Title:  Peptide-coated implants and methods for producing same

United States Patent:  6,280,760

Assignee:  Merck Patent Gesellschaft mit beschraenkter Haftung (Darmstadt, DE)

Filed:  November 22, 1999

PCT NO:  PCT/EP98/02753

102(e) Date:  November 22, 1999

PCT PUB. Date:  November 26, 1998

Foreign Application Priority Data:  May 22, 1997[DE] (197 21 352);  Dec 16, 1997[DE] (197 55 801);  Apr 23, 1998[DE] (198 18 098)

The invention describes the possibility of the biofunctionalization of biomaterials, in particular implants, by their made-to-measure coating with synthesized cell- or tissue-selective RGD peptides which in vitro stimulate the adhesion of mainly those cell species which in each case are intended to accomplish the tissue integration of the appropriate biomaterial.

Description of the Invention

The invention relates to implants of a general nature for the human and animal body, which are coated with peptides which are able to selectively mediate the adhesion of specific cells in the particular environment of the implant. In particular, the invention relates to implants coated with RGD peptides and processes for their preparation.

The invention is based on the principle of targeted adhesion stimulation of selected cell species to surface coatings of biomaterials in general and implants in particular for the purpose of tissue-selective, accelerated and enhanced integration thereof after surgical insertion into the appropriate tissue.

In this manner, various surface parts of an implant can be coated with and made available to various peptides mediating cell adhesion, in particular RGD peptides, which take account of the specific tissue environment into which the implants are inserted.

In this manner, moreover, with respect to tissue engineering the generation of "intelligent" biohybrid organs which carry the biological information for organ regeneration is possible by self-organization by means of specific activation of various cell species by various peptides in different regions of the implant surface.

The term "peptides according to the invention" in the following includes, if not stated otherwise or additionally, all peptides which are able to mediate cell adhesion. Among these, especially those are intended which contain the amino acids arginine (R), glycine (G) and aspartic acid (D) one after the other (RGD peptides). Examples of suitable RGD peptides and suitable peptides not containing RGD are mentioned further below. Furthermore included are corresponding peptides which do not contain the RGD sequence, but nevertheless affect cell adhesion. In the widest sense, the invention also includes non-peptide compounds which qualitatively have the same biological activity as said peptide compounds.

Biomaterials or implants in the sense according to the invention are designated as materials which can be introduced into the human or animal body in order to restore the function of the corresponding functionally damaged natural tissue. These include, for example, hip endoprostheses, artificial knee joints, jaw implants, tendon replacements, skin replacements, vascular prostheses, heart pacemakers, artificial heart valves, breast implants, stents, catheters and shunts.

The integration behaviour of implants in the body still proves problematical. The tissue integration of the materials often proceeds too slowly and too incompletely in order to produce a mechanical stability of the tissue/biomaterial bonding which is adequate for functionality. The composition of the implant surface, which on account of its inadequate interfacial compatibility or biocompatibility prevents an active absorption of surrounding healthy tissue or cells, is often causally responsible for this. This complicates the formation of a stable tissue-implant boundary layer and thus leads to inadequate tissue integration, which in turn results in loosening, tissue resorption, infections, inflammations, allergies, microthrombi formation (restenosis). As a result, revision interventions for the replacement of the implants (e.g. hip endoprostheses, jaw implants, catheters or external fixators) and thus renewed surgical interventions become necessary (Malchau and Herberts, 1996, Prognosis of the Total Hip Arthoplasty, 63. Annual Meeting of the American Academy of Orthopaedic Surgeons, Atlanta; Haddad et al, 1996, The Journal of Bone and Joint Surgery, 78-B:546-549; Collinge et al., 1996, Pin Tract Infections).

Moreover, in particular in the case of hip endoprostheses, so-called aseptic implant loosening proves problematical in which bone cells and thus bony tissue do not, as desired, form the direct connection to the biomaterial, but fibroblasts and connective tissue occur as interfering elements. As a consequence, the prosthesis is lined by connective tissue instead of bony tissue, the resulting stability of the prosthesis-connective tissue bond not being adequate to meet the mechanical demands on the force transmission of an artificial hip joint. As a result, this can lead to loosening of the prosthesis (Pilliar et al., 1986, Clin. Orthop., 208:108-113) and likewise necessitates revision. A further example of undesirable cell types adhering to implants are blood platelets, which can lead to the formation of microthrombi and thus to impaired implant integration (Phillips et al., 1991, Cell 65, 359).

The lack of integrability of biomaterials or implants into the body has a particularly serious effect in the case of complete replacement organs, since here the different cell types come into contact with the implant and the necessary integrability should be targeted. In order to avoid extremely complicated transplantation procedures with the aid of other patients, it is attempted, for example, to accomplish the therapy of functional failure of liver, pancreas, kidney and spleen more and more frequently in the field of tissue engineering by means of so-called biohybrid organs, which consist of carrier materials which are covered with living cells and can be implanted as a functional unit. In most cases, for this purpose functional, healthy cells are included or encapsulated in vitro in resorbable or non-resorbable membranes and transplanted into the patient as artificial biohybrid organs or hollow organs (for example: Lim et al., 1980, Science 210, 908-912; Altman et al., 1982, Horm. Met. Res. Suppl. 12, 43-45; Zekorn et al., 1989, Transplantation Proceedings 21, 2748-2750; Altman et al., 1982, Horm. Met. Res. Suppl. 12, 43-45; EP 0 504 781 B1). However, here too the problems described of the fibrous ensheathing with associated lack of nutrient supply to the transplants, immunological defence reactions due to cell release from the capsules and the formation of blood clots on account of the thrombogenicity of the material surfaces very often occur.

It is known to stimulate the tissue integration of biomaterials/implants by coating thereof with peptides which mediate cell adhesion. For this purpose, on the one hand, those peptides which contain the tripeptide amino acid sequence arginine-glycine-aspartic acid (RGD), or their non-peptide analogues and, on the other hand, cell adhesion-mediating, non-RGD-containing peptides (for examples see below), or their non-peptide analogues, which, as is known, as integral constituents of many proteins, inter alia of the extracellular matrix (e.g. collagen type I, fibronectin, laminin, vitronectin, entactin, osteopontin, thrombospondin) or of the blood clotting cascade (fibrinogen, von Willebrand factor) function as central recognition patterns for the adhesion of eukaryotic cells (e.g.: Pierschbacher and Ruoslahti, 1984; Nature, 309:30-33; Yamada, 1991, J. Biol. Chem., 266:12809-12812). The sequences defined according to the invention are recognized and bound by the respective receptors on the cell surface, the integrins. Since the adhesion of cells to the corresponding proteins is mediated by a large number of different integrins, the integrin expression pattern of a cell species is crucial for their adhesion properties to these proteins. The made-to-measure design and the synthesis of mostly short-chain peptides equipped with the appropriate sequences, which can bind selectively and specifically only to certain integrins, make possible the targeted activation of only those cell species which express these integrins. Thus, for example, RGD peptides are known which bind selectively to alpha_v -integrin receptors and thus are preferably able to stimulate the binding (adhesion) of alpha_v beta₃ -/alpha_v beta₅ -bearing cells (osteoblasts, osteoclasts, endothelial cells) without simultaneously being able to stimulate the adhesion of undesirable cell species, e.g. .alpha._IIb.beta.₃ -bearing blood platelets (Haubner et al., 1996, 7, Am. Chem. Soc., 118:7461). In contrast, other RGD peptides show a reverse effect and preferably bind to .alpha._IIb.beta.₃ -integrin receptors, thus exhibiting selectivity, for example, for blood platelets (Phillips et al., 1991, Cell 65, 359).

The furnishing of implant surfaces with synthetically accessible peptides defined according to the invention is known. In this case, the peptides are attached to the surface to a greater or lesser extent by adsorption or else by covalent bonding. In DE 1 97 06 667, for example, biomaterials are described which relate to bone replacement materials which are based on a porous polymer material which has a surface covering by peptides with an RGD amino acid sequence due to adsorption. In WO 91-05036, metallic prostheses, in particular of titanium or titanium alloys, are furthermore disclosed to whose surfaces peptides, which inter alia can also have RGD sequences, are covalently bonded. Valentini et al. (May 1997, Transactions of the 23rd Annual Meeting of the Society for Biomaterials, New Orleans, USA) describe the covalent binding of RGD peptides to titanium screws provided with a fluorinated ethylenepropylene intermediate layer. Rezania et al. report on the same meeting of silicon dioxide or titanium dioxide surfaces which are coated by means of covalent bonding with amino functional organosilanes and in turn covalently accomplish by means of a heterobifunctional crosslinker the binding of thiol-containing RGD peptides.

These technical solutions, however, do not go into the requirement of making available implants or biomaterials whose surfaces are coated specifically with peptides defined according to the invention, which are selectively tailored to the particular cell type of the tissue surrounding the implant concerned.

It would therefore be desirable to be able to modify biomaterials in such a way that specifically those tissue or cell species which also, after insertion of the implant into the body, should function actively with these, i.e., for example, bone cells in hip endoprostheses or epithelial cells for skin, hair or tooth replacements, are arranged exclusively or preferably for their tissue integration, while at the same time cell species which interfere with this process, for example, blood platelets or fibroblasts which promote the formation of microthrombi or connective tissue capsules, are to be prevented from undergoing selective interaction with the implant.

It would furthermore be a desirable and attractive strategy to coat implants with those peptides defined according to the invention (or their non-peptide analogues) which exclusively or at least preferably stimulate the adhesion of those selected cell types which bear the corresponding complementary integrins, which as a result leads to the accelerated in vivo synthesis of the corresponding selected tissue.

With respect to the development of complete biohybrid organs (skin, blood vessels, urinary passages, bladder, oesophagus, pancreas, liver, spleen, kidney), it would be a decisive advance to be able to activate the desired various cell species in each case for a certain organ by coating different surface parts of an implant with various cell-selective peptides defined according to the invention in a targeted, spatially defined and coordinated manner for carrying out different cellular in vivo processes.

The present invention now describes the possibility of the biofunctionalization of biomaterials, in particular implants for all conceivable organs by coating thereof with synthesized cell- or tissue-selective RGD peptides defined according to the invention, which in vitro stimulate the adhesion mainly of those cell species which in each case should accomplish the tissue integration of the corresponding biomaterial and which at the same time in vitro do not stimulate the adhesion predominantly of those cell species which oppose this process. With the use of such coatings, an accelerated and enhanced integration of various biomaterials/implants can be achieved with improved long-term stability after their insertion into the body.

Moreover, with this concept of the coating of various material surface parts of an implant with different peptides defined according to the invention all possibilities exist for the development of "intelligent", biohybrid organs ("tissue engineering"), which can carry the biological information for the selective activation of various target tissue or target cells and thus can be integrated into the body by self-organization and by this means can enhance tissue integration or can even make it possible for the first time.

The invention thus relates to an implant which is suitable for different human and animal organs, consisting essentially of a carrier matrix and a peptide coating surrounding this matrix, which contains identical or different peptides for the targeted adhesion stimulation of human or animal body cells, which have sequences which recognize binding sites on the integrin receptors responsible for adhesion on human or animal cells, the carrier matrix having reactive groups capable of binding on its surface, which are capable of entering into a stable covalent bond with appropriate functional reactive groups of said peptide layer, the implant being distinguished in that said peptides are arranged in a locally differing manner on the surface of the implant such that on account of their correspondingly different structure-related, cell adhesion-stimulating activity they correspond specifically to the natural different complementary integrin pattern of the tissue cells adjoining them in the particular region into which cells the implant is to be inserted, by means of which a locally differentiated and selective, bioactive coating pattern of the implant surface is present.

The invention further relates to a process for the preparation of implants suitable for organs/tissue based on an inorganic carrier matrix which have a surface which is coated with the cell adhesion-stimulating peptides, said peptides being selective with respect to the complementary integrin pattern of the tissue cells immediately adjoining the implant, which is characterized in that, by methods known per se, (i) the integrin receptor structure of the target cells or of the target tissue in which the implant is to be introduced in vivo is determined in vitro, (ii) the peptides having the appropriate complementary structure are selected or synthesized and, (iii) said peptides are bonded to the relevant surface of the implant.

In particular, the invention relates to processes and implants/biomaterials having the following characteristics: said peptides, in particular RGD peptides, are attached to the implant surface by covalent bonding, if desired via branched, surface-enlarging molecules and/or molecular anchors; preferably RGD peptides are used which can stimulate alpha_v beta₃ -/alpha_v beta₅ -carrying cells, thus in particular, for example, the adhesion of osteoblasts, osteoclasts, endothelial cells, and at the same time are capable of not stimulating the adhesion of blood platelets or fibroblasts; carrier matrices employed are shaped or unshaped parts made of ceramic, polymer material or metal or a biohybrid organ or hollow organ.

At the molecular level, the peptides defined according to the invention are essentially designed from the following constituents:

an amino acid sequence-bearing domain relevant for adhesion (e.g. the RGD sequence mentioned), which selectively recognizes and binds a selected cell species,

a spacer in order to present the cell-recognizing and the recognition sequence-bearing domain to the cells in such a manner that cell binding is only possible from steric standpoints,

a molecular anchor which effects the stable binding of the peptide derivative concerned to the biomaterial or implant surface,

optionally cell adhesion can be increased by additional coupling of the peptides defined according to the invention to branched molecular structures (so-called dendrimers or Tentakels) which exert a surface-enlarging effect, before the binding to the biomaterial surface takes place.

The surface of the biomaterial or implant is to be understood according to the invention as not only the immediate surface of the carrier matrix, but also an additional coating which may be present of, for example, polymeric material, natural or artificial bone materials, proteins or protein derivatives.

Suitable carrier matrices are especially materials made of ceramic, metal, polymer materials (e.g. PMMA) or preferably resorbable bone replacement materials. Resorbable or biodegradable materials made, for example, from polylactides, in particular racemic D,L polylactide compounds or resorbable calcium phosphate or hydroxyapatite mixtures which can bring about the restoration of the original tissue state and such as are disclosed, for example, in WO 96/36562 or EP 0 543 765 are particularly suitable. Depending on the field of use, collagen or agar may also be suitable as a carrier matrix.

The term "biohybrid organ" is to be understood as meaning a customarily inorganic matrix which is loaded with or bonded to living cells in any manner (see above). According to the invention, this is also to be understood as meaning a corresponding arrangement which is free of cells and only contains the corresponding peptides of different types defined according to the invention on different implant surface parts, which, inserted into the defective tissue, are selectively able to activate the surrounding cells. The advantage of such acellular biohybrid organs is that "intelligent", biocompatible implants which can be produced in a cost-efficient and controllable manner carry the biological information for organ regeneration. The integration of such biohybrid organs into the body is then completed by means of self-organization by endogenous regeneration processes, by means of which immunological defence reactions, such as often occur, for example, due to implanted foreign cells or foreign proteins, can be avoided.

According to the invention, the implants as a rule are present in shaped bodies or prostheses, where the shaped body should be tailored to the particular tissue/bone defect. In the case of biohybrid organs, the prostheses can only consist of membranes or films coated with or without corresponding cells and the peptides defined according to the invention or else the arrangements such as are disclosed, for example, in EP 0 504 781.

Suitable peptides which can be employed according to the invention are all peptides and compounds thereof having non-peptide substituents which contain a domain or amino-acid sequence responsible for cell adhesion and which can bind to the implant surfaces via their peptide and non-peptide substituents. In particular, possible corresponding peptides are those having an RGD sequence.

The following list of preferred peptides and peptide compounds are only intended to have exemplary and no limiting character whatsoever, the following abbreviations being used:

Asp(D)=Aspartic acid

Gly(G)=Glycine

Arg(R)=Arginine

Tyr(Y)=Tyrosine

Ser(S)=Serine

Phe(F)=Phenylalanine

Lys(K)=Lysine

DPhe(f)=D-Phenylalanine

Pro(P)=Proline

Leu(L)=Leucine

Ile(I)=Isoleucine

Val(V)=Valine

Glu(E)=Glutamic acid

Thre(T)=Threonine

Ala(A)=Alanine

(a) Examples of Suitable RGD-containing Peptides

RGD (Arg-Gly-Asp),

GRGD (Gly-Arg-Gly-Asp)(SEQ ID NO:1),

GRGDY (Gly-Arg-Gly-Asp-Tyr)(SEQ ID NO:2),

RGDS (Arg-Gly-Asp-Ser)(SEQ ID NO:3),

GRGDS (Gly-Arg-Gly-Asp-Ser)(SEQ ID NO:4),

RGDF (Arg-Gly-Asp-Phe)(SEQ ID NO:5),

GRGDF (Gly-Arg-Gly-Asp-Phe)(SEQ ID NO:6),

cyclo-RGDfK (Arg-Gly-Asp-DPhe-Lysine),

cyclo-RGDfKG (Arg-Gly-Asp-DPhe-Lys-Gly).

(b) Examples of Suitable Non-RGD-containing Peptides

LDV (Leu-Asp-Val),

LGTIPG (Leu-Gly-Thr-Ile-Pro-Gly)(SEQ ID NO:7),

REDV (Arg-Glu-Asp-Val)(SEQ ID NO:8),

IKVAV (Ile-Lys-Val-Ala-Val)(SEQ ID NO:9),

YIGSRG (Tyr-Ile-Gly-Ser-Arg-Gly)(SEQ ID NO:10),

LRE (Leu-Arg-Glu),

PDSGR (Pro-Asp-Ser-Gly-Arg)(SEQ ID NO:11),

DGEA (Asp-Gly-Glu-Ala)(SEQ ID NO:12),

RYVVLPR (Arg-Tyr-Val-Val-Leu-Pro-Arg)(SEQ ID NO:13).

The peptides defined according to the invention can be either linear or cyclic. The abovementioned peptides and peptide sequences can also occur within longer peptides having, depending on the peptide according to the invention selected, approximately a total of 4 to 20 amino acids. Likewise, amino acids which have the D or L configuration or which are C- and/or N-alkylated are also included according to the invention. Cyclic peptides are understood according to the invention as meaning those peptides which are closed to form a ring via an amide bond, preferably no free carboxyl or amino groups being present in the molecule. RGD peptides according to the invention are particularly preferred, in particular those from the abovementioned list and of these particularly the pentapeptide RGDfK, which in its cyclic form is disclosed in DE-A-1 95 38 741 and is specific for osteoblasts, and also the hexapeptide RGDfKG which is likewise present in its cyclic form and is specific for thrombocytes.

Corresponding linear and cyclic peptides defined according to the invention are described, for example, in the following Patent Applications: EP 0 632 053, EP 0 655 462, EP 0 578 083, EP 0 770 622, DE 1 95 38 741. In particular, those peptides are suitable which bind selectively to alpha_v beta₃ -/alpha_v beta₅ -integrin-expressing cell species (e.g. osteoblasts, osteoclasts, endothelial cells) without simultaneously binding to, for example, .alpha._IIb.beta.₃ -bearing cell species (e.g. blood platelets). The peptides and the corresponding derivatives can be easily synthesized by standard methods if they are not obtainable in another way.

In principle, the peptides defined according to the invention can be attached to the surface of the biomaterial by adsorption or covalent bonding. The adsorption method is less well suited when using different peptides on one and the same implant, since the locally selective differing coating of the surface according to the invention can only be effected in a poorly satisfactory manner using this technique.

The coupling of the peptides or their non-peptide analogues to carrier surfaces by covalent bonding mostly by means of so-called molecular anchors is adequately known per se and has been described, i.e., for example, in Singer et al. (1987, J. Cell. Biol. 104: 573); Brandley, Schnaar (1989, Develop. Biol. 135: 74); Massia, Hubbell (1990, Anal. Biochem. 187: 292); Hirano et al. (1991, J. Biomed. Mat. Res. 25: 1523); Lin et al. (1992, Biomaterials 13: 905); Nicol et al. (1992, J. Biomed. Mat. Res. 26: 393); Dee et al. (1995, Tissue Engin. 1: 135), without in this case, however, the coating of implants being gone into in general and in particular in any manner.

The present invention, then, relates to novel applications of coating methods known per se for the preparation of the implants according to the invention, such as, for example, the "Keyloc.RTM." process (EP 0 712 621), which for the first time was employed according to the invention for the coupling of the peptides mentioned (or their non-peptide analogues) to surfaces which contain acryloyl or methacryloyl anchor components, or the "Silicoater.RTM." process (DE-A 42 25 106), which was used here according to the invention for the coupling of the corresponding peptides by means of acryloyl or methacryloyl anchor components, as a rule via an acryloyl/methacryloyl silane derivative intermediate layer (e.g. 3-methacryl-oxypropyl trimethoxysilane) to the corresponding carrier matrices. A further possibility of bonding the peptides defined according to the invention to the surface of the carrier matrix or of the implant consists in the analogous use of a silanization process which is described in DE-A 43 21005, which originally explains the technical teaching for the coating of pearl luster pigments for water-based coating systems for metals and plastics in the automotive and plastics industry. A process for the coating of gold surfaces with thiol group-bearing peptides, which was originally described in another connection (Heuvel et al., 1993, Analytical Biochem. 215: 223) is furthermore suitable according to the invention.

The processes outlined have until now not been employed for the coating of implants for the purpose of bioactivation thereof.

The coupling of the corresponding peptides defined according to the invention to the implant surface takes place according to the invention via appropriate anchor molecules, i.e. the peptide is, as a rule, not directly attached itself to the implant surface. The insertion of such a molecule, defined in greater detail below, especially has the point of taking into account the steric requirements of the biological receptor on the target cells in connection with the binding of the corresponding peptide.

For this purpose, the implant surface must carry appropriate functional groups or reactive units which make possible binding of the corresponding functional group of the anchor molecule. The functional groups which are to be made available on the implant surface in turn depend on the composition of the actual carrier matrix, which differs according to requirement (metal, plastic, bone materials). In the case of metal implants, it is possible, for example, to generate a surface layer reactive for SH radicals of the anchor molecule by vapour deposition with gold. The silanization of metal surfaces according to known processes (see above) likewise leads to reactive surfaces which can enter into compounds with the suitable anchor molecules according to the invention, if appropriate using silane-containing adhesion promoters (see below). Implants made of natural bone or nature-like bone materials (e.g. calcium phosphate cements) can bond anchor molecules which contain a reactive phosphonate group (principle described in Chu, Orgel, 1997, Bioconjugates Chem. 8: 103). Anchor molecules according to the invention, which for their part themselves have a reactive acrylate radical, can in turn be coupled to implants made from acrylate-based plastic (e.g. PMMA) or from other materials having an appropriate plastic coating.

Anchor molecules in the sense of the invention are thus molecules based on modified or substituted alkyl chains or hydrocarbon chains which have at least two different functional groups, one functional group as a rule being a free carboxyl group (free NH₂ group) which generates an amide bond (--CO--NH--) with a free NH₂ group (free carboxyl group) of a side chain of a peptide defined according to the invention, in particular of an RGD peptide, and the other functional group, which is preferably localized at the other end of the C chain of the anchor molecule and brings about direct or indirect bonding to the implant surface, depending on the composition or requirement of the implant surface, preferably being a (meth)acrylate-containing radical or a mercapto group. In principle, it is also possible to use other functional groups which are able to react with the respective reactive groups directly on the implant surface or on a suitable intermediate layer to give a stable bond.

The anchor molecules of the invention, as already indicated above, at the same time have the function of spacers, i.e. besides their outlined linking options have an appropriate optionally specifically tailored length in order to make it possible that the domain responsible for cell adhesion stimulation has the right distance to the target cell so that cell bonding can be improved or even made possible from steric standpoints.

The biological function of the cell-recognizing and the corresponding amino acid sequence-bearing domain was confirmed by way of example by means of a synthetic peptide binding selectively to alpha_v beta₃ -/alpha_v beta₅ -integrin-expressing cell species (e.g. osteoblasts, osteoclasts, endothelial cells) (Haubner et al., 1996, J. Am. Chem. Soc., 118: 7461-7472).

The anchor molecules of the invention preferably have the following linear structures, the peptides defined according to the invention being bonded via the NH₂ group of one of their amino acid side chains, preferably a lysine side chain, to the free carboxyl end of the respective anchor molecule.

(i) Mercapto(amido)carboxylic acid derivatives:

--CO--(CH₂)_k --X--SH,

where X is a single bond or --CO--NH--(CH₂)_l --,

k=2 to 12 and l=2 to 4;

(ii) Acrylamidocarboxylic acid derivatives:

--CO--(CH₂)_m --[NH--CO--(CH₂)_n ]_p --NH--CO--CH=CH₂,

where m,n=2 to 8; p=0 to 2,

(iii) Acrylamido-amidotriethyleneglycolic acid derivatives:

--(CO--CH₂ --O--CH₂ --CH₂ --O--CH₂ --CH₂ --NH)_q --CO--(CH₂)_r --NH--CO--CH=CH₂

where q=1 to 3 and r=2 to 8.

In particular, the following types of specific anchor molecules are preferred:

(ia) --CO--CH₂ --CH₂ --SH (mercaptopropionic acid)

(ib) --CO--CH₂ --CH₂ --CO--NH--CH₂ --CH₂ --SH (mercaptoethyl-amidosuccinic acid)

(iia) --CO--(CH₂)₅ --NH--CO--CH=CH₂ (acrylamidohexanoic acid)

(iib) --CO--(CH₂)₅ --NH--CO--(CH₂)₅ --NH--CO--CH=CH₂ (acrylamidohexanoic acid-amidohexanoic acid)

(iiia) --CO--CH₂ --O--CH₂ --CH₂ --O--CH₂ --CH₂ --NH--CO--(CH₂)₅ --NH--CO--CH=CH₂ (acrylamidohexanoic acid-amidotriethyleneglycolic acid)

(iiib) --(CO--CH₂ --O--CH₂ --CH₂ --O--CH₂ --CH₂ --NH)₂ --CO--(CH₂)₅ --NH--CO--CH=CH₂ (acrylamidohexanoic acid-diamidotriethyleneglycolic acid)

Generally, according to the invention any anchor molecule structures are preferred which have at least six C atoms in the linear C chain. As a matter of fact, it was surprisingly found that this length of the anchor molecule is particularly favourable in order to achieve optimum results with respect to the accelerated and enhanced tissue integration of the implant. The indication of at least six C atoms in the linear chain relates according to the invention to the total length of the molecule between peptide and implant surface. Thus anchor molecules of the structures shown above having a shorter chain (e.g. type ia) are also suitable if still other unmentioned, chain-lengthening coupling components are inserted between peptide and implant surface.

The anchor molecules are bonded in amide form via the carboxyl function to the peptides defined according to the invention by standard methods, by means of which structures of the type peptide-NH--CO-anchor molecule result which, in turn, as presented, are attached to the implant, by means of which, in turn, constructs of the following type result: peptide-NH--CO-anchor molecule-implant (surface). Corresponding implant constructs are preferred which are composed of one of the defined peptides mentioned individually above, in particular RGD peptides, one of the generally and specifically defined anchor molecules individually mentioned above and an appropriately surface-reactive implant. The following implants are particularly preferred:

cyclo-RGDfK NH--CO--thiol derivatives (type: i)--implant,

cyclo-RGDfK NH--CO--acrylate derivatives (type: ii)--implant,

cyclo-RGDfK NH--CO--acrylate-glycol derivatives (type: iii)--implant,

cyclo-RGDfKG NH--CO--acrylate-glycol derivatives (type: iii)--implant,

in which the linear C chain of the entire anchor molecule has at least six C atoms.

Particularly preferred among these are:

cyclo-RGDfK NH--CO--thiol derivative (type: ib)--implant,

cyclo-RGDfK NH--CO--acrylate derivative (type: iia)--implant,

cyclo-RGDfK NH--CO--acrylate derivative (type: iib)--implant,

cyclo-RGDfK NH--CO--acrylate-glycol derivative (type: iiia)--implant,

cyclo-RGDfKG NH--CO--acrylate-glycol derivative (type: iiia)--implant,

cyclo-RGDfK NH--CO--acrylate-glycol derivative (type: iiib)--implant

The preparation of these preferred structures is carried out by standard methods, or is described further below, or in the parallel application of the Applicant filed on the same day, which relates to the peptide anchor structures as such.

As already discussed further above, essentially three alternative routes are followed for the anchoring of the cell- or tissue-selective peptide derivatives described to biomaterial surfaces according to the invention, it being possible for the molecular recognition pattern of the domain carrying the respective RGD sequence to remain selectively unchanged for a certain cell type and the spacer, whereas the molecular anchor can be varied depending on the coupling variants mentioned, for example by:

coupling of thiol peptide derivatives to gold-coated biomaterial surfaces (e.g. to type (i) anchor molecules);

coupling of (meth)acryloyl peptide derivatives to acrylate- or methacrylate-coated biomaterial surfaces (e.g. to type (ii) or (iii) anchor molecules);

coupling of (meth)acryloyl peptide derivatives to silane-coated biomaterial surfaces (e.g. to type (ii) or (iii) anchor molecules) using a (meth)acryloyl silane derivative as an adhesion promoter or intermediate layer (e.g. 3-methacryloxypropyl trimethyloxysilane).

The attainment of the critical minimum length of the anchor molecule for various peptide coupling variants to biomaterial surfaces is carried out by synthesis of the peptides defined according to the invention with the anchor molecules defined according to the invention of a chain length preferably having 6 to 24 C atoms and alternatively different hydrophobic/hydrophilic properties (e.g. by use of numerically different units of --CH₂ -- and/or amidohexanoic acid or ethylene glycol according to methods standard per se and subsequent testing of the biological activity by determination of the cell adhesion in vitro after coating of appropriate biomaterial surfaces).

In the manner described, depending on the material properties of the implant, a suitable coating process can be selected for the conditioning of the surfaces before the actual coupling with the peptide derivatives according to the invention. Moreover, depending on the tissue type or the cell type which is intended to accomplish the integration of the biomaterial/implant, coating with other peptides is possible which in turn activates the integrins of the corresponding target cell species in a targeted manner, such as, for example, alpha₆ beta₄ -integrin from epithelial cells (e.g. for the use of bone, jaw, skin or hair implants) or alpha_IIb beta₃ -integrin from blood platelets. Alpha_v beta₃ -specific RGD peptides have a selectivity for endothelial cells and osteoblasts, as a result of which, for example, they would be suitable for the coating of vascular prostheses or bone implants. By this means, it is possible to realize a suitable bioactivating surface coating for almost any desired organ for implants in the field of bone, vessel, tooth, skin and hair replacement.

Before appropriate implants or biohybrid organs according to the invention can be made available, the peptides suitable for the particular cell type must be tested and determined beforehand for biological activity in an in vitro test system in order to be able to carry out later a specific and selective coating of the implant which is to be inserted into the selected tissue.

The analysis of the integrin receptor structure of the target tissue or of the target cells into which the implant is to be inserted necessary for this purpose is carried out by means of customary, known, immunohistological processes, such as, for example, by means of immunofluorescence or the immunohistochemistry of tissue samples. The antibodies against various integrin receptors or their subunits necessary for this purpose are meanwhile known and available or can be accordingly produced by means of standard methods known per se, such as, for example, suitable immunizations.

The peptides defined according to the invention are covalently coupled in various concentrations to culture surfaces, for example of polystyrene coated with bovine serum albumin (BSA). The material of this test support does not play any essential part in the determination of the suitable peptides. Likewise, the coupling method used here is additionally of little importance. For practical reasons, the coupling in these determinations can also be carried out by means of incubation and adsorption of said peptides on the test support.

Subsequently, the adhesion of selected tissue cell cultures (e.g. osteoblasts) which are able to correspond in their adhesion properties to the cells which are to be activated in the natural tissue in vivo are investigated on the appropriately coated surfaces. The criterion for the selection of suitable cell cultures for the adhesion experiments consists in the comparable integration expression pattern for the target cells in vivo after implantation, for example their alpha_v beta₃ -/alpha_v beta₅ - or alpha_IIb beta₃ -expression, which is verified by means of fluorescence-labelled antibodies against alpha_v beta₃ -, alpha_v beta₅ - or alpha_IIb beta₃ -integrins, or against the alpha_v -, alpha_IIb, the beta₃ - or against the beta₅ -subunits of the integrin receptor by means of a fluorescence-activated cell sorter (FACS). In the case of other target cell species in vivo with different integrin receptor patterns, other antibodies accordingly have to be employed. These are meanwhile known and available or can be produced according to known standard methods, e.g. by means of a suitable immunization.

The various selected cell species are inoculated and incubated on BSA-pretreated polystyrene culture surfaces coated with the peptides under consideration. Non-adherent cells are then washed off.

The binding behaviour of the different, selected cell species on the test surfaces coated with different peptides defined according to the invention corresponds in the positive case, that is to say if an adequate specificity is present, in each case to a titration curve having a maximum adsorption rate of approximately 60 to 100% of the inoculated cells and to a half-maximal cell binding at an RGD peptide concentration in the coating solution of approximately 5 nM to 5 .mu.M.

In a similar manner, as described for the coupling of suitable peptides to BSA-precoated polystyrene surfaces, anchoring strategies to modified or conditioned biomaterial surfaces are possible using various adhesion promoter intermediate layers.

As a summary, the following can be said:

The implants of the prior art have the following disadvantages:

incomplete, slow implant integration into the tissue,

restricted acceptance in the tissue,

inadequate functional stability of the implant/tissue border layer

lack of stimulating action of the implant on tissue neogenesis

non-physiological properties of the implant surface.

As consequences of this further problems result:

aseptic implant loosenings (e.g. fibrous capsule formation)

local formation of microthrombi,

infections,

inflammations,

tissue resorptions,

revisions.

These problems can be largely eliminated by the process made available according to the invention or the implants produced thereby. The subjects according to the invention are distinguished by:

made-to-measure design of adhesion peptides, which are complementary to the integrin expression pattern of the target tissue/target cells;

selective stimulation of the cell adhesion of the target cells which are to accomplish the tissue neogenesis without simultaneously causing the adhesion of the cells which prevent the process;

coatings of higher stability by means of novel peptide anchor molecules;

acceleration and enhancement of the implant integration process into the tissue.

It was possible to show that the critical steric absolute minimum distance between cell recognition sequence on the peptide and uncoated material surface is between 2.0 and 3.5 nm, preferably between 2.5 and 3.5 nm. Maximum coating rates (80-100%) can be achieved with a minimum distance of 3.0 to 5.0 nm.

Claim 1 of 21 Claims

What is claimed is:

1. An implant suitable for a human or animal organ, comprising a carrier matrix and a peptide coating surrounding this matrix which contains different peptides for the targeted adhesion stimulation of human or animal body cells, wherein

said peptides have a length of 3 to 20 amino acids and comprise sequences which recognize binding sites on integrin receptors responsible for adhesion on said human or animal cells,

the carrier matrix has, bound to its surface reactive groups capable of entering into a stable covalent bond with functional reactive groups of said peptides or of anchor molecules for attaching said peptides to the carrier matrix surface,

and said peptides are arranged of the surface of the implant such that their different structure-related, cell adhesion-stimulating activities correspond specifically to the natural different complementary integrin pattern of said human or animal cells in the particular region into which the implant is to be inserted, thereby forming a locally differentiated, selective, bioactive coating pattern on the implant surface.

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