WO1998025643A2

WO1998025643A2 - Neuropeptides as modulators for cell differentiation and proliferation

Info

Publication number: WO1998025643A2
Application number: PCT/EP1997/006901
Authority: WO
Inventors: Konrad Wolf; Irmgard GÜRTNER
Original assignee: Konrad Wolf; Guertner Irmgard
Priority date: 1996-12-13
Filing date: 1997-12-10
Publication date: 1998-06-18
Also published as: DE19781388D2; AU5855598A; DE19652033A1; WO1998025643A3

Abstract

A medicament for treating tissue lesions, differentiation or granulation deficits and metabolic disturbances contains, as active agent, calcitonin-gene related peptide (CGRP) and/or substance P and/or neuropeptide Y (NPY) optionally with usual pharmaceutical inactive ingredients and/or carriers. In order to produce autologous tissue equivalents, precursor cells taken from a patient are cultivated in vitro under addition of CGRP and/or substance P and/or NPY. Such tissue equivalents are used, in accordance with the invention, as implants in the field of therapy of tissue defects, tissue lesions or granulation disturbances.

Description

Neuropeptides as modulators for cell differentiation and proliferation

description

The present invention relates to drugs for the treatment of tissue lesions, differentiation or granulation deficits and calcium metabolism disorders, containing calcitonin-related peptides (CGRP) and / or substance P or / and neuropeptide Y (NPY), and the use of these substances for the production of autologous tissue equi - valents.

When a joint prosthesis is inserted, the current procedure provides for cementing the implant with bone cement or anchoring the implant by driving in the shaft or fixing the socket with additional screws (Degreif et al., Unfallchirurg 99 (1 996), 744-749). The primary location of the implant is ensured by the close contact with the cortical bone (Ludwig et al., Trauma surgeon 99 (1 996), 750-757). If there is a low-replacement camp, there is the option of introducing additional cancellous bone pulp to increase the osteogenic potency in the interface (Rueger et al., Osteologie Volume 4, Supplement 1 (1 995) 35). Demineralized bone matrix is also used.

Bone fleeces have recently been used which are coated with bone morphogenetic protein (BMP) and which stimulate mineralization and osteoblasts in the surrounding tissue (Dee et al., Biomaterials 1 7 (2) (1 996), 209-21 5). The object of the present invention was to enable anchoring of prostheses as effectively as possible, in particular the use of animal auxiliary materials should be avoided and, above all, the use of patient's own auxiliary materials should be made possible. In contrast to the methods known in the prior art using Spongiosa pulp, demineralized bone matrix or collagen vias with BMP, the concept according to the invention is therefore characterized by the use of the neuropeptides CGRP or / and substance P or / and NPY and the use of patented (autologous) precursor cells which are stimulated by these neuropeptides as implants or implant anchors.

The neuropeptides CGRP, substance P and NPY are to be regarded as neurotransmitters. The definition of a neurotransmitter presupposes that various biochemical, physiological and pharmacological criteria are met, such as synthesis and storage in the neuron, transport of the neurotransmitter for axon termination and release at the nerve endings, and ultimately the effect of the substance on the postsynaptic receptor. Hormones belong to different chemical groups. The neuropeptides are short-chain peptides.

An organism's hormones are substances that are synthesized by specific tissues.

They specifically change the activity of the tissue cells in their target organ.

Recent research has shown that the way hormones work involves various principles. Most hormones bind to receptors on the cell surface and trigger a cascade of enzymatic reactions. The adenylate cyclase cascade - which leads to an increased cAMP level and activation of a protein kinase - is an important transmission path. Cyclo-AMP is synthesized by the adenylate cyclase and cleaved by a phosphodieesterase. Cyclic AMP is formed from ATP under the action of an integral membrane protein from adenylate cyclase. Cyclo-AMP is the second messenger of many hormones. The "first messenger" is the hormone itself. The main features of this model include the following definition: Cells have hormone receptors in their plasma membrane. The association of a hormone with its specific receptor in the plasma membrane stimulates adenylate cyclase, which is also bound to the plasma membrane. ^• The increased activity of the adenylate cyclase increases the amount of cAMP in the cytosol.

The cAMP then changes the speed of one or more processes within the cell.

An important property of the second messenger model is that the hormone does not penetrate the cell, but rather exerts its effect on the cell membrane. The biological effects of hormones in the cell are mediated by cAMP and not by the hormone itself.

Cyclo-AMP is created from ATP, a ubiquitous molecule, in a simple reaction that is driven by the subsequent hydrolysis of pyrophosphate. Although cAMP is synthesized from a substance that is at the heart of the metabolism, cAMP itself does not belong to a main metabolic pathway. It is only used as an integrator of metabolism, not as a biosynthetic starting product or as an intermediate in energy production. This means that its concentration can be checked independently. It is also stable unless it is hydrolyzed by a specific phosphodiesterase. Due to the numerous functional groups, the cAMP can bind firmly and specifically to receptor proteins, for example the regulatory subunits of the protein kinase of the muscle, and thus have allosteric effects.

Among the known neuropeptides, the calcitonin gene-related peptide plays the most important role in the early phase of granulation tissue formation for precursor cell stimulation. The cAMP production of osteoblasts can be increased 30 to 50 times by high CGRP doses (Zaidi et al., Quarterly Journal of Experimental Physiology 72 (1 987), 371-408). This is a clear indication of a hormonal character of CGRP.

Substance P has the highest affinity for the neurokinin 1 receptor; in higher concentrations there is also an interaction with the neurokinin receptor type 2 / type 3. The receptor binding to the neurokinin 1 receptor increases in the presence of bivalent cations, such as Mg ^{2 +} and Mn ^{2 +} (Garrett NE, Rapp, PI, Crawys SC, Kidd, BL, Blake, DR, Role of substance P in inflammatery arthritis, (1 992), Annuals of the Rheumatic Diseases, 51: 1 014-1 01 8) .

The NPY binds to a specific class of receptors on the cell surface of which 2 subtypes are known to date: the Y, and the Y ₂ receptor. The Y, receptor is coupled to an inhibitory G protein and thus has an inhibitory effect on adenylate cyclase (Ahmed, M., Bjurholm, B., Theodorsson, E., Schutzberg, M., Kreicbergs, A., Neuropeptide Y- and Vasoactive Intestinal Polypeptide-like Immunreactivity in Adjuvant Arthritis: Effects of Capsaicin Treatment (1 995), Neuropeptides, 29: 33-43).

Apart from a local receptor-bound effect, a systemic effect can also be postulated.

CGRP

In the transplantation of carcinoma cells from the thyroid marrow, Rosenfeld et al., (1981) discovered a permanent change from high to low calcitonin synthesis. As later DNA and RNA analyzes showed, the calcitonin mRNA in these cells was replaced by a similar, but by about 200 nucleotides longer mRNA, which codes for a 1 28 amino acid long precursor (Rosenfeld et al., Nature 304, 7 (1 983), 1 29-1 35). A peptide with 37 amino acids finally emerges from this precursor: the calcitonin gene-related peptide. The calcitonin gene complex codes for a small group of peptides - the calcitonin family - consisting of the calcitonin itself, the katacalcin and finally the CGRP. The calcitonin / CGRP gene complex comprises at least 2 genes, which are referred to as a and β genes.

σ-CGRP gene

The σ-CGRP gene is located on the short arm of chromosome 1 1, between the catalase and the PTH gene (Kittur et al., 1 985: Höppener et al., Ham. Gent. 70 (1 985), 259-263; Alevizaki et al., Federation of European Biochemical Societies 206 (1) (1 986), 47-52). It consists of 6 exons, extends over 6.5 kilobase pairs and is fully transcribed. Exons 1, 2 and 3 are common to calcitonin and CGRP, with the first exon not being translated in any of the peptides. Both have the same coding and non-coding regions at the 5 'end. In humans, both match in the first 225 nucleotides, which corresponds to 75 amino acids (Craiq et al., 1 982; Jonas et al., Proc. Nat. Acad. Be 82 (1 985), 1 994-1 998).

ß-CGRP gene A second calcitonin / CGRP gene was also found on the short arm of chromosome 1 1 in humans and rats, which appears to have arisen from gene duplication (Steenbergh 1985; Amara et al., 1 985, Alevizaki et al., Federation of European Biochemical Societies 206 (1) (1 986), 47-52). While exons 3 and 5 are very similar to the σ gene, the non-coding 5 'and 3' end regions in particular show significant differences. The active neuropeptide consists of 37 amino acids, a disulfide bridge between positions 2 and 7 and a C-terminal amide group. Biochemical processes enable the artificial synthesis of neuropeptides. Precise knowledge of the amino acid sequence is a prerequisite for this. The human neuropeptide CGRP consists of 37 amino acids of a certain amino acid sequence (Brain et al., Nature 31 3 (1 985), 54-56; Alevizacki et al., Federation of European Societies 206 (1) (1986), 47-52 ).

Table I: Sequence of the α- and β-calcitonin gene-related peptide (CGRP)

1 2 3 4 5 6 7 8 9 10 1 1 12 13 σ-CGRP AJa Cys Asp Thr AJa Thr Cys Val Thr His Arg Leu AJa ß-CGRP Aia Cys Thr AJa Thr Cys Val Thr His Arg Leu AJa

14 15 l ό 17 18 19 20 21 22 23 24 25 26 -CGRP Gly Leu Leu Ser Arg Ser Gly Gly Val Val Lys Asn Asn ß-CGRP Gly Leu Leu Sεr Arg Ser "Met Gly Val Val Lys" Ser Asn

27 28 29 30 31 32 .5 ._> 34 35 36 37 -Amid α-CGRP Phe Val Pro Thr Asn Val Gly Ser Lys Ala Phe -NH ß-CGRP Phe Val Pro Thr Asn Val Gly Ser Lys AJa Phe -NH

In humans, the a- and ß-CGRP sequences differ by three amino acids, in the rat by one. Two of the three human deviations appear in places where h (human) CGRP differs from r (rat) CGRP. A comparison of the sequences of different species (rat, salmon, rabbit, goat, guinea pig, etc.) reveals different amino acids at different positions (Zaidi et al., Quarterly Journal of Experimental Physiology 72 (1 987), 371-408).

The presence of a methionine in human ß-CGRP, but not in σ-CGRP, enables the two to be distinguished chromatographically Peptides (Petermann et al., 1 987). Apart from minimal differences, these two CGRP sequences show the same physiological effects, such as vasodilation, reduction of bone resorption by osteoclasts and increase in cardiac activity (Tippins et al., 1,986, Zaidi et al., Quarterly Journal of Experimental Physiology 72 (1,987 ), 371-408).

Table II: Occurrence and effects of calcitonin gene-related peptides (CGRP)

The authors Bernard and Shih (Peptides 1 1 (1 990), 625-632) first described a stimulative effect on bone formation. After adding different doses of calcitonin gene related peptides, they found an increase in the number and size of bone colonies in the tissue culture. There was also an increase in the formation of the bone colonies, as the cell division increased due to the stimulation of the stem cells and the osteoprogenitor cells. Zaidi et al. (Quarterly Journal of Experimental Physiology 72 (1 987), 371-408) found that cAMP production of the osteoblasts can be increased 30- to 50-fold by high CGRP doses. Osteoblasts have specific CGRP receptors to which the adenylate cyclase is coupled (Zaidi et al., Biochemical Biophysiological Research 2, 1 59 (1) (1 989), 68-71). Stimulation of the osteoblast via the CGRP receptor can be achieved using the neuropeptide CGRP (Bjurholm et al., Journal of Bone and Mineral Research 7 (9) (1 992), 101 1 -101 9).

Substance P

The polypeptide Substance P (SP) consists of 1 1 amino acids. It shows a broad spectrum of activity in the body and a widely ramified distribution. It has both neurocrine and paracrine and limited endocrine effects. The sequence of Substance P has been phylogenetically constant for a long time and shows no or only minimal deviations in vertebrates.

Table III: Sequence of substance P

1 2 3 4 5 6 7 8 9 10 11

Amino acid: Arg Pro Lys Pro Gin Gin Phe Phe Gly Leu Met -NH ₂ Substance P belongs to the family of tachykinins in addition to neurokinins A and B. Their common structural feature is the Phe-X-Gly-Leu-Met-NH ₂ sequence, which is uniform at the carboxy end. The tachykinins bind specifically to the so-called neurokinin receptors, Substance P to the receptor subclass Neurokinin-1 (Cascieri et al., 1 992).

In the central nervous system, Substance P acts as a cotransmitter together with various classic neurotransmitters, e.g. GABA, serotonin or noradrenaline. In the brain, SP-positive neurons are found in the basal ganglia (striatonigralis tract, striatopallidal tract) as a paracrine effector in the pituitary system and as a mediator in the limbic system. The neuropeptide has also been detected in cortical interneurons and in spinal projection pathways.

On the one hand, Substance P appears in the periphery as an agonist in pain management. In the nociceptive system we find Substance P represented on all levels, from the first (peripheral) neuron via strand cells or interneurons in the spinal cord to central pathways such as the spinothalamic tract.

On the other hand, the neuropeptide is represented in a wide variety of local control loops. This is how it was found in perivascular nerve fibers of the entire vascular system. In vitro and in vivo experiments demonstrated its strong vasodilatory effect and participation in the control of local blood flow. Substance P is involved in pain transmission, vasodilation and histamine release and in the organism's reaction to inflammatory processes.

SP-positive neurons of the adrenal medulla, carotid body and gastrointestinal tract are assigned to the APUD system (amine precursor uptake and decarboxylation). As cells of the "peripheral endocrine system", they can characteristically both in secrete the bloodstream (endocrine) as well as synaptically (neurocrine) or into the surrounding tissue (paracrine).

In the gastrointestinal tract, Substance P is not only a carrier of the intramural nervous system, but also of extrinsic, vegetative innervation. In addition to the vasodilatory effect, the contraction-stimulating effect of the neuropeptide on the non-vascular, smooth muscles plays a functionally important role in the digestive system. In addition to vasodilation, this results in the second cross-organ reaction to the release of the peptide. Other places of action are the lungs (bronchoconstriction) and presumably the smooth muscles of the urogenital tract (ureter, bladder, uterus).

In the bone, the periosteum, the epiphysis and other bone-cartilage borders have the highest concentration of the neuropeptide. Furthermore, nerve fibers containing SP could be detected in all other bone areas. The sprouting of SP-positive nerve fibers in a heterotopic bone implant suggests that they belong to the innervation of the bone (Bjurholm et al., Arthritis-Rheum. 33 (6) (1 990), 859-865).

The transmitter has a significant influence on the regulation of local blood circulation, the inflammatory response and the nociception. Furthermore, the afferent neurons are said to have an effect on the pressure sensation of the bone (Gronblad et al., 1 984). The removal of mechanoreceptors of the periosteum can, however, as demonstrated by Aro et al., 1 985, on the rat fibula lead to the nonunion of a fracture. Table IV: Occurrence and effects of Substance P

The neuropeptide SUBSTANZ P can stimulate the osteoblast (Bjurholm et al., 1 992)

Neuropeptide Y

Biochemistry of the neuropeptide Y

The neuropeptide Y consists of 36 amino acids. Its sequence shows a tyrosine at the N-terminal and a tyrosinamide at the C-terminal. The neurotransmitter belongs to the peptide family of the better known pancreatic polypeptide (PP) and the neuropeptide YY (NYY).

NPY binds to its own specific class of receptors on the cell surface, of which two subtypes are known to date: the Y1 and the Y2 receptor. The Y1 receptor is coupled to an inhibitory G protein and therefore usually has an inhibitory effect on adenylate cyclase or other second messenger systems.

Occurrence and effect of neuropeptide Y

The neuropeptide Y has so far been detected in the CNS in the pituitary system and in the spinal cord, mainly in the substantia gelatinosa. NPY has an inhibitory effect on the transmission of pain in inhibitory interneurons.

In the periphery, NPY is represented as a co-transmitter in the autonomic nervous system and in cells of the APUD system. The peptide usually appears to have an inhibitory function in the gastrointestinal tract: on the one hand, the inhibition of fEPSP (fast, excitatory, post-synaptic potentials that result in an action potential when summing and consequently a forwarding of arousal) has been achieved in nerve fibers others described an inhibition of gastrointestinal motility. There is also a vasoconstrictive effect. In addition to the abdomen, other parts of the body, such as the heart and cerebral vasculature, also contain perivascular, NPY-containing nerve fibers that cause vasoconstriction.

Occurrence and effect of neuropeptide Y in the bone

NPY fibers innervate the whole bone, preferably transition regions such as the bone-cartilage border or the surrounding soft tissue. Neuropeptide Y has a specific receptor on the osteoblasts and has an inhibitory effect on stimulation of osteoblast activity caused by PTH, noradrenaline or norephinephrine. It completely or partially suppresses an increase in the intracellular cAMP level caused by the substances mentioned above. NPY is therefore involved in modulating bone growth and remodeling.

Table for 2.5.3.4 Occurrence and effect of neuropeptide Y

c

In our own investigations we have found that the neuropeptide hormones lead to massive stimulation of precursor cells.

There are conductive elements, i.e. the ingrowth of the nerve fibers from outside into the fracture gap, and inductive elements, i.e. the nerve stimulation outside the fracture gap or the stimulation of the precursor cells in the fat marrow.

The present invention relates to a medicament for the treatment of tissue lesions, differentiation or granulation deficits and calcium metabolism disorders, which is characterized in that it contains calcitonin gene-related peptides (CGRP) or / and substance P and / or NPY, if appropriate, with conventional pharmaceutical contains table auxiliaries and / or carriers.

The medicament according to the invention makes it possible to combat tissue lesions, fracture healing disorders, differentiation or granulation deficits or calcium metabolism disorders in a variety of forms. This is made possible by the stimulating effect of CGRP, substance P or NPY on cells, whereby both cell division and cell differentiation are promoted. Blood circulation and revascularization are also promoted.

In a preferred embodiment of the invention, the active ingredient, that is to say CGRP or / and substance P or / and NPY, is introduced in or on a biologically or medically suitable carrier. In principle, all carriers known to date are suitable for this, human embryonic collagen type III or other bone substitution material being particularly preferred. However, all endoprosthetic surfaces are suitable for applying the active substances according to the invention to them, or if so is a porous material, to be introduced into it and then to be introduced into wounds or bone gaps or other defects.

Is preferred in the novel drug CGRP and / or be contained in a concentration of 1 0 ^{~ 21} up to 10 ^"3 mol / l substance P or and NPY, which then amount to be administered is preferably in the femtomole range. The stimulation of the precursor cells in the fatty marrow by Neuropeptide hormones lead to a precursor cell proliferation of 3-18 million cells / cm ³ , as shown by our own test results (Wolf et al., 1 997).

The medicament according to the invention can additionally contain other active constituents, growth factors in particular being regarded as preferred, with particularly preferred growth factors being the BMP proteins.

Another object of the present invention is the use of CGRP or / and substance P or / and NPY for the production of autologous tissue equivalents, wherein progenitor cells taken from a patient are cultivated in vitro with the addition of CGRP or / and substance P or / and NPY. In the context of the present invention it was surprisingly found that by adding CGRP or / and substance P or / and NPY to tissue cultures it is possible to cultivate them very effectively and in this way to produce tissue equivalents which make it possible for patient's own material to be grown to obtain a sufficient amount that this can be reimplanted. These tissue equivalents have the advantage that they avoid the risk of disease transmission (HIV transmission, hepatitis transmission, problems with BSE when using animal material) and that rejection reactions only occur in a much smaller form. When using CGRP or / and substance P or / and NPY according to the invention, the active substance is added to the culture medium in a concentration of 10 ^"21 to 10 ^{" 3} mol / l. In addition, the precursor cells can preferably be stimulated to grow biomechanically, electromagnetically or by changing the oxygen partial pressure.

In the use according to the invention, it is also advantageous to use culture medium with autologous blood and / or serum components. As a result, further transmission paths for diseases are excluded, which is a great advantage. In a preferred embodiment of the invention, the precursor cells are applied to a carrier. This carrier facilitates the extraction of the cell culture and also later facilitates the introduction of the autologous tissue equivalents to the place where they are supposed to be effective. It is particularly advantageous here if the carrier consists of resorbable material. The carrier also particularly preferably consists of autologously obtained material (e.g. cancellous bone).

Another object of the invention is the use of the medicament according to the invention for the treatment of tissue lesions, differentiation or granulation deficits, e.g. of bone, cartilage, skin and soft tissues, for the treatment of bone metabolic disorders, in particular calcium metabolic disorders, or for the treatment of metabolic metabolic disorders.

The tissue equivalents produced according to the invention are preferably used as implants in the field of therapy for tissue defects, tissue lesions or granulation disorders. The tissues are preferably soft parts, cartilage or bones. Endoprostheses can also be coated with the tissue equivalent according to the invention. In a further preferred embodiment, one sets also autologous bone matrix too. Finally, erythrocytes or other cells are preferably added during implantation.

The use of the autologous tissue equivalents produced according to the invention or their production is further elaborated in the following:

Surgical debridement is required to prepare the recipient site. The debridement opens larger blood vessels and capillaries. A large collection of erythrocytes and individual granulocytes with platelets fill the room. A fibrin network promotes the migration of new cells. Inflammatory cells such as macrophages, multinucleated leukocytes and mast cells migrate early. The phagocytosis of the immigrating granulocytes and monocytes begins (Sedlarik, wound healing (1 993), publisher K.M. Sedlarik. Publisher: Gustav Fischer-Verlag, Jena-Stuttgart).

Implanted precursor cells begin to proliferate after a few hours. After a few days, the first formation of vascular sinuses shows a capillary sprout. Later, phagocytic cells decrease in favor of fibroblasts. CGRP-positive, substance P-positive and NPY-positive nerve fibers growing from the recipient camp maintain a further stimulation of precursor cell tissue.

CGRP or / and substance P or / and NPY introduced into the wound stimulate the precursor cells already present in the wound and promote the consolidation of granulation tissue and the formation of new capillaries.

Bone gap healing was investigated experimentally (rabbit) on the defined fracture gap model. The granulation tissue removed from the fracture gap contained stem cells or precursor cells in high concentration. CGRP-positive fibers were found after a period of 5 days from the edge of waxing (conductive). The histology of the granulation tissue at this time showed the detection of precursor cells. In addition, neuropeptide-positive fibers (CGRP and substance P) were detected at the same time as the formation of vascular sinusoids and microcapillaries and a precursor cell cluster. NPY-positive nerve fibers were detected in the fat marrow, which is the production site of the precursor cells, after a period of 5 to 10 days. Perivascular, subendothelial, extracellular in the vicinity of the precursor clusters, but also in the fibrin lattice, CGRP- and substance P-positive fibers could be regularly detected (Wolf et al., 60th Annual Meeting of the German Society for Trauma Surgery, Springer Verlag, Heidelberg-New York, 88 (1,996)). The NPY regulates the blood supply to the nerve fibers in the fat marrow.

Resume

The immunocytochemical test results prove that the neuropeptide hormones described are potent stimulators of precursor cell proliferation and differentiation. The investigations with the laser Doppler flowmetry showed an increased blood flow after stimulation by the described neuropeptide hormones for the microcirculation in the granulation tissue of the fracture gap (Wolf et al., 1 997).

The medicament according to the invention or the tissue equivalents can therefore be used:

1 . as a bone cell stimulator for bone defects in benign or malignant bone tumors in fractures with indication for osteosynthesis - in fractures with segmental defects for implant fixation of the prosthetic hip joint, the shoulder joint, the knee, wrist, finger joint and other joint replacement prostheses in the callus - at corrective osteotomy in bone graft with vascular connection to the implant anchor according to prosthetic replacement of hip prostheses, shoulder joint prostheses, knee joint prostheses, wrist prostheses and finger joint prostheses, and other joint replacement prostheses in nonunion (Wrist joint formation after fractures)

2. As a cartilage cell stimulator: for cartilage defects on the hip, knee, shoulder, ankle, hand, finger joint in arthrosis in intra-articular fractures with cartilage detachment in benign or malignant cartilage tumors in autoimmune diseases that cause cartilage damage, e.g. Rheumatoid arthritis

3. as a stimulator of wound healing: with extensive soft tissue defects in diabetes mellitus - with surgery-related wound healing disorders in chronic wounds

There are further possible uses for diseases involving bone metabolism, such as Sudeck's disease, osteoporosis, Paget's disease and hypercalcaemia syndromes for tumor-related osteolysis, pancreatitis etc. A central aspect of the invention is the use of the neuropeptides calcitonin gene related peptides (CGRP or / and substance P or / and NPY). The multiplication and differentiation of progenitor cells, tissues or their equivalents is stimulated. Tissue equivalents are cell aggregates or collagen fiber structures generated in vitro from corresponding progenitor cells. Optionally, combined growth factors, biomechanical stimulation processes and electromagnetic field stimulation processes are used. The local as well as the systemic effect of the CGRP, substance P, NPY on the osteogenesis should be used.

For the combined stimulation of cell proliferation, all growth factors are generally to be mentioned, e.g. IGF, FGF, EGF, TGF alpha, TGF beta, KGF, PDGF, PRP, ILGF, T3 / T4, parathyroid hormone etc. "Bone morphogenic protein" (BMP) represents a family of currently 28 known factors that are specific to biosynthesis of bone material interact (Bjurholm et al., Peptides (9) (1 988), 1 65-1 71; Bjurholm, 1 990).

The biochemical activation cascade of the CGRP or substance P takes place via the adenylate cyclase cascade and leads to an increased cAMP level or to the activation of the protein kinase. There are also points of contact in the biomechanically activated inositol-phosphate cascade. The products inositol, diaglycerol, calmodulin and prostaglandin E cause intracellular calcium influx on the cell membrane via appropriate receptor structures. The NPY has an inhibitory effect on the adenylate cyclase system via the Y receptor.

The cell proliferation is achieved with electromagnetic field stimulation (stimulation 1 994). Here too, cell metabolism processes of regulation and reaction are regulated by appropriate mediators. The growth of the cell culture is said to be influenced by varying the oxygen partial pressure. The oxygenation of the tissue ultimately depends on the vascularization, which is modulated in its different quality by the model variation of the oxygen partial pressure.

In the literature, Bjurholm (1 990) described an occurrence of neuropeptide-positive nerve fibers in combination with particles of a demineralized bone matrix - embedded in rat muscles.

Experimental work has shown that culture medium enriched with autologous serum instead of commonly used fetal calf serum (FCS) led to an improvement in the proliferation rate (donor = recipient). This avoids danger points such as the transmission of hepatitis or HIV (AIDS).

The carrier medium represents a possibility of bringing the cells to the place of their transplantation and embedding them there. Generally, biodegradable, resorbable materials are ideal. Collagen is one of the most commonly used materials. The carrier medium can consist of embryonic, recombinant collagens which are in the solid or liquid phase. The carrier medium can be altered in a corresponding manner as:

Collagen vias

Collagen seeds (granular shape) - collagen pins

Collagen granules collagen powder

Semiliquid form (in combination with other substances, e.g. fibrin or fibrin glue / gel) - capsule (delayed release by a calcium phosphate / apatite

Wrapping) Sponge (calcium apathite sponge, coated with neuropeptide hormone)

Spray (hydrophilic / lipophilic liquid / particle / gas mixture) coating of a solid material (e.g. endoprosthesis or osteosynthetic material)

Alternatively, autologous material can be used, e.g. proliferated fibroblasts that differentiate into fibrocytes and also synthesize collagen material. This collagen material can be used as a carrier medium. Bone wax is also a suitable carrier material. The erythrocytes as such can be used as a carrier medium. The fracture hematoma is also the starting point for the infusion of connective tissue cells in physiological healing, so that the same material is introduced here by analogy.

Claims

Expectations

1 . Medicament for the treatment of tissue lesions, differentiation or granulation deficits and metabolic disorders, characterized in that it contains calcitonin-gene-related peptides (CGRP) or / and substance P or / and neuropeptide Y (NPY) as the active ingredient, optionally with conventional pharmaceutical auxiliaries and / or carriers .

2. Medicament according to claim 1, characterized in that the active ingredient is introduced into or onto a biologically or medically suitable carrier.

3. Medicament according to claim 1 or 2, characterized in that human embryonic collagen type III or another bone substitution material is present as a carrier.

4. Medicament according to one of claims 1 to 3, characterized in that it contains CGRP or / and substance P or / and NPY in a concentration of 10 ^"21 to 1 0 ^'3 mol / l.

5. Medicament according to one of the preceding claims, characterized in that it additionally contains other active ingredients, preferably growth factors.

6. Medicament according to claim 5, characterized in that the further active ingredient is a BMP protein.

7. Use of CGRP or / and substance P or / and NPY for the production of autologous tissue equivalents, characterized in that progenitor cells taken from a patient are cultivated in vitro with the addition of CGRP or / and substance P or / and NPY.

8. Use according to claim 7, characterized in that CGRP or / and substance P or / and NPY are added to the culture medium in a concentration of 1 0 ^"21 to 1 0 ^{" 3} mol / l.

9. Use according to claim 7 or 8, characterized in that the precursor cells are additionally stimulated biomechanically, electromagnetically or by changing the oxygen partial pressure.

1 0. Use according to one of claims 7 to 9, characterized in that culture medium with autologous blood or / and serum components is used.

1 1. Use according to one of claims 7 to 10, characterized in that the cells are applied to a carrier / implant.

1 2. Use according to claim 1 1, characterized in that the carrier consists of resorbable material.

1 3. Use according to claim 1 1 or 1 2, characterized in that the carrier consists of autologous material.

14. Use of a medicament according to one of claims 1 to 6 for the treatment of tissue lesions, differentiation or

Granulation deficits, e.g. of bone, cartilage, skin and soft tissues, for the treatment of bone metabolic disorders or for the treatment of metabolic metabolic disorders.

1 5. Use of tissue equivalents, which were produced according to claims 7 to 1 3 as implants in the field of therapy for tissue defects, tissue lesions or granulation disorders.

1 6. Use according to claim 1 5, wherein the tissues are bones.

1 7. Use according to claim 1 5 or 1 6, characterized in that autologous bone matrix or autologous recombinant carrier material is also added.

1 8. Use according to claim 1 5 to 1 7, characterized in that erythrocytes or other cells are also added.