PL238941B1 - polyRGD peptides obtained by chemical and biotechnological method with antidemarcation and/or neuroprotective effect and a set for determination of their activity - Google Patents

Description

Description of the invention

The invention relates to anti-damage and / or neuroprotective chemical compounds with the amino acid sequence RGD: Arg-Gly-Asp in single or multiple form.

Neurological injuries include all kinds of traumatic and rapid pathological changes in the nervous system and the circulatory system associated with it. Damage to the central nervous system (CNS), i.e. the brain and spinal cord, is particularly dangerous to health and life. In their case, damage causes undesirable structural changes, electrical and chemical transmission dysfunctions, cell death (not only neurons, but also glial cells of various types), resulting in physiological and cognitive changes, as well as those related to feelings, emotions and memory. Such injuries, also called traumas, are most often the result of a mechanical injury (wound) and an ischemic or hemorrhagic stroke (haemorrhage), and in most cases lead to chronic or irreversible encephalopathy. A huge number of cases lead to the death of the patient as a result of direct damage or the occurrence of a number of secondary phenomena that modern medicine is not yet able to cope with.

In the CNS, mechanical (physical) injuries are the most common clinical cases of brain injuries and spinal cord injuries. These include traumatic brain damage (compression injuries) as a result of strong blows, and mechanical brain damage (wounds) with direct deep brain wounds. Both types of injuries most often arise as a result of traffic accidents, falls and other injuries leading to concussion and physical defects, followed by death, or reversible or permanent epileptic seizures, migraines, and disturbances in the functioning of specific areas of the brain. In turn, damage to the spinal cord, which is most often the result of a break in the continuity of the spine (partial or complete cut) or crushing (compression injuries), leads to partial or permanent muscle dysfunction, loss of sensation or lack of autonomic responses in those parts of the body controlled by the nerves of the below the point where the core is broken or damaged.

Strokes are another group of neurological damage that consists of two types of health conditions: ischemic stroke, caused by obstruction of the lumen of a blood vessel in the brain or vessels leading to the brain, and haemorrhagic stroke (stroke), caused by breakage of vessel walls within the brain. In the first case, global and local (focal) strokes can be distinguished. While both of these subtypes of ischemic stroke relate to the deficiency of blood components, neurons and glial cells, they differ in the cause of such vascular dysfunction. Global stroke is caused by systemic hypoxia and cardiac arrest, usually resulting in loss of consciousness, while local stroke is the result of blockage of the vessel lumen by a blood clot or fatty deposits (atherosclerosis). The clinical symptom of this case is tissue death in the immediate vicinity of the ischemic core and penumbra, i.e. a site where reperfusion (re-flow of blood) is possible, provided this is ensured by immediate medical intervention within a few minutes . Brain hypoxia may also occur in newborns in an abnormal course of labor. Stroke (hemorrhagic stroke) is the result of a sudden rupture of blood vessel walls, most often as a result of hypertension or an aneurysm rupture, and leads to a drop in blood pressure and destruction of the parenchyma / nerve tissue in the damaged area of the CNS.

As a result of the above-described mechanical injuries of the brain and spinal cord (both compression / post-stroke injuries as well as after a brain injury or spinal cord cut) and strokes (ischemic or hemorrhagic), intertwining and mutually stimulating processes are induced: 1) excessive neuron stimulation ( excitotoxicity), 2) inflammation stimulated by microglial cells in the CNS or lymphatic cells penetrating from blood vessels, and 3) the phenomenon of cell death (both following the intracellular program called apoptosis and necrotic death). These phenomena are shown in Figs. 1 and 2 - prior art. These three groups of processes take place at different times and with different intensity after injury, as shown in Fig. 1.

A common feature of the neurological lesions mentioned above is the characteristic cascade of the subsequent physiological processes they cause (Fig. 2A). The most important clinical factor underlying these mechanisms is the loss of continuity of blood vessels

And consequently disrupting the blood-brain barrier (BBB). In general, stopping blood flow in the brain results in a lack of oxygen, glucose and nutrients, the so-called energy and respiratory deprivation, leading to immediate death (necrosis) of nerve cells and, in the long term, to programmed cell death (apoptosis) in nearby regions of the brain and general inflammation within the tissue. Energy deprivation is a fundamental metabolic process that causes a self-stimulating series of biochemical and neurochemical reactions. Taking into account the duration of these reactions, short-term (minutes) depolarization processes should be distinguished, leading to the interconnected mechanisms of increasing the intracellular concentration of calcium ions, lowering the pH in the tissue due to the increase in the concentration of lactate, as a product of anaerobic respiration, and, above all, to the dangerous, self-propelling excessive excitation of neurons, i.e. excitotoxicity, as a result of which long-term processes (hours, days) occur, among which inflammation and apoptosis predominate. The key pathological symptom is excitotoxicity resulting from uncontrolled depolarization of neurons and calcium-dependent activation of glutamate receptors, resulting in excessive release of excitatory neurotransmitters (glutamate). Ultimately, the accumulation of glutamate in the synaptic space leads to an avalanche-like stimulation of adjacent neurons. The electrochemical imbalance in synapses, in combination with oxygen deficiency in the tissue, has a direct impact on the inhibition of cellular respiration, i.e. the formation of oxidative stress.

Neurological damage, especially post-stroke complications, leads to many changes in brain vascularization and angiogenesis disorders. Angiogenesis is the process of creating new blood vessels and plays a key role in tissue repair and regeneration. These processes are responsible, among others, of the extracellular matrix (ECM) proteins secreted by cells, responsible for the continuous proliferation, migration, adhesion, maturation and communication between vascular endothelial cells (endothelium). ECM consists mainly of fibrillar proteins (e.g. collagens, elastins), laminins, fibronectins and glycosaminoglycans, heparan sulphates, non-proteoglycan carbohydrates (e.g. hyaluronic acid). In particular, the role of the latter in the process of angiogenesis and wound regeneration has been confirmed in in vitro and in vivo experiments. Fibronectin interacts with collagen and surface integrins, which contributes to the rearrangement of the cytoskeleton, thus enabling the adhesion of fibrins to fibroblasts during blood clotting (thrombogenesis). Integrins are heterodimeric αβ receptors involved in the formation of the cell-to-cell and cell-ECM junctions, which underpin the processes of mesothelial signaling, proliferation, differentiation, growth, and migration. The natural ligands of these receptors display various activity and structural selectivity. Examples are: fibronectin and vitronectin, which bind specifically to the integrin αν | Ή fibrinogen in turn to integrin α ^ β3 and tenascin to ανβι and o ^ Pe; there are also ligand interactions with several different types of integrins, e.g. laminin and osteopontin with integrins. All ECM proteins listed above are characterized by the amino acid sequence Arg-Gly-Asp (aka RGD) flanked by various contiguous sequences. The activity of the RGD sequence is manifested mainly during extracellular signal transduction and proliferation. Moreover, integrins recognizing the RGD motif are recognized as oncomarkers in some tumors with high metastatic potential. Conjugates containing the RGD motif in their structure also show positive properties in stimulating the regeneration of bone, corneal and heart tissues.

RGD-dependent integrins play an important role in synaptic plasticity and nervous system disease. Their task is primarily to ensure interactions between neurons and glial cells in the form of connections between the cytoskeleton of these cells and the extracellular matrix. Such interactions are essential for the proper development of the nervous system. In addition, integrins participate in the coupling of neurotransmitter receptors, which contribute to synaptic plasticity, i.e. the mechanistic ability of the CNS to adapt adaptive changes such as memory and learning, as well as to functional and structural changes manifested as a result of neurological diseases such as epilepsy, trauma, and injuries. or neurodegeneration. However, despite the large amount of experimental evidence supporting the above reports on the role of integrins and ECM in neuropathology, such as multiple sclerosis, gliomas, brain tumors, neurodegenerative diseases, epilepsy, strokes, neurological injuries and peripheral nerve damage, little is known about the molecular mechanisms of these processes.

The specificity of the binding of an RGD sequence to a wide spectrum of receptors depends on its conformation and allosteric specificity to the active site of the integrin in question. Modifications

RGD sequence structures such as C-amidation or N-acetylation provide greater resistance to proteolysis under physiological conditions. In turn, duplication of the active sequence of RGD in the form of an oligopeptide increases the activity of the motif and ensures the controlled release of the RGD tripeptide in the body. In relation to these data, in vivo studies proving the important and positive role of proteins / peptides containing the RGD motif in the treatment of neurological diseases and damage seem interesting. These data indicate the involvement of the RGD sequence in the allosteric blockade of integrin-ECM protein interactions by reducing the number of integrin receptors available. Such activity of peptides containing the RGD sequence results in therapeutic inhibition of epileptic seizures and improvement of cognitive processes in the Alzheimer's disease model. The positive effect of peptides containing the RGD sequence was also demonstrated in the in vivo regeneration of peripheral nerves, where they stimulated the regrowth of nerve fibers. In addition, implantation of collagen fibers containing the RGD sequence into injured rat peripheral nerves resulted in the secretion of trophic factors, Schwann cell proliferation, axonal myelination, and nerve fiber formation in the early days of regeneration. In a rat model of nerve cord injury, polymers modified with RGD sequences provided an optimal and stable scaffold for axonal and nerve regrowth. Products containing the RGD motif were also used as conjugate with cell-penetrating peptides (CPPs) to cross the BBB barrier. In a rat focal ischemic stroke model, it was demonstrated that the intraperitoneally administered RGD-penetratin conjugate efficiently penetrated the BBB into the injured hemisphere of the brain, thereby reducing penumbra edema. Molecular mechanism analysis revealed a significant effect of products containing the RGD motif on extracellular signaling and stimulation of angiogenesis.

RGD-dependent integrins play a key role in signal transduction in the synaptic space. However, despite much experimental data, the details of the mechanisms of neuroprotection and neuroregeneration in which both integrins and ECM proteins are involved, bearing the RGD motif, are still unknown. Due to the involvement of integrins in long lasting physiological processes such as inflammation and apoptosis, it does not appear that blocking these receptors alone is sufficient to provide a therapeutic effect. Hence the need to conduct basic research aimed at understanding the molecular basis of these mechanisms. In recent years, attention has been paid to the significant influence of peptides containing the RGD motif on stimulation, proliferation, differentiation and migration of neural stem cells (NSCs). RGD peptide functionalized biomaterials have been shown to be effective in promoting NSC adhesion to the substrate and their maturation. Similarly, polymer scaffolds containing neurotrophic factors and RGD motifs in their structure immobilize human NSCs, allowing their differentiation.

As mentioned, in neurodamageous diseases, a whole series of simultaneous or sequential processes occur, the direct or indirect effect of which is the death of nerve, glial and other cells (Fig. 2A). Dysregulation of biochemical and electrochemical processes caused by damage manifests itself in energy deprivation, oxidative stress and acidosis, and in general leads to excitotoxicity. This process is the result of uncontrolled activation of calcium-dependent glutamate receptors, manifested by accumulation of glutamate in the synaptic space and self-stimulating neuron stimulation. In vitro techniques to model such processes are described in the literature. The use of a whole series of such techniques or their modification as part of a single, multi-factor diagnostic method / method would bring additional economic and substantive benefits (Fig. 2B). In our model, we predict the use and analysis of the effects of six damage factors, including: glucose deprivation, partial inhibition of cellular respiration and oxidative stress (sodium azide), and excitotoxicity (induced by glutamic acid (including an increase in intracellular calcium ion concentration), NMDA, kainic acid) and acidification (aka acidosis, pH 6.35 due to elevated sodium lactate concentration). The combination of the individual techniques is more efficient than each of them when used alone and avoids various experimental problems. The need to repeatedly isolate cells from the brain can result in developmental and chemical biological variability between sessions and studies. In turn, the composition used by us in the same study of six parallel measurements of physiological responses to 6 key damage factors with CNS damage for one batch (group with exactly the same characteristics) of isolated cells is a simplified in vitro model of neurodamageous diseases, which is specific and efficient.

PL 238 941 Β1 multivariate research method. The parallel application of as many as 6 damage factors is a significant improvement and technological novelty.

The aim of the invention is to provide new compounds allowing to obtain concatameric polysignal proteins, composed of monomeric peptide units, for use in regenerative medicine.

The essence of the invention are chemical compounds with anti-damage and / or neuroprotective properties with the formulas - the sequences are presented in the form of standard one-letter amino acid abbreviations:

H2N-RGD-amide

H ₂ N-RGD-RGD-amide

H ₂ N-RGD-RGD-RGD-amide acetyl-RGD-RGD-RGD-amide

H ₂ N-RGD-RGD-RGD-RGD-amide

H ₂ N-RGD-RGD-RGD-RGD-RGD-amide

H ₂ N-RGD-RGD-RGD-RGD-RGD-RGD-amide

H ₂ N-RGD-RGD-RGD-RGD-RGD-RGD-RGD-amide

H ₂ N-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-amide

H ₂ N-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-amide

H ₂ N-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-amide

IMSRSSP-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-PRRA (abbreviation RGD1), (abbreviation RGD2), (abbreviation RGD3), (abbreviation Ac-RGD3) (abbreviation RGD4), ( abbreviation RGD5), (abbreviation RGD6), (abbreviation RGD7), (abbreviation RGD8), (abbreviation RGD9), (abbreviation RGD10-PM), (abbreviation RGD10-PS).

Structural formulas:

RGD1:

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RGD3:

Ac-RGD3:

RGD4:

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RGD5:

RGD6:

RGD7:

RGD8:

RGD9:

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RGDIO-PM:

RGDIO-PS:

The invention relates to the medical use of the above-mentioned unions.

The method of determining the activity of the anti-damage and / or neuroprotective chemicals defined above at the cellular level, included:

- glucose deprivation

- inhibition of cellular respiration by sodium azide

- acidosis by acidification of pH 6.35 with sodium lactate

- NMDA excitotoxicity

- glutamic acid excitotoxicity

- kainic acid excitotoxicity to the excitable and / or non-excitable cells tested.

The analysis of the activity of chemical compounds and a kit for the determination of the activity of chemical compounds with anti-damage and / or neuroprotective activity are described. The lists include:

a) a chemical selected from: RGD1; RGD2; RGD3; RGD4; AcRGD3, RGD10-PM; RGD10-PS, preferably RGD1,

b) excitable and / or non-excitable cells in a form that allows for homogeneous culture or a culture with a mixed type of cells,

c) media enabling measurements of cell culture viability tests at various concentrations of the test / test compound and measurements of cell viability after the action of 6 damaging and / or neurotoxic factors, however acidification / acidosis is obtained by a medium with a pH of 6.45 and the action of sodium lactate at a concentration of 200 mM or another optimized for a given cell type, energy and respiratory deprivation / glucose deprivation and oxidative stress induced by sodium azide at a concentration of 10 mM or another optimized for a given cell type, and excitotoxicity induced in 3 ways: 400 μΜ glutamic acid, 800 μΜ NMDA and 800 µΜ kainic acid or other concentrations optimized for the type of cell.

Description of the figures:

Fig. 1 is a schematic diagram of the course of major lesions following damage to the central nervous system.

Fig. 2 - shows: (2A) schematically the phenomena occurring at the molecular and cellular level during various lesions within the central nervous system, and (2B) the damage factors modeled in this patent application enabling screening and at the same time a detailed study at the molecular and cellular level of the effectiveness of new substances in the repair of neurons and glial cells. Further, the invention is shown in:

Fig. 3 - Shows an HPLC chromatogram of the RGD1 peptide.

Fig. 4 - Shows the ESI MS spectrum of RGD1.

Figure 5 - Shows an HPLC chromatogram of the RGD2 peptide.

Figure 6. - It shows the ESI MS spectrum of the RGD2 peptide.

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Fig. 7 - Shows an HPLC chromatogram of the RGD3 peptide.

Figure 8. - shows the ESI MS spectrum of the RGD3 peptide.

Fig. 9 - Shows an HPLC chromatogram of the RGD4 peptide.

Fig. 10 - shows the ESI MS spectrum of the RGD4 peptide.

Figure 11 shows the HPLC chromatogram of the RGD10-PM peptide.

Figure 12. It shows the ESI MS spectrum of the RGD10-PM peptide.

Figure 13 - Shows gel (A) and capillary (B) electrophoresis of the RGD10-PS peptide. Descriptions for gel A: mass M-marker, RGD10-PS-Ubq-ubiquitin-cleaved RGD10-PS peptide protein, RGD10-PS - IMSRSSPRGDRGDRGDRGDRGDRGDRGDRGDRGDRGDPRRA peptide cut from ubiquitin. Coomassie staining.

Figure 14. Shows the HPLC chromatogram of the AcRGD3 peptide.

Figure 15. It shows the ESI MS spectrum of the AcRGD3 peptide.

Figure 16. Shows the nucleotide sequence of the cassette with functional regions marked, containing the gene coding for Hiss-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and the expressed amino acid sequence of the fusion protein.

Figure 17 shows the nucleotide sequence of the cassette with functional regions marked, containing the gene encoding Hiss-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein and the expressed amino acid sequence of the fusion protein.

Figure 18. - Shows the genetic map of the expression plasmid construct and the nucleotide and amino acid sequence with the pET28amutSapIKASETARGD functional modules highlighted.

Figure 19. - Shows the genetic map of the expression plasmid construct and the nucleotide and amino acid sequence with pET28amutSapIKASETARGD functional modules highlighted.

Figure 20. - Shows the genetic map of the expression plasmid construct and the nucleotide and amino acid sequence with the functional modules of pET28amutSapIKASETARGDGG highlighted.

Figure 21 shows the genetic map of the expression plasmid construct and the nucleotide and amino acid sequence with the functional modules of pET28amutSapIKASETARGDGG highlighted.

Figure 22 - shows the proteins His6-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein.

22A) shows the result of protein electrophoresis under denaturing conditions (SDS-PAGE, Coomassie Brilliant Blue stained gel) of samples taken from cell extracts and chromatographic fractions of the His6-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein preparation, with a bioinformatically predicted molecular weight 16, 2 kDa, in accordance with the disclosed amino acid sequence (SEQ. 2). Lane M represents the protein molecular weight standards: bovine blood albumin 66 kDa, ovalbumin 40 kDa, carbonic anhydrase 29 kDa, soybean trypsin inhibitor 20/21 kDa, cytochrome C 12 kDa, aprotinin 6.5 kDa; unb lane, unbound cellular proteins; w2, proteins present in the column washing; e3-6, elution fractions from the column.

22B) shows the result of protein electrophoresis in denaturing SDS-PAGE, in 12.5% Tris-Tricine polyacrylamide gel, from samples taken from cell extracts and chromatographic fractions of His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein with bioinformatically predicted a molecular weight of 17.3 kDa. Lane unb, unbound cellular proteins; w2, proteins present in buffer after column washing; e3-6, elution fractions from the column.

22C) from samples of His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein preparation performed under control conditions, i.e. without induction of IPTG culture. Lane M, protein molecular weight standards; unb lane, unbound cellular proteins; w2, proteins present in buffer after column washing; e3-6, elution fractions from the column.

Figure 23 - Shows Hiss-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein.

Figure 24 - Shows Hiss-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein.

Fig. 25 - Shows the result of digestion of Hiss-c-Myc-WYY-Ubiquitin-polysignal protein RGDGG10 with the enzyme YUH1.

Fig. 26 - Shows two techniques for quantifying proteins.

Figure 27. - Shows a scheme of amplification of a synthetic stretch of DNA encoding the RGD 10-mer and RGDGG 10-mer.

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Figure 28.- It shows the directional amplification of a DNA segment encoding the RGD * 10 module with an enzyme-vector system.

Fig. 29 - shows the genetic maps of the duplicator vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Fig. 30 - shows genetic maps of the duplicating vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Fig. 31 - shows the genetic maps of the duplicator vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Fig. 32 - shows the genetic maps of the duplicating vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Figure 33 - shows the genetic maps of the duplicating vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Figure 34 - shows the genetic maps of the duplicator vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Figure 35 - shows the genetic maps of the duplicating vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Fig. 36 - shows the genetic maps of the duplicator vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Figure 37 - shows the genetic maps of the duplicator vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-mer-RGD containing 20, 30, 40, 50, 60, 70, 80, 90, 100 copies of the coding module RGD peptide, with molecular weights:

8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

Figure 38. Shows the Seq sequences. 1; Seq. 2, Seq. 3, Seq. 4.

Figure 39 - shows representative microscopic images of the visualization of marker proteins (epitopes) by immunocytochemistry in cells isolated from rat brain in primary neural culture after the first day of in vitro culture. Top panel (series A): the small arrow shows a cell with a positive reaction to the GFAP protein (Figure A1) characteristic of astrocytes (glial cells), and the tip of the large arrow indicates cells having the DCX protein (dabulcortin) (photo A2) characteristic of young neurons ( neuroblasts), Hoechst staining (picture A3) shows all cells visible in the field of view.

Middle panel (series B): shows the cells indicated by the large arrowhead that undergo cell divisions as visualized by the presence of the Ki67 protein for part of the proliferation period (Photo B2) and shows the selected astrocyte cells indicated by the small arrow currently not dividing (Photo B2). Bottom panel (series C): Shows neural cells showing immunocytochemical reactivity to precursor cells by the presence of the SOX2 marker protein, among which there are cells with division activity like cells at the bottom of image C1. Other cells (non-precursor, non-reactive to Sox2) showing such cell division are also visible (indicated by the large arrowhead in photo C2). Conclusion from the observation: in the primary culture of neuronal cells isolated from the developing cerebral cortex of rats on the first day of culture, there is a large variety of cell types with different proliferative activity.

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Fig. 40 - shows the immunocytochemical detection of characteristic marker proteins for various co-occurring cell populations on the fourteenth day of in vitro culture (14 DIV): with complex normal morphology of mature astrocytes showing the presence of the GFAP protein typical for them, as well as mature neurons showing the presence of their characteristic NeuN proteins. In culture, one can distinguish cells undergoing cell division, showing immunoreactivity for the Ki67 protein, and non-dividing cells not showing such immunoreactivity. In culture on day 14, without the action of the damage factor, no cells undergoing the cell death process (cells non-reactive for caspase 3 protein) are observed.

Fig. 41. Shows the neural culture responses to varying intensities of glucose deprivation damage factor; on this basis, the deprivation effect for 1 hour (gray bar) was selected for further studies allowing the viability of the neural culture at the level of 70% for testing the neuroprotective effect of the tested peptides.

Fig. 42. Shows the neural culture responses to various intensities of the action of the damage factor in the form of inhibition of cellular respiration (oxidative stress); on this basis, the action of sodium azide at a concentration of 10 mM (gray bar) was selected for further studies allowing the viability of the neural culture at the level of about 65% for testing the neuroprotective effect of the tested peptides.

Fig. 43 - shows the neural culture responses to different intensities of the acidification of the medium (acidosis) damage factor; on this basis, the effect of sodium lactate at a concentration of 200 mM (gray bar) was selected for further studies allowing the viability of the neural culture at the level of about 70% for testing the neuroprotective effect of the tested peptides.

Figure 44 shows the neural culture responses to varying intensities of the neuronal overstimulation (excitotoxicity) induced by glutamic acid damage factor; on this basis, the action of glutamic acid at a concentration of 400 μM (gray bar) was selected for further studies, allowing the viability of the neural culture at the level of about 70% for testing the neuroprotective effect of the tested peptides.

Fig. 45 - shows the neural culture responses to varying intensities of the neuronal overstimulation (NMDA-induced excitotoxicity) damage factor; on this basis, NMDA activity at a concentration of 800 μΜ (gray bar) was selected for further studies allowing the viability of the neural culture at about 70% for testing the neuroprotective effect of the tested peptides.

Figure 46. Shows the neural culture responses to varying intensities of the neuronal overstimulation (excitotoxicity) damage factor induced by kainic acid; on this basis, the effect of kainic acid at a concentration of 800 μΜ (gray bar) was selected for further studies, allowing the viability of the neural culture at the level of about 70% for testing the neuroprotective effect of the tested peptides.

Fig. 47 - Shows the effect of RGD1 peptide on survival of neural culture. No cytotoxic effect of the RGD1 peptide was observed at all tested concentrations. The reference peptide is the 50 μΜ TAT peptide marked with a gray bar.

Fig. 48 - Shows the effect of RGD2 peptide on survival of neural culture. Very low cytotoxicity of the RGD2 peptide was observed at all tested concentrations. The reference peptide is the 50 μΜ TAT peptide marked with a gray bar.

Fig. 49 - Shows the effect of RGD3 peptide on survival of neural culture. Low cytotoxicity of the RGD3 peptide was observed. The reference peptide is the 50 μΜ TAT peptide marked with a gray bar.

Fig. 50 - Shows the effect of RGD4 peptide on survival of neural culture. The reference peptide is the 50 μM TAT peptide marked with a gray bar.

Fig. 51 - Shows the effect of AcRGD1 peptide on survival of neural culture. The reference peptide is the 50 μΜ TAT peptide marked with a gray bar.

Fig. 52 - Shows the effect of AcRGD3 peptide on survival of neural culture. The reference peptide is the 50 μΜ TAT peptide marked with a gray bar.

Figure 53. - Shows the effect of RGD10-PM peptide obtained by chemical synthesis on survival of neural culture. The reference peptide is the 50 μΜ TAT peptide marked with a gray bar.

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Figure 54. - Shows the effect of RGD10-PS peptide, obtained by biological synthesis, on survival of neural culture. The reference peptide is the 50 μΜ TAT peptide marked with a gray bar.

Figure 55 - Shows the activity of the neurological peptide.

Figure 56 - Shows the activity of the neurological peptide.

Figure 57. - Shows the activity of the neurological peptide.

Figure 58. - Shows the activity of the neurological peptide.

RGD1

RGD2

RGD3

RGD4 multivariate multivariate multivariate multivariate model model model in in vitro vitro vitro

Figure 59. - Shows the activity of AcRGDI peptide in a multifactorial in vitro model of neurological injury.

Figure 60. - Shows the activity of AcRGD3 peptide in the multifactorial in vitro neurological injury model.

Figure 61. Shows the activity of RGD10-PM peptide, obtained by chemical synthesis, in a multivariate in vitro model of neurological injury.

Figure 62. - Shows the activity of RGD10-PS peptide, obtained by biological synthesis, in the multifactorial in vitro model of neurological injury.

Figure 63 is a schematic view of culturing ex vivo organotypic hippocampal sections and an experiment to study the neuroprotective potential of bioactive compounds. Propidium iodide (PI) was added to the 8-day culture and the sections were preliminarily assessed under a fluorescence microscope to eliminate damaged sections. After removing PI, the excitotoxicity inducing factor NMDA was added together with the fresh medium at a concentration of 100 μM, which was removed after 3 hours, and the bioactive compound that was present in the medium until the end of the experiment. Final observation was made 24 hours from the start of the experiment, after prior addition of PI to visualize dead cells then analyzed under a fluorescence microscope. The percentage of dead cells (positive for propidium iodide staining) was calculated by the formula:

% PI positive cells = (section fluorescence intensity [FI] / max [FI]) χ 100%, and the maximum damage, ie max [FI], was determined by treating NMDA hippocampus sections at a concentration of 250 µM.

Figure 64. - Shows the direct neuroprotective (anti-damage) effect of the RGD1 peptide on ex vivo live sections from rat brain. Shown are representative live sections containing important brain structures called the hippocampus in organotypic culture treated with Mass Neuron Death Factor (NMDA at 100 μΜ), where dying neurons appear as black spots upon staining with propidium iodide, and primary fluorescence microscopy images have been replaced with negative versions:

A) an example of a control section (with saline instead of NMDA given),

B) example of a section 24 hours after exposure to 100 μM NMDA damage factor for 3 hours,

C) Example of a section after treatment with 100 µM NMDA damage factor for 3 hours and then with 100 µM RGD1 solution for 24 hours. Clearly visible confirmation of the neuroprotective (anti-damage) effect of the RGD1 peptide on live sections from rat brain in a model of neurological (excitotoxic) damage.

Fig. 65 - presents a quantitative analysis of the comparison of the cell death study in ex vivo live sections from rat brain after the action of the damage factor (NMDA at 100 μΜ) and after the clearly neuroportive effect of the RGD1 peptide tested at various concentrations. A comparison was also presented that the effect of the RGD1 peptide (100 µM) alone had no negative effect on the quantification of cell mortality compared to the control culture, without the effect of this peptide and device factor. Note the statistically significant differences in mortality between the group with NMDA damage alone and the groups with damage and RGD1 at 10 and 100 μΜ (Tukey's Multiple Comparison Test, P value <0.05).

The following examples illustrate the invention.

PL 238 941 B1

P r z k ł a d 1

Chemical synthesis of RGD peptides.

The peptides RGD1, RGD2, RGD3, RGD4, RGD10-PM and AcRGD3 were synthesized on a solid support by the Fmoc method with an automated synthesizer using TentaGel S RAM amide resin. During the synthesis of acetylated peptide analogs, the respective peptidyl resins were acetylated with N-acetylimidazole in dimethylformamide (DMF) for one hour. After synthesis, the peptides were cleaved from the peptidyl resin with 98% trifluoroacetic acid (TFA) and precipitated with cold diethyl ether. The crude peptides were purified by preparative HPLC using a Maisch C-18 column 250 × 40 mm, grain size 10 µm. A linear gradient of acetonitrile (ACN) was used with 0.08% addition of TFA. The mobile phase flow rate was 25 ml / min. Individual fractions were analyzed by analytical HPLC using a Phenomenex Kinetex XB-C18 column, dimensions 150 × 4.6 mm, grain size 5 μm. A gradient of 0-20% ACN (with 0.08% TFA addition) in 30 min with a flow of 1 ml / min was applied for all peptides. After purification, the peptides were lyophilized and then placed in a 0.01 M HCl solution to exchange trifluoroacetate ions for chloride ions and lyophilized again. The purified peptides were characterized by analytical HPLC and ESI mass spectrometry using a mass spectrometer.

The characterization of the peptides obtained is shown in Figs. 3-15. HPLC chromatograms show the purity of the peptides. All peptides synthesized by solid support synthesis were> 95% pure. On the other hand, mass spectrometry (ESI MS) shows the correct identity of the peptide structure and its homogeneity. The mass spectra show signals corresponding to pseudomolecular ions (M + H) + or to a different degree charged (+2, +3, +4) of a given peptide.

P r z k ł a d 2

A method for obtaining concatameric polysignal proteins, RGD peptide derivatives for pro-regenerative purposes, DNA vectors for carrying out this method and poly RGD polysignal proteins.

The subject of the invention is a method of obtaining polysignal proteins, consisting of a polymerized RGD peptide and its derivatives with pro-regenerative properties, DNA vectors for the implementation of this method, biosynthesized and isolated polysignal proteins, isolation procedures and peptide monomer units used for the construction of polysignal proteins. The proteins obtained according to the invention can find a number of applications in the regeneration of cells, tissues and organs. Polysignal proteins may contain, among others multimers of peptide hormones or biologically active fragments of signaling proteins, stimulating tissue regeneration processes. Polysignal proteins, placed in the wound, can directly stimulate the regeneration process or gradually release, due to auto-degradation or the organism's proteases in the wound, biologically active peptides that stimulate regeneration.

The construction of polysignal proteins was performed using the methods of bioinformatics design of optimized genes, chemical synthesis of the coding DNA, molecular cloning into Escherichia coli bacteria and proprietary technology of directional, enzymatic-vector amplification of a DNA segment, with the continuity of the encoded Open Reading Frame encoding the constructed concatameral polysignal protein. The technology also describes the construction of biological nanostructures in the form of genetically coated polysignal proteins of recombinant bacteriophages. Above technology, is the subject of patent PL 228341 B (BioVentures Institute Sp.z o.o.).

P r z k ł a d 3

Diagram of the method according to the invention.

The selected bioactive RGD peptide with anti-damage and / or neuroprotective action was subjected to further amplification procedures at the DNA level, and as a result of translation of the obtained concatameric genes, it was also amplified at the level of in vivo translation in the form of a concatameric protein. The RGD peptide was amplified in 2 variants: (i) RGD and (ii) RGDGG. The RGDGG variant contained 2 additional G residues. It was assumed that these 2 repeating G residues form unstructured polypeptide segments between the RGD segments, stimulate the RGD motif to autonomously form a bioactive structure and to increase its exposure and availability in solution. Reproduction of the above-mentioned The motifs were made in two ways: (i) at the level of chemical synthesis and (ii) using the above-mentioned patented technology of enzymatic-vector duplication in vitro / in vivo [Skowron et al. 2014-2017]. By applying chemical synthesis it is possible

Typically obtaining only 5-10 copies of an amplified stretch of DNA in a concatamer. Using vector-enzyme technology, it is possible to obtain up to 500 copies of the amplified DNA segment in a concatamer. The construction of RGD polysignal proteins was therefore carried out using a combination of both methods to obtain a spectrum of cloned concatamers with different copy number of monomer units. In approach (i) a multi-module construction of an RGD polysignal fusion protein and RGDGG was used, containing the following modules: (a) Hiss tag - six histidine residues at the N-terminus to apply a high-throughput method of isolating a recombinant protein by metallo-affinity chromatography; (b) a c-Myc tag to use highly sensitive detection of the recombinant protein with dedicated anti-c-Myc antibodies; (c) the amino acid tag WYY to improve detection of the recombinant protein under UV light and during the staining of electrophoretic gels; (d) ubiquitin at the N-terminus of the design protein, after the Hiss and c-Myc tags. The ubiquitin protein is used as a fusion partner to improve the expression level of fusion proteins, reduce the toxicity of the proteins, and improve their in vivo stability. Ubiquitin can optionally be cleaved proteolytically from the isolated fusion protein using the recombinant yeast ubiquitin hydrolase-Hiss enzyme (hereinafter YUH1). This arrangement allows for the cleavage of ubiquitin and the preceding amino acid sequences without leaving additional amino acid residues in the domain of the fusion protein that contains the polysignal protein; (e) a 10-mer RGD or 10-mer RGDGG polysignal protein. In the approach (ii), the DNA section encoding the RGD 10-mer and RGDGG 10-mer was amplified by means of an expression vector, Sap1 endonuclease and DNA ligase as disclosed in patents describing the technology, such as: PL228341, US 10874735, EP 15738474.4 . The obtained higher order concatamers were cloned: (1) into the Sap1 sites of the duplicator-expression vector, creating a fusion of the polysignal protein with the Hiss tag, and (2) into the duplicator-expression vector, into which a synthetic DNA segment was previously cloned, containing the designed gene encoding the Hiss fusion protein -c-Myc-WYY-Ubiquitin-polysignal protein. This vector has internal Sap1 sites, the arrangement of which is designed such that cleavage of the Sap1 DNA releases the polysignal protein, leaving the Hiss-c-Myc-WYY-Ubiquitin cassette in the vector. After cloning of higher order concatamers, they fuse with the Hiss-c-Myc-WYY-Ubiquitin cassette, creating a new fusion protein containing the above-mentioned. cassette, and increased copy numbers of RGD or RGDGG monomeric units in the attached polysignal proteins.

P r z k ł a d 4

Design and molecular cloning of fusion proteins containing ubiquitin, peptide tags and RGD10 and RGDGG10 polysignal proteins.

In the exemplary implementation presented in Figs. 16, 17, 18, 19, DNA cassettes containing the Sap1 amplification module and codon-optimized Escherichia coli bacterial genes encoding the Hiss-c-Myc-WYY-Ubiquitin-protein fusion proteins were designed. RGD10 polysignal (DNA disclosed as Seq. 1, encoded protein disclosed as Seq. 2) and Hiss-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein (DNA disclosed as Seq. 3, encoded protein disclosed as Seq. 4) in bacteria Escherichia coli.

In the exemplary implementation presented in Figs. 20, 21, 22, 23 the cloning of synthetic genes into the commercial vector pET28 (+), having a strong hybrid T7-lac transcription promoter, was performed, transforming the pET28 (+) vector into an expression-duplicating vector carrying the designed synthetic genes in the configuration of the optimal distance from the T7-lac promoter and the Ribosome Binding Site and ATG codon translation initiation signals. The laboratory expression strain Escherichia coli BL21 Star (DE3) was used as a recombinant host for the biosynthesis of the constructed synthetic genes. The nucleotide sequence of the cassette with the functional regions marked, containing the gene coding for Hiss-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and the expressed amino acid sequence of the fusion protein is shown in Fig. 16. The nucleotide sequence of the cassette with the functional regions marked, containing the gene coding for Hiss- The c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein and the expressed fusion protein amino acid sequence are shown in Figure 17. The cassettes were digested with NcoI and Xhol enzymes, allowing the DNA segment to be directed into the pET28 (+) vector digested with Ncol and Xhol. The method of combining the vector DNA and the DNA encoding the cassettes by the bacteriophage T4 DNA ligase was used, characterized in that the transcription start and translation start signals of the pET28a (+) vector were precisely combined with the segments encoding the Open Reading Frames ORF encoding the fusion proteins: Hiss-c- Myc-WYY-Ubiquitin-RGD10 polysignal protein and Fiss-c-Myc-WYY

PL 238 941 B1

-Ubiquitin-RGDGG10 polysignal protein. The obtained ligation mixture was transformed into Escherichia coli DH5a bacteria. The obtained clones were used to isolate plasmid DNA, which was subjected to restriction analysis and DNA sequencing. The plasmid constructs pET28amutSapIKASETARGD and pET28amutSapIKASETARGDGG were selected with the correct sequence, containing the sequences of genes encoding the His6-c-Myc-WYY-Ubiquitin-cDis-signal-cDis-signal-polypeptide-cDis-signal-cDis-signal-cDis-signal cassettes unchanged compared to the in silico designed cassettes. Myc-WYY-Ubiquitin-RGDGG10 polysignal protein and transformed into an expression strain dedicated to the pET28a (+) vector, obtaining clones of Escherichia coli BL21Star (DE3) [pET28amutSapIKASETARGD] and Escherichia coli BL21GASETARGut (DEPIKETARG3). Figures 18 and 19 show the genetic map of the expression plasmid construct and the nucleotide and amino acid sequence with the pET28amutSapIKASETARGD functional modules marked. Figures 20 and 21 show the genetic map of the expression plasmid construct and the nucleotide and amino acid sequence with the functional modules of pET28amutSapIKASETARGDGG marked.

P r z k ł a d 5

Expression of bacteria cloned in Escherichia coli BL21Star (DE3) genes encoding His6-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein fusion proteins.

The constructed clones of Escherichia coli BL21Star (DE3) [pET28amutSapIKASETARGD] and Escherichia coli BL21Star (DE3) [pET28amutSapIKASETARGDGG] disclosed in Example 2 were subjected to exemplary genetic expression by means of bacterial transformation experiments of Escherichia, followed by the culture of bacteria Escherichia (DE3 coli) BL21 ° C on 2xYT microbiological medium modified for high expression purposes with the composition per 1 L (in distilled water): Tryptone, 16 g; yeast extract, 10 g; NaCl 5 g, pH 7.2 adjusted with NaOH; just before using the medium, addition of 20 ml of sterile 1 M glucose solution. After the optical density of the culture was reached OD = 1 and gene expression was induced with 1 mM isopropylthiogalactoside (IPTG), the cultures were grown for another 4 hours. Genetic expression experiments were carried out on a pilot scale in 50 ml of microbial medium, and then on a preparative scale in 1 L of medium. In the exemplary embodiment shown in Fig. 22, His6-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein were isolated from recombinant Escherichia coli cells using the following steps: (and ) the cells were centrifuged for 20 minutes at an angular acceleration of 12,000 g at 4 ° C, then the cell biomass was suspended in lysis buffer [50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 0.75 mg / ml egg lysozyme , 2.5 µg / ml deoxyribonuclease 1, 0.1 mM PMSF protease inhibitor] at 2% of the original recombinant bacterial culture volume and incubated for 10 minutes at 22 ° C; (ii) ultrasonic disintegration was used with cooling the cell suspension in an ice bath and battery debris centrifugation; (iii) applied the supernatant to a Talon metal affinity chromatography column [GE Healthcare Life Sciences, USA] containing immobilized Co ²⁺ ions. The His6-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein hybrid proteins contain the His6 tag, enabling highly specific binding of these proteins to the Talon column and obtaining a very high degree of purification. The Talon column, equilibrated in T buffer [50 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 300 mM NaCl] and containing adsorbed hybrid proteins, was washed with 10 column volumes of W buffer [50 mM sodium phosphate, pH 7.4, 300 mM NaCl; 10 mM imidiazole] was then eluted with 10 column volumes of buffer E [50 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 300 mM NaCl; 150 mM imidiazole]. Fig. 22A; Fig. 22B; Fig. 22C. Comparison of the two preparations disclosed in Fig. 22BC shows massive expression of the recombinant His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein with a bioinformatically predicted 17.3 kDa molecular weight, in accordance with the disclosed amino acid sequence (Seq. 4), but absent. or present in trace amounts in the control formulation. The isolated fusion proteins were frozen in liquid nitrogen and then stored at -80 ° C. In order to finally verify the identity of the recombinant proteins His6-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein were subjected to immunological analysis using antibodies specific for His6 and c-Myc tags, Western Blotting method. In the exemplary embodiment shown in Fig. 23 the protein His6-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and the protein His6-c-Myc-WYY-Ubiquitin shown in Fig. 24

PL 238 941 B1

- RGDGG10 polysignal protein, was subjected to SDS-PAGE electrophoresis on 12.5% Tris-Tricine polyacrylamide gel, and then transferred by electrotransfer in TW buffer [25 mM Tris, 192 mM glycine, pH 8.3, 20% methanol, 0, 04% SDS] onto the PVDF membrane. After the transfer was completed, the membrane was blocked with TBST buffer [50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20] with 5% non-fat dry milk, by gentle shaking for 2 hours at 4 ° C, washed with TBST buffer by shaking 10 minutes and then reacted with anti-Hiss primary rabbit antibodies (VTTF Ltd., Poland) using a 1: 5000 dilution for 18 hours at 4 ° C, shaking in TBST buffer. Unbound antibodies were washed away by shaking the membrane 3 times for 10 minutes in TBST buffer. Bound rabbit antibodies were reacted with enzyme-coupled goat anti-rabbit secondary antibodies at a 1: 10,000 dilution in TBST buffer by shaking the membrane for 1 hour at 22 ° C. Unbound anti-rabbit antibodies were washed away by shaking the membrane 3 times for 10 minutes in TBST buffer. For visualization, the membrane was reacted with Sigma fast BCiP / NBT chromogenic substrates (Sigma, USA) for alkaline phosphatase developed, giving a violet-blue insoluble product at the point of contact with the bound conjugated goat antibodies. In Fig. 23 lane M shows a molecular weight standard of the PageRuler Plus Prestained Protein Ladder proteins (ThermoFisher, USA); K, a control protein with a molecular weight of 130 kDa, containing a His6 tag; Apl, cell lysate applied to the Talon column; w2, washing the column with buffer W; e3, e4, e5, elution of proteins bound to the Talon column with buffer E. The color reaction was shown by the protein bands corresponding to the expected molecular weight of the proteins Hiss-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and Hiss-c-Myc-WYY-Ubiquitin polysignal protein RGDGG10. In the embodiment shown in Fig. 24, an analogous procedure to that shown in Fig. 23 was performed, except for the use of rabbit anti-c-Myc primary antibodies at a 1: 1000 dilution (Novus Biologicals, USA). In Fig. 24, lane M represents the molecular weight standard of the proteins; Apl, cell lysate applied to the Talon column; w2, washing the column with buffer W; e3, e4, elution of proteins bound to the Talon column with buffer E. The color reaction was shown by protein bands corresponding to the expected molecular weight of the Hiss-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein and a small amount of dimers of these proteins, detected on the membrane due to greater sensitivity of c-Myc antibodies. The presence of dimers is probably due to the large number of G residues in the regularly repeated RGD and RGDGG motifs and thus facilitated molecular interactions. This property is desirable when polysignal proteins are used in regenerative medicine, as the formation of such a supramolecular structure lowers the diffusion rate of polysignal protein complexes beyond the drug application area.

P r x l a d 6

Proteolytic cleavage of ubiquitin and peptide tags from polysignal proteins. Isolation and analysis of cut polysignal proteins.

Hiss-c-Myc-WYY-Ubiquitin-RGD10 polysignal protein and Hiss-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein preparations purified as indicated in Example 3 were dialyzed against enzyme reaction buffer YUH1 [50 mM potassium phosphate, pH 7 4, 300 mM NaCl] to perform the procedure for cleaving the Hiss-c-Myc-WYY-Ubiquitin polypeptide segment from polysignal proteins. The exemplary embodiment shown in Fig. 25 discloses the result of digesting the Hiss-c-Myc-WYY-Ubiquitin-polysignal protein RGDGG10 protein with the YUH1 enzyme, using an enzyme to substrate ratio of 1:10 (above), and carrying out a digestion reaction for 16 hours at 4 ° C. In lane M, protein molecular weight standards are indicated; 1, His6-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein; 2, Hiss-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal protein digested with YUH1. Lane 2 shows efficient digestion of the Hiss-c-Myc-WYY-Ubiquitin-RGDGG10 polysignal fusion protein. The evaluation of the efficiency is based on the disappearance of the non-digestible form and the formation of the N-terminal domain Hiss-c-Myc-WYY-Ubiquitin. It is not possible to quantify the released RGDGG10 polysignal protein and the RGD10 polysignal protein due to the lack of aromatic amino acid residues in the sequences of the above-mentioned proteins, the presence of which is necessary for the staining of Coomassie Brilliant Blue proteins on SDS-PAGE electrophoretic gels. Hence, polysignal proteins are not directly observable on SDS-PAGE gels. The truncated polysignal protein RGDGG10 with a molecular weight of 5.5 kDa and the polysignal protein RGD10 with a molecular weight of 4.4 kDa were purified using the steps: (i) chroma

Metal affinity graphing on Talon columns, equilibrated in T buffer [50 mM sodium phosphate, pH 7.4, 300 mM NaCl], was used to bind the truncated N-terminal Hiss-c-Myc-WYY-Ubiquitin domains and undigested an aliquot of the fusion protein; (ii) ultrafiltration fractionation, by sequential use of a centrifuge column with a 10 kDa cut-off (Vivaspin Turbo 15, Sartorius, Germany) - removing the YUH1 enzyme with a molecular weight of 26 kDa and (iii) the use of a 3 kDa cut-off centrifuge column (Amicon Ultra 0.5 mL, MWCO 3 kDa, Millipore, USA), retaining the cut-off RGDGG10 polysignal protein with a molecular weight and the RGD10 polysignal protein. Ultrafiltration was performed in a centrifuge with angular acceleration of 4000 g. The isolated fusion proteins were stored: (i) by freezing in liquid nitrogen and then placing at -80 ° C or (ii) by lyophilization after prior exchange of buffer T into buffer L [50 mM ammonium carbonate, pH 7.4]. Due to the lack of the possibility of quantitative and proportional staining of the protein bands of the RGDGG10 polysignal protein and the RGD10 polysignal protein on SDS-PAGE gels, two techniques for quantifying proteins were used, disclosed in Figs. 11 and 26: (i) high performance molecular filtration chromatography (SEC), made in PBS buffer [10 mM Na ₂ HPO4, 1.8 mM KH2PO4 / 2.7 mM KCI, 137 mM NaCl, pH 7.4] on a Superdex Peptide PC 3.2 / 300 column (GE Healthcare, USA) and HPLC made on column C18. Both techniques show a protein purity of over 90%. The peak appearing at the 3rd minute of elution from the C18 column (Jupiter C18, 5mm, 300A, Phenomenex, USA) corresponds to the elution position of the salts contained in the polysignal protein preparation.

Active sequence of the peptide containing the 10-fold duplicated RGD sequence (containing additionally flanking at the N- and C-terminus sequences resulting from the biotechnological preparation procedure)

IMSRSSPRGDRGDRGDRGDRGDRGDRGDRGDRGDRGDPRRA (hereinafter referred to as RGD10-PS), obtained in a biotechnological process from a bacterial expression system.

P r z k ł a d 7

Construction of higher-order polysignal proteins: DNA amplification reaction, coding for 10-mers of RGD and molecular cloning of higher-order concatamers into a duplicating-expression vector.

As indicated in Example 3, the construction of polysignal proteins with increased copies of RGD and RGDGG monomers requires the use of a patented DNA amplification technology and the construction of concatameric proteins (Skowron et al. 2014-2017). Fig. 27 discloses a scheme of amplification of a synthetic stretch of DNA encoding the RGD 10-mer and RGDGG 10-mer. The cloned DNA cassettes encoding the polysignal proteins indicated in Example 1 were digested with the restriction endonuclease SapI in such a way that the pET28amutSapIKASETARGD and pET28amutSapIKASETARGDGG vectors were cut out of the pET28amutSapIKASETARGDGG vectors, contained between the convergedly directed DNA sequences recognizing their N-terminal coding sequences, Hiss-c-Myc-WYY-Ubiquitin in vectors. Vector DNA digestions were performed at 50 µ! S buffer [20 mM Tris-acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, 100 µg / ml bovine albumin] at 37 ° C for 2 hours. The excised fragments encoding the 10-mer RGD and 10-mer RGDGG were affixed at the 5 'ends with single-stranded DNA sections: CCC and GGG, allowing for ordered ligation of DNA sections in only one orientation with the continuity of the Open Reading Frame, encoding higher-order RGD and RGDGG multimers . The excised fragments were separated by electrophoresis on a 2% agarose gel in TBE buffer (220 mM Tris, 180 boric acid, 5 mM EDTA, pH 8.3) and isolated from the gel by electroelution. The purified fragments were ligated with bacteriophage T4 ligase, controlling the reaction time for 5 minutes to 2 hours at 22 ° C. The ligation products were electrophoresed on a 1.5% agarose gel in TBE buffer and the concatamers of the RGD and RGDGG monomers with a copy number of 20 and more were excised from the gel. DNA was isolated from the excised gel fragments and ligated into the amplifying vector pET21d + _mutSapI, cleaved with the restriction endonuclease Sap1. The duplicator vector pET21d + _mutSapI has a strong T7-lac transcription promoter, translation initiation regions, and a repeater module containing the Hiss-Sapl-Smal-SapI tag segment.

The concatamers cloned into this vector do not fuse with the ubiquitin and the WYY region, only with the Hiss tag. Figure 28 discloses the results of agarose electrophoresis on a 1.5% agarose gel in TBE buffer obtained by amplifying poly-10-mer-RGD DNA concatamers. Lane M, 100 bp DNA size marker (Thermofisher Scientific SM0321); lane S, DNA concatamers containing increasing copies of the RGD10 module.

PL 238 941 B1

Figures 29, 30, 31, 32, 33, 34, 35, 36, 37 disclose genetic maps of the duplicator vector constructs, designated pET21d + _mutSapIRGD_20-100, containing cloned poly-10-merRGD containing 20, 30, 40, 50 , 60, 70, 80, 90, 100 copies of the RGD peptide coding module, with molecular weights: 8.6 kDa, 12.0 kDa, 15.4 kDa, 18.7 kDa, 22.1 kDa, 25.5 kDa, 28.9 kDa, 32.3 kDa, 35.6 kDa.

P r z k ł a d 8

A method of obtaining and maintaining primary neural (i.e. neuronal and glial) cultures of cerebral cortex cells.

Primary neural cortical cultures were obtained from embryos of E18.5 embryonic age from Wistar rats. For this, pregnant females were anesthetized and sacrificed in a carbon dioxide chamber. In some cases, animals were euthanized with isoflurane. Cortical tissues were excised from isolated embryonic brains and washed with HBSS buffer supplemented with a mixture of penicillin and streptomycin and HEPES. The cortical tissue was then trypsinized for 10 minutes at 37 ° C and mechanically homogenized to fragment the tissue into a single cell suspension. The cells were then centrifuged at 300 rcf for 4 minutes, resuspended in HBSS and counted in the Burker chamber to obtain the same cell suspension density in all experiments. Cells were transferred to a Neurobasal or Neurobasal-A culture medium supplemented with 1 mM sodium pyruvate, with a final glucose concentration of 12.5 mM, supplemented with B27 to 2%, L-glutamine up to 200 mM, glutamic acid up to 10 mM, penicillin / streptomycin mixture to 1% and plated on 200,000 cells / well poly-D-lysine coated 24-well culture plates. Neural cultures (cultures of nerve cells or glial cells or a mixture of both) were incubated for 14-15 days in an atmosphere of 5% CO2 (95% air, 98% humidity, 37 ° C). Cultures on days 14-15 in vitro were used in all experiments.

P r z k ł a d 9

A method of characterizing cells in primary neural culture by immunocytochemical detection analysis to demonstrate cell diversity.

To perform a qualitative analysis of the obtained primary cultures, the cells on the first day in vitro (1 DIV) and the fourteenth day in vitro (14 DIV) were fixed with 4% paraformaldehyde, washed with PBS and an immunocytochemical characterization method for the obtained cells was performed using highly specific diagnostic antibodies to visualize the population: neurons (early neurons - DCX and late neurons - NeuN), astrocytes (GFAP), stem cells (SOX2), dividing cells (Ki67) and apoptotic cells (caspase 3). The following primary antibodies were used: rabbit detecting GFAP (Dako Z0334 at 1: 1000), mouse detecting GFAP (Sigma G3893 at 1: 300), rabbit detecting DCX (Abeam ab18723 at 1: 500), murine NeuN ( Millipore MAB11144 at a concentration of 1: 300), goat detecting SOX2 (SantaCruz sc17320 at a concentration of 1: 500), rabbit detecting Ki67 (Abeam ab16667, at a concentration 1: 200), rabbit detecting caspase 3 (CST 9664 at a concentration 1: 125); and all donkey secondary antibodies (Jackson Immunoresearch): with Alexa488 fluorophore and Alexa594 for rabbit antibodies, with Alexa488 fluorophore and Alexa594 for mouse antibodies, with Alexa488 fluorophore and Alexa594 for goat antibodies. The effects of immunodetection were imaged under an epifluorescence microscope, collected with a computerized image analysis system and the fluorescence images were processed into negative versions (dark fields indicate high protein content). Co-visualization of marker proteins with secondary antibodies conjugated to different light-excited fluorophores of different wavelengths allowed for complex analyzes of coexistence or absence of labels in the same cells (with reference to the Hoechst non-selective cellular dye). Representative microscopic images after day 1 of culture are shown in Fig. 39, and analyzes of the location of markers in the mature culture, on day 14 of in vivo culture, are shown in Fig. 40. In the cerebral cortex of rats not only on the first day of culture, but also on the fourteenth day, there is a great diversity of cell types with different proliferative activities and this in vitro model well represents the diversity of neural populations in vivo. The analyzes showed the presence of, among others neuroblasts (immature neurons, immunopositive response to DCX) and neurons (NeuN) and astrocytes (GFAP), and numerous cells with a marker for stem cells (SOX2) and dividing cells (Ki67) were also observed. At the same time on

After cultivation, without the action of damaging factors, cells undergoing the process of cell death (no reaction to caspase 3 in the immunocytochemical method) were not observed.

P r z k ł a d 10

The method of obtaining a multifactorial model of neurological damage for in vitro testing of the neuroprotective and anti-damage efficacy of potential pharmacologically active substances.

The present invention relates to a method of diagnosing the neuroprotective effects of peptides in the model of several co-occurring factors during neurodamageous disorders. The in vitro model of neurological damage was optimized for 14-15 days of primary neural culture of cells isolated directly from the cerebral cortex obtained from Wistar rat embryos and can be used successfully with other types of cells. The model is based on the exposure of cells to damage factors that model biochemical and neurochemical dysfunctions present in diseases and injuries of the nervous system. The model includes the parallel use of potent damage factors such as acidification, i.e. acidosis, respiratory energy deprivation (glucose deprivation and sodium azide-induced oxidative stress) and excitotoxicity independently induced by glutamic acid, NMDA and kainic acid. The damage conditions were optimized in such a way that the potency of the damage factor caused a decrease of 30-40% of responses diagnosed with the CellTiterBlue test (CTB fluorophore in a modified MTT-type biochemical test) in each case in the assessment of cell viability based on a measurable assessment of metabolic functions of mitochondria. For this purpose, cells were incubated with the fluorescent agent CTB for 1-4 hours at 37 ° C (5% CO 2). The survival test itself was optimized previously in 96-well plates coated with poly-D-lysine. The molecular mechanism of the test is the conversion of resazurin (dark blue) to resorufin (pink) in the mitochondria of living cells. The fluorescence intensity of the CTB agent is measured spectrofluorimetrically (560ex / 590em) and is proportional to the number of viable cells. Control (untreated) cell samples represented 100% viability and signals from the remaining wells were referenced to the control and presented as mean ± SD. Statistical analysis included ANOVA with a p value of <0.05 considered statistically significant. Each experiment was performed at least three times.

1) Optimizing the intensity of glucose deprivation as a neurodamage factor to obtain a method for determining the anti-damage and / or neuroprotective efficacy of potential pharmacologically active substances

To optimize glucose deprivation conditions, the culture medium was removed from the cell plates, the cells were immediately washed with 300 μl of PBS (pH 7.4) and incubated with 500 μl of glucose-free and sodium pyruvate-free medium (Neurobasal-A, described above) under standard conditions for 10, 30 minutes and 1, 2, 3 hours. Each plate contained control cells incubated in standard culture medium (with a glucose concentration of 12.5 mM). After an appropriate time, all wells were changed from the glucose-free medium to fresh standard medium (500 μl). Cells were grown under standard conditions and the survival of the culture was measured after 20 hours. As shown in Fig. 41, on this basis, the deprivation effect for 1 hour (gray bar) was selected for further testing, allowing the use of a damage factor to maintain the viability of the neural culture at about 70% for subsequent testing of the neuroprotective effect of the test peptides.

2) Optimization of the intensity of inhibition of sodium azide-induced cell respiration as a neurodamaging factor to obtain a method for determining the anti-damage and / or neuroprotective efficacy of potential pharmacologically active substances. The inhibition of cellular respiration (oxidative stress) was modeled by incubating cells with various concentrations of sodium azide. For this, the culture medium was removed from the cell plates, washed immediately with 300 µl of HBSS and incubated in a culture medium containing sodium azide at concentrations of 1, 5, 10, 20 and 50 mM for 1 hour under standard conditions. After incubation, the medium was changed to a standard (500 µl) and cells were incubated under standard conditions for 20 hours, after which the viability of the culture was measured. As shown in Fig. 42, on this basis, the effect of sodium azide at a concentration of 10 mM (gray bar) was selected for further studies, allowing the use of such damage factor to maintain the viability of the neural culture at about 65-70% for later testing of the neuroprotective effect. tested peptides.

PL 238 941 B1

3) Optimization of the acidosis intensity from pH 6.35 and the action of sodium lactate as a neurodamage factor to obtain a method for determining the anti-damage and / or neuroprotective efficacy of potential pharmacologically active substances

In order to establish acidosis conditions, it was necessary to optimize the pH and the concentration of sodium lactate. After removing the culture medium and washing with 300 μl of PBS (pH 7.4), cells were incubated in 500 μl of Neurobasal at pH: 7.4 (control), 6.5, 6.35, 5.55, 5.2 and 4.6 (established with 0.1 M HCl). The sodium lactate concentration was similarly optimized by incubating the cells in a medium containing sodium lactate at 50, 200 and 1000 mM. Finally, the synergistic effect of both factors was measured. After one hour of incubation, the medium was changed to the standard medium and the cells were incubated for 20 hours. As shown in Fig. 43, on this basis, the effect of sodium lactate at a concentration of 200 mM (gray bar) was selected for further studies, allowing the use of such damage factor to maintain the viability of the neural culture at about 70% for subsequent testing of the neuroprotective effect of the peptides tested.

4) Optimization of the severity of NMDA excitotoxicity, glutamic acid and kainic acid as the three neurodamaging factors for obtaining methods for determining the anti-damage and / or neuroprotective efficacy of potential pharmacologically active substances

The excitotoxicity was optimized by independent use of three common models: excess glutamic acid, NMDA and kainic acid. All samples of excitotoxic agents were prepared just before the experiment in culture media at the following concentrations: 400, 800, 1200, 1600, 2000 μM. Before the administration of the factors, the culture medium was removed from the plates, the cells were washed with 300 µl of PBS (pH 7.4) and then incubated for 1 hour with excitotoxic agents under standard conditions. Cells were incubated with each factor separately. Media was then changed and cells incubated for another 20 hours under standard conditions. As shown in Figs. 44, 45 and 46, respectively, on this basis the effects of glutamic acid at a concentration of 400 μM, NMDA at a concentration of 800 μM and kainic acid at a concentration of 800 μM were selected (bars marked in gray in Figs. 44, 45 and 46) for further research, allowing for the use of damaging factors for such intensity to maintain the viability of the neural culture at the level of about 70% for subsequent testing of the neuroprotective effect of the tested peptides.

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The method of conducting survival tests of primary neural culture and the results of measurements of the influence of polyRGD peptides on survival of neural culture.

Viability of neural culture was quantified using the CellTiterBlue assay (CTB, see Example 10). The fluorescence intensity of the CTB agent was measured spectrofluorimetrically (560ex / 590em) and is proportional to the number of viable cells. Control cell samples (untreated) represented 100% viability and signals from the remaining wells were referenced to the control and presented as mean ± SD. Statistical analysis included ANOVA with a p value of <0.05 considered statistically significant. Each experiment was performed at least three times.

The effect of polyRGD peptides on the survival of primary neural cortical culture was tested by exposing the cells to each peptide for 20 hours dissolved in the culture medium at a concentration of (1, 10, 100, 250 μM) and incubating under standard conditions (5% CO2, 95% air, humidity). 98%, 37 ° C). The exception was the RGD10-PS peptide, which was tested at concentrations: 0.05, 0.1, 0.25, 1, 2.5, 5, 10, 25 µM, and incubated under the conditions described above. A 50 μM TAT peptide solution was used as reference. The tests were performed in 24-well plates coated with poly-D-lysine in a working volume of 500 µl / well. Appropriate peptide solutions were prepared in such a way that no more than 50 µl of medium is replaced in each well with cells, including wells with control cells (untreated with peptide). Each peptide concentration was tested in four wells.

Measurements were performed against the reference (reference) peptide Tat (4957) NH2 (designated as TAT) at a concentration of 50 μM.

In the case of cell survival measurements in control samples (without added peptide), in order to maintain optimal conditions, appropriate volumes of the medium were replaced with new medium. The CellTiterBlue readings for these trials gave an average value labeled 100%, which was then used as a reference (reference) for the measurements of these tests when the individual damage factors were used. It should be emphasized that for

For each damage factor, a separate reference value was obtained, denoted as 100%, within the same measurement session and under the same conditions as for the RGD analogs used.

On the basis of the performed quantitative analysis of the obtained spectrofluorimetric measurements, it was shown that no cytotoxic effect (or only slight cytotoxic effects in individual cases) was demonstrated for the tested concentrations for the peptides:

RGD1 (Fig. 47), RGD2 (Fig. 48), RGD3 (Fig. 49), RGD4 (Fig. 50), AcRGD3 (Fig. 52), RGD10-PM (Fig. 53), RGD10-PS (Fig. 54).

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Neuroprotective effect of RGD peptides in vivo in a model of neurological damage.

The neuroprotective effect of RGD peptides was investigated in an in vivo model of neurological damage, which included: glucose deprivation (Neurobasal-A without the addition of glucose, which was used in any other case at a concentration of 12.5 mM), acidosis (Neurobasal at pH 6.35, sodium lactate at 200 mM), oxidative stress (sodium azide at 10 mM) and excitotoxicity (glutamic acid at 400 μM, NMDA at 800 μM and kainic acid at 800 μM) were analyzed independently. For this purpose, 14-15-day-old primary neural cells were used, cultured in 24-well plates coated with poly-D-lysine in standard medium (Neurobasal, glucose concentration 12.5 mM, 500 μl / well). The activity of each peptide was analyzed on two 24-well plates, each with three damage factors (6 wells for a given factor): the first - glucose deprivation, oxidative stress and acidosis, the second - three inducers of excitotoxicity. There were at least six wells with control cells in each plate. Three wells in each row with a given damage factor were intended for subsequent incubation (after 1 hour exposure to the damage factor) in fresh medium, while the remaining three - for analogous incubation with the given peptide in the medium.

All damage factors were prepared immediately before the experiment. Sterile solutions of RGD1, RGD2, RGD3, RGD4, RGD10-PM and AcRGD3 peptides were prepared in the culture medium at a concentration of 100 µM. The RGD10-PS sample was prepared at a concentration of 1 µg / ml. The TAT peptide reference sample was prepared at a concentration of 50 µM (selected on the basis of in-house optimization). As mentioned, before peptide administration, all cells were exposed to damage factors for 1 hour under standard conditions (5% CO2, 37 ° C). For this purpose, in the case of oxidative stress and excitotoxicity, 250 μ was replaced! in each well and the appropriate factors were added to their final concentration as described above. In the case of glucose deprivation and acidosis, the entire volume of the medium was replaced with the medium containing the given damage factor, after washing the cells with 300 μl of sterile PBS. After a 1 hour incubation, the damage factors were completely removed and the cells were washed with 300 µl! PBS. Then, as explained above, one half of the cell wells were incubated in fresh medium and the other half of the cells in peptide medium. After a 20-hour incubation under standard conditions, the survival test described above was performed. All peptides were tested in at least triplicate with each new batch of isolated cells. The statistics were performed in a manner analogous to the previous experiments.

The results of the anti-damage and / or neuroprotective effects of polyRGD peptides are shown in Figures 55-62. RGD peptides show selective protective properties against particular damage factors. It should be noted that in the case of RGD1, such activity is visible for all damage methods.

The 20-hour incubation in 100 μM RGD1 solution resulted in a significant increase in the viability of the neural culture against all tested damage factors. In the case of the remaining peptides, significant neuroprotective properties against some damage factors were observed.

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Confirmation of the efficacy of the anti-damage action of the RGD1 peptide directly in ex vivo brain sections subjected to NMDA-induced excitotoxicity.

Ex vivo model of neurological damage in rat hippocampal sections

The potential neuroprotective / anti-damage activity of the RGD peptides was verified on the example of the RGD1 peptide, the effect of which was tested directly in rat hippocampal (i.e., hippocampal) sections exposed to the NMDA damage factor. The use of this glutamate receptor agonist induces the proce cascade

In the sections of the hippocampus, that is, the structure of the brain responsible for e.g. for learning and spatial memory and the development of emotions in humans and other mammals, which can be used as a sensitive model to study the pharmacological effect of drugs and enveloping substances. Hippocampal sections were obtained from P7 pups of Wistar rats and grown as organotypic cultures according to a modified Stoppini method. For this purpose, the pups were decapitated, the hippocampuses were carefully isolated from the brains and 400 μm thick tissue sections were obtained from them using a Tissue Chopper. Sections were grown in 6-well plates with culture inserts (4 sections / insert, MILLICELL CM Millipore with 0.4 μm pores and 30 mm diameter), in medium containing 50% Neurobasal, 22% HBSS supplemented with 25% horse serum and 2% B27.1M HEPES; glucose 5 mg / ml; in 1% penicillin and streptomycin. The culture was started in the medium with 25% horse serum, which was gradually (during the following days) replaced with the medium without the serum. Cultures were carried out for 8 days under the conditions of 36 ° C and in an atmosphere of 5% CO2 according to the scheme shown in Fig. 63. On the eighth day of cultivation, a study of the neuroprotective effects of RGD peptides was carried out (Figs. 64 and 65). For this purpose, the sections were divided into four experimental groups:

group 1) control - sections cultured in serum-free medium and without NMDA and without test peptide;

group 2) control + test peptide - sections cultured in serum-free medium together with the test peptide (peptide toxicity test);

group 3) NMDA - sections grown in serum free medium with damaging factor (100 µM NMDA), which were removed after 3 h;

group 4) NMDA + test peptide - sections grown in serum-free medium with damaging factor (100 µM NMDA) and test peptide, after 3 h incubation NMDA was removed and peptide remained until the end of the experiment.

Assessment of neurological damage and neuroprotective effects of RDG1 peptide in sections from rat brain

Sections in all four groups were stained with ~ 1 µM propidium iodide (labeling dead cells) and incubated for 30 minutes to eliminate dead sections. Then the hippocampal cultures in group 4 were exposed to a mixture of 100 μM NMDA and RGD1 at three concentrations: 1, 10, 100 μM (on the day of the experiment, the peptides were suspended in autoclaved water, then filtered and kept on ice at 4 ° C), the sections were then incubated under standard conditions for 3 hours. After this time, the medium in all groups was changed to remove NMDA content by completely replacing the medium with medium without serum and containing the appropriate concentrations of RGD1. The sections were then incubated for 24 hours and then re-stained 30 minutes before the final detection of the damage score with the addition of 6 μM propidium iodide to visualize damaged cells and to assess the neuroprotective effect of the RGD1 peptide, and all sections were quantified using a fluorescence microscope and computerized system. image analysis. The cell mortality of a section was calculated as the ratio of the fluorescence intensity of a given section and the maximum fluorescence which in this case was obtained by treatment with 250 µM NMDA control sections.

The exemplary photos of hippocampal sections in Fig. 64 show the difference in density of stained fluorescent dots corresponding to low neuronal mortality under control conditions (a) and high mortality after 100 µM NMDA application (b). Measurements of the dying cell density prove the neuroprotective and / or anti-damage properties of the RGD1 peptide in an ex vivo model of excitotoxicity occurring, inter alia, in in stroke injuries. As shown in Fig. 65, the RGD1 peptide shows no significant toxicity in this model and, moreover, is protective at a concentration of 10 and 100 µM.

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A kit for the in vitro method of determining the in vitro anti-damage and / or neuroprotective activity of chemical compounds in neural culture cells, which contains 6 damage factors in the phenomena of sudden damage to the nervous system of vertebrates, including humans, and a reference compound for obtaining a method of comparing biological and physiological responses of the tested cells.

The kit is used to determine the in vitro anti-damage and / or neuroprotective activity of chemical compounds against cells derived from neural or non-natural cultures. The proposed diagnostic kit was used successfully in extended experiments

For testing anti-damage activity in cell types other than neurons, e.g. enriched astrocyte cultures. The described kit enables the determination of anti-damage and / or neuroprotective properties in a wide range of different cells that may be involved in the clinical situation and in preclinical research in the repair of damage to the central nervous system. The regenerative potential of a wide range of cells of various origins has been demonstrated, including the participation in the repair of CNS damage for stem cells, tissue-specific stem cells (both progenitors and precursors), progenitors, precursors, juveniles (immature forms) and mature active and inactive forms: neurons, astrocytes, oligodendrocytes, microglia, glia of the olfactory epithelium, adipocytes, hematopoietic and immune cells and others, and may include cells induced to pluripotent properties by natural means (including Very Small Embryonic Like (VSEL) Stem Cells and others) and induced in a human-stimulated way (including so-called induced pluripotent cells (iPS) cells and others) and unlimited as to the species of animals it comes from, including mammalian and human cells. Such positive examples also concern not only primary cultures, also on cell lines, first of all a good example are the relatively numerous publications on the use of various groups of cells in animals and in humans (cells of own or foreign origin) in repairing damage to the central nervous system.

Hence, in the range of cells for which it is possible to use the described kit for the in vitro method of determining neuroprotective and / or anti-damage activity, also non-neuronal cells. Within the scope of the present invention, for non-affected cells (including neurons) we assume the use of at least 4 out of 6 damage factors that mimic at the cellular level key natural processes occurring during sudden damage to the nervous system. For non-excitable cells, within the scope of patent protection, we assume the use of 3 out of the 6 damage factors described (e.g. testing for caninate, glutamate or NMDA excitotoxicity phenomena for non-excitable cells does not have to be correct).

A reference compound should be used in the kit (otherwise also regarded as a reference or calibration compound) that gives positive responses to the damaging factors used. In particular, the reference compound should allow obtaining at least 4 statistically significant positive measurements from the set of 6 in vitro damage factors for excitable cells (e.g. neurons) against the control (cells not exposed to single damage factors). An example of such a compound may be some peptides, an excellent example of which is the RGD1 peptide at a concentration of 100 µM also within the scope of the present claim (Fig. 55), which gives statistically significant changes in anti-damage performance for all 6 in vitro damage factors. For the reference compound used for non-excitable cells, statistically significant changes can be assumed for at least 3 out of a set of 6 factors, all the more that the final common CTB test (modified MTT biochemical test) is described primarily for determining the viability of non-excitable cells.

The use of the kit for a method of determining the in vitro anti-damage and / or neuroprotective activity of chemical compounds in neural culture cells, but is also possible for cells in non-natural culture, includes homogeneous or mixed cultures of various types and sources of vertebrate cells, including human cells.

The suspension of cells used in the kit may contain 1a) freshly isolated cells from biological structures from animals (especially mammals) or, in strict time regime, from tissue derived from human biopsy or postmortem material, enabling the establishment of a mixed primary culture, or 1b) after modification culture, i.e. after the use of selection / differentiating factors in the further stages of culture, the growth may contain only selected fractions of cells (e.g. after adding the cytostatic substance of the AraC type according to an appropriate application scheme, it may be deprived of all dividing cells) or after adding substances supplementing / enriching the primary cells, it may be differentiated to other types even originally absent in the cell mixture. It is also possible to use 1c) freshly isolated cells quickly selected from biological structures on the basis of physical properties (e.g. in size differentiation centrifugation) or biological properties (containing selective surface or intracellular epitopes enabling selection by affinity techniques)

Using selective immunoglobulins conjugated with fluorophores or magnetic particles and separation in the fluorescence signal sorting method or biological or physicochemical affinity column chromatography). Finally, 1d) cell suspensions modified on solid supports by molecular biology techniques, e.g., induced pluripotent cells (iPS) obtained by the person / team providing the kit or obtained from another source. Thus, 2a) primary cell mixtures or 2b) mixtures enriched with specific subpopulations or 2c) homogeneous cultures with one type of cultured cells may be used. A separate type of cells used in the kit can be 3) cell lines, most often with immortalized division characteristics of the population. 4a) excitable cells that are electrically conductive, e.g. adult neurons, neuroblasts (juvenile neurons with different conductivity characteristics such as mature neurons) and others that give physiological responses to stimulus factors, including excitotoxic agents, and 4b) non-excitable non-excitable cells which respond to excitotoxic factors and are responsive to factors of a general nature, such as factors influencing energy and metabolic processes (glucose deprivation, partial oxygen deprivation / oxidative stress, acidosis and lactate effects).

One example of a non-limiting application of the kit for the in vitro determination of the anti-damage and / or neuroprotective activity of chemical compounds is the isolation of a mixture of cell types from selected structures of the developing brain or spinal cord of rats, mice or other mammals isolated in the prenatal period, pre-natal (neonatal) or adults. This was also used in this patent application, where the following were used: cells freshly isolated without modification from selected rat brain structures (1a) or with differentiating modifications (1b), these were cell mixtures (2a) or populations with an enriched content of specific subpopulations (neurons after eliminating on dividing cell media) (2b), excitable cells such as neurons and neuroblasts (4a) were used in a mixture with non-excitable cells such as astrocytes and other types of glial cells (4b).

The cells in the kit are used in the form of a primary live cell suspension or in deep freeze and, after thawing at the appropriate density, are grown in multi-well plates on media coated with laminin or poly-D-lysine as in the previous examples described, or without such coating in "N-Medium "(Composition below) or in other media. Optionally, the independent isolation of neuronal cells from biological structures is possible, which takes place in "Medium D" containing 10% Hank's Balanced Salt Solution in sterile water supplemented with penicillin and streptomycin to a final concentration of 1% and 1% HEPES. Tissue fragments are treated with "E medium" containing D medium with 0.08% trypsin and incubated for several to several minutes at 37 ° C depending on the type of tissue. After the cells are separated by centrifugation and rinsing several times with "Medium D", the cells are cultured typically for 7 to 14 days or for another period at an appropriate density determined by microscopic measurements. Nutrients for neuronal cells ("N medium") with 2% B27 based on Neurobasal, 200 mM L-glutamine, up to 10 mM glutamic acid and 1% penicillin and streptomycin, with a dependent glucose addition on the type of tissue from which the cells were isolated, usually at a final concentration of 12.5 mM with or without sodium pyruvate (usually up to a final concentration of 1 mM). Cells are seeded in 24-well culture plates coated with 200,000 cells / well poly-D-lysine or in 96-well plates coated with 30,000 cells / well with poly-D-lysine. Neural cultures (cultures of nerve cells or glial cells, or a mixture of both) or non-neural cells are incubated for 14-15 days typically in an atmosphere of 5% CO2 (95% air, 98% humidity, 37 ° C) or another.

The kit includes measurements of cell culture survival tests for various concentrations of the tested peptide and measurements of viability after the action of 6 damaging and / or neurotoxic factors.

The model includes parallel use in separate wells with cells derived from one isolation of strong damage factors such as acidification, i.e. acidosis (pH 6.45 and the action of sodium lactate at a concentration of 200 mM or another optimized for a given cell type), energy and respiratory deprivation (deprivation glucose and oxidative stress induced by sodium azide at a concentration of 10 mM or other optimized for the given cell type) and excitotoxicity (400 μM glutamic acid, 800 μM NMDA and 800 μM kainic acid or other concentrations optimized for the given cell type). The damage conditions are conducted in such a way that the potency of the damage factor causes a decrease of 30-40% of responses diagnosed with the CellTiterBlue test (CTB fluorophore in a modified MTT-type biochemical test) or another test for assessing cell viability based on a measurable assessment of metabolic functions of the mitochondria. . For the CTB assay, cells are incubated with the fluorescent agent CTB for 1-4 hours at 37 ° C (5% CO2). The fluorescence intensity of the CTB agent is measured spectrofluorimetrically (560ex / 590em) and is proportional to the number of viable cells. Control (untreated) cell samples account for 100% viability and signals from the remaining wells are referenced to control and presented as mean ± SD.

Control cultures are cultured without the addition of the test substance, and reference cultures are cultured with the addition of a reference substance, usually the RGD1 peptide at a concentration of 100 μΜ or other established for a given cell type.

Reagent literature entries for Example 15

1. Brewer GJ, Cotman CW., Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen. Brain Res. 1989,494 (1): 65-74.

2. Brewer GJ, Torricelli JR, Evege EK, Price PJ., Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. 1993; 35 (5): 567-76.

All chemicals used in the kit have at least the class 'clean for analysis' chemical purity.

Summary

The subject of the present invention is a method of efficient and multifactorial determination and analysis of the anti-damage and / or neuroprotective activity of compounds with potential pharmacological activity, as well as chemical and biotechnological synthesis of polyRGD peptides and RGD peptide analogs and demonstration of biological properties (anti-damage and / or neuroprotective) of these compounds after exposure to the agents. damage.

The obtained peptides showed stability under physiological conditions. The introduction of n-modification in the form of C-amidation and N-acetylation results in increased resistance of the compound to proteolysis, while the multiplication of the sequence of the RGD motif usually reduces its activity, however, allows for a prolonged release of the compound in the body. Both chemical synthesis on a solid support and biotechnological production ensure the purity, specificity and yield of the compounds obtained.

As described above, the RGD motif is an essential component of extracellular matrix proteins recognized by integrins and responsible, inter alia, for synaptic plasticity and the proper functioning of the CNS, and as such can be used as neuroprotectants. A peptide with a single RGD motif as well as polyRGD peptides and their analogs may prove to be an effective tool in the treatment of neurodamageous diseases in the face of the costly production of drugs based on the structure of ECM proteins. Damage to the nervous system, initiated by the mechanical disruption of blood vessels and the blood-brain barrier, leads to a number of metabolic and neurochemical disorders, the final result of which is excessive, self-stimulating excitability of neurons and prolonged inflammation. In the present invention, we propose a set of six such neurodamaging factors as an efficient method for effective screening for neuroprotective compounds and also for detailed analysis of biological activities of potential pharmacologically active injuries in the central nervous system at the cellular level. To demonstrate the neuroprotective properties of RGD peptides, primary neural cultures obtained from the cortex of Wistar rats were first exposed to lesions and then treated with the peptide. None of the tested peptides showed significant cytotoxic effects in the concentration ranges tested, as did the reference compound, which in this case was the TAT peptide. Moreover, the individual tested polyRGD peptides showed anti-damage and / or neuroprotective properties in relation to all or selected damage factors.

The RGD1 peptide showed the broadest spectrum of activity, which may be related to the increased availability of the active sequence, which in the case of multiplied analogs is released only after effective enzymatic hydrolysis. On the other hand, the use of peptides with repeated sequences can provide stability and controlled release over an extended period of time.

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As proved in the course of experiments, most polyRGD peptides have anti-damage and / or neuroprotective properties against the effects of damage caused by excitotoxicity. As mentioned, excitotoxicity occurs as a result of respiratory-energetic deprivation and dysregulation of the expression of integrin receptors in the plasma membrane. These observations were confirmed in an animal model of ischemic stroke. The anti-damage properties of polyRGD analogs are also evident in acidosis and oxidative stress. Nerve cells, and in particular neurons, are susceptible to excessive acidification of the environment caused by the presence of lactic acid, a metabolite of anaerobic respiration resulting from stroke. The pathological consequences of both dysregulation of cellular respiration and oxidative stress, which is a consequence of energy disturbances in the mitochondria, can be alleviated by the action of polyRGD peptides, because the above-mentioned processes are controlled, inter alia, by integrins modulated by other proteins containing in their sequence the RGD motif located in the plasma membrane, which in turn was confirmed in the rat model of Parkinson's disease. To verify RGD1 activity in an ex vivo excitotoxicity model, this peptide was applied to NMDA treated rat hippocampal sections. This experiment further demonstrated the anti-damage and / or neuroprotective properties of the RGD1 peptide.

Literature premises suggest that large proteins containing native RGD motifs (unlike in this application, in which we examine the RGD peptides themselves and their repeats and modifications) probably act through integrins (just activated via RGD sequences) present in dendritic spines during cytoskeleton reorganization in neurons hippocampus dependent on activation of NMDA receptors and calcium and calmodulin dependent kinase type II (CaMKII).

Claims (2)

Patent claims 1. A chemical compound with an anti-damage and / or neuroprotective effect, the core of which is the RGD sequence being the amino acid sequence: Arg-Gly-Asp, the compound having the following sequence: H2N-RGD-amide H2N-RGD-RGD-amide H2N-RGD-RGD-RGD-acetyl amide-RGD-RGD-RGD-amide H2N-RGD-RGD-RGD-RGD-amide H2N-RGD-RGD-RGD-RGD-RGD-amide H2N-RGD-RGD-RGD- RGD-RGD-RGD-amide H2N-RGD-RGD-RGD-RGD-RGD-RGD-RGD-amide H2N-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-amide H2N-RGD-RGD-RGD -RGD-RGD-RGD-RGD-RGD-RGD-amide H2N-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-RGD-amide or IMSRSSP-RGD-RGD-RGD-RGD-RGD-RGD RGD-RGD-RGD-RGD-RGD-PRRA. 2. A chemical compound as defined in claim 1 1 for use as an anti-damage and / or neuroprotective medicament.