JPH07506584A - Use of exogenous glycosaminoglycans or derivatives in the treatment of thrombocytopenia - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] In the treatment of thrombocytopenia Utilization of exogenous glycosaminoglycans or derivatives The present invention provides a method for treating thrombocytopenia. Glycosaminoglycans and similar substances for the preparation of drugs for therapeutic purposes. The invention relates to the use of products and their sublayers, fragments and derivatives.

Glycosaminoglycans (GAGS) are primarily trisaccharide sequence rules. It is a polysaccharide consisting of repeating uronic acids (D-glucuronic acid or L-iduronic acid). -hexosamine (glucosamine or glucosamine)]. these substances exists in several forms.

In other words, heparin, heparan sulfate, chondroitin sulfate (4-8 and 6-3), dermatan sulfate and hyaluronic acid. The first three are sulfuric acid Variously substituted with groups, hyaluronic acid does not contain sulfate groups. various oxide units The glycosidic bond between them varies depending on the shape, and is of ]-4 or 1-3 type. (CasuB, Advanced in Carhohydrate Chemistry Ant Bio Chemistry (Advances in Carbohydr) ate Chemistry and Biochemistry), Akademitsu Academic Press Inc., +985 ．． 43. pp. 51-133 Ruoslahti and Yamaguchi, Ce. ll), 1991.64. (described on pages 867-869).

Glycosaminoglycans are constituents of cells as well as extracellular stroma. The Glyca Glycans carry a significant negative charge and contain many substances of cationic character. Gives the ability to combine. Therefore, the glycans possess several biological activities. have.

Analogs of glycosaminoglycans include dextran sulfate, bentosan polysulfate, and sulfate. Oxidized alginic acid, sulfated lactobionic acid. Polyvinyl sulfate and Sacral It is understood to mean a fart-like substance. These substances are often It also possesses valuable pharmacological properties.

Glycosaminoglycans and more specifically heparin and heparan sulfate fractions, fragments and derivatives of which have or do not have anticoagulant activity Regardless, these are also known.

In fact, glycosaminoglycans, their analogs, as well as their sublayers.

Fragments and derivatives, especially heparin, heparan sulfate and dermatan sulfate , has been the subject of many investigations that have made it possible to demonstrate some biological activity.

These activities include anticoagulant activity, the ability to complex various proteins, activity of the complement system, activity as a regulator or modulator of the development of certain defective or non-defective cells, and vascular morphology. (Casu B, Advanced in Carbohydrate Chemistry Ant Bio Chemistry (Advances in C Arbohydrate Chemistry and Biochemis try), Academic Press I nc, ), +985.43.51-133 pages, Seminar Inthrombosis Seminar in Thrombosisand Haemostasis), 1991. 17. Addendum, l and 2.1-24 Page 6: Developmental Nonheparin Glycosaminoglycans Cerabute 4 Tsuku Agent (Development of no nhep-arin glycosaminoglycans asther apeutic agents), both of whom are guest editors, J., 81. Wale nga, J, Fareed, Teime Medical I Kubri 7 Yards Ink 1. New York (Thieme Medical Publisher sInc, New York): Ruoslahti E, and Yam agUChi Y,, Cell.

1991, 64. Pages 867-869: Lincoln U, Mtibi ー　INT　R.E.B. EM, NiSER, TOU, Carbohydrate 1- (MTPInt, Rev, Sci, Org, Chem, Ser, Two, Carbo hydrate), +976, 7゜pp. 283-312).

Some proprietary medicinal drugs contain glycosaminoglycans or their Contains fragments or derivatives. For example, magnesium heparate is xevalin. A proprietary medical drug marketed in France under the name (Cutheparine■) It is the active predisposition of the product. This is an anticoagulant that suppresses several clotting factors. It is used to prevent and treat thromboembolism and thrombotic conditions. This is also a low molecule Heparin fragments known as heparin also include, for example, natrovaline or dalteparin. active ingredients in drugs such as Nic, Nicsavarin, Parnavarin or Tinzavarin It is also the cause.

These active factors have a well-known antithrombotic effect, especially a strong effect against activated factor X. In addition, “Dicnoyonnare Vidar” (D) has a weak anticoagulant effect. ictionnaire Vidal)”, 1992.-r-Dinoyono Dou Hidal Bali (εd mountain on du Vidal, Paris).

Patent application P-A-0037319 covers such products and their manufacture. The method is described.

Furthermore, the activation of heparin cofactor ■ by heparin and dermatan sulfate , λ1. Researched by Tolfesen (N.O.U.B.N.R. Eevee, Hematol, (Nouv, Rev.

Fr, llematol, ) 1984. 26. No, 4. 233- 237 pages). This author describes the effects of heparin and dermatan sulfate on clotting factors. describes the impact and suggests the use of these two products in this same field. Ru.

Patent application EP-A-0199033 also describes prevention of venous thrombosis or arteriosclerosis development. Or the therapeutic dermatan sulfate and heparan sulfate II I! I! f ,; Describes the use of combinations of stones.

Dex sulfate) is also used as an anticoagulant (J, E, P, Reyn.

lds, “Martinsor The Extra Pharmacavia CMar+1n Dale, The Extra Pharmacopeia) 1989. The The Pharmaceutical Press Press), p. 1564). Furthermore, patent application EP-A-027031 7 is a low molecular weight sulfur compound that exerts inhibitory activity against cell fusion and viral adsorption. Acid dextran derivatives are described and used for the treatment of diseases caused by viral infections; Discloses the value of these products in prevention.

Sodium hyaluronate is used during surgical procedures in the anterior chamber of the eye. J Used as a drug. This product provides mechanical protection to the septum and prevents adhesions from forming. (“Dicjionnai Vidar”) re Vidal)”, 1992.Edition Dou Vidal Bali (Edition du Vidal, Paris)) o This same product It is also used to treat arthritis (J, E, F, Reynolds, “Mar. Tinzor, The Extra 7 Furuma Pier (λ1 artindale, The Extra Pharmacopeia)” 1989. The Pharmaceutical Press ), p. 1617).

Bentosan polysulfate is called Hemoclar [F] (Hemoclar @l). The active predisposing agent of a proprietary medical product marketed in France under the name It also has fibrinolytic properties and has an activity in the release of lipoprotein base. Exercise (“Dictionary Vid” al)”, 1992. Nibuinoran Do Bidart Paris (Edition du Vidal, Paris), and “The Merck Index” (T he NIerck Index)'', +989. Merck & Two.

ink,.

(Wlerck and Co, Inc.,) Marway N.G. United States of America, (Rahway N, J,, USA,) 1131 pages, n o, 7090).

Chondroitin sulfate is a physiological component of the cornea and is used for symptomatic treatment of lachrymal insufficiency. be done. This naturally occurring mucopolysaccharide glycoprotein compound has a long-term effect on the hydrophobic cells of the eye. (= 1 cause of wear and therefore maintains effective protection ("disk") Dictionnaire Vidal”, N19 2. Nibuinolan 1-”Upidard, Paris (Edition du Vi dal, Paris)). Chondroitin sulfate is also associated with cardiac ischemia and osteoporosis. Also used (J, E, F, Reynolds, “Martinsall Zaek Stra Pharmacabia (Martindale, The Extra P pharmacopeia)”, 1989. The Pharmacy Group The Pharmaceutical l’ress, page 1556 ). Elsewhere in this document, this product is said to increase white matter activity 1'1 to prevent is also described (“The Klerck Index”). dex)”, 1989. Merck Antoko Inc. 6. (λIerck and Co, Inc.,) Marway N.J.

United States of America, (Rahway N.J., USA,) 344 pages, no. , 2217).

Patent application BP-A-0287477 describes the inhibition of angiogenesis or smooth muscle cells. can be used to modulate certain physiological systems, such as inhibiting the proliferation of It describes low molecular weight heparins with

Patent application εP-A-0244298 discloses that heparin exists in natural heparin and specific affinity for cationic or anionic cell growth factors that distinguish at least 5 monosaccharide units (glucosamine derivatives and describes oligosaccharides containing rononic acid). These fragments are produced during cell division. It has been described as having regulatory activity on cell differentiation as well as muscle tissue. , accelerate the repair of heart or skin tissue, and promote healing of traumatic lesions. and whether angiogenesis is pathological, either alone or in combination with steroids. Applies to the deterrence process.

Patent US-3466365 covers polyvinyl sulfate derivatives with antiviral activity. Describes thorium or potassium salts.

Lactobionic acid poly(H-sulfate) derivatives having anti-sleeving activity and the like The salts are described in patent US-4258034.

Mononuclear cell formation involves the development of hematopoietic stem cells into megakaryotic cells, the proliferation of mononuclear cell precursors, and involves the maturation of megakaryocytes that lead to the development of platelets (klazur E , λ (0, megakaryocyte boiesis and platelet production (&Iegakaryocytopoiesis and platele) tproduction): A Review E-XP Hematol ( Review, EXIl, llemajol), 1987゜15.　 Pages 340-350: ) Loffman R,.

Regulat-io 9 years old megakaryocyte boiesis n or megakaryocytopoiesis), Brad (tllo od), 1989.74°+196-1212). Platelets are involved in certain physiological processes , especially plays an extremely important role in blood coagulation (Caen J, P., Cro nberg S, and Kubisz P,.

Platelet Physiology and Pathology ysiology and pathology), - knee yoke strut International (New York, 5tratton) ational), 1977).

Huge nucleus! I? I follicle formation is induced by mononuclear cell colony stimulating factor (Dini-CSF), Ikin-3 (lL3), inleukin-6 (rL6), granulocyte-macropher Dicolony stimulating factor (GλI-C5F) and erythroboietin (EPO) has been shown to be directly regulated by a number of hematopoietic growth factors such as . Mononuclear cell formation is also caused by interleukin-1 and interleukin-11. However, it is indirectly controlled (IJazur, p. 1212).

Among these factors, IL3. EPO and GM-CSF in normal subjects It has been used in vivo in both patients and patients. Stimulation of megakaryotic cell 1a formation by these factors Efficacy is limited, especially in cases of aplastic anemia and secondary hematopoiesis deficiency ( Peters W, P, The Miniroid Coro 211 Stimulating Factors (The myeloid colony-stimulati) ng factors), Preface and Compendium, Seminars in Hematroaro, 66 6-676). This reveals the existence of another mechanism governing mononuclear cell formation. I have to. It should be noted that each growth factor has different activities. mosquito Therefore, excessive administration of these drugs may cause unexpected and untoward side effects. Therefore, it cannot be taken into consideration (1!etcalf D,, the Consifen Su of Fcess Levels Talents Hematopoietisoku Growth Fa The Consequences of Excess 1eve ls of haematopoietic growth factors), P.R., J., Hematol (Br, J., Ilematol).

1990, pp. 75.1-3). Hematopoietic bone marrow is important in their woody and functional , a complex system formed by different cells. The organization consists of 5 cell compartments. It can be thought of as i.e., stem cells, hematopoietic progenitor cells, and maturation of the entire hematopoietic system. Hematopoietic cells, microenvironment and auxiliary cells. After a series of deterioration and maturation, stem cells become Contains selected cell precursors: gives rise to progenitor cells, which in turn are transferred to blood cells as described above. Produce a plate.

All we know is that closed room fi targeting the growth factor CJI-CSF. 1M to demonstrate the presence of endogenous broaoglycans that play a role in granule formation. and tGordon^1.71 etc. 1. Convert Mentalization Talent A hematopoiete ink growth factor (GM-CSF) pie Glycosaminoclycans in the bone marrow microenvironment ( ”Compartmentalixation ofa haematopoi etic growth factor (G^human 3F) by glycos aminoglycansin the bone marrow micro environment+”, Neci+-(Nature).

+987.326.403-405 pages; Roberts R, et al., Heparan Sulph-1 Bound Growth Factors, A Mechanism Fo - Stromal Cell Mediated Hemoboiesis ifate bound growth factors, A meshan ism for stromal cellmediated haemopo iesis), Neci+-(Nature), 1988.332.376- 378 pages).

However, the effects of exogenous glycosaminoglycans on the cell-forming system are unclear. If there is no information, there is no information.

Inadvertently, if exogenous glycosaminoglycans, their analogues, and Once the debris, fragments and derivatives are administered to animals or humans, they are In more detail, in the system investigated, i.e., a cell system in which hematopoiesis is completely different from the myeloid system. , as a specific inducer or modulator of the regulation of mononuclear cell formation in humans or mice. and can be used in therapy for the treatment of thrombocytopenia. It has now been proven that this can be done.

Thrombocytopenia is a well-known condition, especially during anti-cancer chemotherapy. At present, this state There is no effective and economical treatment for curing the rash. Therefore, this pathology It is of paramount importance to find specific and economical means of combating the current situation.

Accordingly, the present invention provides exogenous glycosaminoglycans, analogs thereof, and sublayers thereof. , fragments and derivatives for the production of medicaments for the treatment of thrombocytopenia. It concerns its use as a special inducer of nuclear cell formation.

Non-split heparins that can be used to manufacture these drugs are Pig or bovine heparin extracted from mucous membranes or any other organ.

As an example, low molecular weight heparin that can be used is natrovaline calcium (INN). CY 222 + Laboratories Choy, France (Labort-oir es CHOAY, France) + patent PR-2440376 and EP -0037319).

PKIO+69, also known as Nick Sabalin (INN). ! Laboratories Farmka, France (Labortoires Pt1ARLluKA, F Lance): Patent application EP-00401441, Tartvaline (INN) KABI VITRIIl, Sweden ); Thrombosis Earl Enis.

(Thrombosis Res,) (1986) 42.435-447), Parnanocrin or 0P2123 + Obocrin, Italy (OPOCRIN, Italy); patent application εP-0121067 and WO861067291, RD 118851 Hepar Industries, Ohio, USA ( IIEPA RINDUSTRIES, 0hio, USA): Patent application EP -0244235, patent GB-2002406, and GB-20680111. and tinzaparin (INN) or LHN-1 (Novo Industries) , chin mark (NOVOINDUSTRIES, Denmark; patent application B P-0244234, and EP-02442351.

These products are all based on literature (Haemostasis).

1988 Yu8. sup, pp. 3.3-15), and these Most are active factors in proprietary medical products that are already available to physicians.

Thus, for example, natrovaline calcium is a proprietary medical product, fraxoparin ( It is the active factor of Fraxiparine■), and Daltenorin is a proprietary medical product. Fraxiparin is the active factor and enoxane (phosphorus is the proprietary medical product Labnoc) The active factors of Lovenox@) and Flexan (C1exan■) Yes, Teinzaparin is a proprietary medical product called Logiparin (Logiparin @). It is an active factor for Innohep and Innohep, and it is also an active factor for Varna. Parin is the proprietary medical product Fluxam (Fluy+am■) and Minidalton ( This is the activation factor of Minidalton@). For fragments of non-anticoagulant heparin and oversulfated heparin derivatives are those used in the preparation of the medicament according to the present invention. and are described in patent applications EP-0287477 and EP-0214879, respectively. It is stated.

O-acylated heparin derivatives are described in patent application BP-0356275. Ru. Other heparin fragments (hexasatsucaride, octasatucaride, etc.) are patent application E. It is described in P-0244298.

Chondroitin sulfate, dermatan sulfate, hyaluronic acid, and heparan sulfate It is a commercially available product.

In one particular embodiment of the invention, the glycosaminoglycans used chondroitin sulfate 4-3 and 6-3, dermatan sulfate and their are divisions, fragments and derivatives.

In another particular embodiment of the invention, the glycosaminoglycans used The classes are hyaluronic acid and its sublayers, fragments and derivatives.

In yet another particular embodiment of the invention, the glycosaminoglycans used Cans include heparin, heparan sulfate, and their fractions, fragments, and derivatives. be.

In one particularly advantageous embodiment of the invention, the glycosaminoglycans used The classes include heparin and its fractions, fragments and derivatives.

In another advantageous embodiment of the invention, glycosaminoids, such as the following products: Noglycan analogs are used. i.e. dextran sulfate, polysulfate vent San, sulfated artinic acid, sulfated lactobionic acid. Polyvinyl sulfate and steel It's Kralfaat.

Preference is given to using dextran sulfate as well as bentosan polysulfate.

According to the present invention, drugs having no anticoagulant activity or glycosaminochlicans, Advantageously manufactured using analogs thereof, as well as scraps, fragments and derivatives thereof .

Low-molecular-weight heparins, such as natrovaline calcium, are exogenous glycosaminoglycans. It is very preferably used as a lubricant.

In human and mouse in-vivo experiments, normal mice and chemical In vivo experiments conducted on mice that were rendered stunted by therapeutic treatments showed that Cosaminoglycans, their analogues, and their sublayers, fragments, and derivatives are macrokaryotic. It has been shown to be a clear modulator of cell formation.

Stimulatory or modulating effects on the formation of these eye cells and their effects In view of i1M, these products therefore reduce the risk of thrombocytopenia in each It can be used for the treatment of.

That is, - Around each PIT+ mechanism +lIt hypocytopenia (idiopathic II: Immune hematopoiesis (antipeniasis) of purpura and other tumors.

– especially during recovery from chemotherapy (cancer, leukemia, AIDS, etc.) or bone marrow transplantation. Myeloid thrombocytopenia. The impact on bone marrow recovery, especially on mononuclear cells and platelets, is significant. It is said.

Blood H! 1λIK-C3F activity is observed in patients undergoing chemoIM therapy or bone marrow transplantation. It has been shown that the ratio is extremely high. Eazur E, M, etc.

- Pond-type thrombocytopenia, e.g. gray platelet syndrome, which affects the dosage of drugs used These factors include the patient's age and weight, the method and frequency of administration, and the severity of thrombocytopenia. and related treatments.

1. Glycosaminoglycans, their analogues, or their scraps, fragments, or derivatives Body if administered intravenously, intramuscularly or subcutaneously? l per kg, and 1 It varies from 40 μg to 1 mg per dosage unit. For oral administration, body weight It is necessary to administer 40 μg to 10 mg per IKg and per 1 dose. every In the case of parenteral administration, the daily dose is 40 μg to 2 mg per IKg of body weight, and For oral administration, the dose varies from 40 μg to 20 mg.

The present invention can be used for the treatment of thrombocytopenia and One or several types of glycans, their analogues, or their scraps, fragments, or derivatives in combination with a suitable inert excipient for the treatment of thrombocytopenia. Also covers compositions of drugs present as active agents in combination with other compounds that may be present. are doing.

Various pharmaceutical forms, such as injectable solution single tablets, coated tablets, gelatin capsules. herbal medicine products suitable for sublingual administration.

Nanoparticles, microcapsules and solutions for nasal sprays may be considered. Wear. For oral administration, it is preferred to use enteral formulations.

The above drug forms are illustrative and not meant to be limiting. different drug forms If so, other means of administration may be considered.

The invention is illustrated by the following examples containing experimental results obtained in various drug tests. I decided to.

test product Chotroitin monosulfate (6-3), dermatan sulfate, Henonoran sulfate.

Hyaluronic acid ((Sigma Chemical, USA) SigmaChemi cal, USA).

- Sold under the name of Cutheparine O and has a bio of A Non-branched heparin supplied by sedra -Heparin fragment *Natrovaline calcium, a proprietary medical product fragi supplied by Choay Institute in Paris Active factor of ciparin.

*CY222: Described in patent EP-0037319 (Example 1) and A product supplied by Elf 5anofi.

*Product A: One piece of heparin with almost no anticoagulant effect. 11 for this product To do this, heparin was first subjected to a controlled oxidation using periodic acid, and then A demolding table was carried out by adding a strong base. Finally, the product obtained by polymerization was applied for patent application EP- 0287477 (reduced by the method described in Example I). Product A was 6500 Da It has an average molecular weight of 2.3 and a sulfation degree of 2.3. Product A is Elf of Paris Supplied by Elf5anofi.

Wood product B This was produced by the method described in patent application EP-0244298. Hexasaccharide made from heparin. This compound has been applied for the above patent application. The Elf of Paris, Zanofi, is clearly mentioned in ofi) was co-funded by f.

-dermatan sulfate fragment These products were first obtained by demolding dermatan sulfate with periodic acid. Tollefsen D, 8 (1, NV 8 FF Hematol (Nuv, Rev, Fr, Hematol), l98 4.26. No.

4, pp. 233-237). These are clearly defined numbers of monosaccharide units. Consisting of (IJ from L-iduronic acid and D-galactosamine), As shown below. That is, the wood product consists of 0.3 units of force.

Wood product D: Consists of 5 monosatsurido units.

Wood product E: Consists of 7 monosanocalito units.

Wood product F: Consists of 9 monosatsurido units.

*Product G consists of monosasancalito units.

Wood product Hl consists of 13 monosaccharide units.

All of these products are provided by Elf 5anofi in Paris. was provided.

Monoglycosaminoglycan analogs *Sigma Chemical, USA [1, dextran sulfate phased.

*Polysulfuric acid supplied by Bali Elf Sanofi Bentosan.

Example 1 Influence on stimulation of eye cell formation in vivo Mouse femoral bone marrow in a mononuclear cell culture system.

Glycosaminoglycans, analogs thereof, and sublayers, fragments and derivatives thereof The effect on the formation of plucked cells was tested in vivo using a culture system of plucked cell precursors. did. (Han 2. C, etc. 1. C, R.

Ni No Ni De Ni No Eye (C, R, Acad, Sci, ) Par is.

1991, vol. 313.5 series II+, pp. 553-558).

In this culture system, mononuclear cells from mouse femoral bone marrow were collected from mouse Ba1b/c (1FFAcRE[]o). 2. Deplete 10' mouse mononuclear cells. At least three sets of 10% bovine citrate plasma and 1% bovine serum albumin. and 10% mercapto-ethanol (final concentration 10-'M) and 0.34 Contains mg/ml calcium chloride and 10% hypotrophic serum or Add it to a volume of 1 ml of basic medium (α medium) without adding it.

Various amounts of the test product were diluted in a volume of 0.2 ml of alpha medium and each of the various concentrations A product solution is added to the plasma coagulum formed after plasma coagulation. stunted growth of pigs Serum is used for bone marrow culture of these mice. Mononuclear cells and their mice Colonies are confirmed by staining with acetylcoronestase (Ja ckson C, W.

Blood (circa 1973.42.413-42).

■-Results of combination of heparin and heparan sulfate-Effects on formation of eye cell colonies Sound test products, especially non-layered standard heparin, fraxiparin and hepara sulfate was used at various doses shown in Table 1.

Results are based on three separate experiments performed in triplicate. - Mean numbers ± SEM are shown. Statistical significance (*) is relative to p<0.05 Steutint was recognized as W1 using the assay.

(Margin below) Table■ The results shown in Table I are based on standard heparin, Fraxiparin (Fraxiparin). e■ and heparan sulfate are especially rich in the activity known as AIK-CSF. In the presence of stunted pig serum from irradiated pigs, the plasma clot culture system The results show that scales significantly improve the formation of mononuclear cells. On the other hand, They do not significantly stimulate G&I-CFU formation, regardless of the presence of hypotrophic serum. Not intense.

-1 Shadow on the maturation and size of nuclear cells and their colonies W: 500 or more 1 - colonies as well as 100 mononuclear cells were analyzed. Unit + + l’ff! ! Gender (*) is for p<0.05. The results of this test I are shown in Table ■.

Standard heparin, Fraxiparin (Fraxiparine@) and sulfuric acid Paran promotes maturation and increases the size of mononuclear cells and their colonies be able to.

2-Product A, product B and resultant product A with CY222, i.e. periodic acid Product B, which is heparin that has been deformed and has extremely low anticoagulant activity, is Heparin. The regular hexasaccharides and CY222 are also found in plasma coagulation systems. can significantly stimulate the formation of eye-pick cell colonies in cells. these tess i., each cell was cultured in the presence of pig stunted serum.

The results reported in Table ■ are based on three separate heel-neck experiments conducted in three sets. The average number of colonies per cell is 5EII. Statistical significance (*) is p<o , for os.

3-Vt acid fundroitin, hyaluronic acid and dermatan sulfate and their Results from fragments Each product was tested at varying doses from O to 100 μg/ml.

The results of this survey are shown in Table ■.

(Margin below) Sulfur chondroitin, &Itl'jl [dermatan and their fragments and human Aluronic acid, at concentrations of 10-100 μg/mf, r? present added to murine bone marrow cultures under conditions significantly stimulated the formation of eye cell colonies. It can be intense.

The results of testing with 4-glycosaminoglycan analogs are shown in Table ■. .

(*) p<0.05 The results in table It has been shown that it stimulates the formation of eye cell colonies in culture.

Example 2 Influence on stimulation of mononuclear cell formation in living organisms: Culture system for human mononuclear bone marrow cells test The mononuclear cell precursor culture system used was described by Han Z, C, et al., Exp, ) Ie. Major.

+989.17.46-52.

Human mononuclear bone marrow cells were collected from fil individuals (various samples were At least three sets of 2.10' mononuclear bone marrow cells (collected with the consent of Ntia) 10% bovine plasma with citrate, 1% bovine serum albumin, and merca 10% ethanol (R final concentration 10-4 Nl) and calcium chloride 0. 10% stunted serum in a volume of 11 ml of base J3 containing 34 H/ml. Add it to something with or without it. human hypoplastic tlI t is used for human bone marrow culture. Heparin and heparan after plasma coagulation Add to a volume of 0.2 ml of alpha medium on the formed plasma clot (see Example 1). (see).

Human target cell colonies directed against platelet glycoprotein 11h/1lla Confirmed by immunoperoxidase staining using a monoclonal antibody (ll an Z, c, etc. 9. Exp, hematol (Exp, llemaj ol, ), 1989. j7.46-521゜The test product is non-separated IN heparin. and heparan sulfate.

The doses adopted were 1 to 5 ml/ml for heparin and 1 to 5 ml/ml for heparan sulfate. The concentration ranged from 10 to 50 μg/ml. The people hired for this study, iJ, are plastic Zuma♂! This is the block method. The results reported in Table Vl are based on two experiments for t-1 nuclear cells. Showing Ronnie's number of junior uniforms SIEM. Heparin and heparan sulfate, humans The presence of hypotrophic serum is due to the shape of the eye cell colony in regular bone marrow culture. It can be seen that the formation is 1. intense.

These results indicate that if heparin and heparan sulfate were administered at a dose of 51 U/ml, At 0 μg/mf, it induces the growth of human eye cells and shows scales. .

Example 3 Interaction of standard heparin and fraquinparin with growth stamps that affect mononuclear cell formation research for Effects of standard heparin and frachimbaline on the formation of eye cell colonies in mice was investigated in the presence of various growth factors. The results shown in Figure 1 show that at least 3f ! Average number of mononuclear cell colonies based on separate experiments for lfs ± S [λ1 is shown There is. Statistical significance (*) is for p<o, os.

PqKj (heparin and fracinoparin are h) improve the activation ability of hypoplastic blood is replaced by other growth factors In this case, no effect of heparin on mononuclear cell formation is recognized. This example is an analogy IL3 (Genzyme, USA), IL6 (Genzyme.

USA), G&I-C3F (Genzyme, USA) or Ep.

(Amersham, France).

IL3 or 1 of the monoclonal antibody directed against murine 113 or IL5. 16, hypoplasia h'l and fraxiparin, and hypoplasia Mouse megakaryocyte (LIK) colonies induced by whole serum and heparin The influence on the formation of The implementation procedure used is the one described in Example 1. is the same as The results shown in Table ■ below represent the mean ± SEM of 2 to 3 experiments. ing.

Addition of monoclonal antibodies directed against murine IL3 or IL6 Fraxiparin or standard heparin It can be seen that phosphorus activity is not suppressed. This means that the effect of heparin is on IL3 and This suggests that it is unrelated to 116.

(Margin below) Table■ Example 4 In vivo testing on normal mice l- hematopoietic and mononuclear cells and their colony size Rin's! Effect on L-form Fraxiparin [Yes], 0.4.20.40.2 00 and +0001U/kg twice a day for 4r+ (Balb/c) mice were injected subcutaneously. “Control” mice are Acid acid I! l# agent (PBS.

Only Eurobio was administered. The animal received the last injection. Two days later, he was sacrificed due to dislocation of his tibia.

11-CFU, GM-CFU and number of mononucleated cells, as well as blood counts, fl ank, C, etc., C, R, A.C.A.D., Nis.C.I.

Paris (C, R, Acad, Sci, Paris), 1991. 313 ．． Tested using the method described on pages 553-558.

Bone marrow cells were collected from the Ozeki bone, and blood was collected from the heart.

Bone marrow cells are mononuclear cell precursors or in the presence of CFU and granule single cell precursors or GM-CFU; Five sets were recultured.

The number of mononuclear cells (λ1K) was not cultured, but after extraction from the femur, Bone marrow cells inoculated into culture dishes were evaluated. Platelet and white blood cell counts and hemoglobin levels were measured using an automatic hematopoietic counter.

The results shown in Tables ■ and ■ represent the mean ± SEM. Each group has 5 or more More than one mouse included. Statistical significance (*) is relative to p<0.05 It is.

(Margin below) As can be seen, the stimulatory effect of 111 [fragiciparin 0 on platelet production] The effect lasts for at least 511 days after the last 11 shots. These results indicate that in vivo Fraxiparin significantly increases the production of mononuclear cells and platelets. It shows.

3-Other glycosaminoglinos or similar products by 1 minute layer or section 11 111 [Dermatan sulfate, Dex I-lan sulfate (5000D a) Heparin, product A and product B were added to the mouse in the same manner as fragiciparin ■. Injected at a concentration of 10 μg per injection per mouse. bone marrow and blood Samples were collected 2 days after the final injection. The results of this investigation are shown in Table X1.

(Margin below) As in the case of frackinbaline, the test product tested mononuclear cells as well as platelets (product It can be seen that the production of A) can be promoted.

Example 5 5-Fragicipari affecting medullary dysfunction induced by injection of fluoride Balb/ c) R gong, 150 mg/kg to mice (5-10-7 mice for each lit) of 5-fluoride was injected.

After 211, Fraquinparin @ (4o+u/kg) or Product B (4010/k The first one was injected subcutaneously (once a day for 4 days). For “control” mice Phosphate buffer (PBS Eurobio) was used. The animal glares at the neck [1] I made it a sacrifice of 1'1. 1.1 The medullary 11a is obtained from the femur, and the blood is... The viscera was obtained by I.I., f; pulp cells were incubated at a concentration of 5.10'/ml for 1 week. In order to test the number of IK-CFU in the presence of stunted growth in pigs, To celebrate, the 5th group played 'if R' again. The number of mononuclear cells (MK) was not cultured. Alternatively, the bone marrow cells that had been extracted from the femur and then inoculated into a culture dish were evaluated. TafHaguchi, Z, C, etc., C, R, ADSCI, Paris (C,R,Acad,Sci,Paris), 1991.313゜553- 558 pages). Automatically calculates platelet and white blood cell counts and hemoglobin levels. Measured with a blood counter.

The results shown in Table Xll display SE&+ above the mean and are not statistically significant. (ne) is for P<0.05.

(Margin below) 1q results showed that fraquinoparin was administered to mice treated with 5-fluorouracil. This shows that administration of this drug accelerates the recovery of plucked cells. Thus, Frachinobari On days 2 and 3 after injection of N0, the number of MK-CPUs and isolated cells was , are markedly increased in the bone marrow as well as the platelet count.

Dextran sulfate (5000 ml a) and product B were administered filOμli/injection. Mice treated with 5-fluoride uracil after being shot with fragicivalin■ was injected into.

Administration of these two products also accelerated platelet recovery two days after the final injection.

Example 6 Specialities of the action of glycosaminoglycans Supplementary experiments for light B1 [on heparin] Protamine sulfate (Laboratories Choi Bali (L) is the main antidote for The effect of aboratoires Choay Paris) is described in Example 1. Tested in murine bone marrow cultures in the described implementation summary. Protamine is 50-100μg /ml completely neutralizes the activity of Fraxiparin.

Heparinase 1 Sigma United States (Sjgma, USA)] and Co. Introitinase ABC (Sigma, USA) 1 When added at a concentration of 0.1-1 μg/ml, formation of rough cell colonies may be inhibited. Significantly deter. These results indicate that glycosaminoglycans are 1 for the cells. clearly shown to be responsible for the observed activity.

The results of these studies are compared in Tables X1] and X[V, respectively. these indicates the mean ± SEM of 3 experiments. Statistical significance (ne) is p < 0.05 It is for.

(Margin below) Table X lit Effects of protamine sulfate on the effects of fraxivalin in mouse bone marrow I3 nutrition influence Table XIV Hebarinase and chontroitinase AB contribute to colony formation on λ1 in living organisms Shadow of C 9! l (2,10' cells) Jun Funny / 2 * 105111! ! P CT/FR93100458 ,-,,,,,,,-Continuation of PCT/FR93100458 front page (72) Inventors: Lormeaux, Jean-Claude France, Kremlin Piset Tor F -94270 Roux Carnot 16 (72) Inventor: Pettitou, Maurice France, Paris F-75013 Roux Doujabero 65

Claims (13)

[Claims] 1. Exogenous glycosaminoglycans, their analogues, and their scraps, fragments, or is the use of recognized conductors for the manufacture of drugs for the treatment of thrombocytopenia. 2. Exogenous glycosaminoglycans such as heparin, heparan sulfate, analogs or The use according to claim 1, characterized in that is a scrap, fragment or derivative thereof. 3. Claim characterized in that the exogenous glycosaminoglycan is low molecular weight heparin Use as described in Section 1 or 2. 4. Exogenous glycosaminoglycans include chondroitin sulfate 4-S and chondroitin sulfate. Chin 6-S, dermatan sulfate or their scraps, fragments or recognized conductors. The use according to claim 1, characterized in that: 5. If the exogenous glycosaminoglycan is hyaluronic acid or its debris, fragments or The use according to claim 1, characterized in that is a derivative. 6. Glycosaminoglycan analogs include dextran sulfate, pentosan polysulfate, and sulfur Oxidized alginic acid, sulfated lactobionic acid, polyvinyl sulfate and sucralfa 2. Use according to claim 1, characterized in that it is selected from the following. 7. Exogenous glycosaminoglycans, their analogues, and their scraps, fragments, and Any one of claims 1 to 6, wherein the derivative has no anticoagulant effect. Use as described in section. 8. Exogenous glycosaminoglycans, their analogues, or their scraps, fragments or A drug for the treatment of thrombocytopenia characterized by the presence of a recognized conductor as an active predisposition agent composition. 9. Exogenous glycosaminoglycans, their analogs or their scraps, fragments or derivatives The conductor can be used as an active predisposing agent in combination with other compounds that can be used to treat thrombocytopenia. A pharmaceutical composition for treating thrombocytopenia, characterized in that the composition is present in 10. Exogenous glycosaminoglycans, their analogues, and their scraps and fragments administration of an effective amount of the compound selected from the compounds and derivatives to humans in need. A method for treating thrombocytopenia, characterized by: 11. At a daily dose of 40 μg to 2 mg per kg of body weight, 11. The method according to claim 10, characterized in that the product is administered parenterally. 12. Externalization at a daily dose of 40 μg to 20 mg/kg body weight 11. The method according to claim 10, wherein the compound is administered orally. 13. Exogenous glycosaminoglycans, their analogues, and their scraps and fragments Effective amounts of compounds selected from the characterized in that it is administered in combination with an effective amount of the compound to a human in need thereof and treatment methods for thrombocytopenia.