Method of producing recombinant protein tat-hoxb4h for use as hemopoiesis stimulator in vivo

FIELD: chemistry.

SUBSTANCE: invention discloses a pharmaceutical composition which contains TAT-HOXB4 protein as an effective component. Said composition has stimulating effect on production of hematopoietic stem cells. More specifically, the recombinant protein TAT-HOXB4 enhances acceptance of intramedullary transplants, hematopoietic reconstruction, repopulation and number of circulating stem cells, specifically after chemotherapy or exposure.

EFFECT: higher protein output and stability.

24 cl, 11 dwg, 2 tbl, 9 ex

 

The present invention relates to a new and unobvious method for producing a labeled C-terminal histidine TAT-NOHW hybrid protein (TAT-NAHUN)containing at least 6 histidine residues at the C-end. The method of obtaining provides unexpected advantages in the form of higher stability and output, which provide for the successful function of this protein in vivo.

The growing interest in regenerative medicine is supported by finding of organ-specific stem or self-sustaining cells. The most studied self-sustaining population of cells is the hematopoietic stem cells (HSC), because they are an innovative means of treating diseases from cancer to metabolic disease and immunodeficiency.

The formation of blood cells, whereby erythrocytes and leukocytes are replaced by dividing found in the bone marrow HSC, called hematopoiesis. HSC have key abilities for self-maintenance and differentiation into Mature cells of both lymphoid. and the myeloid lineage. However, the genetic mechanisms responsible for the control over the results of self-maintenance and differentiation of HSC divisions, remain largely unknown.

Currently, transplantation of HSC from the bone marrow of an adult, mo is ilikuwa peripheral blood, and umbilical cord blood (UCB), used in clinical trials for treatment of hematopoietic cancers (leukemias and lymphomas). and in order to facilitate the recovery of the immune system after high-dose chemotherapy megamarketinc types of cancer. However, for effective transplantation need a significant amount of HSC from a variety of sources, and there may arise the need for expansion.

HSC can be taken from bone marrow, peripheral blood and UCB. Extraction of bone marrow cells is through surgery and is accompanied by painful sensations, which makes it less acceptable method. The use of peripheral blood cells is also a problem because of the difficulty of obtaining suitable HSC from hemopoiesis poses a risk to a patient suffering from a disease or chemotherapy. UCB relatively easier to obtain, and the quality of HSC much higher, but the quantity obtained with this method HSC is still limited. The amount of extracted every way cells is enough for the child, but may not be enough for an adult. To overcome this potential problem to the transplantation of HSC really need a new approach, which would contribute to proliferation of HSC in the laboratory by means of intervention in the process of self-maintenance of stem cells.

As mentioned, transcription factors play a key role in the regulation of gene expression and differentiation in stem cells (Orkin. S.H. Nature Reviews Genetics 1. 57-64, 2000). A transcription factor by binding to various cellular processes on a specific gene target, and this regulation also depends on its cellular concentration. Previously, it was found that the group of transcription factors called DNA-binding homeobox (NOH). plays a major role in embryogenesis. Recently it was discovered that the family of NOH is also involved in the development of HSC (S. Buske et, J. Hematol. 71, 301-308, 2000). Regulation of HSC self-sustaining through transcription factor NOH studied Dr. guy Sovago from the University of Montreal. Group Sovago proved that homeobox-containing gene NOHW extremely important for the regulation of HSC self-sustaining due to its ability to maintain populations of HSC in the bone marrow. First, genes NOH, expressed in blood cells, was observed in human cell lines and mouse. Some types of genes NOH ubiquitously expressed in various cell types, whereas other types are specifically expressed in certain cell types or at certain times during development. For example, eight members of the cluster of human NOHW expressed at an early stage of development of the erythrocyte. However, such genes NOHW. as NAHW and NAHW, also expressively in T-cells and b-cells. Group Sovago confirmed that nine genes of NOH. eight genes NAHW and four gene NOHS expressed in CD34+-bone marrow cells. Among these CD34+-bone marrow cells NOHW. NAHW and NOHA most enriched in cells-the precursors of erythrocytes. However, in CD34+cells no genes NOH is not expressed. Recently it was proved that homeobox containing a human gene B4 (NAHW) effectively increases HSC in the form of a retroviral or recombinant protein. Recombinant proteins TAT-NOHW was used to increase stem cells in laboratory scale without the risk of retroviral insertion or coculture with stromal cells in the bone marrow (see Krosl, J., and others, Nature Medicine 9, 1428-1432, 2003). Therefore, protein NAHW is commonly used as a stimulant expansion of HSC in vitro (see figure 1).

According to the latest data of exogenous NOHW can be delivered into the cell, adding the label to the TAT protein sequence to the N-end NOHW. This TAT-sequence reverses the direction of the transfer NOHW from extracellular to intracellular. After logging into the cytosol NOHW can refold into its native conformation with the chaperone HSP90. TAT-NOHW can stimulate 2-6x proliferatio HSC (Amsellem S. and others, Nature Medicine 9, 1423-1427, 2003; Krosl, J., and others, Nature Medicine 9, 1428-1432, 2003). However, the yield of recombinant protein TAT-NOHW of Ki is echnol coli (E. coli) using the usual technique of cleaning is too low.

In order to increase the yield of recombinant protein TAT-NOHW was developed a way to create protein TAT-NAHUN with an additional six histidine residues, marked With ' -end, which resulted in 3-to 4-fold increase in output compared to the original protein after purification. The resultant recombinant protein (TAT-NAHUN) contains 6 histidine residues at the C-end. This method was described in the application PCT/CN 2006/000646.

It is established that the recombinant protein TAT-NAHUN can be used for the expansion of stem cells in peripheral blood or UCB person, and an increased number of stem cells is still inherent in pluripotency. In addition, human stem cells treated with the recombinant protein TAT-NAHUN were introduced in the bone marrow of the patient with diabetes without obesity mice with severe combined immune deficiency (NOD-SCID), and human leukocytes were found in peripheral blood leukocytes, indicating the restoration of the mouse immune system and hematopoiesis.

However, recombinant proteins TAT-NAHUN never been used as a stimulator of hematopoiesis in vivo, namely, to enhance the recovery of hematopoiesis, expansion, repopulation of the bone marrow, as well as to increase the number of circulating stem cells in the peripheral blood, especially after chemotherapy liblucene. Krosl and others (2003) and Amsellem and others (2003) were not able to obtain large amounts of highly stable protein NOHW used in clinical studies for the expansion of HSC. In the present invention the total amount of protein TAT-NAHUN obtained after purification, in most cases ranges from 6-10 mg from 1 liter of culture, whereas the total protein TAT-NOHW obtained after purification with 1 liter of culture by the prior art typically ranges from 1-2 mg Plasmid of chat-ON-NOHW used for expression of the protein TAT-NAHW by prior art, was a gift from Dr. guy Sovago, University of Montreal, Canada. Method of purification protein TAT-NAHUN with the present invention clearly indicates the increase in the yield of protein required for appointment in vivo. Krosl and others (2003) also stated that most of their protein TAT-NOHW was lost after 4 hours incubation in medium with serum. The present invention shows a very high stability of the protein TAT-NAHUN even after 4 weeks, which is a key factor in the use of protein TAT-NOHW in clinical studies.

The present invention is based on a new and unobvious method of obtaining TAT-NAHUN protein with high yield and stability, as well as on the conclusion that if the appointment needs it the object of recombinant protein T IS T-NAHUN increases the number of HSC in the bone marrow, and in the peripheral blood in vivo.

One aspect of the invention relates to a method of obtaining protein TAT-NAHUN. The method includes: (a) obtaining a host cell containing a vector encoding the protein; (b) the expression of the protein in the cell is the owner; (C) the collection of the crude solution expressed protein; (d) purification of the protein from the solution through the following stages: (i) introducing the solution into HisTrap column; (ii) washing HisTrap column; (iii) elution partially purified protein from the HisTrap column in order to obtain a solution of partially purified protein; (iv) making a solution of partially purified protein in MonoSP column; (v) washing the column MonoSP; (vi) elution of purified protein from the column MonoSP in denatured form; (e) refolding the protein, lirovannomu in denaturirovannyj form, with the aid of hydrophobic compounds through the following stages: (i) the consolidation of lirovannomu denatured protein and a solution of hydrophobic compounds in order to obtain a solution containing a protein and a hydrophobic compounds; (ii) the demineralization solution containing a protein and a hydrophobic compound, to obtain a desalted solution; (iii) the removal of hydrophobic compounds from the desalted solution using ultrafiltration.

Another aspect of the invention relates to a method for amplification of activation in peripheral blood cells HSC from the bone marrow of the object. SPO is about includes: (a) appointment effective amount of the protein TAT-NOJUN, retrieved described in this document ways, needs it the object, and (b) the provision by protein TAT-NAHUN increase in the absolute number of hematopoietic stem cells in the bone marrow of the object to enhance the activation of hematopoietic stem cells in the peripheral blood of the object.

Another aspect of the invention relates to a method for reducing the recovery time of the patient undergoing HSC transplantation, radiation or chemotherapy. The method includes: (a) appointed effective amount of the protein TAT-NAHUN obtained are described in this document, means, needs it the object, and (b) the provision by protein TAT-NAHUN increase in the absolute number of HSC in the bone marrow of the object.

Another aspect of the invention relates to pharmaceutical compositions for increasing activation in peripheral blood cells HSC from the bone marrow in need of this object. The pharmaceutical composition according to the invention includes an effective amount received is described herein by way of protein TAT-NAHUN sufficient to increase the absolute number of HSC in the bone marrow of the object, which increases the activation of HSC cells in the peripheral blood of the object.

The pharmaceutical composition according to the invention it is possible to enter the patient undergoing autologous HSC, in aiming the reduction in recovery time after transplantation of HSC.

The pharmaceutical composition according to the invention can be immune to enter granulocyte colony-stimulating factor (G-CSF) to the patient instead of G-CSF for HSC activation in the peripheral blood.

The pharmaceutical composition according to the invention can be supplied by the donor HSC, thereby fence for transplantation of a sufficient number HSC much less invasive way of peripheral blood instead of bone marrow mentioned donor.

Another aspect of the invention relates to the treatment of diseases caused by hereditary deficiency of HSC, through a systematic technique for effective amount of recombinant protein TAT-NAHUN obtained are described in this document the methods or pharmaceutical compositions of the same composition suffering from disease of the object. Thus the introduced recombinant protein TAT-NAHUN increases the absolute number of HSC in the bone marrow of the object.

Another aspect of the invention relates to a method for reducing the recovery time after transplantation of HSC through systematic technique for effective amount of recombinant protein TAT-NAHUN obtained are described in this document the methods or pharmaceutical compositions of the same composition needy in this object.

Another aspect of the invention relates to a method of stimulation of no is of HSC last radiation or chemotherapy patient through a systematic technique for effective amount of recombinant protein TAT-NOJUN, retrieved described in this document the methods or pharmaceutical compositions of the same composition needy in this object.

The foregoing features and advantages of the present invention may become apparent from the following detailed description, accompanied by figures of graphic materials, which are presented next.

Figure 1 presents a schematic depiction of HSC activation of CD34 cells in the peripheral blood (PB) by expansion in vivo;

Figure 2 - schematic representation of the construction and cloning of RTT-NAHUN in a modified vector RET b;

Figure 3 illustrates the DNA sequence of RTT-NAHUN where additional 6 histidine residues introduced at the N - and C-ends of RTT-NAHUN are underlined, and TAT promoted to the foreground.

Figure 4 shows the protein sequence of the protein TAT-NAHUN.

Figure 5 illustrates the cleaning 10%SDS-polyacrylamide gel (1.5 mm) protein TAT-NAHUN. SDS-polyacrylamide gel was dyed Kumasi blue. The strip 1, molecular weight markers (M), 0.3 μg of protein; strip 2, cell lysate from reinducing cells BL21 (DE3) pLysS. expressing the protein TATE-NOJUN, 1 μg protein; strip 3, cell lysate from induced cells BL21 (DE3) pLysS expressing the protein TAT-NAHUN with 1 mmol IPTG, 1 μg protein; strip 4, purified TAT-NOJUN, 0.7 mg of protein; Oska 5, purified TAT-NAHW (0.2 mg protein). Plasmid of chat-ON-NOHW used for expression of the protein TAT-NAHW (strip of 5) was a gift from Dr. guy Sovago from the University of Montreal Canada. Equal volume fractions collected from MonoSP column, put in the strips 4 and 5.

6 illustrates SDS-polyacrylamide gel analysis of the stability of the purified protein TAT-NAHUN stored in phosphate buffered saline (SFR) for 0 h (a) and 16 h (In) at 4°C. M denotes molecular weight markers, a 0 indicates incubation for 0 hour at 4°C. 16 denotes incubation for 16 hours at 4°.

Fig.7 illustrates the stability of the protein TAT-NAHUN stored at 4°C and -20°C in STR and buffer storage. 1MDM analyzed on 10%SDS-polyacrylamide with staining of Kumasi. Arrows indicate bands of protein TAT-NAHUN.

Figa illustrates the stimulating effect of G-CSF on the number of stem cells CD34+in the bone marrow of mice by analysis on a flow cytometer.

Figv illustrates the impact SFR on the number of stem cells CD34+in the bone marrow of mice in the result of analysis on a flow cytometer.

Figs illustrates a stimulating effect on the number of stem cells CD34+in the bone marrow of mice by analysis on a flow cytometer.

Figa illustrates a stimulating effect on the number with Volovich cells CD34 +in the bone marrow of rhesus macaques analysis on a flow cytometer.

Figv illustrates the stimulating effect of TAT-NAHUN along with G-CSF on the number of stem cells CD34+in the bone marrow of rhesus macaques analysis on a flow cytometer.

Figs illustrates the stimulating effect of TAT-NAHUN on the number of stem cells CD34+in the bone marrow of rhesus macaques analysis on a flow cytometer.

Fig.9D illustrates the impact SFR on the number of stem cells CD34+in the bone marrow of rhesus macaques analysis on a flow cytometer.

Figure 10 illustrates the effect of protein TAT-NAHUN on hematopoietic recovery in mice NOD-SCID.

11 illustrates the effect of protein TAT-NAHUN on hematopoietic recovery in mice Balb/c mice after chemotherapy with cisplatin.

Next follows a detailed description.

I. Protein TAT-NAHUN

The present invention relates to a new and unobvious method for producing a protein TAT-NOHW with providing unexpected benefits of increased stability and output, which allows the assignment of this protein in vivo. Protein TAT-NAHUN is a construct that includes three elements: TAT, HOXB4 and his-tag tag. HOXB4 is a member of the family of NOH of transcription factors and promotes the expansion of HSC. TAT lets share HOXB4 be transported into the cell. His-tag tag accepts races is no initial increased output from recombinant sources of expression, although further method of obtaining increases the yield of protein. rtat-NAHUN constructed as shown in figure 2, and figure 3 presents the DNA sequence. Recombinant protein TAT-NAHUN is a hybrid protein TAT-NOHW with an additional six histidine residues marked on the s-end (see figure 4).

Unless otherwise indicated, the amino acid sequence of the protein (i.e., its "primary structure" or "native sequence") can be recorded with the amino end at the carboxy-end. In non-biological systems (for example, using solid phase synthesis), the primary structure of a protein (which also includes the sites of disulfide bonds (cysteine) links) can be defined by the user.

"Division" refers to the change in the amino acid or nucleotide sequences due to the absence of one or more amino acid residues or nucleotides. The terms "insertion" or "addition" refers to changes in the amino acid or nucleotide sequences, leading to the addition of one or more amino acid or nucleotide, respectively, and also to the molecule or to report this, compared with the standard sequence, for example, a sequence found in a naturally occurring molecule. "Substitution" refers to substitution of one or more amino acids of the Il is nucleotides by different amino acids or nucleotides, respectively.

Sequences similar or homologous (e.g., at least about 85%sequence identity) disclosed in this invention sequences are also related to this application. In some embodiments of the invention, the sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Conversely, the substantial identity exists when can hybridisierung segments of nucleic acid under the selected hybridization conditions (for example, a very stringent hybridization conditions) to the addition circuit. The nucleic acid may be present in whole cells, cell lysate, or in a partially purified or substantially pure form.

Calculations of "homology" or "sequence identity" between two sequences (the terms are used herein in an interchangeable manner) is made as follows. Sequence uravnivayutsya for optimal comparison purposes (for example, you can enter the gaps in one or both of the first and second amino acid or nucleotide sequences for optimal alignment and non-homologous sequences for comparison can be ignored). In a preferred embodiment, the length of the aligned for comparison a standard sequence with the hat at least 30%, preferably at least 40%, more preferably at least 50%, and still more preferably not less than 60%, and even more preferably not less 70%, 80%, 90%, 100% the length of the standard sequence. Then compare amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then in this position the molecules are identical (as used herein, an amino acid or Oleinikova "identity" is equivalent to amino acid or nucleinate "homology"). The percentage identity between the two sequences is a function of the number of partial sequences of identical positions with regard to the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

Comparison of sequences and determination of percent identity between two sequences can be accomplished through the use of a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined by using the algorithm of Needlman-In is : INAH ((1970) J. Mol. Biol. 48:444-453)included in the GAP program in the GCG software package (available at http://www.gcg.com) using either matrix Blossum 62 or matrix RAM and weight break 16, 14, 12, 10, 8, 6 or 4 and weight length 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the percent identity between two nucleotide sequences is determined by using the GAP program in the GCG software package (available at http://www.gcg.com) using matrix NWSgapdna, CMP and weight of the gap 40, 50, 60, 70 or 80, as well as weight, length 1, 2, 3, 4, 5 or 6. Particularly preferred set of parameters (and the one that can be used when uncertainty practitioner and in what settings could be applied to determine if a molecule is within a consistent identity or homology limitation of the invention) is estimated matrix Blossum 62 with a fine gap 12, a fine length of the gap 4, and a fine gap of the shear frame 5. The percentage identity between two amino acid or nucleotide sequences can also be determined by using the algorithm of E. Myers and U. Miller ((1989) CABIOS, 4:11-17), included in the ALIGN program (version 2.0) using a table of residue weight RAM, fine length of the gap 12 and the fine gap 4.

II. How to create recombinant protein TAT-NAHUN

A. It is onirovanie and expression

The prior art systems for cloning and protein expression in a variety of cells of the host. Suitable for the production of cell proteins are described, for example, in (Fernandez et al. Gene Expression Systems, Academic Press, eds., 1999). Briefly speaking, a suitable cell host include mammalian cells, insect cells, plant cells, yeast cells or prokaryotic cells such as E. coli. Known from the prior art mammalian cells for heterologous expression of protein include lymphocytic cell lines (e.g., NSO), cells NECK, ovarian cells of the Chinese hamster (Cho), COS cells, HeLa cells, kidney cells of baby hamsters, oocyte cells, and cells from a transgenic animal, for example breast epithelial cells. Suitable vectors can be chosen or designed to contain appropriate regulatory sequences, including stimulating sequence, the terminal sequences, polyadenylated sequences, enhancer sequences, gene markers and other sequences. The vectors may also include a plasmid or viral core. More detailed information can be found in (Sambrook el al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989). Many installed used with vectors of methods, including manipulation, preparation, mutage the ez, sequencing and transfection of DNA, described in Current Protocols in Molecular Biology, Second Edition, Ausubel el al. eds., John Wiley & Sons, 1992).

The following characteristic provides a method of introducing a nucleic acid into the cell host. For eukaryotic cells, suitable transfection method may include the use of calcium phosphate, DEAE-dextran, electroporation, transfection using liposomes and transduction using retrovirus or other virus, such as cowpox virus or baculovirus. For bacterial cells suitable technique may include the transformation of the calcium chloride, electroporation and transfection using bacteriophage. Introduction DNA may be accompanied by the method of selection (e.g., resistance to drug) for selection of cells containing the nucleic acid.

C. Purification and refolding

Protein TAT-NAHUN can initially be allocated from the recombinant host cell of any appropriate and well known from the field of technology tools. For example, the protein can be isolated from the cell supernatant, if the protein is capable of secreting or it can be isolated from cell lysate.

Protein TAT-NAHUN can be cleaned through the use of chromatographic methods, including: (a) the introduction of cellular lysate or cell supernatant, if the protein is secreted into the solution, HisTrap column; (b) p is mivka HisTrap column buffer; (C) elution partially purified protein from the HisTrap column; (d) payment received from the HisTrap column partially purified protein in MonoSP column; (e) rinsing MonoSP column buffer; (f) elution of purified protein TAT-NAHUN of MonoSP column.

Cellular lysate or cell supernatant, if a protein capable of secreting in solution, can be cleared by centrifugation at 20000×g for 30 minutes at 4°C, and the supernatant is adjusted to 10 mmol of imidazole and entered into chelat forming HisTrap column (Amersham Pharmacia), for example, Ni-separate as filler. The column can be washed with urea 8 mol, HEPES 20 mmol, DTT 0.5 mmol, NaCl 10 mmol pH of 0.8 and imidazole 10 mmol in order to remove unbound proteins. Partially purified protein TAT-NOHW can be eluted from the column HisTrap high concentration of imidazole and salts.

For further purification of the partially purified derived from the HisTrap column protein can be entered in MonoSP column (Amersham Pharmacia), for example, SP-separate as filler. The column can be washed with urea 4 mol, HEPES 20 mm, NaCl 50 mmol pH 6.5 in order to remove unbound proteins. Associated TAT-NAHUN can be eluted by high salt content. The information collected during this procedure, the purified protein TAT-NAHUN is in a denatured form.

Next, suirvey of MonoSP column denatured protein TAT-NAHUN can re-SVER is the folder with the use of hydrophobic compounds by: (i) the consolidation of lirovannomu denatured protein and a solution of hydrophobic compounds to form a solution, containing protein and hydrophobic compounds; (ii) the demineralization solution containing a protein and a hydrophobic compound, to obtain a desalted solution; and (iii) the removal of hydrophobic compounds from the desalted solution using ultrafiltration.

Used in the present invention, the term "hydrophobic compound" refers to any hydrophobic compounds that can protect the desired protein from the formation of insoluble compounds during removal of denatured salt. Suitable for use in the present invention, the hydrophobic compound is described in (Oganesyan et al. Suitable considered are hydrophobic compounds include, but are not limited to, Triton X-100, tween-20 or polybenzene compounds. Pharmagenomics (2004) 71, 22-26). The ultrafiltration or buffer exchange can be accomplished using centricon or hub stir-cell. Conditions for ultrafiltration or buffer exchange can vary by specialists in this field, depending on the types of the desired protein.

In one embodiment of the present invention the hydrophobic compound in demineralized containing denatured protein NAHUN solution is removed by 5-10-fold buffer exchange (each manufactured by centrifugation at 1000 to 2500×g for 10 minutes) with a solution containing large hydrophobic compounds in concentrations from low to high, for example beta cycle is dextrin, where denatured protein NAHUN subjected to refolding in the corresponding native form.

In one embodiment, the purified protein TAT-NAHUN can be stored in a commercially available medium IMDM (Wednesday Dulbecco modification Iscove; HyClone; buffer storage 1) at 4°C or -20°C.

In another embodiment, the purified protein TAT-NAHUN can be stored in a commercially available medium DMEM (Wednesday Needle in the modification Dulbecco; HyClone; buffer storage 2) at 4°C or -20°C.

In yet another embodiment, the label on His To-end protein TAT-NAHUN can be removed prior to the appointment in vivo.

In another embodiment, the label on His N-end protein TA-T-NAHUN can be removed prior to the appointment in vivo.

In yet another embodiment, both His label at the N - and C-ends can be removed prior to the appointment in vivo.

C. preparation of a pharmaceutical composition

Protein TAT-NAHUN can be used as pharmaceutical compositions in combination with an acceptable carrier with a pharmaceutical point of view. This composition, in addition to protein TAT-NAHUN and media that may contain various diluents, fillers, salts, buffers, stabilizers, sigalda agents and other well-known from the prior art substances. The term "acceptable from a pharmaceutical point of view" means a non-toxic substance that does not impede the effectiveness of the biological the banking activity of the active(s) component(s). The characteristics of the media may vary depending on how the destination.

Especially, it is preferable to prepare the composition in the form of dosage units for simplicity and uniformity of dosage. Used in this description of the dosage form unit refers to physically discrete units suitable as a single dose for treatment of the object;

each unit contains a predetermined quantity of active compound calculated to achieve the desired therapeutic effect in combination with the required pharmaceutical carrier. Requirements for the form of dosage units of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular achieved therapeutic effect, but also from the limitations of the prior art, for example, active compounds for the treatment of humans.

Typical routes of administration include, without limitation, oral, local, parenteral (e.g., sublingual or transbukkalno), sublingual, rectal, vaginal, and intranasal. Used in this description, the term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, vnutriobolochechnoe, intermetallide, intraurethral methods the s injection or infusion. The pharmaceutical composition is selected so that it contains active components could be in a biologically available form in the introduction of this composition to the patient. Enter the patient's compositions take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the present invention in the form of an aerosol can contain multiple dosage units.

When administered orally effective therapeutic standpoint protein TAT-NOJUN, the binder may be in the form of tablets, capsules, powder, solution or elixir. With the introduction in the form of tablets pharmaceutical composition may additionally contain a solid carrier type of gelatin or adjuvant. In tablet, capsule, and powder contain about 5-95% of binder, and preferably about 25-90% of the binder. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethylcellulose. May be present colouring and/or flavouring agents. Can be used sheath-floor. When introduced in liquid form can be added to a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut Mac is on, mineral oil, soybean oil, or sesame oil, and synthetic oil. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, and glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When introduced in liquid form, the pharmaceutical composition contains about 0.5-90% by weight of binder, and preferably about 1-50% of the binder.

For intravenous, cutaneous or subcutaneous injection effective therapeutic point of view, the number of protein TAT-NOJUN, the binder may be in the form of a parenterally acceptable aqueous solution without pyrogen. Duly given pH, the preparation of such parenterally acceptable protein solutions, isotonicity, stability and the like is in the range known from the technical field. In some embodiments, the implementation of a pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection may include, in addition to the binding agent, isotonic agent, as for example, injection of sodium chloride, injection of ringer's solution. injection, dextrose and sodium chloride injections Laktionova of ringer's solution or other known from the prior art tools. The pharmaceutical composition according to the present invented the Yu may also contain stabilizers, preservatives, buffers, antioxidants, or other well-known to experts in the field of technology Supplement.

When applying the method of treatment or use of the present invention, an effective therapeutic point of view, the amount of protein TAT-NAHUN is inserted object, such as a mammal (e.g. human). Used in this description, the term "effective therapeutic point of view, the amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to demonstrate a compelling benefit to the patient, for example the reduction in intensity of symptoms, a cure, or an increase in the degree of cure under such conditions. When applied to an individual, only one input active component, this term refers only to this one component. When applied to a combination, the term refers to combined amounts of the active components, leading to a therapeutic effect, regardless of the combined, sequential or simultaneous administration.

The amount of protein TAT-NAHUN in the pharmaceutical compositions of the present invention may depend on the nature and severity of the condition, which is being treated and on the nature of the earlier of the last patient treatment, age and sex of the patient. In the end, the, the doctor can determine the number of active component for the treatment of each individual patient. Initially, the attending physician may enter a low dose of the active component and to observe the response reaktsii patient. Higher doses of the active ingredient you can enter up until the patient will not achieve an optimal therapeutic effect, and at this point the dosage is not increased. It is assumed that different used for the application of the method according to the present invention the pharmaceutical composition may contain from about 1 μg to 1 mg protein TAT-NAHUN per kilogram of body weight. Examples of dosage ranges that you can enter the object, you can choose from the following: 1 μg/kg to 1 mg/kg, 1 μg/kg to 0.5 mg/kg, 1 μg/kg to 0.1 mg/kg, 10 mg/kg 0.5 mg/kg, 10 μg/kg to 0.1 mg/kg, 100 µg 0.5 mg/kg, 250 μg/kg, 0.5 mg/kg Hereinafter, examples of dosage ranges that you can choose from 50 mg to 100 mg, 100 µg 50 mg, 500 μg to 50 mg, 1 mg - 50 mg Duration of intravenous therapy using the pharmaceutical compositions of the present invention may vary depending on the severity of the disease, condition and potential of a specific reaction of each individual patient. In one embodiment, it is assumed that the duration of each application of protein TAT-NAHUN can be in the range is from 12 to 24 hours of continuous intravenous administration. In another implementation, the duration of protein TAT-NAHUN can last as long as you continue a course of radiation therapy or chemotherapy patient. Protein TAT-NOHW you can enter in the range of 10-100 µg/kg intravenously twice a day from 4.5 to 5 days. One cycle of treatment may be sufficient to increase HSC in vivo. Ultimately, the attending physician can determine the appropriate duration of intravenous therapy using the pharmaceutical compositions of the present invention.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50(lethal dose for 50% of the population) and ED50(effective dose from medical point of view at 50% of the population). The dose ratio between toxic and therapeutic effects is a healing factor, which can be expressed as the ratio LD50/ED50. Obtained from analysis of cell culture and animal studies can be used in the assessment of a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that includes the ED50little toxicity or without is the th. The dosage may vary within this range depending on the dosage form and route of administration. Effective therapeutic point of view, the dose of the protein TAT-NAHUN can initially be assessed on the basis of the analysis of cell culture. The dose can be determined in animal models to achieve a range of concentrations of circulating plasma, including IC50(i.e. the concentration of the test protein, which reaches premaxillae inhibition of symptoms)as determined in cell culture. Levels in plasma may be measured, for example, liquid chromatography high resolution. The effect of any given dosage can be controlled acceptable biological analysis. Examples of acceptable biological assays include, but are not limited to, measurement of stem cells CD34+in mononuclear cells by using antibodies with a fluorescent label (e.g., FITC) to stem cells CD34+measurement of the percentage of LY5 cells in the peripheral blood or bone marrow HSC flow cytometry. It is assumed that polynucleotides and proteins of the present invention will demonstrate one or more of the manifestations of the following biological activities (including those associated with outlined in this description of the analysis). Described in relation to protein p. the present invention manifestation or activity will be ensured through the assignment or use of such proteins, and through the assignment or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or acceptable for introduction of DNA vectors).

III. Ways to stimulate hematopoiesis in vivo

A. Patients who need treatment

The pharmaceutical composition according to the invention can be used for the treatment of diseases, which include, but are not limited to, autoimmune disorders, immune deficiency, and hematologic disorders. In addition, the pharmaceutical compositions according to the invention can be used to improve the rehabilitation period after transplantation of hematopoietic stem cells.

The pharmaceutical composition according to the invention includes, but is not limited to, the treatment of patients suffering from or susceptible to lymphoma, leukemia, Hodgkin's disease and myeloproliferative violations. In addition, hereditary diseases caused by deficiency of hematopoietic stem cells and gipoplasticheskaya anemia can be treated by the pharmaceutical composition of the present invention.

Further, the pharmaceutical composition according to the present invention can be assigned to a donor hematopoietic stem cells and patients with immune granulocyte colony-stimulating factor.

In one embodiment of the invention white is TAT-NAHUN is the only active substance, input for activation of hematopoietic stem cells, and fluorouracil inside the body (5-FU) is not introduced to the donor, whether in the form of a scheme of preliminary treatment or combination therapy.

Associated with the increased survival of cells additional diseases or conditions that can be treated by the pharmaceutical compositions according to the invention, include, but are not limited to, progression and/or metastasis of malignancy and is associated with conditions such as leukemia (including acute leukemias (such as acute lymphoblastic leukemia, acute myeloblastic leukemia (including myeloblastic, promyelocytic, monocytic leukemia, and erythromelas)and chronic leukemias (such as chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), myelodysplastic syndrome true polycythemia, lymphoma (e.g., Hodgkin's disease and the disease is not-Hodgkin's lymphoma), mnozhestvennaya myeloma, waldenstrom's disease (macroglobulinemia), as well as tumors, including, but not limited to, sarcomas and carcinomas type fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, ehotelier.com, lymphangiosarcoma, lymphangiosarcoma, synovioma, Ewing tumors, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma cancer, pancreatic cancer, breast cancer, the aka the ovaries, prostate cancer, squamous cell cancer, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, carcinoma of the sebaceous glands, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchogenic cancer, kidney cancer, hepatoma, carcinoma of the bile ducts, choriocarcinoma, seminolcasinos, embryonal cancer, Wilms tumor, cervical cancer, testicular tumor, squamous cell lung cancer, bladder cancer, epithelial cancer, gliomas, astrocytomas, medulloblastomas, craniopharyngioma, ependymomas, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrogliomas, meningioma, melanoma, neuroblastoma, and retinoblastoma.

The present invention is not limited to the particular methodology, protocols, cell lines, species or genera of animals, as well as the following reagents. Used to describe specific accomplishments terminology is not intended to limit the scope of the present invention, which may be limited by the attached claims. Used in this description forms the singular include references to the plural, unless the context clearly indicated otherwise. Thus, for example, reference to "a cell" is a reference to one or more cells, and provides for the matter of well-known experts in this field technique equivalents.

Unless otherwise specified, all materials used in this description of the technical and scientific terms have the same value, which is usually understood by the average person skilled in the art to which this invention relates. Although any similar or equivalent to those described in this invention, the methods, devices, and substances can be used in practice or testing of the invention, there is described the preferred methods, devices, and substances. Everything mentioned in the description of the publication and patents are hereby incorporated into this invention by reference for purposes of describing and disclosing, for example, described in the publications of interpretation and methodology that could be used in connection with the present described invention. The above and found throughout the text of the publication is provided solely for the purpose of disclosure to the emergence of legal protection of this application. Nothing described in this invention should not be construed as an admission that the inventors are not entitled to use prior to the disclosure of the invention in effect prior to the invention.

Definition

"A stem cell is a pluripotent or multipotent cell with the ability to renew themselves, to remain undifferentiated and become differentiated. Stem cells mo the ut share without restriction, at least during the life of the animal in which they are located. Stem cells are not terminal differentiated, i.e. they are not at the end of the path of differentiation. When dividing stem cells, each daughter cell can remain a stem cell, or it may go the way of terminal differentiation. "Chimeric" a stem cell is a stem cell with a part of his DNA, owned by heterologous organism.

"Hematopoietic" cell is a cell involved in the process of hematopoiesis, i.e. in the formation of Mature blood cells from precursor cells. The adult hematopoi occurs in the bone marrow. In the early development of hematomas happening in different areas during different stages of development; primitive blood cells appear in the yolk SAC, and subsequently, blood cells are produced in the liver, spleen and bone marrow. Hematopoi subjected to comprehensive regulation, including regulation by hormones, such as erythropoietin; growth factors such as colony-stimulating factors, and cytokines. for example interleukins.

Used in this description, the term "vector" is intended to refer to the nucleic acid molecule capable of transporting another nucleic acid to which it is associated. One type of vector is a "plasmas is Yes" which refers to a loop of circular double-stranded DNA, which can be ligitamate additional segments of DNA. Another type of vector is a viral vector, into which additional DNA segments can be ligitamate in the viral genome. Certain vectors are capable of Autonomous replication in a cell host, in which they are introduced (e.g., bacterial vectors with bacterial origin of replication and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) can be integrated into the genome of a host cell upon introduction into the cell of the host, and thus, replicating along with the genome of the host. Moreover, certain vectors are capable of directing the expression of genes with which they are effectively connected. Such vectors are referred to as "recombinant expression vectors" (or simply, "expression vectors"). In General, expression vectors of utility in recombinant methods DNA are often in the form of plasmids. In the present description of the invention "plasmid" and "vector" can be used in an interchangeable manner, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of ex-pressione vectors, such as viral vectors (e.g., defective retro virus replication, adenovirus and the Deno-associated viruses), which serve equivalent functions.

Used in this description, the term "transformation" refers to the insertion of exogenous polynucleotide in a cage-the owner, regardless for the insertion of ways: for example, transformation by direct uptake, transfection, infection, etc. Specific ways transfection see below. Exogenous polynucleotide can be saved as reintegrating vector, such as episome, or Vice versa, can be integrated into the host genome.

In General, the term "protein" refers to any polymer of two or more individual amino acids (naturally occurring or not) through a peptide bond, what happens when associated with the α-carbon of one amino acid (or amino acid residue) carbon atom of carboxyl group of the carboxylic acid becomes covalently bound to the atom of aminatta amino groups bound to the α-carbon of the adjacent amino acids. These peptide bonds involving atoms (i.e. atoms of the α-carbon, the carbon atoms of carboxyl (and their replacement oxygen atoms), and the atoms of aminatta (and replacing them with hydrogen atoms)) form a "polypeptide backbone" of the protein. In addition, as used in this description, the term "protein" is understood to include the terms "polypeptide" and "peptide" (which occasionally can be used in mutual the replacement procedure in this description). Similarly, protein fragments, analogs, derivatives and options, can be referred to as "proteins" and think "protein", unless otherwise specified. The term "fragment" of a protein refers to a polypeptide that includes less than all of the amino acid residues of the protein. According to "fragment" of a protein can be a form of protein, truncated on aminocore, carboxylic and/or internally (such as natural splicing), and may also be a variant and/or derivative. "Domain" of a protein is a fragment, which includes amino acid residues of the protein, which requires giving the biochemical activity of the corresponding naturally occurring protein.

Used to describe nucleic acid molecules, the term "re-combinatii" means polynucleotide genomic viral cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or actions not associated with all polynucleotides or part of it with which is associated in nature. Used in relation to a protein or polypeptide, the term "recombinant" means a polypeptide obtained by expression of recombinant polynucleotide.

"Isolated", "purified", "substantially isolated" or "substantially pure" molecule (such as a polypeptide or polynucleotide) is the molecule that was affected so that it prisutstvie the shaft in a higher concentration, than in nature. For example, the object protein isolated, purified, substantially isolated or substantially purified when removed not less than 50%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more substances with neobyachnym protein with which it is associated in nature. Used in this description of "isolated", "purified", "substantially isolated" or "substantially pure" molecule includes a recombinant molecule.

Mouse "SCID" is a mouse model for severe combined syndrome immunodeficita (SCID), which causes severe disorders in the development of the immune system. In these mice, there is an insufficient number of T - and b-lymphocytes, or they are completely absent. The SCID mutation impairs recombination of antigen receptor genes, thereby causing a lack of functional T - and b-lymphocytes. Other types of hematopoietic cells develop and function normally. Mouse SCID willingly support normal lymphocyte differentiation and can be reconstituted with normal lymphocytes from isogenic or allogeneic mice, or human lymphocytes. These mice also support the growth of allogeneic and xenogeneic tumors. Therefore, SCID mice, which allow disseminated growth of several human tumors, in particular, hematologic disorders, and malignant melanomas, can be used to research the development of malignancy.

The terms "object", "person", "owner" and "patient" are used herein in an interchangeable manner to send live specimens, including human and non-human animals. The object, for example, can be the body with immune cells that are able to respond to antigenic stimulation and stimulating inhibitory signal transduction through binding to surface-cell receptor. The object can be a mammal, the mammal is a human or mammal is a non-man, for example dogs, cats, pigs, cows, sheep, goats, horses, rats and mice. The term "object" does not exclude individuals who are completely normal in relation to disease or normal in all respects.

The term "treatment" refers to therapeutic or preventive measure. You can assign a treatment object, which suffers from disease or, ultimately, can be ill in order to prevent, cure, delay, reduce the severity, as well as reducing the intensity of one or more symptoms of the disease or recurrent disease, or in order to prolong the survival of the object beyond the expected in the absence of such treatment.

The term "effective therapeutic point of view, the amount" means the amount of the test compound, which is able to detect the desired reaction, for example, biological or medical reaction is human tissue, system, animal or human, which, for example, a researcher, veterinarian, medical doctor or other Clinician.

Examples

The following examples should in any case be considered merely as illustrative, and not limiting of the remaining part of the description. Without further elaboration, it is considered that specialist in this area of engineering on the basis of this description may use the present invention in full.

Example 1: Construction of the plasmid pET21b-His-pTAT-HOXB4-His

(a) Modification of the plasmid pET21b containing signal peptide TAT and His label with N - and C-end

The expression vector pET21b containing the label with His M-end and a TAT signal peptide was obtained by introducing oligonucleotides 5'T-TATGCACCACCACCACCACCACTACGGCCGCAAGAAACGCCGCCAGCGCC GCCGGCG-3' (sense) and 5'-CTAGCGGCGCTGGCGGCGTTTCTTGCGGCC GTAGTGGTGGTGGTGGTGGTGCA-3' (antimal) in plasmid pET21b. Label with His From-the end is already present in the vector pET21b.

(b) Cloning NOHW in the modified expression vector pET21b

The DNA fragment containing the open reading frame (ORF) HOXB4 and additionally 6 his-tag coding sequence was obtained by PCR amplification (PRC - polymerase chain reaction) using plasmids MGC54130 (GeneDiscovery, Taipei, Taiwan, catalog number 5533346) as a matrix, and a fragment of HOXB4 cDNA obtained by PCR, was subcloning the n in the modified expression vector pET21b. Construction of plasmids and nonlegislative sequence shown in figure 2 and 3.

Example 2: Expression of recombinant protein TAT-NAHUN in E. coli

The expression vector pET21b-His-TAT-HOXB4-His was transformed into E. coli strain BL21 (DE3) pLysS (Novagen). Transformed cells were grown overnight at 37°C. cultures were diluted to an initial OD600 of 0.05 in. Then the cultures were grown to OD600 of 0.5 at 37°C with induction with 1 mm isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37°C for 3 hours with vigorous shaking.

Example 3: Purification of recombinant TAT protein-NAHUN

After induction, cells were collected by centrifugation and re-suspended in buffer A (8 mol of urea, 20 mmol HEPES, 0.5 mmol DTT and 100 mmol NaCl, pH 8.0). The cell suspension was performed three times through a French press cell lysate was cleared by centrifugation at 20000×g for 30 minutes at 4°C, supernatant was adjusted with imidazole 10 mmol and was placed in hepatoblastoma HisTrap column (Amersham Pharmacia). Associated proteins were suirable the imidazole 50, 100 and 250 mmol in buffer A. Fractions with protein TAT-NAHUN put in MonoSP column buffer (urea 4 mol, HEPES 20 mmol NaCl and 50 mmol, pH 6.5) and was suirable NaCl 1.5 mol and HEPES 20 mm (pH 8.0).

Example 4: Resaturate recombinant protein TAT-NAHUN

Protein TAT-NAHUN in buervenich fractions was solubilizer is n and are denatured in solution, containing denatured salt (e.g., guanidine hydrochloride), and then was mixed with buffer (D-PBS-T (1% Triton X-100 in 2x PBS). The ratio of protein solution TAT-NAHUN to the buffer (D-PBS-T was 1:4. The resulting mixture was added into a test tube filter hub "Centricon 10K (50 ml or 15 ml), pretreated with water (10 ml or 3 ml), and then centrifuged at 3000 rpm for 10 minutes At this stage denatured salt is replaced by buffer (D-PBS-T, in which Triton X-100 is able to communicate with the hydrophobic region of the protein NAHUN.

This stage ultrafiltration or buffer exchange using "Centricon 10K was performed ten times with a solution containing various concentrations of beta-cyclodextrin (twice every 1 mmol, 2 mmol, 3 mmol, 4 mmol) and 5 mmol of beta-cyclodextrin in buffer storage IMDM by centrifugation at 1000 to 2500×g for 10 min). Remaining in the tube-filter "Centricon 10K sample was collected and stored at -80°C.

The homogeneity of the purified protein TAT-NAHUN was analyzed on SDS-polyacrylamide gel, after spent painting Kumasi (see figure 5). As shown in figure 5, purified using HisTrap columns and MonoSP protein TAT-NAHUN was obtained from 3 - to 4-fold output compared with the same protein TAT-NOHW. Plasmid of chat-ON-NOHW was a gift from Dr. guy Sovago of MES is alsogo University of Canada. This plasmid was transformed into BL21 (DE3) pLysS (Novagen), and protein purification of TAT-NOHW was produced in accordance with the description in (Krosl et al, 2003).

Example 5: the Stability of recombinant protein TAT-NAHUN

The stability of the protein TAT-NAHUN was measured by analysis with SDS-polyacrylamide gel. After storage protein TAT-NAHUN can be degraded into fragments of 30 KD and 10 KD. As shown in Fig.6, obtained by the method according to this invention, the protein TAT-NAHUN was stable even after 16 hours when stored in phosphate buffered saline at 4°C.

Further, the stability stored at 4°C and -20°C in phosphate buffered saline protein TAT-NAHUN was analyzed by electrophoresis on 10%polyacrylamide gel, after spent painting Kumasi. As shown in Fig.7, when using IMDM as buffer storage, the stability of the protein TAT-NAHUN remained even after 4 weeks.

Example 6: Effect of recombinant TAT protein-NAHUN on hematopoesis in wild type mice Balb/c

Mouse Balb/c mice were used to study the potential impact of recombinant protein TAT-NAHUN activation in peripheral blood HSC from the bone marrow. Recombinant protein TAT-NAHUN in SFR was injected subcutaneously four times a day for 4 days. In order to study the response to the dose of the experimental group (n=21) had a dose in the range from 1 μg, 5 mcg, 10 mcg, 15 mcg ... to 100 μg per kg of body weight of the mouse. A separate group of control mice received only phosphate buffered saline, and the other group of control mice were injected subcutaneously twice daily for 4 days, the dose of 5 μg per 1 kg of G-CSF/mouse.

All mice took peripheral blood and analyzed on a flow cytometer to determine the content of stem cells CD34+in the mononuclear cell (MNC). The results are presented in table 1 as mean value ± standard deviation.

Table
Group (experimental / control)Protein TAT-NAHUN (ug/kg)CD34+/MNC (%)
110±0,03
250,3±0,05
3100,45±0,03
4150,42±0,01
5200,38±0,05
625 0,41±0,02
7300,35±0,21
8350,33±0,11
9400,29±0,16
10450,46±0,01
11500,45±0,02
12550,42±0,06
13600,44±0,02
14650,41±0,04
15700,41±0,03
16750,49±0,01
17800,45±0,04
18850,46±0,07
19900,44±0,02
20 950,41±0,01
211000,42±0,05
Control (SFR)00,002
Control (G-CSF)00,5±0,03

The percentage of CD34+/MNC in the peripheral blood taken from treated mice are shown in table 1. Protein TAT-NOJUN, which was treated experimental group 3-21 (received a dose of 10 mg or higher per kg of weight), showed almost the same action as in the control group, which was treated with G-CSF.

Bone marrow from mice treated with TAT-NAHUN (experimental group 3 received a dose of 10 mcg per kg of body weight), mice treated with G-CSF, as well as mice, which were injected SFR, was further phenotypicaly using antibodies labeled fluoresceinisothiocyanate (FITZ) to CD34+(Becton Dickinson), and analyzed on a flow cytometer. Bone marrow from mice treated with TAT-NAHUN (see figs), was more rich in stem cells CD34+than bone marrow from mice that were injected G-CSF (see figa) and SFR (see figv). Therefore, these results indicate that the introduction of recombinant TAT protein-NAHUN leads to an increased number SC and in the bone marrow, and in the peripheral blood in mice.

Example 7: Effect of recombinant TAT protein-NAHUN on hematopoesis rhesus macaques

To study the efficiency of recombinant protein TAT-NAHUN the monkeys were used adult male rhesus macaques. Experimental group I (n=5) was injected intravenously 10 μg per 1 kg of body weight of recombinant protein TAT-NAHUN four times a day for 4 days. Experimental group II (n=5) was injected intravenously 10 μg per 1 kg of body weight of recombinant protein TAT-NAHUN four times per day) was subcutaneously injected with 5 μg per 1 kg of G-CSF for 4 days. The control group received only SFR, and control group II were injected subcutaneously twice daily for 4 days, the dose of 5 μg per 1 kg of G-CSF. All monkeys blood samples were taken and analyzed on a flow cytometer to obtain the percentage of stem cells CD34+in the mononuclear cell (MNC). The results are presented in table 2.

Table 2
Group (experimental / control)CD34+/MNC (%)
I. TAT-NAHUN (10 µg/kg)0,62
II. TAT-NAHUN (10 mg /kg) + G-CSF (5 µg /kg)0,38
Control 1 (SFR)0,07
Control 2 (G-CSF g, 5 mg /kg)0,28

As shown in table 2, monkey (experimental group I), processed protein TAT-NOJUN, showed a much better activated impact than monkeys treated with G-CSF (control group II). Monkeys treated with TAT protein-NAHUN and G-CSF (experimental group)showed slightly better impact than monkeys treated with G-CSF (control group II).

Samples of bone marrow from monkeys treated with TAT-NOJUN, G-CSF or SFR were then phenotypically using antibodies labeled fluoresceinisothiocyanate (FITZ) to CD34+(Becton Dickinson), and analyzed on a flow cytometer. Samples of bone marrow from monkeys treated with TAT-NAHUN (see figs), were more rich in stem cells CD34+than samples of bone marrow from monkeys, which was administered G-CSF (see figa), TAT-NAHUN+G-CSF (see figv) and SFR (see fig.9D).

Example 8: Effect of recombinant TAT protein-NAHUN on hematopoietic recovery in mice NOD-SCID

Human cells 104Lin-/CD34+were introduced irradiated (2,5 Gy) mice NOD-LtSz-scid/scid (NOD-SCID) as well As cells 105CD34. Mice were randomly allocated into two GRU the groups: one group (n=28) was injected intravenously 10 μg per 1 kg of body weight of recombinant protein TAT-NAHUN twice a day, and the other (n=28) received SR twice a day. The presence of human CD45 cells+in peripheral blood cells of all mice were periodically measured by flow cytometry after transplantation. Hematopoietic recovery was evaluated by the number of mice, human cells CD45+, which reached a level of > 0.1% in peripheral blood (PC) after transplantation. As shown in figure 10, enhanced hematopoietic recovery was observed in mice which were injected with recombinant TAT protein-NAHUN.

Example 9: Effect of recombinant TAT protein-NAHUN on gammapathies recovery in Balb/c mice after chemotherapy with cisplatin

Mice five weeks of age, Balb/c repeatedly intravenously injected cisplatin, while the number of Ly5 cells (murine CD45) in the peripheral blood of mice has not decreased to approximately 10% of the original amount. Treated with cisplatin mice were randomly allocated into two groups: one group (n=28) was injected intravenously 10 μg per 1 kg of body weight of recombinant protein TAT-NAHUN twice a day, and the other (n=28) twice a day received SFR. The presence of Ly5 cells in the peripheral blood cells of all mice were periodically measured by flow cytometry after transplantation. The degree of hematopoietic recovery was estimated as a percentage of the number of Ly5 cells is peripheral blood to the original number. As shown in figure 11, enhanced hematopoietic recovery was observed in mice which were injected with recombinant TAT protein-NAHUN.

Used in these experiments, animal models recognized in the field of engineering as a predictive results that will be obtained on the patient-the person (see, for example, Broxmeyer et al. (2005) The Journal of Experimental Medicine, 201, 1207-1318: Larochelle et al. (2006) Blood 107, 3772-3778).

Although the invention is explained various options for implementation, it should be understood that it is possible to perform many other possible modifications and variants, without leaving the substance and the scope of protection of the invention.

1. The method of obtaining protein TAT-NOJUN, comprising the following stages:
(a) obtaining a host cell E. coli, transformed with the expression vector containing the DNA fragments coding sequence for the signal peptide TAT, His label-end and open-reading frames NOHW;
(b) expression of recombinant protein TAT-NAHUN in the cell host E.coli;
(c) lysis of the cells by passing through a French press and centrifuged to collect the crude solution expressed protein;
(d) purification of the protein from solution by the following steps:
(i) the introduction of the solution into the column HisTrap;
(ii) rinsing HisTrap column washing buffer containing urea 8 mol, HEPES 20 mmol, DTT 0.5 mmol, NaCl 100 mmol, pH 8.0 and the imide is angry 10 mmol;
(iii) chromatography of partially purified protein from the HisTrap column using an eluting buffer containing 50-250 mmol of imidazole, 8 mol of urea, 20 mmol HEPES, 0.5 mmol DTT, 100 mmol NaCl at pH 8.0 to obtain a partially purified protein solution;
(iv) the introduction of partially purified protein solution in MonoSP column;
(v) rinsing MonoSP column washing buffer containing urea 4 mol, HEPES 20 mmol NaCl and 50 mmol at pH 6.5;
(vi) elution of purified protein from the column MonoSP in denatured form using an eluting buffer containing NaCl, 1.5 mol and HEPES 20 mmol, pH 8,0;
(e) refolding the protein, lirovannomu in denatured form, using hydrophobic compounds, such as Triton X-100, Tween-20 or polyanaline connection due to the following stages:
(i) solubilization and protein denaturing, lirovannomu in denatured form, in the solution containing salt type, guanidine hydrochloride;
(ii) the consolidation of solubilizing denatured protein and a solution of hydrophobic compounds to obtain a solution containing a protein and a hydrophobic compounds;
(iii) desalting of the solution containing the protein and hydrophobic compounds to obtain a desalted solution;
(iv) removal of hydrophobic compounds from the desalted solution by ultrafiltration with the use of a solution containing C is CODEXTER.

2. The method according to claim 1, characterized in that the partially purified protein from the HisTrap column elute eluting buffer solution containing imidazole 50-100 mmol.

3. The method according to claim 1, characterized in that the partially purified protein from the HisTrap column elute eluting buffer solution containing imidazole 100-250 mmol.

4. The method according to claim 1, characterized in that the ultrafiltration is performed with the use of the tube-filter concentrator centricon or hub stir-cell.

5. The method according to claim 1, characterized in that the purified protein is stored in IMDM or DMEM.

6. The method according to claim 1, characterized in that mark His s-end of the protein TAT-NAHUN removed.

7. Pharmaceutical composition for increasing the absolute number of cells HSC in the bone marrow object containing an effective amount of the protein TAT-NAHUN obtained by the method according to claim 1, and a pharmaceutically acceptable carrier.

8. The composition according to claim 7, characterized in that the object is a donor HSC.

9. The composition according to claim 7, characterized in that the object is a patient, subjected to autologous transplantation of HSC cells.

10. The composition according to claim 7, characterized in that the object is a patient unresponsive to G-CSF.

11. The composition according to claim 10, characterized in that the object suffers from a disease caused by a hereditary deficiency of HSC cells.

12. The composition according to claim 11, Otley is audacia fact, the object suffers from hereditary gipoplasticheskaya anemia.

13. The composition according to claim 7, characterized in that the object suffers hematological disorders, solid tumors, or immunological disorders.

14. The composition according to item 13, wherein the hematological breach selected from the group of: lymphoma, leukemia, Hodgkin's disease or myeloproliferative disorders.

15. The composition according to claim 7, characterized in that the protein TAT-NAHUN does not contain a label with His end.

16. Pharmaceutical composition for reducing the time of recovery of the object past the transplantation of hematopoietic stem cells HSC, radiation or chemotherapy containing an effective amount of the protein TAT-NAHUN obtained by the method according to claim 1, and a pharmaceutically acceptable carrier.

17. The composition according to item 16, characterized in that the need of the object is the donor cells HSC.

18. The composition according to item 16, characterized in that the need of the object is a patient that underwent autologous transplantation of HSC cells.

19. The composition according to item 16, characterized in that the need of the object is a patient unresponsive to G-CSF.

20. The composition according to claim 19, characterized in that the object suffers from a disease caused by a hereditary deficiency of HSC.

21. The composition according to claim 20, characterized in that the object suffers from hereditary is apoplastically anemia.

22. The composition according to item 16, characterized in that the object suffers hematological disorders, solid tumors, or immunological disorders.

23. The composition according to item 22, wherein the hematological breach selected from the group of: lymphoma, leukemia, Hodgkin's disease or myeloproliferative disorders.

24. The composition according to item 16, wherein the protein TAT-NAHUN does not contain a label with His end.



 

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SUBSTANCE: invention can be used in medical and biologic industry for preparing antineoplastic drugs. Plasmid DNA pFK2 providing synthesis of recombinant analogue of human kappa casein fragment, in Escherichia coli cells is designed; and a method for preparing a recombinant product with using it is described. The recombinant analogue of human kappa-casein fragment recovered from Escherichia coli cells transformed by recombinant plasmid DNA pFK2 has molecular weight of approximately 16 kDa; consists of residual methionine, human kappa-casein fragment with 24 on 134 amino acid residue and C-terminal histidine path and exhibits apoptotic activity in relation to malignant cells.

EFFECT: higher anticancer activity of the compounds.

3 cl, 6 dwg, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.

EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.

8 cl, 10 dwg, 14 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.

EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.

3 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.

EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.

17 cl, 1 dwg, 7 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: in dissolvent, which contains from 55% to 70% of water (wt/wt), precursor of insulin or precursor of insulin derivative is exposed to fermentative splitting at alkaline values of pH. In process of fermentative splitting, they use tripsin or lysil-specific protease, preferably Achromobacter lyticus protease I. Then without separation of intermediate product from reaction mixture, mentioned intermediate product is fermentatively complemented with nucleophilic compound, which represents aminoacid ether, aminoacid amide, peptide, peptide ether or peptide amide in reaction mixture, having water content in the range from 10% to 50% of water (wt/wt), at acidic values of pH, close to neutral pH value. If required, protective group (s) is/are removed.

EFFECT: preparation of insulin compound from its precursor by efficient improved method.

24 cl, 5 ex

FIELD: medicine.

SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.

EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.

16 cl, 3 tbl, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of producing recombinant protein human albumin-interleukin-2 or recombinant protein human albumin-alpha 16-interferon, modified by attachment of human albumin. The method involves technology of culturing yeast strain Pichia pastoris PS106/pPIC9HAbIL-2 or yeast strain Pichia pastoris PS106/pPIC9HAbIFNa-16 in modified culture medium BMGY, after which induction synthesis of target proteins is carried out at low temperature. Further, cells are removed and the medium is concentrated. Target proteins are then precipitated using ammonium sulphate or polyethyleneglycol 3350. Target proteins are then separated by gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents. Finally, affinity chromatography is then done on Cibacron F3GA sorbent.

EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.

3 cl, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.

EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.

29 cl, 5 dwg, 6 tbl, 7 ex

FIELD: medicine; microbiology.

SUBSTANCE: way is intended for reception of functionally active LF form, the basic toxic protein defining cellular disturbances, leading to death of an organism at infection with a malignant anthrax bacterium. For realisation of the way a recombinant plasmid pETHIS-LF (7816 items) is designed, containing a full-size gene of the lethal factor (LF) of malignant anthrax under the control of the promotor of bacteriophage T7 and to a determinant of ampicillin tolerance. The plasmide provides effective synthesis of LF protein of malignant anthrax merged with sequence of six Histidinums for clearing with the metal-chelate chromatography. The strain Escherichia coli BL-HISLF is designed using transformation of the specified plasmid DNA in the strain E.coli BL21 (DE3), synthesizing active LF protein. The target product is separated with the way including clearing on a metal-chelate sorbent with the subsequent additional clearing of the LF protein by gel-filtration.

EFFECT: reception of active recombinant protein LF on the simplified technology and with a high output of synthesised protein of the lethal factor.

3 cl, 3 dwg, 3 ex

FIELD: genetic engineering.

SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.

EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.

9 cl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention is aimed at compound in accordance with formula (R2R3)-B1-A1-c(A2-A3-A4-A5-A6-A7-A8-A9)-A10-A11-A12-A13-B2-B3-R1, which act as ligands for one or several melanocortin receptors, their pharmaceutically acceptable salts.

EFFECT: elaboration of method of peptide application and obtaining pharmaceutical compositions, which include said peptides.

132 cl, 4 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention is aimed at compound in accordance with formula (R2R3)-B1-A1-c(A2-A3-A4-A5-A6-A7-A8-A9)-A10-A11-A12-A13-B2-B3-R1, which act as ligands for one or several melanocortin receptors, their pharmaceutically acceptable salts.

EFFECT: elaboration of method of peptide application and obtaining pharmaceutical compositions, which include said peptides.

132 cl, 4 tbl, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in various industries. The method involves the following. Initial material, e.g. cartilaginous tissue of fish, bird, shellfish or mammals is ground up. The ground initial material immersed in a 0.0025-0.05 n solution of an alkali metal salt. The obtained extract is centrifuged to separate the solid and oil phase. The obtained liquid phase is centrifuged to obtain a filtrate. The obtained filtrate is washed with distilled water which is 6 times more than the volume of the filtrate with subsequent separation and concentration of the obtained end product.

EFFECT: invention enables production of proteoglycan in unaltered undecomposed form in a short period of time.

1 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: invention can be used in medical and biologic industry for preparing antineoplastic drugs. Plasmid DNA pFK2 providing synthesis of recombinant analogue of human kappa casein fragment, in Escherichia coli cells is designed; and a method for preparing a recombinant product with using it is described. The recombinant analogue of human kappa-casein fragment recovered from Escherichia coli cells transformed by recombinant plasmid DNA pFK2 has molecular weight of approximately 16 kDa; consists of residual methionine, human kappa-casein fragment with 24 on 134 amino acid residue and C-terminal histidine path and exhibits apoptotic activity in relation to malignant cells.

EFFECT: higher anticancer activity of the compounds.

3 cl, 6 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel glucagons-like peptide-1 (GLP-1) derivatives which have a prolonged activity profile, their pharmaceutical compositions and use of the compounds in preparing a medicinal agent for treating or preventing hyperglycemia, diabetes 2 or obesity. The compound is a therapeutic GLP-1 polypeptide bonded to a residue bonded with albumin through a hydrophilic separator.

EFFECT: higher effectiveness of derivatives and treatment method.

26 cl, 66 ex

FIELD: chemistry.

SUBSTANCE: invention relates to granulocyte colony-stimulating factor peptide conjugates formed by linkage through an intact glycosyl linker group of (polyethylene glycol) PEG fragments. Said conjugates are obtained from glycosylated and non-glycosylated peptides under the effect of glycosyl transferase. Glycosyl transferase links a fragment of modified sugar to an amino acid or glycosyl peptide residue.

EFFECT: improved method.

35 cl, 24 dwg, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention discloses a method of producing isotope-modified peptides and proteins, based on introduction of a stable 18O isotope (isotopic tag) into carboxyl groups of peptides and proteins by holding in a solution simultaneously containing H218O and trifluoroacetic acid.

EFFECT: method enables to obtain isotope-modified tags which are resistant to destruction and suitable for quantitative mass-spectrometric analysis with retention of the initial structure of the analysed compounds.

1 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: thymus-specific protein T101 consisting of 84 amino acids is recovered from human thymus. The recovered full-length peptide T101 includes the signal peptide consisting of 33 amino acids and the peptide sequence T101 consisting of 51 amino acids and exhibiting immunomodulating activity. The full-length peptide T101, and also peptide fragments and derivatives are used as a part of a pharmaceutical composition for treating autoimmune and inflammatory diseases.

EFFECT: invention allows preparing polypeptide capable to stimulate lymphocyte proliferation of human peripheral blood, to inhibit the tumour growth and to modulate the immune system.

10 cl, 15 dwg, 1 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: thymus-specific protein T101 consisting of 84 amino acids is recovered from human thymus. The recovered full-length peptide T101 includes the signal peptide consisting of 33 amino acids and the peptide sequence T101 consisting of 51 amino acids and exhibiting immunomodulating activity. The full-length peptide T101, and also peptide fragments and derivatives are used as a part of a pharmaceutical composition for treating autoimmune and inflammatory diseases.

EFFECT: invention allows preparing polypeptide capable to stimulate lymphocyte proliferation of human peripheral blood, to inhibit the tumour growth and to modulate the immune system.

10 cl, 15 dwg, 1 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns development of a diagnostic test system in immunochip format and a method of simultaneous and differential detection of reaginic antibodies and antibody spectrum to diagnostically significant mmunologically relevant proteins Treponema pallidum of G (IgG) and M (IgM) classes. The diagnostic test system in immunochip format for differential serum diagnostics of syphilis consists of an immunosorbent with separately immobilised antigens Treponema pallidum Tp15, Tp17, TmpA, Tp47, conjugate and reactants required to detect an antigen-antibody complex. Cardiolipin is additionally immobilised on the immunosorbent; antigens Treponema pallidum and cardiolipin are immobilised at least, in two repetitions, and conjugate consists of mixed antispecies human IgG antibodies and human IgM antibodies modified by two phosphors with different spectral characteristics. On the immunosorbent, there can be additionally immobilised at least, one antigen and/or peptide Treponema pallidum specified e.g. of Tp39, Tp41, Tp42, Tp44.5, Tp92, Tp0453 at least in two repetitions. Cardiolipin can represent an oxidised cardiolipin derivative bounded with protein. The differential serum syphilis diagnostic technique with using the diagnostic test system in immunochip format involves application of human IgM modified by two phosphors with different spectral characteristics. On the immunosorbent, there can be additionally immobilised at least one antigen and/or peptide Treponema pallidum, specified e.g. of Tp39, Tp41, Tp42, Tp44.5, Tp92, Tp0453 at least in two repetitions. Cardiolipin can represent an oxidised cardiolipin derivative bounded with protein. The differential serum syphilis diagnostic technique with using the diagnostic test system in immunochip format implying that a cultivation solution for check and test samples is introduced on the immunosorbent with separately immobilised antigens Treponema pallidum and cardiolipin; the check and test samples are introduced; the prepared mixture is incubated at temperature 20-42°C for 15-60 min to prepare the antigen-antibody complex; the immunosorbent is rinsed; then a conjugate solution of mixed human IgG antibodies and human IgM antibodies modified by two phosphors with different spectral characteristics is introduced on the immunosorbent; it is followed with incubation at temperature 20-42°C for 15-60 min; the immunosorbent is rinsed, dried to detect the prepared antigen-antibody complex.

EFFECT: improved diagnostic accuracy.

20 cl, 5 dwg, 2 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to (i) essentially crystalline melagatran in the form of hydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 21.1, 10.5, 7.6, 7,0, 6.7, 6.4, 6.2, 5.7, 5.4, 5.3, 5.22, 5,19, 5.07, 4.90, 4.75, 4,68, 4.35, 4.19, 4.00, 3.94, 3.85, 3.81, 3.73, 3.70, 3.63, 3.52, 3.39, 3.27, 3,23, 3.12, 3.09, 3.06, 2.75, 2.38, and 2.35 Å and/or water content 4.3%; and (ii) essentially crystalline melagatran in the form of anhydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 17.8, 8.9, 8.1, 7.5, 6.9, 6.3, 5.9, 5.6, 5.5, 5.4, 5.3, 5.2, 5.0, 4.71, 4.43, 4.38, 4.33, 4.14, 4.12, 4.05, 3.91, 3.73, 3.61, 3.58, 3.56, 3.47, 3.40, 3.36, 3,28, 3.24, 3.17, 3.09, 3.01, 2.96, 2.83, 2.54, 2.49, 2.41, 2.38, and 2.35 Å. Invention also relates to a method for preparation of indicated form, a method for interconversion of anhydrite form, to use of indicated compounds as pharmaceutical agent, and to preparation of drugs. Pharmaceutical preparation is suitable for treatment of condition, in case of which inhibition of thrombin is needed or desirable. Invention provides a method for treatment of such condition.

EFFECT: increased chemical stability and solid state stability as compared to amorphous forms of melagatran.

14 cl, 4 dwg, 3 tbl, 9 ex

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