ES2705602T3 - Compositions of purified amphiphilic peptides and uses thereof - Google Patents

Abstract

A composition comprising an aqueous solution of at least 2% and up to 5% by weight of amphiphilic peptide chains, wherein the peptide chains are each n-RADARADARADARADA-c (RAD16-I), for use in a method for facilitating hemostasis by direct injection at a wound site prior to gelation where the ions migrate to the peptide solution of the surrounding tissue and cause it to form a hemostatic gel.

Description

DESCRIPTION

Compositions of purified amphiphilic peptides and uses thereof

Field of the invention

The present invention relates to compositions of amphiphilic peptides of high purity.

BACKGROUND OF THE INVENTION

In general, the body is able to regenerate injured tissue to produce new tissue that has properties similar to the original tissue. For example, small cuts heal without permanent scarring, and clean fractures in the bone are cured by the formation of new bone that joins the two bone fragments. However, connective tissue cells and other organic cells depend on the anchor; they require scaffolding to show normal physiological behavior. When the tissue damage is extensive or there are large gaps, it is possible that the cells that migrate to the wound do not find the proper anchoring and produce scar tissue to close the gap between the healthy tissue at the edges of the wound. Scar tissue does not have the same mechanical and biological properties as the original tissue. For example, the scar tissue on the skin is not as flexible as the original tissue. The scar tissue in the bone is not as strong as the uninjured bone and often provides a weak spot where it is easier to break the bone again. Some tissues, such as articular cartilage, do not regenerate naturally, and scarring only comes from the formation of scar tissue.

To date, a substantial effort has been made to develop materials to replace or aid the regeneration of various tissues. These materials sometimes exploit the ability of small wounds to heal through regeneration in tissues where large wounds are healed by scarring. Therefore, the materials are packaged in a wound site that helps close the gap between the edges of a wound and attempts to prevent scar formation before tissue regeneration proceeds. Although various synthetic materials and derivatives have been developed naturally for the regeneration of tissues, these materials sometimes exhibit immune incompatibility and inadequate distribution of tension. In addition, the use of animal material, such as cow skin or the cartilage of pigs or sharks, has raised the concern of possible contamination by infectious agents, such as prions. Therefore, improved materials of biological origin which have improved compatibility, present a reduced risk of contamination and provide adequate biomechanical characteristics for tissue repair are desirable.

WO 03/096972 A2 and WO 02/062961 A2 describe compositions comprising self-assembled peptide hydrogels for use in tissue repair.

WO 03/084980 A2 describes a composition comprising an aqueous solution of amphiphilic peptide chains, in which the peptide chains have a hydrophilic and hydrophobic part and in which the peptide chains self-assemble as a gel, for use in surgery reconstructive and to stop the blood flow in the vessels.

WO 2005/014615 A2 discloses compositions comprising self-assembly peptides comprising (a) a first amino acid domain that mediates self-assembly, wherein the domain comprises hydrophobic and hydrophilic alternative amino acids that are complementary, structurally compatible and self-assembled in a macroscopic structure when they are present in unmodified form; and (b) a second amino acid domain that does not auto-assemble in isolation.

In addition, the 3D Matrix Japan website describes a peptide biomaterial called PuraMatrix ™, which comprises a peptide with the amino acid sequence n-RADARADARADARADA-c (3D Matrix Japan website: established via "WayBackMachine", April 15, 2005, URL: http: // www.3d-matrix.co.jp/pr01.html; "WayBackMachine", April 16, 2005, URL: http: //www.3d-matrix.co.jp/pr02. html; "WayBackMachine", April 16, 2005, URL: http: //www.3d-matrix.co.jp/ pr03.html).

Definitions

"Scaffolding" means a three-dimensional structure capable of supporting cells. The cells may be encapsulated by the scaffolding or they may be arranged in a layer on a scaffolding surface. The scaffold beta secondary structure of the scaffolding can be confirmed using standard circular dichroism for detect a minimum absorbance at approximately 218 nm and a maximum at approximately 195 nm. The scaffolding is formed by the self-assembly of peptides which may include L-amino acids, D-amino acids, natural amino acids, non-natural amino acids, or a combination thereof. If L-amino acids are present in the scaffold, scaffold degradation produces amino acids that can be reused by the host tissue. It is also contemplated that the peptides may be covalently linked to a compound, such as a chemoattractant or a therapeutically active compound. The peptides can be chemically synthesized or purified from natural or recombinant sources, and the amino and carboxy termini of the peptides can be protected or unprotected. The peptide scaffold can be formed from one or more molecular species other than peptides that are complementary and structurally compatible with each other. Peptides containing mismatched pairs, such as the repulsive pairing of two similarly charged residues of adjacent peptides, can also form scaffolds if the disruptive force is dominated by stabilizing interactions between the peptides. Scaffolds are also referred to herein as peptide structures, peptide hydrogel structures, peptide gel structures, or hydrogel structures.

By "complementary" is meant capable of forming interactions of ionic bonds or hydrogen bonds between hydrophilic residues from adjacent peptides in the scaffolding, as illustrated in Figure 1, each hydrophilic residue in a peptide is hydrogen-bonded or paired ionically with a hydrophilic residue in an adjacent peptide or exposed to solvent.

By "structurally compatible" is meant capable of maintaining a sufficiently constant intrapeptidal distance to allow the formation of scaffolding. The variation in intrapeptide distance is less than 4, 3, 2, or 1 angstrom. It is also contemplated that greater variations in the intrapeptidal distance may not prevent the formation of scaffolding if efficient stabilizing forces are present. This distance can be calculated based on molecular modeling or based on a simplified procedure that has been previously published (U.S. Patent No. 5,670,483). In this method, the intrapeptide distance is calculated by taking the sum of the number of unbranched atoms in the side chains of each amino acid in a pair. For example, the intrapeptide distance for a lysine-glutamic acid ion pair is 5 4 = 9 atoms, and the distance for a hydrogen bond glutamine-glutamine pair is 4 4 = 8 atoms. Using a conversion factor of 3 angstroms per atom, the variation in the intrapeptide distance of the peptides having lysine-glutamic acid pairs and glutamine-glutamine pairs (eg, 9 versus 8 atoms) is 3 angstroms.

The term "pure" is used to indicate the extent to which the peptides described herein are exempt from other chemical species, including deletion adducts of the peptide in question and peptides of different lengths.

The term "biologically active agent" is used to refer to agents, compounds, or entities that alter, inhibit, activate, or otherwise affect biological or biochemical events. Such agents can be naturally or synthetically derived. Biologically active agents include classes of molecules (eg, proteins, amino acids, peptides, polynucleotides, nucleotides, carbohydrates, sugars, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, growth factors, chemoattractants, etc.) which are commonly found in cells and tissues, whether the molecules themselves exist naturally or are artificially created (for example, by synthetic or recombinant methods). Biologically active agents also include drugs, for example, anticancer, analgesic, and opioid substances. Preferably, although not necessarily, the drug is one that has already been considered safe and effective for use by the appropriate government agency or agency. For example, drugs for human use listed by the FDA under 21 CFR §§330.5, 331 to 361, and 440 to 460; drugs for veterinary use listed by the DA under 21 CFR §§500 to 589 are all considered acceptable for use in accordance with the present invention. Additional exemplary biologically active agents include, but are not limited to, anti-AIDS substances, anti-cancer substances, immunosuppressants (eg, cyclosporin), anti-viral agents, enzyme inhibitors, neurotoxins, hypnotics, antihistamines, lubricants, tranquilizers, anticonvulsants, relaxants. muscle and anti-Parkinson, antispasmodic and contractive muscle agents including channel blockers, miotics and anticholinergics, anti-glaucoma compounds, anti-parasite agents, anti-protozoa, and / or anti-fungal, modulators of cell-extracellular matrix interactions including inhibitors of cell growth and anti-adhesion molecules, vasodilator agents, inhibitors of DNA, RNA or protein synthesis, antihypertensive agents, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, Ophthalmic compounds, prostaglandins, targeting agents, neurotransmitters, proteins, cell response modifiers, and vaccines.

As used herein, a hydrogel such as a peptide hydrogel is "stable with respect to mechanical or physical agitation" if, when subjected to mechanical agitation, it retains substantially the physical properties (such as elasticity, viscosity, etc.), which characterized the hydrogel before physical agitation. The hydrogel does not need to maintain its shape or size and can fragment into smaller pieces when subjected to mechanical agitation, although it is still considered stable with respect to mechanical or physical agitation. The term "stable" does not have this meaning except when used with this phrase.

As used herein, the term "nanofiber" refers to a fiber having a diameter of nanoscale dimensions. Typically, a nanoscale fiber has a diameter of 500 nm or less. According to one aspect of the disclosure, a nanofiber has a diameter less than 100 nm. According to some other aspects of the disclosure, a nanofiber has a diameter less than 50 nm. According to some other aspects of the disclosure, a nanofiber has a diameter of less than 20 nm. According to some other aspects of the disclosure, a nanofiber has a diameter between 10 and 20 nm. According to some other aspects of the disclosure, a nanofiber has a diameter between 5 and 10 nm. According to some other aspects of the disclosure, a nanofiber has a diameter less than 5 nm.

The term "nanoscale environment scaffolding" refers to a scaffold comprising nanofibers. According to certain aspects of the disclosure, at least 50% of the fibers comprising the scaffolding are nanofibers. According to certain aspects of the disclosure, at least 75% of the fibers comprising the scaffolding are nanofibers. According to certain aspects of the disclosure, at least 90% of the fibers comprising the scaffolding are nanofibers. According to certain aspects of the disclosure, at least 95% of the fibers comprising the scaffolding are nanofibers. According to certain aspects of the disclosure, at least 99% of the fibers comprising the scaffolding are nanofibers. Of course, the scaffolding may also comprise constituents other than fibers, for example, water, ions, growth-inducing and / or differentiating agents such as growth factors, therapeutic agents, or other compounds, which may be found in solution in the scaffolding and / o attached to scaffolding.

Summary of the invention

The invention relates to a composition for use as defined in claim 1. An aqueous solution comprising peptide chains is described herein. The solution can form a hydrogel that is stable with respect to mechanical agitation. The solution may be injectable and may have a pH between about 4.5 and about 8.5. The peptides can be self-assembled in a matrix in the solution. Self-assembly does not need to be stable with respect to mechanical agitation. The solution may contain at least 0.1 mM of an electrolyte.

The solution with the electrolyte can be injectable. The peptides can be adapted and constructed to be capable of self-assembly after injection. The concentration of the peptide chains in the aqueous solution can be at least about 2%, at least about 3%, at least about 4%, at least about 5% by weight. The aqueous solution or peptide chains may further include one or more biologically active agents.

A method for preparing a peptide chain is also described herein. The method includes providing amino acids comprising a group on the side chain that is attached to a protecting group, assembling the amino acids into an amphiphilic peptide chain, removing the protecting group by reacting the peptide chain with an acid, and exchanging the acid for a salt of non-fluorinated acetate. The non-fluorinated acetate salt can be selected from sodium acetate and potassium acetate. The acid can be trifluoroacetic acid. Also described herein is a composition that includes an aqueous solution of more than 2% amphiphilic peptide chains of at least 8 amino acids in length and having alternative hydrophilic and hydrophobic amino acids. The solution can, but does not need to be mechanically stable with respect to the self-assembly of the peptides.

Also described herein is a macroscopic scaffold comprising amphiphilic peptide chains, wherein the peptide chains have alternating hydrophobic and hydrophilic amino acids, are complementary and structurally compatible, and self-assemble into a macroscopic beta-scaffold scaffolding, and in which the amphiphilic peptide chains are in an aqueous solution, and in which at least about 75% of the peptide chains have the same sequence. The macroscopic scaffolding does not need to be mechanically stable with respect to physical agitation. The amphiphilic peptide chains can be in an aqueous solution containing an electrolyte, and the scaffolding can be mechanically stable with respect to physical agitation. Peptide chains can be adapted and constructed to be capable of self-assembly after injection. A biologically active agent can be encapsulated within the scaffolding.

Also disclosed herein is a method for administering a biologically active agent to a patient which includes providing an aqueous solution of amphiphilic peptides, adding a predetermined amount of the biologically active agent to the aqueous solution, and adding a predetermined amount of an electrolyte to the aqueous solution, in which, after adding the predetermined amounts, the aqueous solution forms a hydrogel. The biologically active agent and the electrolyte can be combined in a single aqueous solution and added to the aqueous solution of the amphiphilic peptides simultaneously. The biologically active agent can be selected from an anti-inflammatory, an antibiotic, an anti-cancer agent, an analgesic, an opioid, a drug, a growth factor, a protein, an amino acid, a peptide, a polynucleotide, a nucleotide, a carbohydrate, a sugar, a lipid, a polysaccharide, a nucleoprotein, a glycoprotein, a lipoprotein, a steroid, a chemoattractant and any combination of the above. The method may further include injecting the hydrogel at a predetermined site in a patient. For example, the method may include injecting the aqueous solution into a patient before adding a predetermined amount of electrolyte and after adding a predetermined amount of said biologically active agent, wherein adding a predetermined amount of electrolyte comprises allowing the ions to migrate to the patient. the solution injected from surrounding tissue. The method may include injecting the aqueous solution at a predetermined site in a patient before adding a predetermined amount of electrolyte and after adding a predetermined amount of said biologically active agent, wherein the injected aqueous solution is stable with respect to migration from the default site

A kit for administering a peptide composition to a patient is also described herein. The kit includes a purified peptide composition, which may be in the form of an aqueous solution, and at least one element selected from the group consisting of: an electrolyte, a buffer, a delivery device, a container suitable for mixing the peptide composition with one or more different agents. The delivery device can be, for example, a catheter, a needle, a syringe or a combination of any of these. The kit may further include instructions for use, for example, for preparing the peptide composition, for mixing the peptide composition with other agents, for introducing the peptide composition into a subject, etc.

A kit for administering a biologically active agent to a patient is also described herein. The kit includes a purified peptide composition, which may be in the form of an aqueous solution comprising amphiphilic peptides, and the biologically active agent, which may be provided pre-mixed with the peptides or separately. The kit may also include an electrolyte, a buffer, a delivery device, instructions, etc. The biologically active agent may be present in nanospheres, microspheres, etc. (also called nanocapsules, microcapsules, etc.). Numerous methods and reagents are known in the art for making such spheres, capsules, etc., which encapsulate a biologically active agent. For example, standard polymers and methods for making sustained-release and / or pH-resistant drug formulations can be used. A kit for administering cells to a patient is also described herein. The kit includes a purified peptide composition, which may be in the form of an aqueous solution comprising the peptides. The kit can include cells. The kit can include any of the items listed in the description of the above kits. The kit may also include instructions for the preparation of the cells to be administered using the kit. For example, instructions can describe how to grow cells, how to collect cells from a subject, how to mix cells with peptides, types of cells suitable for use, etc. The aqueous solution can have a shelf life of at least nine weeks under predetermined conditions. The aqueous solution may also include an electrolyte.

Brief description of the drawing

The invention is described with reference to the various figures of the drawing, in which,

Figure 1 is a schematic illustration of the interactions between the peptides in the peptide scaffold. Various peptides with amino acid sequences of alternating hydrophobic and hydrophilic moieties self-assemble to form a stable beta-scaffold scaffold when exposed to physiologically equivalent electrolyte solutions (U.S. Pat. Nos. 5,955,343 and 5,670,483). Peptide scaffolds are stabilized by numerous peptide interactions. For example, the side chains of amino acids positively charged and negatively charged peptides Adjacent pairs form complementary ion pairs, and other hydrophilic residues such as asparagine and glutamine, participate in hydrogen bonding interactions. Hydrophobic groups in adjacent peptides participate in the van der Waals interactions. The amino and carbonyl groups in the peptide backbone also participate in intermolecular hydrogen bonding interactions.

Figure 2 is a series of photomicrographs depicting, for a rat calvarial defect 28 days after treatment, A) normal tissue (uninjured), B) a control calvarial defect (untreated), C) treatment with a solution 3% of non-assembled peptide chains, D) treatment with COLLAPLUG ™, E) treatment with a 3% solution of peptide chains assembled in the presence of NaCl, F) treatment with a 3% solution of peptide chains assembled in medium, treatment with a 3% solution of unassembled peptide chains combined with blood in a 1: 1 ratio, H) treatment with a combination of COLLAGRAFT ™ and blood, I) treatment with a 3% solution of assembled peptide chains combined with phosphate tricalcium in a ratio of approximately 1: 1 (solution volume / weight of TCP), J) treatment with a combination of tricalcium phosphate and blood (1: 1), and K) treatment with a 1% solution of pep chains tídicas assembled in the middle.

Detailed description

Peptides can be prepared by providing amino acids including a group on the side chain that is attached to a protecting group, assembling amino acids on an amphiphilic peptide, and removing the protecting group by reacting the peptide with an acetate salt in the absence of trifluoroacetic acid or any of its salts.

Self-assembling peptides

Reference is made to those indicated in U.S. Patent Nos. 5,670,483 and 5,955,343, and U.S. Patent Application No. 09 / 778,200. These peptide chains consist of alternating hydrophilic and hydrophobic amino acids which are capable of self-assembly to form a macroscopic structure of beta sheet extremely stable in the presence of electrolytes, such as monovalent cations. The peptide chains are complementary and structurally compatible. The side chains of the peptide chains in the structure are divided into two faces, a polar face with charged ionic side chains and a non-polar face with alanines or other hydrophobic groups. These ionic side chains are self-complementary with each other, since positively charged and negatively charged amino acid residues can form complementary ion pairs. Therefore, these peptide chains are called ionic self-complementary peptides or type I self-assembly peptides. If the ionic residues are alternated with a positively charged and negatively charged residue (- + - + - + - +), the peptide chains are described as "module I"; if the ionic residues alternate with two positively charged and two negatively charged residues (- ++ - ++), the peptide chains are described as "module II". Exemplary peptide sequences include those listed in Table 1. Both D-amino acids and L-amino acids can be used to produce peptide chains. They can be mixed in the same chain, or peptide compositions can be prepared with mixtures of individual chains that only include D-amino acids and L-amino acids.

Table 1. Self-assembly peptides

Name Sequence (n -> c) Module RAD164 n-RADARADARADARADA-c I RGDA16-I n-RADARGDARADARGDA-c I RADA8-I n-RADARADA-c I RAD16-11 n-RARADADARARADADADA-c II RAD8-II n-RARADADA -c II EAKA164 n-AEAKAEAKAEAKAEAK-c I EAKA8-I n-AEAKAEAK-c I RAEA16-I n-RAEARAEARAEARAEA-c I RAEA8-I n-RAEARAEA-c I KADA164 n-KADAKADAKADAKADA-c I KADA8-I n-KADAKADA -c I KLD12 n-KLDLKLDLKLDL-c

EAH164I n-AEAEAHAHAEAEAHAH-c II EAH8-II n-AEAEAHAH-c II EFK16-II n-FEFEFKFKFEFEFKFK-c II EFK8-II n-FEFKFEFK-c I Name Sequence (n -> c) Module KFE12 n-FKFEFKFEFKFE-c

KFE8 n-FKFEFKFE-c

KFE16 n-FKFEFKFEFKFEFKFE-c

KFQ12 n-FKFQFKFQFKFQ-c

KIE12 n-IKIEIKIEIKIE-c

KVE12 n-VKVEVKVEVKVE

ELK16-II n-LELELKLKLELELKLK-c II ELK8-II n-LELELKLK-c II EAK16-II n-AEAEAKAKAEAEAKAK-c II EAK12 n-AEAEAEAEAKAK-c IV / II EAK8-II n-AEAEAKAK-c II KAE16-IV n- KAKAKAKAEAEAEAEA-c IV EAK16-IV n-AEAEAEAEAKAKAKAK-c IV RAD16-IV n-RARARARADADADADA-c IV DAR16-IV n-ADADADADARARARAR-c IV DAR16-IV * n-DADADADARARARA-c IV DAR324V n- (ADADADARARARARAR) 2-c IV EHK16 n-HEHEHKHKHEHEHKHK-c N / A EHK8-I n-HEHEHKFIK-c N / A VE20 * n-EVVEVEVEVEVEVEVEVE-c N / A RF20 * n-RFRFRFRFRFRFRFRFRFRF-c N / AN / A represents not applicable

* These peptides form a p-sheet when incubated in a solution containing NaCl, however, it has not been observed that they self-assemble to form macroscopic scaffolds .__________________________________

Many sequences of self-complementary peptides of moduli I and II, such as EAK16, KAE16, RAD16, RAE16 and KAD16, have been previously analyzed (Table 1). Module I autocomplementary ionic peptide sequences containing 16 amino acids, such as EAK16-IV, KAE16-IV, DAR16-IV and RAD16-IV have also been studied. If the residues loaded in these peptide self-assembly chains are replaced (ie, the positively charged lysines are replaced by positively charged arginines and the negatively charged glutamates are replaced by negatively charged aspartates), there are essentially no significant effects in the self-assembly process. However, if the positively charged residues, lysine and arganine, are replaced by negatively charged residues, aspartate and glutamate, the peptide chains can no longer self-assemble to form macroscopic scaffolds; however, they can still form a beta sheet structure in the presence of salt. Other hydrophilic residues, such as asparagine and glutamine, which form hydrogen bonds can be incorporated into the peptide chains instead of, or in addition to, charged residues. If the alanins in the peptide chains are changed to more hydrophobic residues, such as leucine, isoleucine, phenylalanine or tyrosine, these peptide chains have a greater tendency to self-assemble and form peptide matrices with greater resistance. Some peptides having compositions and lengths similar to the peptide chains mentioned above form alpha helices and random coils in place of beta sheets and do not form macroscopic structures. Therefore, in addition to self-complementarity, it is likely that other factors are important for the formation of macroscopic scaffolds, such as the length of the chain, the degree of intermolecular interaction, and the ability to form stepped matrices.

Other self-assembling peptide chains can be generated by changing the amino acid sequence of any self-assembling peptide chain to a single amino acid residue or to multiple amino acid residues. In addition, the incorporation of specific cellular recognition ligands, such as RGD or RAD, into the peptide scaffold can promote the proliferation of the encapsulated cells. In vivo, these ligands can also attract cells from outside scaffolding to the scaffold, where they can invade the scaffolding or otherwise interact with the encapsulated cells. To increase the mechanical strength of the resulting scaffolds, the cysteines can be incorporated into the peptide chains to allow the formation of disulfide bonds, or the residues with aromatic rings can be incorporated and crosslinked by exposure to UV light. The in vivo half-life of the scaffolds can also be modulated by the incorporation of protease cleavage sites in the scaffolding, which allows the scaffolding to degrade enzymatically. Combinations of any of the above alterations can also be made in the same peptide scaffold.

Self-assembled nanoscale structures can be formed with varying degrees of stiffness or elasticity. If you want to be bound by any theory, low elasticity can be an important factor in allowing cells migrate towards the scaffolding and communicate with each other once they reside on the scaffolding. The peptide scaffolds described herein typically have a low elastic modulus, in the range of 1-10 kPa as measured in a standard cone-plate rheometer. Such low values allow deformation of the scaffolding as a result of cellular contraction, and this deformation can provide the means for cell-cell communication. In addition, such modules allow the scaffolding to transmit physiological stresses to the cells that migrate therein, stimulating the cells to produce tissue that is closer in the microstructure to the native tissue than in the scar. The rigidity of the scaffolding can be controlled by a variety of means including changes in peptide sequence, changes in peptide concentration and changes in peptide length. Other methods for increasing rigidity may also be used, such as attaching a biotin molecule to the amino or carboxy terminus of the peptide chains or between the amino and carboxy termini, which can then be crosslinked.

The peptide chains capable of crosslinking can be synthesized using standard f-moc chemistry and purified using high pressure liquid chromatography (Table 2). The formation of a peptide scaffold can be initiated by the addition of electrolytes as described herein. Hydrophobic residues with aromatic side chains can be crosslinked by exposure to UV radiation. The extent of crosslinking can be controlled precisely by the predetermined duration of exposure to UV light and the concentration of the predetermined peptide chain. The extent of crosslinking can be determined by light scattering, gel filtration or scanning electron microscopy using standard methods. In addition, the extent of crosslinking can also be examined by HPLC or scaffold mass spectrometry analysis after digestion with a protease, such as matrix metalloproteases. The strength of the scaffolding material can be determined before and after crosslinking, as described herein.

TABLE 2 n i i r r i l ion

Aggrecan processing sites, such as those underlined in Table 3, can optionally be added to the amino or carboxy terminus of the peptides or between the amino and carboxy termini. Likewise, other matrix metalloprotease (MMP) cleavage sites, such as those of collagenases, can be introduced in the same manner. Peptide scaffolds formed from these peptide chains, alone or in combination with peptides capable of crosslinking, can be exposed to various proteases for various periods of time and at various concentrations of proteases and peptides. The degradation rate of the scaffolds can be determined by HPLC, mass spectrometry or NMR analysis of the digested peptide chains released in the supernatant at various time points. Alternatively, if radiolabelled peptide chains are used for scaffold formation, the amount of radiolabeled material released in the supernatant can be measured by scintillation counting. For some embodiments, the structure of the beta sheet of the assembled peptide chains is degraded fast enough so that it is not necessary to incorporate cleavage sites in the peptide chains.

TABLE 3 S n i i n n ii r n ag ag e

If desired, the peptide scaffolds can also be formed in a predetermined shape or volume. To form a scaffold with a desired geometry or dimension, an aqueous peptide solution is added to a preformed casting mold, and the peptide chains are induced to self-assemble into a scaffold by the addition of an electrolyte, as described herein. The resulting geometry and dimensions Scaffolding of macroscopic peptides is governed by the concentration and amount of peptide solution applied, the concentration of electrolyte used to induce the scaffolding assembly, and the dimensions of the casting apparatus.

If desired, peptide scaffolds can be characterized using various biophysical and optical instruments, such as circular dichroism (CD), dynamic light scattering, infrared Fourier transform (FTIR), atomic force microscopy (ATM), electron microscopy. sweep (SEM), and transmission electron microscopy (TEM). For example, biophysical methods can be used to determine the degree of secondary structure of the beta sheet in the peptide scaffold. In addition, the size of the filaments and pore, the diameter of the fiber, the length, the elasticity and the volume fraction can be determined by quantitative analysis of scanning and transmission electron microscopy images. The scaffolds can also be examined using various standard mechanical testing techniques to measure the degree of swelling, the effect of pH and electrolyte concentration on scaffold formation, the level of hydration under various conditions, and the tensile strength.

Peptide production

Peptide chains for use with the invention can be produced using techniques well known to those skilled in the art, including solution phase synthesis and solid phase synthesis. These techniques can be optimized for the production of the peptide chains described herein at levels of purity which provide the resulting composition with surprising properties, including, but not limited to, shelf life, rate of degradation, solubility and mechanical characteristics.

In one aspect of the disclosure, peptide chains for use with the invention are produced using solid phase peptide synthesis techniques. The synthesis can be carried out at room temperature in a glass reactor vessel, for example, with a thick porous glass disk in the bottom. The reactor facilitates the addition of amino acid derivatives, solvents and reagents used in the reaction. One skilled in the art will recognize that the size of the reactor depends on the amount of resin used for the synthesis. The reactor vessel may be equipped with a mechanical agitator or placed on a platform agitator to efficiently mix the peptide-resin complex with the reaction solution.

One skilled in the art will be familiar with a variety of resins that can be used to support peptide chains as they are synthesized. An exemplary resin for use with the techniques of the invention is Rink amide MBHA resin, available from Glycopep Chemicals, Inc. (Chicago, IL). MBHA resin is a polystyrene resin cross-linked with 1% DVB derived divinylbenzene with 0.4-0.8 mmole / gram of

where Fmoc is N-alpha- (9-fluorenylmethyloxycarbonyl) and Nle is norleucine, which is used as a marker to determine the level of substitution in the resin. Although the level of substitution of the polymer does not significantly affect the quality of the product, it significantly affects the time and cost of manufacturing. A too high substitution level can result in long coupling times, while a too low substitution level increases the amount of resin required to perform the reaction and the volume of solvents required for the washing steps. Additional resins include, but are not limited to , resin chloromethylpolystyrene (CMS) resin, 4-hidroxifenoximetilpoliestireno, M ^s resin Rink amide (

), and sarcosine dimethyl acrylamide resin (

) all available at Polymer Laboratories. These resins can be purchased with a particular amino acid already attached to the resin.

When the peptide chains are synthesized from the C-terminal amino acid, both the reactive side chain and the alpha amino group of the amino acid that is added to the peptide chain must be protected. A labile protecting group such as Fmoc can be used to block the alpha amino group, while a stable protecting group can be used to block reactive side chains. In one aspect of the disclosure, the labile protecting group is removed with piperidine, and the stable protecting group is removed with strong acid, as described below. Exemplary protecting groups for amino acid side chains include, but are not limited to, 2,2,4,6,7-pentamethylhydrobenzofuran (Pbf), tert-butoxy (OtBu), trityl (Trt), tert-butyl (tBu) and Boc. (tert-butoxycarbonyl). One skilled in the art will recognize which protecting groups are appropriate for the specific amino acids. Amino acids with existing protecting groups are commercially available, for example, in Advanced ChemTech, Louisville, KY.

To add an amino acid to a growing peptide chain, the alpha amino group at the end of the resin-terminated chain is acylated by the next activated amino acid that is added to the peptide chain. In one aspect of the disclosure, the protected amino acid and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) are dissolved in DMF. N-methylmorpholine (NMM) is added to the solution, which is then added to the resin. In general, reagents for acylation are selected to optimize the reaction conditions and to facilitate removal after coupling.

The mixture of HBTU / NMM / resin is allowed to react for an appropriate interval, for example, at least one hour, after which the degree of reaction can be determined using the TNBS test (Hancock, et al., Anal. Biochem. , 1976, 71, 260) and / or the ninhydrin test (eg, sample reaction with ninhydrin, amines colorimetrically identified). If residual amino groups are detected, the coupling reaction can be repeated using half the amount of reagent required for the initial coupling reaction. The unreacted alpha amino acids still present after the repeat reaction can be covered with acetic anhydride to avoid deletion sequences in the following cycles.

After adding each amino acid, the labile Fmoc protecting group can be cleaved from the alpha amino function of the N-terminal amino acid in the growing peptide by treating the resin twice with a 20% solution of piperidine in DMF or a mixture of DMF with Water. In this aspect of the disclosure, the stable protecting group is resistant to elimination under these conditions and remains attached to the amino acid.

After both the coupling reaction and the deprotection reaction, the resin can be washed with DMF to remove excess reagent. The DMF is an excellent solvent for the reagents used in the coupling step and also has excellent swelling properties. Alternate solvents can also be used to produce peptide chains for use with the invention. Such solvents should be selected to minimize the risk of side reactions while efficiently removing the excess reagent from the reaction mixture. The rinsing times should be sufficient to allow complete contact of the peptide resin with the solvent and for the extraction of the reagents. One skilled in the art will recognize that the washing steps may be repeated, but that repeated washing will increase the manufacturing cost.

After the last amino acid in the sequence has been added, the N-terminus of the peptide can be protected with acetic anhydride and the entire chain can be released from the resin. Then any side chain protecting group is cleaved. This can be achieved by treatment of the peptide complex with trifluoroacetic acid in the presence of sequestrants, for example, water, phenol and / or triisopropylsilane. The sequestrators trap the reactive cations during cleavage and avoid the alkylation of reactive side chains.

In an alternative aspect of the disclosure, the peptide chains are synthesized using techniques in the solution phase. The synthesis in the solution phase is based on the gradual addition of protected amino acids to the growing peptide chain. This method is faster than solid phase methods for peptide chains having a repeating unit, such as RAD16. For example, four mer RADA can be produced in several reaction vessels. In one of the reaction vessels, the C-terminal amino acid is deprotected and the N-terminal acid is protected. The addition of the four unprotected C-terminal mer to a second reaction vessel doubles the length of the peptide in a single reaction step. The process is repeated to produce a 12-mer. One skilled in the art will recognize that several combinations of four mers with each other will result in a rapid production of 16-mer.

The completed peptides can be purified using standard peptide purification techniques, including gel filtration and high performance liquid ion exchange chromatography (HPLC). In one aspect of the disclosure, the peptide chains are purified using reverse phase HPLC with PLRP-S, a polystyrene cross-linked with DVB available from Polymer Laboratories, Inc., Amherst, MA. The separation is based on hydrophobic interactions between the peptide and the polymer. The interaction of the peptide chains with the resin is specific enough so that the peptide chains of similar structures can be easily separated using the resin support. In general, the column is washed with aqueous acetic acid to equilibrate it. The crude peptide is loaded onto the column and eluted using a gradient of acetic acid / water and acetonitrile / acetic acid / water. In one embodiment, 0.1% acetic acid and 80% acetonitrile are used, but one skilled in the art will recognize that the proportions can be adjusted to optimize processing times and separation between the desired peptide and various impurities. Also, the gradient may be a 0.5% -1.0% per minute change (e.g., an increase in the acetonitrile solution), but may be adjusted to optimize the separation between the reaction product and the impurities. The purification method also exchanges acetate for trifluoroacetate, converting the peptide to the acetate salt form. The product exchanged with acetate is more biocompatible than the fluorinated product. Unexpectedly, the use of sodium acetate also produces a more highly purified product with fewer elimination adducts.

As the fractions are collected, the content of the fraction can be controlled by analytical reverse phase HPLC eluted with 0.1% TFA in aqueous acetonitrile. Any reverse phase HPLC support that can be used to purify the peptide product can also be used to analyze the quality of the eluted fractions. In one aspect of the disclosure, a C18 column can be used (for example, using silica derived with C18 chains). The fractions of the purification column that meet the desired purity specification can be combined and lyophilized. Fractions with lower purity can be processed again. Fractions that can not be processed again to obtain a higher purity material can be discarded.

The purification step separates the peptide chains having the desired sequence from various types of by-products. The first is the excess of reagents, of which the desired peptide sequence is easily separated because it has a different chemical structure and a higher molecular weight. The second includes deletion adducts, peptide chains similar in sequence to the desired peptide but having one or more missing amino acids. It has been unexpectedly found that the improved removal of the elimination adducts increases the solubility of the desired peptide chains in aqueous media and changes various properties of the self-assembled scaffolds. In some embodiments, the final product may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% pure.

Preparation of the peptide solution and its characteristics

The purified product can be stored as a powder or re-dissolved in an aqueous solution. The peptide product is significantly more soluble in water than the previous formulations, and concentrations greater than 2% or 3% can be achieved. Agitation, for example, the use of sonication or a stirring table, helps the peptide chains to come into solution. The concentration of peptide chains in solution may vary depending on the desired application. Peptide chains in solution self-assemble spontaneously in scaffolds through electrostatic interactions. The self-assembled peptide chains form a hydrogel that remains ductile and capable of flowing after the application of an appropriate stimulus.

The peptide solution can have a storage life of at least one year with or without added electrolyte (see below).

The peptide solution can be sterilized by radiation, if desired. In one aspect of the disclosure, the peptide solution is stable with respect to exposure to gamma radiation at about 35 Kgrey. If it is sufficient, less radiation can be used for sterilization, for example, approximately 25 Kgrey. Peptide solutions produced using the techniques described herein are stable with respect to radiation exposure and do not undergo structural alterations upon exposure to sterilization radiation. Of course, the peptide solutions can also be sterilized by injection through a 0.45 micron filter.

Formation of a self-assembled peptide scaffold and its properties

Peptide solutions can be formed in a stable scaffold by exposure to a monovalent saline solution. Sufficient electrolyte is added to the solution to initiate self-assembly of the peptides in a macroscopic beta sheet structure. In a certain aspect of the disclosure, the concentration of the added electrolyte is at least 5, 10, 20 or 50 mM. Smaller concentrations, for example, 0.1 to 1 mm, or larger concentrations may also be used. The choice of concentration depends partly on the desired ionic strength of the peptide gel and also affects the rate of gelation. Suitable electrolytes include, but are not limited to, Li +, Na +, K +, and Cs +. The electrolyte causes the peptide chains to self-assemble into a scaffold that is stable with respect to mechanical agitation.

At low concentrations, the addition of electrolyte to the peptide solution results in an agar-type gel having a limited liquid flow and high flexibility. The mechanical behavior of hydrogels of low peptide concentration is similar to that of gelatin. While it is highly flexible, the hydrogel is also brittle. Instead of plastically deforming, the gel breaks into separate pieces or fractures. It has been found that these properties do not duplicate at higher concentrations. Rather, gels of higher concentration remain ductile and coherent and are susceptible to plastic deformation. With a consistency similar to that of long chain hyaluronic acid, they can be injected through a 30 gauge needle.

At higher concentrations, the peptide chain solutions behave like non-Newtonian fluids. In addition, the solution becomes more viscous with time. For example, a 3% solution showed an elastic limit of 20-30 Pa and a viscosity of less than 40 cP approximately one hour after mixing, increasing the yield strength to 50-65 Pa after 5-6 hours. After two weeks, the yield strength increased to 100-160 Pa, and the viscosity increased to less than 200 cP.

Ductile gel behaves like an injection molded material. Peptide hydrogels that pass through a needle fill the desired space with a single coherent bolus instead of a tangled, coiled mass. That is, the material is assembled both on the scale of the individual peptide chains and on a macroscale such as a gel. It provides both an injectable material to facilitate administration and a continuous fibrous network that facilitates cell growth and proliferation.

It has also been found that peptide gels of high purity (for example, with respect to the chain composition) exhibit accelerated degradation compared to gels of lower purity. In fact, gels produced using the techniques of the invention exhibit faster degradation in vivo than collagen or hyaluronic acid. For example, a 20011.1 bolus of 1% RAD16 produced using the techniques of the invention and implanted in vivo was degraded and excreted in 28 days. More than 90% of a 70 pl bolus of 1% RAD16 was degraded and excreted in 14 days. Higher concentration gels have longer degradation times.

In a certain aspect of the disclosure, high purity gels are also stable at physiological pH. The gels can be brought to physiological pH by balancing them with a buffer solution. For example, a layer of buffered solution having the desired pH can be loaded above or below a layer of a peptide gel produced using the techniques of the invention. This approach has been used, for example, to balance peptide scaffolds with a phosphate-buffered saline or tissue culture medium before use of the scaffolds for cell culture. After the gel and buffer solution have equilibrated for a certain time, the pH of the gel is easily tested by removing the solution and placing a pH paper strip on the gel. One skilled in the art will recognize that it may be necessary to repeat the process several times to bring the gel to the desired pH. As an alternative, or, in addition, an excess of buffer can be used. A rocker can also be used to accelerate the buffer and gel balance. Gels at physiological pH can retain the physical properties they had at a lower pH. They can still be injected or combined with cells. Of course, such gels are much more compatible with physiological tissue and cells at physiological pH than at pH 2-3.

According to a certain aspect of the disclosure, it is desirable to modify the pH of a peptide solution, for example, to raise the pH to a physiological pH of ~7-8.5 before introducing the peptide solution into a subject (see below). ) or before encapsulating the cells. The peptide solutions modified with pH can also be used for other purposes, for example, for tissue culture or for encapsulating biologically active agents. It may be desirable to achieve the pH change relatively quickly, for example, without the need for a prolonged equilibrium with buffer solution and / or multiple changes of the buffer solution.

Several different buffers were tested to evaluate their ability to raise the pH of a 1% aqueous solution of peptide. The RAD16-I peptide was used for this study, and the following buffers were tested:

1. Ammonium acetate (prepared in the laboratory)

2. sodium citrate (prepared in the laboratory)

3. sodium acetate (prepared in the laboratory)

4. sodium bicarbonate (clinical grade manufactured by Abbott, Inc.),

5. HEPES (research grade manufactured by Stem Cell Technologies, Inc.)

6. Tris-HCl (prepared in the laboratory)

7. Tromethamine, also called THAM (clinical grade manufactured by Abbott, Inc.).

8. Phosphate buffered saline solution without Ca or Mg (Gibco-BRL)

All these buffers could increase the pH of the peptide solution without destroying the ability of the peptide chains to assemble in scaffolding, either before or after the addition of an electrolyte. The assembled scaffolds can also be balanced with a buffer to raise the pH. Tables 4 and 5 summarize the results obtained in these experiments. In the tables, "Pre-salt" refers to experiments in which an electrolyte was added to the peptide composition before the addition of the buffer solution, and "Post-salt" refers to experiments in which it was added an electrolyte to the peptide composition after the addition of the buffer. The "Pre-salt" and "Post-salt" columns indicate the final concentration of the buffer and the pH of the peptide solution. The listed buffers allow the peptide gel to reach pH between 4.5 and 8.5. The resulting peptide gels were not stable with respect to mechanical agitation. For example, when the buffer was stirred in an assembled gel, the peptide structure was disassembled and became liquid and aqueous. A similar phenomenon was observed when the electrolyte was stirred in non-buffered solutions. Said pH values are compatible with the in vivo uses of the peptide solutions and hydrogels, since they are diverse between pH values, for example, 6.0, 6.5, 7.0, 7.5, 8.0 , etc.

In both sets of experiments, the pH of the peptide solutions and gels was determined by contacting the peptide solution or assembled peptide scaffold with pH paper that had been calibrated against standard pH calibration solutions. The peptide solution was removed from a container containing it and sprayed on pH paper with a pipette or, in the case of peptide gels that remained assembled in the presence of the buffer solution, the buffer solution was removed first and the gel The peptide was removed after the container using a spatula and contacted with the pH paper.

It is noted that the experimental data indicated in the present document is provided for exemplary purposes and is not intended to limit the invention.

T l 4: R l - T m n m z l n n i i n i i l 1:

T l: R l - r i i n m n r l r l l i i n i i:

Use of self-assembled peptide scaffolds as tissue fillings

The peptide gels described herein provide a matrix to which the cells can adhere and on which they can migrate into a wound site. The peptide scaffolds described herein comprise a network of nanofibers with intervening spaces instead of a solid matrix. Such a structure can allow cell infiltration and cell-cell interaction in a way that more closely resembles the configuration of cells within the body than is allowed by other culture techniques and materials. Instead of simply healing from the edges, the entire area of the wound can regenerate simultaneously as the cells migrate towards the center of the scaffolding.

High purity gels can be injected directly into the wound sites to facilitate wound healing. For example, they can be injected into biopsy sites or wound sites created by the removal of a tumor. Gels can also be used to facilitate healing in chronic wounds, such as skin lesions and diabetic ulcers. The use of peptide hydrogels to facilitate healing in nerve tissue is discussed in U.S. Application No. 10 / 968,790. Of course, peptide gels can be used to promote tissue regeneration in wound sites not surgically created. Because the gels can be injected, there is no need to enlarge the wound site beyond what was originally needed to remove the diseased tissue. In addition, the gel does not need to be molded to adapt to the wound site. Rather, it simply fills the wound site the way a liquid fills a container into which it is poured. Because the injected gel forms a coherent bolus instead of single strands or threads, it easily penetrates the nooks and crannies of the edges of an irregular wound.

Peptide gels can be used as bulking agents. For example, gels or peptide solutions can be injected under the skin to fill the tissue depressions resulting from the scars. They can also be used instead of injections of collagen or hyaluronic acid to fill sagging skin and fill in wrinkles. Large dimples can also be the result of large subcutaneous wounds, for example, severe injuries to the skull or jaw. The peptide gels can be used to fill said dimples, whether or not the remodeling of the gel is desired in natural tissue or a capsule of fibrous tissue. For example, peptidic gels can be used for a breast augmentation. It is not necessary for the gel to be remodeled in the breast tissue. As described herein, the gels can be injected subcutaneously to make the skin stretch. For example, when a flap of skin is needed for surgery, the skin can be produced by autologously injecting a bolus of gel under the skin near the surgical site or elsewhere in the body. The gel bolus pressure stretches the existing skin. To relieve pressure, the body produces more skin in the area of the bolus, just as the body produces new skin to adapt to a pregnancy. Additional gel can be added over time to increase the size of the bolus.

The internal tissues can also be increased with peptide gels. For example, gels can be injected into the urethra to prevent reflux or correct incontinence. In a related application, the gels described herein can be used for embolization. For example, gels can be injected into blood vessels around a tumor or vessels that have been cut during surgery to stop the flow of blood. The use of peptide gels as hemostatic agents is discussed in United States Provisional Application No. 60 / 674,612. Alternatively, or in addition, gels can be injected between the tissues, especially after surgery, to prevent adhesion. Gels injected into open wounds can help prevent the adhesion of the dressings to the underlying tissues. Alternatively, the gels can be injected under the abdominal periosteum to prevent the formation of adhesions after abdominal surgery.

The gels can also be injected into the heart muscle to stimulate muscle production in the thin cardiac walls, as discussed in Patent Publication No. 20040242469. Without being limited by a particular theory, it is believed that the peptide gel injected into Certain tissue sites create a permissive cavity for cell growth and tissue development. The pH of the gel can be adjusted to further promote cell growth and extracellular matrix production, or growth factors can be added to the peptide gel to promote specific cellular behavior.

The techniques described herein can also be used to cure orthopedic defects. For example, gels produced using the techniques described herein can be arranged around dental implants to close any gap between the implant and the surrounding tissue and promote internal growth in the implants. Gels can be injected into cartilage defects or osteochondral defects to prevent scar formation. Gels can also be used to coat internal surfaces or fill pores in ceramic scaffolds. For example, a three-dimensional block of calcium phosphate or hydroxyapatite can be infused with a peptide solution before or after gelation. The infusion can be assisted by pressing the peptide solution or by drawing a vacuum to promote infiltration. Alternatively, or, in addition, the ceramic particles, especially the crystalline, semi-crystalline or amorphous calcium phosphate materials, can be combined with a peptide solution to form an injectable composition. Figure 2 shows photomicrographs of calvarial bone defects filled with 1% and 3% phosphate buffered solutions (PBS) of peptide chains produced as described herein. Both peptide concentrations facilitated bone healing superior to COLLAGRAFT ™, a product of collagen / tricalcium phosphate, or COLLAPLUG ™, an absorbable collagen product. The peptide solution facilitated the development of bone tissue, with little or no void at the defect site, whereas defects filled with COLLAGRAFT ™ developed fibrous scar tissue, as well as an untreated control defect. Better results were achieved with higher concentrations, combinations of the peptide gel with tricalcium phosphate, and the assembly of the gel using an electrolyte. The untreated control showed very little scarring and minimal new bone (Figure 2B). The 3% non-assembled peptide chain solution resulted in a gap of approximately half the size of the empty control, with more osteoid, good vascularization and no inflammatory reaction (Figure 2C). Treatment with Collaplug resulted in discontinuous bone islands and fibrous scar formation (Figure 2D).

Treatment with a 3% solution of peptide chain assembled in NaCl resulted in a thicker continuous bone (Figure 2E), whereas treatment with the same solution assembled in medium resulted in discontinuous bone and tissue formation fibrous (Figure 2F). When a 3% solution without assembly was used in combination with blood, the defect site was detectable but much smaller than the original defect (Figure 2G). The combination of Collagraft and blood resulted in the development of fibrous tissue and a very thin bone. The implant was reabsorbed very slowly (Figure 2H). A 3% solution of peptide chains assembled in medium and combined with tricalcium phosphate facilitated the continuous and complete healing of the defect site (Figure 2I). A 1: 1 mixture of TCP and blood provided discontinuous healing; the fragments were surrounded by osteoblasts, but were slowly reabsorbed (Figure 2J). A 1% solution of peptide chains assembled in medium was liquid and was not ideal for implantation or combination with other materials; however, it facilitated the development of continuous bone at the defect site, although bone was not as thick as after treatment with higher concentration solutions (Figure 2K).

The gels described herein also find utility in ophthalmic applications. For example, gels can be used for short or long term repair of retinal detachment. The gel is injected into the eyeball, where the extra pressure presses the retina against the wall of the eye. Because the gel is transparent, lasers can still be used to permanently fix the retina in place. In the prior art therapeutic techniques, patients often have to keep their heads in awkward positions to retain an air bubble in place against the retina. The gels described herein exert a constant hydrostatic pressure. As a result, it is expected that, in applications where prior art therapies require that an air bubble be disposed against a particular point in the retina, replacing the air bubble with an incompressible gel can alleviate at least part of the the difficulty of convalescence after retinal surgery. Alternatively, or in addition, the peptide gels described herein may be used for scleral buckling procedures. The gel is arranged against the outer surface of the eye to push the sclera toward the middle of the eye.

In any of the applications described above, peptide solutions can be injected prior to gelation. In fact, the peptide solutions can be injected prior to gelation in any application in which the ions can migrate to the peptide solution from the surrounding tissue and cause it to gel. For example, when a long or narrow gauge needle is required, or when it is advantageous for the gel to infiltrate dense tissue at the edges of a wound site, the pre-gelation injection provides a more fluid material that will generate less return pressure. during injection and can penetrate into dense fibrous tissues and between mineralized bone fibrils. Instead of flowing away from the side of the wound, the fluid undergoes some self-assembly even before exposure to an electrolyte. Spontaneous self-assembly of peptide chains has been observed in solutions with concentrations of peptide chains of up to 5%. This holds the material in place without the need for a cover or tissue flap. Of course, when the ionic strength of the fluid at the injection site is insufficient, the peptide solution can be injected after gelation or it can be followed by an electrolyte solution.

Optionally, one or more biologically active agents, for example, therapeutically active compounds or chemoattractants can be added to the peptide gels. Examples of such compounds include synthetic organic molecules, organic molecules of natural origin, nucleic acid molecules, biosynthetic proteins such as chemokines, and modified proteins of natural origin. Growth factors are also foreseen for use in this embodiment of the invention, alone or in combination with other biologically active agents. Exemplary growth factors include, but are not limited to, cytokines, epidermal growth factor, nerve growth factor, transforming growth factor alpha and beta, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor. , hematopoietic growth factor, heparin-binding growth factor, acid fibroblast growth factor, basic fibroblast growth factor, hepatocyte growth factor, brain-derived neurotrophic factor, keratinocyte growth factor, bone morphogenetic protein, or a growth factor derived from cartilage.

For example, biologically active agents may also be added to the gels to recruit cells at a wound site or to promote extracellular matrix production or vascularization development. The gels may also include compounds selected to reduce inflammation after implantation or to control pain. Because the gels described herein degrade relatively quickly, analgesics may be added, for example, without fear of analgesics being continuously administered at the wound site for months. In addition, potent analgesics can be administered locally at the wound site without having to administer them systematically. Systemic administration leaves patients lethargic, and the Long-term opioid administration increases the risk of future dependence on drugs. Local administration leaves patients on alert and may continue for much longer periods of time.

Because the gel is injectable, it can be used for repeated and localized administration of the drug. For example, it can be used to administer anti-cancer drugs to a patient for long-term therapy. Traditional chemotherapy techniques distribute toxic drugs throughout the patient's body. The techniques of the invention can be used to administer chemotherapeutic agents to a specific site and release them rapidly or over a period of days or weeks. In place of repeated systemic administration, the gels containing the desired agent can be periodically administered to a desired site. Alternatively, or in addition, anticancer agents can be administered locally to a site from which a tumor has just been excised. Peptide gels can be used as drug stores to store drugs and release them for long periods of time. Even biological products, such as proteins, are stabilized by peptide scaffolding and can be released over a period of days, weeks or months without losing their potency. Peptide gels can be produced with encapsulated biologically active agents and stored for long periods of time. That is, it is not necessary to store the added agent and the peptide separately and mix them before administration. When the agent itself is ionic or is commonly stored in ionic media, the addition of the biologically active agent to a peptide solution will cause the solution to gel, trapping the agent. Alternatively, the agent can be combined with an ionic solution and added to a peptide solution. It has been found that antibodies added to an ungelled peptide solution gel the peptide solution and also remain stable, without diffusing out of the gel, for at least nine weeks. The extended shelf life of a gel-drug mixture increases comfort for the healthcare provider and reduces the risk of contamination and infection by mixing materials or transferring material from one vial to another. In some cases, it is possible to teach patients to inject themselves. When repeated administration is indicated for a prolonged period of time, self-administration can help the patient reduce hospital visits. In an alternative possibility, the peptide solution and the added agent combine without added electrolyte. After injection, the ions migrate to the solution from the surrounding tissue to gel the peptide solution and encapsulate the agent.

As also described herein, biologically active agents can bind directly to the peptide chains. For example, said agents can be attached to the ends of the peptide chains during peptide synthesis. Alternatively, or in addition, they may be linked through aggrecan processing sites, such as those described in relation to Table 3. While there are agents that are large enough to interfere with the self-assembly of the peptide chains, they do not. they are necessarily unsuitable for use with the compositions of the invention. Rather, the agent will simply form a bulge in the beta-sheet structure of the assembled peptide chains.

The concentration or composition of the materials that are combined with the gels described herein may vary. For example, a gel capsule containing a particular molecule at a given concentration can be produced. That capsule may be encapsulated within a larger capsule or in a matrix containing several similar capsules. The larger capsule or matrix may contain a different molecule, for example, an additional biologically active agent, or a different concentration of the same material.

The cells can also be encapsulated within the scaffolds, as described, for example, in pending applications together with the present U.S.S.N. 09 / 778,200, entitled "Peptide Scaffold Encapsulation of Tissue Cells and Uses Thereof", filed on February 6, 2001, and in U.S.S.N. 10/877068, entitled "Self-Assembling Peptides Incorporating Modifications", filed on June 25, 2004.

As also described herein, the purified peptide product can be provided as part of a kit. The kit may include a purified peptide composition in dry or in solution form, and one or more of an electrolyte, a buffer, a delivery device, a container suitable for mixing the peptide composition with one or more additional agents, instructions for preparing the Peptide composition for use, instructions for mixing the peptide composition with other agents, and instructions for introducing the peptide composition into a subject. The delivery device can be, for example, a catheter, a needle, a syringe or a combination of any of these. When the kits are provided with an aqueous solution of peptide chains, the solution may have a storage life of at least nine weeks under predetermined conditions and may include an electrolyte.

Such kits can administer a biologically active agent in addition to the purified peptide composition. The

Claims (8)

A composition comprising an aqueous solution of at least 2% and up to 5% by weight of amphiphilic peptide chains, wherein the peptide chains are each n-RADARADARADARADA-c (RAD16-I), for its use in a method to facilitate hemostasis by direct injection into a wound site prior to gelation where the ions migrate to the peptide solution of the surrounding tissue and cause it to form a hemostatic gel. The composition for use according to claim 1, wherein said method for facilitating hemostasis comprises stopping blood flow in blood vessels that have been cut during surgery. 3. The composition for use according to claim 2, wherein the blood flow is stopped by embolization. 4. The composition for use according to claim 1, wherein said composition infiltrates dense tissue at the edges of the wound site where the ions migrate to the peptide solution of the surrounding tissue and cause it to form a hemostatic gel. The composition for use according to any one of the preceding claims, wherein the concentration of the peptide chains in the aqueous solution is greater than 2% by weight. 6. The composition for use according to claim 5, wherein the concentration of the peptide chains in the aqueous solution is at least about 3% by weight. The composition for use according to claim 5, wherein the concentration of the peptide chains in the aqueous solution is at least about 4% by weight. The composition for use according to any one of the preceding claims, wherein the composition is at least 75%, at least 80%, at least 85%, at least 90%, at least the 95% or at least 99% pure.