WO2023025914A1 - Compositions comprising igf-1, igfbp-2 and insulin, and use thereof - Google Patents
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- WO2023025914A1 WO2023025914A1 PCT/EP2022/073729 EP2022073729W WO2023025914A1 WO 2023025914 A1 WO2023025914 A1 WO 2023025914A1 EP 2022073729 W EP2022073729 W EP 2022073729W WO 2023025914 A1 WO2023025914 A1 WO 2023025914A1
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1754—Insulin-like growth factor binding proteins
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Definitions
- compositions comprising IGF-1, IGFBP-2 and insulin and their use
- the present invention relates to compositions and kits for specifically influencing the energy metabolism in muscle and adipose tissue.
- the invention teaches a composition or kit that provides the three components insulin, IGF-1 and IGFBP-2.
- the three components can be present in the composition or the kit as a protein or can be encoded by at least one nucleic acid contained in the composition or the kit, the nucleic acid preferably being RNA.
- the invention discloses the use of the composition or kit in a method for increasing the intramuscular fat content in meat of farm animals or in v/yro culture.
- composition or kit according to the invention can also be a pharmaceutical composition or a pharmaceutical kit used in the treatment of insulin resistance, muscular dystrophy, the maintenance and regeneration of neuronal functions and the treatment and/or prevention of neuropathy Can be found.
- present invention discloses the use of the pharmaceutical composition and pharmaceutical kit of the invention for treating an injury to a muscle, ligament and/or tendon in a subject.
- the meat of the Japanese Kobe beef (also known as Wagyu beef) is world famous for its exceptionally high meat quality.
- the Wagyu meat is characterized by a particularly tender structure and an exceptionally high intramuscular fat content, which is perceived as fine marbling of the meat.
- the excellent quality of the Wagyu meat requires an extraordinarily costly and time-consuming rearing of the cattle.
- the animals receive a special feed mix made from selected ingredients and are then allowed to socialise roam freely in small herds on meadows and pastures.
- Wagyu cattle live significantly longer than conventional beef cattle before they are ready for slaughter and are usually smaller and lighter when they are slaughtered.
- the effort that goes into raising Wagyu cattle in order to obtain meat of the highest quality is reflected in the price: Wagyu beef is considered the most expensive meat in the world.
- the invention discloses a composition or kit providing IGF-1, insulin and IGFBP-2, wherein
- the IGF-1, insulin and/or IGFBP-2 is present as a protein, or (b) at least one nucleic acid is present which codes for IGF-1, insulin and/or IGFBP-2.
- Insulin is a hormone that works with glucagon to regulate the concentration of glucose in the blood. While glucagon causes an increase in the concentration of glucose in the blood, insulin promotes the uptake of glucose by muscle and fat cells, which leads to a reduction in blood sugar levels. To do this, insulin must dock to a specific insulin receptor, which is expressed as a transmembrane protein. The interaction of insulin with its receptor triggers a cascade of phosphorylation reactions, which ultimately leads to the activation of specific glucose transporter proteins, thus allowing glucose to enter cells. Fully matured insulin consists of an A chain and a B chain and is normally produced in the ß-cells within the islets of Langerhans in the pancreas. It is only released into the blood in response to the ingestion of carbohydrate-rich food.
- the insulin-like growth factor 1 (English insulin-like growth factor 1) is a growth factor with high sequence homology to insulin, which is mainly formed in the liver with the participation of the growth hormone somatotropin. IGF-1 production occurs throughout life, but peaks during the growth spurt of puberty. IGF-1 is able to stimulate the growth of almost every type of cell in the body, including by stimulating the AKT kinase signaling pathway. At the same time, IGF-1 is a potent inhibitor of programmed cell death. However, due to its growth-promoting and apoptosis-inhibiting function, overexpression of IGF-1 has also been implicated in the development and progression of cancer.
- IGFBP-2 insulin-like growth factor binding proteins
- IGFBP-2 in addition to its inhibitory function, can also trigger activation of the AKT kinase signaling pathway together with IGF-1 and consequently cause the migration and proliferation of vascular smooth muscle cells (Shen et al. 2012).
- IGFBP-2 appears to stimulate the receptor protein tyrosine phosphatase ß (RPTPß), which leads to dephosphorylation and thus inactivation of the lipid phosphatase PTEN.
- RPTPß receptor protein tyrosine phosphatase ß
- the IGF-1 binds to its specific receptor, thereby activating vimentin. Vimentin then also binds to RPTPß.
- PTEN phosphatidylinositol(3,4,5)-trisphosphate
- the invention takes advantage of these findings and provides a novel approach whereby a combination of IGF-1 and IGFBP-2 can be used to promote muscle growth and/or fat storage by activating AKT signaling and promoting increased insulin effectiveness to stimulate purposefully.
- a relevance of a combined activity of IGF-1 and IGFBP-2 for the effectiveness of insulin has not yet been described.
- the invention provides a composition or kit that provides insulin along with IGF-1 and IGFBP-2 as components and can be administered, for example, to a subject or to in vitro culture to measure the effect of the provided insulin within the subject or to potentiate the culture.
- IGF-1 in the form of a composition or a kit with IGFBP-2 as an additional growth-promoting component, the dose of the provided IGF-1 can also be reduced compared to pure IGF-1 preparations in order to avoid uncontrolled cell growth and the formation prevent malignant diseases.
- composition according to the invention or the kit according to the invention can comprise or provide further ingredients.
- components only refers to insulin, IGF-1 and IGFBP-2.
- the kit may comprise a single compartment in which the three different components are present together as proteins and/or the at least one nucleic acid encoding one or more of the components.
- the components or the at least one nucleic acid can be divided into different compartments of the kit and, optionally, only brought into contact with one another before or during use of the kit.
- the kit or composition can be stored at a temperature of 2-6°C for several days to several weeks, eg 1 day to 4 weeks, 1 day to 3 weeks, 1 day to 2 weeks or 1 day to 7 days.
- the kit or composition can be stored at 2-6°C for less than 2 weeks.
- the kit or composition can be stored at -80°C for a period of several weeks to several months before use, e.g. B. from 4 weeks to 24 months, from 2 months to 20 months, from 4 months to 16 months, from 8 months to 14 months or for about 12 months.
- the kit or composition can be stored at -80°C for even longer periods of time before use, e.g. B. for 3 years, for 4 years, for 5 years, for 6 years, for 7 years, for 8 years, for 9 years or even for up to 10 years. Lyophilization of components and resuspension prior to administration are also possible.
- At least one of the three components i.e. insulin, IGF-1 or IGFBP-2, is present as a protein in the composition or the kit according to the invention.
- the composition according to the invention or the kit according to the invention comprises two or else all three components as proteins.
- the three components can in this case be present in equal parts in the composition or kit, ie approximately in an equimolar ratio.
- the provision of the growth hormone IGF-1 could be associated with an increased risk of cancer, it can be advantageous within the meaning of the invention if the IGF-1 protein is present in a lower proportion in the composition or the kit according to the invention than the other two components.
- the relative IGF-1 content in the composition or the kit according to the invention By reducing the relative IGF-1 content in the composition or the kit according to the invention, its anabolic, ie growth-promoting potential can be restricted.
- a larger relative IGF-1 content in the composition or kit enhances the formation and regeneration of the body's own tissues, which may be desirable depending on the use of the composition or kit.
- the ratio between insulin, IGF-1 and IGFBP-2 can deviate from the 1:1:1 ratio.
- the ratio of the three components in the composition or kit to one another can be 0.1-10:0.1-10:0.1-10.
- the physiologically effective doses for IGF-1, IGFBP-2 or insulin are 10-1000 ng/mL, 30-2000 ng/mL, or 10-1000 ng/mL serum or culture medium. Such concentrations can also be used according to the invention.
- an IGF-1 concentration of 10-1000 ng/mL eg 20-500 ng/mL, 25-100 ng/mL or 25-50 ng/mL
- preferably about 25 ng/mL can be used.
- the concentration of IGFBP2 can be, for example, 30-2000 ng/mL, preferably 100-500 ng/mL.
- a concentration of 5-1000 ng/mL of insulin can be used, preferably about 10-600 ng/mL, 20-200 ng/mL or 50-70 ng/mL, or about 60 ng/mL.
- -Additional effects are possible through further increases in concentration, for example because insulin can also bind to the IGF-1 receptor at higher concentrations.
- the components are provided as fully translated and correctly folded polypeptides in the composition or kit.
- the components present as proteins are preferably completely processed.
- the insulin is preferably provided without the 31 amino acid C peptide or the 24 amino acid signal sequence, and is therefore neither preproinsulin (signal peptide - B chain - C peptide - A chain) or proinsulin (B chain - C-peptide - A chain) but as fully matured insulin (B chain - A chain) in the composition or kit.
- the components are provided in an incompletely processed form.
- composition according to the invention or the kit according to the invention can also comprise only preproinsulin or proinsulin, but also any desired mixture of preproinsulin, proinsulin and/or matured insulin.
- preproinsulin or proinsulin for example, can also comprise only preproinsulin or proinsulin, but also any desired mixture of preproinsulin, proinsulin and/or matured insulin.
- not all of the components of the composition or kit of the invention are provided as a protein.
- only one or two of the three components can be provided as a protein.
- the other components are provided by means of at least one nucleic acid which encodes these components. Accordingly, the components of the composition according to the invention or of the kit according to the invention encoded by the at least one nucleic acid are only produced after they have been used in a cell of the target tissue.
- the composition according to the invention or the kit according to the invention comprises at least one nucleic acid which codes for IGF-1, insulin and/or IGFBP-2.
- the at least one nucleic acid thus encodes at least one component of the composition or kit, while the other components are provided as a protein, as described herein.
- the at least one nucleic acid can also code for more than one component of the composition or kit, eg for two or all three of the components.
- the at least one nucleic acid can encode insulin and IGF-1, while IGFBP-2 is provided as a protein. All three components, ie IGF-1, insulin and IGFB-2, are preferably encoded by at least one nucleic acid.
- one, two or all three components in the composition or kit are both provided as a protein, as well as in the form of at least one nucleic acid encoding at least one of the components.
- the respective components of the kit according to the invention or the composition according to the invention are encoded by separate nucleic acids.
- a first nucleic acid codes for insulin
- a second nucleic acid for IGF-1 and a third nucleic acid for IGFBP-2 are encoded by separate nucleic acids.
- one and the same nucleic acid can also code for several components of the composition according to the invention or the kit according to the invention.
- a first nucleic acid can encode both insulin and IGF-1
- a second nucleic acid encodes IGFBP-2.
- a single nucleic acid encodes all three components, ie both insulin and IGF-1 and IGFBP-2.
- the at least one nucleic acid of the kit according to the invention or the composition according to the invention is DNA.
- the DNA can be, for example, a DNA vector, e.g. a plasmid derived from bacteria or baker's yeast, a DNA vector of viral origin or a minicircle, which comprises at least one transgene coding for at least one of the components of the composition according to the invention or the kit according to the invention.
- a DNA vector e.g. a plasmid derived from bacteria or baker's yeast
- a DNA vector of viral origin or a minicircle which comprises at least one transgene coding for at least one of the components of the composition according to the invention or the kit according to the invention.
- the person skilled in the art is readily able to select a suitable DNA vector for providing the at least one transgene.
- the at least one transgene can be the respective gene for insulin, IGF-1 or IGFBP-2, which occurs endogenously in the organism to be treated with the kit according to the invention or the composition according to the invention, or originates from the same organism from which an in vitro culture to be treated has been derived.
- the composition or the kit according to the invention is intended for use in a human subject, for example, the at least one transgene is preferably the human gene for insulin, IGF-1 and/or IGFBP-2 (and/or the respective human protein).
- the at least one transgene is preferably the human gene for insulin, IGF-1 and/or IGFBP-2 (and/or the respective human protein).
- there is also cross-reactivity between species such that, for example, mouse IGFBP2 is also effective in humans.
- bovine or mouse proteins or the genes coding for them can be used, also in different combinations thereof. Functional variants are also known from the prior art. Human sequences are specified by way of example.
- the insulin from the kit according to the invention or the composition according to the invention can, for example, have an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or have 100% sequence identity to or consist of the NCBI reference sequence with accession number: NP_000198.1.
- the IGF-1 from the kit according to the invention or the composition according to the invention can, for example, have an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least Have or consist of 99% or 100% sequence identity to the NCBI reference sequence with the accession number: NP_000609.1.
- the IGFBP-2 from the kit according to the invention or the composition according to the invention can, for example, have an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to the NCBI reference sequences with the approval numbers: NP_000588.2 and/or NP_001297588 or consist of one of these sequences.
- the protein used is functional, i.e. it is able, in cooperation with the other components of the composition according to the invention, to trigger an increase in the intracellular fat content in an in vitro culture of bovine muscle cells.
- the at least one transgene is preferably the bovine gene for insulin, IGF-1 and/or IGFBP-2 (and/or the respective bovine protein).
- the transgene may include any endogenous control elements (e.g. promoter) and/or introns.
- the transgene may be operably linked to a heterologous promoter and/or be devoid of introns. It can also include only a few selected introns or artificial or alien introns that do not normally occur in one of the genes mentioned.
- the heterologous promoter can be a constitutive promoter that ensures constant and sustained expression of the transgene.
- the expression of the transgene can also be controlled by a tissue-specific promoter, so that the Is transgenically expressed only in a specific target tissue, for example in muscle cells or in nerve cells, preferably in muscle cells. In this way, any undesired side effects that may occur in a subject with a systemic application of the kit according to the invention or the composition according to the invention can be avoided. Controlled, exclusively tissue-specific expression of IGF-1 can, for example, reduce the risk of non-specific cell growth in other, undesired tissues associated with systemic overexpression of IGF-1.
- the heterologous promoter can also be an inducible promoter, for example a Tet-inducible promoter, which initiates the expression of the transgene only in the presence of a corresponding chemical or physical stimulus.
- an inducible promoter for the expression of at least one component of the kit according to the invention or the composition according to the invention can be particularly useful, for example, if the composition or the kit is intended for use in an in vitro culture.
- a single DNA e.g. the plasmid
- a single DNA allows for the expression of three separate mRNA molecules that can be separately translated into the three components to be provided by the composition or kit.
- This approach just like the alternative described above, according to which the three components are each expressed by separate nucleic acids, i.e. three different DNA vectors, has the advantage that the individual components of the composition according to the invention or the kit according to the invention have different strengths due to the targeted selection Promoters can be expressed at different levels within the target tissue.
- an increased risk of cancer can be limited by driving IGF-1 expression only by means of a weak and/or inducible promoter, while the expression of the other components can be controlled by selecting strong and/or constitutive promoters. Conversely, a desired cell growth can be promoted by targeted tissue-specific overexpression of IGF-1.
- the transgenes coding for the individual components of the composition according to the invention or the kit according to the invention can also be combined to form a single operon which is under the control of a common promoter ters stands.
- the transcription of the operon would correspondingly result in the production of a single polycistronic mRNA molecule, which can then be split into three separate mRNAs, for example by means of targeted RNA splicing, and translated separately.
- the genes for insulin, IGF-1 and IG-FBP-2 can be sequentially cloned within an operon on the DNA vector and separated from each other by an internal ribosomal entry site (IRES).
- the translation of the correspondingly produced polycystronic mRNA can be initiated simultaneously at several positions, ie independently of the presence of a 5′ cap end structure, so that the three components can be synthesized simultaneously using the same mRNA as a template. Since the expression efficiency is usually reduced due to the introduced IRES sequences for each subsequent gene, it can be advantageous if the IGF-1 gene is inserted at the last position within the operon. Thus, the expression levels for IGF-1 were lower than for the other two components, which may reduce an increased risk of cancer. Alternatively, expression of multiple components from a single operon can be achieved through the use of interleaved 2A peptide sequences.
- the transcribed mRNA is translated into a single long polypeptide, which subsequently breaks down into multiple proteins due to the intervening 2A peptides.
- Such and similar vector design strategies for the simultaneous expression of several proteins from the prior art are known to a person skilled in the art.
- the at least one nucleic acid of the composition according to the invention or the kit according to the invention is preferably an RNA.
- the RNA may be an RNA vector of viral origin, e.g., a lentiviral vector.
- the at least one nucleic acid is an mRNA which encodes at least one of the components insulin, IGF-1 or IGFBP-2.
- each component can be encoded by a separate mRNA.
- the at least one mRNA according to the invention encodes several components, e.g. two or all three components, which are to be provided by the composition according to the invention or the kit according to the invention. Expression of multiple different components from a single mRNA can be achieved, for example, using the purposefully interposed IRES or 2A peptide sequences, as described herein.
- the mRNA can be a natural mRNA, ie mRNA that has been expressed by a cell and subsequently isolated.
- natural mRNAs have the disadvantage that that they have poor stability and can elicit an undesirable immune response when administered to a subject or in vitro culture. Therefore, in a preferred embodiment, the mRNA according to the invention is produced synthetically as a chemically modified mRNA.
- the at least one mRNA can be a nucleoside-modified mRNA in which individual nucleosides have been replaced by other natural modified nucleosides or by synthetic nucleoside analogs.
- one or more uridine nucleosides can be replaced by pseudouridine or N 1 -methylpseudouridine.
- cytosine within the at least one mRNA can be replaced by 5-methyl-cytosine.
- the use of such nucleoside analogues influences the secondary structure of the modified mRNA, so that it can no longer be recognized and combated by the immune system.
- the stability of the at least one modified mRNA can also be increased by the targeted selection of suitable untranslated regions (UTR) at the 3' and 5' end of the mRNA and the use of other nucleoside analogs, such as /V 6 -methyladenosine, / ⁇ / 6,2 '-O-dimethyladenosine, 8-oxo-7,8-dihydroguanosine or /V 4 -acetylcytidine are increased.
- UTR untranslated regions
- the at least one mRNA of the kit according to the invention or the composition according to the invention is preferably introduced into a suitable, physiologically harmless carrier.
- suitable carriers include, for example, lipid nanoparticles, which are also used in mRNA-based vaccines, among other things.
- Other suitable carriers for introducing the at least one mRNA into a target cell are also known to the person skilled in the art from the prior art and can include, for example, minicells, RNA-based nanoparticles or dendromers.
- the carriers can be equipped with specific targeting molecules that enable targeted transport of the at least one mRNA to a desired tissue within the subject treated with the composition or the kit according to the invention, e.g. to muscle cells and/or nerve cells.
- the at least one mRNA within the composition according to the invention or the kit according to the invention can also be administered without a carrier.
- the nucleic acid is preferred for expression in the target organism and/or the target tissue Codon optimized.
- individual triplet codons of the nucleotide sequence are replaced by synonymous codons in the course of an in vitro mutagenesis in order to adapt the expression rate of the individual components to the preferred codon usage of the target organism or the target tissue.
- the present invention further discloses the use of the composition according to the invention or the kit according to the invention for increasing the intramuscular fat content in meat, preferably from livestock, optionally from bovines, or in in v/yro culture.
- an increase in intramuscular fat content in meat means that the meat quantitatively has an increased proportion of adipose tissue within muscle tissue in response to the use of the composition or kit of the invention disclosed herein.
- the fatty tissue can preferably be distributed particularly evenly within the meat.
- the expert speaks of a particularly fine and even marbling of the meat.
- the meat of the Kobe beef (also known as Wagyu beef) is known in particular for its particularly tender structure and such fine marbling with small veins of fat. Due to its high intramuscular fat content, Wagyu beef is particularly tender and tasty and is therefore considered a delicacy worldwide.
- a study by Connolly et al., 2020 recently revealed a possible link between the fine marbling of Wagyu beef and increased sugar levels in the cattle serum.
- a particularly efficient supply of carbohydrates to the muscles of Wagyu beef can therefore promote the unusually high intramuscular fat content of the meat.
- Living beings have a complex energy metabolism in order to provide sufficient energy for their survival. The energy gained is needed to maintain normal bodily functions and to build up body components. It is therefore important that living beings can access the body's own energy stores at any time if necessary in order to be able to provide the necessary energy.
- Glucose can be metabolized by cells during glycolysis to produce energy in the form of adenosine triphosphate (ATP).
- ATP adenosine triphosphate
- the breakdown of glucose also supplies the building blocks for the biosynthesis of many other compounds that are essential for survival.
- glucose pyruvate which is required as a starting material for the production of fatty acids, among other things.
- Fatty acids in turn serve cells as a starting material for the biosynthesis of lipids or fats.
- Lipids are very high-energy compounds that are stored by the body in the form of storage or depot fat in the cells of the adipose tissue, the so-called adipocytes.
- the fat reserves serve as a long-term energy store and can, for example, supply a healthy person with energy for weeks.
- Adipocytes can absorb glucose provided by the diet from the blood and thus replenish or increase the fat reserves.
- glycogen a polysaccharide made up of glucose monomers.
- glycogen is more of a short- to medium-term energy store that only supplies the muscles with energy for a few hours, depending on the intensity of the load.
- Tissues use different mechanisms to take up glucose. Above all in cells of the central nervous system, hepatocytes, the intestinal mucosa and the epithelial cells of the kidneys, glucose uptake takes place by means of insulin-independent glucose transporters. In contrast, muscle and fat tissue contains the glucose transporter Glut4, an insulin-dependent channel that enables the transport of glucose from the circulation into fat and muscle cells. The mechanism of insulin action is mediated via the insulin receptor and the AKT kinase signaling pathway.
- the present invention reveals that administering the composition according to the invention or the kit according to the invention to a subject increases their insulin sensitivity and thus the uptake of glucose in muscle and fat cells can be promoted in a targeted manner.
- the present invention consequently allows tissue-specific influencing of the energy metabolism, particularly in muscle and fat tissue, and thereby enables selective hormonal control of muscle and fat accumulation.
- the composition according to the invention or the kit according to the invention is intended for use in farm animals.
- livestock generally includes animals that are used economically by humans due to certain abilities or for aesthetic reasons (https://de.wikipedia.org/wiki/Nutztier).
- the word “livestock” refers in particular to animals that are used as suppliers of food, in particular as suppliers of meat. Farm animals within the meaning of the invention are therefore primarily animals for slaughter, such as members of the Bovinae family, pigs, sheep, goats, horses, zebras, camels, llamas, alpacas, poultry, hares or rabbits.
- the glucose uptake from the blood into the muscle and adipose tissue of these animals can be selectively increased, as described above. Accordingly, the intramuscular fat content of the animals can be increased, which has a positive effect on the quality and tenderness of the meat produced by these livestock.
- the farm animals are preferably members of the Bovinae family. This subfamily of bovids includes, for example, cattle, bison, buffalo and all antelope species.
- the farm animals are particularly preferably cattle, most preferably native cattle breeds such as the Angler cattle, the Ansbach-Triesdorfer cattle, the Brown Swiss, the Angus, e.g. the German Angus, the German Holstein Rotbunt/Schwarzbunt, the German black and white lowland cattle, the German Shorthorn, the Fleckvieh, the Gelbvieh, the Glanrind, the Deutschenwalder or the Limpurger.
- these animals can provide meat of a quality and tenderness that resembles or even corresponds to the meat from the exclusive Japanese Wagyu cattle.
- the hormonal, tissue-specific influencing of energy metabolism and selective control of muscle and fat accumulation described here can also have a positive effect on the meat quality of other slaughter animal species listed here.
- the meat of animals which some consumers generally tend to avoid due to its high firmness and dryness, can be made more tender and thus taste stimulating through increased fat deposits.
- the use of the composition according to the invention or the kit according to the invention in livestock does not only affect the quality of the meat produced.
- use of the composition or kit also contributes to improving feed efficiency in livestock.
- a hormonally controlled, increased amount of fat in the muscles of pigs reduces energy losses during rearing, which allows for more cost-effective husbandry. Therefore, the use of the composition according to the invention or the kit according to the invention is also of interest in those farm animals that do not necessarily serve as meat suppliers. Increased feed efficiency also optimizes the keeping of dairy cows or sheep as wool suppliers, for example.
- the composition according to the invention or the kit according to the invention is intended for use in an in vitro culture.
- the targeted in vitro cultivation of tissue with the aim of synthetically producing meat for human consumption on an industrial scale has made rapid progress in recent years.
- the production of in v/Yro meat is based on cell culture methods, in particular on tissue engineering methods such as 3D cell culture and tissue engineering (https://de.wikipedia.org/wiki/ln-vitro-Fleisch) .
- the production of such “laboratory meat” has the potential to overcome a variety of ethical and environmental issues associated with factory farming in the meat industry.
- An important challenge in the field of in v/yro meat farming is to produce synthetic meat with a high nutritional value that can also convince in terms of taste.
- the use of the composition according to the invention or the kit according to the invention in the in v/yro culture can therefore also have a positive effect on the quality and tenderness of the synthetically produced meat.
- the present invention thus also provides a method for increasing the intramuscular fat content in meat, preferably from farm animals or from v/yro-grown meat.
- the method comprises a step in which the composition or the kit according to the invention is administered to the livestock or crop.
- the intramuscular fat content can be increased in an in vitro culture of bovine muscle cells by cultivating bovine cells, preferably bovine cells, with the composition according to the invention.
- the invention also relates to an in vitro culture of muscle cells, eg bovine muscle cells, in a cell culture medium, the cell culture medium comprising the proteins of the composition according to the invention. These are preferably of exogenous origin, for example produced by genetic engineering. It can be bovine proteins.
- the composition or the kit or at least one of the components of the kit is administered to the animal systemically, ie injected into the bloodstream.
- Local application of the composition according to the invention or at least one component of the kit into the muscle and/or fat tissue of the animal is preferred.
- the composition of the invention may be incorporated into a suitable carrier, such as a capsule, for oral ingestion by the animal and administered to the animal along with the animal's usual diet.
- a suitable carrier such as a capsule
- Such a capsule is provided with an enteric coating, i. H. a suitable polymeric barrier to prevent dissolution in the gastric environment.
- the enteric coating should be chosen so that it remains stable in the low pH of gastric acid but dissolves once it enters the more alkaline environment of the small intestine. Suitable coatings are e.g. B. known in the art.
- composition according to the invention or the kit according to the invention via a gastric juice-resistant capsule thus enables a targeted absorption of the components into the blood circulation via the intestinal wall and can avoid unnecessary stress for the animals.
- Intranasal administration of at least one of the components of the kit or of the composition is also possible.
- composition according to the invention or the kit according to the invention can be administered by medical professionals.
- the composition according to the invention or the kit according to the invention can preferably be administered to the livestock by the farm animal owner himself, i.e. even in the absence of a medically trained specialist or a medically trained specialist.
- composition or the kit is to be administered to an in vitro culture in the course of the method according to the invention
- the manner of administration depends on the form of the individual components provided. It can be administered by direct injection into the cell culture, for example into meat that has already been generated in vitro.
- the composition or kit can be incorporated into the cell medium to be taken up by the cell, for example by endocytosis.
- Nucleic acids encoding one or more components can also be introduced into the cells by transfection. Both chemical transfection methods such as lipofection or calcium phosphate precipitation or physical methods using microinjection or electroporation are suitable for this purpose. Procedure for introducing exogenous proteins Ine or nucleic acids in cell and tissue cultures are well known from the prior art.
- the subject matter of the invention is therefore also a cell, such as a muscle or fat cell or a progenitor cell, such as a mesenchymal stem cell, which comprises the nucleic acid ⁇ ) of the kit according to the invention, the nucleic acids not being present in a form that is present in wild-type cells of this cell type.
- composition or kit can be administered once to the farm animal and/or in v/yro culture. However, the administration can also be repeated several times, e.g. in the form of a longer-term hormone cure.
- the composition or kit may be designed to be administered on a monthly, weekly or even daily basis.
- composition or kit disclosed herein can also be used for therapeutic purposes in a subject. Accordingly, the composition or kit of the invention can also be a pharmaceutical composition or kit which can be administered to a subject for the purpose of therapy or prevention of a disease. Such a pharmaceutical composition or a pharmaceutical kit according to the invention can additionally comprise at least one pharmaceutically acceptable excipient.
- a subject to be treated with the pharmaceutical composition or the pharmaceutical kit is preferably an animal.
- the subject may be a farm animal, as described herein, or an animal used in racing or manual labor, such as a horse, camel, dog, cattle, buffalo, or elephant, or a domestic animal, such as a cat, dog, or a rabbit.
- the subject to be treated with the pharmaceutical composition or kit is a human.
- the pharmaceutical composition or the pharmaceutical kit according to the invention can be intended for use in the treatment of insulin resistance, preferably severe insulin resistance.
- the physiological insulin production needed to maintain normal blood glucose levels is approximately 20-40 International Units (IU) of insulin per day. If the required insulin production is above these values for several days, one can speak of insulin resistance. "Severe insulin resistance” is generally assumed to be a long-term secretion of more than 200 IU insulin per day. Insulin resistance is associated with numerous diseases and is indicative of developing type 2 diabetes mellitus.
- the simultaneous provision of the components insulin, IGF-1 and IGFBP-2 by the composition or the kit disclosed herein causes an increased effectiveness of the insulin.
- the insulin efficacy thus improved may be sufficient to overcome insulin resistance in a subject.
- the pharmaceutical composition or the pharmaceutical kit according to the invention can be administered subcutaneously into the subcutaneous fatty tissue with the aid of a pen, a syringe or an insulin pump, but also intramuscularly or intravenously.
- the administration can preferably be carried out by a patient suffering from insulin resistance himself. Alternatively, however, administration can also be carried out by trained specialists or a doctor. Because the composition or kit as described herein enhances the activity of the AKT kinase signaling pathway, its use may increase the risk of malignancy. For this reason, the regular use of the pharmaceutical composition according to the invention or the pharmaceutical kit according to the invention for the treatment of insulin resistance should preferably only take place in the presence of particularly severe insulin resistance, i.e. if the required insulin production is permanent, but at least continuously over several days at over 200 IU insulin per day lies.
- the risk of AKT-induced tumor formation can be reduced if the composition according to the invention or the pharmaceutical kit according to the invention also comprises an mTOR inhibitor such as metformin.
- Metformin also increases insulin sensitivity itself and can inhibit the formation of new glucose in the liver.
- it can also be advantageous to use the pharmaceutical composition according to the invention or the pharmaceutical kit according to the invention in the treatment of less severe forms of insulin resistance.
- the subject matter of the present invention is therefore also an administration means, such as a pen, a syringe or an insulin pump, which comprises the composition according to the invention or the kit according to the invention.
- a multi-chamber syringe can also be used. This can also be useful if the composition is lyophilized, with a further chamber then usually comprising water for injection (optionally also a buffer).
- the pharmaceutical composition or kit increases glucose uptake by muscle and fat cells and stimulates the anabolic activity of the AKT kinase pathway, it is also useful in the treatment of muscle weakness, particularly muscular dystrophy.
- Muscular dystrophy refers to a particular group of inherited muscle disorders associated with muscle weakness and/or wasting. All muscular dystrophies are characterized by ongoing (progressive) degeneration of the muscles, accompanied by remodeling processes. The individual muscular dystrophies differ in terms of the type of inheritance, the main body regions affected, the age at onset and the course.
- a selection of the muscular dystrophies that can be treated using the pharmaceutical composition or kit disclosed herein includes Duchenne muscular dystrophy, Becker-Kiener muscular dystrophy, Emery-Dreifuss muscular dystrophy type 1-5, scapuloperoneal muscular dystrophy, reducing body myopathy (RBM) , LGMD1A-E, LGMD2A-Q, Congenital Merosin Deficiency, Congenital Myopathy with Integrin Alpha 7 Deficiency, Congenital Muscular Dystrophy 1C, Congenital Muscular Dystrophy Fukuyama Type, Walker-Warburg Syndrome, Muscle Eye Brain Disease, Rigid Spine syndrome, Ullrich myopathy, Bethlem myopathy, Bethlem myopathy, distal muscular dystrophies, myofibrillar myopathy type 1-6, myotonic dystrophy type 1/2, facioscapulohumeral muscular dystrophy and scapuloperoneal muscular dystrophy or other degenerative diseases such as amyotrophic lateral
- RNA-based hormone therapy approaches for muscle weakness and/or injuries currently only envisages the administration of IGF-1.
- the combination of IGF-1 with IGFBP-2 and insulin described here can significantly increase the cellular response. Because of the higher effect lower IGF-1 doses are used than with the pure IGF-1 preparations, which minimizes the risk of malignancy (eg due to systemic effects of IGF-1).
- compositions or the kit according to the invention Due to the growth-promoting properties of the composition or the kit according to the invention, its use for the therapy of growth disorders, i.e. a body length deviating from the norm in children and adolescents, is also conceivable. Thus, regular use of the composition according to the invention or the kit according to the invention in affected subjects can counteract hyposomy (short stature).
- composition or kit disclosed herein is suitable for use in treating an injury to a muscle, ligament and/or tendon of a subject, preferably a muscle.
- the subject is preferably an animal.
- the subject can be a human being, e.g., a professional athlete or ambitious amateur athlete, for whom the shortest possible recovery time after a muscle, ligament, or tendon injury is advantageous.
- the subject may be a horse, camel, dog, or bird of prey, such as an animal used in athletic competitions, for example, that is at increased risk of muscle, ligament, or tendon injury.
- the injury may be acute, i.e., a sudden and violent injury that impairs the affected subject's ability to function.
- the injury can also be a chronic injury, ie an injury which originated a long time ago and which has never completely healed, or whose condition is progressively deteriorating.
- the injury is an acute injury.
- the muscle injury can be, for example, a muscle strain, a hamstring tear, a muscle tear, a bruise or a laceration.
- a ligament or tendon injury includes a tendon or ligament tear or strain.
- the pharmaceutical composition or the pharmaceutical kit according to the invention is preferably intended for use in the treatment of a muscle injury.
- the present invention also provides the pharmaceutical composition or the pharmaceutical kit described herein for use in the maintenance and regeneration of neuronal functions.
- the neuronal functions can be functions of both central and peripheral nerve cells.
- the pharmaceutical composition according to the invention or the kit according to the invention can also be used for the treatment and/or prevention of a neuropathy.
- Scientific studies indicate that insulin resistance can contribute to the pathophysiology and clinical symptoms of various neurodegenerative diseases such as Alzheimer's (Watson et al., 2003) or Parkinson's (Hong et al., 2020).
- the composition or kit of the invention can also help prevent nerve damage resulting from high blood sugar levels, such as is the case with diabetic neuropathy.
- the pharmaceutical composition or kit described herein can be administered to a subject either systemically or locally.
- a systemic application can be useful, for example, in the treatment of insulin resistance.
- the pharmaceutical composition or kit is applied locally in the treatment and/or prevention of the diseases and/or injuries described herein. If the composition or the kit according to the invention is to be used to treat a muscle injury, local application of the composition or the kit directly to the injured muscles is advantageous. Local application of the composition according to the invention or of the kit is also preferred in the treatment of muscular dystrophy, a neuronal dysfunction or neuropathy in order to minimize the risk of developing malignant diseases.
- the administration of the pharmaceutical composition or the pharmaceutical kit for the treatment of any of the medical conditions described herein can be carried out once. In alternative embodiments, multiple administrations of the pharmaceutical composition or kit may be necessary to effect an improvement in the medical condition being treated. Thus, the pharmaceutical composition or kit may need to be administered to the subject at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 50 times, or at least 100 times before an improvement in his condition occurs. In some embodiments, administration must be on a regular basis for as long as the subject's condition persists, possibly throughout life.
- the treatment of the subject with the pharmaceutical composition or the pharmaceutical kit according to the invention can also be combined with other therapeutic approaches or, in particular, in the case of injuries on muscles or the holding apparatus, to support a wide variety of rehabilitation measures.
- the present invention teaches a completely new approach for specifically influencing the energy metabolism in muscle and fat tissue in animals and humans.
- the compositions and kits disclosed herein provide a combination of insulin, IGF-1 and IGFBP-2 that can be used to tailor insulin activity and thus glucose uptake in a tissue-specific manner.
- the use of the compositions and kits according to the invention thus makes it possible to increase the intramuscular fat content.
- the targeted fat deposit can increase the quality of meat and the feed efficiency of livestock.
- an increased energy storage capacity of the muscle tissue is achieved, which increases the regeneration potential of the muscles.
- the activation of the AKT signaling pathway mediated by the compositions and kits also promotes cell growth.
- Insulin-like growth factor (IGF) binding protein 2 functions coordinately with receptor protein tyrosine phosphatase ß and the IGF-I receptor to regulate IGF-l-stimulated signaling. Mol. Cell. Biol. 32, 4116-4130. Shen et al. (2015) The Coordinate Cellular Response to Insulin-like Growth Factor-I (IGF-I) and Insulin-like Growth Factor-binding Protein-2 (IGFBP-2) Is Regulated through Vimentin Binding to Receptor Tyrosine Phosphatase ß (RPTPß). J Biol Chem. 290 , 11578-11590.
- FIG. 1 Human recombinant IGF-1 at low doses improves cellular sensitivity to insulin signalling.
- Human embryonic kidney fibroblasts stably transfected with an expression vector for mouse IGFBP2 cDNA (Höflich et al. 1998, FEBS Letters 434: p329-334) were treated with various concentrations (0-6000 ng/mL) of human insulin in the presence and absence of recombinant human IGF-1 (25 ng/mL). After 20 minutes incubation with the peptides, the cells were harvested and the phosphorylation of AKT was assayed.
- AKT kinase phosphorylation is used to illustrate that the effect of administered insulin can be potentiated by a combination of IGF-1 and IGFBP-2 within cells.
- Human embryonic kidney fibroblasts stably transfected with an expression vector for mouse IGFBP2 cDNA were treated with various concentrations (0-6000 ng/mL) of human insulin in the presence and absence of recombinant human IGF-1 (25 ng/mL). After 20 minutes incubation with the peptides, cells were harvested and AKT phosphorylation was determined as described in Walz et al., 2021 (Development of a Sensitive Bioassay for the Analysis of IGF-Related Activation of AKT/mTOR Signaling in Biological Matrices. Cells 10( 3):482.doi:10.3390/cells10030482), tested. Results and discussion
- IGF-1 is able to stimulate the growth of almost every type of cell in the body, including by stimulating the AKT kinase signaling pathway. It has also been shown in the past that IGFBP-2, together with IGF-1, can trigger activation of the AKT kinase signaling pathway and consequently cause the migration and proliferation of vascular smooth muscle cells (Shen et al. 2012).
- IGFBP-2 expressed by the cells transfected with the expression vector attached itself to the cell membranes, it was not possible to precisely determine the expression level of IGFBP-2 within the scope of the study. However, from a previous study by Walz et al., 2021, it is known that IGFBP-
- IGF-1-dependent AKT phosphorylation in HEK293-2 cells from a concentration of 100-500 ng/mL. It is assumed that the concentration of IGFBP-2 in the experiment shown in Figure 1 does not exceed this dose. It was also striking that supplementing the IGFBP-2-expressing cells with a high insulin concentration of 6000 ng/mL only led to a low AKT phosphorylation, regardless of whether the cells were additionally treated with IGF-1 or not.
Abstract
The present invention relates to compositions and kits for influencing, in a targeted manner, energy metabolism in muscular and fatty tissue. In detail, the invention teaches a composition or a kit which provides the three components insulin, IGF-1 and IGFBP-2. The three components can be present in the composition or the kit as protein or can be encoded by at least one nucleic acid contained in the composition or the kit, the nucleic acid preferably being RNA. The invention further discloses the use of the composition or the kit in a method for increasing the intramuscular fat content in the meat of farm animals or in culture in vitro. The composition or kit according to the invention can also be a pharmaceutical composition or a pharmaceutical kit, which can be used in treating insulin resistance, muscular dystrophy, in preserving and regenerating neural functions and in treating and/or preventing neuropathy. Finally, the present invention discloses the use of the pharmaceutical composition and the pharmaceutical kit according to the invention in treating injuries to muscles, ligaments and/or tendons in a subject.
Description
Forschungsinstitut für Nutztierbiologie (FBN) Research Institute for Livestock Biology (FBN)
IGF-1 , IGFBP-2 und Insulin umfassende Zusammensetzungen und ihre Verwendung Compositions comprising IGF-1, IGFBP-2 and insulin and their use
Die vorliegende Erfindung betrifft Zusammensetzungen und Kits zur gezielten Beeinflussung des Energiestoffwechsels in Muskel- und Fettgewebe. Im Detail lehrt die Erfindung eine Zusammensetzung oder ein Kit, die bzw. das die drei Komponenten Insulin, IGF-1 und IGFBP- 2 bereitstellt. Die drei Komponenten können in der Zusammensetzung oder dem Kit als Protein vorliegen oder durch mindestens eine in der Zusammensetzung oder dem Kit enthaltene Nukleinsäure kodiert werden, wobei es sich bei der Nukleinsäure bevorzugt um RNA handelt. Ferner offenbart die Erfindung die Verwendung der Zusammensetzung oder des Kits in einem Verfahren zur Erhöhung des intramuskulären Fettgehalts in Fleisch von Nutztieren oder in in v/Yro-Kultur. Bei der erfindungsgemäßen Zusammensetzung oder dem Kit kann es sich zudem um eine pharmazeutische Zusammensetzung bzw. um ein pharmazeutisches Kit handeln, die/das bei der Behandlung von Insulinresistenz, Muskeldystrophie, der Erhaltung und Regeneration neuronaler Funktionen und der Behandlung und/oder Prävention einer Neuropathie Verwendung finden kann. Schließlich offenbart die vorliegende Erfindung die Verwendung der erfindungsgemäßen pharmazeutischen Zusammensetzung und des pharmazeutischen Kits zur Behandlung einer Verletzung eines Muskels, Bandes und/oder einer Sehne in einem Subjekt. The present invention relates to compositions and kits for specifically influencing the energy metabolism in muscle and adipose tissue. In detail, the invention teaches a composition or kit that provides the three components insulin, IGF-1 and IGFBP-2. The three components can be present in the composition or the kit as a protein or can be encoded by at least one nucleic acid contained in the composition or the kit, the nucleic acid preferably being RNA. Furthermore the invention discloses the use of the composition or kit in a method for increasing the intramuscular fat content in meat of farm animals or in v/yro culture. The composition or kit according to the invention can also be a pharmaceutical composition or a pharmaceutical kit used in the treatment of insulin resistance, muscular dystrophy, the maintenance and regeneration of neuronal functions and the treatment and/or prevention of neuropathy Can be found. Finally, the present invention discloses the use of the pharmaceutical composition and pharmaceutical kit of the invention for treating an injury to a muscle, ligament and/or tendon in a subject.
In Deutschland lag der jährliche Fleischverbrauch im Jahr 2020 bei knapp 60 kg pro Kopf, wobei der größte Teil des Fleischkonsums auf Schweinefleisch entfällt. Obwohl der Fleischkonsum in den letzten Jahren stetig zurückging, ist seit 2005 eine kontinuierlich steigende Nachfrage nach Rindfleisch festzustellen (https://www.landwirtschaft.de/landwirtschaft-ver- stehen/haetten-sies-gewusst/infografiken). Diese Entwicklung geht mit wachsenden Ansprüchen an die Qualität von Nahrungsmitteln einher. So legen immer mehr Konsumenten einen gesteigerten Wert auf eine hohe Fleischqualität, die sich durch eine besonders zarte Fleischkonsistenz und einen ansprechenden Geschmack auszeichnet. In Germany, annual meat consumption in 2020 was just under 60 kg per capita, with pork accounting for the majority of meat consumption. Although meat consumption has steadily declined in recent years, there has been a continuous increase in demand for beef since 2005 (https://www.landwirtschaft.de/landwirtschaft-ver-standing/haetten-sies-gewusst/infographics). This development is accompanied by growing demands on the quality of food. More and more consumers are placing increasing value on high meat quality, which is characterized by a particularly tender meat consistency and an appealing taste.
Insbesondere das Fleisch des japanischen Kobe-Rinds (auch als Wagyu-Rind bekannt) ist für seine außergewöhnlich hohe Fleischqualität weltberühmt. Das Wagyu-Fleisch zeichnet sich durch eine besonders mürbe Struktur und einen außergewöhnlichen hohen intramuskulären Fettgehalt aus, der als feine Marmorierung des Fleisches wahrgenommen wird. Die ausgezeichnete Qualität des Wagyu-Fleischs erfordert allerdings eine außergewöhnlich kosten- und zeitaufwändige Aufzucht der Rinder. So erhalten die Tiere in ihren ersten Lebensmonaten einen speziellen Futtermix aus ausgewählten Zutaten und dürfen sich anschließend
frei in kleinen Herden auf Wiesen und Weiden aufhalten. Darüber hinaus leben Wagyu-Rin- der bis zur Schlachtreife deutlich länger als konventionelle Fleischrinder und sind bei ihrer Schlachtung in der Regel kleiner und leichter. Der Aufwand, der für die Aufzucht der Wagyu- Rinder betrieben wird, um Fleisch von allerhöchster Güte und Qualität zu erhalten, äußert sich dementsprechend im Preis: Das Fleisch des Wagyu-Rinds gilt als das teuerste Fleisch der Welt. In particular, the meat of the Japanese Kobe beef (also known as Wagyu beef) is world famous for its exceptionally high meat quality. The Wagyu meat is characterized by a particularly tender structure and an exceptionally high intramuscular fat content, which is perceived as fine marbling of the meat. However, the excellent quality of the Wagyu meat requires an extraordinarily costly and time-consuming rearing of the cattle. In the first months of life, the animals receive a special feed mix made from selected ingredients and are then allowed to socialise roam freely in small herds on meadows and pastures. In addition, Wagyu cattle live significantly longer than conventional beef cattle before they are ready for slaughter and are usually smaller and lighter when they are slaughtered. The effort that goes into raising Wagyu cattle in order to obtain meat of the highest quality is reflected in the price: Wagyu beef is considered the most expensive meat in the world.
Es liegt auf der Hand, dass eine derartige ressourcenintensive Aufzucht von Nutztieren zur Fleischproduktion für den deutschen Massenmarkt nicht in Frage kommt. In Deutschland dominiert bei den Rindern die Laufstallhaltung. Lediglich ein Drittel erhält regelmäßigen Weidezugang, wobei dieser Anteil in den letzten Jahren kontinuierlich gesunken ist (https://www.landwirtschaft.de/landwirtschaft-verstehen/haetten-sies-gewusst/infografiken). Konventionelle Mastverfahren zielen zudem auf eine möglichst große Gewichts- und Größenzunahme der Schlachttiere in möglichst kurzer Zeit ab. In Zeiten von Massentierhaltung wird es somit zunehmend schwer, die Qualitätsansprüche vieler deutscher Fleischkonsumenten zufriedenzustellen It is obvious that such a resource-intensive rearing of livestock for meat production is out of the question for the German mass market. In Germany, freestall housing dominates for cattle. Only a third receive regular access to pasture, although this proportion has fallen continuously in recent years (https://www.landwirtschaft.de/landwirtschaft-verständigen/haetten-sies-gewusst/infografiken). Conventional fattening methods also aim to increase the weight and size of the animals for slaughter as much as possible in the shortest possible time. In times of factory farming, it is becoming increasingly difficult to satisfy the quality requirements of many German meat consumers
Nichtdestotrotz gilt auch in Deutschland bei der Rindermast die Maßgabe, dass ein hoher intramuskulärer Fettgehalt der Nutztiere wünschenswert ist. Ein erhöhter Fettansatz in Nutztieren dient nicht nur einer hohen Qualität des produzierten Fleisches, sondern kann auch die Futtereffizienz der Tiere erheblich steigern. Die selektive Kontrolle des Fett- und Muskelansatzes hat somit auch das Potential, die Zucht und Haltung von Nutztieren kostengünstiger zu gestalten. Nevertheless, the stipulation that a high intramuscular fat content of livestock is desirable also applies to cattle fattening in Germany. An increased amount of fat in livestock not only ensures a high quality of the meat produced, but can also significantly increase the feed efficiency of the animals. The selective control of fat and muscle growth therefore also has the potential to make the breeding and husbandry of livestock more cost-effective.
Es besteht somit ein Bedarf an neuartigen Verfahren und Ansätzen, die eine selektive gewebsspezifische Regulation des Energiestoffwechsels erlauben, um eine gezielte Kontrolle und Förderung des Fett- und Muskelansatzes, etwa eine Steigerung der intramuskulären Fetteinlagerung, zu ermöglichen. There is therefore a need for novel methods and approaches that allow selective tissue-specific regulation of the energy metabolism in order to enable targeted control and promotion of fat and muscle accumulation, such as an increase in intramuscular fat storage.
Gelöst wird die erfindungsgemäße Aufgabe durch den Gegenstand der vorliegenden Erfindung, insbesondere der Ansprüche. The object according to the invention is achieved by the subject matter of the present invention, in particular the claims.
Die Erfindung offenbart eine Zusammensetzung oder ein Kit, welche(s) IGF-1 , Insulin und IGFBP-2 bereitstellt, wobei The invention discloses a composition or kit providing IGF-1, insulin and IGFBP-2, wherein
(a) das IGF-1 , Insulin und/oder IGFBP-2 als Protein vorliegt, oder
(b) mindestens eine Nukleinsäure vorliegt, welche für IGF-1 , Insulin und/oder IGFBP-2 kodiert. (a) the IGF-1, insulin and/or IGFBP-2 is present as a protein, or (b) at least one nucleic acid is present which codes for IGF-1, insulin and/or IGFBP-2.
Insulin ist ein Hormon, das gemeinsam mit Glucagon die Konzentration von Glucose im Blut reguliert. Während Glucagon eine Erhöhung der Glucosekonzentration im Blut bewirkt, fördert Insulin die Aufnahme der Glucose durch Muskel- und Fettzellen, was zu einer Senkung des Blutzuckerspiegels führt. Hierzu muss Insulin an einen spezifischen Insulinrezeptor andocken, der als Transmembranprotein exprimiert wird. Durch die Interaktion des Insulins mit dessen Rezeptor wird eine Kaskade von Phosphorylierungsreaktionen ausgelöst, die letztlich zur Aktivierung spezifischer Glucose-Transporterproteine führt, und somit den Eintritt der Glucose in die Zellen ermöglicht. Vollständig gereiftes Insulin besteht aus einer A-Kette und einer B-Kette und wird normalerweise in den ß-Zellen innerhalb der Langerhans-Inseln der Bauspeicheldrüse gebildet. Es wird erst als Reaktion auf die Aufnahme kohlenhydratreicher Nahrung in das Blut ausgeschüttet. Insulin is a hormone that works with glucagon to regulate the concentration of glucose in the blood. While glucagon causes an increase in the concentration of glucose in the blood, insulin promotes the uptake of glucose by muscle and fat cells, which leads to a reduction in blood sugar levels. To do this, insulin must dock to a specific insulin receptor, which is expressed as a transmembrane protein. The interaction of insulin with its receptor triggers a cascade of phosphorylation reactions, which ultimately leads to the activation of specific glucose transporter proteins, thus allowing glucose to enter cells. Fully matured insulin consists of an A chain and a B chain and is normally produced in the ß-cells within the islets of Langerhans in the pancreas. It is only released into the blood in response to the ingestion of carbohydrate-rich food.
Der insulinähnliche Wachstumsfaktor 1 (englisch insulin-like growth factor 1 ) ist ein Wachstumsfaktor mit hoher Sequenzhomologie zu Insulin, der hauptsächlich unter Mitwirkung des Wachstumshormons Somatotropin in der Leber gebildet wird. Die IGF-1 Produktion erfolgt ein Leben lang, erreicht jedoch ihr Maximum während des Wachstumsschubes in der Pubertät. IGF-1 ist in der Lage, das Wachstum nahezu jeder Zellart des Körpers anzuregen, u.a. indem es u.a. den AKT-Kinasesignalweg stimuliert. Gleichzeitig ist IGF-1 ein wirksamer Inhibitor des programmierten Zelltods. Aufgrund seiner wachstumsfördernden und Apoptose-hemmenden Funktion wird eine Überexpression von IGF-1 jedoch auch mit der Entstehung und dem Fortschreiten von Krebs in Verbindung gebracht. The insulin-like growth factor 1 (English insulin-like growth factor 1) is a growth factor with high sequence homology to insulin, which is mainly formed in the liver with the participation of the growth hormone somatotropin. IGF-1 production occurs throughout life, but peaks during the growth spurt of puberty. IGF-1 is able to stimulate the growth of almost every type of cell in the body, including by stimulating the AKT kinase signaling pathway. At the same time, IGF-1 is a potent inhibitor of programmed cell death. However, due to its growth-promoting and apoptosis-inhibiting function, overexpression of IGF-1 has also been implicated in the development and progression of cancer.
Die anabolen Funktionen des IGF-1 werden unter anderem durch die Familie der Insulin- like Growth Factor Binding Proteins (IGFBP) reguliert. IGFBP-2 ist das zweithäufigste IG- FBP und wird in verschiedenen Geweben exprimiert, wo es beispielsweise die Bindung von IGF-1 an dessen spezifischen Rezeptor unterdrücken kann und somit die IGF-1 Aktivität inhibiert. The anabolic functions of IGF-1 are regulated, among other things, by the family of insulin-like growth factor binding proteins (IGFBP). IGFBP-2 is the second most common IG-FBP and is expressed in various tissues where it can, for example, suppress the binding of IGF-1 to its specific receptor and thus inhibit IGF-1 activity.
In der Vergangenheit konnte gezeigt werden, dass IGFBP-2 neben seiner inhibitorischen Funktion auch gemeinsam mit IGF-1 eine Aktivierung des AKT-Kinasesignalwegs auslösen und folglich die Migration und Vermehrung von vaskulären glatten Muskelzellen bewirken kann (Shen et al. 2012). IGFBP-2 scheint hierzu die Rezeptorprotein-Tyrosinphosphatase ß (RPTPß) zu stimulieren, was zu einer Dephosphorylierung und somit Inaktivierung der Lipidphosphatase PTEN führt. Gleichzeitig bindet das IGF-1 an dessen spezifischen Rezeptor,
und aktiviert dadurch Vimentin. Vimentin bindet im Anschluss ebenfalls an RPTPß. Die koordinierte Stimulation von RPTPß durch IGFBP-2 und Vimentin und die damit einhergehende Inaktivierung von PTEN verhindert die Dephosphorylierung von Phosphatidylinositol(3,4,5)- trisphosphat (PIP3) und ermöglicht infolgedessen die Aktivierung der wachstumsfördernden AKT-Kinase (Shen et al., 2012, Shen et al., 2015). In the past it could be shown that IGFBP-2, in addition to its inhibitory function, can also trigger activation of the AKT kinase signaling pathway together with IGF-1 and consequently cause the migration and proliferation of vascular smooth muscle cells (Shen et al. 2012). IGFBP-2 appears to stimulate the receptor protein tyrosine phosphatase ß (RPTPß), which leads to dephosphorylation and thus inactivation of the lipid phosphatase PTEN. At the same time, the IGF-1 binds to its specific receptor, thereby activating vimentin. Vimentin then also binds to RPTPß. The coordinated stimulation of RPTPß by IGFBP-2 and vimentin and the concomitant inactivation of PTEN prevents the dephosphorylation of phosphatidylinositol(3,4,5)-trisphosphate (PIP3) and consequently enables the activation of the growth-promoting AKT kinase (Shen et al. , 2012, Shen et al., 2015).
Weitere Studien konnten zeigen, dass eine supprimierte PTEN-Aktivität und eine Aktivierung von AKT nicht nur das Zellwachstum fördern, sondern auch gegen Hyperglykämie und Insulinresistenz schützen und eine erhöhte Insulinsensitivität bewirken (z.B. Li et al., 2017). Further studies have shown that suppressed PTEN activity and activation of AKT not only promote cell growth, but also protect against hyperglycemia and insulin resistance and cause increased insulin sensitivity (e.g. Li et al., 2017).
Die Erfindung macht sich diese Erkenntnisse zu Nutze und stellt einen neuartigen Ansatz zur Verfügung, wonach eine Kombination von IGF-1 und IGFBP-2 verwendet werden kann, um Muskelwachstum und/oder Fetteinlagerung durch die Aktivierung der AKT-Signalisierung und der Förderung einer erhöhten Insulinwirksamkeit gezielt anzuregen. Tatsächlich wurde eine Relevanz einer kombinierten Aktivität von IGF-1 und IGFBP-2 für die Wirksamkeit von Insulin bislang noch nicht beschrieben. Die Erfindung stellt eine Zusammensetzung bzw. ein Kit zur Verfügung, das/die Insulin gemeinsam mit IGF-1 und IGFBP-2 als Komponenten bereitstellt und beispielsweise einem Subjekt oder einer in vitro Kultur verabreicht werden kann, um die Wirkung des bereitgestellten Insulins innerhalb des Subjekts oder der Kultur zu potenzieren. Aufgrund der Bereitstellung von IGF-1 in Form einer Zusammensetzung oder eines Kits mit IGFBP-2 als weitere, wachstumsfördernde Komponente kann zudem die Dosis des bereitgestellten IGF-1 im Vergleich zu reinen IGF-1 Präparaten herabgesetzt werden, um ein unkontrolliertes Zellwachstum und die Entstehung maligner Erkrankungen vorzubeugen. The invention takes advantage of these findings and provides a novel approach whereby a combination of IGF-1 and IGFBP-2 can be used to promote muscle growth and/or fat storage by activating AKT signaling and promoting increased insulin effectiveness to stimulate purposefully. In fact, a relevance of a combined activity of IGF-1 and IGFBP-2 for the effectiveness of insulin has not yet been described. The invention provides a composition or kit that provides insulin along with IGF-1 and IGFBP-2 as components and can be administered, for example, to a subject or to in vitro culture to measure the effect of the provided insulin within the subject or to potentiate the culture. Due to the provision of IGF-1 in the form of a composition or a kit with IGFBP-2 as an additional growth-promoting component, the dose of the provided IGF-1 can also be reduced compared to pure IGF-1 preparations in order to avoid uncontrolled cell growth and the formation prevent malignant diseases.
Die erfindungsgemäße Zusammensetzung bzw. das erfindungsgemäße Kit kann weitere Inhaltstoffe umfassen oder bereitstellen. Im Kontext der Erfindung bezieht sich der Begriff „Komponenten“ jedoch lediglich auf Insulin, IGF-1 und IGFBP-2. The composition according to the invention or the kit according to the invention can comprise or provide further ingredients. In the context of the invention, however, the term "components" only refers to insulin, IGF-1 and IGFBP-2.
Das Kit kann ein einziges Kompartiment umfassen, in dem die drei verschiedenen Komponenten als Proteine und/oder die mindestens eine Nukleinsäure, die für eine oder mehrere der Komponenten kodiert, gemeinsam vorliegen. Alternativ können die Komponenten bzw. die mindestens eine Nukleinsäure in unterschiedliche Kompartimente des Kits aufgeteilt vorliegen und, optional, erst vor oder während der Verwendung des Kits miteinander in Kontakt gebracht werden. Das Kit oder die Zusammensetzung kann bei einer Temperatur von 2-6°C für mehrere Tage bis mehrere Wochen gelagert werden, z.B. 1 Tag bis 4 Wochen, 1 Tag bis 3 Wochen, 1 Tag bis 2 Wochen oder 1 Tag bis 7 Tage. Vorzugsweise kann das Kit oder die Zusammensetzung bei 2-6°C für weniger als 2 Wochen gelagert werden. Für eine langfristige
Lagerung kann das Kit oder die Zusammensetzung bei -80°C für einen Zeitraum von mehreren Wochen bis zu mehreren Monaten vor der Verwendung gelagert werden, z. B. von 4 Wochen bis zu 24 Monaten, von 2 Monaten bis zu 20 Monaten, von 4 Monaten bis zu 16 Monaten, von 8 Monaten bis zu 14 Monaten oder für etwa 12 Monate. In einigen Ausführungsformen kann das Kit oder die Zusammensetzung bei -80°C für noch längere Zeiträume vor der Verwendung gelagert werden, z. B. für 3 Jahre, für 4 Jahre, für 5 Jahre, für 6 Jahre, für 7 Jahre, für 8 Jahre, für 9 Jahre oder sogar für bis zu 10 Jahre. Auch eine Lyophilisierung von Komponenten und eine Resuspendierung vor Verabreichung sind möglich. The kit may comprise a single compartment in which the three different components are present together as proteins and/or the at least one nucleic acid encoding one or more of the components. Alternatively, the components or the at least one nucleic acid can be divided into different compartments of the kit and, optionally, only brought into contact with one another before or during use of the kit. The kit or composition can be stored at a temperature of 2-6°C for several days to several weeks, eg 1 day to 4 weeks, 1 day to 3 weeks, 1 day to 2 weeks or 1 day to 7 days. Preferably, the kit or composition can be stored at 2-6°C for less than 2 weeks. For a long term Storage The kit or composition can be stored at -80°C for a period of several weeks to several months before use, e.g. B. from 4 weeks to 24 months, from 2 months to 20 months, from 4 months to 16 months, from 8 months to 14 months or for about 12 months. In some embodiments, the kit or composition can be stored at -80°C for even longer periods of time before use, e.g. B. for 3 years, for 4 years, for 5 years, for 6 years, for 7 years, for 8 years, for 9 years or even for up to 10 years. Lyophilization of components and resuspension prior to administration are also possible.
Wie oben bereits erwähnt, liegt in einer ersten Ausführungsform der Erfindung mindestens eine der drei Komponenten, d.h. Insulin, IGF-1 oder IGFBP-2, als Protein in der erfindungsgemäßen Zusammensetzung oder dem erfindungsgemäßen Kit vor. In einer weiteren Ausführungsform umfasst die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit zwei oder auch alle drei Komponenten als Proteine. As already mentioned above, in a first embodiment of the invention at least one of the three components, i.e. insulin, IGF-1 or IGFBP-2, is present as a protein in the composition or the kit according to the invention. In a further embodiment, the composition according to the invention or the kit according to the invention comprises two or else all three components as proteins.
Die drei Komponenten können in diesem Fall zu gleich Teilen in der Zusammensetzung oder dem Kit vorliegen, d.h. ungefähr in einem äquimolaren Verhältnis. Da eine Bereitstellung des Wachstumshormons IGF-1 jedoch mit einem erhöhten Krebsrisiko einhergehen könnte, kann es im Sinne der Erfindung vorteilhaft sein, wenn das IGF-1 Protein zu einem geringeren Anteil in der erfindungsgemäßen Zusammensetzung oder dem erfindungsgemäßen Kit vorliegt als die beiden übrigen Komponenten. Durch Verringerung des relativen IGF-1 Anteils in der erfindungsgemäßen Zusammensetzung bzw. dem Kit kann dessen anaboles, d.h. wachstumsförderndes Potential eingeschränkt werden. Auf der anderen Seite verstärkt ein größerer relativer IGF-1 -Anteil in der Zusammensetzung oder dem Kit den Aufbau und die Regeneration von körpereigenem Gewebe, was je nach Verwendung der Zusammensetzung oder des Kits wünschenswert sein kann. Das Verhältnis zwischen Insulin, IGF-1 und IGFBP-2 kann von dem Verhältnis 1 :1 :1 abweichen. So kann das Verhältnis der drei Komponenten in der Zusammensetzung oder dem Kit zueinander bei 0,1-10:0,1-10:0,1-10 liegen. Die physiologisch wirksamen Dosen für IGF-1 , IGFBP-2 bzw. Insulin liegen bei 10-1000 ng/mL, 30-2000 ng/mL, bzw. bei 10-1000 ng/mL Serum oder Kultur-Medium. Derartige Konzentrationen können auch erfindungsgemäß eingesetzt werden. So kann z.B. eine Konzentration von IGF-1 von 10- 1000 ng/mL (z.B. 20-500 ng/mL, 25-100 ng/mL oder 25-50 ng/mL), bevorzugt etwa 25 ng/mL eingesetzt werden. Die Konzentration von IGFBP2 kann z.B. 30-2000 ng/mL bevorzugt 100- 500 ng/m betragen. Von Insulin kann z.B. eine Konzentration von 5-1000 ng/mL eingesetzt werden, bevorzugt ca. 10-600 ng/mL, 20-200 ng/mL oder 50-70 ng/mL, oder etwa 60 ng/mL. -Durch weitere Konzentrationserhöhungen sind zusätzliche Effekte möglich, zum Beispiel
dadurch, dass Insulin bei höheren Konzentrationen auch an den IGF-1 Rezeptor binden kann. The three components can in this case be present in equal parts in the composition or kit, ie approximately in an equimolar ratio. However, since the provision of the growth hormone IGF-1 could be associated with an increased risk of cancer, it can be advantageous within the meaning of the invention if the IGF-1 protein is present in a lower proportion in the composition or the kit according to the invention than the other two components. By reducing the relative IGF-1 content in the composition or the kit according to the invention, its anabolic, ie growth-promoting potential can be restricted. On the other hand, a larger relative IGF-1 content in the composition or kit enhances the formation and regeneration of the body's own tissues, which may be desirable depending on the use of the composition or kit. The ratio between insulin, IGF-1 and IGFBP-2 can deviate from the 1:1:1 ratio. Thus the ratio of the three components in the composition or kit to one another can be 0.1-10:0.1-10:0.1-10. The physiologically effective doses for IGF-1, IGFBP-2 or insulin are 10-1000 ng/mL, 30-2000 ng/mL, or 10-1000 ng/mL serum or culture medium. Such concentrations can also be used according to the invention. For example, an IGF-1 concentration of 10-1000 ng/mL (eg 20-500 ng/mL, 25-100 ng/mL or 25-50 ng/mL), preferably about 25 ng/mL, can be used. The concentration of IGFBP2 can be, for example, 30-2000 ng/mL, preferably 100-500 ng/mL. For example, a concentration of 5-1000 ng/mL of insulin can be used, preferably about 10-600 ng/mL, 20-200 ng/mL or 50-70 ng/mL, or about 60 ng/mL. -Additional effects are possible through further increases in concentration, for example because insulin can also bind to the IGF-1 receptor at higher concentrations.
Im Kontext der Erfindung bedeutet „als Protein vorliegen“, dass die Komponenten als vollständig translatierte und korrekt gefaltete Polypeptide in der Zusammensetzung oder dem Kit bereitgestellt werden. Bevorzugt sind die als Protein vorliegenden Komponenten zudem vollständig prozessiert. So wird das Insulin beispielsweise bevorzugt ohne das 31 Aminosäuren umfassende C-Peptid oder die 24 Aminosäure umfassende Signalsequenz bereitgestellt, liegt somit weder als Präproinsulin (Signalpeptid - B-Kette - C-Peptid - A-Kette) oder als Proinsulin (B-Kette - C-Peptid - A-Kette), sondern als vollständig gereiftes Insulin (B-Kette - A-Kette) in der Zusammensetzung oder dem Kit vor. In anderen Ausführungsformen werden die Komponenten in nicht vollständig prozessierter Form bereitgestellt. So kann die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit beispielsweise auch anstatt Insulin lediglich Präproinsulin oder Proinsulin, aber auch eine beliebige Mischung aus Präproinsulin, Proinsulin und/oder gereiftem Insulin, umfassen. Entsprechendes gilt für die übrigen Komponenten der Zusammensetzung oder des Kits. In the context of the invention, "existing as a protein" means that the components are provided as fully translated and correctly folded polypeptides in the composition or kit. In addition, the components present as proteins are preferably completely processed. For example, the insulin is preferably provided without the 31 amino acid C peptide or the 24 amino acid signal sequence, and is therefore neither preproinsulin (signal peptide - B chain - C peptide - A chain) or proinsulin (B chain - C-peptide - A chain) but as fully matured insulin (B chain - A chain) in the composition or kit. In other embodiments, the components are provided in an incompletely processed form. For example, instead of insulin, the composition according to the invention or the kit according to the invention can also comprise only preproinsulin or proinsulin, but also any desired mixture of preproinsulin, proinsulin and/or matured insulin. The same applies to the other components of the composition or kit.
In einer weiteren Ausführungsform werden nicht alle Komponenten der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits als Protein bereitgestellt. Beispielsweise können lediglich eine oder zwei der drei Komponenten als Protein bereitgestellt werden. Die übrigen Komponenten werden dagegen mittels mindestens einer Nukleinsäure bereitgestellt, die für diese Komponenten kodiert. Entsprechend werden die durch die mindestens eine Nukleinsäure kodierten Komponenten der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits erst nach deren Verwendung in einer Zelle des Zielgewebes hergestellt. In another embodiment, not all of the components of the composition or kit of the invention are provided as a protein. For example, only one or two of the three components can be provided as a protein. In contrast, the other components are provided by means of at least one nucleic acid which encodes these components. Accordingly, the components of the composition according to the invention or of the kit according to the invention encoded by the at least one nucleic acid are only produced after they have been used in a cell of the target tissue.
Im Umkehrschluss bedeutet dies, dass die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit in einer Ausführungsform mindestens eine Nukleinsäure umfasst, welche für IGF-1 , Insulin und/oder IGFBP-2 kodiert. Die mindestens eine Nukleinsäure kodiert somit für mindestens eine Komponente der Zusammensetzung bzw. des Kits, während die übrigen Komponenten als Protein, wie hierin beschrieben, bereitgestellt werden. Die mindestens eine Nukleinsäure kann auch für mehr als eine Komponente der Zusammensetzung bzw. des Kits kodieren, z.B. für zwei oder alle drei der Komponenten. Beispielsweise kann die mindestens eine Nukleinsäure für Insulin und IGF-1 kodieren, während IGFBP-2 als Protein bereitgestellt wird. Bevorzugt werden alle drei Komponenten, also IGF-1 , Insulin und IGFB-2, durch mindestens eine Nukleinsäure kodiert. In einer Ausführungsform der Erfindung werden eine, zwei oder alle drei Komponenten in der Zusammensetzung oder dem Kit sowohl
als Protein, als auch in Form mindestens einer Nukleinsäure bereitgestellt, die für mindestens einer der Komponenten kodiert. Conversely, this means that in one embodiment the composition according to the invention or the kit according to the invention comprises at least one nucleic acid which codes for IGF-1, insulin and/or IGFBP-2. The at least one nucleic acid thus encodes at least one component of the composition or kit, while the other components are provided as a protein, as described herein. The at least one nucleic acid can also code for more than one component of the composition or kit, eg for two or all three of the components. For example, the at least one nucleic acid can encode insulin and IGF-1, while IGFBP-2 is provided as a protein. All three components, ie IGF-1, insulin and IGFB-2, are preferably encoded by at least one nucleic acid. In one embodiment of the invention, one, two or all three components in the composition or kit are both provided as a protein, as well as in the form of at least one nucleic acid encoding at least one of the components.
In einer Ausführungsform werden die jeweiligen Komponenten des erfindungsgemäßen Kits oder der erfindungsgemäßen Zusammensetzung durch separate Nukleinsäuren kodiert. So kodiert eine erste Nukleinsäure für Insulin, eine zweite Nukleinsäure für IGF-1 und eine dritte Nukleinsäure für IGFBP-2. Alternativ kann ein und dieselbe Nukleinsäure auch für mehrere Komponenten der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits kodieren. Beispielsweise kann eine erste Nukleinsäure sowohl für Insulin und IGF-1 kodieren, während eine zweite Nukleinsäure für IGFBP-2 kodiert. In einer weiteren Ausführungsform kodiert eine einzige Nukleinsäure für alle drei Komponenten, also sowohl für Insulin als auch für IGF-1 und IGFBP-2. In one embodiment, the respective components of the kit according to the invention or the composition according to the invention are encoded by separate nucleic acids. Thus, a first nucleic acid codes for insulin, a second nucleic acid for IGF-1 and a third nucleic acid for IGFBP-2. Alternatively, one and the same nucleic acid can also code for several components of the composition according to the invention or the kit according to the invention. For example, a first nucleic acid can encode both insulin and IGF-1, while a second nucleic acid encodes IGFBP-2. In another embodiment, a single nucleic acid encodes all three components, ie both insulin and IGF-1 and IGFBP-2.
Bei der mindestens einen Nukleinsäure des erfindungsgemäßen Kits oder der erfindungsgemäßen Zusammensetzung handelt es sich in einer ersten Ausführungsform um DNA. Bei der DNA kann es sich beispielsweise um einen DNA-Vektor handeln, z.B. um ein aus Bakterien oder Backhefe abgeleitetes Plasmid, einen DNA-Vektor viralen Ursprungs oder einen Minicircle, welcher mindestens ein T ransgen umfasst, das für mindestens eine der Komponenten der erfindungsgemäßen Zusammensetzung bzw. des erfindungsgemäßen Kits kodiert. Der Fachmann ist ohne weiteres in der Lage, einen geeigneten DNA-Vektor für die Bereitstellung des mindestens einen Transgens auszuwählen. In a first embodiment, the at least one nucleic acid of the kit according to the invention or the composition according to the invention is DNA. The DNA can be, for example, a DNA vector, e.g. a plasmid derived from bacteria or baker's yeast, a DNA vector of viral origin or a minicircle, which comprises at least one transgene coding for at least one of the components of the composition according to the invention or the kit according to the invention. The person skilled in the art is readily able to select a suitable DNA vector for providing the at least one transgene.
Bei dem mindestens einen Transgen kann es sich um das jeweilige Gen für Insulin, IGF-1 oder IGFBP-2 handeln, welches endogen in dem mit dem erfindungsgemäßen Kit oder der erfindungsgemäßen Zusammensetzung zu behandelndem Organismus vorkommt, oder aus dem gleichen Organismus stammt, aus dem eine zu behandelnde in vitro Kultur abgeleitet wurde. Gleiches gilt natürlich für die Anwendung in Proteinform. Sofern die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit beispielsweise für die Anwendung in einem menschlichen Subjekt vorgesehen ist, handelt es sich bei dem mindestens einen T ransgen bevorzugt um das menschliche Gen für Insulin, IGF-1 und/oder IGFBP-2 (und/oder das jeweilige menschliche Protein). Wie im Stand der Technik bekannt und durch die vorliegende Anmeldung bestätigt, gibt es jedoch auch eine Kreuzreaktivität zwischen den Spezies, so dass z.B. Maus-IGFBP2 auch im Menschen wirksam ist. Es können also z.B. Rinder- oder Maus-Proteine oder die dafür kodierenden Gene eingesetzt werden, auch in unterschiedlichen Kombinationen davon. Funktionelle Varianten sind ebenfalls aus dem Stand der Technik bekannt.
Beispielhaft sind humane Sequenzen spezifiziert. Das Insulin aus dem erfindungsgemäßen Kit oder der erfindungsgemäßen Zusammensetzung kann z.B. eine Aminosäuresequenz mit mindestens 50%, mindestens 60%, mindestens 70%, mindestsens 75%, mindestens 80%, mindestens 85%, mindestens 90%, mindestens 95%, mindestens 99% oder 100% Sequenzidentität zu der NCBI Referenzsequenz mit der Zulassungsnummer: NP_000198.1 aufweisen oder aus dieser bestehen. The at least one transgene can be the respective gene for insulin, IGF-1 or IGFBP-2, which occurs endogenously in the organism to be treated with the kit according to the invention or the composition according to the invention, or originates from the same organism from which an in vitro culture to be treated has been derived. The same applies, of course, to the application in protein form. If the composition or the kit according to the invention is intended for use in a human subject, for example, the at least one transgene is preferably the human gene for insulin, IGF-1 and/or IGFBP-2 (and/or the respective human protein). However, as is known in the art and confirmed by the present application, there is also cross-reactivity between species, such that, for example, mouse IGFBP2 is also effective in humans. Thus, for example, bovine or mouse proteins or the genes coding for them can be used, also in different combinations thereof. Functional variants are also known from the prior art. Human sequences are specified by way of example. The insulin from the kit according to the invention or the composition according to the invention can, for example, have an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or have 100% sequence identity to or consist of the NCBI reference sequence with accession number: NP_000198.1.
Das IGF-1 aus dem erfindungsgemäßen Kit oder der erfindungsgemäßen Zusammensetzung kann z.B. eine Aminosäuresequenz mit mindestens 50%, mindestens 60%, mindestens 70%, mindestsens 75%, mindestens 80%, mindestens 85%, mindestens 90%, mindestens 95%, mindestens 99% oder 100% Sequenzidentität zu der NCBI Referenzsequenz mit der Zulassungsnummer: NP_000609.1 aufweisen oder aus dieser bestehen. The IGF-1 from the kit according to the invention or the composition according to the invention can, for example, have an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least Have or consist of 99% or 100% sequence identity to the NCBI reference sequence with the accession number: NP_000609.1.
Das IGFBP-2 aus dem erfindungsgemäßen Kit oder der erfindungsgemäßen Zusammensetzung kann z.B. eine Aminosäuresequenz mit mindestens 50%, mindestens 60%, mindestens 70%, mindestsens 75%, mindestens 80%, mindestens 85%, mindestens 90%, mindestens 95%, mindestens 99% oder 100% Sequenzidentität zu den NCBI Referenzsequenzen mit den Zulassungsnummern: NP_000588.2 und/oder NP_001297588 aufweisen oder aus einer dieser Sequenzen bestehen. The IGFBP-2 from the kit according to the invention or the composition according to the invention can, for example, have an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to the NCBI reference sequences with the approval numbers: NP_000588.2 and/or NP_001297588 or consist of one of these sequences.
Auch im Falle eine Sequenzidentität von weniger als 100% ist das eingesetzte Protein funktional, d.h. es ist in der Lage, in Zusammenwirkung mit den anderen Komponenten der erfindungsgemäßen Zusammensetzung eine Erhöhung des intrazellulären Fettgehalts in einer in vitro-Kultur boviner Muskelzellen auszulösen. Even in the case of a sequence identity of less than 100%, the protein used is functional, i.e. it is able, in cooperation with the other components of the composition according to the invention, to trigger an increase in the intracellular fat content in an in vitro culture of bovine muscle cells.
Sofern die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit beispielsweise für die Anwendung in einem Rind vorgesehen ist, handelt es sich bei dem mindestens einen T ransgen bevorzugt um das Rinder-Gen für Insulin, IGF-1 und/oder IGFBP-2 (und/oder das jeweilige bovine Protein). Das Transgen kann sämtliche endogenen Kontrollelemente (z.B. Promotor) und/oder Introns umfassen. Alternativ kann das Transgen auch operabel mit einem heterologen Promoter verbunden sein und/oder frei von Introns sein. Es kann auch lediglich einige ausgewählte Introns umfassen oder künstliche bzw. artfremde Introns, die normalerweise nicht in einem der genannten Gene vorkommen. If the composition according to the invention or the kit according to the invention is intended for use in cattle, for example, the at least one transgene is preferably the bovine gene for insulin, IGF-1 and/or IGFBP-2 (and/or the respective bovine protein). The transgene may include any endogenous control elements (e.g. promoter) and/or introns. Alternatively, the transgene may be operably linked to a heterologous promoter and/or be devoid of introns. It can also include only a few selected introns or artificial or alien introns that do not normally occur in one of the genes mentioned.
Der heterologe Promoter kann ein konstitutiver Promoter sein, der eine konstante und fortwährende Expression des Transgens gewährleistet. Alternativ kann die Expression des Transgens auch durch einen gewebsspezifischen Promoter kontrolliert werden, sodass das
Transgen lediglich in einem konkreten Zielgewebe exprimiert wird, z.B. in Muskelzellen oder in Nervenzellen, bevorzugt in Muskelzellen. Auf diese Weise lassen sich gegebenenfalls unerwünschte Nebenwirkungen umgehen, die mit einer systemischen Anwendung des erfindungsgemäßen Kits oder der erfindungsgemäßen Zusammensetzung in einem Subjekt auftreten können. Eine kontrollierte, ausschließlich gewebsspezifische Expression von IGF-1 kann beispielsweise das mit der systemischen Überexpression von IGF-1 verbundene Risiko eines unspezifischen Zellwachstums in anderen, unerwünschten Geweben reduzieren. Alternativ kann der heterologe Promoter auch ein induzierbarer Promoter, z.B. ein Tet-induzier- barer Promoter sein, welcher die Expression des T ransgens nur in Gegenwart eines entsprechenden chemischen oder physikalischen Stimulus initiiert. Die Verwendung eines induzierbaren Promoters zur Expression der mindestens einen Komponente des erfindungsgemäßen Kits oder der erfindungsgemäßen Zusammensetzung kann beispielsweise dann besonders sinnvoll sein, wenn die Zusammensetzung oder das Kit für die Anwendung in einer in vitro Kultur vorgesehen ist. The heterologous promoter can be a constitutive promoter that ensures constant and sustained expression of the transgene. Alternatively, the expression of the transgene can also be controlled by a tissue-specific promoter, so that the Is transgenically expressed only in a specific target tissue, for example in muscle cells or in nerve cells, preferably in muscle cells. In this way, any undesired side effects that may occur in a subject with a systemic application of the kit according to the invention or the composition according to the invention can be avoided. Controlled, exclusively tissue-specific expression of IGF-1 can, for example, reduce the risk of non-specific cell growth in other, undesired tissues associated with systemic overexpression of IGF-1. Alternatively, the heterologous promoter can also be an inducible promoter, for example a Tet-inducible promoter, which initiates the expression of the transgene only in the presence of a corresponding chemical or physical stimulus. The use of an inducible promoter for the expression of at least one component of the kit according to the invention or the composition according to the invention can be particularly useful, for example, if the composition or the kit is intended for use in an in vitro culture.
Sofern die gleiche DNA für mehrere Komponenten der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits kodiert, kann eine einzelne DNA, z.B. das Plasmid, mehrere Transgene umfassen, deren Expression jeweils durch einen eigenen Promoter reguliert wird. Folglich ermöglicht eine einzige DNA die Expression dreier separater mRNA Moleküle, die getrennt voneinander in die drei durch die Zusammensetzung oder das Kit bereitzustellenden Komponenten translatiert werden können. Dieser Ansatz, genau wie die oben beschriebene Alternative, wonach die drei Komponenten jeweils von separaten Nukleinsäuren, d.h. drei unterschiedlichen DNA-Vektoren, exprimiert werden, hat den Vorteil, dass die einzelnen Komponenten der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits durch die gezielte Auswahl unterschiedlich starker Promotoren in unterschiedlichen Mengen innerhalb des Zielgewebes exprimiert werden können. So kann z.B. ein erhöhtes Krebsrisiko dadurch beschränkt werden, dass die IGF-1 Expression lediglich mittels eines schwachen und/oder induzierbaren Promoters angetrieben wird, während die Expression der übrigen Komponenten durch die Auswahl starker und/oder konstitutiver Promotoren gesteuert werden kann. Umgekehrt kann ein erwünschtes Zellwachstums durch eine gezielte gewebsspezifische Überexpression von IGF-1 gefördert werden. If the same DNA encodes several components of the composition according to the invention or the kit according to the invention, a single DNA, e.g. the plasmid, can comprise several transgenes, the expression of which is regulated in each case by its own promoter. Thus, a single DNA allows for the expression of three separate mRNA molecules that can be separately translated into the three components to be provided by the composition or kit. This approach, just like the alternative described above, according to which the three components are each expressed by separate nucleic acids, i.e. three different DNA vectors, has the advantage that the individual components of the composition according to the invention or the kit according to the invention have different strengths due to the targeted selection Promoters can be expressed at different levels within the target tissue. For example, an increased risk of cancer can be limited by driving IGF-1 expression only by means of a weak and/or inducible promoter, while the expression of the other components can be controlled by selecting strong and/or constitutive promoters. Conversely, a desired cell growth can be promoted by targeted tissue-specific overexpression of IGF-1.
Die für die einzelnen Komponenten der erfindungsgemäßen Zusammensetzung bzw. des erfindungsgemäßen Kits kodierenden Transgene können alternativ auch zu einem einzelnen Operon zusammengefasst werden, welches unter der Kontrolle eines gemeinsamen Promo-
ters steht. In einer solchen Ausführungsform würde die Transkription des Operons entsprechend zur Herstellung eines einzelnen polyzistronischen mRNA-Moleküls führen, das anschließend beispielsweise mittels gezielter RNA-Spleißung in drei separate mRNAs aufgeteilt und separat translatiert werden kann. Alternativ können die Gene für Insulin, IGF-1 und IG- FBP-2 hintereinander innerhalb eines Operons auf den DNA Vektor kloniert werden und jeweils durch eine interne ribosomale Eintritsstelle (IRES) voneinander getrennt werden. Hierdurch kann die Translation der entsprechend hergestellten polyzystronischen m RNA an mehreren Positionen, d.h. unabhängig von der Gegenwart einer 5’Cap-Endstruktur, gleichzeitig initiiert werden, sodass die drei Komponenten simultan mit derselben mRNA als Vorlage synthetisiert werden können. Da sich die Expressionseffizienz in der Regel aufgrund der eingeführten IRES-Sequenzen für jedes weitere nachfolgende Gen verringert, kann es vorteilhaft sein, wenn das IGF-1 -Gen an letzter Position innerhalb des Operons eingefügt wird. Somit fielen die Expressionslevel für IGF-1 geringer aus als für die beiden übrigen Komponenten, was ein erhöhtes Krebsrisiko verringern kann. Eine Expression mehrerer Komponenten von einem einzelnen Operon kann alternativ durch den Einsatz von zwischengelagerten 2A-Pep- tid-Sequenzen erzielt werden. In einer solchen Ausführungsform wird die transkribierte mRNA in ein einzelnes langes Polypeptid translatiert, welches anschließend aufgrund der zwischengelagerten 2A-Peptide in mehrere Proteine zerfällt. Einem Fachmann sind solche und ähnliche Vektordesign-Strategien zur gleichzeitigen Expression mehrerer Proteine aus dem Stand der Technik bekannt. Alternatively, the transgenes coding for the individual components of the composition according to the invention or the kit according to the invention can also be combined to form a single operon which is under the control of a common promoter ters stands. In such an embodiment, the transcription of the operon would correspondingly result in the production of a single polycistronic mRNA molecule, which can then be split into three separate mRNAs, for example by means of targeted RNA splicing, and translated separately. Alternatively, the genes for insulin, IGF-1 and IG-FBP-2 can be sequentially cloned within an operon on the DNA vector and separated from each other by an internal ribosomal entry site (IRES). As a result, the translation of the correspondingly produced polycystronic mRNA can be initiated simultaneously at several positions, ie independently of the presence of a 5′ cap end structure, so that the three components can be synthesized simultaneously using the same mRNA as a template. Since the expression efficiency is usually reduced due to the introduced IRES sequences for each subsequent gene, it can be advantageous if the IGF-1 gene is inserted at the last position within the operon. Thus, the expression levels for IGF-1 were lower than for the other two components, which may reduce an increased risk of cancer. Alternatively, expression of multiple components from a single operon can be achieved through the use of interleaved 2A peptide sequences. In such an embodiment, the transcribed mRNA is translated into a single long polypeptide, which subsequently breaks down into multiple proteins due to the intervening 2A peptides. Such and similar vector design strategies for the simultaneous expression of several proteins from the prior art are known to a person skilled in the art.
Bevorzugt handelt es sich bei der mindestens einen Nukleinsäure der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits um eine RNA. Die RNA kann beispielsweise ein RNA-Vektor viralen Ursprungs sein, z.B. ein lentiviraler Vektor. In einer bevorzugten Ausführungsform ist die mindestens eine Nukleinsäure jedoch eine mRNA, die für mindestens eine der Komponenten Insulin, IGF-1 oder IGFBP-2 kodiert. Wie hierin ausgeführt, kann jede Komponente durch eine separate mRNA kodiert werden. Optional kodiert die mindestens eine erfindungsgemäße mRNA jedoch für mehrere Komponenten, z.B. zwei oder alle drei Komponenten die durch die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit bereitgestellt werden sollen. Die Expression mehrerer unterschiedlicher Komponenten von einer einzelnen mRNA kann beispielsweise mittels gezielt zwischengeschalteter IRES- oder 2A-Peptidsequenzen erreicht werden, wie hierin beschrieben. The at least one nucleic acid of the composition according to the invention or the kit according to the invention is preferably an RNA. For example, the RNA may be an RNA vector of viral origin, e.g., a lentiviral vector. In a preferred embodiment, however, the at least one nucleic acid is an mRNA which encodes at least one of the components insulin, IGF-1 or IGFBP-2. As detailed herein, each component can be encoded by a separate mRNA. Optionally, however, the at least one mRNA according to the invention encodes several components, e.g. two or all three components, which are to be provided by the composition according to the invention or the kit according to the invention. Expression of multiple different components from a single mRNA can be achieved, for example, using the purposefully interposed IRES or 2A peptide sequences, as described herein.
Die mRNA kann eine natürliche mRNA sein, d.h., eine mRNA, die durch eine Zelle exprimiert und anschließend isoliert wurde. Derartige natürliche mRNAs haben jedoch den Nachteil,
dass sie eine geringe Stabilität aufweisen und bei Verabreichung in ein Subjekt oder eine in vitro Kultur eine unerwünschte Immunreaktion auslösen können. Daher wird die erfindungsgemäße mRNA in einer bevorzugten Ausführungsform als chemisch modifizierte mRNA synthetisch hergestellt. Zum Beispiel kann es sich bei der mindestens einen mRNA um eine Nukleosid-modifizierte mRNA handeln, bei der einzelne Nukleoside durch andere natürliche modifizierte Nukleoside oder durch synthetische Nukleosid-Analoga ersetzt wurden. So können in der mindestens einen mRNA ein oder mehrere Uridin-Nukleoside durch Pseudouridin oder N1-Methylpseudouridin ersetzt werden. Alternativ oder zusätzlich kann Cytosin innerhalb der mindestens einen mRNA durch 5-Methyl-Cytosin ersetzt werden. Der Einsatz derartiger Nukleosid-Analoga beeinflusst die Sekundärstruktur der modifizierten mRNA, sodass diese schlechter oder nicht mehr durch das Immunsystem erkannt und bekämpft werden kann. Die Stabilität der mindestens einen modifizierten mRNA kann ferner durch die gezielte Auswahl geeigneter untranslatierter Regionen (UTR) am 3‘- und 5‘-Ende der mRNA sowie den Einsatz weiterer Nukleosidanaloga, wie beispielsweise /V6-methyladenosine, /\/6,2'-O-di- methyladenosine, 8-oxo-7,8-dihydroguanosine oder /V4-acetylcytidine erhöht werden. Dem Fachmann sind unterschiedliche Verfahren und Werkzeuge zur synthetischen Herstellung einer erfindungsgemäßen mRNA aus dem Stand der Technik bekannt. The mRNA can be a natural mRNA, ie mRNA that has been expressed by a cell and subsequently isolated. However, such natural mRNAs have the disadvantage that that they have poor stability and can elicit an undesirable immune response when administered to a subject or in vitro culture. Therefore, in a preferred embodiment, the mRNA according to the invention is produced synthetically as a chemically modified mRNA. For example, the at least one mRNA can be a nucleoside-modified mRNA in which individual nucleosides have been replaced by other natural modified nucleosides or by synthetic nucleoside analogs. Thus, in the at least one mRNA, one or more uridine nucleosides can be replaced by pseudouridine or N 1 -methylpseudouridine. Alternatively or additionally, cytosine within the at least one mRNA can be replaced by 5-methyl-cytosine. The use of such nucleoside analogues influences the secondary structure of the modified mRNA, so that it can no longer be recognized and combated by the immune system. The stability of the at least one modified mRNA can also be increased by the targeted selection of suitable untranslated regions (UTR) at the 3' and 5' end of the mRNA and the use of other nucleoside analogs, such as /V 6 -methyladenosine, /\/ 6,2 '-O-dimethyladenosine, 8-oxo-7,8-dihydroguanosine or /V 4 -acetylcytidine are increased. The person skilled in the art is familiar with different methods and tools for the synthetic production of an mRNA according to the invention from the prior art.
Für die Applikation wird die mindestens eine mRNA des erfindungsgemäßen Kits oder der erfindungsgemäßen Zusammensetzung bevorzugt in einen geeigneten, physiologisch unbedenklichen Carrier eingebracht. Geeignete Carrier umfassen beispielsweise Lipidnanopartikel, die u.a. auch bei mRNA-basierten Vakzinen zum Einsatz kommen. Weitere geeignete Carrier für das Einbringen der mindestens einen mRNA in eine Zielzelle sind dem Fachmann ebenfalls aus dem Stand der Technik bekannt und können beispielsweise Minizellen, RNA basierte Nanopartikel oder Dendromere umfassen. In einigen Ausführungsformen können die Carrier mit spezifischen Targeting-Molekülen ausgestattet sein, die einen zielgerichteten Transport der mindestens einen mRNA zu einem gewünschten Gewebe innerhalb des mit der erfindungsgemäßen Zusammensetzung oder dem erfindungsgemäßen Kit behandelten Subjekts ermöglicht, z.B. zu Muskelzellen und/oder Nervenzellen. Alternativ kann die mindestens eine mRNA innerhalb der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits jedoch auch ohne Carrier verabreicht werden. For the application, the at least one mRNA of the kit according to the invention or the composition according to the invention is preferably introduced into a suitable, physiologically harmless carrier. Suitable carriers include, for example, lipid nanoparticles, which are also used in mRNA-based vaccines, among other things. Other suitable carriers for introducing the at least one mRNA into a target cell are also known to the person skilled in the art from the prior art and can include, for example, minicells, RNA-based nanoparticles or dendromers. In some embodiments, the carriers can be equipped with specific targeting molecules that enable targeted transport of the at least one mRNA to a desired tissue within the subject treated with the composition or the kit according to the invention, e.g. to muscle cells and/or nerve cells. Alternatively, however, the at least one mRNA within the composition according to the invention or the kit according to the invention can also be administered without a carrier.
Unabhängig davon, ob die mindestens eine in der erfindungsgemäßen Zusammensetzung bzw. dem erfindungsgemäßen Kit vorliegende Nukleinsäure eine DNA oder RNA ist, ist die Nukleinsäure bevorzugt für die Expression in dem Zielorganismus und/oder dem Zielgewebe
Codon-optimiert. Hierbei werden vereinzelte Triplettcodons der Nukleotidsequenz im Zuge einer in vitro Mutagenese durch synonyme Codons ersetzt, um die Expressionsrate der einzelnen Komponenten an die bevorzugte Codonverwendung des Zielorganismus oder des Zielgewebes anzupassen. Irrespective of whether the at least one nucleic acid present in the composition or the kit according to the invention is DNA or RNA, the nucleic acid is preferred for expression in the target organism and/or the target tissue Codon optimized. Here, individual triplet codons of the nucleotide sequence are replaced by synonymous codons in the course of an in vitro mutagenesis in order to adapt the expression rate of the individual components to the preferred codon usage of the target organism or the target tissue.
Die vorliegende Erfindung offenbart ferner die Verwendung der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits zur Erhöhung des intramuskulären Fettgehalts in Fleisch, bevorzugt von Nutztieren, optional von Bovinen, oder in in v/Yro-Kultur. The present invention further discloses the use of the composition according to the invention or the kit according to the invention for increasing the intramuscular fat content in meat, preferably from livestock, optionally from bovines, or in in v/yro culture.
Im Kontext der Erfindung bedeutet „eine Erhöhung des intramuskulären Fettgehalts in Fleisch“, dass das Fleisch in Reaktion auf die hierin offenbarte Verwendung der erfindungsgemäßen Zusammensetzung oder des Kits quantitativ einen erhöhten Anteil von Fettgewebe innerhalb des Muskelgewebes aufweist. Zudem kann das Fettgewebe bevorzugt besonders gleichmäßig innerhalb des Fleisches verteilt sein. Der Fachmann spricht in diesem Kontext von einer besonders feinen und gleichmäßigen Marmorierung des Fleisches. In the context of the invention, "an increase in intramuscular fat content in meat" means that the meat quantitatively has an increased proportion of adipose tissue within muscle tissue in response to the use of the composition or kit of the invention disclosed herein. In addition, the fatty tissue can preferably be distributed particularly evenly within the meat. In this context, the expert speaks of a particularly fine and even marbling of the meat.
Das Fleisch des Kobe-Rinds (auch als Wagyu-Rind bekannt) ist insbesondere durch seine besonders mürbe Struktur und eben eine solche feine Marmorierung mit Fettäderchen bekannt. Aufgrund seines hohen intramuskulären Fettgehalts ist das Fleisch aus Wagyu-Rin- dern besonders zart und schmackhaft und wird daher weltweit als Delikatesse angesehen. Eine Studie von Connolly et al., 2020 offenbarte erst kürzlich einen möglichen Zusammenhang zwischen der feinen Marmorierung des Wagyu-Fleisches und einem erhöhten Zuckergehalt in dem Serum der Rinder. Eine besonders effiziente Versorgung der Muskeln des Wagyu-Rinds mit Kohlenhydraten kann somit den ungewöhnlich hohen intramuskulären Fettgehalt des Fleisches begünstigen. The meat of the Kobe beef (also known as Wagyu beef) is known in particular for its particularly tender structure and such fine marbling with small veins of fat. Due to its high intramuscular fat content, Wagyu beef is particularly tender and tasty and is therefore considered a delicacy worldwide. A study by Connolly et al., 2020 recently revealed a possible link between the fine marbling of Wagyu beef and increased sugar levels in the cattle serum. A particularly efficient supply of carbohydrates to the muscles of Wagyu beef can therefore promote the unusually high intramuscular fat content of the meat.
Lebewesen verfügen über einen komplexen Energiestoffwechsel, um ausreichend Energie für ihr Überleben bereitzustellen. Die gewonnene Energie wird für die Aufrechterhaltung der normalen Körperfunktionen und den Aufbau von Körperbestandteilen benötigt. Es ist daher wichtig, dass Lebewesen bei Bedarf jederzeit auf körpereigene Energiespeicher zurückgreifen können, um die notwendige Energie bereitstellen zu können. Living beings have a complex energy metabolism in order to provide sufficient energy for their survival. The energy gained is needed to maintain normal bodily functions and to build up body components. It is therefore important that living beings can access the body's own energy stores at any time if necessary in order to be able to provide the necessary energy.
Der natürlich vorkommende Einfachzucker Glucose ist ein wichtiger Energieträger. Glucose kann durch Zellen im Zuge der Glykolyse metabolisiert werden, um Energie in Form von A- denosintriphosphat (ATP) zu gewinnen. Der Abbau von Glucose liefert zudem auch die Bausteine für die Biosynthese vieler weiterer überlebenswichtiger Verbindungen. Beispielsweise
entsteht bei der Glycolyse von Glucose Pyruvat, welches als Ausgangsstoff u.a. für die Herstellung von Fettsäuren benötigt wird. Fettsäuren dienen Zellen wiederum als Ausgangsstoff für die Biosynthese von Lipiden bzw. Fetten. Lipide sind sehr energiereiche Verbindungen, die durch den Körper in Form von Speicher- oder Depotfett in die Zellen des Fettgewebes, den sogenannten Adipozyten, eingelagert werden. Die Fettreserven dienen als Langzeitenergiespeicher und können z.B. einen gesunden Menschen über Wochen mit Energie versorgen. Adipozyten können über die Ernährung bereitgestellte Glucose aus dem Blut aufnehmen und somit die Fettreserven auffüllen oder vergrößern. The naturally occurring simple sugar glucose is an important source of energy. Glucose can be metabolized by cells during glycolysis to produce energy in the form of adenosine triphosphate (ATP). The breakdown of glucose also supplies the building blocks for the biosynthesis of many other compounds that are essential for survival. For example formed during the glycolysis of glucose pyruvate, which is required as a starting material for the production of fatty acids, among other things. Fatty acids in turn serve cells as a starting material for the biosynthesis of lipids or fats. Lipids are very high-energy compounds that are stored by the body in the form of storage or depot fat in the cells of the adipose tissue, the so-called adipocytes. The fat reserves serve as a long-term energy store and can, for example, supply a healthy person with energy for weeks. Adipocytes can absorb glucose provided by the diet from the blood and thus replenish or increase the fat reserves.
Im Gegensatz zu Adipozyten speichern Muskelzellen aus dem Blut aufgenommene Glucose in Form von Glykogen, einem aus Glucose-Monomeren aufgebauten Polysaccharid. Im Gegensatz zu Fett, welches der langfristigen Energiespeicherung dient und den Körper über mehrere Wochen mit Energie versorgen kann, stellt Glykogen eher einen kurz- bis mittelfristiger Energiespeicher dar, der den Muskeln je nach Belastungsintensität lediglich für einige Stunden Energie liefert. Unlike adipocytes, muscle cells store glucose taken from the blood in the form of glycogen, a polysaccharide made up of glucose monomers. In contrast to fat, which is used for long-term energy storage and can supply the body with energy for several weeks, glycogen is more of a short- to medium-term energy store that only supplies the muscles with energy for a few hours, depending on the intensity of the load.
Gewebe nutzen unterschiedliche Mechanismen zur Aufnahme von Glukose. Vor allem in Zellen des zentralen Nervensystems, Hepatozyten, der Darmmukosa, und den Epithelzellen der Niere erfolgt die Glucoseaufnahme mittels insulinunabhängiger Glucosetransporter. Im Gegensatz dazu findet sich im Muskel- und im Fettgewebe mit dem Glucosetransporter Glut4 ein insulinabhängiger Kanal, der den Transport von Glucose aus der Zirkulation in die Fett- und Muskelzellen ermöglicht. Der Mechanismus der Insulin-Wirkung wird hierbei über den Insulin-Rezeptor und den AKT-Kinasesignalweg vermittelt. Tissues use different mechanisms to take up glucose. Above all in cells of the central nervous system, hepatocytes, the intestinal mucosa and the epithelial cells of the kidneys, glucose uptake takes place by means of insulin-independent glucose transporters. In contrast, muscle and fat tissue contains the glucose transporter Glut4, an insulin-dependent channel that enables the transport of glucose from the circulation into fat and muscle cells. The mechanism of insulin action is mediated via the insulin receptor and the AKT kinase signaling pathway.
Die vorliegende Erfindung offenbart, dass durch die Verabreichung der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits an ein Subjekt dessen Insulinsensiti- vität erhöht und somit die Aufnahme von Glucose in Muskel- und Fettzellen gezielt gefördert werden kann. Von einer „Erhöhung“ der Insulinsensitivität kann gesprochen werden, wenn die Insulinwirksamkeit, also die Fähigkeit des Insulins, die Aufnahme von Glucose aus dem Blut in die Muskel- und Fettzellen zu fördern, nach der Verabreichung der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits stärker ausfällt als davor. Die vorliegende Erfindung erlaubt folglich eine gewebsspezifische Beeinflussung des Energiestoffwechsels insbesondere in Muskel- und Fettgewebe und ermöglicht dadurch eine selektive hormonelle Kontrolle des Muskel- und Fettansatzes. The present invention reveals that administering the composition according to the invention or the kit according to the invention to a subject increases their insulin sensitivity and thus the uptake of glucose in muscle and fat cells can be promoted in a targeted manner. One can speak of an "increase" in insulin sensitivity if the insulin effectiveness, i.e. the ability of insulin to promote the absorption of glucose from the blood into the muscle and fat cells, after administration of the composition or the kit according to the invention is greater than before. The present invention consequently allows tissue-specific influencing of the energy metabolism, particularly in muscle and fat tissue, and thereby enables selective hormonal control of muscle and fat accumulation.
In einer wichtigen Ausführungsform der Erfindung ist die erfindungsgemäße Zusammensetzung bzw. das erfindungsgemäße Kit für die Verwendung in Nutztieren vorgesehen. Unter
dem Begriff „Nutztier“ werden im Allgemeinen Tiere zusammengefasst, die aufgrund bestimmter Fähigkeiten oder aus ästhetischen Gründen vom Menschen wirtschaftlich genutzt werden (https://de.wikipedia.org/wiki/Nutztier). Im Kontext der vorliegenden Erfindung bezieht sich das Wort „Nutztier“ jedoch insbesondere auf Tiere, die als Nahrungslieferanten, insbesondere als Fleischlieferanten genutzt werden. Nutztiere im Sinne der Erfindung sind somit vornehmlich Schlachttiere, wie beispielsweise Mitglieder der Familie der Bovinae, Schweine, Schafe, Ziegen, Pferde, Zebras, Kamele, Lamas, Alpakas, Geflügel, Hasen oder Kaninchen. In an important embodiment of the invention, the composition according to the invention or the kit according to the invention is intended for use in farm animals. Under The term "livestock" generally includes animals that are used economically by humans due to certain abilities or for aesthetic reasons (https://de.wikipedia.org/wiki/Nutztier). In the context of the present invention, however, the word “livestock” refers in particular to animals that are used as suppliers of food, in particular as suppliers of meat. Farm animals within the meaning of the invention are therefore primarily animals for slaughter, such as members of the Bovinae family, pigs, sheep, goats, horses, zebras, camels, llamas, alpacas, poultry, hares or rabbits.
Durch die Bereitstellung der drei Komponenten Insulin, IGF-1 und IGFBP-2 mit Hilfe des erfindungsgemäßen Kits bzw. der erfindungsgemäßen Zusammensetzung lässt sich, wie oben beschrieben, die Glucoseaufnahme aus dem Blut in das Muskel- und Fettgewebe dieser Tiere selektiv erhöhen. Entsprechend kann der intramuskuläre Fettgehalt der Tiere gesteigert werden, was sich positiv auf die Qualität und Zartheit des produzierten Fleisches dieser Nutztiere auswirkt. By providing the three components insulin, IGF-1 and IGFBP-2 using the kit according to the invention or the composition according to the invention, the glucose uptake from the blood into the muscle and adipose tissue of these animals can be selectively increased, as described above. Accordingly, the intramuscular fat content of the animals can be increased, which has a positive effect on the quality and tenderness of the meat produced by these livestock.
Bevorzugt handelt es sich bei den Nutztieren um Mitglieder der Familie der Bovinae. Diese Unterfamilie der Hornträger umfasst z.B. Rinder, Bisons, Büffel und sämtliche Antilopenarten. Besonders bevorzugt handelt es sich bei den Nutztieren um Rinder, am meisten bevorzugt um einheimische Rinderrassen wie dem Angler-Rind, dem Ansbach-Triesdorfer-Rind, dem Braunvieh, dem Angus, z.B. dem deutschen Angus, dem deutschen Holstein Rot- bunt/Schwarzbunt, dem deutschen schwarzbunten Niederungsrind, dem deutschen Shorthorn, dem Fleckvieh, dem Gelbvieh, dem Glanrind, dem Hinterwälder oder dem Lim- purger. Durch die gezielte Erhöhung des Fettgehalts in den Muskeln dieser Rinderrassen mit Hilfe der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits können diese Tiere Fleisch von einer Qualität und Zartheit bereitstellen, das dem Fleisch aus den exklusiven japanischen Wagyu-Rindern ähnelt oder gar entspricht. The farm animals are preferably members of the Bovinae family. This subfamily of bovids includes, for example, cattle, bison, buffalo and all antelope species. The farm animals are particularly preferably cattle, most preferably native cattle breeds such as the Angler cattle, the Ansbach-Triesdorfer cattle, the Brown Swiss, the Angus, e.g. the German Angus, the German Holstein Rotbunt/Schwarzbunt, the German black and white lowland cattle, the German Shorthorn, the Fleckvieh, the Gelbvieh, the Glanrind, the Hinterwalder or the Limpurger. By specifically increasing the fat content in the muscles of these breeds of cattle with the aid of the composition or the kit according to the invention, these animals can provide meat of a quality and tenderness that resembles or even corresponds to the meat from the exclusive Japanese Wagyu cattle.
Die hierin beschriebene hormonelle gewebsspezifische Beeinflussung des Energiestoffwechsels und selektive Kontrolle des Muskel- und Fettansatzes kann sich ebenfalls positiv auf die Fleischqualität weiterer, hierin aufgeführter Schlachttierarten auswirken. So kann unter Umständen das Fleisch von Tieren, das von einigen Konsumenten aufgrund seiner hohen Festigkeit und Trockenheit grundsätzlich eher gemieden wird, durch eine erhöhte Fetteinlagerungen zarter und somit geschmacklich anregender gestaltet werden.
Die Verwendung der erfindungsgemäßen Zusammensetzung bzw. des erfindungsgemäßen Kits in Nutztieren wirkt sich jedoch nicht lediglich auf die Qualität des erzeugten Fleisches aus. In einer weiteren Ausführungsform trägt die Verwendung der Zusammensetzung oder des Kits auch zur Verbesserung der Futtereffizienz bei Nutztieren bei. So verringert ein hormonell gesteuerter, erhöhter Fettansatz in der Muskulatur von Schweinen beispielsweise Energieverluste während der Aufzucht, was eine kostengünstigere Haltung erlaubt. Daher ist die Verwendung der erfindungsgemäßen Zusammensetzung bzw. des erfindungsgemäßen Kits auch in solchen Nutztieren von Interesse, die nicht zwangsläufig als Fleischlieferanten dienen. So optimiert eine erhöhte Futtereffizienz auch die Haltung beispielsweise von Milchkühen oder Schafen als Wolllieferanten. The hormonal, tissue-specific influencing of energy metabolism and selective control of muscle and fat accumulation described here can also have a positive effect on the meat quality of other slaughter animal species listed here. Under certain circumstances, the meat of animals, which some consumers generally tend to avoid due to its high firmness and dryness, can be made more tender and thus taste stimulating through increased fat deposits. However, the use of the composition according to the invention or the kit according to the invention in livestock does not only affect the quality of the meat produced. In another embodiment, use of the composition or kit also contributes to improving feed efficiency in livestock. For example, a hormonally controlled, increased amount of fat in the muscles of pigs reduces energy losses during rearing, which allows for more cost-effective husbandry. Therefore, the use of the composition according to the invention or the kit according to the invention is also of interest in those farm animals that do not necessarily serve as meat suppliers. Increased feed efficiency also optimizes the keeping of dairy cows or sheep as wool suppliers, for example.
In einer weiteren Ausführungsform der Erfindung ist die erfindungsgemäße Zusammensetzung bzw. das erfindungsgemäße Kit für die Verwendung in einer in vitro Kultur vorgesehen. Die gezielte in vitro Züchtung von Gewebe mit der Maßgabe, Fleisch zum menschlichen Verzehr im industriellen Maßstab synthetisch herzustellen, hat in den letzten Jahren rasante Fortschritte gemacht. Die Erzeugung von in v/Yro-Fleisch basiert auf den Methoden der Zellkultur, insbesondere auf den Methoden der Gewebezüchtung wie die 3D-Zellkultur und das Tissue Engineering (https://de.wikipedia.org/wiki/ln-vitro-Fleisch). Die Herstellung von derartigem „Laborfleisch“ besitzt das Potential, eine Vielzahl ethischer und ökologischer Probleme, die mit der Massentierhaltung in der Fleischindustrie verbunden sind, zu überwinden. Eine wichtige Herausforderung auf dem Gebiet der in v/Yro-Fleischzucht besteht darin, synthetisches Fleisch mit hohem ernährungsphysiologischem Wert herzustellen, das zudem geschmacklich überzeugen kann. Die Verwendung der erfindungsgemäßen Zusammensetzung bzw. des erfindungsgemäßen Kits in der in v/Yro-Kultur kann sich daher auch positiv auf die Qualität und die Zartheit des synthetisch hergestellten Fleischs auswirken. In a further embodiment of the invention, the composition according to the invention or the kit according to the invention is intended for use in an in vitro culture. The targeted in vitro cultivation of tissue with the aim of synthetically producing meat for human consumption on an industrial scale has made rapid progress in recent years. The production of in v/Yro meat is based on cell culture methods, in particular on tissue engineering methods such as 3D cell culture and tissue engineering (https://de.wikipedia.org/wiki/ln-vitro-Fleisch) . The production of such “laboratory meat” has the potential to overcome a variety of ethical and environmental issues associated with factory farming in the meat industry. An important challenge in the field of in v/yro meat farming is to produce synthetic meat with a high nutritional value that can also convince in terms of taste. The use of the composition according to the invention or the kit according to the invention in the in v/yro culture can therefore also have a positive effect on the quality and tenderness of the synthetically produced meat.
Die vorliegende Erfindung stellt somit auch ein Verfahren zur Erhöhung des intramuskulären Fettgehalts in Fleisch bereit, bevorzugt von Nutztieren oder von in v/Yro-gezüchtetem Fleisch. Das Verfahren umfasst einen Schritt, bei dem man die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit an das Nutztier oder der Kultur verabreicht. So kann z.B. in einer in vitro-Kultur boviner Muskelzellen der intramuskuläre Fettgehalt erhöht werden, indem man bovine Zellen, bevorzugt Rinder-Zellen, mit der erfindungsgemäßen Zusammensetzung kultiviert. Gegenstand der Erfindung ist auch eine in vitro-Kultur von Muskelzellen,
z.B. bovinen Muskelzellen, in einem Zellkulturmedium, wobei das Zellkulturmedium die Proteine der erfindungsgemäßen Zusammensetzung umfasst. Diese sind bevorzugt exogenen Ursprungs, z.B. gentechnisch hergestellt. Es kann sich um bovine Proteine handeln. The present invention thus also provides a method for increasing the intramuscular fat content in meat, preferably from farm animals or from v/yro-grown meat. The method comprises a step in which the composition or the kit according to the invention is administered to the livestock or crop. For example, the intramuscular fat content can be increased in an in vitro culture of bovine muscle cells by cultivating bovine cells, preferably bovine cells, with the composition according to the invention. The invention also relates to an in vitro culture of muscle cells, eg bovine muscle cells, in a cell culture medium, the cell culture medium comprising the proteins of the composition according to the invention. These are preferably of exogenous origin, for example produced by genetic engineering. It can be bovine proteins.
In einer Ausführungsform wird die Zusammensetzung oder das Kit bzw. mindestens eine der Komponenten des Kits dem Tier systemisch verabreicht, also etwa in die Blutzirkulation injiziert. Bevorzugt ist eine lokale Applikation der erfindungsgemäßen Zusammensetzung oder mindestens einer Komponente des Kits in das Muskel- und/oder Fettgewebe des Tieres.In one embodiment, the composition or the kit or at least one of the components of the kit is administered to the animal systemically, ie injected into the bloodstream. Local application of the composition according to the invention or at least one component of the kit into the muscle and/or fat tissue of the animal is preferred.
Optional kann die erfindungsgemäße Zusammensetzung oder mindestens eine Komponente davon, etwa Insulin und/oder IGFBP-2, für eine orale Aufnahme durch das Tier in einen geeigneten Träger, beispielsweise einer Kapsel eingearbeitet und dem Tier gemeinsam mit der üblichen Nahrung verabreicht werden. Eine derartige Kapsel ist mit einem magensaftresistenten Überzug, d. h. einer geeigneten Polymerbarriere zur Verhinderung der Auflösung im Magenmilieu, beschichtet. Die magensaftresistente Beschichtung sollte so gewählt werden, dass sie bei niedrigem pH-Wert der Magensäure stabil bleibt, sich aber auflöst, sobald sie in die alkalischere Umgebung des Dünndarms gelangt. Geeignete Überzüge sind z. B. in der Fachwelt bekannt. Die orale Verabreichung der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits über eine magensaftresistente Kapsel ermöglicht somit eine gezielte Aufnahme der Komponenten in die Blutzirkulation über die Darmwand und kann unnötigen Stress für die Tiere vermeiden. Auch eine intranasale Verabreichung mindestens einer der Komponenten des Kits oder der Zusammensetzung ist möglich. Optionally, the composition of the invention, or at least one component thereof, such as insulin and/or IGFBP-2, may be incorporated into a suitable carrier, such as a capsule, for oral ingestion by the animal and administered to the animal along with the animal's usual diet. Such a capsule is provided with an enteric coating, i. H. a suitable polymeric barrier to prevent dissolution in the gastric environment. The enteric coating should be chosen so that it remains stable in the low pH of gastric acid but dissolves once it enters the more alkaline environment of the small intestine. Suitable coatings are e.g. B. known in the art. The oral administration of the composition according to the invention or the kit according to the invention via a gastric juice-resistant capsule thus enables a targeted absorption of the components into the blood circulation via the intestinal wall and can avoid unnecessary stress for the animals. Intranasal administration of at least one of the components of the kit or of the composition is also possible.
Die Administration der erfindungsgemäßen Zusammensetzung oder des erfindungsgemäßen Kits kann durch medizinisches Fachpersonal erfolgen. Bevorzugt kann die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit jedoch durch den Nutztierhalter selbst, d.h. auch in Abwesenheit eines medizinisch geschulten Fachmanns bzw. einer medizinisch geschulten Fachfrau dem Nutztier verabreicht werden. The composition according to the invention or the kit according to the invention can be administered by medical professionals. However, the composition according to the invention or the kit according to the invention can preferably be administered to the livestock by the farm animal owner himself, i.e. even in the absence of a medically trained specialist or a medically trained specialist.
Für den Fall, dass die Zusammensetzung oder das Kit im Zuge des erfindungsgemäßen Verfahrens einer in vitro Kultur verabreicht werden soll, hängt die Art und Weise der Verabreichung von der bereitgestellten Form der einzelnen Komponenten ab. Die Verabreichung kann mittels direkter Injektion in die Zellkultur erfolgen, etwa in bereits in vitro generiertes Fleisch. Alternativ kann die Zusammensetzung oder das Kit in das Zellmedium eingebracht werden, um durch die Zelle z.B. mittels Endozytose aufgenommen zu werden. Nukleinsäuren, die für eine oder mehrere Komponenten kodieren, können auch mittels Transfektion in die Zellen eingeschleust werden. Hierzu bieten sich sowohl chemische Transfektionsverfah- ren wie Lipofektion oder Calcium-Phosphat-Präzipitation oder physikalische Verfahren mit Hilfe von Mikroinjektion oder Elektroporation an. Verfahren zur Einbringung exogener Prote-
ine oder Nukleinsäuren in Zell- und Gewebskulturen sind aus dem Stand der Technik hinlänglich bekannt. In diesem Fall ist es hilfreich, die Zellen vor oder am Anfang der in vitro- Kultur zu transfizieren, so dass alle Abkömmlinge der transfizierten Zellen die vorteilhaften Nukleinsäuren tragen. Gegenstand der Erfindung ist damit auch eine Zelle, etwa eine Muskeloder Fettzelle oder eine Vorläuferzelle, etwa eine mesenchymale Stammzelle, die die Nukleinsäure^) des erfindungsgemäßen Kits umfasst, wobei die Nukleinsäuren in nicht einer Form vorliegen, die in Wildtyp-Zellen dieses Zelltyps vorliegt. In the event that the composition or the kit is to be administered to an in vitro culture in the course of the method according to the invention, the manner of administration depends on the form of the individual components provided. It can be administered by direct injection into the cell culture, for example into meat that has already been generated in vitro. Alternatively, the composition or kit can be incorporated into the cell medium to be taken up by the cell, for example by endocytosis. Nucleic acids encoding one or more components can also be introduced into the cells by transfection. Both chemical transfection methods such as lipofection or calcium phosphate precipitation or physical methods using microinjection or electroporation are suitable for this purpose. Procedure for introducing exogenous proteins Ine or nucleic acids in cell and tissue cultures are well known from the prior art. In this case it is helpful to transfect the cells before or at the beginning of the in vitro culture so that all progeny of the transfected cells carry the beneficial nucleic acids. The subject matter of the invention is therefore also a cell, such as a muscle or fat cell or a progenitor cell, such as a mesenchymal stem cell, which comprises the nucleic acid ^) of the kit according to the invention, the nucleic acids not being present in a form that is present in wild-type cells of this cell type.
Die Zusammensetzung oder das Kit kann dem Nutztier und/oder der in v/Yro-Kultur einmalig verbreicht werden. Die Verabreichung kann jedoch auch mehrmals wiederholt werden, z.B. in Form einer längerfristigen Hormonkur. So kann die Zusammensetzung oder das Kit beispielsweise für eine sich monatlich, wöchentlich oder sogar täglich wiederholende Verabreichung vorgesehen sein. The composition or kit can be administered once to the farm animal and/or in v/yro culture. However, the administration can also be repeated several times, e.g. in the form of a longer-term hormone cure. For example, the composition or kit may be designed to be administered on a monthly, weekly or even daily basis.
Die hierin offenbarte Zusammensetzung bzw. das Kit kann auch zu therapeutischen Zwecken in einem Subjekt eingesetzt werden. Folglich kann es sich bei der erfindungsgemäßen Zusammensetzung oder dem erfindungsgemäßen Kit auch um eine pharmazeutische Zusammensetzung oder ein pharmazeutisches Kit handeln, die bzw. das einem Subjekt zu Therapiezwecken oder zur Prävention einer Erkrankung verabreicht werden kann. Eine derartige erfindungsgemäße pharmazeutische Zusammensetzung oder ein pharmazeutisches Kit kann zusätzlich mindestens einen pharmazeutisch akzeptablen Hilfsstoff umfassen. The composition or kit disclosed herein can also be used for therapeutic purposes in a subject. Accordingly, the composition or kit of the invention can also be a pharmaceutical composition or kit which can be administered to a subject for the purpose of therapy or prevention of a disease. Such a pharmaceutical composition or a pharmaceutical kit according to the invention can additionally comprise at least one pharmaceutically acceptable excipient.
Im Kontext der Erfindung ist ein Subjekt, das mit der pharmazeutischen Zusammensetzung oder dem pharmazeutischen Kit behandeln werden soll, bevorzugt ein Tier. Das Subjekt kann ein Nutztier sein, wie hierin beschrieben, oder ein Tier, das in Rennen oder für körperliche Arbeit eingesetzt wird, etwa ein Pferd, Kamel, Hund, Rind, Büffel oder Elefant, oder ein Haustier, etwa eine Katze, ein Hund oder ein Kaninchen. Am meisten bevorzugt ist das Subjekt, das mit der pharmazeutischen Zusammensetzung oder dem pharmazeutischen Kit behandelt werden soll, ein Mensch. In the context of the invention, a subject to be treated with the pharmaceutical composition or the pharmaceutical kit is preferably an animal. The subject may be a farm animal, as described herein, or an animal used in racing or manual labor, such as a horse, camel, dog, cattle, buffalo, or elephant, or a domestic animal, such as a cat, dog, or a rabbit. Most preferably, the subject to be treated with the pharmaceutical composition or kit is a human.
In einer Ausführungsform kann die erfindungsgemäße pharmazeutische Zusammensetzung oder das erfindungsgemäße pharmazeutische Kit zur Verwendung bei der Behandlung von Insulinresistenz, bevorzugt schwerer Insulinresistenz vorgesehen sein. In one embodiment, the pharmaceutical composition or the pharmaceutical kit according to the invention can be intended for use in the treatment of insulin resistance, preferably severe insulin resistance.
Von einer Insulinresistenz wird gesprochen, wenn insbesondere die Muskulatur, das Fettgewebe und/oder die Leber eines Organismus, z.B. eines Menschen, lediglich vermindert auf
das Hormon Insulin anspricht. Dies kann geschehen, wenn der insulinabhängige Glut4 Glucosetransporter in Gegenwart von Insulin nur einen unzureichenden Glucosetransport in das Muskel- und Fettgewebe vermittelt. We speak of insulin resistance when, in particular, the musculature, fatty tissue and/or the liver of an organism, eg a human being, is merely reduced the hormone insulin responds. This can happen when the insulin-dependent Glut4 glucose transporter mediates insufficient glucose transport into muscle and adipose tissue in the presence of insulin.
In einem gesunden Menschen liegt die physiologische Insulinproduktion, die benötigt wird, um normale Blutzuckerwerte aufrechtzuerhalten, bei ungefähr 20-40 Internationalen Einheiten (I.E.) Insulin pro Tag. Liegt die benötigte Insulinproduktion über mehrere Tage über diesen Werten, so lässt sich von einer Insulinresistenz sprechen. Eine „schwere Insulinresistenz“ wird im Allgemeinen bei einer längerfristigen Ausschüttung von über 200 I.E. Insulin pro Tag angenommen. Eine Insulinresistenz ist mit zahlreichen Erkrankungen verbunden, und deutet auf eine sich entwickelnde Typ-2-Diabetes-mellitus-Erkrankung hin. In a healthy individual, the physiological insulin production needed to maintain normal blood glucose levels is approximately 20-40 International Units (IU) of insulin per day. If the required insulin production is above these values for several days, one can speak of insulin resistance. "Severe insulin resistance" is generally assumed to be a long-term secretion of more than 200 IU insulin per day. Insulin resistance is associated with numerous diseases and is indicative of developing type 2 diabetes mellitus.
Wie beschrieben, bewirkt die simultane Bereitstellung der Komponenten Insulin, IGF-1 und IGFBP-2 durch die hierin offenbarte Zusammensetzung bzw. das Kit eine erhöhte Wirksamkeit des Insulins. Die so verbesserte Insulinwirksamkeit kann ausreichen, um eine Insulinresistenz in einem Subjekt zu überwinden. As described, the simultaneous provision of the components insulin, IGF-1 and IGFBP-2 by the composition or the kit disclosed herein causes an increased effectiveness of the insulin. The insulin efficacy thus improved may be sufficient to overcome insulin resistance in a subject.
Die erfindungsgemäße pharmazeutische Zusammensetzung bzw. das pharmazeutische Kit kann, ähnlich wie bei eine Insulintherapie, mit Hilfe eines Pens, einer Spritze oder einer Insulinpumpe subkutan in das Unterhaut-Fettgewebe, aber auch intramuskulär oder intravenös verabreicht werden. Similar to insulin therapy, the pharmaceutical composition or the pharmaceutical kit according to the invention can be administered subcutaneously into the subcutaneous fatty tissue with the aid of a pen, a syringe or an insulin pump, but also intramuscularly or intravenously.
Bevorzugt kann die Verabreichung durch einen unter Insulinresistenz leidenden Patienten selbst vorgenommen werden. Alternativ kann die Verabreichung jedoch auch durch geschultes Fachpersonal oder einen Arzt erfolgen. Da die Zusammensetzung bzw. das Kit, wie hierin beschrieben, die Aktivität des AKT-Kinasesignalwegs verstärkt, kann deren/dessen Verwendung das Risiko einer malignen Erkrankung erhöhen. Aus diesem Grund sollte die regelmäßige Verwendung der erfindungsgemäßen pharmazeutischen Zusammensetzung bzw. des erfindungsgemäßen pharmazeutischen Kits zur Behandlung von Insulinresistenz bevorzugt nur bei Vorliegen einer besonders schweren Insulinresistenz erfolgen, also wenn die erforderliche Insulinproduktion dauerhaft, zumindest jedoch kontinuierlich über mehrere Tage hinweg bei über 200 I.E. Insulin pro Tag liegt. Unter Umständen kann die Gefahr von AKT- induzierter Tumorbildung verringert werden, wenn die erfindungsgemäße Zusammensetzung bzw. das erfindungsgemäße pharmazeutische Kit ferner einen mTOR-lnhibitor wie Metformin umfasst. Metformin steigert zudem selbst die Insulinsensitivität und kann die Glucose-Neubildung der Leber hemmen. Insbesondere in Kombination mit derartigen geeigneten mTOR-
Inhibitoren kann eine Verwendung der erfindungsgemäßen pharmazeutischen Zusammensetzung bzw. des erfindungsgemäßen pharmazeutischen Kits bei der Behandlung auch von weniger schwerwiegenden Formen von Insulinresistenz vorteilhaft sein. The administration can preferably be carried out by a patient suffering from insulin resistance himself. Alternatively, however, administration can also be carried out by trained specialists or a doctor. Because the composition or kit as described herein enhances the activity of the AKT kinase signaling pathway, its use may increase the risk of malignancy. For this reason, the regular use of the pharmaceutical composition according to the invention or the pharmaceutical kit according to the invention for the treatment of insulin resistance should preferably only take place in the presence of particularly severe insulin resistance, i.e. if the required insulin production is permanent, but at least continuously over several days at over 200 IU insulin per day lies. Under certain circumstances, the risk of AKT-induced tumor formation can be reduced if the composition according to the invention or the pharmaceutical kit according to the invention also comprises an mTOR inhibitor such as metformin. Metformin also increases insulin sensitivity itself and can inhibit the formation of new glucose in the liver. Especially in combination with such suitable mTOR Inhibitors, it can also be advantageous to use the pharmaceutical composition according to the invention or the pharmaceutical kit according to the invention in the treatment of less severe forms of insulin resistance.
Gegenstand der vorliegenden Erfindung ist damit auch ein Verabreichungsmittel, wie ein Pen, eine Spritze oder eine Insulinpumpe, welches die erfindungsgemäße Zusammensetzung oder das erfindungsgemäße Kit umfasst. Insbesondere Im Falle eines Kits kann auch eine Mehrkammernspritze verwendet werden. Dies kann auch sinnvoll sein, wenn die Zusammensetzung lyophilisiert ist, wobei eine weitere Kammer dann üblicherweise Wasser zur Injektion (optional auch einen Puffer) umfasst. The subject matter of the present invention is therefore also an administration means, such as a pen, a syringe or an insulin pump, which comprises the composition according to the invention or the kit according to the invention. In the case of a kit in particular, a multi-chamber syringe can also be used. This can also be useful if the composition is lyophilized, with a further chamber then usually comprising water for injection (optionally also a buffer).
Da die pharmazeutische Zusammensetzung bzw. das pharmazeutische Kit in erhöhtem Maße die Glucoseaufnahme durch Muskel- und Fettzellen anregt und die anabole Aktivität des AKT-Kinasesignalwegs stimuliert, eignet sie sich auch zur Verwendung bei der Behandlung von Muskelschwäche, insbesondere bei Muskeldystrophie. Because the pharmaceutical composition or kit increases glucose uptake by muscle and fat cells and stimulates the anabolic activity of the AKT kinase pathway, it is also useful in the treatment of muscle weakness, particularly muscular dystrophy.
Muskeldystrophie bezeichnet eine besondere Gruppe erblicher Muskelerkrankungen, die mit einer Muskelschwäche und/oder Muskelschwund verbunden sind. Alle Muskeldystrophien sind durch fortschreitende (progressive) Degeneration der Muskulatur, einhergehend mit Umbauprozessen, gekennzeichnet. Die einzelnen Muskeldystrophien unterscheiden sich hinsichtlich der Art des Erbgangs, der hauptsächlich betroffenen Körperregionen, des Erkrankungsalters und des Verlaufs. Eine Auswahl der Muskeldystrophien, die mit Hilfe der hierin offenbarten pharmazeutischen Zusammensetzung bzw. des pharmazeutischen Kits behandeln werden können, umfasst Muskeldystrophie Duchenne, Muskeldystrophie Becker-Kiener, Emery-Dreifuss-Muskeldystrophie Typ 1-5, Skapuloperoneale Muskeldystrophie, Reducing body myopathy (RBM), LGMD1A-E, LGMD2A-Q, Kongenitaler Merosinman- gel, Kongenitale Myopathie mit lntegrin-alpha-7-Mangel, Kongenitale Muskeldystrophie 1C, Kongenitale Muskeldystrophie Typ Fukuyama, Walker- Warburg-Syndrom, Muskel-Auge- Gehirn-Krankheit, Rigid-Spine-Syndrom, Ullrich-Myopathie, Bethlem-Myopathie, Bethlem- Myopathie, Distale Muskeldystrophien, Myofibrilläre Myopathie Typ 1-6, Myotone Dystrophie Typ 1/2, Fazioskapulohumerale Muskeldystrophie und Skapuloperoneale Muskeldystrophie oder andere degenerative Erkrankungen wie die amyotrophe Lateralsklerose. Muscular dystrophy refers to a particular group of inherited muscle disorders associated with muscle weakness and/or wasting. All muscular dystrophies are characterized by ongoing (progressive) degeneration of the muscles, accompanied by remodeling processes. The individual muscular dystrophies differ in terms of the type of inheritance, the main body regions affected, the age at onset and the course. A selection of the muscular dystrophies that can be treated using the pharmaceutical composition or kit disclosed herein includes Duchenne muscular dystrophy, Becker-Kiener muscular dystrophy, Emery-Dreifuss muscular dystrophy type 1-5, scapuloperoneal muscular dystrophy, reducing body myopathy (RBM) , LGMD1A-E, LGMD2A-Q, Congenital Merosin Deficiency, Congenital Myopathy with Integrin Alpha 7 Deficiency, Congenital Muscular Dystrophy 1C, Congenital Muscular Dystrophy Fukuyama Type, Walker-Warburg Syndrome, Muscle Eye Brain Disease, Rigid Spine syndrome, Ullrich myopathy, Bethlem myopathy, Bethlem myopathy, distal muscular dystrophies, myofibrillar myopathy type 1-6, myotonic dystrophy type 1/2, facioscapulohumeral muscular dystrophy and scapuloperoneal muscular dystrophy or other degenerative diseases such as amyotrophic lateral sclerosis.
Die aktuelle Entwicklung beispielsweise von RNA-basierten Hormon-Therapie-Ansätzen bei Muskelschwäche und/oder -Verletzungen sieht aktuell lediglich die Verabreichung von IGF- 1 vor. Durch die hierin beschriebene Kombination des IGF-1 mit IGFBP-2 und Insulin kann die zelluläre Antwort erheblich gesteigert werden. Aufgrund der höheren Wirkung können
niedrigere IGF-1 Dosen verwendet werden als bei den reinen IGF-1-Präparaten, wodurch die Gefahr einer Malignität (z.B. durch systemische Effekte des IGF-1 ) minimiert wird. The current development, for example, of RNA-based hormone therapy approaches for muscle weakness and/or injuries currently only envisages the administration of IGF-1. The combination of IGF-1 with IGFBP-2 and insulin described here can significantly increase the cellular response. Because of the higher effect lower IGF-1 doses are used than with the pure IGF-1 preparations, which minimizes the risk of malignancy (eg due to systemic effects of IGF-1).
Aufgrund der wachstumsfördernden Eigenschaften der erfindungsgemäßen Zusammensetzung bzw. des Kits ist auch deren/dessen Verwendung zur Therapie von Wachstumsstörungen, d.h. , eine von der Norm abweichende Körperlänge bei Kindern und Jugendlichen denkbar. So kann durch eine regelmäßige Verwendung der erfindungsgemäßen Zusammensetzung bzw. des erfindungsgemäßen Kits in betroffenen Subjekten einer Hyposomie (Kleinwuchs) entgegengewirkt werden. Due to the growth-promoting properties of the composition or the kit according to the invention, its use for the therapy of growth disorders, i.e. a body length deviating from the norm in children and adolescents, is also conceivable. Thus, regular use of the composition according to the invention or the kit according to the invention in affected subjects can counteract hyposomy (short stature).
Zudem eignet sich die hierin offenbarte pharmazeutische Zusammensetzung bzw. das pharmazeutische Kit zur Verwendung bei der Behandlung einer Verletzung eines Muskels, Bandes und/oder einer Sehne eines Subjekts, bevorzugt eines Muskels. In addition, the pharmaceutical composition or kit disclosed herein is suitable for use in treating an injury to a muscle, ligament and/or tendon of a subject, preferably a muscle.
Wie oben beschrieben, ist das Subjekt bevorzugt ein Tier. Das Subjekt kann z.B. ein Mensch sein, z.B. ein professioneller Sportler oder ambitionierter Amateursportler, für den eine möglichst kurze Regenerationszeit nach einer Muskel-, Bänder-, oder Sehnenverletzung vorteilhaft ist. Gleichsam kann das Subjekt auch ein Pferd, ein Kamel, ein Hund oder ein Raubvogel sein, also ein Tier, dass beispielsweise bei sportlichen Wettkämpfen eingesetzt wird und einem erhöhten Risiko unterliegt, eine Verletzung der Muskeln, Bänder oder Sehnen zu erleiden. Die Verletzung kann akut sein, d.h. eine plötzliche und heftige Verletzung, die das betroffene Subjekt in seiner Leistungsfähigkeit einschränkt. Bei der Verletzung kann es sich alternativ auch um eine chronische Verletzung handeln, also eine Verletzung, deren Ursprung beispielsweise längere Zeit zurückliegt und die nie vollständig ausgeheilt ist, oder deren Zustand sich zunehmend verschlechtert. In einer bevorzugten Ausführungsform ist die Verletzung eine akute Verletzung. Die Muskelverletzung kann beispielsweise eine Muskelzerrung, ein Muskelfaserriss, ein Muskelriss, eine Prellung odereine Schnittwunde sein. Eine Bänder- bzw. Sehnenverletzung umfasst einen Sehnen- oder Bänderriss oder eine Dehnung. Bevorzugt ist die erfindungsgemäße pharmazeutische Zusammensetzung bzw. das pharmazeutische Kit für die Verwendung bei der Behandlung einer Muskelverletzung vorgesehen. As described above, the subject is preferably an animal. For example, the subject can be a human being, e.g., a professional athlete or ambitious amateur athlete, for whom the shortest possible recovery time after a muscle, ligament, or tendon injury is advantageous. Similarly, the subject may be a horse, camel, dog, or bird of prey, such as an animal used in athletic competitions, for example, that is at increased risk of muscle, ligament, or tendon injury. The injury may be acute, i.e., a sudden and violent injury that impairs the affected subject's ability to function. Alternatively, the injury can also be a chronic injury, ie an injury which originated a long time ago and which has never completely healed, or whose condition is progressively deteriorating. In a preferred embodiment, the injury is an acute injury. The muscle injury can be, for example, a muscle strain, a hamstring tear, a muscle tear, a bruise or a laceration. A ligament or tendon injury includes a tendon or ligament tear or strain. The pharmaceutical composition or the pharmaceutical kit according to the invention is preferably intended for use in the treatment of a muscle injury.
Schließlich stellt die vorliegende Erfindung die hierin beschriebene pharmazeutische Zusammensetzung bzw. das pharmazeutische Kit auch zur Verwendung bei der Erhaltung und Regeneration von neuronalen Funktionen bereit. Bei den neuronalen Funktionen kann es sich sowohl um Funktionen zentraler als auch peripherer Nervenzellen handeln.
In einer bevorzugten Ausführungsform der Erfindung kann die erfindungsgemäße pharmazeutische Zusammensetzung oder das erfindungsgemäße Kit auch zur Behandlung und/ oder Prävention einer Neuropathie verwendet werden. So deuten wissenschaftliche Studien darauf hin, dass eine Insulinresistenz zur Pathophysiologie und den klinischen Symptomen diverser neurodegenerativer Erkrankungen wie Alzheimer (Watson et al., 2003) oder Parkinson (Hong et al., 2020) beitragen kann. Die Zusammensetzung bzw. das Kit der Erfindung kann zudem zur Prävention von Nervenschädigungen beitragen, die auf hohe Blutzuckerwerte zurückzuführen sind, wie dies z.B. bei einer diabetischen Neuropathie der Fall ist. Finally, the present invention also provides the pharmaceutical composition or the pharmaceutical kit described herein for use in the maintenance and regeneration of neuronal functions. The neuronal functions can be functions of both central and peripheral nerve cells. In a preferred embodiment of the invention, the pharmaceutical composition according to the invention or the kit according to the invention can also be used for the treatment and/or prevention of a neuropathy. Scientific studies indicate that insulin resistance can contribute to the pathophysiology and clinical symptoms of various neurodegenerative diseases such as Alzheimer's (Watson et al., 2003) or Parkinson's (Hong et al., 2020). The composition or kit of the invention can also help prevent nerve damage resulting from high blood sugar levels, such as is the case with diabetic neuropathy.
Unabhängig von der konkreten medizinischen Indikation kann die hierin beschriebene pharmazeutische Zusammensetzung oder das pharmazeutische Kit einem Subjekt entweder systemisch oder lokal verabreicht werden. Eine systemische Applikation kann beispielsweise bei der Behandlung eine Insulinresistenz sinnvoll sein. In einer bevorzugten Ausführungsform wird die pharmazeutische Zusammensetzung oder das pharmazeutische Kit jedoch bei der Behandlung und/oder Prävention der hierin beschriebenen Erkrankungen und/oder Verletzungen lokal appliziert. Sofern die erfindungsgemäße Zusammensetzung oder das Kit zur Behandlung einer Muskelverletzung verwendet werden soll, ist eine lokale Applikation der Zusammensetzung oder des Kits direkt in den verletzten Muskeln vorteilhaft. Auch bei der Behandlung einer Muskeldystrophie, einer neuronalen Funktionsstörung oder Neuropathie ist eine lokale Applikation der erfindungsgemäßen Zusammensetzung oder des Kits bevorzugt, um das Risiko der Entstehung maligner Erkrankungen zu minimieren. Regardless of the specific medical indication, the pharmaceutical composition or kit described herein can be administered to a subject either systemically or locally. A systemic application can be useful, for example, in the treatment of insulin resistance. In a preferred embodiment, however, the pharmaceutical composition or kit is applied locally in the treatment and/or prevention of the diseases and/or injuries described herein. If the composition or the kit according to the invention is to be used to treat a muscle injury, local application of the composition or the kit directly to the injured muscles is advantageous. Local application of the composition according to the invention or of the kit is also preferred in the treatment of muscular dystrophy, a neuronal dysfunction or neuropathy in order to minimize the risk of developing malignant diseases.
Die Verabreichung der pharmazeutischen Zusammensetzung oder des pharmazeutischen Kits zur Behandlung eines der hierin beschriebenen medizinischen Zustände kann einmalig erfolgen. In alternativen Ausführungsformen kann eine mehrmalige Verabreichung der pharmazeutischen Zusammensetzung oder des pharmazeutischen Kits notwendig sein, um eine Verbesserung des zu behandelnden medizinischen Zustands zu erwirken. So kann es erforderlich sein, dass die pharmazeutische Zusammensetzung oder das Kit dem Subjekt mindestens 2 Mal, mindestens 3 Mal, mindestens 4 Mal, mindestens 5 Mal, mindestens 10 Mal, mindestens 50 Mal oder mindestens 100 Mal verabreicht werden muss, bevor eine Verbesserung seines Zustands eintritt. In manchen Ausführungsformen muss die Verabreichung regelmäßig erfolgen, solange der Zustand des Subjektes vorliegt, unter Umständen auch ein Leben lang. Die Behandlung des Subjekts mit der erfindungsgemäßen pharmazeutischen Zusammensetzung bzw. dem pharmazeutischen Kit kann ferner begleitend mit anderen Therapieansätzen verbunden werden oder, insbesondere im Fall von Verletzungen
an Muskeln oder dem Halteapparat, zur Unterstützung verschiedenster Rehabilitationsmaßnahmen eingesetzt werden. The administration of the pharmaceutical composition or the pharmaceutical kit for the treatment of any of the medical conditions described herein can be carried out once. In alternative embodiments, multiple administrations of the pharmaceutical composition or kit may be necessary to effect an improvement in the medical condition being treated. Thus, the pharmaceutical composition or kit may need to be administered to the subject at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 50 times, or at least 100 times before an improvement in his condition occurs. In some embodiments, administration must be on a regular basis for as long as the subject's condition persists, possibly throughout life. The treatment of the subject with the pharmaceutical composition or the pharmaceutical kit according to the invention can also be combined with other therapeutic approaches or, in particular, in the case of injuries on muscles or the holding apparatus, to support a wide variety of rehabilitation measures.
Zusammenfassend lehrt die vorliegende Erfindung einen vollkommen neuartigen Ansatz zur gezielten Beeinflussung des Energiestoffwechsels in Muskel- und Fettgewebe von Tieren und Menschen. Die hierin offenbarten Zusammensetzungen und Kits stellen eine Kombination aus Insulin, IGF-1 und IGFBP-2 bereit, mit Hilfe derer die Insulinwirksamkeit und somit die Glucoseaufnahme gewebespezifisch gesteuert werden kann. Die Verwendung der erfindungsgemäßen Zusammensetzungen und Kits ermöglicht somit eine Steigerung des intramuskulären Fettgehalts. Der gezielte Fettansatz kann die Qualität von Fleisch und die Futtereffizienz von Nutztieren erhöhen. Zudem wird eine vergrößerte Energiespeicherkapazität des Muskelgewebes erreicht, die das Regenerationspotential der Muskulatur erhöht. Die durch die Zusammensetzungen und Kits vermittelte Aktivierung des AKT-Signal- wegs fördert ferner das Zellwachstum. In summary, the present invention teaches a completely new approach for specifically influencing the energy metabolism in muscle and fat tissue in animals and humans. The compositions and kits disclosed herein provide a combination of insulin, IGF-1 and IGFBP-2 that can be used to tailor insulin activity and thus glucose uptake in a tissue-specific manner. The use of the compositions and kits according to the invention thus makes it possible to increase the intramuscular fat content. The targeted fat deposit can increase the quality of meat and the feed efficiency of livestock. In addition, an increased energy storage capacity of the muscle tissue is achieved, which increases the regeneration potential of the muscles. The activation of the AKT signaling pathway mediated by the compositions and kits also promotes cell growth.
Im Rahmen der Erfindung ist der Begriff " ungefähr" im Sinne von "+/- 10 %" zu verstehen. Bezieht sich der Begriff " ungefähr" auf einen Bereich, so bezieht er sich sowohl auf die untere als auch auf die obere Grenze des Bereichs. "Ein" bedeutet "ein oder mehrere", wenn nicht ausdrücklich anders angegeben. Sämtliche hier zitierte Literatur wird hiermit vollständig einbezogen. Die vorliegende Erfindung wird durch das folgende Beispiel weiter veranschaulicht, aber nicht beschränkt. In the context of the invention, the term "approximately" is to be understood in the sense of "+/−10%". When the term "approximately" refers to a range, it refers to both the lower and upper limits of the range. "A" means "one or more" unless expressly stated otherwise. All literature cited here is hereby incorporated in its entirety. The present invention is further illustrated, but not limited, by the following example.
Literatur literature
Li et al. (2017) PTEN, Insulin Resistance and Cancer. Curr Pharm Des. 23(25), 3667-3676.Li et al. (2017) PTEN, Insulin Resistance and Cancer. Curr Pharm Des. 23(25), 3667-3676.
Watson et al. (2003) The role of insulin resistance in the pathogenesis of Alzheimer's disease: implications for treatment. CNS Drugs 17(1), 27-45. Watson et al. (2003) The role of insulin resistance in the pathogenesis of Alzheimer's disease: implications for treatment. CNS Drugs 17(1), 27-45.
Hong et al. (2020) Insulin Resistance Promotes Parkinson’s Disease through Aberrant Expression of a-Synuclein, Mitochondrial Dysfunction, and Deregulation of the Polo-Like Kinase 2 Signaling. Cells 9(3). https://www.landwirtschaft.de/landwirtschaft-verstehen/haetten-sies-gewusst/infografikenHong et al. (2020) Insulin Resistance Promotes Parkinson's Disease through Aberrant Expression of a-Synuclein, Mitochondrial Dysfunction, and Deregulation of the Polo-Like Kinase 2 Signaling. Cells 9(3). https://www.landwirtschaft.de/landwirtschaft-verständigen/haetten-sies-gewusst/infographics
Shen et al. (2012) Insulin-like growth factor (IGF) binding protein 2 functions coordinately with receptor protein tyrosine phosphatase ß and the IGF-I receptor to regulate IGF-l-stimu- lated signaling. Mol. Cell. Biol. 32, 4116-4130.
Shen et al. (2015) The Coordinate Cellular Response to Insulin-like Growth Factor-I (IGF-I) and Insulin-like Growth Factor-binding Protein-2 (IGFBP-2) Is Regulated through Vimentin Binding to Receptor Tyrosine Phosphatase ß (RPTPß). J Biol Chem. 290, 11578-11590.Shen et al. (2012) Insulin-like growth factor (IGF) binding protein 2 functions coordinately with receptor protein tyrosine phosphatase ß and the IGF-I receptor to regulate IGF-l-stimulated signaling. Mol. Cell. Biol. 32, 4116-4130. Shen et al. (2015) The Coordinate Cellular Response to Insulin-like Growth Factor-I (IGF-I) and Insulin-like Growth Factor-binding Protein-2 (IGFBP-2) Is Regulated through Vimentin Binding to Receptor Tyrosine Phosphatase ß (RPTPß). J Biol Chem. 290 , 11578-11590.
Connolly et al. (2020) Changes in the blood metabolome of Wagyu crossbred steers with time in the feedlot and relationships with marbling. Scientific reports 10, 18987. https://de.wikipedia.org/wiki/Nutztier https://de.wikipedia.org/wiki/ln-vitro-Fleisch Connolly et al. (2020) Changes in the blood metabolome of Wagyu crossbred steers with time in the feedlot and relationships with marbling. Scientific reports 10, 18987. https://de.wikipedia.org/wiki/Nutztier https://de.wikipedia.org/wiki/ln-vitro-Fleisch
Walz et al. (2021) Development of a Sensitive Bioassay fort he Analysis of IGF-Related Activation of AKT/mTOR Signaling in Biological Matrices. Cells 10(3):482. doi: 10.3390/cellsl 0030482 Walz et al. (2021) Development of a Sensitive Bioassay for the Analysis of IGF-Related Activation of AKT/mTOR Signaling in Biological Matrices. Cells 10(3):482. doi: 10.3390/cellsl 0030482
Legende Legend
Fig. 1 : Humanes rekombinantes IGF-1 in niedrigen Dosen verbessert die zelluläre Empfindlichkeit für die Insulinsignalisierung. Humane embryonale Nierenfibroblasten, die mit einem Expressionsvektor für Maus-IGFBP2-cDNA (Höflich et al. 1998, FEBS Letters 434: p329-334) stabil transfiziert wurden, wurden mit verschiedenen Konzentrationen (0- 6000 ng/mL) von humanem Insulin in Gegenwart und Abwesenheit von rekombinantem humanem IGF-1 (25 ng/mL) inkubiert. Nach 20 Minuten Inkubation mit den Peptiden wurden die Zellen geerntet und die Phosphorylierung von AKT getestet. Figure 1: Human recombinant IGF-1 at low doses improves cellular sensitivity to insulin signalling. Human embryonic kidney fibroblasts stably transfected with an expression vector for mouse IGFBP2 cDNA (Höflich et al. 1998, FEBS Letters 434: p329-334) were treated with various concentrations (0-6000 ng/mL) of human insulin in the presence and absence of recombinant human IGF-1 (25 ng/mL). After 20 minutes incubation with the peptides, the cells were harvested and the phosphorylation of AKT was assayed.
Beispiel Example
In dem folgenden Beispiel wird anhand der Phosphorylierung der AKT-Kinase veranschaulicht, dass die Wirkung von verabreichtem Insulin durch eine Kombination von IGF-1 und IGFBP-2 innerhalb von Zellen verstärkt werden kann. In the following example, AKT kinase phosphorylation is used to illustrate that the effect of administered insulin can be potentiated by a combination of IGF-1 and IGFBP-2 within cells.
Materialien und Methoden Materials and Methods
Humane embryonale Nierenfibroblasten, die mit einem Expressionsvektor für Maus-IGFBP2- cDNA (Höflich et al. 1998, FEBS Letters 434: p329-334) stabil transfiziert wurden, wurden mit verschiedenen Konzentrationen (0-6000 ng/mL) von humanem Insulin in Gegenwart und Abwesenheit von rekombinantem humanem IGF-1 (25 ng/mL) inkubiert. Nach 20 Minuten Inkubation mit den Peptiden wurden die Zellen geerntet und die Phosphorylierung von AKT wie in Walz et al., 2021 (Development of a Sensitive Bioassay fort he Analysis of IGF-Related Activation of AKT/mTOR Signaling in Biological Matrices. Cells 10(3):482. doi: 10.3390/cells10030482) beschrieben, getestet.
Ergebnisse und Diskussion Human embryonic kidney fibroblasts stably transfected with an expression vector for mouse IGFBP2 cDNA (Höflich et al. 1998, FEBS Letters 434: p329-334) were treated with various concentrations (0-6000 ng/mL) of human insulin in the presence and absence of recombinant human IGF-1 (25 ng/mL). After 20 minutes incubation with the peptides, cells were harvested and AKT phosphorylation was determined as described in Walz et al., 2021 (Development of a Sensitive Bioassay for the Analysis of IGF-Related Activation of AKT/mTOR Signaling in Biological Matrices. Cells 10( 3):482.doi:10.3390/cells10030482), tested. Results and discussion
IGF-1 ist in der Lage, das Wachstum nahezu jeder Zellart des Körpers anzuregen, u.a. indem es den AKT-Kinasesignalweg stimuliert. In der Vergangenheit konnte ferner gezeigt werden, dass IGFBP-2 gemeinsam mit IGF-1 eine Aktivierung des AKT-Kinasesignalwegs auslösen und folglich die Migration und Vermehrung von vaskulären glatten Muskelzellen bewirken kann (Shen et al. 2012). IGF-1 is able to stimulate the growth of almost every type of cell in the body, including by stimulating the AKT kinase signaling pathway. It has also been shown in the past that IGFBP-2, together with IGF-1, can trigger activation of the AKT kinase signaling pathway and consequently cause the migration and proliferation of vascular smooth muscle cells (Shen et al. 2012).
Auf Grundlage dieser Erkenntnisse fragten sich die Erfinder, ob eine Kombination von IGF-1 und IGFBP-2 auch die Wirksamkeit von Insulin gezielt verstärken kann. Da auch die Insulinsignalisierung zu einer Aktivierung des AKT-Kinasesignalwegs führt, untersuchte man daher, wie sich eine Kombination aus IGF-1 , IGFBP-2 und Insulin auf die Phosphorylierung der AKT Kinase auswirkt. Wie aus Fig. 1 hervorgeht, zeigten Zellen, die rekombinantes Maus-IGFB-2 exprimierten und mit 0-6000 ng/mL Insulin supplementiert wurden, in Abwesenheit von IGF-Based on these findings, the inventors wondered whether a combination of IGF-1 and IGFBP-2 could also specifically enhance the effectiveness of insulin. Since insulin signaling also leads to activation of the AKT kinase signaling pathway, it was therefore investigated how a combination of IGF-1, IGFBP-2 and insulin affects the phosphorylation of the AKT kinase. As shown in Figure 1, in the absence of IGF-2, cells expressing recombinant mouse IGFB-2 supplemented with 0-6000 ng/mL insulin showed
1 lediglich eine geringe AKT-Phosphorylierung. Wenn die Zellen jedoch zusätzlich mit 25 ng/mL IGF-1 inkubiert wurden, erhöhte sich die AKT-Phosphorylierung deutlich. Die Aktivierung von AKT fiel am stärksten aus, wenn die Zellen mit einer Kombination aus 60 ng/mL Insulin und 25 ng/mL IGF-1 behandelt wurden. Da sich das durch die mit dem Expression- vektor-transfizierten Zellen exprimierte IGFBP-2 an die Zellmembranen anlagerte, war es im Rahmen der Untersuchung nicht möglich, die Expressionslevel des IGFBP-2 genau zu bestimmen. Allerdings ist aus einer früheren Studie von Walz et al., 2021 bekannt, dass IGFBP-1 shows only minor AKT phosphorylation. However, when cells were additionally incubated with 25 ng/mL IGF-1, AKT phosphorylation increased significantly. AKT activation was greatest when cells were treated with a combination of 60 ng/mL insulin and 25 ng/mL IGF-1. Since the IGFBP-2 expressed by the cells transfected with the expression vector attached itself to the cell membranes, it was not possible to precisely determine the expression level of IGFBP-2 within the scope of the study. However, from a previous study by Walz et al., 2021, it is known that IGFBP-
2 die IGF-1 abhängige AKT-Phosphorylierung in HEK293-2 Zellen bereits ab einer Konzentration von 100-500 ng/mL stark erhöht. Es ist anzunehmen, dass die Konzentration von IGFBP-2 in dem in Fig. 1 gezeigten Versuch diese Dosis nicht übersteigt. Auffallend war ferner, dass eine Supplementierung der IGFBP-2 exprimierenden Zellen mit einer hohen Insulin- Konzentration von 6000 ng/mL lediglich zu einer geringen AKT-Phosphorylierung führte, unabhängig davon, ob die Zellen zusätzlich mit IGF-1 behandelt wurden oder nicht. 2 strongly increases IGF-1-dependent AKT phosphorylation in HEK293-2 cells from a concentration of 100-500 ng/mL. It is assumed that the concentration of IGFBP-2 in the experiment shown in Figure 1 does not exceed this dose. It was also striking that supplementing the IGFBP-2-expressing cells with a high insulin concentration of 6000 ng/mL only led to a low AKT phosphorylation, regardless of whether the cells were additionally treated with IGF-1 or not.
Zusammengefasst legen die gezeigten Experimente somit nahe, dass sich IGFBP-2 in Kombination bereits mit einer verhältnismäßig geringen Dosis von IGF-1 merklich auf die Wirkung von Insulin auswirken kann.
In summary, the experiments shown suggest that IGFBP-2 in combination with a relatively low dose of IGF-1 can have a noticeable effect on the effect of insulin.
Claims
1. Eine Zusammensetzung oder ein Kit, welches IGF1 , Insulin und IGFBP-2 bereitstellt, wobei 1. A composition or kit providing IGF1, insulin and IGFBP-2, wherein
(a) das IGF1 , Insulin und/oder IGFBP-2 als Protein vorliegt, oder (a) the IGF1, insulin and/or IGFBP-2 is present as a protein, or
(b) mindestens eine Nukleinsäure vorliegt, welche für IGF1 , Insulin und/oder IGFBP-2 kodiert. (b) at least one nucleic acid is present which codes for IGF1, insulin and/or IGFBP-2.
2. Zusammensetzung oder Kit nach Anspruch 1 , dadurch gekennzeichnet, dass es sich um eine pharmazeutische Zusammensetzung oder ein pharmazeutisches Kit handelt. 2. Composition or kit according to claim 1, characterized in that it is a pharmaceutical composition or a pharmaceutical kit.
3. Zusammensetzung oder Kit nach einem der Ansprüche 1-2, wobei mindestens eine Komponente als Protein vorliegt, wobei bevorzugt IGF1 , Insulin und IGFBP-2 als Protein vorliegen. 3. Composition or kit according to any one of claims 1-2, wherein at least one component is present as a protein, with IGF1, insulin and IGFBP-2 preferably being present as a protein.
4. Zusammensetzung oder Kit nach einem der Ansprüche 1-3, wobei mindestens eine Nukleinsäure vorliegt, welche für IGF1 , Insulin und/oder IGFBP-2 kodiert, wobei bevorzugt IGF1 , Insulin und IGFB-2 durch mindestens eine Nukleinsäure kodiert werden. 4. Composition or kit according to any one of claims 1-3, wherein at least one nucleic acid is present which codes for IGF1, insulin and/or IGFBP-2, with preferably IGF1, insulin and IGFB-2 being coded by at least one nucleic acid.
5. Zusammensetzung oder Kit nach einem der Ansprüche 1-4, wobei die Nukleinsäure RNA ist. 5. A composition or kit according to any one of claims 1-4, wherein the nucleic acid is RNA.
6. Verwendung der Zusammensetzung oder des Kits nach einem der Ansprüche 1 -5 zur Erhöhung des intramuskulären Fettgehalts in Fleisch, bevorzugt von Nutztieren, optional von Bovinen, oder in in v/tro-Kultur. 6. Use of the composition or kit according to any one of claims 1-5 for increasing the intramuscular fat content in meat, preferably from livestock, optionally from bovines, or in in v/tro culture.
7. Verwendung der Zusammensetzung oder des Kits nach einem der Ansprüche 1-5 zur Verbesserung der Futtereffizienz bei Nutztieren. 7. Use of the composition or kit according to any one of claims 1-5 for improving feed efficiency in livestock.
8. Verfahren zur Erhöhung des intramuskulären Fettgehalts in Fleisch, bevorzugt von Nutztieren oder in vitro, einen Schritt umfassend, bei dem man die Zusammensetzung oder das Kit nach einem der Ansprüche 1-5 dem Nutztier oder der Kultur verabreicht. 8. A method for increasing the intramuscular fat content in meat, preferably of livestock or in vitro, comprising a step in which the composition or kit according to any one of claims 1-5 is administered to the livestock or the culture.
25
Pharmazeutische Zusammensetzung oder pharmazeutisches Kit nach einem der Ansprüche 2-5 zur Verwendung bei der Behandlung von Insulinresistenz, bevorzugt schwerer Insulinresistenz. Pharmazeutische Zusammensetzung oder pharmazeutisches Kit nach einem der Ansprüche 2-5 zur Verwendung bei der Behandlung einer Muskelschwäche, bevorzugt einer Muskeldystrophie ausgewählt aus der Gruppe umfassend Muskeldystrophie Duchenne, Muskeldystrophie Becker-Kiener, Emery-Dreifuss-Muskel- dystrophie Typ 1-5, Skapuloperoneale Muskeldystrophie, Reducing body myopathy (RBM), LGMD1A-E, LGMD2A-Q, Kongenitaler Merosinmangel, Kongenitale Myopathie mit lntegrin-alpha-7-Mangel, Kongenitale Muskeldystrophie 1C, Kongenitale Muskeldystrophie Typ Fukuyama, Walker- Warburg-Syndrom, Muskel-Auge- Gehirn-Krankheit, Rigid-Spine-Syndrom, Ullrich-Myopathie, Bethlem-Myopathie, Bethlem-Myopathie, Distale Muskeldystrophien, Myofibrilläre Myopathie Typ 1-6, Myotone Dystrophie Typ 1/2, Fazioskapulohumerale Muskeldystrophie und Skapuloperoneale Muskeldystrophie oder andere degenerative Erkrankungen wie die amyotrophe Lateralsklerose. Pharmazeutische Zusammensetzung oder pharmazeutisches Kit nach einem der Ansprüche 2-5 zur Verwendung bei der Erhaltung und Regeneration von neuronalen Funktionen, bevorzugt zur Behandlung und/oder Prävention einer Neuropathie, optional einer diabetischen Neuropathie. Pharmazeutische Zusammensetzung oder pharmazeutisches Kit nach einem der Ansprüche 2-5 zur Verwendung bei der Behandlung einer Verletzung eines Muskels, Bandes und/oder einer Sehne eines Subjekts, bevorzugt eines Muskels. Pharmazeutische Zusammensetzung oder pharmazeutisches Kit nach einem der Ansprüche 2-5 zur lokalen Verabreichung, optional zur Verwendung nach einem der Ansprüche 10-12.
25 A pharmaceutical composition or pharmaceutical kit according to any one of claims 2-5 for use in the treatment of insulin resistance, preferably severe insulin resistance. Pharmaceutical composition or pharmaceutical kit according to any one of claims 2-5 for use in the treatment of muscle weakness, preferably a muscular dystrophy selected from the group comprising Duchenne muscular dystrophy, Becker-Kiener muscular dystrophy, Emery-Dreifuss muscular dystrophy type 1-5, scapuloperoneal muscular dystrophy , Reducing body myopathy (RBM), LGMD1A-E, LGMD2A-Q, Congenital merosin deficiency, Congenital myopathy with integrin alpha-7 deficiency, Congenital muscular dystrophy 1C, Congenital muscular dystrophy Fukuyama type, Walker-Warburg syndrome, Muscle-eye-brain -Disease, Rigid Spine Syndrome, Ullrich Myopathy, Bethlem Myopathy, Bethlem Myopathy, Distal Muscular Dystrophies, Myofibrillar Myopathy Type 1-6, Myotonic Dystrophy Type 1/2, Facioscapulohumeral Muscular Dystrophy and Scapuloperoneal Muscular Dystrophy or other degenerative diseases such as the amyotrophic lateral sclerosis. Pharmaceutical composition or pharmaceutical kit according to any one of claims 2-5 for use in the maintenance and regeneration of neuronal functions, preferably for the treatment and/or prevention of a neuropathy, optionally a diabetic neuropathy. A pharmaceutical composition or kit according to any one of claims 2-5 for use in treating an injury to a muscle, ligament and/or tendon of a subject, preferably a muscle. A pharmaceutical composition or pharmaceutical kit according to any one of claims 2-5 for local administration, optionally for use according to any one of claims 10-12.
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| DE102021122096.3A DE102021122096A1 (en) | 2021-08-26 | 2021-08-26 | Compositions comprising IGF-1, IGFBP-2 and insulin and their use |
| DE102021122096.3 | 2021-08-26 |
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| WO1998006423A1 (en) * | 1996-08-13 | 1998-02-19 | Genentech, Inc. | Composition comprising insulin and insulin-like growth factor-i (igf-i) |
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| US6121416A (en) | 1997-04-04 | 2000-09-19 | Genentech, Inc. | Insulin-like growth factor agonist molecules |
| US20020028764A1 (en) | 2000-09-04 | 2002-03-07 | Aarhus Amt. | Treatment of acute and chronic liver disease |
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| US5756264A (en) * | 1991-11-06 | 1998-05-26 | Baylor College Of Medicine | Expression vector systems and method of use |
| WO1998006423A1 (en) * | 1996-08-13 | 1998-02-19 | Genentech, Inc. | Composition comprising insulin and insulin-like growth factor-i (igf-i) |
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