td> RGDT (SEQ ID NO: 102)</td>

<td></td><td> 45.</td><td> RGDV (SEQ ID NO: 103)</td>

<td></td><td> 46.</td><td> RGES (SEQ ID NO: 104)</td>

<td> 50</td><td> 47.</td><td> SDGR (SEQ ID NO: 105)</td>

<td></td><td> 48.</td><td> SDGRG (SEQ ID NO: 106)</td>

<td></td><td> 49.</td><td> YRGDS (SEQ ID NO: 107)</td>

<td></td><td> 50.</td><td> EGVNDNEEGFFSAR (SEQ ID NO: 108)</td>

<td> 55</td><td> 51.</td><td> YADSGEGDFLAEGGGVR (SEQ ID NO: 109)</td>

EP 2 581 097 Β1 (continued)

52. Glp-GVNDNEEGFFSARY (SEQ ID NO: 110)

Pmp = pyridoxamine phosphate

Mpr = 3-mercaptopropionyl

Deamino-Pen = deamino penicillamine

Pen = penicillamine

Asu = amino succinyl

OEt = ethoxy

Me = methyl

Cit = citruline [0043] Other useful self-assembling peptides can be generated, for example, which differ from those exemplified by a single amino acid residue or by multiple amino acid residues (e.g., by inclusion or exclusion of a repeating quartet). For example, one or more cysteine residues may be incorporated intő the peptides, and these residues may bond with one another through the formation of disulfide bonds. Structures bonded in this manner may have increased mechanical strength relatíve to structures made with comparable peptides that do nőt include cysteine residues and thus are unable to form disulfide bonds.

[0044] The amino acid residues in the self-assembling peptides can be naturally occurring or non-naturally occurring amino acid residues. Naturally occurring amino acids can include amino acid residues encoded by the standard genetic eode as well as non-standard amino acids (e.g., amino acids having the D-configuration instead ofthe L-configuration), as well as those amino acids that can be formed by modifications of standard amino acids (e.g. pyrrolysine or selenocysteine). Non-naturally occurring amino acids are nőt found or have nőt been found in natúré, bút can be incorporated intő a peptide chain. Suitable non-naturally occurring amino acids include, bút are nőt limited to, D-alloisoleucine(2R,3S)2-amino-3-methylpentanoic acid, L-cycIopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. Other examples of nonnaturally occurring amino acids can be found in textbooks or on the worldwide web (e.g., a site is maintained by the California Institute of Technology which displays structures of non-natural amino acids that have been successfully incorporated intő functional proteins). Non-natural amino acid residues and amino acid derivatives described in U.S. Patent Application Publication No. 2004/0204561 to Ellison.

[0045] Self-assembling peptides can be chemically synthesized or purified from natural or recombinantly-produced sources by methods well known in the art. For example, peptides can be synthesized using standard f-moc chemístry and purified using high pressure liquid chromatography (HPLC).

[0046] Self-complementary peptides such as EAKA16-I (SEQ. ID NO. 410), RADA16-I (SEQ. ID NO. 1), RAEA16-I (SEQ. ID NO. 58), and KADA16-I (SEQ. ID NO. 59) are described in Zhang, S., et al. ((1999) Peptide self-assembly in functional polymer Science and engineering. Reactive &amp; Functional Polymers, 41,91-102). The self-assembling peptides comprise a sequence of amino acid residues conforming to one or more of Formulas l-IV:

<td> ((Xaa^neu-Xaa⁺)x(Xaa^neu-Xaa^_)y)_n</td><td> (I)</td>

<td> (Xaa^neu-Xaa)x(Xaa^neu-Xaa⁺)y)_n</td><td> (II)</td>

<td> ((Xaa⁺-Xaa^neu)x(Xaa-Xaa^neu)y)_n</td><td> (III)</td>

<td> ((Xaa-Xaa^neu)x(Xaa⁺-Xaa^neu)y)_n</td><td> (IV)</td>

Xaa^neu represents an amino acid residue having a neutrai charge; Xaa⁺ represents an amino acid residue having a positive charge; Xaa represents an amino acid residue having a negative charge; x and y are integers having a value of 1,2, 3, or 4, independently; and n is an integer having a value of 1-5. Peptides with modulus I (i.e., peptides having alternate positively and negatively charged R groups on one side (e.g., the polarface ofthe β-sheet) are described by each of Formulas l-IV, where x and y are 1. Peptides of modulus II (i.e., peptides having two residues bearing one type of charge (e.g., a positive charge) followed by two residues bearing another type of charge (e.g., a negative charge)) are described by the same formulas where both x and y are 2. Examples of peptides of modulus III (i.e. peptides having three residues bearing one type of charge (e.g., a positive charge) followed by three residues bearing another type of charge (e.g., a negative charge)) include, bút are nőt limited to, RARARADADADA (SEQ. ID NO. 112) and RARARARADADADADA (SEQ. ID NO. 113).

[0047] Other hydrophilic residues that form hydrogen bonds including, bút nőt limited to, asparagine and glutamine, may be incorporated intő the peptides. Ifthe alanine residues in the peptides are changed to more hydrophobic residues,

EP 2 581 097 Β1 such as leucine, isoleucine, phenylalanine or tyrosine, the resulting peptides have a greater tendency to self-assemble and form peptide matrices with enhanced strength. Somé peptides that have similar amino acids sequences and lengths as the peptides described herein form alpha-helices and random-coils, rather than beta-sheets, and do nőt form macroscopic structures. Thus, in addition to self-complementarity, other factors are likely to be important fór the formation of macroscopic structures, such as the peptide length, the degree of intermolecular interaction, and the ability to form staggered arrays.

[0048] Peptide-based structures can be formed of heterogeneous mixtures of peptides (i.e., mixtures containing more than one type of peptide conforming to a given formula or to two or more ofthe formulas). In somé embodiments, each of the types of peptides in the mixture are able to self-assemble alone. In other embodiments, one or more of each type of peptide would nőt, alone, self-assemble bút the combination of heterogeneous peptides may self-assemble (i.e., peptides in the mixture are complementary and structurally compatible with each other). Thus, either a homogeneous mixture of self-complementary and self-compatible peptides of the same sequence or containing the same repeating subunit, or a heterogeneous mixture of different peptides, which are complementary and structurally compatible to each other, can be used.

[0049] In a preferred embodiment, one or more short amino acid sequences that assists in self-assembly (referred to as assembly assist sequences) can be added to a homogeneous or heterogeneous mixture of amino acid sequences that alone do nőt self-assemble. The assembly assist sequences contain amino acids that are complementary with the amino acids in the sequences in the mixture. The assembly assist sequences may contain any number of amino acids. Preferably, the assembly assist sequences contain at least 4 amino acids. The assembly assist sequences may contain a flexible linker between the amino acids that assists in self-assembly. Fór example, the assembly assist sequence may contain a pair, a triad, or a quartetof assembly assisting amino acids at the termini ofthe sequence which are connected via a flexible linker. Suitable assembly assist sequences include, bút are nőt limited to, RADA (SEQ ID NO: 57) and EAKA (SEQ ID NO: 1111).

[0050] Suitable linkers include, bút are nőt limited to, ether based tethers such as polyethylene glycol (PEG), N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP, 3- and 7-atom spacer), long-chain- SPDP (12-atom spacer), (Succinimidyloxycarbonyl-a-methyl-2-(2-pyndyldithio) toluene) (SMPT, 8-atom spacer), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC, 11-atom spacer) and Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, (sulfo-SMCC, 11-atom spacer), m-Maleimidobenzoyl-N hydroxysuccinimide ester (MBS, 9-atom spacer), Ν-(γmaleimidobutyryloxy)succinimide ester (GMBS, 8-atom spacer), N-(y-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBS, 8-atom spacer), Succinimidyl 6-((iodoacetyl) amino) hexanoate (SIAX, 9-atom spacer), Succinimidyl 6-(6-(((4iodoacetyl)amino)hexanoyl)amino)hexanoate (SIAXX, 16-atom spacer), and p-nitrophenyl iodoacetate (NPIA, 2-atom spacer). One ordinarily skilled in the art alsó will recognize that a number of other linkers, with different numbers of atoms, may be used.

[0051] The compositions described herein regardless of the precise form (e.g., whether in a liquid form or molded) and regardless ofthe overall compositions (e.g., whether combined with another agent, contained within a device, or packaged in a kit) can include a mixture of one or more peptide chains.

[0052] Self-assembled structures can be formed that have varying degrees of stiffness or elasticity. The structures typically have a low elastic modulus (e.g., a modulus in the rangé of about 0.01 to about 1000 kPa, preferably from about 1 to about 100 kPa, more preferably from about 1 to about 10 kPa as measured by standard methods, such as in a standard cone-plate rheometer). Low values may be preferable, as they permit structure deformation as a result of movement, in response to pressure, in the event of cell contraction. More specifically, stiffness can be controlled in a variety ofways, including by changing the length, sequence, and/or concentration ofthe precursor molecules (i.e., selfassembling peptides). Other methods fór increasing stiffness can alsó be employed. Fór example, one can attach, to the precursors, biotin molecules or any other molecules that can be subsequently cross-linked or otherwise bonded to one another. The molecules (e.g., biotin) can be included at an N- or C-terminus ofa peptide or attached to one or more residues between the termini. Where biotin is used, cross-linking can be achieved by subsequent addition of avidin. Biotin-containing peptides or peptides containing other cross-linkable molecules are within the scope of the present invention. Fór example, amino acid residues with polymerizable groups, including bút nőt limited to vinyl groups, may be incorporated and cross-linked by exposure to UV light. The extent of crosslinking can be precisely controlled by applying the radiation fór a predetermined length of time. The extent of crosslinking can be determined by lightscattering, gél filtration, or scanning electron microscopy using methods well known in the art. Furthermore, crosslinking can be examined by HPLC or mass spectrometry analysis of the structure after digestion with a protease, such as mátrix metalloproteases. Matéria! strength may be determined before and after cross-linking. Regardless of whether crosslinking is achieved by a Chemical agent or light energy, the molecules may be cross-linked in the course of creating a mold or when peptide-containing Solutions are applied to the body. Further, self-assembling peptide chains can be crosslinked to form a spider web-type pattern to reinforce the matéria! in vivő. The crosslinks serve to reinforce the matéria! providing increased rigidity and strength. Fór example, the self-assembling peptides can be applied to a wound, wherein the periphery ofthe self-assembled peptides is functionalized with polymerizable groups. Upon crosslinking,

EP 2 581 097 Β1 the periphery of the self-assembled peptides becomes more rigid, anchoring the peptides to the wound site, while the interior of self-assembled peptides remains flexible to move as the body moves.

[0053] The half-life (e.g., the in vivő half-life) ofthe structures can alsó be modulated by incorporating protease or peptidase cleavage sites intő the precursors that subsequently form a given structure. Proteases or peptidases that occur naturally in vivő or that are introduced (e.g., by a surgeon) can then promote degradation by cleaving their cognate substrates.

[0054] Combinations of any ofthe modifications described here can be made. For example, self-assembling peptides that include a protease cleavage site and a cysteine residue and/or a cross-linking agent, kits and devices containing them, and methods of using them can be utilized.

[0055] The peptide structures formed from any self-assembling peptides made by any process can be characterized using various biophysical and optical techniques, such as circular dichroism (CD), dynamic light scattering, Fourier transform infrared (FTIR), atomic force (tension) microscopy (ATM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). For example, biophysical methods can be used to determine the degree of betasheet secondary structure in the peptide structure. Filament and poré size, fiber diameter, length, elasticity, and volume fraction can be determined using quantitative image analysis of scanning and/or transmission electron micrographs. The structures can alsó be examined using several standard mechanical testing techniques to measure the extent of swelling, the effect of pH and ion concentration on structure formation, the level of hydration under various conditions, the tensile strength, as well as the manner in which various characteristics change over the period of time required for the structures to form and degrade. These methods allow one of ordinary skill in the art to determine which ofthe various alternatives and peptides described herein are most suitable for use in the various methods, and allow optimization of the various processes.

[0056] In another embodiment, the self-assembling peptides can anchor or interact with the structural extracellular mátrix (ECM) at the edges of blood vessels and/or tissues are described herein. These self-assembling peptides typically have hydrophobic and/or hydrophilic sections which allow the matéria! to react or interact with the glycoproteins found in the ECM.

[0057] Preferably, the self-assembling peptides when they breakdown, do nőt cause any secondary toxicity. Further, the break down product of the self-assembling peptides would be suitable for the growth and repair of the surrounding tissues.

1. Other Self-Assembling Materials [0058] Another embodiment provides self-assembling peptides having a segmentof residues having a positive charge under physiological conditions joined to a segment of residues having a negative charge under physiological conditions. The segment of positively or negatively charged residues can include about 2 to about 50 amino acid residues, typically about 3 to about 30 residues, more typically about 10 to about 20 amino acid residues. In another embodiment, about half ofthe residues ofthe self-assembling peptide are positively charged and the other half ofthe self-assembling peptide has negatively charged amino acid residues. A combination of these peptides can self-assemble by matching the positive end of a first self-assembling peptide to the negative end of a second self- assembling peptide. The negative end of the first self-assembling peptide will match up or align with the positive end ofthe second self-assembling peptide. The selfassembling peptides will stack-up or aggregate based on opposite ends ofthe self-assembling peptides being attacked based on charge at physiological compositions. One representative embodiment provides a self-assembling peptide having thefollowing sequence RRRR -DDDD (SEQ ID NO: 114) or GGGG-SSSS (SEQ ID NO: 115).

[0059] In still another embodiment, the self-assembling peptide has a first hydrophobic region operably linked to a first hydrophilic region. The first hydrophobic region can include a segment of amino acid residues that have hydrophobic side chains under physiological conditions. The first hydrophilic region can include a segment of amino acid residues that have hydrophilic side chains under physiological conditions. In this embodiment, the hydrophobic ends of the selfassembling peptides would assemble with other hydrophobic ends and the hydrophilic ends would assemble with other hydrophilic ends. Assembly can be controlled by altering the environment ofthe peptides. Such materials could be used to coat the inside ofa lumen. The hydrophobic ends would likely interact with the ECM ofthe lumen surface sealing the surface while the hydrophilic ends extend out towards the center ofthe lumen. Fluids would continue to flow through the lumen. As the self-assembling peptides degrade and/or are removed from the lumen surface, peptides would flow in from other areas and again anchor to the lumen surface, thus the composition acts a reservoir providing new peptides as needed. Alternatively, additional self-assembling peptides could be administered to replace peptides that have worn or been degraded. In another embodiment, the self-assembling peptides can be used as dynamic patches, for example, in the treatment of ulcers or for use in the intestine.

[0060] Another embodiment provides a self-assembling peptide that contains a segment of residues that have either a positive or negative charge under physiological conditions. Representative amino acid sequences for positively charged self-assembling peptides include, bút are nőt limited to, KKKK (SEQ ID NO: 116), RRRR (SEQ ID NO: 117), or HHHH

EP 2 581 097 Β1 (SEQ ID NO: 118). Representative amino acid sequences for negatively charged self-assembling peptides include, bút are nőt limited to, DDDD (SEQ ID NO: 119) or EEEE (SEQ ID NO: 120). When combined, a string of positively charged amino acid residues will align parallel and opposite with a string of negatively charged amino acid residues. In certain embodiments, strings of positively charged amino acids will alternate with strings of negatively charged amino acids to for a multilayered structure.

[0061] Still another embodiment provides self-assembling peptides that have a combination of hydrophilic polar amino acid residues and hydrophobic non-polar amino acid residues under physiological conditions. The one or more hydrophilic residues can alternate with one or more hydrophobic residues. For example, the amino acid sequence of a representative self-assembling peptide can be GQGQ (SEQ ID NO: 121), GGQQGG (SEQ ID NO: 122), GQQGQQG (SEQ ID NO: 123), GGQGGQGG (SEQ ID NO: 124), etc. It will be appreciated that the partitioning ofthe self-assembling peptide intő a polar or non-polar environment can be controlled by altering the ratio of hydrophobic amino acid residues to hydrophilic amino acid residues, wherein a ratio greater than 1:1 indicates that the peptide partitions more in hydrophobic conditions compared to hydrophilic conditions. A ratio of less than 1:1 indicates that the peptide partitions more in hydrophilic conditions compared to hydrophobic conditions.

[0062] Combinations of any ofthe modifications described here can be made. For example, self-assembling peptides that include a protease cleavage site and a cysteine residue and/or a cross-linking agent, kits and devices containing them, and methods of using them can be utilized. The compositions can be used to prevent or limit movement of a bodily fluid, to stabilize tissue or cells, orto prevent contamination when administered to a site in need thereof. The compositions can be in the form of a dry powder, a wafer, a disk, a tablet, a eapsule, a liquid, a gél, a cream, a foam, an ointment, an émulsion, a coating on a stent, catheter or other medical implant, the peptides incorporated intő a microparticle, a polymeric mátrix, a hydrogel, a fabric, a bandages, a suture, or a sponge.

B. Formation of Self-assembling Peptides [0063] Prior to self-assembly, the self-assembling peptides may be contained in (e.g., dissolved in) a solution that is substantially free of ions (e.g., monovalent ions) or that contains a sufficiently low concentration of ions to prevent significant self-assembly (e.g., a concentration of ions less than 10, 5, 1, or 0.1 mM). Self-assembly may be initiated or enhanced at any subsequent time by the addition ofan ionic solute or diluent to a solution ofthe matéria! or by a change in pH. For example, NaCI at a concentration of between approximately 5 mM and 5 M can induce the assembly of macroscopic structures within a short period oftime (e.g., within a few minutes). Lower concentrations of NaCI may alsó induce assembly bút at a slower rate. Alternatively, self-assembly may be initiated or enhanced by introducing the peptides (whether dry, in a semi-solid gél, or dissolved in a liquid solution that is substantially free of ions) intő a fluid (e.g., a physiological fluid such as blood or gastric juice) or an area (e.g., a body cavity such as the nőse or mouth or a cavity exposed by a surgical procedure) comprising such ions. The gél does nőt have to be preformed prior to application to the desired site. Generally, self-assembly is expected to occur upon contacting the peptides with such a solution in any manner.

[0064] A wide variety of ions, including anions and cations (whether divalent, monovalent, or trivalent), can be used. For example, one can promote a phase transition by exposure to monovalent cations such as Li⁺, Na⁺, K⁺, and Cs⁺. The concentration of such ions required to induce or enhance self-assembly is typically at least 5 mM (e.g., at least 10, 20, or 50 mM). Lower concentrations alsó facilitate assembly, although at a reduced rate. When desired, self-assembling peptides can be delivered with a hydrophobic matéria! (e.g. a pharmaceutically-acceptable oil) in a concentration that permits self-assembly, bút at a reduced rate. When self-assembling peptides are mixed with a hydrophobic agent such as an oil or lipid the assembly ofthe peptides forms different structures. The structures will appear like ice on a layer of oil. In somé cases when another matéria! is added, the matéria! will assemble intő various other three dimensional structures that may be suitable for loading of a therapeutic agent. The hydrophilic part of the molecule will assemble in such away as to minimize hydrophobic-hydrophilic interaction, thereby creating a barrier between the two environments. Several experiments have shown that the self-assembling peptides will align on the surface of the oil like ice on water with the hydrophobic part of the molecule toward the surface and the hydrophilic portion of the molecule facing away from the oil, or will form toroidal-like structures with the hydrophobic matéria! contained inside. This type of behavior enables the encapsulation of therapeutics or other molecule of interested for delivery in the body.

[0065] In another embodiment, the composition may contain a salt scavenger to drive assembly to a preferred configuration. For example, circular dichroism (CD) experiments indicate that the assembly dynamics can be controlled using salt scavengers or salt enhancement to increase the formation of β-sheets, α-helices, or more random configurations. The compositions may optionally contain an indicator showing the configuration ofthe assembly (e.g., α-helix, βsheet, lattice, etc.).

[0066] Alternatively, somé ofthe self-assembling peptides described herein do nőt require ions to self-assemble bút may self-assemble due to interactions with a solvent, hydrophobic interactions, side chain interactions, hydrogen bonding, and the like.

EP 2 581 097 Β1 [0067] Depending on the formulation and desired properties of the macroscopic structure (e.g., the stiffness of the scaffold or the rate of its formation), the concentration of precursors (i.e., self-assembling peptides) can vary from approximately 0.01 % w/v (0.1 mg/ml) to approximately 99.99% w/v (999.9 mg/ml), inclusive. For example, the concentration priorto scaffold formation can be between approximately 0.1% (1 mg/ml) and 10% (100 mg/ml), inclusive (e.g., about 0.1%-5%; 0.5%-5%; 1.0%; 1.5%; 2.0%; 2.5%; 3.0%; or 4.0% or more). The precursors (i.e., self-assembling peptides) can be formulated as powders and administered in a powder form or resuspended. If dry, the peptides can then self-assemble following contact with bodily fluids (e.g., at a site of injury).

[0068] The self-assembling peptides can be formed within regularly or irregularly-shaped molds, which may include a body cavity óra portion ofthe body (e.g., the lumen ofa blood véssél) or which may be an inért matériái such as plastic or glass. The structures or scaffolds can be made to conform to a predetermined shape or to have a predetermined volume. To form a structure with a predetermined shape or volume (e.g., a desired geometry or dimension, including thin sheets or films), an aqueous solution ofthe self-assembling peptides is placed in a pre-shaped casting mold, and the peptides are induced to self-assemble by the addition of a plurality of ions. Alternately, the ions may be added to the solution shortly before piacing the solution intő the mold, provided that care is taken to piacé the solution intő the mold before substantial assembly occurs. Where the mold is a tissue (e.g., the lumen of a blood véssél or other compartment, whether in situ or nőt), the addition of an ionic solution may nőt be necessary. The resulting self-assembled peptides characteristics, the time required for assembly, and the dimensions ofthe macroscopic structure that forms are governed by the concentration and amount of solution that is applied, the concentration of ions used to induce assembly ofthe structure, and the dimensions ofthe casting apparátus. The scaffold can achieve a gel-like or substantially solid form at room temperature, and heat may be applied to facilitate the molding (e.g., one can heat a solution used in the molding proeess (e.g., a precursor-containing solution) to a temperature ranging up to about body temperature (approximately 37°C)). Once the scaffold has reached the desired degree of firmness, it can be removed from the mold and used for a purpose described herein. Alternatively, the self-assembling peptides described herein may be used to anchor hőst tissue to a tissue mátrix or scaffold. For example, the self-assembling peptides described herein can be used as a glue to anchor hőst tissue that is to be regenerated to a tissue mátrix or scaffold to ensure that the mátrix or scaffold stays in piacé in the Iocal environment to which it is injected or implanted. Tissue matrices and scaffolds are well known in the art and can be prepared from synthetic, semisynthetic, and/or natural materials.

[0069] Peptides that assemble and/or undergo a phase transition (e.g., a transition from a liquid state to a semi-solid, gél, etc.) when they come in contact with the body or an ionic solution are useful in preventing the movement of bodily substances. Self-assembly or phase transition is triggered by components found in a subject’s body (e.g., ions) or by physiological pH and is assisted by physiological temperatures. Self-assembly or phase transition can begin when the compositions are exposed to or brought intő contact with a subject’s body and may be facilitated by the Iocal application of heat to the area where the composition has been (or will be) deposited. Based on studies to date, self-assembly occurs rapidly upon contact with internál bodily tissues without the application of additional heat. The time required for effective assembly and/or phase transition can occur in 60 seconds or less following contact with a subject’s internál tissues or to conditions similar to those found within the body (e.g., in 50, 40, 30, 20, or 10 seconds or less). In somé circumstances, such as where the concentration of self-assembling peptides in the composition is low or where the movement of the bodily substance is substantial, self-assembly or phase transition may take longer to achieve the desired effect, forexample, up to a minute, 5 minutes, 10 minutes, 30 minutes, an hour, or longer. Forexample, a solution containing a self-assembling peptide applied to sites of blood véssél transection in the brain, liver, or musele provided complete hemostasis within times as short as 10 seconds following application. lon-containing Solutions may be preferred when the compositions are used to protect a subject from contamination, as phase transitions do nőt occur, or do nőt readily occur, when non-ionic Solutions contact intact skin.

[0070] The compositions can form structures that are substantially rigid (e.g., solid or nearly solid) or that assume a definite shape and volume (e.g., structures that conform to the shape and volume of the location to which a liquid composition was administered, whether in vivő or ex vivő). The solidified matéria! may be somewhat deformable or compressible after assembly or phase transition, bút will nőt substantially flow from one area to another, as compositions at a different point along the liquid to solid continuum may do, which may be due, at least in part, to their ability to undergo phase transitions. As a result, the compositions can be used to prevent the movement of a bodily substance in a subject in need thereof. Self-assembly can be achieved in vivő or ex vivő by exposure to conditions within a certain rangé of physiological values (e.g., conditions appropriate for cell or tissue culture) or non-physiological conditions. Non-physiological conditions refers to conditions within the body or at a particular site that deviate from normál physiological conditions at that site. Such conditions may result from trauma, surgery, injury, infection, or a disease, disorder, or condition. For example, a puncture wound in the stomach generally resuits in a decrease in the pH as stomach acid flows intő the wound site. The peptides described herein shouid self-assemble under such conditions. While liquid formulations are readily dispensed, the compositions administered may alsó be in a gél form that may become stiffer upon contact with the subject’s body.

[0071] The concentration of the self-assembling peptides in any given formulation can vary and can be between

EP 2 581 097 Β1 approximately 0.1% (1 mg/ml) and 10% (100 mg/ml), inclusive. For example, the concentration ofthe self-assembling peptides (e.g., in a liquid formulation) can be approximately 0.1-3.0% (1-30 mg/ml) (e.g., 0.1-1.0%; 1.0-2.0%; 2.0-3.0% or 1.0-3.0%). The concentration of self-assembling peptides can be higher in stock Solutions and in solid (e.g., powdered) formulations. In solid preparations, the concentration of self-assembling peptides can approach 100% (e.g., the concentration of self-assembling peptides can be 95, 96, 97, 98, 99% or more (e.g., 99.99%) ofthe composition). Whether in liquid or solid form, the self-assembling peptides can be brought to the desired concentration prior to use by addition of a diluent (e.g., deionized water), powder, wetting agent, or a therapeutic, diagnostic or prophylactic agent.

[0072] Regardless ofthe precise natúré ofthe self-assembling peptides, upon exposure to conditions such as those described herein, the peptides can form membranous two-or three-dimensional structures includíng a stable macroscopic porous mátrix having ordered or non-ordered interwoven nanofibers (e.g., fibers approximately 10-20 nm in diameter, with a poré size ofabout 50-100 nm in a lineardimension). Three-dimensional macroscopic matrices can have dirnensions large enough to be visible under low magnification (e.g., about 10fold or less), and the membranous structures can be visible to the naked eye, even iftransparent. Although three-dimensional, the structures can be exceedingly thin, includíng a limited number of layers of molecules (e.g., 2, 3, or more layers of molecules). Typically, each dimension of a given structure will be at least 10 μπι in size (e.g., two dirnensions ofat least 100-1000 μπι in size (e.g., 1-10 mm, 10-100 mm, or more)). The relevant dirnensions may be expressed as length, width, depth, breadth, height, radius, diameter, or circumference in the case of structures that have a substantially regular shape (e.g., where the structure is a sphere, cylinder, cube, orthe like) or an approximation of any ofthe foregoing where the structures do nőt have a regular shape. [0073] The self-assembling peptides can form a hydrated matéria! when contacted with water under conditions such as those described herein (e.g., in the presence of a sufficient concentration (e.g., physiological concentrations) of ions (e.g., monovalent cations)). The self-assembling peptides may have a high water content (e.g., approximately 95% or more (e.g., approximately 97%, 98%, 99% or more)), and the compositions can be hydrated bút nőt substantially selfassembled. A given value may be approximate in recognition ofthe fact that measurements can vary depending, for example, on the circumstances underwhich they are made and the skill ofthe person taking the measurement. Generally, a first value is approximately equal to a second when the first fails within 10% of the second (whether greater than or less than) unless it is otherwise clear from the context that a value is nőt approximate or where, for example, such value would exceed 100% ofa possible value.

[0074] The properties and mechanical strength of the structures or scaffolds can be controlled as required through manipuiation ofthe components therein. For example, the stiffness of an assembled gél can be increased by increasing the concentration of self-assembling peptides therein. Alternatively, it may be desirable for different parts of the selfassembling peptides to have different mechanical properties. For example, it may be advantageous to decrease the stability of all or part of the self-assembling peptides by manipulating the amino acid sequence. This may be desirable when the self-assembling peptides are used to fill a void, such thatthe edges ofthe peptides self-assemble to attach to the tissue site while the rest ofthe peptides flows out intő the void. The sequences, characteristics, and properties of the peptides and the structures formed by them upon self-assembly are discussed further below.

[0075] The compositions can be formulated as concentrated stocksor in dry form, and these can be diluted or dissolved to form biocompatible compositions, which are substantially non-toxic to biological cells in vitro or in vivő. For example, the compositions can contain materials in quantities that do nőt elicit a significant deleterious effect on the recipient’s body (e.g., a prohibitively severe immunological or inflammatory reaction, or unacceptable scar tissue formation). [0076] When a solution containing non-assembled peptides is Iáid down on a biological tissue, the peptides having sufficient proximity to the tissue assemble, causing the solution to gél. Any solution that remains distant from the tissue remains liquid, as the self-assembling peptides have nőt yet been exposed to conditions that promote their assembly. As the self-assembling peptides are disturbed (e.g., by performing a surgical procedure), liquid matéria! appears to gél as it comes intő sufficient contact with the body. At times, the compositions can take on characteristics ranging from a liquid to those of a solid, appearing gél- or salve-like or as a slurry.

C. Modification of Self-Assembling Materials to Target Specific Tissues [0077] The self-assembling peptides further contain a tissue specific component. The tissue specific component can be peptides that are specific for eye, brain, or skin cells. For example, cell surface carbohydrates are major components ofthe outer surface of mammalian cells and are very often characteristicof cell types. lt is assumed that cell type-specific carbohydrates are involved in cell-cell interaction. The tissue specific component can therefore, target these cell specific surface carbohydrates.

[0078] Additionally, hydrophobic tails can be added to the self-assembling peptides. The tails can interact with cell membranes, thus anchoring the self-assembling peptides on to the cell surface. Table 3 shows a list of peptides with hydrophobic tails. Hydrophilic tails can alsó be added to the peptides, in addition to hydrophobic tails, to facilitate interaction with the ECM of different vessels or tissues, such as the bladder.

EP 2 581 097 Β1

Table 3. Hydrophobic Tails

<td> 1</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> D</td><td> G</td><td> D</td><td> G</td><td> D</td><td> G</td><td> D</td><td> G</td><td> D</td><td> G</td><td> D</td><td> (SEQ. IDNO. 126)</td>

<td> 2</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> E</td><td> G</td><td> E</td><td> G</td><td> E</td><td> G</td><td> E</td><td> G</td><td> E</td><td> G</td><td> E</td><td> (SEQ. IDNO. 127)</td>

<td> 3</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> K</td><td> G</td><td> K</td><td> G</td><td> K</td><td> G</td><td> K</td><td> G</td><td> K</td><td> G</td><td> K</td><td> (SEQ. IDNO. 128)</td>

<td> 4</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> (SEQ. IDNO. 129)</td>

<td> 5</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> H</td><td> (SEQ. IDNO. 130)</td>

<td> 6</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> D</td><td> A</td><td> D</td><td> A</td><td> D</td><td> A</td><td> D</td><td> A</td><td> D</td><td> A</td><td> D</td><td> (SEQ. IDNO. 131)</td>

<td> 7</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> E</td><td> A</td><td> E</td><td> A</td><td> E</td><td> A</td><td> E</td><td> A</td><td> E</td><td> A</td><td> E</td><td> (SEQ. IDNO. 132)</td>

<td> 8</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> K</td><td> A</td><td> K</td><td> A</td><td> K</td><td> A</td><td> K</td><td> A</td><td> K</td><td> A</td><td> K</td><td> (SEQ. IDNO. 133)</td>

<td> 9</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> (SEQ. IDNO. 134)</td>

<td> 10</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> H</td><td> (SEQ. IDNO. 135)</td>

<td> 11</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> D</td><td> V</td><td> D</td><td> V</td><td> D</td><td> V</td><td> D</td><td> V</td><td> D</td><td> V</td><td> D</td><td> (SEQ. IDNO. 136)</td>

<td> 12</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> E</td><td> V</td><td> E</td><td> V</td><td> E</td><td> V</td><td> E</td><td> V</td><td> E</td><td> V</td><td> E</td><td> (SEQ. IDNO. 137)</td>

<td> 13</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> K</td><td> V</td><td> K</td><td> V</td><td> K</td><td> V</td><td> K</td><td> V</td><td> K</td><td> V</td><td> K</td><td> (SEQ. IDNO. 138)</td>

<td> 14</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> (SEQ. IDNO. 139)</td>

<td> 15</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> H</td><td> (SEQ. IDNO. 140)</td>

<td> 16</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> D</td><td> L</td><td> D</td><td> L</td><td> D</td><td> L</td><td> D</td><td> L</td><td> D</td><td> L</td><td> D</td><td> (SEQ. IDNO. 141)</td>

<td> 17</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> E</td><td> L</td><td> E</td><td> L</td><td> E</td><td> L</td><td> E</td><td> L</td><td> E</td><td> L</td><td> E</td><td> (SEQ. IDNO. 142)</td>

<td> 18</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> K</td><td> L</td><td> K</td><td> L</td><td> K</td><td> L</td><td> K</td><td> L</td><td> K</td><td> L</td><td> K</td><td> (SEQ. IDNO. 143)</td>

<td> 19</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> (SEQ. IDNO. 144)</td>

<td> 20</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> H</td><td> (SEQ. IDNO. 145)</td>

<td> 21</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> D</td><td> I</td><td> D</td><td> I</td><td> D</td><td> I</td><td> D</td><td> I</td><td> D</td><td> I</td><td> D</td><td> (SEQ. IDNO. 146)</td>

<td> 22</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> E</td><td> I</td><td> E</td><td> I</td><td> E</td><td> I</td><td> E</td><td> I</td><td> E</td><td> I</td><td> E</td><td> (SEQ. IDNO. 147)</td>

<td> 23</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> K</td><td> I</td><td> K</td><td> I</td><td> K</td><td> I</td><td> K</td><td> I</td><td> K</td><td> I</td><td> K</td><td> (SEQ. IDNO. 148)</td>

<td> 24</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> (SEQ. IDNO. 149)</td>

<td> 25</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> H</td><td> (SEQ. IDNO. 150)</td>

<td> 26</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> D</td><td> M</td><td> D</td><td> M</td><td> D</td><td> M</td><td> D</td><td> M</td><td> D</td><td> M</td><td> D</td><td> (SEQ. IDNO. 151)</td>

<td> 27</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> E</td><td> M</td><td> E</td><td> M</td><td> E</td><td> M</td><td> E</td><td> M</td><td> E</td><td> M</td><td> E</td><td> (SEQ. IDNO. 152)</td>

<td> 28</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> K</td><td> M</td><td> K</td><td> M</td><td> K</td><td> M</td><td> K</td><td> M</td><td> K</td><td> M</td><td> K</td><td> (SEQ. IDNO. 153)</td>

<td> 29</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> (SEQ. IDNO. 154)</td>

<td> 30</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> H</td><td> (SEQ. IDNO. 155)</td>

<td> 31</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> D</td><td> F</td><td> D</td><td> F</td><td> D</td><td> F</td><td> D</td><td> F</td><td> D</td><td> F</td><td> D</td><td> (SEQ. IDNO. 156)</td>

<td> 32</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> E</td><td> F</td><td> E</td><td> F</td><td> E</td><td> F</td><td> E</td><td> F</td><td> E</td><td> F</td><td> E</td><td> (SEQ. IDNO. 157)</td>

<td> 33</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> K</td><td> F</td><td> K</td><td> F</td><td> K</td><td> F</td><td> K</td><td> F</td><td> K</td><td> F</td><td> K</td><td> (SEQ. IDNO. 158)</td>

<td> 34</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> (SEQ. IDNO. 159)</td>

<td> 35</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> H</td><td> (SEQ. IDNO. 160)</td>

<td> 36</td><td> W</td><td> W</td><td> W</td><td> W</td><td> W</td><td> D</td><td> W</td><td> D</td><td> W</td><td> D</td><td> W</td><td> D</td><td> W</td><td> D</td><td> W</td><td> D</td><td> (SEQ. IDNO. 161)</td>

<td> 37</td><td> W</td><td> W</td><td> W</td><td> W</td><td> W</td><td> E</td><td> W</td><td> E</td><td> W</td><td> E</td><td> W</td><td> E</td><td> W</td><td> E</td><td> W</td><td> E</td><td> (SEQ. IDNO. 162)</td>

<td> 38</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> K</td><td> w</td><td> K</td><td> w</td><td> K</td><td> w</td><td> K</td><td> w</td><td> K</td><td> w</td><td> K</td><td> (SEQ. IDNO. 163)</td>

<td> 39</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> (SEQ. IDNO. 164)</td>

<td> 40</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> H</td><td> (SEQ. IDNO. 165)</td>

<td> 41</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> D</td><td> P</td><td> D</td><td> P</td><td> D</td><td> P</td><td> D</td><td> P</td><td> D</td><td> P</td><td> D</td><td> (SEQ. IDNO. 166)</td>

<td> 42</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> E</td><td> P</td><td> E</td><td> P</td><td> E</td><td> P</td><td> E</td><td> P</td><td> E</td><td> P</td><td> E</td><td> (SEQ. IDNO. 167)</td>

<td> 43</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> K</td><td> P</td><td> K</td><td> P</td><td> K</td><td> P</td><td> K</td><td> P</td><td> K</td><td> P</td><td> K</td><td> (SEQ. IDNO. 168)</td>

<td> 44</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> (SEQ. IDNO. 169)</td>

<td> 45</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> H</td><td> (SEQ. IDNO. 170)</td>

<td> 46</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> R</td><td> A</td><td> D</td><td> A</td><td> R</td><td> A</td><td> D</td><td> A</td><td> R</td><td> A</td><td> D</td><td> (SEQ. IDNO. 171)</td>

<td> 47</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> D</td><td> A</td><td> D</td><td> A</td><td> R</td><td> A</td><td> R</td><td> (SEQ. IDNO. 172)</td>

<td> 48</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> E</td><td> A</td><td> K</td><td> A</td><td> E</td><td> A</td><td> K</td><td> A</td><td> E</td><td> A</td><td> K</td><td> (SEQ. IDNO. 173)</td>

<td> 49</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> E</td><td> A</td><td> E</td><td> A</td><td> K</td><td> A</td><td> K</td><td> A</td><td> E</td><td> A</td><td> E</td><td> (SEQ. IDNO. 174)</td>

<td> 50</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> R</td><td> A</td><td> E</td><td> A</td><td> R</td><td> A</td><td> E</td><td> A</td><td> R</td><td> A</td><td> E</td><td> (SEQ. IDNO. 175)</td>

EP 2 581 097 Β1

<td> 51</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> R</td><td> A</td><td> R</td><td colspan="2"> (continued) A E</td><td> A</td><td> E</td><td> A</td><td> R</td><td> A</td><td> E</td><td> (SEQ. IDNO. 176)</td>

<td> 52</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> K</td><td> A</td><td> D</td><td> A</td><td> K</td><td> A</td><td> D</td><td> A</td><td> K</td><td> A</td><td> D</td><td> (SEQ. IDNO. 177)</td>

<td> 53</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> E</td><td> A</td><td> H</td><td> A</td><td> E</td><td> A</td><td> H</td><td> A</td><td> E</td><td> A</td><td> H</td><td> (SEQ. IDNO. 178)</td>

<td> 54</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> E</td><td> A</td><td> E</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> E</td><td> A</td><td> E</td><td> (SEQ. IDNO. 179)</td>

<td> 55</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> (SEQ. IDNO. 180)</td>

<td> 56</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> D</td><td> A</td><td> D</td><td> (SEQ. IDNO. 181)</td>

<td> 57</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> R</td><td> A</td><td> D</td><td> A</td><td> D</td><td> A</td><td> D</td><td> (SEQ. IDNO. 182)</td>

<td> 58</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> H</td><td> A</td><td> D</td><td> A</td><td> H</td><td> A</td><td> D</td><td> A</td><td> H</td><td> A</td><td> D</td><td> (SEQ. IDNO. 183)</td>

<td> 59</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> H</td><td> A</td><td> H</td><td> (SEQ. IDNO. 184)</td>

<td> 60</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> H</td><td> A</td><td> D</td><td> A</td><td> D</td><td> A</td><td> H</td><td> A</td><td> D</td><td> A</td><td> D</td><td> (SEQ. IDNO. 185)</td>

<td> 61</td><td> A</td><td> A</td><td> A</td><td> A</td><td> A</td><td> H</td><td> A</td><td> E</td><td> A</td><td> E</td><td> A</td><td> H</td><td> A</td><td> E</td><td> A</td><td> E</td><td> (SEQ. IDNO. 186)</td>

<td> 62</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> R</td><td> G</td><td> D</td><td> G</td><td> R</td><td> G</td><td> D</td><td> G</td><td> R</td><td> G</td><td> D</td><td> (SEQ. IDNO. 187)</td>

<td> 63</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> D</td><td> G</td><td> D</td><td> G</td><td> R</td><td> G</td><td> R</td><td> (SEQ. IDNO. 188)</td>

<td> 64</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> E</td><td> G</td><td> K</td><td> G</td><td> E</td><td> G</td><td> K</td><td> G</td><td> E</td><td> G</td><td> K</td><td> (SEQ. IDNO. 189)</td>

<td> 65</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> E</td><td> G</td><td> E</td><td> G</td><td> K</td><td> G</td><td> K</td><td> G</td><td> E</td><td> G</td><td> E</td><td> (SEQ. IDNO. 190)</td>

<td> 66</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> R</td><td> G</td><td> E</td><td> G</td><td> R</td><td> G</td><td> E</td><td> G</td><td> R</td><td> G</td><td> E</td><td> (SEQ. IDNO. 191)</td>

<td> 67</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> E</td><td> G</td><td> E</td><td> G</td><td> R</td><td> G</td><td> E</td><td> (SEQ. IDNO. 192)</td>

<td> 68</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> K</td><td> G</td><td> D</td><td> G</td><td> K</td><td> G</td><td> D</td><td> G</td><td> K</td><td> G</td><td> D</td><td> (SEQ. IDNO. 193)</td>

<td> 69</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> E</td><td> G</td><td> H</td><td> G</td><td> E</td><td> G</td><td> H</td><td> G</td><td> E</td><td> G</td><td> H</td><td> (SEQ. IDNO. 194)</td>

<td> 70</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> E</td><td> G</td><td> E</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> E</td><td> G</td><td> E</td><td> (SEQ. IDNO. 195)</td>

<td> 71</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> (SEQ. IDNO. 196)</td>

<td> 72</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> D</td><td> G</td><td> D</td><td> (SEQ. IDNO. 197)</td>

<td> 73</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> R</td><td> G</td><td> D</td><td> G</td><td> D</td><td> G</td><td> D</td><td> (SEQ. IDNO. 198)</td>

<td> 74</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> H</td><td> G</td><td> D</td><td> G</td><td> H</td><td> G</td><td> D</td><td> G</td><td> H</td><td> G</td><td> D</td><td> (SEQ. IDNO. 199)</td>

<td> 75</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> H</td><td> G</td><td> H</td><td> (SEQ. IDNO. 200)</td>

<td> 76</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> H</td><td> G</td><td> D</td><td> G</td><td> D</td><td> G</td><td> H</td><td> G</td><td> D</td><td> G</td><td> D</td><td> (SEQ. IDNO.201)</td>

<td> 77</td><td> G</td><td> G</td><td> G</td><td> G</td><td> G</td><td> H</td><td> G</td><td> E</td><td> G</td><td> E</td><td> G</td><td> H</td><td> G</td><td> E</td><td> G</td><td> E</td><td> (SEQ. IDNO. 202)</td>

<td> 78</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> R</td><td> V</td><td> D</td><td> V</td><td> R</td><td> V</td><td> D</td><td> V</td><td> R</td><td> V</td><td> D</td><td> (SEQ. IDNO. 203)</td>

<td> 79</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> D</td><td> V</td><td> D</td><td> V</td><td> R</td><td> V</td><td> R</td><td> (SEQ. IDNO. 204)</td>

<td> 80</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> E</td><td> V</td><td> K</td><td> V</td><td> E</td><td> V</td><td> K</td><td> V</td><td> E</td><td> V</td><td> K</td><td> (SEQ. IDNO. 205)</td>

<td> 81</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> E</td><td> V</td><td> E</td><td> V</td><td> K</td><td> V</td><td> K</td><td> V</td><td> E</td><td> V</td><td> E</td><td> (SEQ. IDNO. 206)</td>

<td> 82</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> R</td><td> V</td><td> E</td><td> V</td><td> R</td><td> V</td><td> E</td><td> V</td><td> R</td><td> V</td><td> E</td><td> (SEQ. IDNO. 207)</td>

<td> 83</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> E</td><td> V</td><td> E</td><td> V</td><td> R</td><td> V</td><td> E</td><td> (SEQ. IDNO. 208)</td>

<td> 84</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> K</td><td> V</td><td> D</td><td> V</td><td> K</td><td> V</td><td> D</td><td> V</td><td> K</td><td> V</td><td> D</td><td> (SEQ. IDNO. 209)</td>

<td> 85</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> E</td><td> V</td><td> H</td><td> V</td><td> E</td><td> V</td><td> H</td><td> V</td><td> E</td><td> V</td><td> H</td><td> (SEQ. IDNO.210)</td>

<td> 86</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> E</td><td> V</td><td> E</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> E</td><td> V</td><td> E</td><td> (SEQ. IDNO.211)</td>

<td> 87</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> (SEQ. IDNO. 212)</td>

<td> 88</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> D</td><td> V</td><td> D</td><td> (SEQ. IDNO.213)</td>

<td> 89</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> R</td><td> V</td><td> D</td><td> V</td><td> D</td><td> V</td><td> D</td><td> (SEQ. IDNO. 214)</td>

<td> 90</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> H</td><td> V</td><td> D</td><td> V</td><td> H</td><td> V</td><td> D</td><td> V</td><td> H</td><td> V</td><td> D</td><td> (SEQ. IDNO.215)</td>

<td> 91</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> H</td><td> V</td><td> H</td><td> (SEQ. IDNO.216)</td>

<td> 92</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> H</td><td> V</td><td> D</td><td> V</td><td> D</td><td> V</td><td> H</td><td> V</td><td> D</td><td> V</td><td> D</td><td> (SEQ. IDNO.217)</td>

<td> 93</td><td> V</td><td> V</td><td> V</td><td> V</td><td> V</td><td> H</td><td> V</td><td> E</td><td> V</td><td> E</td><td> V</td><td> H</td><td> V</td><td> E</td><td> V</td><td> E</td><td> (SEQ. IDNO.218)</td>

<td> 94</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> R</td><td> L</td><td> D</td><td> L</td><td> R</td><td> L</td><td> D</td><td> L</td><td> R</td><td> L</td><td> D</td><td> (SEQ. IDNO.219)</td>

<td> 95</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> D</td><td> L</td><td> D</td><td> L</td><td> R</td><td> L</td><td> R</td><td> (SEQ. IDNO. 220)</td>

<td> 96</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> E</td><td> L</td><td> K</td><td> L</td><td> E</td><td> L</td><td> K</td><td> L</td><td> E</td><td> L</td><td> K</td><td> (SEQ. IDNO. 221)</td>

<td> 97</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> E</td><td> L</td><td> E</td><td> L</td><td> K</td><td> L</td><td> K</td><td> L</td><td> E</td><td> L</td><td> E</td><td> (SEQ. IDNO. 222)</td>

<td> 98</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> R</td><td> L</td><td> E</td><td> L</td><td> R</td><td> L</td><td> E</td><td> L</td><td> R</td><td> L</td><td> E</td><td> (SEQ. IDNO. 223)</td>

<td> 99</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> E</td><td> L</td><td> E</td><td> L</td><td> R</td><td> L</td><td> E</td><td> (SEQ. IDNO. 224)</td>

<td> 100</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> K</td><td> L</td><td> D</td><td> L</td><td> K</td><td> L</td><td> D</td><td> L</td><td> K</td><td> L</td><td> D</td><td> (SEQ. IDNO. 225)</td>

<td> 101</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> E</td><td> L</td><td> H</td><td> L</td><td> E</td><td> L</td><td> H</td><td> L</td><td> E</td><td> L</td><td> H</td><td> (SEQ. IDNO. 226)</td>

EP 2 581 097 Β1 (continued)

<td> 102</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> E</td><td> L</td><td> E</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> E</td><td> L</td><td> E</td><td> (SEQ. IDNO.227)</td>

<td> 103</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> (SEQ. IDNO.228)</td>

<td> 104</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> D</td><td> L</td><td> D</td><td> (SEQ. IDNO.229)</td>

<td> 105</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> R</td><td> L</td><td> D</td><td> L</td><td> D</td><td> L</td><td> D</td><td> (SEQ. ID NO.230)</td>

<td> 106</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> H</td><td> L</td><td> D</td><td> L</td><td> H</td><td> L</td><td> D</td><td> L</td><td> H</td><td> L</td><td> D</td><td> (SEQ. IDNO.231)</td>

<td> 107</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> H</td><td> L</td><td> H</td><td> (SEQ. IDNO.232)</td>

<td> 108</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> H</td><td> L</td><td> D</td><td> L</td><td> D</td><td> L</td><td> H</td><td> L</td><td> D</td><td> L</td><td> D</td><td> (SEQ. IDNO.233)</td>

<td> 109</td><td> L</td><td> L</td><td> L</td><td> L</td><td> L</td><td> H</td><td> L</td><td> E</td><td> L</td><td> E</td><td> L</td><td> H</td><td> L</td><td> E</td><td> L</td><td> E</td><td> (SEQ. IDNO.234)</td>

<td> 110</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> R</td><td> I</td><td> D</td><td> I</td><td> R</td><td> I</td><td> D</td><td> I</td><td> R</td><td> I</td><td> D</td><td> (SEQ. IDNO.235)</td>

<td> 111</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> D</td><td> I</td><td> D</td><td> I</td><td> R</td><td> I</td><td> R</td><td> (SEQ. IDNO.236)</td>

<td> 112</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> E</td><td> I</td><td> K</td><td> I</td><td> E</td><td> I</td><td> K</td><td> I</td><td> E</td><td> I</td><td> K</td><td> (SEQ. IDNO.237)</td>

<td> 113</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> E</td><td> I</td><td> E</td><td> I</td><td> K</td><td> I</td><td> K</td><td> I</td><td> E</td><td> I</td><td> E</td><td> (SEQ. IDNO.238)</td>

<td> 114</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> R</td><td> I</td><td> E</td><td> I</td><td> R</td><td> I</td><td> E</td><td> I</td><td> R</td><td> I</td><td> E</td><td> (SEQ. IDNO.239)</td>

<td> 115</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> E</td><td> I</td><td> E</td><td> I</td><td> R</td><td> I</td><td> E</td><td> (SEQ. IDNO.240)</td>

<td> 116</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> K</td><td> I</td><td> D</td><td> I</td><td> K</td><td> I</td><td> D</td><td> I</td><td> K</td><td> I</td><td> D</td><td> (SEQ. IDNO.241)</td>

<td> 117</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> E</td><td> I</td><td> H</td><td> I</td><td> E</td><td> I</td><td> H</td><td> I</td><td> E</td><td> I</td><td> H</td><td> (SEQ. IDNO.242)</td>

<td> 118</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> E</td><td> I</td><td> E</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> E</td><td> I</td><td> E</td><td> (SEQ. IDNO.243)</td>

<td> 119</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> (SEQ. IDNO.244)</td>

<td> 120</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> D</td><td> I</td><td> D</td><td> (SEQ. IDNO.245)</td>

<td> 121</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> R</td><td> I</td><td> D</td><td> I</td><td> D</td><td> I</td><td> D</td><td> (SEQ. IDNO.246)</td>

<td> 122</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> H</td><td> I</td><td> D</td><td> I</td><td> H</td><td> I</td><td> D</td><td> I</td><td> H</td><td> I</td><td> D</td><td> (SEQ. IDNO.247)</td>

<td> 123</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> H</td><td> I</td><td> H</td><td> (SEQ. IDNO.248)</td>

<td> 124</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> H</td><td> I</td><td> D</td><td> I</td><td> D</td><td> I</td><td> H</td><td> I</td><td> D</td><td> I</td><td> D</td><td> (SEQ. IDNO.249)</td>

<td> 125</td><td> I</td><td> I</td><td> I</td><td> I</td><td> I</td><td> H</td><td> I</td><td> E</td><td> I</td><td> E</td><td> I</td><td> H</td><td> I</td><td> E</td><td> I</td><td> E</td><td> (SEQ. IDNO.250)</td>

<td> 126</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> R</td><td> M</td><td> D</td><td> M</td><td> R</td><td> M</td><td> D</td><td> M</td><td> R</td><td> M</td><td> D</td><td> (SEQ. IDNO.251)</td>

<td> 127</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> D</td><td> M</td><td> D</td><td> M</td><td> R</td><td> M</td><td> R</td><td> (SEQ. IDNO.252)</td>

<td> 128</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> E</td><td> M</td><td> K</td><td> M</td><td> E</td><td> M</td><td> K</td><td> M</td><td> E</td><td> M</td><td> K</td><td> (SEQ. IDNO.253)</td>

<td> 129</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> E</td><td> M</td><td> E</td><td> M</td><td> K</td><td> M</td><td> K</td><td> M</td><td> E</td><td> M</td><td> E</td><td> (SEQ. IDNO.254)</td>

<td> 130</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> R</td><td> M</td><td> E</td><td> M</td><td> R</td><td> M</td><td> E</td><td> M</td><td> R</td><td> M</td><td> E</td><td> (SEQ. IDNO.255)</td>

<td> 131</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> E</td><td> M</td><td> E</td><td> M</td><td> R</td><td> M</td><td> E</td><td> (SEQ. IDNO.256)</td>

<td> 132</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> K</td><td> M</td><td> D</td><td> M</td><td> K</td><td> M</td><td> D</td><td> M</td><td> K</td><td> M</td><td> D</td><td> (SEQ. IDNO.257)</td>

<td> 133</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> E</td><td> M</td><td> H</td><td> M</td><td> E</td><td> M</td><td> H</td><td> M</td><td> E</td><td> M</td><td> H</td><td> (SEQ. IDNO.258)</td>

<td> 134</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> E</td><td> M</td><td> E</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> E</td><td> M</td><td> E</td><td> (SEQ. IDNO.259)</td>

<td> 135</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> (SEQ. IDNO.260)</td>

<td> 136</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> D</td><td> M</td><td> D</td><td> (SEQ. IDNO.261)</td>

<td> 137</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> R</td><td> M</td><td> D</td><td> M</td><td> D</td><td> M</td><td> D</td><td> (SEQ. IDNO.262)</td>

<td> 138</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> H</td><td> M</td><td> D</td><td> M</td><td> H</td><td> M</td><td> D</td><td> M</td><td> H</td><td> M</td><td> D</td><td> (SEQ. IDNO.263)</td>

<td> 139</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> H</td><td> M</td><td> H</td><td> (SEQ. IDNO.264)</td>

<td> 140</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> H</td><td> M</td><td> D</td><td> M</td><td> D</td><td> M</td><td> H</td><td> M</td><td> D</td><td> M</td><td> D</td><td> (SEQ. IDNO.265)</td>

<td> 141</td><td> M</td><td> M</td><td> M</td><td> M</td><td> M</td><td> H</td><td> M</td><td> E</td><td> M</td><td> E</td><td> M</td><td> H</td><td> M</td><td> E</td><td> M</td><td> E</td><td> (SEQ. IDNO.266)</td>

<td> 142</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> R</td><td> F</td><td> D</td><td> F</td><td> R</td><td> F</td><td> D</td><td> F</td><td> R</td><td> F</td><td> D</td><td> (SEQ. IDNO.267)</td>

<td> 143</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> D</td><td> F</td><td> D</td><td> F</td><td> R</td><td> F</td><td> R</td><td> (SEQ. IDNO.268)</td>

<td> 144</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> E</td><td> F</td><td> K</td><td> F</td><td> E</td><td> F</td><td> K</td><td> F</td><td> E</td><td> F</td><td> K</td><td> (SEQ. IDNO.269)</td>

<td> 145</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> E</td><td> F</td><td> E</td><td> F</td><td> K</td><td> F</td><td> K</td><td> F</td><td> E</td><td> F</td><td> E</td><td> (SEQ. IDNO.270)</td>

<td> 146</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> R</td><td> F</td><td> E</td><td> F</td><td> R</td><td> F</td><td> E</td><td> F</td><td> R</td><td> F</td><td> E</td><td> (SEQ. IDNO.271)</td>

<td> 147</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> E</td><td> F</td><td> E</td><td> F</td><td> R</td><td> F</td><td> E</td><td> (SEQ. IDNO.272)</td>

<td> 148</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> K</td><td> F</td><td> D</td><td> F</td><td> K</td><td> F</td><td> D</td><td> F</td><td> K</td><td> F</td><td> D</td><td> (SEQ. IDNO.273)</td>

<td> 149</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> E</td><td> F</td><td> H</td><td> F</td><td> E</td><td> F</td><td> H</td><td> F</td><td> E</td><td> F</td><td> H</td><td> (SEQ. IDNO.274)</td>

<td> 150</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> E</td><td> F</td><td> E</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> E</td><td> F</td><td> E</td><td> (SEQ. IDNO.275)</td>

<td> 151</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> (SEQ. IDNO.276)</td>

<td> 152</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> D</td><td> F</td><td> D</td><td> (SEQ. IDNO.277)</td>

EP 2 581 097 Β1 (continued)

<td> 153</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> R</td><td> F</td><td> D</td><td> F</td><td> D</td><td> F</td><td> D</td><td> (SEQ. IDNO.278)</td>

<td> 154</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> H</td><td> F</td><td> D</td><td> F</td><td> H</td><td> F</td><td> D</td><td> F</td><td> H</td><td> F</td><td> D</td><td> (SEQ. IDNO.279)</td>

<td> 155</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> H</td><td> F</td><td> H</td><td> (SEQ. IDNO.280)</td>

<td> 156</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> H</td><td> F</td><td> D</td><td> F</td><td> D</td><td> F</td><td> H</td><td> F</td><td> D</td><td> F</td><td> D</td><td> (SEQ. IDNO.281)</td>

<td> 157</td><td> F</td><td> F</td><td> F</td><td> F</td><td> F</td><td> H</td><td> F</td><td> E</td><td> F</td><td> E</td><td> F</td><td> H</td><td> F</td><td> E</td><td> F</td><td> E</td><td> (SEQ. IDNO.282)</td>

<td> 158</td><td> W</td><td> w</td><td> w</td><td> w</td><td> w</td><td> R</td><td> W</td><td> D</td><td> W</td><td> R</td><td> W</td><td> D</td><td> W</td><td> R</td><td> W</td><td> D</td><td> (SEQ. IDNO.283)</td>

<td> 159</td><td> W</td><td> w</td><td> w</td><td> w</td><td> w</td><td> R</td><td> W</td><td> R</td><td> W</td><td> D</td><td> W</td><td> D</td><td> W</td><td> R</td><td> W</td><td> R</td><td> (SEQ. IDNO.284)</td>

<td> 160</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> E</td><td> w</td><td> K</td><td> w</td><td> E</td><td> w</td><td> K</td><td> w</td><td> E</td><td> w</td><td> K</td><td> (SEQ. IDNO.285)</td>

<td> 161</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> E</td><td> w</td><td> E</td><td> w</td><td> K</td><td> w</td><td> K</td><td> w</td><td> E</td><td> w</td><td> E</td><td> (SEQ. IDNO.286)</td>

<td> 162</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> R</td><td> w</td><td> E</td><td> w</td><td> R</td><td> w</td><td> E</td><td> w</td><td> R</td><td> w</td><td> E</td><td> (SEQ. IDNO.287)</td>

<td> 163</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> E</td><td> w</td><td> E</td><td> w</td><td> R</td><td> w</td><td> E</td><td> (SEQ. IDNO.288)</td>

<td> 164</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> K</td><td> w</td><td> D</td><td> w</td><td> K</td><td> w</td><td> D</td><td> w</td><td> K</td><td> w</td><td> D</td><td> (SEQ. IDNO.289)</td>

<td> 165</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> E</td><td> w</td><td> H</td><td> w</td><td> E</td><td> w</td><td> H</td><td> w</td><td> E</td><td> w</td><td> H</td><td> (SEQ. IDNO.290)</td>

<td> 166</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> E</td><td> w</td><td> E</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> E</td><td> w</td><td> E</td><td> (SEQ. IDNO.291)</td>

<td> 167</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> (SEQ. IDNO.292)</td>

<td> 168</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> D</td><td> w</td><td> D</td><td> (SEQ. IDNO.293)</td>

<td> 169</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> R</td><td> w</td><td> D</td><td> w</td><td> D</td><td> w</td><td> D</td><td> (SEQ. IDNO.294)</td>

<td> 170</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> H</td><td> w</td><td> D</td><td> w</td><td> H</td><td> w</td><td> D</td><td> w</td><td> H</td><td> w</td><td> D</td><td> (SEQ. IDNO.295)</td>

<td> 171</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> H</td><td> w</td><td> H</td><td> (SEQ. IDNO.296)</td>

<td> 172</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> H</td><td> w</td><td> D</td><td> w</td><td> D</td><td> w</td><td> H</td><td> w</td><td> D</td><td> w</td><td> D</td><td> (SEQ. IDNO.297)</td>

<td> 173</td><td> w</td><td> w</td><td> w</td><td> w</td><td> w</td><td> H</td><td> w</td><td> E</td><td> w</td><td> E</td><td> w</td><td> H</td><td> w</td><td> E</td><td> w</td><td> E</td><td> (SEQ. IDNO.298)</td>

<td> 174</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> R</td><td> P</td><td> D</td><td> P</td><td> R</td><td> P</td><td> D</td><td> P</td><td> R</td><td> P</td><td> D</td><td> (SEQ. IDNO.299)</td>

<td> 175</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> D</td><td> P</td><td> D</td><td> P</td><td> R</td><td> P</td><td> R</td><td> (SEQ. IDNO.300)</td>

<td> 176</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> E</td><td> P</td><td> K</td><td> P</td><td> E</td><td> P</td><td> K</td><td> P</td><td> E</td><td> P</td><td> K</td><td> (SEQ. IDNO.301)</td>

<td> 177</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> E</td><td> P</td><td> E</td><td> P</td><td> K</td><td> P</td><td> K</td><td> P</td><td> E</td><td> P</td><td> E</td><td> (SEQ. IDNO.302)</td>

<td> 178</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> R</td><td> P</td><td> E</td><td> P</td><td> R</td><td> P</td><td> E</td><td> P</td><td> R</td><td> P</td><td> E</td><td> (SEQ. IDNO.303)</td>

<td> 179</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> E</td><td> P</td><td> E</td><td> P</td><td> R</td><td> P</td><td> E</td><td> (SEQ. IDNO.304)</td>

<td> 180</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> K</td><td> P</td><td> D</td><td> P</td><td> K</td><td> P</td><td> D</td><td> P</td><td> K</td><td> P</td><td> D</td><td> (SEQ. IDNO.305)</td>

<td> 181</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> E</td><td> P</td><td> H</td><td> P</td><td> E</td><td> P</td><td> H</td><td> P</td><td> E</td><td> P</td><td> H</td><td> (SEQ. IDNO.306)</td>

<td> 182</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> E</td><td> P</td><td> E</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> E</td><td> P</td><td> E</td><td> (SEQ. IDNO.307)</td>

<td> 183</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> (SEQ. IDNO.308)</td>

<td> 184</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> D</td><td> P</td><td> D</td><td> (SEQ. IDNO.309)</td>

<td> 185</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> R</td><td> P</td><td> D</td><td> P</td><td> D</td><td> P</td><td> D</td><td> (SEQ. IDNO.310)</td>

<td> 186</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> H</td><td> P</td><td> D</td><td> P</td><td> H</td><td> P</td><td> D</td><td> P</td><td> H</td><td> P</td><td> D</td><td> (SEQ. IDNO.311)</td>

<td> 187</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> H</td><td> P</td><td> H</td><td> (SEQ. IDNO.312)</td>

<td> 188</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> H</td><td> P</td><td> D</td><td> P</td><td> D</td><td> P</td><td> H</td><td> P</td><td> D</td><td> P</td><td> D</td><td> (SEQ. IDNO.313)</td>

<td> 189</td><td> P</td><td> P</td><td> P</td><td> P</td><td> P</td><td> H</td><td> P</td><td> E</td><td> P</td><td> E</td><td> P</td><td> H</td><td> P</td><td> E</td><td> P</td><td> E</td><td> (SEQ. IDNO.314)</td>

<td> 190</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> R</td><td> s</td><td> D</td><td> s</td><td> R</td><td> s</td><td> D</td><td> s</td><td> R</td><td> s</td><td> D</td><td> (SEQ. IDNO.315)</td>

<td> 191</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> D</td><td> s</td><td> D</td><td> s</td><td> R</td><td> s</td><td> R</td><td> (SEQ. IDNO.316)</td>

<td> 192</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> E</td><td> s</td><td> K</td><td> s</td><td> E</td><td> s</td><td> K</td><td> s</td><td> E</td><td> s</td><td> K</td><td> (SEQ. IDNO.317)</td>

<td> 193</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> E</td><td> s</td><td> E</td><td> s</td><td> K</td><td> s</td><td> K</td><td> s</td><td> E</td><td> s</td><td> E</td><td> (SEQ. ID NO.318)</td>

<td> 194</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> R</td><td> s</td><td> E</td><td> s</td><td> R</td><td> s</td><td> E</td><td> s</td><td> R</td><td> s</td><td> E</td><td> (SEQ. IDNO.319)</td>

<td> 195</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> E</td><td> s</td><td> E</td><td> s</td><td> R</td><td> s</td><td> E</td><td> (SEQ. IDNO.320)</td>

<td> 196</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> K</td><td> s</td><td> D</td><td> s</td><td> K</td><td> s</td><td> D</td><td> s</td><td> K</td><td> s</td><td> D</td><td> (SEQ. IDNO.321)</td>

<td> 197</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> E</td><td> s</td><td> H</td><td> s</td><td> E</td><td> s</td><td> H</td><td> s</td><td> E</td><td> s</td><td> H</td><td> (SEQ. IDNO.322)</td>

<td> 198</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> E</td><td> s</td><td> E</td><td> s</td><td> H</td><td> s</td><td> H</td><td> s</td><td> E</td><td> s</td><td> E</td><td> (SEQ. IDNO.323)</td>

<td> 199</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> R</td><td> (SEQ. IDNO.324)</td>

<td> 200</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> D</td><td> s</td><td> D</td><td> (SEQ. IDNO.325)</td>

<td> 201</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> R</td><td> s</td><td> D</td><td> s</td><td> D</td><td> s</td><td> D</td><td> (SEQ. IDNO.326)</td>

<td> 202</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> H</td><td> s</td><td> D</td><td> s</td><td> H</td><td> s</td><td> D</td><td> s</td><td> H</td><td> s</td><td> D</td><td> (SEQ. IDNO.327)</td>

<td> 203</td><td> s</td><td> s</td><td> s</td><td> s</td><td> s</td><td> H</td><td> s</td><td> H</td><td> s</td><td> H</td><td> s</td><td> H</td><td> s</td><td> H</td><td> s</td><td> H</td><td> (SEQ. IDNO.328)</td>

EP 2 581 097 Β1

<td> 204</td><td> S</td><td> s</td><td> s</td><td> s</td><td> s</td><td> H</td><td> S</td><td> D</td><td colspan="2"> (continued) S D</td><td> S</td><td> H</td><td> S</td><td> D</td><td> S</td><td> D</td><td> (SEQ. IDNO.329)</td>

<td> 205</td><td> S</td><td> s</td><td> s</td><td> s</td><td> s</td><td> H</td><td> S</td><td> E</td><td> S</td><td> E</td><td> S</td><td> H</td><td> S</td><td> E</td><td> S</td><td> E</td><td> (SEQ. IDNO. 330)</td>

<td> 206</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> R</td><td> T</td><td> D</td><td> T</td><td> R</td><td> T</td><td> D</td><td> T</td><td> R</td><td> T</td><td> D</td><td> (SEQ. IDNO.331)</td>

<td> 207</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> D</td><td> T</td><td> D</td><td> T</td><td> R</td><td> T</td><td> R</td><td> (SEQ. IDNO.332)</td>

<td> 208</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> E</td><td> T</td><td> K</td><td> T</td><td> E</td><td> T</td><td> K</td><td> T</td><td> E</td><td> T</td><td> K</td><td> (SEQ. IDNO.333)</td>

<td> 209</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> E</td><td> T</td><td> E</td><td> T</td><td> K</td><td> T</td><td> K</td><td> T</td><td> E</td><td> T</td><td> E</td><td> (SEQ. IDNO.334)</td>

<td> 210</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> R</td><td> T</td><td> E</td><td> T</td><td> R</td><td> T</td><td> E</td><td> T</td><td> R</td><td> T</td><td> E</td><td> (SEQ. IDNO.335)</td>

<td> 211</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> E</td><td> T</td><td> E</td><td> T</td><td> R</td><td> T</td><td> E</td><td> (SEQ. IDNO.336)</td>

<td> 212</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> K</td><td> T</td><td> D</td><td> T</td><td> K</td><td> T</td><td> D</td><td> T</td><td> K</td><td> T</td><td> D</td><td> (SEQ. IDNO.337)</td>

<td> 213</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> E</td><td> T</td><td> H</td><td> T</td><td> E</td><td> T</td><td> H</td><td> T</td><td> E</td><td> T</td><td> H</td><td> (SEQ. IDNO.338)</td>

<td> 214</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> E</td><td> T</td><td> E</td><td> T</td><td> H</td><td> T</td><td> H</td><td> T</td><td> E</td><td> T</td><td> E</td><td> (SEQ. IDNO.339)</td>

<td> 215</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> R</td><td> (SEQ. IDNO. 340)</td>

<td> 216</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> D</td><td> T</td><td> D</td><td> (SEQ. IDNO.341)</td>

<td> 217</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> R</td><td> T</td><td> D</td><td> T</td><td> D</td><td> T</td><td> D</td><td> (SEQ. IDNO.342)</td>

<td> 218</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> H</td><td> T</td><td> D</td><td> T</td><td> H</td><td> T</td><td> D</td><td> T</td><td> H</td><td> T</td><td> D</td><td> (SEQ. IDNO.343)</td>

<td> 219</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> H</td><td> T</td><td> H</td><td> T</td><td> H</td><td> T</td><td> H</td><td> T</td><td> H</td><td> T</td><td> H</td><td> (SEQ. IDNO.344)</td>

<td> 220</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> H</td><td> T</td><td> D</td><td> T</td><td> D</td><td> T</td><td> H</td><td> T</td><td> D</td><td> T</td><td> D</td><td> (SEQ. IDNO.345)</td>

<td> 221</td><td> T</td><td> T</td><td> T</td><td> T</td><td> T</td><td> H</td><td> T</td><td> E</td><td> T</td><td> E</td><td> T</td><td> H</td><td> T</td><td> E</td><td> T</td><td> E</td><td> (SEQ. IDNO.346)</td>

<td> 222</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> R</td><td> c</td><td> D</td><td> c</td><td> R</td><td> c</td><td> D</td><td> c</td><td> R</td><td> c</td><td> D</td><td> (SEQ. IDNO.347)</td>

<td> 223</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> D</td><td> c</td><td> D</td><td> c</td><td> R</td><td> c</td><td> R</td><td> (SEQ. IDNO.348)</td>

<td> 224</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> E</td><td> c</td><td> K</td><td> c</td><td> E</td><td> c</td><td> K</td><td> c</td><td> E</td><td> c</td><td> K</td><td> (SEQ. IDNO.349)</td>

<td> 225</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> E</td><td> c</td><td> E</td><td> c</td><td> K</td><td> c</td><td> K</td><td> c</td><td> E</td><td> c</td><td> E</td><td> (SEQ. IDNO. 350)</td>

<td> 226</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> R</td><td> c</td><td> E</td><td> c</td><td> R</td><td> c</td><td> E</td><td> c</td><td> R</td><td> c</td><td> E</td><td> (SEQ. IDNO.351)</td>

<td> 227</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> E</td><td> c</td><td> E</td><td> c</td><td> R</td><td> c</td><td> E</td><td> (SEQ. IDNO.352)</td>

<td> 228</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> K</td><td> c</td><td> D</td><td> c</td><td> K</td><td> c</td><td> D</td><td> c</td><td> K</td><td> c</td><td> D</td><td> (SEQ. IDNO.353)</td>

<td> 229</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> E</td><td> c</td><td> H</td><td> c</td><td> E</td><td> c</td><td> H</td><td> c</td><td> E</td><td> c</td><td> H</td><td> (SEQ. IDNO.354)</td>

<td> 230</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> E</td><td> c</td><td> E</td><td> c</td><td> H</td><td> c</td><td> H</td><td> c</td><td> E</td><td> c</td><td> E</td><td> (SEQ. IDNO.355)</td>

<td> 231</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> R</td><td> (SEQ. IDNO.356)</td>

<td> 232</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> D</td><td> c</td><td> D</td><td> (SEQ. IDNO.357)</td>

<td> 233</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> R</td><td> c</td><td> D</td><td> c</td><td> D</td><td> c</td><td> D</td><td> (SEQ. IDNO.358)</td>

<td> 234</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> H</td><td> c</td><td> D</td><td> c</td><td> H</td><td> c</td><td> D</td><td> c</td><td> H</td><td> c</td><td> D</td><td> (SEQ. IDNO.359)</td>

<td> 235</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> H</td><td> c</td><td> H</td><td> c</td><td> H</td><td> c</td><td> H</td><td> c</td><td> H</td><td> c</td><td> H</td><td> (SEQ. IDNO. 360)</td>

<td> 236</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> H</td><td> c</td><td> D</td><td> c</td><td> D</td><td> c</td><td> H</td><td> c</td><td> D</td><td> c</td><td> D</td><td> (SEQ. IDNO.361)</td>

<td> 237</td><td> c</td><td> c</td><td> c</td><td> c</td><td> c</td><td> H</td><td> c</td><td> E</td><td> c</td><td> E</td><td> c</td><td> H</td><td> c</td><td> E</td><td> c</td><td> E</td><td> (SEQ. NO. ID362)</td>

<td> 238</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> R</td><td> Y</td><td> D</td><td> Y</td><td> R</td><td> Y</td><td> D</td><td> Y</td><td> R</td><td> Y</td><td> D</td><td> (SEQ. IDNO.363)</td>

<td> 239</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> D</td><td> Y</td><td> D</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> (SEQ. ID NO.364)</td>

<td> 240</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> E</td><td> Y</td><td> K</td><td> Y</td><td> E</td><td> Y</td><td> K</td><td> Y</td><td> E</td><td> Y</td><td> K</td><td> (SEQ. IDNO.365)</td>

<td> 241</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> E</td><td> Y</td><td> E</td><td> Y</td><td> K</td><td> Y</td><td> K</td><td> Y</td><td> E</td><td> Y</td><td> E</td><td> (SEQ. IDNO.366)</td>

<td> 242</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> R</td><td> Y</td><td> E</td><td> Y</td><td> R</td><td> Y</td><td> E</td><td> Y</td><td> R</td><td> Y</td><td> E</td><td> (SEQ. IDNO.367)</td>

<td> 243</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> E</td><td> Y</td><td> E</td><td> Y</td><td> R</td><td> Y</td><td> E</td><td> (SEQ. IDNO.368)</td>

<td> 244</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> K</td><td> Y</td><td> D</td><td> Y</td><td> K</td><td> Y</td><td> D</td><td> Y</td><td> K</td><td> Y</td><td> D</td><td> (SEQ. IDNO. 125)</td>

<td> 245</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> E</td><td> Y</td><td> H</td><td> Y</td><td> E</td><td> Y</td><td> H</td><td> Y</td><td> E</td><td> Y</td><td> H</td><td> (SEQ. IDNO. 369)</td>

<td> 246</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> E</td><td> Y</td><td> E</td><td> Y</td><td> H</td><td> Y</td><td> H</td><td> Y</td><td> E</td><td> Y</td><td> E</td><td> (SEQ. IDNO. 370)</td>

<td> 247</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> (SEQ. IDNO. 371)</td>

<td> 248</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> D</td><td> Y</td><td> D</td><td> (SEQ. IDNO. 372)</td>

<td> 249</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> R</td><td> Y</td><td> D</td><td> Y</td><td> D</td><td> Y</td><td> D</td><td> (SEQ. NO. ID373)</td>

<td> 250</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> H</td><td> Y</td><td> D</td><td> Y</td><td> H</td><td> Y</td><td> D</td><td> Y</td><td> H</td><td> Y</td><td> D</td><td> (SEQ. IDNO. 374)</td>

<td> 251</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> H</td><td> Y</td><td> H</td><td> Y</td><td> H</td><td> Y</td><td> H</td><td> Y</td><td> H</td><td> Y</td><td> H</td><td> (SEQ. IDNO. 375)</td>

<td> 252</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> H</td><td> Y</td><td> D</td><td> Y</td><td> D</td><td> Y</td><td> H</td><td> Y</td><td> D</td><td> Y</td><td> D</td><td> (SEQ. IDNO. 376)</td>

<td> 253</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> Y</td><td> H</td><td> Y</td><td> E</td><td> Y</td><td> E</td><td> Y</td><td> H</td><td> Y</td><td> E</td><td> Y</td><td> E</td><td> (SEQ. IDNO. 377)</td>

<td> 254</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> R</td><td> N</td><td> D</td><td> N</td><td> R</td><td> N</td><td> D</td><td> N</td><td> R</td><td> N</td><td> D</td><td> (SEQ. IDNO. 378)</td>

EP 2 581 097 Β1

<td> 255</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> R</td><td> N</td><td> R</td><td colspan="2"> (continued) N D</td><td> N</td><td> D</td><td> N</td><td> R</td><td> N</td><td> R</td><td> (SEQ. IDNO.378)</td>

<td> 256</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> E</td><td> N</td><td> K</td><td> N</td><td> E</td><td> N</td><td> K</td><td> N</td><td> E</td><td> N</td><td> K</td><td> (SEQ. IDNO.380)</td>

<td> 257</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> E</td><td> N</td><td> E</td><td> N</td><td> K</td><td> N</td><td> K</td><td> N</td><td> E</td><td> N</td><td> E</td><td> (SEQ. IDNO.381)</td>

<td> 258</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> R</td><td> N</td><td> E</td><td> N</td><td> R</td><td> N</td><td> E</td><td> N</td><td> R</td><td> N</td><td> E</td><td> (SEQ. IDNO.382)</td>

<td> 259</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> E</td><td> N</td><td> E</td><td> N</td><td> R</td><td> N</td><td> E</td><td> (SEQ. IDNO.383)</td>

<td> 260</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> K</td><td> N</td><td> D</td><td> N</td><td> K</td><td> N</td><td> D</td><td> N</td><td> K</td><td> N</td><td> D</td><td> (SEQ. IDNO.384)</td>

<td> 261</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> E</td><td> N</td><td> H</td><td> N</td><td> E</td><td> N</td><td> H</td><td> N</td><td> E</td><td> N</td><td> H</td><td> (SEQ. IDNO.385)</td>

<td> 262</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> E</td><td> N</td><td> E</td><td> N</td><td> H</td><td> N</td><td> H</td><td> N</td><td> E</td><td> N</td><td> E</td><td> (SEQ. IDNO.386)</td>

<td> 263</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> R</td><td> (SEQ. IDNO.387)</td>

<td> 264</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> D</td><td> N</td><td> D</td><td> (SEQ. IDNO.388)</td>

<td> 265</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> R</td><td> N</td><td> D</td><td> N</td><td> D</td><td> N</td><td> D</td><td> (SEQ. IDNO.389)</td>

<td> 266</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> H</td><td> N</td><td> D</td><td> N</td><td> H</td><td> N</td><td> D</td><td> N</td><td> H</td><td> N</td><td> D</td><td> (SEQ. IDNO.390)</td>

<td> 267</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> H</td><td> N</td><td> H</td><td> N</td><td> H</td><td> N</td><td> H</td><td> N</td><td> H</td><td> N</td><td> H</td><td> (SEQ. IDNO.391)</td>

<td> 268</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> H</td><td> N</td><td> D</td><td> N</td><td> D</td><td> N</td><td> H</td><td> N</td><td> D</td><td> N</td><td> D</td><td> (SEQ. IDNO.392)</td>

<td> 269</td><td> N</td><td> N</td><td> N</td><td> N</td><td> N</td><td> H</td><td> N</td><td> E</td><td> N</td><td> E</td><td> N</td><td> H</td><td> N</td><td> E</td><td> N</td><td> E</td><td> (SEQ. IDNO.393)</td>

<td> 270</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> R</td><td> Q</td><td> D</td><td> Q</td><td> R</td><td> Q</td><td> D</td><td> Q</td><td> R</td><td> Q</td><td> D</td><td> (SEQ. IDNO.394)</td>

<td> 271</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> D</td><td> Q</td><td> D</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> (SEQ. IDNO.395)</td>

<td> 272</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> E</td><td> Q</td><td> K</td><td> Q</td><td> E</td><td> Q</td><td> K</td><td> Q</td><td> E</td><td> Q</td><td> K</td><td> (SEQ. ID NO.396)</td>

<td> 273</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> E</td><td> Q</td><td> E</td><td> Q</td><td> K</td><td> Q</td><td> K</td><td> Q</td><td> E</td><td> Q</td><td> E</td><td> (SEQ. IDNO.397)</td>

<td> 274</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> R</td><td> Q</td><td> E</td><td> Q</td><td> R</td><td> Q</td><td> E</td><td> Q</td><td> R</td><td> Q</td><td> E</td><td> (SEQ. IDNO.398)</td>

<td> 275</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> E</td><td> Q</td><td> E</td><td> Q</td><td> R</td><td> Q</td><td> E</td><td> (SEQ. IDNO.399)</td>

<td> 276</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> K</td><td> Q</td><td> D</td><td> Q</td><td> K</td><td> Q</td><td> D</td><td> Q</td><td> K</td><td> Q</td><td> D</td><td> (SEQ. IDNO.400)</td>

<td> 277</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> E</td><td> Q</td><td> H</td><td> Q</td><td> E</td><td> Q</td><td> H</td><td> Q</td><td> E</td><td> Q</td><td> H</td><td> (SEQ. IDNO.401)</td>

<td> 278</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> E</td><td> Q</td><td> E</td><td> Q</td><td> H</td><td> Q</td><td> H</td><td> Q</td><td> E</td><td> Q</td><td> E</td><td> (SEQ. IDNO.402)</td>

<td> 279</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> (SEQ. IDNO.403)</td>

<td> 280</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> D</td><td> Q</td><td> D</td><td> (SEQ. IDNO.404)</td>

<td> 281</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> R</td><td> Q</td><td> D</td><td> Q</td><td> D</td><td> Q</td><td> D</td><td> (SEQ. IDNO.405)</td>

<td> 282</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> H</td><td> Q</td><td> D</td><td> Q</td><td> H</td><td> Q</td><td> D</td><td> Q</td><td> H</td><td> Q</td><td> D</td><td> (SEQ. IDNO.406)</td>

<td> 283</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> H</td><td> Q</td><td> H</td><td> Q</td><td> H</td><td> Q</td><td> H</td><td> Q</td><td> H</td><td> Q</td><td> H</td><td> (SEQ. IDNO.407)</td>

<td> 284</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> H</td><td> Q</td><td> D</td><td> Q</td><td> D</td><td> Q</td><td> H</td><td> Q</td><td> D</td><td> Q</td><td> D</td><td> (SEQ. IDNO.408)</td>

<td> 285</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> H</td><td> Q</td><td> E</td><td> Q</td><td> E</td><td> Q</td><td> H</td><td> Q</td><td> E</td><td> Q</td><td> E</td><td> (SEQ. IDNQ.409)</td>

[0079] The self-assembling peptides are generally linear sequences. However, the self-assembling peptides can be in the form of non-linear sequences, optionally containing hydrophobic tails, which interact with the ECM. In one embodiment, the sequence is in the form of a rake, wherein the tines of the rake are the hydrophobic sequences, which interact with the ECM to anchor the self-assembling peptides to the tissue or véssél. The handle of the rake contains a sequence that self-assembles. In another embodiment, the handle ofthe rake is a hydrophobic sequence which interacts with the ECM and the tines of the rake are sequences that self-assemble. The self-assembling sequences may selfassemble alone or in the presence of one or more assembly assist sequences.

D. Therapeutic, Prophylactic and Diagnostic Agents [0080] The formulations may alsó include other therapeutic, prophylactic or diagnostic agents. In a preferred embodiment, these may be anti-inflammatory agents, vasoactive agents, anti-infective agents, anesthetics, growth factors, vitamins, nutrients, and/or cells.

[0081] These can be peptides or proteins, polysaccharides or saccharides, nucleíc acids nucleotides, proteoglycan, lipid, carbohydrate, or a small molecule, typically an organic compound, having multiple carbon-carbon bonds that may be isolated from natúré or prepared via Chemical synthesis. Small molecules have relatively low molecular weights (e.g., less than about 1500 g/mol) and are nőt peptides or nucleíc acids. The substance can alsó be a biomolecule, which is a molecule such as a peptide, proteoglycan, lipid, carbohydrate, or nucleíc acid having characteristics typical of molecules found in living organisms. Like small molecules, biomolecules can be naturally occurring or may be artificial (i.e., they may be molecules that have nőt been found in natúré). For example, a protein having a sequence that has nőt been found in natúré (e.g., one that does nőt occur in a publicly available databaseof sequences) orthat has a known sequence

EP 2 581 097 Β1 modified in an unnatural way by a humán hand (e.g., a sequence modified by altering a post-translational process such as glycosylation) is an artificial biomolecule. Nucleic acid molecules encoding such proteins (e.g., an oligonucleotide, optionally contained within an expression vector) are alsó biomolecules and can be incorporated intő the compositions described herein. For example, a composition can include a piuraiity of self-assembling materials and cells that express, or that are engineered to express, a protein biomolecule (by virtue of containing a nucleic acid sequence that encodes the protein biomolecule).

[0082] Many different therapeutic, prophylactic or diagnostic agents can be incorporated intő the formulation. Representative vasoconstrictors include epinephrine and phenylephrine; representative coloring agents include arsenazo III, chlorophosphonazo III, antipyrylazo 111, murexide, Eriochrome Black T, Eriochrome Blue SE, oxyacetazo I, carboxyazo III, tropolone, methylthymol blue, and Mordant Black 32; representative anesthetic agents include benzocaine, bupivacaine, butamben picrate, chloroprocaine, cocaine, curare, dibucaine, dyclonine, etidocaine, lidocaine, mepivacaine, pramoxine, prilocaine, procaine, propoxycaine, ropivacaine, tetracaine, or combinations thereof. Local application ofthe anesthetic agent may be all that is required in somé situations, for example, for a burn or other wound to the skin, including decubitus ulcers; wounds, such as cancersores; orforminimally invasivesurgeries. Combining local anesthetics with the self-assembling peptides, whether combined by virtue of being present in the same formulation or by virtue of co-administration, can help contain the anesthetic within the body and reduce the amount entering the circulation. [0083] Vasoconstrictors such as phenylephrine can be included to prolong the effect of local anesthesia (e.g., 0.1-0.5% phenylephrine). Analgesic agents other than a local anesthetic agent, such as steroids, non-steroidal anti-inflammatory agents like indomethacin, platelet activating factor (PAF) inhibitors such as lexipafant, CV 3988, and/or PAF receptor inhibitors such as SRI 63-441.

[0084] An anti-infective or antimicrobial agent (e.g., an antibiotic, antibacterial, antiviral, or antifungal agent) can be included for either systemic or local administration. Examples include β-lactam antibiotics such as penicillins and cephalosporins; other inhibitors of cell wall synthesis such as vancomycin; chloramphenicol; tetracyclines; macrolides; clindamyin; streptogramins; aminoglycosides; spectinomycin; sulfonamides; trimethoprim; quinolones; amphotericin B; flucytosine; azoles such as ketoconazole, itraconazole, fluconazole, clotrimazole, and miconazole; griseofulvin; terbinafine; and nystatin. The antimicrobial can be topically administered (e.g., to treat skin infections or burns) or to help prevent infection at a site of catheter insertion (e.g., an intravenous catheter). Suitable topical antimicrobials include kanamycin, neomycin, bacitracin, polymixin, topical sulfonamides such as mafenide acetate or silver sulfadiazine, and gentamicin sulfate. The antimicrobial can alsó be a broad-spectrum agent. For example, a second, third, or fourth generation cephalosporin can be used. These agents may be active against a wide rangé of bacteria including both gram positive and gram-negative species. Such antibacterial agents may be particularly appropriate where the present scaffolds are used to inhibit movement of intestinal contents such as during intestinal resection or other surgery that purposefully or accidentally disturbs the integrity ofthe intestinal wall. One of ordinary skill in the art will be able to select appropriate antimicrobial agents by considering factors such as the patient’s history (e.g., any history of an allergic reaction to such agents), the location to which the peptides are to be applied, and the type of infectious agent likely to be present. Compositions containing antimicrobial agents can prevent infections in a variety of ways including: (1) killing the infectious agent due to the activity ofthe antimicrobial agent; (2) preventing infection by assembly ofthe peptides to form a barrier which blocks infiltration ofthe infectious agent intő the tissue by blocking the tissue specific sequence on the infectious agent from interacting with the tissue; (3) causing the infectious agent to change its orientation with respect to the tissue due to the charge ofthe self-assembling matéria! and thus block infiltration ofthe infectious agent intő the tissue; (4) encapsulating the infectious agent within the self-assembling peptides to prevent infiltration of the infectious agent; and combinations thereof. The self-assembling peptides can alsó be used to prevent contamination or infection by other biologics and/or hazardous materials.

[0085] Any ofthe compositions described herein, whether they contain only self-assembling peptides and one or more bioactive molecules (and whether in a liquid, semi-solid, or solid form), can include a coloring agent. Suitable coloring agents include commercially available food colorings, natural and synthetic dyes, and fluorescent molecules. Preferably, the coloring agent is nontoxic or is included at such low concentrations as to minimize any toxic effect. The use of a coloring agent allows for improved visualization of an area that is covered by a structure or scaffold and can facilitate removal, if such removal is desired. The coloring agent can be one that changes color when it comes intő contact with a contaminated area (e.g., a color change may be triggered by the contamination itself (e.g., by the blood or bacteria present at a wound site)). For example, a metabolic product of a bacterium may trigger a color change. Conditions such as pH or redox state induced by contaminants may alsó be detected. Exemplary indicators include arsenzazo III, chlorophosphonazo III, antipyrylazo III, murexide, Eriochrome Black T and Eriochrome Blue SE for Mg²⁺, oxyacetazo I, carboxyazo III, tropolone, methylthymol blue, and Mordant Black 32. AlamarBlue, a redox indicator, and phenol red are alsó of use in the compositions and methods. In another embodiment, the coloring agent may be in the form of a nanoparticle which reflects one wavelength of light and upon aggregation (i.e., self-assembly ofthe peptide) reflects a different wavelength of light.

[0086] Many other active agents can be included in the compositions. For example, a number of growth factors can

EP 2 581 097 Β1 be included to accelerate one or more aspects of healing (e.g., angiogenesis, cell migration, process extension, and cell proliferation). These types of compositions can be included as others can, by virtue of inclusion in the compositions or by virtue of co-administration in the present methods. Examples include vascular endothelial growth factor (VEGF), a transforming growth factor (TGF) such as transforming growth factor p, a piatelet derived growth factor (PDGF), an epidermal growth factor (EGF), a nerve growth factor (NGF), an insulin-like growth factor (e.g., insulin-like growth factor I), a glial growth factor (GGF), a fibroblast growth factor (FGF), etc. It will be appreciated that in many cases these terms refer to a variety of different molecular species. For example, several transforming growth factor R species are known in the art. One of ordinary skill in the art will be guided in the selection of an appropriate growth factor by considering, for example, the site at which the composition isto be administered. For example, an EGF can be included in compositions applied to the skin; an NGF and/or GGF can be included in compositions applied to nerves or the nervous system; and so forth.

[0087] The growth factor or another agent can be a chemotactic substance, which has the ability, in vivő or in cell culture, to recruit cells to a site at which the substance is present. The cells recruited may have the potential to contribute to the formation of new tissue or to repair existing, damaged tissue (e.g., by contributing structurally and/or functionally to the tissue (e.g., by providing growth factors or contributing to a desirable immuné response)). Certain chemotactic substances can alsó function as proliferation agents (e.g., neurotropic factors such as NGF or BDNF).

[0088] The compositions can alsó be used in combination with or instead of compounds such as cyanoacrylates, oxidized cellulose, fibrin sealants, collagen gél, thrombin powder, microporous polysaccharide powders, clotting factors (e.g., Factor V, Factor Vili, fibrinogen, or prothrombin) and zeolite powders.

[0089] In one embodiment, vitamins may be added to theself-assembling peptides such as vitamin K after liver surgery. In addition, other vitamins can be added to facilitate the reconstruction of tissue or skin when applied topically in combination with the matéria!. This could be after injury or in the normál course of topical hydration.

[0090] The one or more therapeutic, diagnostic and/or prophylactic agents can be administered simultaneously with the self-assembling peptides in the same formulation, administered simultaneously in separate formulations, or sequentially. Alternatively, the aetive agent(s) can be covalently coupled to the self-assembling peptide.

[0091] It will be understood that therapeutic molecules are generally administered in an effective amount in order to achieve a clinically significant result, and effective dosages and concentrations are known in the art. These dosages and concentrations can guide the selection of dosages and concentrations in the present context. Bioactive molecules can be provided at a variety of suitable concentrations and in suitable amounts (e.g., in the microgram or milligram rangé, or greater). For guidance, one can consult texts such as Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Ed., and Katzung, Basic and Clinical Pharmacology.

Cells [0092] Where cells are delivered to a patient (e.g., to promote tissue healing), utologous cells can be used. In one embodiment, the cells could be hematopoietic cells from the patient, dispersed in the self-assembling peptides and implanted. In another embodiment, the cells can be cord red blood cells.

[0093] Molded scaffolds as described above, liquid compositions, gels, solids (e.g. powders) or other semi-solid embodiments may include one or more additional substances such as bioactive molecules or cells. In somé instances, the cell may secrete the bioactive molecule either naturally or following genetic engineering (e.g., to express and/or secrete a recombinant protein). The structures described herein are able to support cell attachment, viability, and growth; these have been observed when cells are cultured on the surface ofthe matéria! or when cells grow within the matéria! (e.g., when encapsulated). In addition, the structures are able to serve as substratesfor neurite growth and synapse formation when neurons are grown on or within them. Thus, bioactive molecules and cells can be encapsulated within the peptide structures and maintain substantial function and viability when so encapsulated (see, e.g., U.S.S.N. 09/778,200 and 10/196,942).

E. Formulations [0094] In the preferred embodiment, the formulation is a liquid or reconstitutable powder, applied topically. In one embodiment, the formulation is provided as a dry or lyophilized powder which can be administered directly as a powder which hydrates at the site of application, or suspended or dissolved in a liquid, most preferably aqueous, and applied as a spray, paint, or injection óra hydrogel such as chitin, collagen, alginate, or synthetic polymer. In another embodiment, the formulation is administered as a compressed wafer, disc, or tablet. In still another embodiment, the formulation is provided as a coating on a device, for example a stent or a catheter, which may be dissolved in an aqueous solution and dried on the device, or mixed with a polymeric carrier and applied to the device. In yet another embodiment, the formulation is provided in a bandage.foam or mátrix, in which the peptides may be dispersed orabsorbed. The formulation could alsó be in the form of sutures, tape, or adhesive.

EP 2 581 097 Β1 [0095] Conventionally, local anesthetics are delivered by topical administration (e.g., formulated as anointment, cream, or solution) or injected intő an area where the nerve fibers one wishes to block reside. The formulation may be administered to a burn or ulcer, especially when formulated with anesthetics, anti-inflammatory agents, growth factors, and antiinfectives, in the form of a foam, mátrix or bandage, to stop bleeding or loss of interstitial fluid.

[0096] One or more ofthe compositions described herein can be assembled in kits, togetherwith instructions fór use. Fór example, the kits can include a biocompatible composition including self-assembling peptides (or a concentrated solution or powdered formulation thereof, togetherwith a diluent) and a vasoconstrictor, a coloring agent, oran analgesic or anesthetic agent and instructions fór their combination (if nőt already combined) and use (e.g., dilution and administration). The kits can further include one or more ofthe additional agents described herein. These agents can be present within a peptide-based composition or packaged separately, and they can include one or more types of biological cells, an antibiotic or other therapeutic, collagen, an anti-inflammatory agent, a growth factor, or a nutrient. The kit may alsó include one or more of a syringe (e.g., a barrel syringe or a bulb syringe), a needle, a pipette, gauze, sponges, or cotton, swabs, a bandage, a nosebleed plug, a disinfectant, surgical thread, scissors, a scalpel, a sterilé fluid, a spray canister, including those in which a liquid solution issprayed through a simpíe hand pump, a sterilé Container, or disposable gloves. [0097] The formulation can be administered as appropriate fór treatment ofone or more disorders. Fór example, the formulation may be applied to repair an injury or dealing surgery of the lung or dura, or following an epidural or spinal táp, to stop leakage of cerebrospinal fluid. The formulation may be dispersed in a suture or adhesive fór administration at the time of or as released following suturing or gluing of a wound, thereby limiting bleeding, loss of tissue fluids, or other fluids such as those produced by parenchymal tissues such as the liver, pancreas, and gastrointestinal tract. The formulation may be applied to any site of bleeding, in a bandage, gauze, sponge, or other matériái, fór immediate control of bleeding, or released later to control bleeding if the initial treatment such as suturing or pressure is insufficient. Dried fabric, dehydrated foams or hydrogels, or bandages containing the formulation may be part of first aid kids fór treatment of injuries, fór example, in war, at accident sites, or clinics where rapid treatment may be required and storage space is limited.

[0098] In somé embodiments, compositions including self-assembling peptides can be associated with surgical sponges. Fór example, liquid compositions can be drawn intő commercially available sponges prior to or during their use. Studies indicate that hemostasis can be satisfactorily achieved without traditional sponges, bút there may be instances where including compositions containing a self-assembling matéria! may be beneficial (e.g., where a patient is experiencing profound bleeding or where the goal of treatment is temporary stabilization). The compositions employed can include any ofthe non-fibrous agents described herein. The sponges can be any known in the art, including woven and non-woven sponges and those designed specifically fór dental or ophthalmic surgeries. See, e.g., U.S. Patent Nos. 4,098,728; 4,211,227; 4,636,208; 5,180,375; and 6,711,879.

[0099] In embodiments featuring bandages or dressings, the bandage or dressing can include a first layer of sufficient shape and size to cover a wound or a substantial portion thereof (e.g., the most injured portion ofthe tissue or the area bleeding most profusely). The first layer can have a top surface, a bottom surface, and a perimeter that is, optionally, wholly or partially covered with an adhesive. A second layer of the bandage or dressing can be detachably affixed to the bottom surface of the first layer, optionally excluding the perimeter or any part of the perimeter bearing adhesive, and can include a liquid or non-liquid composition (e.g., a gél, pásté, foam, cream, ointment, or powdered composition) including self-assembling peptides. The composition will come in contact with the wound upon application ofthe bandage or dressing and is transferable from the bandage or dressing to the wound site upon removal ofthe first layer orthe first and second layers. In simpler configurations, the composition comprising self-assembling peptides can be associated with the bottom ofthe first layer (e.g., interior to the adhesive perimeter), and the second layer can be omitted. In either case, either the first and/or second layers can include a transparent window, through which somé or all ofthe underlying wound can be viewed. The composition including the self-assembling peptides can be added to the bandage before it is packaged or just before use. In another embodiment, the formulation may include a further physical barrier, such as a layer of Silicon film, to prevent loss of fluid by drying, after the active flow of fluids has been stopped by application of the formulation.

[0100] The formulations may alsó be administered as immediate or controlled release formulations. A delayed release dosage form is one that releases a drug (or drugs) at a time other than promptly after administration. An extended release dosage form is one that allows at least a twofold reduction in dosing frequency as compared to the drug presented as a conventional dosage form (e.g. as a solution or prompt drug-releasing, conventional solid dosage form). A modified release dosage form is one fór which the drug release characteristics of time, course and/or location are chosen to accomplish therapeutic or convenience objectives nőt offered by conventional dosage forms such as Solutions, ointments, or promptly dissolving dosage forms. Delayed release and extended release dosage forms and their combinations are types of modified release dosage forms.

[0101] Matrix-forming materials are materials which form strong, viscous gels upon hydration and provide control of drug diffusion and release. In hydrophilic mátrix systems, matrix-forming materials are uniform ly incorporated throughout the tablet. Upon contact with water, the outer tablet layer is partially hydrated, forming a gél layer. The rate of diffusion

EP 2 581 097 Β1 of the drug(s) out of the gél layer and the rate of erosion of the gél layer determine overall tablet dissolution and drug delivery rates. Examples of mátrix forming materials include cellulose ethers that are water-soluble such as methylcellulose, ethyl cellulose and hydroxypropyl methylcellulose.

[0102] Formulations are prepared using a pharmaceutically acceptable barrier composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. The term carrier includes bút is nőt limited to diluents, binders, lubricants, disintegrators, fillers, matrix-forming compositions and coating compositions.

[0103] Carrier alsó includes all components ofthe coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. The delayed release dosage formulations may be prepared as described in references such as Pharmaceutical dosage form tablets, eds. Liberman et al. (New York, Marcel Dekker, Inc., 1989), Remington—The Science and practice of pharmacy, 20th ed., Lippincott Williams &amp; Wilkins, Baltimore, Md., 2000, and Pharmaceutical dosage forms and drug delivery systems, 6th Edition, Ansel et al., (Media, Pa.: Williams and Wilkins, 1995) which provides Ínformation on carriers, materials, equipment and processes for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0104] Examples of suitable coating materials include, bút are nőt limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name Eudragit™ (Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides. Additionally, the coating matéria! may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, poré formers and surfactants. Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, bút are nőt limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants.

[0105] Diluents, alsó termed fillers, are typically necessary to increase the búik of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, bút are nőt limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pre-gelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powder sugár.

[0106] Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, bút are nőt limited to, starch, pre-gelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and syntheticgums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone. Somé ofthe materials, which are suitable as binders, can alsó be used as matrix-forming materials such as hydroxypropyl-methylcellulose, ethyl cellulose, and microcrystalline cellulose.

[0107] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, bút are nőt limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil. [0108] Disintegrants are used to facilitate dosage form disintegration or breakup after administration, and generally include, bút are nőt limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pre-gelatinized starch, clays, cellulose, alginine, gums or eross linked polymers, such as crosslinked PVP (Polyplasdone™ XL from GAP Chemical Corp).

[0109] Stabilizers are used to inhibit orretard drug decomposition reactions which include, by way of example, oxidative reactions.

[0110] Surfactants may be anionic, cationic, amphotericornonionic surface active agents. Suitable anionic surfactants include, bút are nőt limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium salts of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, bút are nőt limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer™ 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amidé. Examples of amphoteric surfactants include sodium N-dodecyl-p-alanine, sodium N-lauryl-p-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

EP 2 581 097 Β1 [0111] If desired, the tablets, beads, granules or particles may alsó contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, and preservatives.

[0112] In one type of formulation, the self-assembling peptides can be utilized as a shaving cream or hand lotion additive, to form a barrier for loss of fluids and as a barrier to adhesions and contamination.

[0113] Extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in Remington-The Science and practice of pharmacy (20th ed., Lippincott Williams &amp; Wilkins, Baltimore, MD, 2000). A diffusion system typically consists of two types of devices, a reservoir and a mátrix, and is well known and described in the art. The mátrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier intő a tablet form. The three major types of materials used in the preparation of mátrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcellulosessuch as hydroxypropyl-cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and Carbopol™ 934, polyethylene oxides and mixtures thereof. Fatty compounds include, bút are nőt limited to, various waxes such as carnauba wax and glyceryl tristearate and wax-type substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof. In certain embodiments, the plastic matéria! is a pharmaceutically acceptable acrylic polymer, including bút nőt limited to, acrylicacid and methacrylicacid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymer poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers. In certain embodiments, the acrylic polymer is comprised of one or more ammonio methacrylate copolymers. Ammonio methacrylate copolymers are well known in the art, and are described in NF XVII as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.

[0114] Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semipermeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.

[0115] An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads. Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, ordry granulation. Theirformulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inért powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugár. Powdered cellulose derivatives are alsó useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can alsó be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can alsó serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils. Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In the congealing method, the drug is mixed with a wax matéria! and either spray-congealed orcongealed and screened and processed.

[0116] The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies. The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility ofthe coating, and will generally represent about 10 wt. % to 50 wt. % relatíve to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used tostabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % ofthe polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerolmonostearates may alsó be used. Pigments such as titanium dioxide may alsó be used. Small quantities ofan anti-foaming agent, such as a silicone (e.g., simethicone), may alsó be added to the coating composition.

EP 2 581 097 Β1

Polymeric Matrices [0117] Both non-biodegradable and biodegradable matrices can be used for delivery ofthe self-assembling peptides, although biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In somé cases linear release may be most useful, although in others a pulse release or búik release may provided more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers. [0118] Representative synthetic polymers that can be used for delivery include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, polyvinyl chloride, polystyrene and polyvinylpyrrolidone.

[0119] Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof. Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly (lactide-co-caprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, Chemical derivatives thereof (substitutions, additions of Chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In generál, these materials degrade either by enzymatic hydrolysis or exposure to water in vivő, by surface or búik erosion.

[0120] Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethylmethacrylates), poly(butylmethacrylate), poly (isobutyl methacrylate), poly (hexylmethacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0121] The mátrix can be in the form of microparticles such as microspheres, where peptides are dispersed within a solid polymeric mátrix or microcapsules, where the core is ofa different matériái than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in natúré. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab orfilm, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gél such as a hydrogel. The polymer can alsó be in the form ofa coating or part ofa stent or catheter, vascular graft, or other prosthetic device.

[0122] The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art.

[0123] Bioerodible microspheres can be prepared using any ofthe methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release 5,13-22 (1987); Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci. 35, 755-774 (1988). The selection ofthe method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by Mathiowitz, et al., Scanning Microscopy 4,329-340 (1990); Mathiowitz, et al., J. Appl. Polymer Sci. 45, 125-134 (1992); and Benita, et al., J. Pharm. Sci. 73, 1721-1724 (1984). In solvent evaporation, described for example, in Mathiowitz, et al., (1990), Benita, and U.S. Patent No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent. The peptide either in soluble form or dispersed as fine particles, is added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface active agent such as poly(vinyl alcohol). The resulting emulsion is stirred until most ofthe organic solvent evaporates, Ieaving solid microspheres. In generál, the polymer can be dissolved in methylene chloride. Microspheres with different sizes (1-1000 microns) and morphologies can be obtained by this method which is useful for relatively stable polymers such as polyesters and polystyrene. However, labile polymers such as polyanhydrides may degrade due to exposure to water. For these polymers, hot melt encapsulation and solvent removal may be preferred.

[0124] In hot melt encapsulation, the polymer is first melted and then mixed with the solid particles of peptides. The mixture is suspended in a non-miscible solvent such as Silicon oil and, with continuous stirring, heated to 5°C above the

EP 2 581 097 Β1 meiting point ofthe polymer. Once the émulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microspheres are washed bydecantation with petróleum ether to give a free-flowing powder. Microsphereswith diameters between one and 1000 microns can be obtained with this method. The external surface of spheres prepared with this technique is usually smooth and dense. This procedure is useful with water labile polymers, bút is limited to use with polymers with moleeular weights between 1000 and 50000. Solvent removal was primarily designed for use with polyanhydrides. In this method, the drug is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then suspended in oil, such as Silicon oil, by stirring, to form an émulsion. Within 24 hours, the solvent diffuses intő the oil phase and the émulsion droplets harden intő solid polymer microspheres. Unlike solvent evaporation, this method can be used to make microspheres from polymers with high meiting points and a wide rangé of moleeular weights. Microspheres having a diameter between one and 300 microns can be obtained with this procedure. The external morphology ofthe spheres is highly dependent on the type of polymer used. In spray drying, the polymer is dissolved in methylene chloride (0.04 g/ml). A known amount of active drug is suspended (if insoluble) or co-dissolved (if soluble) in the polymer solution. The solution or the dispersion is then spray-dried. Double walled microspheres can be prepared according to U.S. Patent No. 4,861,627 to Mathiowitz.

[0125] Hydrogel microspheres made of gel-type polymers such as alginate or polyphosphazines or other dicarboxylic polymers can be prepared by dissolving the polymer in an aqueous solution, suspending the matéria! to be incorporated intő the mixture, and extruding the polymer mixture through a microdroplet forming device, equipped with a nitrogén gas jet. The resulting microspheres fali intő a slowly stirring, ionic hardening bath, as described, for example, by Salib, et al., Pharmazeutische Industrie 40-11 A, 1230 (1978). Chitosan microspheres can be prepared by dissolving the polymer in acidic solution and crosslinking with tripolyphosphate. For example, carboxymethylcellulose (CMC) microsphere are prepared by dissolving the polymer in an acid solution and precipitating the microspheres with lead ions. Alginate/polyethylene imide (PEI) can be prepared to reduce the amount of carboxyl groups on the alginate microcapsules.

[0126] Other delivery systems including films, coatings, pellets, slabs, and devices can be fabricated using solvent or melt casting, and extrusion, as well as standard methods for making composites. The polymer can be produced by first mixing monomers and peptides as described by Sawhney, et al., and polymerizing the monomers with UV light. The polymerization can be carried out in vitro as well as in vivő.

F. Devices for Administration [0127] The liquid formulations may be provided in a syringe or pipette having a barrel containing a composition including self-assembling peptides and a means for expelling the composition from an open tip ofthe syringe or pipette (e.g., a plunger or bulb). The syringe may consist ofone or more compartments, so that mixing ofthe self-assembling peptides with one or more other agents occurs at the time of application. The compartments may alsó contain an excipient such as a matéria! forming a hydrogel or adhesive in one compartment and the self-assembling peptides in the other compartment. In another embodiment, one compartment may contain lyophilized powder or particles of self-assembling peptides, and another compartment may contain solution to dissolve or hydrate the peptides, or other powders to mix with the self-assembling peptides for dry application. The composition within the barrel can further include any of the non-fibrous agents described herein (e.g., one or more of a vasoconstrictor, a coloring agent, an anesthetic or analgesic agent, an antibiotic or other therapeutic, collagen, an anti-inflammatory agent, a growth factor, or a nutrient).

[0128] The self-assembling peptides can be applied as a coating by spraying or dipping the device intő the peptides, the peptides can be impregnated intő a bandage, gauze or other absorbent matériái, the peptides can be mixed with a polymeric matéria!. The self-assembling peptides can alsó be formulated as a pharmaceuticai foam. Pharmaceuticai foams are pressurized dosage forms that, upon valve actuation, emit a fine dispersion of liquid and/or solid materials in a gaseous médium. In one embodiment, the foam contains the self-assembling peptides, in liquid or solid form, optionally in combination with one or more active agents. Suitable propellants include, bút are nőt limited to, hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA227), hydrocarbons, and carbon dioxide.

III. Methods of Administration

A. Sites of Administration [0129] The self-assembling peptides can be applied to a variety of different surfaces to prevent or control fluid passage or to function as a barrier. The amount of self-assembling peptides is determined in part by the function of the peptides in controlling fluid fiow, as well as the properties of any other materials or structures associated with the self-assembling peptides, alone or in combination with other bioactive materials. The self-assembling peptides can be used to stop the movement of fluids in or out of tissues/organs.

[0130] In a first embodiment, the self-assembling peptides are used to prevent or control bleeding. The self-assembling

EP 2 581 097 Β1 peptides may be applied as a powder, liquid, a gél, or as part ofa substrate such as a bandage or membráné. This may be applied to a blood véssél, either within the lumen, for example at the time of angioplasty, administered by or as a coating on a stent or catheter, or exteriőr to the véssél, typically at the site of anastomosis. The self-assembling peptides may be applied to tissues before, during or after surgery, to prevent bleeding, which is especially problematic with tissue such as liver, kidney or spleen, or other surgeries where there is a high riskof transfusion, orto seal and protect a tissue, for example, which is for transplantation or reattachment. In another embodiment, the self-assembling peptides can be used as a shaving cream additive where they can act as hemostatic agents to stop bleeding due to razor cuts, a barrier to prevent contamination of razor cuts and/or a lubricant.

[0131] The self-assembling peptides can be used to stop the flow of fluids other than blood. The self-assembling peptides can be applied to burns to stop leakage of interstitial fluid. The self-assembling peptides can be applied to the dura or lung as a dural or lung sealant. In one embodiment the self-assembling peptides can be used to repair a lung after a puncture wound, thereby restoring its ability to function.

[0132] The self-assembling peptides can alsó be utilized in generál órai surgery, periodontistry, and generál dentistry, as a barrier.

[0133] The use ofthe self-assembling peptides in individuals with impaired coagulation (hemophilia, von Willebrands, vitamin K, protein Sor protein Cdeficiency.fulminant hepatitis, disseminated intravascularcoagulation (DIC), hemolyticuremic syndrome (HÚS)) is alsó an important utility since the mechanism of action is independent of the normál coagulation pathway. For example, the compositions can be used to replace a damaged semi-permeable barrier, such as in cells, to restore the Iocal environment and facilitate cell survival and repair.

[0134] In another embodiment, the self-assembling peptides are applied, typically by spraying or injection, to the exteriőr of a tissue such as a tumor, to prevent breakage or metastasis at the time of surgery. The self-assembling peptides control bleeding during tumor resection, as well as limits metastasis. This alsó minimizes the immuné response that can be caused by a laser during tumor resection. The self-assembling peptides are alsó useful in holding loose tumors together so that nothing is left behind when they are resected. There are several types of tumors that are notoriously hard to resect because they are nőt held together tightly (i.e. they are nőt solid masses). The self-assembling peptides are expected to be particularly useful in tumor resection in the brain, and may be useful in a dose-response manner for subeutaneous tumor resection. This may make it easier to resect melanomas in the skin because it appears that the self-assembling peptides alsó facilitate skin healing. Further, self-assembling peptides containing a targeting segment specific for a tumor can be attached to the tumor causing the tumor to aggregate so that it can be resected. The self-assembling peptides can alsó immobilize cells that break away from the tumor during resection to stop or slow metastases. The self-assembling peptides can alsó include a marker reactive with certain types of antigens on the tumor cell surface, producing a colorimetric change to show that all of the cells have been removed or there are more that need to be resected. The addition of an indicator to the self-assembling peptides as well as the ability of the peptides to act like a bio-barrier could reduce the need for second and third operations as well as complications due to outside contamination intő the surgical field. The self-assembling peptides can alsó be used to deliver materials such as DNA to the site of injury for an extended period of time in vitro and for multiple treatments in vivő. Another advantage of the self-assembling peptides is that they can be injected and gél in piacé, so that the peptides can be applied and reapplied during surgery, as necessary.

[0135] In still another embodiment, the self-assembling peptides are particularly well suited to functioning as a barrier to prevent contamination, either tothe tissue orfrom the tissue, forexample, during intestinal surgery. The self-assembling peptides may be applied to prepare an internál site prior to surgery, especially sites such as the sinus cavities, and for surgeries such as transurethral and transvaginal surgery. The self-assembling peptides shouid alsó be particularly useful in cardiovascular surgery, where both barrier and hemostasis properties can be of value, for example, for heart valve patients who are prone to adverse consequences such as valve ring abseesses (coat valve, add antibiotic), endocarditis (coat valve), aortic root dissection (provide immediate hemostasis). In yet another embodiment, a mixture of complementary amino aeids sequences that do nőt self-assemble may be applied to tissue to block infection of the tissue by bacteria. The fact that the sequences are complementary shouid result in the formation of a more effective barrier than complementary sequences that self-assemble.

[0136] The self-assembling peptides in combination with a metál such as silver has anti-adhesive properties and can inhibit angiogenesis. Accordingly, it may be useful in decreasing scarring and adhesions. The self-assembling peptides are applied after surgery, or to an injury such as a burn, to decrease scarring, fluid loss, and limit infection. This has further application in plastic surgery, especially for protection of areas cleaned and debrided prior to closure or skin transplant, for example, in abdominoplasty, face lifts, flap donor sites, latissimus dorsi for breast reconstruction.

[0137] In still another embodiment, the self-assembling peptides are administered as a slurry that can be drunk by a patient to reduce stomach bleeding, for example, from an ulcer, or decrease acidity. Alternatively, the self-assembling peptides could be provided as an enema or suppository to treat hemorrhoids or to fill in diverticula. In yet another embodiment, the self-assembling peptides can be used to prevent infertility due to adhesions in the fallopian tubes or vas deferens.

EP 2 581 097 Β1 [0138] Self assembly is nőt irreversible, contained substanees can be released. For example, the molecules or cells can be released from the structures in vivő (e.g., small molecules can diffuse away and larger molecules and cells can be released as the structures degrade).

[0139] In still another embodiment, the self-assembling peptides are used as a neuroprotective to minimize damage and scarring following neural injury. Peptide-based structures promote repair and regeneration of neural tissue (e.g., when self-assembling peptides are applied to a lesion in the brain as described in U.S.S.N. 10/968,790). The small size of the fibers within the scaffolds and/or the open weave structure of the materials permits extension of cell processes and allows adequate diffusion of nutrients and waste products in a manner that provides unique advantages for neural tissue regeneration.

[0140] In the course ofpromoting wound repair, the compositions may nőt only improve the final outcome (e.g., reduced scar formation resulting in an outcome that more closely resembles the original tissue), bút alsó reduce the time required for healing. These results could nőt have been predicted on the basis ofthe results achieved following application to the injured Central nervous system, given the substantial differences between neural and non-neural tissues.

[0141] Finally, the self-assembling peptides could be used as nanodrapes to prevent cross contamination. For example, the self-assembling peptides could be applied as a coating to the outside ofthe body and then induced to selfassemble. The self-assembled peptides may stop the movement of liquids intő the body, thus reducing the possibility of cross contamination.

B. Effective Dosages [0142] In generál, the amount of self-assembling peptides required will vary depending on various factors such as the size or extent of an injury (which can, in turn, be expressed in terms of the length of an incision, the caliber or number of damaged blood vessels, the degree of a burn, the size and depth of an ulcer, abrasion, or other injury). The amount may vary, for example, from a few microliters to several milliliters or more, e.g., tens or hundreds of milliliters. The device used to deliver the self-assembling peptides will vary in accordance with the amount. For example, a syringe can be conveniently used to deliver smaller amounts, whereas a tűbe or squeezable bottle would be more suitable for larger amounts. An effective amount (whether in reference to a scaffold, precursors thereof, or another bioactive molecule present in the formulation), means the amount necessary to elicit an improved or desired biological response.

[0143] As will be appreciated by those of ordinary skill in this art, the effective amount of self-assembling peptides may vary depending on such factors as the desired biological endpoint, the peptides to be delivered, the natúré ofthe site to which the peptides are delivered, and the natúré ofthe condition for which the agent is administered. For example, an effective amount of a composition for accelerating hemostasis may be an amount sufficient to decrease the amount of blood lost between the time that bleeding begins and the time when bleeding ends by at least 25% relatíve to the amount of blood lost following treatment with cold saline or no treatment. An effective amount of a composition for accelerating hemostasis may alsó be an amount sufficient to decrease the time required to achieve cessation of visible bleeding by at least 25% relatíve to the time required following treatment with cold saline or no treatment. An effective amount of a composition for promoting wound healing may be an amount sufficient to decrease the time required to achieve a predetermined percent reduction in the size of a lesion by at least 25% relatíve to the time required in the absence of such treatment.

[0144] The amount of the composition provided can vary depending on the severity of the subject’s condition and should be sufficient to inhibit the unwanted movement to an extent that benefits the subject. The bodily substance can be blood, cerebrospinal fluid, pus, serous exudate, bile, pancreatic juice, or a substance normally contained within the gastrointestinal tract (e.g., the stomach or intestine), or urinary tract.

C. How Administered [0145] The composition can be provided on the surface ofthe subject’s body and/or provided within a cavity generated by force (e.g., by unexpected trauma or a surgical procedure). In this way the unwanted movement of bodily substanees can be inhibited in the context ofa wide rangé of situations, includíng traumatic injury, a medical condition (e.g., a chronic or prolonged medical condition associated with bleeding), or surgical procedures (e.g., orthopedic surgery, dental surgery, cardiac surgery, ophthalmic surgery, or plastic or reconstructive surgery). For example, where the unwanted movement ofthe bodily substance is the result of trauma, thesubject may have a partly or completely severed body part, a laceration, abrasion, or puncture wound. Where the compositions are applied to a surface ofthe body, they may nőt only inhibit the unwanted movement ofa bodily substance, bút alsó help protect the subject from contamination. For example, applying self-assembling peptides to the skin will impede the movement of an unwanted foreign substance on the skin or hair intő a wound. When the unwanted movement ofthe bodily substance results from a chronic medical condition, the subject may experience recurrent bleeding. For example, the subject may be experiencing bleeding in connection with varicose veins, includíng telangiectases, hemorrhoids, bleeding in the lungs (due, for example, to lung cancer, bronchitis,

EP 2 581 097 Β1 or a bacterial or viral disease, including pneumonia or influenza), or esophageal varices. Medical conditions associated with recurrent bleeding can be treated with the compositions described herein, including those that contain self-assembling peptides and a vasoconstrictor (e.g., phenylephrine, which can constitute about 0.25-0.5% ofthe composition). Where bleeding occurs in the oropharynx or lungs, the compositions can be administered through a metered dose inhaler. If the patient’s condition has deteriorated to the point where artificial ventilation is required, the compositions may be administered through a respirator or by lavage.

[0146] The unwanted movement ofthe bodily substance can alsó take piacé during a surgical procedure, and that procedure can involve an incision within the subject’s nervous system, eye, ear, nőse, mouth, pharynx, respiratory system, cardiovascular system, digestive system, urinary system, reproductive system, musculoskeletal system, liver, or integument. The methods can be carried out regardless of whether or nőt the movement ofthe bodily substance was intentional. The compositions described herein can be applied before or after the unwanted movement occurs (e.g., during a surgical procedure before the intentional transection of a blood véssél or after an unintentional transection of a blood véssél). Fór example, the surgical procedure can be carried out with the intent to repair an aneurysm, impede bleeding within the brain, to treat esophageal varices, to treat an ulcer orto inhibit the loss of gastric contents or intestinal contents (e.g., from a swollen or ruptured appendix). The surgical procedure can involve resecting a portion of the subject’s intestine. Other procedures that can be carried out with the assistanceof compositions including self-assembling peptides include arteriography, cardiac catheterization, insertion of a stent, assistance with a natural birth or birth by Caesarean section, hysterectomy, organ transplant, joint replacement, or excision of an intervertebral disk. These procedures are representative. The surgical procedure can be performed with the assistance of an endoscope or laparoscope, and the compositions can be delivered independently or from a ehamber situated within these devices and connected to a distal end by a passage fór release onto the subject’s tissues. Where the patient has an ulcer, that ulcer can be an esophageal, gastric, duódénál, diabetic, or decubitus ulcer. More generally, the compositions can be applied to any disrupted area of the skin, and any of the methods described herein can include a step of identifying a patient in need of treatment.

[0147] A self-assembling peptide nanofiber scaffold (SAPNS) can provide a transparent environment fór the surgical field, while alsó creating an opticaíly clear liquid that allows operation through the resultant liquid and gél mix. The surgical field is often obseured with blood and debris during an operation. In addition, clearing debris from the surgical field usually requires irrigating the site with saline. Saline is only a temporary solution and needs to be continuously applied to maintain a clear surgical field. This poses several issues: any contamination in existence will easily spread; a small opening will require alternating between irrigation and operating; and during intestinal operations use of saline can result in a massive infection leading to post-operative complications. Using the SAPNS fór biological confinement will reduce post operatíve complications in endoscopic and open surgical procedures. Efficacy has been demonstrated on brain, spinal cord, gastrointestinal tract, liver, muscle, arteries and veins.

[0148] Fór example, a partial resection is currently performed as follows. The surgeon performs a partial resection of the intestine to remove a precancerous area. The incision is made and the intestines are gently lifted out ofthe intraperitoneal cavity and placed on the table next to the patient. The offending area is resected and the two ends of the intestine are then ligated together. Before the intestines are pút back in the body there is a colostomy bag connected to the upper end ofthe intestine and the area ofthe operation is disinfected. The intestines are replaced in the abdomen and are sewn back up. Adrain is placed in the abdomen to make sure there is no leakage or bleeding. In contrast, using the self-assembling peptides, a partial resection is performed as follows. The doctor opens the abdomen and finds the offending part ofthe intestine. It is isolated with additional liquid that is poured intő the intraperitoneal cavity to isolate it from the rest of intraperineal cavity. The surgeon reaches through the gél that was formed by the liquid and resects the intestine. The two ends are ligated together and the area is checked fór any changes in color. The gél alsó has an indicator die that changes to blue if there is any leakage of gastric fluids or bacteria. All of the blue is removed with suction. A little more self-assembling peptides are sprayed around the area ofthe repair and the abdomen is sewn up. [0149] Scar treatment: Experiments have demonstrated that application ofthe self-assembling matéria! can be used to block formation of scarring in the Central nervous system (CNS). Administration of the self-assembling peptides at the site of the lesion blocks the stable formation of a scar, which can permit regeneration through that site; removing the scar that develops in the Central nervous system (CNS) permits axons to grow across the injury site.

[0150] Chelation enhanced wound healing: The self-assembling peptides can be used fór the delivery of a chelator such as írón to a site so it can be used by the body in the local environment to rebuild basement membráné. In tissues that do nőt contain enough írón, the delivery of írón in a stable form will help healing and the rebuilding of tissue. Metals with a cystine or cystine like residue can be incorporated in the nanomaterial so there is little or no steric hinderance with the assembly of the mátrix in-vivo or in-vitro.

[0151] In summary, the self-assembling peptides can be used to create a clean local environment to perform surgery; isolate structures and migration of contaminates; inflate structures fór surgical procedures, i.e. intestine; surround structures that are being removed that may leak, i.e. appendix, patch holes in body; allow fór surgery in dirty environments; used with scope procedures to surround the organ before the operation to contain any leakage; used to create a barrier

EP 2 581 097 Β1 to prevent adhesions while performing abdominal surgery; and used fór form a gasket between the scope and the insertion point ofthe scope. Benefits during surgery are that the self-assembling peptides are optically clear, has a long shelf life at room temperature, can be operated through it, shortens prep time, eliminates counting sponges, isolates each structure in the surgical field, shortens ciean up time of the operating room, shortens surgical time, reduces or eliminates cross contamination caused by other irrigants, the matéria! is biocompatible, the breakdown products are natural and are absorbed by the body. The self-assembling peptides are easy to manipulate, can be injected at the location needed, shouid eliminate Staphylococcal infections, may be abie to reduce the cost of surgical theater disposables paper, reduce biohazard bags since the matéria! can be boiled to sterilize after the procedure to yield steam. Since the self-assembling peptides are clear it shouid enable the surgeon to operate faster because the operating field is clear of blood. The elimination of wound packing to control bleeding could reduce the operating time as much as 50% in a complicated case. Post-op infection, due to secondary infection, may be reduced by the use ofthe self-assembling peptides since it can coat the wound during and after surgery, thus reducing contamination from foreign bodies. Postop care may be abie to use the self-assembling peptides to reduce infection due to drainage by slowing the spread of particulate matéria! in the abdomen or chest cavity.

[0152] While the compositions can be removed from a site of application (e.g., a bleeding véssél) at any time, a physician may wish to allow them to remain in piacé even afterthe initial goal of promoting hemostasis has been achieved in orderto promote wound healing.

[0153] The compositions include self-assembling peptides, and those peptides can include amino acid residues that are naturally occurring and that can be absorbed by the body. The compositions are nőt difficult to manipulate, and they can be easily dispensed on an as-needed basis. Their features (e.g., stiffness) can be altered readily by altering the concentrations of components therein (e.g., by altering the concentration of self-assembling peptides in a given composition). As the resulting, assembled structure does nőt significantly impair one’s view of an underlying tissue, and does nőt have to be removed before a procedure can be earried out. Fór example, a physician can assess a burn or other surface trauma that has been treated in the field with a composition described herein. In the operating room, a surgeon can make an initial incision through the matéria! and can continue to operate with standard equipment, such as scalpels and clamps, or more modern means, such as lasers, in an internál field to which the compositions may alsó have been applied. Another advantage may be realized in time, as use ofthe compositions can decrease the time required to prepare a patient fór surgery. As the compositions can be applied around the site of an incision and form a coating to protect against infectious agents, there is less need to shave a patient’s skin, apply drapes, and apply disinfectants. [0154] Given the structural integrity ofthe assembled scaffolds, they can be removed from an area in which they have formed if desired. Thus, an assembled scaffold can be removed by, fór example, suction, or by lifting it away with an instrument such as forceps, or wiping it away with a swab or gauze. Fór example, the scaffold can be removed after hemostasis is achieved or in the course of cleaning a wound. Based on studies to date, the scaffold or a majority thereof can be removed without damaging the underlying tissue. Where the assembled scaffolds are formed ex vivő, they can be removed from a mold and used subsequently (e.g., implanted in a tissue or tissue void). The compositions shouid reduce the amount of matéria! that requires disposal or cleaning afterward (e.g., surgical drapes, sponges, and other biohazards).

[0155] Nanodrapes can be used to replace traditional paper orcloth drapes, by limiting infection following application directly to the patient, fór example, by spraying or otherwise coating the patient or the area around the surgical incision. Currently a patient is prepared fór surgery by shaving, scrubbing, disinfecting and draping after positioning on the surgical table. Then bactericide and tape is applied to the area where the surgery is to be performed. The self-assembling composition can be applied in piacé of drapes by spraying the warm liquid onto the body where it self-assembles intő a thin second skin. This matéria! has a poré size that is smaller than any bacteria can fit through, so it protects from any airborne contaminants, and because the one milliméter thick matéria! can contain a mild anti-bactericide, that clings to the body Iike a second skin. The self-assembling peptides can alsó have a hydrating component fór the skin so it does nőt dry out. There is no worry about getting the self-assembling peptides intő the wound site because they will be broken down by the body. Color can be added so it is easier to determine if the self-assembling peptides have all been washed off after the operation.

[0156] These self-assembling peptides can alsó be used to prevent the introduction of foreign bodies within the humán body and/or on the surface of the humán body. The self-assembling peptides can prevent the introduction of bacteria, fungi, viruses, spores and/or other infectious agents by creating a barrier that prevents the passage of these materials. [0157] A scaffold (e.g., a nanoscale structured matéria!) can be provided by introducing, to a subject, a precursor of the scaffold at a location, or in the vicinity of a location, where the scaffold is desired (e.g., to control movement or leakageofa bodily substance, to protect a wound, orto promote tissue repair). Precursors (i.e., self-assembling peptides) are provided in the vicinity of a location when they are provided at a position that is close enough to the targeted area (e.g., a bleeding véssél, a diseased section ofthe digestive tract, or an area of burned skin) that they reach the targeted area in an effective amount. The precursors, which may be homogenous or heterogeneous (e.g., one may apply a single type of self-assembling peptide óra mixture oftwo or more different such peptides), can be contained within a composition

EP 2 581 097 Β1 and, upon contact with physiological conditions, assemble to form the scaffold (e.g., a nanoscale structured matériái). Thus, the precursors can assemble in situ (i.e., within the body of a subject at or in the vicinity of administration). [0158] The nanoscale structured matéria! may include, or its assembly may involve, additiönai components present in situ, (e.g., ions). Thus, precursors such as self-assembling peptides can be applied in a solution that is substantially free of ions (e.g., substantially free of monovalent cations) and self-assemble to form a macroscopic structure when they come in contact with such ions in the body (e.g., in a bodily substance such as blood, gastrointestinal contents, and the like). Fór example, a solution containing precursors can be applied at, or in the vicinity of, a site of gastric or intestinal perforation or a site where a surgical incision has been or will be made.

[0159] The scaffold can alsó be provided in the form of a gél, as the precursors (i.e., self-assembling peptides) can be assembled prior to introducing a composition to a targeted area (e.g., the site at which an incision will be made fór a surgical procedure). The assembled structure may assume any convenient shape.

[0160] The scaffold can alsó be provided by providing precursors in the form of a dry powder. A dry powder will have a reiatively low liquid content (e.g., sufficiently low that the particles therein are readily dispersible). Self-assembling peptides provided in the form of a dry powder will assemble when they come intő contact with a bodily fluid containing monovalent cations, and a solution containing such ions may be added if desired to altér the rate at which the scaffold forms or its stiffness. Self-assembling peptides may be provided as emulsions or, as described above, molded intő preformed shapes that can be inserted intő a body cavity or wound site in a manner similar to the manner that surgical sponges are currently used. If desired, a binder can be added to a dry powder which is then formed intő a desired shape. Regardless of the precise manner in which the scaffold is assembled (e.g., whether by bringing a liquid formulation containing precursors intő contact with the body or a dry powder intő contact with an ion-containing solution ex vivő), the formed scaffolds can assume a desired shape. Where the size and shape is such that the scaffold fii Is the lumen of a blood véssél, the scaffold can be used a vascular plug.

[0161] A preventative measure can be carried out before a subject experiences an unwanted event (e.g., before an injury occurs or before bleeding begins). Thus, the site of administration can be a site of potential movement or potential leakage, and the application can be made to prevent or minimize such movement or leakage should it occur. When used in the context of a therapeutic procedure or treatment, the compositions can reverse, alleviate, or inhibit the progress of a condition (e.g., a state, syndrome, disease, óra sign, symptom, or manifestation ofsuch). Methods oftreating a subject are generally carried out once the subject is recognized as having a condition amenable to treatment, and any ofthe methods described herein, whether best described as prophylactic or therapeutic, can include a step of identifying an amenable subject.

[0162] As the compositions described here can be used to inhibit movement of a bodily substance in a subject, including movement within or from the epidermis, the compositions can be employed in the context of performing surgery and may be described as new methodsfor performing surgery or generating a surgical field. The methods, whether performed in the context of surgery or nőt, can include a step of identifying a subject in need of treatment and a step of providing a nanoscale structured matériái, or a precursor thereof, at or in the vicinity of a site where unwanted movement has occurred or is expected to occur. Fór example, one can identify a patient who is about to undergo a surgical procedure and provide a biocompatible composition comprising self-assembling peptides and a vasoconstrictor, a coloring agent, or a local anesthetic agent to a site at which an incision or other invasive maneuver will be made or has been made. The bodily substance that is affected may be a fluid such as blood or a blood product, serous exudate (an inflammationassociated exudate composed largely of plasma, which typically appears as a clear or amber-colored fluid), pus, gastric juice, urine, bile, cerebrospinal fluid (CSF), pancreatic juice, and the like. The bodily substance may be viscous, sludgelike or semi-solid bút will generally exhibit an ability to flow or move. Substances of this natúré include the contents of the gastrointestinal tract. The composition may be removed after application (e.g., after hemostasis is achieved or an operation on the bowel is complete) or may be left in piacé. Fór example, the compositions can be applied to accelerate hemostasis or inhibit movement of intestinal contents during surgery and somé or all of the scaffold may be left in piacé when the operation is complete. This provides a substantial advantage relatíve to the use of sponges and other materials that must be removed prior to closure. The compositions can be removed in a variety of ways (e.g., by wiping orbysuction). [0163] The compositions can alsó be applied to shield an underlying area (e.g., an area of burned or otherwise injured skin orother tissue) and can, therefore, help to prevent contaminants (e.g., foreign substances) from coming intő contact with the area (i.e., the compositions can be used as a barrier or shield). A physician or other health-care provider can examine a wound through the self-assembling peptides, and a surgeon can operate through it, while it is in piacé. Contaminating substances that have landed on the self-assembling peptides during the procedure could then be removed by virtue of removing the peptides.

[0164] The compositions can be administered to stabilize a wound prior to definitive treatment (e.g., while the victim is awaiting transport to a hospital or during transít). The compositions are similarly useful where operations are conducted under conditions of less than optimál sterility (e.g., in field hospitals or in areas of the world where access to sterilé operating rooms is limited). The compositions and methods have the potential to significantly reduce the likelihood of contamination in instances such as these.

EP 2 581 097 Β1 [0165] The self-assembling peptides can alsó be locally applied in combination with anesthetic in the local area where a procedure is to take piacé and can be applied at a higher concentration to reduce organ movement during surgery. This may reduce cognitive deficits to older patients by reducing the generál anesthetic load. A thin layer can be sprayed on the tissue or skin where the surgeon is operating. It can be applied separately or together, administering specific anesthetic for specific organs. Skin has different receptors than intestines and the need for a specific anesthetic is needed for each ofthe organs. Intestines need tostop moving during surgery while the blood and blood véssél contraction need to remain constant.

[0166] Treatment and prevention ofbleeding'. Any individual who has an increased risk of suffering undesirable bleeding, which may or may nőt be excessive or immediately life-threatening, can be treated with the compositions described herein. These individuals include those with blood clotting disorders such as hemophilia, patients who are receíving anticoagulant therapy, patients who suffer recurrent nosebleeds, and individuals undergoing surgery, particularly major surgery or procedures that involve accessing an artery. Without limitation, the surgery or procedure can be an operation on the nervous system, eye, ear, nőse, mouth, pharynx, respiratory system, cardiovascular system, digestive system, urinary system, musculoskeletal system, integumentary (skin) system, or reproductive system. Specific examples of surgeries and procedures in which the compositions can be used include arteriography, angiocardiography, cardiac catheterization, repairofobstetric laceration, removalofcoronary arteryobstruction, insertionofstent, Caesareansection, hysterectomy, reduction of fracture, coronary artery bypass graft, cholecystectomy, organ transplant, totál joint (e.g., knee, hip, ankle, shoulder) replacement, appendectomy, excision or destruction of intervertebral disk, partial excision ofthe large intestine, mastectomy, or prostatectomy.

[0167] Accident victims, individuals engaged in combat, and women giving birth are alsó at risk of experiencing significant blood loss. The compositions can be applied to a site of obstetric bleeding (e.g., within the uterus, vagina, or neighboring tissue) in order to accelerate hemostasis. For example, the compositions can be applied to a placental tear or used to pack the uterus to control bleeding. As with other indications, compositions applied to the reproductive tract can be removed or left in piacé. Spontaneous hemorrhage, aneurysm rupture, esophageal varices, gastric ulcers, ulcers ofthe upper portion ofthe intestine (e.g., duódénál ulcers) are alsó medical conditions in which considerable bleeding can occur, and these individuals can alsó be treated as described here.

[0168] The precise source ofthe bleeding can vary and can be from any blood véssél in the artéria! or venous system (e.g., an artery, arteriole, capillary or capillary bed, venule, or vein). The size of the véssél may rangé from large (e.g., the compositions can inhibit bleeding from the aorta, the iliacorfemoral artery, or a portai vein) to small (e.g., a capillary), and the véssél may be located anywhere in the body (e.g., in a solid organ such as liver, the stomach, intestine, skin, muscle, boné, the lungs, orthe reproductive system).

[0169] The time normally required for blood clotting can be prolonged when plasma levels of clotting factors and/or platelets are low or in cases in which an individual has received an anticoagulant (e.g., warfarin or heparin). Bleeding frequently persists for considerably longer than the average clotting time when there is more than minimál damage to blood véssél íntegrity. Based on the studies, it is expected that the compositions will cause hemostasis in a period of time that is less than, and in at least somé cases much less than, the average blood clotting time. Although the compositions are nőt limited to those that achieve hemostasis in any given time (and uses such as protecting an area from eontamination or promoting tissue healing are independent of this function), the compositions may confer a benefit to a bleeding subject in as little as five seconds following application. Other compositions can exert an effect in about 10, 15, or20 seconds following application. The effective period can be characterized in a manner other than absolute time. For example, compositions may reduce the time required to achieve hemostasis by between 25% and 50%; between 50% and 75%; or between 75% and 100% relatíve to the time required when iced saline is applied. The time required to achieve hemostasis can be reduced by approximately 2-, 3-, 4-, or 5-fold relatíve to the time required when iced saline is applied.

[0170] The self-assembling peptides’ concentration may be selected with reference to variables such as the caliber ofthe véssél, the extent to which it has been injured, and the force with which blood is exiting (or would exit upon injury). Higher peptide concentrations will be desirable to promote hemostasis from a major véssél (e.g., the aorta, brachiocephalic, carotid, subclavian, celiac, superior mesenteric, renal, iliac, femoral, or popliteal arteries). Useful concentrations can rangé from between approximately 0.1-10% (e.g., 1-10%; 0.5-5%; 1-4%; 0.1-2%; 0.1-3%; 0.1-4%; 0.1-5%; and 1-8% (e.g., about 1,1.5, 2, 2.5, 3, 4, 5, 6, or 7%). Any subrange, or any specific value within any ofthe aforesaid ranges, can be used. Any ofthe aforementioned concentrations may alsó be used for the other indications described herein. [0171] As noted, bleeding can be due to any of a large number of different causes and can be internál or external. The compositions can be applied regardless ofthe cause or the natúré ofthe cause (e.g. whether caused by a disease process or intentional or accidental trauma). The compositions can be used to achieve hemostasis in a confined space (e.g., inside a hollow organ) or at or near the body’s surface. For example, the compositions can be applied to a partly or completely severed body part such as a limb ordigit. In that event, the compositions may be serving multiple functions; they may nőt only promote hemostasis, bút alsó protect the wounded tissue from contaminants and promote tissue healing. More specifically, the compositions can be applied to a wound, left in piacé for a period of time sufficient to

EP 2 581 097 Β1 achieve hemostasis and for blood clotting to occur, and then removed. Contaminating matéria! such as particulates and infectious agents adhered to the peptide gél would be removed with it. A sterilé dressing may then be applied. Of course the compositions can be applied for purposes of cleaning a wound, preventing contamination, or promoting tissue healing even after hemostasis has been achieved or in situations in which accélération of hemostasis is nőt needed.

[0172] When used to treat a nosebleed, the compositions are inserted intő the appropriate nostril and can be left in piacé until the bleeding has subsided. The compositions can be easily removed by suction (e.g., using an eyedropper or syringe) or may be removed by other physical means, including simply blowing the nőse.

[0173] The compositions can alsó be left in piacé on a wound, and a dressing can be applied over the composition. Since the composition itself is easily removed, its presence under the dressing can help prevent the dressing from sticking to the damaged tissue. If desired, a bandage having a transparent portion may be used so the injured site can be viewed through the transparent portion ofthe bandage and the peptide structure below. This would allow a physician to monitor the progress ofthe healing without removing the dressing. Modified bandages are described further below and are within the scope ofthe present invention.

[0174] Many medical procedures involve vascular puncture, which can be followed by significant bleeding. A selfassembling peptide composition can be applied to the wall ofa punctured véssél, e.g., during withdrawal of an instrument used to puncture the véssél. A vascular plug formed from self-assembling peptides provides an alternative to existing vascular plugs and devices such as those described in U.S. Patent Nos. 5,192,302; 5,222,974; 5,645,565; and 6,663,655. The vascular plug can be formed in situ (e.g., at a site of vascular puncture), or can be preformed and applied to the site. [0175] More generally, compositions comprising nanostructured materials or precursors thereof (i.e., self-assembling peptides) can be used forsealing any passage through tissue. The present methods therefore include methods ofsealing a passage through tissue by applying a composition comprising a nanoscale structured matéria! (e.g., self-assembling amphiphilic peptides) to one or both ends of the passage or to its interior. The tissue can be, for example, the wall of a blood véssél, the wall of an organ, subcutaneous tissue, or adipose tissue. Sealing the passage can result in hemostasis. The passage can alsó be a fistula (i.e., an abnormal connection between two organs or body structures or between an organ or structure and the external world). If desired, a surgeon can apply the compositions to the interior of a tubular structure such as the intestine or a blood véssél, resect and ligate the intestine or blood vesse! in the gél, and evacuate the gél from the interior ofthe structure to restore continuity ofthe structure and allow reperfusion ofthe area with blood or other body substances. The self-assembling peptides may alsó be used to limit reperfusion injury. For example, the self-assembling peptides can be administered post ischemia, such as to patients who have been treated with a thrombolytic agent. The self-assembling peptides may alsó be used to limit reperfusion injury through the reestablishment of blood tissue barriers prior to, during, and/or after reperfusion. For example, the self-assembling peptides may be used to re-establish blood tissue barrierthrough the internál coating of portions ofthe circulatory system. This may be beneficial in diseases such as ischemic infarction, hemorrhagic stroke, or reperfusion injury. Finally, the self-assembling peptides may be used to limit reperfusion injury through the reestablishment of the integrity of the vascular structure prior to, during, and/or after reperfusion.

[0176] Forsurgical applications, the wound or any part ofthe surgical field can be packed with a composition comprising self-assembling peptides. This approach can be used instead of wound packing as it is conventionally performed during surgery. As the compositions contain biocompatible and biodegradable matéria!, they can be left in piacé, thereby avoiding the need for removal at the end ofthe procedure and avoiding the need for a subsequent operation for this purpose. Biodegradable materials can be broken down physically and/or chemically within cells or within the body of a subject (e.g., by hydroiysis under physiological conditions or by natural biological processes such as the action of enzymes present within cells or within the body) to form smaller Chemical species which can be metabolized and, optionally, reused, and/or excreted or otherwise disposed of. Preferably, the biodegradable compounds are biocompatible.

[0177] Gastrointestinal bleeding, which can occur as a consequence of ulcers orangiodysplasia, is a relatively common and serious condition that can be fatal if left untreated. Bleeding esophageal varices, and bleeding gastric or duódénál ulcers can be particularly severe. A number of endoscopic therapeutic approaches have been developed to achieve hemostasis, such as the injection of sclerosing agents, the attachment of mechanical hemostatic devices, and contact electrocautery techniques. The compositions can be administered at, or in the vicinity of, an ulcer or a site of bleeding in the esophagus, stomach, small intestine, or large intestine. Bleeding in the distal portion ofthe large intestine, rectum, or anus (e.g., hemorrhoids) can alsó be treated in this manner.

[0178] Rupture of an aneurysm can represent a catastrophic event with rapidly fatal consequences. Ruptured aortic aneurysms can rapidly result in exsanguination despite prompt medical attention. Ruptured intracranial aneurysms frequently havedevastating consequences. The compositions and methods ofthe invention can be used to treat bleeding from a ruptured aneurysm in an essentially similar manner to the way in which they are used to treat bleeding due to other causes (e.g., by application of self-assembling precursors or a preformed structure to the site of bleeding). Given the often severe consequences of aneurysm rupture, surgical repair is often attempted. The compositions can be applied in the context of any attempted repair (e.g., during open surgery or endovascular repair (e.g., with piacement of a graft

EP 2 581 097 Β1 and/or stent)). More specifically, the present methods include treating an aneurysm by introducing a composition comprising a nanoscale structured matéria! or precursor thereof (e.g., a composition comprising self-assembling peptides) into the aneurysm (e.g., into the aneurysm sac). Once any bleeding is under better control, the aneurysm may then be repaired using any suitable technique. Presence ofthe peptide structure within the aneurysm sac reduces the chance of leakage or rupture prior to or during these other procedures. The scaffold can be left in piacé. Further, the presence ofthe matéria! in the aneurysm sac may promote healing ofthe aneurysm.

[0179] Inhibiting movement or leakage of cerebrospinal fluid (CSF)'. The dura mater is the tough, outermost, fibrous membráné that covers the brain and spinal cord, and lines the inner surface ofthe skull. Leakage of CSF is a significant complication following injury, surgery, or other procedures in which the dura mater is penetrated, including inadvertent penetration in the course of administering an anestheticto the epidural space. Such leakage can lead to serious sequelae, such as severe headaches, infection, and meningitis. The composition can inhibit movement or leakage of CSF in a subject in need thereof after application at, or in the vicinity of, a site of unwanted movement or leakage of CSF. The compositions can be applied over sutures following dura mater surgery to help prevent CSF from leaking out of the incision site. The compositions can alsó be used to inhibit movement or leakage offluid from the ear drum.

[0180] Inhibiting leakage of contents ofthe gastrointestinal tract'. The compositions can inhibit the movement of gastrointestinal contents. For example, the structures can prevent leakage of gastrointestinal contents following gastric or intestinal perforation or during surgery (see Example 4). The structures can be used to isolate such bodily substances and prevent their spread within the peritoneal cavity, thereby minimizing contamination and the risk of subsequent Chemical peritonitis and/or infection. Gastric contents, which contain digestivesecretions ofthe stomach glands consisting chiefly of hydrochloric acid, mucin, and enzymes such as pepsin and lipase, can cause injury and/or infection if released into the peritoneal cavity. Release of intestinal contents into the peritoneal cavity represents a frequent event during surgery on the intestine and can alsó occur in cases of intestinal perforation or a ruptured appendix. The composition can be used to inhibit leakage of gastrointestinal contents into the peritoneal cavity. The site of movement can be a site of gastric or intestinal damage caused by a disease process or a surgical incision. The compositions can be applied to the exteriőr of any organ in the digestive system (e.g., the stomach, or small or large intestine) or can be injected or otherwise introduced into their interior. The compositions can be administered in the course of resecting a segment of the intestine. For example, one can fill a segment of intestine that extends from a first point to a second point with a present composition and resect a portion ofthe intestine that lies between the first and second points. In one embodiment, the self-assembling peptides may be used to treat heartburn. For example, the self-assembling peptides can be formulated as a solution, suspension, or emuision (such as a drink orshake), gél, tablet, wafer, capsule, etc. that is administered orally in orderto coat portions ofthe gastrointestinal tract. The formulations can be used to: stop the movement of bodily fluids including, gastricjuices and blood; coat the GI tract, and/or stop the progression of ulcers, erosion, and inflammation. The formulations may be used to prevent damage to the esophagus from acid reflux disease. The formulations may alsó be used to help the repair of cells in the esophagus that were damaged by acid reflux, other diseases or disorders, and/or therapeutic interventions. The formulations may be used to help the repair of primary and secondary ulcers and erosions to the mucosa. The formulations may be used to deliver therapeutic, prophylactic, and/or diagnostic agents to portions of the GI tract as needed. For example, the self-assembling peptides may be used to deliver agents to reestablish the flóra and fauna of the GI tract which have been deleted to radiation treatment and/or disease or trauma. [0181] In a related method, one can use the compositions to remove intestinal contents that have been released into the peritoneal cavity. The method includes applying a liquid composition to the released intestinal contents, allowing the liquid composition to undergo a phase transition, and then removing the gel-like or semi-solid composition. These steps can be repeated onceor more until the surgeon is satisfied with the amount of intestinal contents that have been removed from the peritoneal cavity. In another related method, the compositions can be applied to ulcers to act as a barrier to prevent acid from contacting the surface ofthe stomach.

[0182] One can similarly inhibit movement ofthe contents of other internál organs (e.g., organs in the biliary or urinary systems). For example, one can inhibit movement of bile, pancreatic juice (i.e., secretions ofthe exocrine portion ofthe pancreas that contain digestive enzymes), or urine and/or decontaminate or clean an area into which bile, pancreatic juice, or urine have been released by application and subsequent removal ofthe compositions to the site. The methods thus have broad application tosurgeriesforrepairing or otherwise treating intestinal, biliary, and/or urinary system defects. [0183] Wound healing'. Studies alsó indicate that the compositions have the ability to enhance healing, particularly of an epithelial layer or muscle, and can therefore be administered to treat a site of tissue damage. For example, one can appiy a composition including self-assembling peptides to the site of tissue damage. The compositions appear to both increase the rate of tissue repair and inhibit formation of scar tissue. The compositions can be used for either acute or chronic wound care. For example, they can be applied to skin wounded in any manner (e.g., lacerated or burned) and to lesions such as diabetic ulcers and pressure sores. In the case of burns, a self-assembling composition, optionally containing a debridement agent could be administered to the burn site. Debridement could occur in or through the selfassembling peptides thus reducing the amount of abrasion to the site and minimizing or eliminating contamination of the surrounding tissue or environment. The assembly of the self-assembling peptides alsó forms a barrier which can

EP 2 581 097 Β1 prevent infection and/or contamination ofthe burn.

[0184] The self-assembling peptides may alsó be useful in inhibiting or preventing the formation of scar tissue by chelating iron and other metál ions that act as cofactors fór the formation of scar tissue. As the wound heals, the pH of the wound site drops and the concentration of iron increases. Scarring and scar tissue can block the growth of axons. Self-assembling peptides can block the formation of scar tissue by chelating iron at the wound site, likely through the presence of electronegative and/or negatively charged functional groups which can complex positively charged metál ions. The removal or complexation of iron in a wound prevents the stable formation of collagen IV. The ability to control the wound environment allows one to control the rate and extent of healing.

[0185] This ability of self-assembling peptides to chelate metál ions may alsó be useful in preventing bacterial or fungal infections by chelating metals, which are cofactors fór bacteria and fungi. Infections can alsó be prevented by coating a tissue with a layer of self-assembling peptides, thus preventing the bacteria or fungus from latching on to the tissue. [0186] These self-assembling peptides can be used to maintain hydration and nutrition to patients that have had burns or in cases the outer skin has been breached due to abrasion or burn.

[0187] In another case the self-assembling peptides can be used to maintain body temperature when the patient is covered with the peptides by means of external heat or cooling source.

Tissue Regeneration [0188] Drug delivery vehicle to the intrathecal space: The self-assembling peptides described herein may be used to deliver therapeutic and/or imaging agents to the intrathecal space. Examples of therapeutic agents include, bút are nőt limited to, anti-inflammatory agents and agents to stimulate nerve/spinal cord regeneration. Hydrogel matéria! have been used to attempt to delivery ofone or more active agents to the intrathecal space. However, these materials can be limited by their slow polymerization times, which allows fór the matéria! to diffuse away before the matéria! polymerizes to form the gél. The self-assembling peptides described herein can be designed to self-assembly quickly so that the peptides do nőt diffuse away.

[0189] Cartilage repair: The self-assembling peptides described herein may be used fór cartilage repair. The selfassembling peptides would typically be injected intő the site where cartilage repair is needed. The self-assembling peptides can be used alone or in combination with cells and/or growth factors.

[0190] Boné regeneration: The self-assembling peptides described herein may be used to prepare composite materials fór boné regeneration. Fór example, the self-assembling peptides can act as a carrier fór inorganic materials, such as calcium phosphate or hydroxyapatite, organíc materials, such as growth factors, and/or boné grafts. Inorganic materials such as calcium phosphate can be remodeled by the osteoclast resorption mechanism to regenerate boné. The selfassembling peptides may alsó be injected under the periosteum to stimulate boné growth as a means fór creating boné grafts in vivő. Alternatively, the self-assembling peptides described may be used forguided boné regeneration therapies which limit fibrous in growth. Fór example, the self-assembling peptides described herein may be used in dental procedures as molds which are placed over the tooth Socket to prevent fibrous tissues from growing intő the Socket space. [0191] Oxygen Delivery: The self-assembling peptides described herein may alsó be used to deliver oxygen to the lungs and/or other organs. Fór example, the self-assembling peptides can be superoxygenated to provide oxygen support/perfusion fór patients suffering from pulmonary hemorrhage and other lung diseases.

[0192] Delivery Methods, Devices, and Kits\ A variety of devices can be used to introduce the compositions to a target area of the body. The devices can be simpíe, such as a syringe, and such devices can be provided together with the compositions in kits. The composition can be locally delivered at or near a target area in the body by injection (e.g., using a needle and syringe), or with a catheter, cannula, or by dispensing (e.g., pouring) from any suitably-sized véssél. The compositions can be delivered with the assistance of imaging guidance (e.g., stereotactic guidance) if necessary. Alternately, a matéria! can be wetted with the composition and then used to apply a composition to an area of tissue. [0193] Fór storage and shipping, self-assembling peptides can be dissolved in a suitable solvent (e.g., an aqueous médium such as sterilé water, and stored fór long periods of time prior to use). Peptide-containing Solutions have been stored fór up to two years without substantial loss of activity. If partial self-assembly occurs after a prolonged period of time, physical agitation (e.g., sonication) can be used to restore the self-assembling peptides to a more liquid state prior to administration. Alternatively, the self-assembling peptides can be applied as a gél. If desired, a small amount of ions (e.g., monovalent cations) can be added to a solution prior to application. This may speed the process of gél formation. Alternately, monovalent cations can be applied after the solution has been administered.

[0194] Kits containing syringes of various capacities or vessels with deformable sides (e.g., plastic vessels or plasticsided vessels) that can be squeezed to force a liquid composition out ofan orifice are provided. In one embodiment, the syringe or véssél contains multiple compartments, one containing monovalent ions, and the other self-assembling peptides, which are mixed at the time of administration, through a common needle. An endoscope can be used to deliver the compositions fór treatment of a hollow organ (e.g., the esophagus, stomach, intestine, etc.) or body cavity (e.g., during minimally invasive surgery). Minimally invasive surgery refers to an approach to surgery whereby operations are

EP 2 581 097 Β1 performed with specialized Instruments designed to be inserted through small incisions or natural body openings, often performed with endoscopic visualization. Examples include laparoscopic surgery, arthroscopic surgery, and endovascular surgery. An endoscope is typically a long, flexible tube-like device. In addition to allowing visualization of internál structures, many endoscopes have additiönai diagnostic (e.g. biopsy) and therapeutic capabilities (e.g. delivery of therapeutic agents) through special channels. Colonoscopes, sigmoidoscopes, bronchoscopes, cystoscopes, and laparoscopes, are variants of an endoscope having features making them particularly well suited for viewing certain organs, structures, or cavities. Any of these devices can be used to deliver the compositions. Kits may be packaged including an endoscope and a véssél containing a solution comprising self-assembling peptides. Suitable endoscopes are known in the art and are wideiy available. Endoscopes are currently in use to deliver sclerosing agents to sites of esophageal bleeding.

[0195] Kits can include self-assembling peptides and one or more of: a syringe, a needle, thread, gauze, a bandage, a disinfectant, an antibiotic, a local anesthetic, an analgesic agent, surgical thread, scissors, a scalpel, a sterilé fluid, and a sterilé véssél. The peptides can be in solution or dry (e.g., as a dry powder). Components of the kit may be packaged individually and are sterilé. The kits are generally provided in a Container, e.g., a plastic, cardboard, or metál Container suitable for commercial sale. The kit may be styled as a first aid kit, in which case it will typically have a Symbol such as a red cross on the exteriőr. Any ofthe kits can include instructions for use.

Examples

Example 1: Self-Assembling Peptide Matéria! Accelerates Hemostasis in the Brain [0196] Complete transection of a branch ofthe superior sagittal sinus in the brains of rats and hamsters was performed after removing a portion of the skull overlying the transected tissue. Animals were anesthetized with an i.p. injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). All surgical procedures were conducted under an operating microscope. Twenty-two animals, including 10 aduit hamsters and 12 young aduit female Spraque-Dawley rats (200-250g), were treated with either iced saline or 20 μΙ of a 1 % peptide solution at the site ofthe sinus branch transection. The matéria! was prepared by dissolving RADA16-1 (n-RADARADARADARADA-c; SEQ ID NO: 1) peptide in sterilé water, and the peptide-containing solution was applied to the injured tissue with a 31 gauge needle attached to a 2 cc syringe.

[0197] The experiment was videotaped with a time stamp and was replayed one frame at a time to evaluate the length oftime required for the peptide solution to form a gél, which effectively impeded bleeding. Hemostasis was assessed visually, and complete hemostasis was defined as the complete lack of movement of blood from the wound site. Complete hemostasis was aehieved within 10 seconds of the application of the peptide solution in all cases.

[0198] A series of pictures was taken of an aduit rat in which a portion ofthe overlying skull was removed and one of the veins of the superior sagittal sinus was transected and then treated with a peptide-containing solution. The initial picture shows the exposed brain and veins ofthe superior sagittal sinus; the next picture shows the cutting ofthe vein; the next picture shows bleeding from the ruptured vein; and the final picture shows the same area five seconds after the peptide solution was applied. Complete hemostasis was aehieved.

[0199] Durations were measured from the start of application of peptide solution to the completion of hemostasis after transection ofthe veins leading to the sinus in the brains of aduit rats. Complete hemostasis was aehieved in an average of 8.3 seconds. In the saline Controls, cessation of bleeding was never aehieved. The saline control experiment was terminated at the same time point in order to prevent the animals from bleeding to death.

[0200] Similar resuits have been obtained following complete transection ofthe superior sagittal sinus. A higher concentration of peptide (e.g., approximately 3% - 4%) was used in the latter experiment in order to achieve hemostasis. The three saline control cases continued to bleed after 20 seconds. In the control animals, the iced saline was removed and the peptide solution was applied, resulting in complete hemostasis almost immediately.

[0201] A totál of 22 rats and 64 hamsters have been subjected to experiments in which peptide-containing Solutions effectively aehieved hemostasis within 10 seconds following application to a site intracranial bleeding.

Example 2: Self-Assembling Peptide Matéria! Accelerates Hemostasis Following Femoral Artery Transection [0202] The sciatic nerve and the adjacent femoral artery were exposed in aduit rats, and the femoral artery was transected. Twelve rats were treated by application of 20 μΙ of a 1 % solution of RADA16-I peptide (SEQ ID NO:1) to the site of transection using a glass pipette attached to a syringe body, while Controls were treated by applying cold saline to the site of transection. In all treated cases, hemostasis was aehieved in less than 10 seconds. The saline control cases continued to bleed until the experiment was terminated at 110 seconds. In these control animals, subsequent replacement of the cold saline with the peptide solution resulted in almost immediate achievement of complete hemostasis.

[0203] A series of pictures was taken in an aduit rat in which the femoral artery was transected. In the picture taken

EP 2 581 097 Β1 first, the sciatic nerve and the femoral artery are exposed. The next pieture shows the cutting ofthe artery, and the next pieture shows bleeding. After about five seconds, complete hemostasis was observed in the area of a clear gél formed by the assembled peptides in the presence of blood and plasma. The assembled matéria! can be suctioned off the site easily if desired. Complete hemostasis was maintained for the duration of the test (1 hour).

[0204] Complete hemostasis was achieved in less than 10 seconds. In the saline Controls, hemostasis was never reached.

[0205] Muscle trauma experiments showed immediate hemostasis after 1-2 cm incisions were made in the muscle on the back of a rat. The spinotrapezius muscles on the back of the rats were exposed and a deep cut was made in the muscle, after which 1% peptide solution (RADA16-I) (SEQ ID NO:1) was applied in the cut. Within 10 seconds, all bleeding had stopped. With the application of iced saline alone, control animals continued to bleed after 20 seconds. [0206] This procedure was duplicated in the muscle ofthe hind limb (porteocaudalis and musculus tibialis cranialis) and similar results were obtained. Between 1 % to 100% peptide (RADA16-1) (SEQ ID NO:1) was applied to limb wounds, and hemostasis was achieved in all cases. However when an artery orvéin was transected 2% or higher matéria! was needed to bring about hemostasis. With the application of iced saline alone, control animals continued to bleed after 20 seconds.

Example 3: Self-Assembling Peptide Matéria! Accelerates Hemostasis in Liver [0207] To further demonstrate the ability of peptide-containing structures to halt bleeding of a véssél having relatively low pressure, the intraperitoneal cavity ofan aduit rat was opened, the liver was exposed, and the lobus minister laterális received a rostral to caudal cut completely transecting a portion ofthe liver. Profuse bleeding ensued. A 1% peptide solution (RADA16-1) (SEQ ID NO:1) was applied to the cut and in its vicinity using a 27 gauge needle and 4 cc syringe. All bleeding stopped within 10 seconds. A series of pictures was obtained. The first shows exposure ofthe liver; in the second, the liver is separated, and profuse bleeding is evident; and in the third, the two portions ofthe liver are allowed to come back together, and the bleeding continues. After treating the site with 1 % peptide solution (applied topically and in the cut), all bleeding stopped within 10 seconds. A clear area was observed between the two halves of the lobus minister laterális. This procedure was repeated several times with the same result.

[0208] A similar experiment demonstrated the ability of the peptide structures to halt bleeding of a véssél in the liver having a higher pressure. A series of pictures illustrate the experiment. The first depicts the opened intraperitoneal cavity and exposed liver; in the second, the lobus sinister laterális received a transverse cut completely transecting a portion of the liver and a major branch of the portai vein; and the third shows profuse bleeding from the site of injury. The cut was treated with 4% peptide solution applied topically and in the cut. All bleeding stopped within 10 seconds. The lower part of the lobus minister laterális was pulled downward to show that the peptide structure is in the cut. The site did nőt bleed even when subjected to this physical stress. Ten minutes later, there was still no bleeding. Thus, application of 4% peptide solution brings about complete hemostasis in a high pressure bleeding environment in less than 10 seconds. [0209] Treatment with a 2% or3% peptide solution was tested in thesame type of experiment and complete hemostasis was alsó achieved. Treatment with a 1% solution resulted in partial cessation of bleeding. In addition, 30 seconds after treatment the excess peptide structure was wiped away from the injury site and hemostasis was maintained. This procedure was repeated several times with the same result.

[0210] In other experiments % ofthe lobé in the lower right quadrant ofthe lobus sinistras laterialis was removed, and the margin was treated with a topical application of 2% peptide (RADA16-I) (SEQ ID NO:1) to the site of injury. Bleeding stopped in less than 10 seconds. One minute later the peptide was removed, and complete hemostasis was achieved at the margin of the liver.

Example 4: Self-Assembling Peptide Matéria! to Present Adhesions [0211] The liver of 18 aduit rats was exposed under deep anesthesia, the upper right lobé was punched by a 4mm punch, then the wound was treated with 3% NHS-1. Animals were allowed tosurvive2d, 7d, 14d, 6w, and 8w respectively, then the animals were anesthetized again and the punched lobé of liver was dissected and processed for H&amp;E staining. In addition a set of Controls were treated with either saline or cautery.

[0212] 4mm liver punch biopsy experiment with control and filling of punch with 3% RADA16-I (SEQ ID NO:1). All of the Controls had adhesions on both surfaces while the treated had no adhesions. In the 2week, 6 week and 8 week Controls adhesions were cut away on the upper and lower surfaces. On all the 3% RADA16-I (SEQ ID NO:1) treated cases there were no adhesions on the upper or lower surface of the liver.

Example 5: Self-Assembling Peptide Matéria!

[0213] The intestine of an aduit rat was perforated with a small cut at the level of the duodenum that resulted in the

EP 2 581 097 Β1 leakage of gastric fluid intő the intraperitoneal cavity. When the site was treated with 2% peptide (RADA16-I) (SEQ ID NO:1) solution all leakage of gastric fluids from the intestine stopped. An additional volume of 2% peptide solution was injected intő the duodenum at the level of the injury. This prevented all leakage from the intestine for one hour, the duration of the procedure. In the control cut at the level of the duodenum, the wall of the intestine inverted and gastric fluids continued to leakfrom the site of injury when left untreated. When the site was treated with peptide solution 15 minutes after the injury, the peptide treatment alsó stopped all leakage from this injury site. In addition, the treatment stopped the progression ofthe intestinal wall inversion.

Example 6: Self-Assembling Peptide Matéria! Accelerates Healing of Skin Wounds [0214] To demonstrate the ability ofthe self-assembling peptides to enhance wound healing, animals were subjected to punch biopsies of the skin and subcutaneous tissue. The regions from which the biopsies were taken were either treated by a single application of self-assembling peptide (RADA16-I) (SEQ ID NO:1) solution or were left untreated. The wounds were left unbandaged. A series of pictures of a 4 mm punch biopsy healing test in which injured animals were treated with the self-assembling peptide and compared to matching cases with no treatment illustrates the results. The wounds were photographed on day 0,day 1, day 4, and day 7. The treated wounds healed much faster as evidenced bythe contraction ofthe wound site in all three punches as early as day 1. Treatment with the peptide appeared to speed healing by as much as 5 days in somé cases. In all cases, shrinkage ofthe wound site happened faster in the treated cases.

Example 7; Compositions containing Lidocaine [0215] RADA16-I (SEQ ID NO:1) mixed with lidocaine and the mixture was applied to the skin of aduit rats before applying a pin prick. It is a 5% mix of lidocaine and RADA16-I(SEQ ID NO:1). Applied on the skin and left for the duration ofthe testing. When mixed with a self-assembling peptide, the response to pin prick was muted four times longer than the response was muted using lidocaine alone. In addition, we applied Solutions of self-assembling peptides and lidocaine to the intestines of two rats while performing intestinal surgery. The solution reduced peristalsis for the duration of the surgery with no apparent sídé effects to the animals.

The following paragraphs provide a generál description ofthe invention.

1. A method of preventing or limiting formation of adhesions comprising administering to a site in need thereof, in the absence ofor after bleeding or leakage of fluid has been substantially stopped, a self-assembling matéria! which forms a barrier to, or otherwise prevents, the formation of adhesions.

2. The method of paragraph 1, wherein the self-assembling matéria! comprises peptides having a sequence of amino acid residues conforming to one or more of Formulas l-IV:

((Xaa^neu-Xaa⁺)x(Xaa^neu -Xaa-)y)_n (I) ((Xaa^neu -Xaa^_)x(Xaa^neu-Xaa⁺)y)_n (II) ((Xaa⁺-Xaa^neu)x(Xaa^_-Xaa^neu)y)_n (Ili) ((Xaa^_-Xaa^neu)x(Xaa⁺-Xaa^neu)y)_n (IV) wherein Xaa^neu represents an amino acid residue having a neutral charge; Xaa⁺ represents an amino acid residue having a positive charge; Xaa^- represents an amino acid residue having a negative charge; x and y are integers having a value of 1,2, 3, or 4, independently; and n is an integer having a value of 1-5.

3. The method of paragraph 2, wherein the self-assembling peptides comprise a sequence of amino acid residues conforming to Formula Ili or Formula IV.

4. The method of paragraph 3, wherein Xaa represents alanine; Xaa⁺ represents arginine or lysine; and Xaa represents aspartic acid or glutamic acid.

5. The method of paragraph 2, wherein the amino acid residues include naturally occurring amino acid residues.

6. The method of paragraph 2, wherein the amino acid residues are naturally occurring amino acid residues.

EP 2 581 097 Β1

7. The method of paragraph 1, wherein the self-assembling materials are selected from the group consisting of peptidomimetics, nucleotidomimetics, di- and triblock copolymers, N-alkylacrylamides, and dendimers.

8. The method of paragraph 7, wherein the self-assembling matériái is a peptidomimetic.

9. The method of paragraph 8, wherein the peptidomimetic is selected from the group consisting of α-peptides, βpeptides, γ-peptides, δ-peptides, and oligomers having backbones which can adopt helical or sheet conformations.

10. The method of paragraph 4, wherein the oligomers having backbones which can adopt helical or sheet conformations are selected from the group consisting of compounds having backbones utilizing bipyridine segments, compounds having backbones utilizing solvophobic interactions, compounds having backbones utilizing side chain interactions, compounds having backbones utilizing hydrogen bonding interactions, and compounds having backbones utilizing metál coordination.

11. The method of paragraph 7, wherein the self-assembling matériái is a nucleotidomimetic.

12. The method of paragraph 11, wherein the nucleotidomimetic is selected from the group consisting of isomeric oligonucleotides, modified carbohydrates, oligonucleotides with modified nucleotide linkages, and nucleotides with alternative nucleobases.

13. A method of form ing a barrier to movement of fluids, cells, tissue, biomolecules, orgas, comprising administering to a site in need thereof a self-assembling matériái, wherein the self-assembling matéria! is selected from the group consisting of peptidomimetics, nucleotidomimetics, di- and triblock copolymers, N-alkylacrylamides, and dendimers.

14. The method of paragraph 13, wherein the self-assembling matéria! is a peptidomimetic.

15. The method of paragraph 14, wherein the peptidomimetic is selected from the group consisting of a-peptides, β-peptides, γ-peptides, δ-peptides, and oligomers having backbones which can adopt helical or sheet conformations.

16. The method of paragraph 13, wherein the oligomers having backbones which can adopt helical or sheet conformations are selected from the group consisting of compounds having backbones utilizing bipyridine segments, compounds having backbones utilizing solvophobic interactions, compounds having backbones utilizing side chain interactions, compounds having backbones utilizing hydrogen bonding interactions, and compounds having backbones utilizing metál coordination.

17. The method of paragraph 13, wherein the self-assembling matéria! is a nucleotidomimetic.

18. The method of paragraph 17, wherein the nucleotidomimetic is selected from the group consisting of isomeric oligonucleotides, modified carbohydrates, oligonucleotides with modified nucleotide linkages, and nucleotides with alternative nucleobases.

19. A method for regeneration or repair of tissue or cells forming tissue comprising administering to the tissue or cells self-assembling materials, wherein the self-assembling materials are selected from the group consisting of peptidomimetics, nucleotidomimetics, di- and triblock copolymers, N-alkylacrylamides, and dendimers, alone or in combination with a pharmaceutically acceptable carrier.

20. The method of paragraph 19, wherein the self-assembling matéria! is a peptidomimetic.

21. The method of paragraph 20, wherein the peptidomimetic is selected from the group consisting of a-peptides, β-peptides, γ-peptides, δ-peptides, and oligomers having backbones which can adopt helical or sheet conformations.

22. The method of paragraph 19, wherein the oligomers having backbones which can adopt helical or sheet conformations are selected from the group consisting of compounds having backbones utilizing bipyridine segments, compounds having backbones utilizing solvophobic interactions, compounds having backbones utilizing side chain interactions, compounds having backbones utilizing hydrogen bonding interactions, and compounds having backbones utilizing metál coordination.

23. The method of paragraph 19, wherein the self-assembling matéria! is a nucleotidomimetic.

EP 2 581 097 Β1

24. The method of paragraph 23, wherein the nucleotidomimetic is selected from the group consisting of isomeric oligonucleotides, modified carbohydrates, oligonucleotides with modified nucleotide linkages, and nucleotides with alternative nucleobases.

25. The method of paragraphs 1, 13 or 19 further comprising providing one or more therapeutic, prophylactic, diagnostic agent, or cells.

26. The method of paragraphs 1, 13 or 19 further comprising providing with the self-assembling materials a pharmaceutically acceptable carrier or mátrix or support matéria! for administration onto or intő the body.

27. The method of paragraph 26 wherein the materials are administered as a dry powder, wafer, disk, tablet, capsule, liquid, gél, cream, foam, ointment, emulsion, suspension, solution, as a coating on a medical device or implant, or incorporated intő micropartides, polymeric matrices, hydrogels, textilé, suture, orsponges.

28. The method of paragraph 19 comprising applying to a cardiovascular or neurological defect, injury or infarct to permit or promote repair of the defect, injury or infarct.

29. The method of paragraph 19 comprising applying to a region of boné with a defect or injury to permit or promote repair of the defect or injury.

30. The method of paragraph 19 comprising applying to visceral organs with a defect or injury to permit or promote repair of the defect or injury.