A GROWTH FACTOR IN CONNECTION WITH ARTIFICIAL TRANSPLANTS

Abstract: This invention utilizes the biological activity of the copper complex of the tripeptide glycyl-L-histidyl-L-lysine. This peptide is covalently bound to artificial transplants where it has a chemoattractive effect and also acts when it is released by hydrolysis of the transplants. The peptide increases fibroblastic collagen synthesis thereby enabling a more rapid replacement of the transplants with human tissue.

Description: Title: 1 A growth factor in connection with artificial trans¬ plants. Technical field: This invention utilizes the biological activity of the copper complex of the tripeptide glycyl-L-histidyl- -L-lysine. This peptide is covalently bound to artifi¬ cial transplants where it has a chemoattractive effect and also acts when it is released by hydrolysis of the transplants. The peptide increases fibroblastic colla¬ gen synthesis thereby enabling a more rapid replace¬ ment of the transplants with human tissue. Background: The evolution of ulticellular organisms is de¬ pendent upon the ability of cells to communicate with each other and with their environment. One method the cells use for this communication is to release pep- tides that induce a specific activity in the receiving cell. Such a peptide with biological activity is the human plasma growth factor copper-binding tripeptide glycyl-L-histidyl-L-lysine (GH -Cu 2+) . L Pickart and S Lovejoy describe the properties of the peptide GHK-Cu 2+ .in Methods in Enzymology, Vol 147 (1987) pp 314-328, Academic Press. It plays a physio¬ logical role in the healing of wounds by stimulation the complex course of events necessary for the forma¬ tion of new tissues such as angiogenesis and axon and dendrite growth in neurons. The peptide has also a chemoattractive effect oil cells necessary for wound- healing such as macrophages, onocytes, mast cells and capillary endothelial cells. Collagen is a fibrous protein that constitutes a quarter of the total amount of protein in the human body. It is the major fibrous element of skin, bone, tendons,cartilage, ligaments and blood vessels. Colla¬ gen is synthesized by fibroblasts, a type of cell lo¬ calized in the area surrounding other cells and tissues. In a publication in FEBS Letters 238 (1988) 343- -346, F-X Maguart et al present data showing that GHK-Cu 2+ stimulates collagen synthesis m cultures of fibroblasts. This stimulation is observed at a peptide concentration as low as 10 -12M and reaches a maximum at 10 -9M where the increase in collagen synthesis is about 80 %. 2+ In European Patent Appliction 0190736, GHK-Cu with a modified C-terminal carboxyl group is used as an ointment -for faster healing of wounds. GHK-Cu 2+ possesses a significant superoxide dis u- tase-like activity with a rate constant of about 25 % of the activity of enzymatic Cu,Zn-superoxide dismuta- se on a molar basis. When wounds and damaged tissue are present, cells from the immune system invade the injured area and large quantities of toxic oxygen radicals are released to kill invading bacteria. These radicals also destroy intact tissue which starts a vicious circle where more radicals are released, thus delaymg healing. GHK-Cu 2+ -superoxide dismutase activ¬ ity detoxifies the tissuedestroying superoxide anions. Aggregation of blood platelets is the first stage of thrombosis. GHK-Cu 2+ inhibits this aggregation and it also inhibits the hormone thromboxane which causes thrombosis. The structure of the -tripeptide GHK-Cu 2+ i.s shown in Fig 1. The affinity of the peptide for copper is very high with a pK for the dissociation constant of about 16. For biological activity the ε-amino group on the side-chain of the lysine must be free. The technical problem: The use of artificial materials in surgery is gradually increasing. In orthopedic surgery, trans¬ plants are used for soft parts such as muscles, tendons and ligaments. In the case of ligaments, the preferred material used has been poly propylen bands which are unfortunately not degradable, and results have been unfavorable in the long run. When degradable material is used to replace ligaments, it is of utmost impor¬ tance that this material is biocompatible. Polymers of lactic acid and glycolic acid are degraded to non- toxic products that living tissues tolerate. In the human body they are hydrolysed to their monomers. Lactic acid is metabolized to carbondioxide and water in the citric acid cycle while glycolic acid is excre¬ ted with the urine or is oxidized to pyruvate and is also metabolized in the citric acid cycle resulting in the same products as for lactic acid. These harmless products are very advantageous when compared with the hydrolysis products of many other polymers. Polymers of lactic and glycolic acid have been used for long time as resorbable suture material. When this material is used for artificial ligaments the main problem is that they hydrolyse so rapidly that the body is not allowed enough time to replace them with its own tissue where collagen constitutes the main part. The rapid hydroliεis could be compen¬ sated for if one could increase the rate of collagen synthesis. The solution: The invention presented here is that material used for transplants incorporates the copper-complex of the 2+ tripeptide glycyl-L-histidyl-L-lysme GHK-Cu . As may be seen in Fig 1, the C-terminal carboxyl group is not involved in the copper binding and for that reason is suitable for the covalent binding of the peptide to transplants. With well-known peptide synthesis, the free car¬ boxyl group of the peptide is coupled to a polymer containing free primary amino groups or to a polymer which can be modified to contain free amino groups. Since the peptide has maximal activity at a concentra- t on as low as 10 -9M where it almost doubles the fibroblasts synthesis of collagen, it is fully suffi¬ cient that one peptide molecule is coupled to a poly¬ mer molecul having a molecul weight of 500 000. Thought of chemoattraction and when the peptide GHK-Cu 2+ is released by hydrolysis of the transplant in the tissues, very specific biological processes are started involving many closely coordinated reactions that must be in balance in order to allow healing to occur. This includes an increase in collagen synthesis, an increase of tissue-protective superoxide dismutase activity, and a chemoattractive effect on among others mast cells and capillary endothelial cells which accu¬ mulate at the transplant -site, there stimulating new formation of blood vessels and the flow nourishment to the area. Best mode of carrying out the invention: Example 1. The free carboxyl group of poly-L-lactic acid are activated with carbodiimide and then allowed to react with an excess of a diamine, NH 2 (CH 2 ) NH 2 where n = = 2-6 depending upon the length of the desired spacer- arm. The carbodiimide will be dicyclohexylcarbodiimid if the reaction is carried out in organic solution, or l-ethyl-3-(3-dimethylaminopropyl-)carbodiimide if the reaction is performed in a water solution. The tripeptide NH 2 -glycyl-L-histidyl-L-lysine-C00H is blocked on the amino group of glycine, the imidazole group of histidine and the ε-amino group of lysine with 9-fluorenylmethyl chloroformate (FM0C-C1) . The free carboxyl group of lysine is activated with car- boldiimide as described above and allowed to react with the free amino group of the derivative of poly- -L-lactic acid [poly-L-lactic acid-C-NH(CH 2 ) -NH . After removal of the protective FMOC-groups with piperidine and the addition of copper (II) acetate the desired product is obtained, the structure of which is shown in Fig 2. In this figure the diamine NH„ (CH 2 ) 2 NH 2 has been used. The same methods of synthesis are also valid for poly-DL-lactic acid, poly-D-lactic acid, polyglycolic acid and their co-polymers. Above, FMOC has been used as the blocking group. Another blocking agent is the tert-butoxycarbonyl group (t-BOC-) . Deblocking after the synthesis is in this case performed with dilute acid (25% trifluoro- acetic acid) . The peptide glycyl-L-histidyl-L-lysine is commer¬ cially available. Other examples Another way for synthesis is by usual peptide synthesis coupling the amino blocked L-lysine, L-histi- dine and glycine one after the other to the amino de¬ rivative of the polymer. Lactic acid and glyσolic acid are α-hydroxy- carboxylic acids. A number of other α-hydroxycarboxylic acids are useful for homo- or co-polymerization. Examples are α-hydroxybutyric -acid, α-hydroxysiobutyric acid, α-hydroxyvaleric acid, cc*-hydroxyisovaleric acid. To increase the amount of peptider per polymer mole¬ cule, one can treat the poly-α-hydroxycarboxylic acid with mild hydrolysis to increase the available number of carboxyl groups. Among β-hydroxycarboxylic acid D- β -hydroxybutyric acid has been used as a polymer for transplants (Bri¬ tish Patent 1034123) . Polymer based on p-dioxanon has also been used as transplants. This has the formula where R and R' represent hydrogen, methyl- or ethyl- group and n the degree of polymerization. Synthetic biodegradable polyester amides have been described by T H Barrows et al at 3M Center, St Paul, Minnesota, USA. These polymers consist of units with the formula -0-CH„-C-NH-(CH„) -NH-C-CH -0-C-(CH ) -C 2 ii 2 ~ n II z i IiI y 0 0 0 n All of these polymers can be coupled covalently to GHK-Cu2+ with the samemethods described above. Another method is to covalently couple GHK-Cu 2+ to other biocompatible materials, which may or may not be resorbed, that contain an amino group or a site where an amino group can be introduced. For example glass or silicic acid can be silylated with 3-amino- propyl-triethoxysilone. In this case a great number of GHK-Cu 2+ could be bound to the glass. When metal transplants are to be made the surface can first be covered with a protein, for instance serumalbumin, and the amino groups bridge-bound with glutardialdehyde. After introduction of new amino groups, the GHK-CU 2+ can be bound to the protein. Fig 1 shows the three-dimensional structure of GHK-Cu 2+. The side-chains of lysine and histidine and the amino terminal of glycin are necessary for the copper-binding and for the biological activity. One of the hydrogens of the " -carbon in the glycin can be ex¬ changed with another radical without disturbing the conformation of the copper-complex. Proposals for new tripepticles can, for example, be Ala-His-Lys, Val- His-Lys or Leu-His-Lys. One advantage of this is that we can use the D-form of the amino-terminal amino acid which renders the peptide more resistant to proteoly- tic hydrolysis. In the practical use of the invention reinforcing material can of course be used. This material can be degredable or not degradable. Although the invention has been described here with reference to certain examples, it should be obser¬ ved the invention is by no means restricted to such specific characteristics since closely related varia¬ tions and modifications are obvious for the specialist in the field. Claims: 1. A method of increasing natural collagen synthesis at the site of an artifical transplant by immobilizing a biologically active agent on the transplant, c h a¬ r a c t e r i z e d b y the fact that this agent is a growth factor for collagen and that it is the copper- complex of the tripeptide glycyl-L-histidyl-L-lysine which is immobilized because the carboxyl-terminal

Claims: carboxyl group of lysine is covalently bound to the transplant. 2. A method according to claim 1 c h a r a c t e¬ r i z e d b the fact that the transplant consists of poly-L-lactic acid, polyglycolic acid or copolymer of this, where the carboxyl group of the polymer has first been bound by a peptidebond to a diamine, pref¬ erably 1,6,-hexandiamine, and where the free amino group of the product is bound by a peptide bond to the carboxyl group of the lysine in the copper complex of 2+ the tripeptide glycyl-L-histidy-L-lysine (GHK-Cu ) . 3. A method accordning to claim 2 c h a r a c t e¬ r i z e d b y the fact that the transplants are homo- or copolymers of the L-hydroxy-derivatives of glycolic acid, lactic acid, butyric acid, isobutyric acid, valeric acid or isovaleric acid. 4. A method according to claim 2 c h a r a c t e¬ r i z e d b y the fact that the transplant is a polymer based on p-dioxanone consisting of units with the formula R R' I I -0-CH-C-O-CH-C- I u R R O n where R and R' indicate hydrogen, methyl or ethyl group and n the degree of polymerization.

Inventors: STRID LARS, STRID, LARS
Applicants: STRID LARS, STRID, LARS
Application year: 1991
Main classification: A61L27
IPC codes: A61K38, A61L27