Agent for treating myelofibrosis

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the chemical-pharmaceutical industry and represents a substance-delivering carrier for the substance delivery to a bone marrow cell producing an extracellular matrix containing retinoid as a delivering agent, wherein the substance is a therapeutic agent, which inhibits the beginning, progression and/or recurrence of myelofibrosis.

EFFECT: invention provides the effective inhibition of the beginning, progression and recurrence of myelofibrosis.

10 cl, 14 dwg, 8 ex

 

Field of the invention

The present invention relates to a carrier transporting substance to purposefully producing extracellular matrix of the bone marrow cells, as well as a means for the treatment of myelofibrosis and method for the treatment of myelofibrosis with use of the drug that controls the activity and growth producing extracellular matrix cells in the bone marrow.

The level of technology

Myelofibrosis is a General term referring to diseases that cause extensive diffuse fibrosis in the bone marrow, and includes primary myelofibrosis, whose etiology is unknown, and secondary myelofibrosis with underlying illness.

Primary myelofibrosis is a chronic myeloproliferative disorder that is characterized by involvement in the pathological process of fibrosis of the bone marrow of the entire body and extramedullary hematopoiesis in liver and spleen, as well as a manifestation of leukoerythroblastosis in which immature granulocytes and erythroblasts appear in the peripheral blood. The main feature of primary myelofibrosis is considered to be monoclonal proliferation of hematopoietic cells due to genetic anomalies involving the Jak2 gene mutation that occurred at the level of hematopoietic stem cells. Various cytokines, produced prooperirovanna hematopoietic cell�mi (mainly by megakaryocytes), act on the cells of the bone marrow stroma, causing the proliferation of reactive polyclonal stroma cells of the bone marrow, leading to bone marrow fibrosis, osteosclerosis and angiogenesis. This leads to the typical clinical symptoms, such as ineffective hematopoiesis, the appearance of dcritical in the peripheral blood, leukoerythroblastic and extramedullary hematopoiesis causing splenomegaly.

About 40% of primary myelofibrosis have a gene mutation in Jak2, a tyrosine kinase required for signal transduction of cytokines, leading to constitutive activation of Jak2, even in the absence of stimulation by cytokines. In addition to Jak2, there are several cases of gene mutations in the c-mp1 (receptor thrombopoetin).

Now consider that primary myelofibrosis difficult to cure with medical therapy, and that the only effective therapy is allogeneic hematopoietic stem cell transplant. However, the mortality associated with transplantation, is from 25% to 48%, limiting the overall rate of survival to about 50%. Recently, it was widely reported the use of non-destructive stem cell transplant bone marrow (mini-transplant) with less treatment-related, toxicity has so far been studied only a limited number of cases, and associated long-term forecasts still have� to know.

As drug therapy, although palliative, was shown the effectiveness of anabolic hormones such as danazol and Primobolan, inhibitors of angiogenesis such as thalidomide and lenalidomide, antineoplastic drugs, such as hydroxyurea, anagrelide, imatinib, 2-chlorodeoxyadenosine, melphalan, busulfan and etoposide and other drugs, such as erythropoietin for anemia, thrombocytopenia and splenomegaly (see non-patent literature 1 and 2).

On the other hand, secondary myelofibrosis occurs on the background of diseases such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, polycythemia Vera, primary thrombocytopenia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, carcinoma, systemic lupus erythematosus and progressive systemic sclerosis, or radiation sickness, and demonstrates similar with primary myelofibrosis picture of the bone marrow. Treatment of secondary myelofibrosis is focused on the improvement of the underlying disease. However, many of these major diseases are difficult to cure radically. Thus, there is an urgent need to reduce adverse effects of the myelofibrosis.

In this regard, there have been many studies to develop medicines against mi�of latebrosa. The result reported that results have been obtained, successful to a certain extent, in the study of animal models or in clinical trials, for example, with the use of the JAK2V617F tyrosine kinase inhibitors, inhibitors of TGF-β, such as soluble TGF-β receptor, NFκB inhibitors, such as bortezomib, inhibitors of DNA methyltransferase, such as decitabine, inhibitors discontiuation such how trichostatin A, VEGF inhibitors such as PTK787/ZK222584 and bevacizumab (see non-Patent literature 1), and some types of antibodies to human lymphocytes (see Patent literature 1). However, none of these drugs is not satisfactory and it was desirable to develop other means for the treatment of myelofibrosis.

[Patent literature 1] JP A No. 8-002799

[Patent literature 2] WO 2006/068232

[Non-patent literature 1] Hematology Am Soc Hematol Educ Program. 2007; 2007:346-54

[Non-patent literature 2] The Journal of The Japanese Society of Internal Medicine, Vol.96, No. 7, July 10, 2007, pp.1398-1404

The aim of the present invention is to provide a new treatment for myelofibrosis and method for the treatment of myelofibrosis.

In the process of studying new therapeutic agents for the treatment of myelofibrosis, the researchers found that the myelofibrosis can be effectively treated by administering a composition in which the inhibitor of extracellular products �of atrixo is delivered to the carrier, containing retinoid.

Although it was known that the media containing vitamin a, can deliver the drug in stellate cells, which accumulate vitamin a (see Patent literature 2), its relationship with myelofibrosis was completely unknown to date. Also there were no reports that the myelofibrosis could be cured with a composition containing as active ingredient an inhibitor of production of extracellular matrix. Therefore, these study results is quite amazing.

Namely, the present invention concerns the following:

(1) Delivering the substance to the carrier for delivery of substances to the cell of the bone marrow that produce extracellular matrix containing a retinoid as a directing agent.

(2) a Carrier under paragraph (1), where the retinoid contains retinol.

(3) a Carrier under paragraph (1) or (2), where the content of retinoid is 0.2-20 wt.% of the total weight of the carrier.

(4) the Carrier according to any one of items (1) to(3), where the carrier has a form of liposome, and the molar ratio of retinoid to the lipid contained in the liposome is 8:1 - 1:4.

(5) a Composition for the treatment of myelofibrosis, containing a drug that controls the activity or growth of bone marrow cells that produce extracellular matrix.

(6) the Composition according to item (5), further comprising �Eitel on any of the items(1)-(4).

(7) the Composition according to item (5) or (6), where a drug that controls the activity or growth of bone marrow cells that produce extracellular matrix selected from the group consisting of a means for inhibiting activity or production of a bioactive substance selected from the group consisting of gelatinase And, gelatinase and In the angiotensinogen gene inhibitor of cellular activity, growth inhibitor, an apoptosis-inducing means, as well as miRNAs (siRNA), ribozyme, antisense nucleic acid, and DNA/RNA chimeric polynucleotide that targets at least one of the molecules that form the extracellular matrix, or molecules involved in the production or secretion of these molecules that form the extracellular matrix, and a vector that expresses the indicated miRNA, a ribozyme, antisense nucleic acid and DNA/RNA chimeric polynucleotide.

(8) the Composition according to item (7), where a molecule involved in the production or secretion forming extracellular matrix molecules, is HSP47.

(9) the Composition according to any one of paragraphs (5) to(8), where the drug and carrier are mixed at a place of medical treatment or nearby.

(10) a Kit for preparing a composition according to any one of paragraphs (6) through(9) containing one or more containers that contain either individually or in combination of pharmaceutical�any means, that controls the activity or growth of bone marrow cells that produce extracellular matrix, a retinoid and, if necessary, a substance which is a component carrier different from retinoid.

(11) miRNAs targeting a portion of the nucleotide sequence SEQ ID NO:13, wherein the portion is selected from the sections from the position 1130 to the position 1145, position from 1485 to position 1500, the position of the position 1501 to 1516, position from 1654 to the position of 1678 and position from 1951 to 1978 position of the nucleotide sequence SEQ ID NO:13.

(12) miRNAs on item 11, consisting of any of the following combinations A to E of the sense chain and antisense chain:

A: the combination of 5'-UGGAUGGGAAAGAUGCAGAAGAAGGAG-3' (sense chain, SEQ ID NO:1) and 3'-UAACCUACCCUUUCUACGUCUUCUUCC-5' (antisense chain, SEQ ID NO:2),

In: the combination of 5'-UGUCUGAGUGGGUAUUUUUAGACAGAG-3' (sense chain, SEQ ID NO:3) and 3'-UAACAGACUCACCCAUAAAAAUCUGUC-5' (antisense chain, SEQ ID NO:4),

With the combination of 5'-GAUGCGAGAUGAGUUGUAGAGUCCAAG-3' (sense chain, SEQ ID NO:5) and 3'-UACUACGCUCUACUCAACAUCUCAGGU-5' (antisense chain, SEQ ID NO:6),

D: a combination of 5'-CAGAACUGCCCAUCCUUAAAAUGAUAG-3' (sense chain, SEQ ID NO:7) and 3'-UAGUCUUGACGGGUAGGAAUUUUACUA-5' (antisense chain, SEQ ID NO:8),

E: the combination of 5'-GAGACAAGAUGCGAGAUGAGUUGUAAG-3' (sense chain, SEQ ID NO:9) and 3'-UACUCUGUUCUACGCUCUACUCAACAU-5' (antisense chain, SEQ ID NO:10).

Although the exact mechanism of action of the composition for the treatment of myelofibrosis of the present invention is not yet completely elucidated, the mechanism is approached�Xia as follows: retinoid in the composition acts as a directing agent to produce extracellular matrix of the bone marrow cells, such as bone marrow fibroblasts, and delivers to such cells active ingredients such as pharmaceutical agents that control the activity or growth producing extracellular matrix of bone marrow cells, thereby showing the effect against myelofibrosis.

As the active ingredients can be effectively delivered to the site of action and further to the target cells, through the use of media according to the present invention, it became possible treatment, suppression of progression and prevent the development of myelofibrosis, including primary myelofibrosis, treatment which until now was difficult; thus, the carrier of the present invention makes a significant contribution to medicine and veterinary medicine.

In addition, since the composition of the present invention contains as active agent a drug that controls the activity or growth producing extracellular matrix of bone marrow cells, whose efficiency in relation to myelofibrosis was not known, the treatment of myelofibrosis may occur by a mechanism different from the currently known. Therefore, it is expected the improvement of pathological conditions that cannot be cured by conventional drugs mechanism of action, and increase therapeutic effect proofmaster the use of conventional drugs.

Moreover, the carrier of the present invention may be combined with any pharmaceutical drugs (for example, with existing therapeutic sredstvami against myelofibrosis) to increase the effectiveness of their actions; therefore, a wide range of its application is also useful from the point of view of technology of pharmaceutical production, facilitating the production of effective therapeutic agents.

Brief description of the drawings

Fig.1 shows photographs showing images of the bone marrow of patients with idiopathic myelofibrosis. Patient 1 and Patient 2 demonstrated trabecular thickening staining GOE (left column), hyperplasia of the reticular fiber staining on Gitter (Gitter staining) (Central column) and the deposition of collagen staining by Athan (Azan staining) (right column).

Fig.2 shows a diagram showing the pathogenesis of myelofibrosis in a murine model.

Fig.3 shows photographs showing images of bone marrow TRO transgenic mice, age 4 and 7 months. The left column shows the staining of the GOE, in the Central column shows staining Gittery, and the right column shows staining for the Azan.

Fig.4 shows photographs showing images of bone marrow TRO transgenic mice, age and 12 months. The left column shows the staining of the GOE, in the Central column shows staining Gittery, and the right column shows staining for the Azan.

Fig.5 shows photographs showing the transition to trabecular thickening in TRO transgenic mice. The upper left panel is the staining of the GOE bone marrow 4-month mice (4M), upper right panel - age 7 months (7M), lower left panel - the age of 9 months (9M), and lower right panel - the age of 12 months (12M).

Fig.6 shows a photograph showing the morphology of primary cultured bone marrow fibroblasts TRO mice received under the inverted microscope (magnification x400).

Fig.7 shows diagrams illustrating the results of analysis of expression of Vimentin and α-SMA by using flow cytometry in primary cultured fibroblasts, bone marrow TRO mice. The vertical axis shows the number of cells.

Fig.8 shows images of a Western blot showing the effect of different miRNAs on the expression of HSP47 HSP47 in NIH3T3 (A) and primary cultures of bone marrow fibroblasts TRO mice (Primary fibroblasts and In (C).

Fig.9 shows graphs that show the effect of vitamin A (VA) on the introduction enclosed in liposomes miRNAs HSP47 (Lip-siRNA) in primary-cultured bone marrow fibroblasts TRO mice. (A) and (B) until�yaut the results of analyses using flow cytometry and image under fluorescent microscope, respectively.

Fig.10 is a diagram showing the impact of miRNAs HSP47 in collagen secretion in primary cultured fibroblasts, bone marrow TRO mice.

Fig.11 shows images of the samples under a microscope, stained Gittery that demonstrate the impact of miRNAs on HSP47 fibrillation bone marrow TRO mice in vivo.

Fig.12 shows a graph showing the quantification of hyperplasia of reticular fibers in TRO mice using miRNAs HSP47 by image analysis. The vertical axis shows the ratio of points reticular fibers and all points in each field.

Fig.13 shows image samples under a microscope, stained by the Azan, which demonstrate the impact of miRNAs on HSP47 fibrillation bone marrow TRO mice in vivo.

Fig.14 shows image samples under a microscope by staining the GOE that demonstrate the impact of miRNAs on HSP47 fibrillation bone marrow TRO mice in vivo.

Description of embodiments of the present inventions

The present invention relates to delivering a substance to a carrier for delivery of a substance to produce extracellular matrix cell of bone marrow containing a retinoid as a directing agent.

In the present invention, producing extracellular matrix cell bone marrow� specially limited only by reference, that this cell is present in the bone marrow and are capable of producing extracellular matrix, and is typically the fibroblast bone marrow. For fibroblast bone marrow characterized by the expression of α-SMA (alpha-smooth muscle actin). In the present invention the fibroblast bone marrow is one of identified, for example, immunoablative using labelled anti-α-SMA antibodies.

In the present invention, the retinoid is not limited in a special way, except for a mention that it contributes to the delivery of a substance to produce extracellular matrix cell of the bone marrow, and its examples include derivatives of retinoids, such as retinol (vitamin a), etretinate, tretinoin, isotretinoin, adapalene, acitretin, tazarotene, and retinol palmitate, as well as analogs of vitamin A, such as fenretinide (4-HPR, 4-hydroxyphenylethylamine) and bexarotene.

The retinoid according to the present invention is one of those substances that facilitate the specific delivery of a substance to produce extracellular matrix cell of the bone marrow. A mechanism to facilitate retinoid upon delivery of the substance is not yet fully clear; however, for example, it is believed that the retinoid, is specifically associated with retinol-binding protein (RBP), is captured inside the bone marrow cells that produce extracellular Matri�with, via a specific receptor presented on the surface of the cell.

A retinoid is a member of the class of compounds having a skeleton in which four units of isoprenoid way connected "head-to-tail" (see G. P. Moss, "Biochemical Nomenclature and Related Documents", 2nd Ed. Portland Press, pp.247-251 (1992)). Vitamin a is a typical descriptor retinoid, which qualitatively demonstrates the biological activity of retinol. A retinoid that can be used in the present invention is not specifically limited, and examples include derivatives of retinoids, such as retinol, retinal, retinoic acid, an ester of retinol and a fatty acid, an ester of aliphatic alcohol and retinoic acid, etretinate, tretinoin, isotretinoin, adapalene, acitretin, tazarotene and retinol palmitate, and analogues of vitamin A, such as fenretinide (4-HPR) and bexarotene.

Of them middle retinol, retinal, retinoic acid, an ester of retinol and a fatty acid (such as retinyl acetate, retinyl palmitate, retinyl stearate and retinyl laurate) and an ester of aliphatic alcohol and retinoic acid (such as etirement) are preferred from the viewpoint of the efficiency of specific delivery of a substance to the bone marrow cells that produce extracellular matrix.

All isomers of retinol, including CIS-TRANS isomers, are included in the scope of this and�gaining. A retinoid may be substituted by one or more substitutes. The retinoid according to the present invention includes the retinoid in the dedicated form, as well as in the form of a solution or mixture with the environment, which can dissolve or retain the retinoid.

The carrier of the present invention can be formed from retinoid or can be obtained by linking retinoid with forming a carrier component different from retinoid, or by inclusion of retinoid to a component, which component carrier different from retinoid. Therefore, the media of the present invention may contain a component, which component carrier different from retinoid. This special component is not limited, and can be used any known component in the field of medicine or pharmacy, but preferred are those that can surround a retinoid or contact a retinoid.

Examples of such components include lipids, e.g. phospholipids, such as glycerophospholipid, sphingolipid, such as sphingomyelin, Sterol, such as cholesterol, vegetable oil such as soybean oil or poppy seed oil, mineral oil and lecithin, such as egg yolk lecithin, but these examples are not limited. Among them are preferred are those that can form liposomes, such as finely�th a phospholipid such as lecithin, semisynthetic phospholipid, such as dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPE), dilauroyllecithin (DLPC) and cholesterol.

Particularly preferred component is a component that can avoid capture by the reticuloendothelial system, and its examples include cationic lipids such as N-(α-trimethylammonium acetyl)-didodecyl-D-glutamate chloride (TMAG), N,N',N",N"'-tetramethyl-N,N',N",N"'-metropolitician (TMTPS), 2,3-valerossi-N-[2(sprintersexual)ethyl]-N,N-dimethyl-1-propanamine triptorelin (DOSPA), N-[1-(2,3-valerossi)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dictatorially chloride (DODAC), deterimine bromide (DDAB), 1,2-valerossi-3-trimethylammonium propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), 1,2-demeritorious-3-dimethylhydroxylamine (DMRIE), and 0,0'-ditetradecyl-N-(α-trimethylammonium acetyl)diethanolamine chloride (DC-6-14).

The binding of retinoid with the carrier of the present invention or the conclusion of retinoid it is also possible by linking retinoid with or placing it in a component carrier different from retinoid, using chemical and/or physical way. Alternatively, the retinoid may be linked to or enclosed in nositelya the present invention, by mixing retinoid and component carrier different from retinoid, in the preparation of media. The number of retinoid associated with or prisoner in the media of the present invention may be in relation to the mass of the components that are part of media, from 0.01% to 100%, preferably from 0.2% to 20% and more preferably from 1% to 5%. A retinoid may be linked to or enclosed in the carrier before adding the drug to the carrier; or a carrier, the retinoid and the drug can be mixed simultaneously; or a retinoid may be mixed to the carrier, the drug carrier, and so on. Therefore, the present invention also relates to a method for producing compositions that are specific to producing extracellular matrix cell of the bone marrow, where the method includes the step of linking retinoid with any existing binding a drug carrier or encapsulating the drug carrier, such as liposomal composition, such as DaunoXome®, Doxil, Caelyx® or Myocet®.

The carrier of the present invention may be in any form, required to move a substance or object to a given cell in bone marrow that produce extracellular matrix, and the examples above, but without limiting to this, include macromolecular�th micelle, liposome, an emulsion, microspheres and nanospheres. In the present invention, among these forms liposomal form is preferred from the viewpoint of high efficiency of delivery, a wide choice of the delivered material and services received, and so forth, and particularly preferred cationic liposomes including cationic lipid. In the case where the medium is in the form of liposomes is preferred that the mole ratio of retinoid to other components of the liposomes from 8:1 to 1:4, more preferably from 4:1 to 1:2, even more preferably from 3:1 to 1:1 and particularly preferably 2:1, whereas the binding efficacy of retinoid with or opinions in the media.

The carrier of the present invention may contain the medium to be transported inside, can be attached to the outer part of the transported substance or may be mixed with the transported substance, yet it contains a retinoid in such a form that the retinoid is capable of acting as a guide or agent. The phrase "act as the directing agent" herein means containing a retinoid carrier reaches and/or is captured by the cell-target, i.e., the cell of the bone marrow that produce extracellular matrix, faster and/or more than nesadurai a retinoid carrier, and it can be easily confirmed with POM�means, for example, adding a labeled media or media containing the label, to the culture of target cells and the analysis of the distribution of label after a certain period of time. Structurally, this requirement may be satisfied, for example, if a retinoid, at least in part, turns on the external part of the composition containing the carrier, not later than the time when it reaches the target cell. Exposed or not a retinoid on the outer part of the composition can be estimated by the interaction of the composition with a substance that specifically binds to a retinoid, such as rathinasamy protein (RBP), and assessment of its binding with the song.

The substance or object is delivered to the data carrier, in a special way is not limited, and preferably has such a size that can physically move around in the body from the place of introduction to the place of destruction, where there is a target cell. Therefore, the media of the present invention can convey not only the substance, such as atom, molecule, compound, protein, or nucleic acid, but also an object such as a vector, a viral particle, cell, releasing the medicament, the system comprising one or more elements, or micro-machines. A substance or object, preferably tend to have some effect on the target cell, napiermatt the target cell or control (for example, potentiate or inhibit) the activity of growth of the target cells.

Thus, in one embodiment of the present invention, that is delivered to the carrier, is a drug that controls the activity or growth of bone marrow cells that produce extracellular matrix". The activity of bone marrow cells that produce extracellular matrix, according to the present invention applies to different activities, such as secretion, absorption or migration exhibited by the cell of the bone marrow that produce extracellular matrix, and the present invention the activity preferably is an activity involved in the onset, progression and/or relapse of myelofibrosis. Examples of such activities include, but without limiting to this, the production/secretion of biologically active substances, such as gelatinase and gelatinase IN (MMR and MMR respectively) and angiotensinogen gene and a component of the extracellular matrix, such as collagen, proteoglycan, tenascin, fibronectin, thrombospondin, osteopontin, osteonectin and elastin.

Thus, a drug that controls the activity and growth of bone marrow cells that produce extracellular matrix, can be any drug that directly or indirectly suppresses the physical�th, chemical and/or physiological effect of the specified cells associated with the onset, progression and/or recurrence of myelofibrosis and including but without limitation to this: the medication, the overwhelming activity or production of the above biologically active substance, an inhibitor of MMP, such as batimastat, and antibodies and fragments of antibodies that neutralize the above biologically active substance, and the substance suppressing the expression of the above biologically active substances, such as miRNAs (siRNA), a ribozyme, antisense nucleic acid (including RNA, DNA, NCP (peptide nucleic acid) or a mixture thereof), or a substance having a dominant negative effect such as a dominant negative mutant, or a vector expressing them, or a drug inhibiting the production or secretion of the above component of the extracellular matrix, such as a substance suppressing the expression of a component of the extracellular matrix, such as miRNAs (siRNA), a ribozyme, antisense nucleic acid (including RNA, DNA, NCP or a mixture thereof), or a substance having a dominant negative effect such as a dominant negative mutant, or a vector expressing them, an inhibitor of cell activity, such as a sodium channel blocker, cell growth inhibitors, such as an alkylating agent (�the ark, as ifosfamide, nimustine, nikopoli, dacarbazine, melphalan and ranimustine), antitumor antibiotics (such as idarubicin, epirubicin, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitoxantrone and mitomycin C), an antimetabolite (such as gemcitabine, enocitabine, cytarabine, tegafur/uracil, tegafur/gimeracil/oteracil potassium, doxifluridine, hydroxyurea, fluorouracil, methotrexate, and mercaptopurine), an alkaloid, such as etoposide, irinotecan hydrochloride, vinorelbine ditartrate, docetaxel hydrate, paclitaxel, vincristine sulfate, vindesine sulfate, vinblastine sulfate, and platinum complexes such as carboplatin, cisplatin and nedaplatin and as inducers of apoptosis, such as compound 861, gliotoxin, lovastatin and Beractant. Moreover, "a drug that controls the activity and growth of bone marrow cells that produce extracellular matrix" of the present invention may be any drug that directly or indirectly promote the physical, chemical and/or physiological actions of bone marrow cells that produce the extracellular matrix that are directly or indirectly related to the suppression of the beginning, progression and/or recurrence of myelofibrosis.

Among medicines regulatory activity and cell growth coast�wow brain producing extracellular matrix" of the present invention preference is given to drugs that inhibit the production/secretion of a component of the extracellular matrix, such as collagen, proteoglycan, tenascin, fibronectin, thrombospondin, osteopontin, osteonectin and elastin, and particularly preferably inhibitors of heat shock Protein 47 (HSP47), including miRNAs (siRNA) against HSP47.

Delivered by carrier substance according to the invention includes, without limitation to this, drugs that inhibit the onset, progression and/or relapse of myelofibrosis, and drugs that have not been mentioned, and examples include, without limitation, anabolic hormones, such candanoza and Primobolan, angiogenesis inhibitors such as thalidomide and lenalidomide, antineoplastic drugs, such as hydroxyurea, anagrelide, imatinib, 2-chlorodeoxyadenosine, melphalan, busulfan and etoposide, erythropoietin, the JAK2V617F tyrosine kinase inhibitors, inhibitors of TGF-β, such as a soluble receptor of TGF-β, and NFκB inhibitors, such as bortezomib, an inhibitor of DNA methyltransferase, such as decitabine, an inhibitor distanceinmiles, such as trichostatin A, VEGF inhibitors such as PTK787/ZK222584 and bevacizumab, and antitumor lymphocyte antibodies described in the Patent literatu�re 1, the above.

Deliver a carrier material according to the invention may be labeled and the unlabeled. Tagging makes possible the monitoring of the success or failure of transportation, or increase and decrease in target cells, and so forth, and is especially useful on the level of testing/research. The label can be selected from any label known to those skilled in the art, such as, for example, any radioisotope, magnetic material, a substance which binds to the labeled substance (e.g., antibody), a fluorescent substance, a fluorophore, a chemiluminescent substance and the enzyme, and so on.

In the present invention, the phrase "to produce extracellular matrix cell of the bone marrow or for delivery to producing extracellular matrix cell of the bone marrow" means that it is ideal for use in relation to producing the extracellular matrix of the bone marrow cells as a target cell, and this includes, for example, the ability to deliver substances to the cell more quickly, effectively and/or in greater numbers than to other cells, such as normal cells. For example, the carrier of the present invention can deliver a substance to produce extracellular matrix cell of the bone marrow with speed and/or efficiency of 1.1 times or more, 1.2 times higher or more than 1.3 times or more, 1.5 times or more, 2 times or more, and even 3 times higher or more, compared with other cells.

The present invention also relates to a composition for controlling the activity or growth producing extracellular matrix of bone marrow cells, or for the treatment of myelofibrosis, a composition containing a drug that controls the activity or growth producing extracellular matrix of bone marrow cells, and the present invention also relates to use in obtaining said compositions of the drug that controls the activity or growth producing extracellular matrix of bone marrow cells. The drug may be contained in the composition by itself or in combination with a pharmaceutically acceptable carrier. A composition according to the present invention may be aimed at producing extracellular matrix cell of the bone marrow, which will be targeted for effective delivery to a target cell. A method of targeting a special way is not restricted, except that it needs to facilitate the delivery compositions of the present invention to produce extracellular matrix cell of the bone marrow, such as fibroblast, bone marrow, and examples include the addition of retinoid. Accordingly, the preferred embodied�e of this invention includes a retinoid as a directing agent, and more preferably includes a carrier containing the above mentioned retinoid, as the guide agent.

The present invention includes primary myelofibrosis myelofibrosis and secondary myelofibrosis. Secondary myelofibrosis includes, without limitation to this, myelofibrosis, which is secondary to the disease, such as acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, polycythemia Vera, primary thrombocytopenia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, carcinoma, systemic lupus erythematosus and progressive systemic sclerosis or radiation sickness.

Myelofibrosis of the present invention may be diagnosed by any means known to those skilled in the art. The most characteristic pathology of myelofibrosis is fibrillation bone marrow, and it can be determined to some extent by the failure to collect the aspirate of bone marrow by bone marrow aspiration ("dry needling"). The final diagnosis can be made by confirming fibrillation bone marrow and/or enhancement of trabeculae with bone marrow biopsy (see Fig.1). Advanced primary myelofibrosis may show anemia, hepatosplenomegaly, and the advent of leukoerythroblastosis,�kinotitas, such as dakryzei, progenitor cells, microthrombosis and megakaryocytes in peripheral blood, increased serum lactate dehydrogenase (LDH), increased hepatic-splenic uptake in scintigraphy of the bone marrow, the trend of random bleeding, bloating, fever, General malaise, weight loss, etc. In secondary myelofibrosis symptoms of primary diseases often come to the fore. Symptoms specific to the underlying disease, well known to specialists in this field.

In the compositions according to the present invention, when included in the media, the retinoid is present in such a form that acts as a directing agent, the host can deliver a substance within itself, can be attached to the outer part of the transported substances, or may be mixed with the transported substance. Therefore, depending on the method of administration and method of releasing the drug, and so forth, the composition may be covered with a suitable material, such as, for example, enteric-coated or material with adjustable time decay, or may be included in the right system releasing the drug.

A composition according to the present invention may be administered by various routes including both oral and parenteral route, and examples of et�include th, but without limiting to this, oral, intravenous, intramuscular, subcutaneous, local, intrapulmonary, inside the airway, intratracheal, intrabronhialno, nasal, rectal, intra-arterial, intraportal, intraventricular, intramedullary, inside the lymph node, vnutripoliticheskie, intracerebral, intrathecal, intracerebroventricularly, transmucosally, percutaneous, intranasal, intraperitoneal, and intrauterine path, and can be made in the form of a dosage form suitable for each route of administration. Such a dosage form and a method of producing can be selected as appropriate from any known dosage forms and methods (see, for example, Hyojun Yakuzaigaku (Standard Pharmaceutics), Ed. by Yoshiteru Watanabe et al., Nankodo, 2003).

Examples of dosage forms suitable for oral method of administration include, but without limitation to this, powder, granules, tablet, capsule, liquid, suspension, emulsion, gel and syrup, and examples of dosage forms suitable for parenteral method of administration include injection, such as injection, suspension for injection emulsion for injection, and injection form, prepared immediately before introduction. Formulations for parenteral administration may be in such form, as an aqueous or non-aqueous sterile from�tonic solution or suspension.

The composition of the present invention may contain one or more of any other medicines that can treat myelofibrosis or facilitate its onset, progression and/or relapse, and/or symptoms, or may be used in combination with such drugs. Examples of such drugs include, without limitation, for example, anabolic hormones such as danazol and Primobolan, an inhibitor of angiogenesis such as thalidomide and lenalidomide, an antitumor drug such as hydroxyurea, anagrelide, imatinib, 2-chlorodeoxyadenosine, melphalan, busulfan and etoposide, erythropoietin, the JAK2V617F tyrosine kinase inhibitors, inhibitors of TGF-β, as, for example, a soluble receptor of TGF-β, and NFκB inhibitors, such as bortezomib, an inhibitor of DNA methyltransferase, such as decitabine, an inhibitor distanceinmiles, such as trichostatin A, an inhibitor of VEGF, such as PTK787/ZK222584 and bevacizumab, and antitumor lymphocyte antibodies described in Patent literature 1 above. When used in the combination composition of the present invention may be administered simultaneously, before or after other medicines. The method of administration may be the same or different. For example, the composition of the present invention may be introduced parenterally, whereas others� drug substance can be administered orally, and so on.

The carrier or composition according to the present invention may be provided in any form, but from the point of view of preservation stability, it is preferable presentation in a form that can be prepared in place of medical treatment or beside him, the doctor and/or pharmacist, nurse, or other health care provider. In this case, the carrier or composition according to the present invention is provided in the form of one or more containers containing at least one component necessary for the preparation and cooking is carried out before use, for example, within 24 hours prior to use, preferably within 3 hours before using and more preferably immediately prior to use. During preparation, usually in the preparation area available reagent, solvent, equipment for cooking, etc., which can be applied accordingly.

Thus, the present invention also relates to a kit for the preparation of the carrier or the composition, wherein the kit comprises one or more containers which contain or separate, or in combination a retinoid, and/or deliver the substance, and/or a substance which is a component carrier different from retinoid, as well as component, t he�for my carrier or composition, presented in the form of this set. The kit of the present invention may contain, in addition to the above specified, instructions, electronic media such as CD or DVD relating to methods of preparation and introduction of the carrier and compositions of the present invention. Moreover, the kit of the present invention may contain all components for the final receiving media or compositions of the present invention, but not necessarily to contain all the components. Thus, in the kit of the present invention does not require the contents of the reagent or solvent, which are usually available at the venue of medical treatment or in the experimental room, and so on, such as, for example, sterile water, physiological saline or glucose solution.

In addition, the present invention relates to a method for controlling the activity or growth producing extracellular matrix of bone marrow cells, or to a method for the treatment of myelofibrosis, where the method includes introducing an effective amount of the aforementioned composition to a subject in need of it. An effective amount according to the present invention, a method for the treatment of myelofibrosis, a represents, for example, the amount that suppresses start or relapse of myelofibrosis, relieves its symptoms, or retards or ustanavli�AET its progression and preferably is a number, which prevents a start or relapse of myelofibrosis or cures him. Also preferably, the amount which does not cause any side effect that is greater than the benefit from the introduction. Such amount may be appropriately determined using a test in vitro, using cell culture or by test on animals, such as mouse, rat, dog or pig, and such test techniques well known to those skilled in the art. Moreover, the dose of retinoid contained in the carrier, and dose of medicines used in the method according to the present invention are known to those skilled in the art, or can be properly determined using the aforementioned tests, and so on.

Specific dose of the composition administered in the method according to the present invention, can be determined by taking into account different conditions in relation to the patient in need of treatment, such as the severity of symptoms, General health of patient, age, body weight, sex, diet, time and frequency of administration, concomitant drug, susceptibility to treatment, and the appropriateness of therapy for a given patient, and so on.

The method of administration includes various ways, including both oral and parenteral route, for example, such as oral, intravenous, intramuscular, subcutaneous, local, intrapulmonary, inside the airway, intratracheal, intrabronhialno, nasal, rectal, intra-arterial, intraportal, intraventricular, intramedullary, inside the lymph node, vnutripoliticheskie, intracerebral, intrathecal, intracerebroventricularly, transmucosally, percutaneous, intranasal, intraperitoneal, and intrauterine path.

Frequency of administration varies depending on the properties of the used composition and the above-mentioned conditions of the patient, and may be, for example, several times a day (i.e. 2, 3, 4, 5 or more times per day), once daily, once every few days (or once every 2, 3, 4, 5, 6 or 7 days and so on), several times a week (e.g., 2, 3, 4 times and so on in the week), once a week, or every few weeks (i.e., every 2, 3, 4 weeks and so on).

In the method according to the present invention, the term "patient" means any living individual, preferably an animal, more preferably a mammal and more preferably human. In the present invention, the patient may be healthy or sick in some disorder, and if the treatment of myelofibrosis assigned, it usually means that a patient has a disease myelofibrosis or subjected to risk of developing myelofibrosis. When the assigned preventive� primary myelofibrosis, for example, typical examples include, without limitation, patients who have a mutation in the Jak2 gene and/or c-mpl. When the assigned secondary prevention of myelofibrosis, typical examples include, without limitation, the patients suffering from such diseases as acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, polycythemia Vera, primary thrombocytopenia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, carcinoma, systemic lupus erythematosus and progressive systemic sclerosis or a patient who was exposed to radiation.

Moreover, the term "treatment" includes all types acceptable from a medical point of view prophylactic and/or therapeutic interventions for the treatment, temporary remission or prevention of the disorder. For example, the term "treatment" includes acceptable from a medical point of view the intervention for different purposes, including delay or stop the progression of myelofibrosis, regression, or elimination of the lesion, prevention of onset or prevention of recurrence of myelofibrosis.

The present invention also relates to a method of applying the above carrier for delivery of the drug to produce extracellular matrix cell of the bone marrow. This method includes, but without limiting to this, for example, stage d�the Addendum to the bearer of the delivered material, the stage of introduction or addition of a carrier, the carrier deliver the substance in the body or environment, such as culture medium, which contains producing extracellular matrix cell of bone marrow. These stages can be properly achieved in accordance with any known method or this method, as described in this description of the invention. The delivery method may be combined with another delivery method, for example another method of targeted delivery to bone marrow. Moreover, the method includes variant embodiment, performed in vitro, and a variant of an embodiment in which target is producing the extracellular matrix of the bone marrow cell inside the body.

The present invention also relates to a new miRNAs (siRNA) against mouse HSP47, preferably one that aims to part selected from plots of the position 1130 to the position 1145, position from 1485 to position 1500, the position of the position 1501 to 1516, position from 1654 to the position of 1678 and position from 1951 to 1978 position sequence SEQ. ID NO:13. Although the methods of construction and receipt of miRNAs against a specific portion of a gene, to suppress the expression of a specified gene, is known in the art, as a rule, it is impossible to predict the area of the gene that should be targeted, and this can only be set through the experiment. In this Fig�thenia, thanks to the strong inhibitory effects on the expression of HSP47 is preferred miRNAs targeting the area from the position 1501 in position 1516, and miRNAs targeting the area from 1951 to position the position of the 1978 sequence SEQ. ID NO:13. Some preferred examples of new miRNAs of the present invention consist of the following combinations of the sense chain and antisense chain.

Sequence A: combination:

5'-UGGAUGGGAAAGAUGCAGAAGAAGGAG-3' (sense, SEQ ID NO:1) and

3'-UAACCUACCCUUUCUACGUCUUCUUCC-5' (antisense, SEQ ID NO:2).

Sequences: combination:

5'-UGUCUGAGUGGGUAUUUUUAGACAGAG-3' (sense, SEQ ID NO:3) and

3'-UAACAGACUCACCCAUAAAAAUCUGUC-5' (antisense, SEQ ID NO:4).

Sequences: combination:

5'-GAUGCGAGAUGAGUUGUAGAGUCCAAG-3' (sense, SEQ ID NO:5), and

3'-UACUACGCUCUACUCAACAUCUCAGGU-5' (antisense, SEQ ID NO:6).

Sequence D: combination:

5'-CAGAACUGCCCAUCCUUAAAAUGAUAG-3' (sense, SEQ ID NO:7), and

3'-UAGUCUUGACGGGUAGGAAUUUUACUA-5' (antisense, SEQ ID NO:8).

Sequence E: combination:

5'-GAGACAAGAUGCGAGAUGAGUUGUAAG-3' (sense, SEQ ID NO:9) and

3'-UACUCUGUUCUACGCUCUACUCAACAU-5' (antisense, SEQ ID NO:10).

Among them, sequences C and D are particularly preferred due to their strong inhibitory effect on the expression of HSP47.

miRNAs of the present invention may have a natural structure of RNA, or can also have various modifications aimed at improving stable�spine in vivo or affinity to the target sequence. Such modification includes, without limitation to this, modified by the terminal amino group, thiol group, cholesterol, long-chain alkyl, a sugar chain or peptide, and so on, the formation of the AP-site, the introduction of modified nucleic acids such as locked nukleinovoi acid (LNA), and peptide nucleic acid (NCP), a nucleotide modified at the 2' position of sugar, such as 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro-modified nucleotide.

miRNAs of the present invention is extremely useful for suppressing the expression of HSP47 in mice and to suppress collagen production that is associated with the expression of HSP47, and may be particularly suitable for use in research, experiments and tests on mice.

Examples

Example 1: Confirmation of the pathology in a mouse model of myelofibrosis.

Thrombopoetin And (SRW) transgenic mouse obtained Dr. Kazuya Shimoda and Dr. Mine Harada (below may also be known as TRO mouse; see Leukemia Research 29:761-769, 2005), was used as a mouse model of myelofibrosis. In this mouse TRO excessively produced in the cell, transfetsirovannyh TPO gene, resulting in the proliferation of megakaryocytes in the bone marrow. Spread megakaryocytes produce in excess transforming growth factor-beta (TGF-β), which stimulates the bone marrow fibroblasts that�obstet secretion of collagen by fibroblasts and leads to fibrillation bone marrow (see Fig.2)

TRO mouse (courtesy of Kyushu University Animal Center) were bred under normal rearing conditions, and then were subjected to euthanasia at the age of 4, 7, 9, or 12 month, their bone marrow was collected for preparation of tissue samples, which were painted by staining with hematoxylin-eosin (he) staining on Gittery or staining on Azan, respectively, and images of bone marrow were obtained using an optical microscope. The results are shown in Fig.3 through 5.

Fibrillation was not visible at the age of 4 months (4M). However, at the age of 7 months (7M), although thickening of the trabeculae are not visible (GE), hyperplasia of reticular fibers (Getter) and deposition of collagen (Adhan) was observed (see Fig.3).

At the age of 9 months (9M) and 12 months (12M) was markedly thickened trabeculae (GE), was also observed hyperplasia of reticular fibers (Getter) and deposition of collagen (the Adhan). Moreover, fibrillation bone marrow and thickening of the trabeculae progressed (increased) in 12M compared to 9M (see Fig.4).

In the GOE samples of bone marrow TRO mouse trabecular thickening began with the age of 9 months (9M), and further increased at 12 months of age (see Fig.5).

Accordingly, the development of symptoms of myelofibrosis was confirmed at TRO mice.

Example 2: Production of miRNAs.

miRNAs targeting murine HSP47 (miRNAs SP47), were obtained in the form of drugs that inhibit the activity of bone marrow cells that produce extracellular matrix. More specifically, five miRNAs HSP47 (miRNAs HSP47 from A to E) having the following sequences, and random miRNAs were synthesized and were used in the experiments described further in this document. miRNAs HSP47 were purchased from iGENE Therapeutics, Inc. (Tokyo), and target sequences of miRNAs HSP47 were designed based on the Refseq database (GenBank Accession No. NM_009825), registered in November 2006. Random miRNAs was also purchased from iGENE Therapeutics, Inc. (product name: scramble dsRNA).

miRNAs AND HSP47:

5'-UGGAUGGGAAAGAUGCAGAAGAAGGAG-3' (sense, SEQ ID NO:1)

3'-UAACCUACCCUUUCUACGUCUUCUUCC-5' (antisense, SEQ ID NO:2)

miRNAs IN HSP47:

5'-UGUCUGAGUGGGUAUUUUUAGACAGAG-3' (sense, SEQ ID NO:3)

3'-UAACAGACUCACCCAUAAAAAUCUGUC-5' (antisense, SEQ ID NO:4)

miRNAs WITH HSP47:

5'-GAUGCGAGAUGAGUUGUAGAGUCCAAG-3' (sense, SEQ ID NO:5)

3'-UACUACGCUCUACUCAACAUCUCAGGU-5' (antisense, SEQ ID NO:6)

miRNAs-D HSP47:

5'-CAGAACUGCCCAUCCUUAAAAUGAUAG-3' (sense, SEQ ID NO:7)

3'-UAGUCUUGACGGGUAGGAAUUUUACUA-5' (antisense, SEQ ID NO:8)

miRNAs-E HSP47:

5'-GAGACAAGAUGCGAGAUGAGUUGUAAG-3' (sense, SEQ ID NO:9)

3'-UACUCUGUUCUACGCUCUACUCAACAU-5' (antisense, SEQ ID NO:10)

Random miRNAs:

5'-CGAUUCGCUAGACCGGCUUCAUUGCAG-3' (sense, SEQ ID NO:11)

3'-UAGCUAAGCGAUCUGGCCGAAGUAACG-5' (antisense, SEQ ID NO:12)

Also miRNAs were obtained, labelled on the 5 conce fluorescent dye�it 6'-carboxyfluorescein.

Example 3: Properties of primary cultured bone marrow fibroblasts TRO mice.

Primary culture of fibroblasts of bone marrow was obtained by culturing bone marrow cells from TRO mice aged 4 to 6 weeks in MEM (minimum essential medium eagle, Sigma) with addition of 15% fetal calf serum (FCS) for 4 weeks. Fig.6 shows the morphology of cells obtained under the inverted microscope. The cells are spindle shaped, typical of fibroblasts. Fig.7 shows the results of flow-cytometric analysis using antibodies to markers of mesenchymal cells to Vimentin and α-SMA, respectively (anti-Vimentin antibody (Santa Cruz Biotechnology) and anti-α-SMA antibody (Santa Cruz Biotechnology)). Watched the expression of both markers, demonstrating that the cells obtained from the culture were typical bone marrow fibroblasts. In the analyses used a flow cytometer FACS calibur (Becton Dickinson), and measurement data were analyzed using CellQuest software (Becton Dickinson).

Example 4: Effect of miRNAs on HSP47 NIH3T3 cell (cell line of mouse fibroblasts).

1×105NIH3T3 cells suspended in the medium Needle, modified Dulbecco (DMEM, Life Technologies) with 10% calf serum (CS) and were sown in 6-well culture plates. After 24 hours NIH3T3 cells at 50-60% conflue�particularly miRNAs were transfected by HSP47, with the use of Lipotrust (Hokkaido System Science Co., Ltd.). Specifically, 20Nm Lipotrust and 50 nm random miRNAs or miRNAs HSP47 mixed on the vortex and used for transfection. Transfetsirovannyh NIH3T3 cells were cultured for 4 hours in medium OPTI-MEM without serum (GIBCO). Then NIH3T3 cells were washed with DMEM and then cultured for 24 hours in DMEM with 10% CS, and extracted protein. Then the expression of HSP47 was analyzed by Western blot. Namely, a protein extracted from NIH3T3 cells was fractionally using 4/20 SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane. First, it treated or the first antibodies against HSP47 (Stressgen) or the first antibody against β-actin (Cell Signaling Technology), then treated with a second antibody, conjugated with peroxidase (Oncogene Research Product), and finally showed using ECL (Amersham Life Science).

The result showed that 24 hours after the transfer of miRNAs HSP47 in NIH3T3 cells miRNAs-and-D HSP47 had a stronger effect compared to A, b and E (see Fig.8A). Accordingly, we applied HSP47 miRNAs-C and-D as miRNAs HSP47 in future experiments.

Example 5: Effect of miRNAs on HSP47 primary cultured fibroblasts bone marrow obtained from TRO mice.

5×105primary cultured fibroblasts of bone marrow obtained from TRO mice, suspended in MEM with addition of 15% FC, and were sown in 6-well culture plates. After 24 hours, bone marrow fibroblasts at 50-60% of confluently miRNAs were transfected by HSP47, using Lipotrust Specifically, 20 nm Lipotrust and 50 nm random miRNAs or 5-50 nm miRNAs-C or-D HSP47 mixed on the vortex and was used for transfection, or they were further mixed with 40 nm of vitamin A (retinol, Sigma) or vortex after 5 minutes, and used for trospective. Fibroblasts bone marrow, travelround or vitamin a-conjugated or vitamin a-unconjugated liposomes with HSP47 miRNAs, were cultured for 4 hours in OPTI-MEM without serum. Then these bone marrow fibroblasts were washed with MEM, and further cultured for 48 hours in MEM with addition of 15% FCS, and were extracted with protein. In different experiments, the bone marrow fibroblasts, transfetsirovannyh 50 nm vitamin a-conjugated liposomes with miRNAs-D HSP47 (VA-Lip-HSP47 miRNAs-D; here next "vitamin a-conjugated" and "liposomes" can be abbreviated as "VA" and "Lip", respectively), were cultured for 4 hours in OPTI-MEM without serum and washed with MEM, and then further cultured for 24 to 96 hours in MEM with addition of 15% FCS prior to protein extraction. Then extracted protein were analyzed for the expression of HSP47 using Western blot. Specifically, extracted from fibroblasts of bone marrow protein was fractionally using 4/20 SDS-polyacrylamide selelect�of forese (SDS-PAGE) and transferred onto nitrocellulose membrane. Then, similarly to Example 4, the membrane was treated with the first antibodies against HSP47 or β-actin, then treated with a second antibody, conjugated with peroxidase, and finally showed using ECL. The results are shown in Fig.8B and 8C.

When used in primary-cultured bone marrow fibroblasts, no miRNAs WITH HSP47 (Fig.8, A) or HSP47 miRNAs-D (Fig.8, B) alone showed no effect. However, when conjugation with vitamin A (VA), was confirmed by demonstration of the effect of miRNAs WITH HSP47 (VA-Lip-HSP47 miRNAs-C) at a concentration of 50 nm or higher, while the VA-Lip-HSP47 miRNAs-D showed the effect at a concentration of 25 nm or higher. Accordingly, it became clear that it is necessary to apply the VA-Lip-HSP47 miRNAs for effective transfer of HSP47 miRNAs in primary cultured fibroblasts, bone marrow, and which VA-Lip-HSP47 miRNAs-D has a stronger inhibitory effect on HSP47 compared with VA-Lip-HSP47 miRNAs-C. Thus, it was decided to apply in the experiments VA-Lip-HSP47 miRNAs.

In addition, it became clear that the effect of VA-Lip-HSP47 miRNAs-D (50 nm) can be stored up to 72 hours (see Fig.8C).

Example 6: Effect of vitamin A (VA) on the introduction enclosed in liposomes miRNAs HSP47 (Lip-siRNA miRNAs) in primary-cultured bone marrow fibroblasts obtained from TRO mice.

Primary cultured bone marrow fibroblasts obtained from TRO mice, transfi�Aravali Lip-HSP47 miRNAs or VA-Lip-HSP47 miRNAs, conjugated with the 50nm carboxyfluorescein (FAM) (Lip-HSP47 miRNAs-FAM or VA-Lip-HSP47 miRNAs-FAM) in ART-MEM culture medium with addition of 10% CS, in the presence or absence of 10 mg/ml interministeriale protein antibody (anti-RBP-Ab, BD Pharmingen), and analyzed using flow cytometry after 30 minutes. Mean intensity of fluorescence (SIF) of the transfected cells was measured using FACS calibur and analyzed using CellQuest software (Becton Dickinson). 5×105primary cultured fibroblasts of bone marrow obtained from TRO mice were sown in 6-well culture plates. After 24 hours added 50nm VA-Lip-HSP47 miRNAs-FAM or Lip-HSP47 miRNAs-FAM. These cells were cultured for 30 minutes in ART-MEM plus 10% FCS, and washed in PBS and subsequently fixed with 4% paraformaldehyde (25°C, 15 minutes). After fixation, the nucleus of these cells was stained with DAPI for 1 minute. Intracellular localization of FAM was evaluated using a fluorescence microscope. Typical pattern of flow cytometry and a typical result subcellular localization of FAM-labeled miRNAs shown in Fig.9A and 9B respectively. Seth, as shown in Fig.9A. It is clear that in comparison with Lip-HSP47 miRNAs VA-Lip-HSP47 miRNAs shows higher transfection efficiency in primary cultured bone marrow fibroblasts obtained from TRO m�Shay (A and b). In addition, since the introduction of the VA-Lip-HSP47 miRNAs was partially suppressed anti-RBP-Ab (A), it was suggested that a partial seizure VA-Lip-HSP47 miRNAs can be carried out via the receptor for RBP (Retinol Binding Protein) fibroblasts bone marrow.

Example 7: the Effect of HSP47 miRNAs on the secretion of collagen primary cultured bone marrow fibroblasts obtained from TRO mice.

5×105primary cultured bone marrow fibroblasts suspended in MEM with addition of 15% FCS, and seeded on 6-well culture plates. After 24 hours introduced 50nm or Lip-miRNAs random (miRNAs SLC), Lip-HSP47 miRNAs-D (miRNAs (D), VA-Lip-miRNAs random (VA-miRNAs SLC) or VA-Lip-HSP47 miRNAs-D (VA-miRNAs (D). After 4 hours, the culture medium was replaced with MEM with the addition of 15% FCS, before culturing for 48 hours. Then the culture medium was removed and replaced with OPTI-MEM without serum, before culturing for 4 hours. After that, first, measured the collagen content in the culture supernatant using a kit Sircol™ Collagen Assay (Biocolor). Namely, the culture supernatant was mixed with the dye solution for 30 minutes, then the solution was centrifuged at 10,000 × g for 10 minutes. After removal of unbound dye solution was added 1 ml of alkaline reagent to a linked dye and stirred on a vortex for 10 minutes, determined quantitative�e made on the basis of the absorption coefficient, measured using an absorption spectrometer (540 nm). Secondly, the amount of collagen deposited on the fibroblasts attached to the culture plate was quantified by addition of the dye Sirius red and the measurement of the absorption coefficient using an absorption spectrometer (540 nm).

The result is shown in Fig.10. Please note that "Culture Supernatant" in the drawing shows the content of collagen in the culture supernatant of fibroblasts, while "Cup (culture supernatant of deleted)" shows the amount of collagen deposited on fibroblasts after removing the culture supernatant, and "Total" shows the sum of "Culture Supernatant" and "Cup (culture supernatant of deleted)", respectively. Data are presented as the mean of 3 cultures ± SD (*P<0.05).

As a result, it became clear that the amount secreted in the culture supernatant of collagen primary cultured fibroblasts transfected by VA-Lip-HSP47 miRNAs-D was significantly less compared with other cells; that cells transfected with VA-Lip-HSP47 miRNAs-D, the amount of collagen deposition on a culture plate was smaller, but not significantly, compared with other cells; and that in transfected VA-Lip-HSP47 miRNAs-D fibroblasts, the amount of collagen secreted into the supernatant ku�of Tory fibroblasts and the number pending on the fibroblast of collagen, after removal of the culture supernatant was significantly less compared with other cells.

Example 8: the Effect of miRNAs on HSP47 fibrillation bone marrow TRO mice in vivo.

We studied the effect of VA-Lip-HSP47 miRNAs-D in vivo to improve fibrillation bone marrow of mice at the age of 7 months. Intravenously via the retro-orbital plexus, was administered 12.5 mg miRNAs-D on the mouse HSP47 using a tuberculin syringe, a day, a total of 4 doses of injections. Namely to 12.5 mg/mouse miRNAs (8 µl) and 12.5 nm Lipotrust (12,5 µl) either with or without 25 nm of vitamin A (2,5 μl) is then added RNase pure PBS to a total volume of 100 μl, which was administered to mice as a single dose. Mice were euthanized 8 days after the initiation of miRNAs-D HSP47, and was harvested bone marrow for the preparation of tissue samples, each of which was stained with hematoxylin-eosin (he) staining, the staining on Gittery or Adhan, and image bone marrow was examined under an optical microscope. The results are shown in Fig.11-14.

Painted by Gitter samples after treatment (Fig.11) it became clear that the hyperplasia of reticular fibers in bone marrow was significantly improved in two of the mice, which were injected with VA-Lip-miRNAs HSP47 (VA-Lip-miRNAs HSP47 (1) and (2)), compared with mice without treatment (No treatment), mice which were injected with Lip-miRNAs HSP47 (Lip-consider� HSP47) and mice which were injected with random VA-Lip-miRNAs (VA-Lip-miRNAs SLC). In addition, the level of improvement hyperplasia of reticular fibers using miRNAs HSP47 was measured and calculated using software KS-400 (Carl Zeiss). Namely, it was selected 10 optical fields of the samples after treatment, stained Gittery, and was measured by the ratio of points reticular fibers against all points in each field (the percentage of the area retikulinovye fibrosis) as an index of hyperplasia of reticular fibers. It was confirmed that the hyperplasia of reticular fibers in the bone marrow was significantly improved in mice, which were injected with VA-Lip-miRNAs HSP47-D (VA-miRNAs (D), compared with mice without treatment (No treatment), mice, which were injected with Lip-miRNAs HSP47 (miRNAs (D), and mice that were administered a random VA-Lip-miRNAs (VA-miRNAs SLC) (see Fig.12).

In addition, on the basis of Azan-stained samples after treatment, as shown in Fig.13, it was found that the collagen hyperplasia in bone marrow was significantly improved in two of the mice, which were injected with VA-Lip-miRNAs HSP47 (VA-Lip-miRNAs HSP47 (1) and (2)), compared to mice without treatment (No treatment), mice, which were injected with Lip-miRNAs HSP47 (Lip-miRNAs HSP47), and mice that were administered a random VA-Lip-miRNAs (VA-Lip-miRNAs SLC).

Moreover, on the basis of colored GE samples after treatment, as shown in Fig.14, it was found that trabecular thickening was significantly improved in two mice, which�first injected with VA-Lip-miRNAs HSP47 (VA-Lip-miRNAs HSP47 (1) and (2)), compared with mice without treatment (No treatment), mice, which were injected with Lip-miRNAs HSP47 (Lip-miRNAs HSP47), and mice that were administered a random VA-Lip-miRNAs (VA-Lip-miRNAs SLC).

Based on these results, it was suggested that it may be useful for the treatment of myelofibrosis with miRNAs targeting HSP47. Also due to the fact that miRNAs mainly act in the cytoplasm, these results suggest that retinoid acts as directing agent to produce extracellular matrix cell of bone marrow and effectively delivers the drug to the cell, thereby significantly improving the pathology of myelofibrosis.

1. Delivering the substance to the carrier for delivery of substances to the cell of the bone marrow that produce extracellular matrix containing a retinoid as a directing agent, where the substance is a medicament that suppresses the onset, progression and/or relapse of myelofibrosis.

2. Media of claim 1, wherein the retinoid contains retinol.

3. The carrier according to claim 1 or 2, where the content of retinoid is 0.2-20 wt.% of the total weight of the carrier.

4. The carrier according to claim 1 or 2, where the carrier has a form of liposome, and the molar ratio of retinoid to the lipid contained in the liposome is 8:1-1:4.

5. Composition for the treatment of myelofibrosis, containing a drug, which�OE suppresses the beginning, the progression and/or relapse of myelofibrosis, and a carrier according to any one of claims. 1-4.

6. A composition according to claim 5, where medicine that suppresses the onset, progression and/or relapse of myelofibrosis, is a drug that inhibits the activity or growth of bone marrow cells that produce extracellular matrix.

7. A composition according to claim 6, where a drug which inhibits the activity or growth of bone marrow cells that produce extracellular matrix, is selected from the group consisting of a means for inhibiting activity or production of a bioactive substance selected from the group consisting of gelatinase And, gelatinase and In the angiotensinogen gene inhibitor of cellular activity, growth inhibitor, an apoptosis-inducing means, and miRNA, ribozyme, antisense nucleic acid and DNA/RNA chimeric polynucleotide that focus on at least one of the molecules that form the extracellular matrix, or molecules, involved in the production or secretion of these molecules that form the extracellular matrix, and a vector that expresses the indicated miRNA, a ribozyme, antisense nucleic acid and DNA/RNA chimeric polynucleotide.

8. A composition according to claim 7, where the molecule is involved in the production or secretion forming extracellular matrix molecules, made�is HSP47.

9. A composition according to any one of claims. 5-7, where the drug and carrier are mixed at a place of medical treatment or nearby.

10. Kit for preparing a composition according to any one of claims. 5-9 containing one or more containers that contain either individually or in combination the drug that suppresses the onset, progression and/or relapse of myelofibrosis, a retinoid and a substance which is a component carrier different from retinoid.



 

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2 cl, 2 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: method relates to medicine, namely to surgery. Figured longitudinal cut is performed on pre-planned lines with formation of trapezoidal and triangular cutaneous-subcutaneous flaps of palm surface of finger. Cuts coincide or intersect transversal folds at angle not less than 45 degrees. Changed fascia is excised. 0.25% novocaine solution is introduced into the centre of formed flaps. Wound is sewn without skin defect. Antihypohant and antioxidant therapy is carried out.

EFFECT: method of combined surgical treatment prevents formation of rough scar, ensures good cosmetic effect.

1 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of veterinary. Method includes detection, instrumental examination of injured place and treating impact on the region of injury. In carrying out aspiration of bone marrow from sternum of injured animal, preliminarily sternum localisation is detected by palpation. Local anesthesia is performed and in the course of it trepanopunctures of spongy substance from sternum bone are carried out with medical needle for bone marrow biopsy, provided with mandrin and lateral holes on its end, with 1.5-2 cm step to the depth of penetration into sternum bone up to 1 cm. Bone marrow is sampled in volume 250-500 ml. In realisation of each puncture, aspiration of bone marrow dose is performed with syringe, containing mixture of NaCl 0.9% solution and heparin, it is filtered and all doses of bone marrow are collected into sterile airtight plastic bag, which contains 43 ml of anticoagulant CPDA. After that, bone marrow is separated with isolation of mesenchymal stem cell concentrate by means of apparatus for cell separation Sepax S-100 with volume 20-50 ml. Under control of injured place with ultrasound devices and/or thermography, boundaries of place to be treated and points of cell material introduction are marked with a marker, after which it is injected into the mass of injured ligament or tendon. After introduction of stem cells, injured segment of extremity is immobilised.

EFFECT: method increases efficiency and reduces treatment terms.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical composition for treatment of osteoarthritis, which includes clodronic acid and hyaluronic acid or their salts as active components and appropriate fillers.

EFFECT: invention also relates to method of treatment of osteoarthritis, which includes intra-joint introduction of efficient quantity of combination of clodronic acid and hyaluronic acid or their salts.

18 cl, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to therapy and can be used for undifferentiated connective displasy (UCD) correction. Method involves introduction of the medicinal agent intended for normalising gut organisms and containing bifidus bacteria in therapeutic doses 20-30 min before meals within 3-4 weeks.

EFFECT: when prescribed, the medicinal agent containing bifidus bacteria allows for direct connective metabolism control thus lowering and improving activity of the antioxidant systems.

6 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns to new diamides of pyrimidine-4,6-dicarboxylic acid of I formula, selective inhibitors of collagenases possessing properties which concern to the metalloproteinase superfamily and the matrix metalloproteinases. The bonds render influence on hyperactivity of the matrix metalloproteinase-13 (MMP-13) and thus do not render influence on MMP-3 and MMP-8. In the formula I R1 means an atom of hydrogen, R2 means - (C1-C6)-alkyl where alkyl is unitary replaced by phenyl where phenyl is replaced 1) -(C0-C6)-alkyl-C(O)-N(R9)-(R10), where R9 and R10 identical or different and independently from each other mean i) atom of hydrogen or ii) - (C1-C6)-alkyl or R9 and R10 together with atom of nitrogen to which they are bound, form 5, 6-links the sated cycle, and instead of one or two other atoms of carbon there can be also a heteroatom from an oxygen row, sulphur and nitrogen, and in case of nitrogen atoms of nitrogen independently from each other can be unsubstituted or substituted with (C1-C6)-alkyl, 2) -(C0-C6)-alkyl-C(O)-NH-SN, 3) -O-(C0-C6)-alkyl-C(O)-N(R9)-(R10) where R9 and R10 have the specified above value, 4) -(C0-C6)-alkyl-C(O)-N (R8)-(C0-C6)-alkyl-N(R9)-(R10) where R8 means hydrogen, R9 and R10 have the specified above value, 5) -(C0-C6)-alkyl-C(O)-N(R8)-(C0-C6)-alkyl-Het, and R8 has the specified above value, and Het means the sated or nonsaturated monocyclic heterocyclic system with number of links from 3 to 6 which contains in a cycle of 1 or 2 identical or different heteroatoms from a number nitrogen, oxygen and sulphur and unsubstituted or one-, two- or triple independently from each other is replaced by halogen, b) hydroxy,) -(C1-C6)-alkyl, and alkyl is unsubstituted or one-, two- or triple is substituted by halogen, d)=0,e)-Het, R4 and R5 or R5 and R6 together with atom of Carboneum to which they are bound, independently from each other form 5 or 6-unit cycle which is sated and contains one or two heteroatoms from an oxygen row.

EFFECT: obtaining of bonds which can find application for treatment of degenerate diseases of joints, such as osteoarthritis, rheumatic disease.

7 cl, 3 tbl, 117 ex

FIELD: medicine.

SUBSTANCE: method involves applying three courses of apitoxitherapy given every other day. One bee sting is given in one session at the first and the third day, two bee stings are given at the fifth and the seventh day, three bee stings are given at the ninth and eleventh day, four bee stings are given at the thirteenth and fifteenth day, five bee stings are given at the seventeenth and nineteenth day at the first treatment course. The second treatment course is given later after 3-4 days long pause. Two bee stings are given at the first and the third day, three bee stings are given at the fifth and the seventh day. Four bee stings are given at the ninth and eleventh day. Five bee stings are given at the thirteenth and fifteenth day. Six bee stings are given at the seventeenth and nineteenth day. The third treatment course is given later after 3-4 days long pause. Three bee stings are given at the first and the third day, four bee stings are given at the fifth and the seventh day. Five bee stings are given at the ninth and eleventh day. Six bee stings are given at the thirteenth and fifteenth day. Seven bee stings are given at the seventeenth and nineteenth day.

EFFECT: reduced treatment laboriousness; prevented allergic response occurrence.

FIELD: medicine.

SUBSTANCE: invention relates to application of pharmaceutical and cosmetic compositions containing placental growth factor-1 (PLGF-1) and respective pharmaceutical and cosmetic compositions for treatment of pathologies associated with excess production and deposition of sclerotic collagen, dermatosclerosis, skin aging and loss of hair. It was discovered that PLGF-1 is capable of angiogenesis enhancing in skin and hypodermic tissues and vitals connective tissues.

EFFECT: effective composition for improvement of skin tonus and appearance.

21 cl, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention represents an encapsulated liposomal antiviral agent based on human interferon alpha-2b for vaginal application, characterised by the fact that each capsule is made in the form of a hollow coating, which encloses a powder excipient and liposomes distributed in the excipient, and sodium alginate, a water-soluble polymer gel former; the excipient consists of lactose, sodium chloride, 12-aqueous disodium hydrogen phosphate and sodium dihydrogen phosphate, whereas each of the liposomes represents a hollow coating containing lecithin, cholesterol and alpha-tocopherol, and a nucleus inside the coating and containing recombinant human interferon alpha-2; the ingredients of the agents are taken in a certain ratio, mg.

EFFECT: maintaining the storage activity of recombinant human interferon alpha-2 and prolonged action in vaginal application.

2 cl, 3 dwg, 6 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry and represents a method for reducing extracellular matrix producing cells in lungs or suppressing an increase of the extracellular matrix producing cells in lungs, involving administering into an individual a composition containing (i) a carrier containing retinoid as a targeting agent, and (ii) a pro-drug specified in a group consisting of siRNA, RNA enzyme, anti-sense nucleic acid and DNA/RNA chimeric polynucleotide, which is targeted on HSP47, and vectors expressing said siRNA, RNA enzyme, anti-sense nucleic acid and/or DNA/RNA chimeric polynucleotide.

EFFECT: invention provides using retinoic acid as a targeting agent for the drug delivery to the extracellular matrix producing cells in the lungs.

8 cl, 6 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel crystalline modification (R)-DOPC, which can be applied in pharmaceutical industry. Claimed is novel crystalline form of DOPC and method of its obtaining, as well as its application as component in obtaining medications. Claimed method consists in the fact that crystallisation of (R)-DOPC is carried out in aprotic solvent.

EFFECT: claimed crystalline form of DOPC is characterised by improved stability.

14 cl, 5 ex, 2 tbl, 11 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a pharmaceutical composition for treating bladder cancer. The above composition contains an effective amount of valrubicin and dimethyl sulphoxide, as well as polyethoxylated castor oil or one or more substances specified in trimethyl chitosan, mono-N-carboxymethyl chitosan, N-diethylmethyl chitosan, sodium caprylate, cytochalasin B, IL-1, polycarbophil, Carbopol 934P, N-sulphate-N,O-carboxymethyl chitosan, Zonula occludens toxin, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, and represents a dosage form for intra-bladder administration by instillation. The invention also refers to liposomal pharmaceutical compositions containing valrubicin, and methods of treating bladder cancer involving administering the above compositions.

EFFECT: invention reduces bladder irritation and increases the clinical effectiveness in bladder cancer.

12 cl, 3 dwg, 3 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of invention refers to medicine, namely to oncology, and can be used in treating cancer in a patient. The method involves administering at least one encapsulated chemotherapeutic agent, and at least one amphiphilic block copolymer in this patient. What is also presented is a composition, a kit for treating cancer in the patient and using the amphiphilic block copolymer.

EFFECT: group of inventions provides potentiating the encapsulated chemotherapeutic agent by stimulating the active chemotherapeutic agent release from liposomes by the use of the amphiphilic block copolymer, which is poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, in the composition.

10 cl, 11 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: inventions refers to medicine, namely to pulmonology, and can be used for treating a pulmonary disorder in a patient. That is ensured by administering an effective dose of the nebulised liposomal amikacin formulation of 100 to 2,500 mg daily within the cycle of treatment, which involves the period of administration from 15 to 75 days and the following withdrawal period from 15 to 75 days. The cycle of treatment repeats at least twice.

EFFECT: invention provides improving the pulmonary function, which is supported for at least 15 days after the termination of treatment, and increasing the one-second forced expiratory volume (FEV1).

28 cl, 16 tbl, 11 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry and represents a composition for the external treatment and prevention of infections caused by the type 1, 2 herpes virus and bacterial complications caused by the herpetic infection, containing lysozyme, peroxidase, povyargol as active ingredients, escin and glycyrrhizinic acid or its salts as anti-inflammatory ingredients, liposomes on the basis of high-active hydrated lecithin in a combination with cholesterol as carriers and pharmaceutically acceptable carriers and excipients, with the ingredients of the composition being taken in certain proportions, wt %.

EFFECT: invention provides extending the range of products for treating and preventing the infections caused by type 1, 2 herpes virus and bacterial complications caused by the herpetic infection.

4 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmaceutics and represents a pharmaceutical composition for parenteral administration containing sub-micron particles of dosocahexaenoic acid ester dispersed in a water phase with the use of mixture of at least two surfactants specified in a) at least one fatty acid polyoxyethylene ester and b) at least one phospholipide derivative, as well as a method for preparing the above pharmaceutical composition.

EFFECT: invention provides higher pharmacological activity.

14 cl, 3 dwg, 3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: stabiliser includes modified chitosan which is obtained by modifying chitosan particles located in an emulsion of an organic solvent - water, with pH 6.0-6.5, by first reacting a mixture consisting of a carboxylic acid in an organic solvent and a condensing agent, and then with an organic base, wherein the carboxylic acid used is either palmitic acid or stearic acid or dodecanoic acid, the condensing agent used is a mixture of hydroxysuccinimide and an aliphatic carbodiimide or formaldehyde and an aliphatic isocyanide, and the organic base used is triethylamine.

EFFECT: effective liposome composition stabiliser which can be obtained using a simple method.

8 cl, 3 tbl, 5 ex, 7 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a carrier applicable for the local drug delivery. A drug is enclosed in a carrier, and the carrier comprises a coating able to release an enclosed drug as a result of a local stimulus. The coating additionally surrounds the contrast agent MR 19F which changes its detectability after being released from the carrier. The invention refers to a method for the drug delivery to an MRT-controlled individual, wherein the method involves administering the above carrier into the individual enabling the carrier releasing the drug, and forming MR 19F images with the use of a contrast produced by the contrast agent MR 19F.

EFFECT: invention enables monitoring the beginning of the drug release from the carrier.

18 cl, 11 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: declared is a pharmaceutical composition for oral administration, containing methisoprinol, methyl n-hydroxybenzoate, propyl-n-hydroxybenzoate, saccharose or maltose, glycerol, citrus flavouring and purified water.

EFFECT: composition has a pleasant flavour and storage-stable.

2 ex, 1 tbl, 5 dwg

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