Agent for increasing reserved stem cell number in organism

FIELD: medicine.

SUBSTANCE: invention refers to radiation synthesis nanotechnology for the creation of a low-immunogenicity agent to increase the reserved stem cell number in an organism, and can be used in regenerative medicine. The agent represents polymer immobilised hyaluronidase introduced in the dissolved low-molecular water-soluble polymer pre-exposed to ionising radiation in dosage 1-5 Mrad, to final concentration 70 UN in 1 ml.

EFFECT: invention allows producing immobilised hyaluronidase which is characterised by substantially higher ability to increase the reserved stem cell number in an organism and effective both in parenteral, and in oral introduction.

4 tbl, 5 ex

 

The invention relates to the field of experimental biology and medicine, specifically to pharmacology and cellular technologies, and can be used in regenerative medicine.

There are ways to increase the content of progenitor cells of different classes in certain tissues using linear-restrictively and randalstown growth factors [1].

Known for the ability to grow the pool of stem cells in the body with the help of the native drug hyaluronidase [2, 3].

The disadvantages of this tool are the side effects and complications [4, 5]associated with the immunogenicity of hyaluronidase, which represents the protein preparation. In particular, systemic and local allergic reactions of varying intensity (up to anaphylactic shock) when it is administered parenterally, representing the only possible target of this enzyme. In this regard, the importance of the problem of creating funds with the ability to increase the pool of stem cells in the body, which would have no adverse effects observed with parenteral use of native hyaluronidase.

The problem solved by this invention is the creation of tools that increase the reserve of stem cells in the body that do not have immunogen the STI, effective as parenteral and oral administration.

This object is achieved by immobilization of hyaluronidase on the native low molecular mass media with ionizing radiation. As the carrier used biologically indifferent substance is a water - soluble polymer with a molecular weight of 400-4000 Yes.

The most preferred method of immobilization is the impact on the polymeric carrier and the biologically active compound with a directed stream of accelerated electrons with energy of 2.5 MeV electrons, absorbed dose from 2 to 10 kGy, the speed of the set dose of 1.65 kGy/hour.

As the water-soluble polymer used polyethylene, gidroxiatilkrahmal, polyvinylpyrrolidone, etc. with a molecular weight of from 400 to 4000 Da.

New in the present invention is the creation of tools, which is a hyaluronidase, mobilisierung using ionizing radiation, increasing the reserve stem cells in the body that do not have immunogenicity and effective as parenteral and oral administration.

We use the original tool immobilizovannoi using ionizing radiation at low-molecular carrier hyaluronidase was developed and received the Institute of pharmacology WITH the Russian Academy of medical Sciences (Tomsk) with whom jointly with LLC "Scientific Future Management" (Novosibirsk).

Currently very promising for the treatment of various diseases look how cell therapy. The most physiological and reasonable approach to solving problems of regenerative medicine is pharmacological stimulation of endogenous stem cells (SC) [6]. However, the regulators of the functions of the IC in the vast majority are protein compounds for use as drugs for systemic use is very limited due to their high immunogenicity [4, 5]. There are ways to overcome the immunogenicity of protein drugs with glucocorticoid hormones, antihistamines, etc. However, the use of these drugs is often accompanied by the development of unwanted side effects and complications specific to them [6, 7].

At the same time a significant reduction in immunogenicity substances observed in enteral medication. However, the means protein in the gastrointestinal tract are split under the influence of proteolytic enzymes, which makes this way of introducing them into the body ineffective. At the same time, there are data on the reduction of immunogenicity and increase the stability of proteins to proteases during their immobilization on low-molecular-weight carriers [8]. Know the ten drug - logidata representing conjugate hyaluronidase ("Lydasum") and polyoxidonium obtained by chemical synthesis [9]. However, information about the possibility of reducing the immunogenic properties of hyaluronidase, as well as ways to create a drug with high hyaluronidase activity is effective, including when administered, due to the immobilization of enzyme molecules on the native low molecular weight with the use of ionizing radiation, to date, does not exist. Moreover, no data exists about the pharmacological benefits of the drugs obtained by immobilization of biologically active substances using ionizing radiation, before the connections formed by the conjugation of the same original substance, but by chemical means. That is not aware of the existence of qualitative differences of the final product is related to the manner of its production: the technology of chemical synthesis (which are longido) and electron beam technology immobilization (with which you are obtaining the offer). At the same time, it is known that the process of manipulation of heterogeneous substances at the molecular level [10, 11], including the use of physical factors with high energy, can be accompanied by a significant change is mi stereochemical structure of the source substances, and, ultimately, lead to modification of their properties, the nature of which is often unpredictable.

The fact immobilization of hyaluronidase on the media using ionizing radiation to a new technical result: the creation of tools that increase the reserve of stem cells in the body that do not have immunogenicity and effective parenteral and oral administration, for the specialist is not obvious.

The essential features of the claimed showed together new properties that are not derived explicitly from the prior art in this field. The present invention can be used in experimental biology and medicine with access to practical health care. Identical set of features in the study of the prior art in the patent and scientific and medical literature is not found.

Based on the above, you should consider the claimed solution meets the criteria of "Novelty", "Inventive step", "Industrial applicability".

The experiments were conducted on mice-male CBA/CaLac in the amount of 106 pieces, weighing 18-20 g, 27 Guinea pigs of either sex weighing 200-250 g Animals obtained from the nursery of the Department of experimental biomedical modeling, Institute of pharmacology, SB RAMS.

Example 1

10% aqueous solution of p is litlington with a molecular weight of 400 Da was irradiated with a stream of accelerated electrons at a dose of 1.5 Mrad. The processing performed by the bremsstrahlung radiation generated by the accelerator ILU-6 with the electron energy of 2.5 MeV, the absorbed dose of 2 kGy, the speed of the set dose of 1.65 kGy/hour. In the irradiated solution was made hyaluronidase ("Lydasum", FSUE "NPO Microgen" MOH) to a final concentration of 70 IU hyaluronidase in 1 ml of 10% polyethylene glycol. The mixture was stirred 10 minutes and got the final immobilizovannyi the drug in the form of a slightly opalescense solution. Yield is 96,2%.

Example 2

5% aqueous solution of hydroxyethyl-starch with a molecular weight of 1.5 kDa was irradiated with a stream of accelerated electrons at a dose of 1.5 Mrad. The processing performed by the bremsstrahlung radiation generated by the accelerator ILU-10 with the electron energy of 2.5 MeV, the absorbed dose of 10 kGy, the speed of the set dose of 1.65 kGy/hour. In the irradiated solution was made hyaluronidase ("Lydasum", FSUE "NPO Microgen" MOH) to a final concentration of 70 IU hyaluronidase in 1 ml of 5% hydroxyethyl-starch. The mixture was stirred 10 minutes and got the final immobilizovannyi the drug in the form of a slightly opalescense solution. Yield is 97.3%.

Example 3

Studied specific activity imgd. As the comparison drug used "Longido" (NPO Petrovax Pharm. Russia), which is a chemical conjugate of hyaluronidase ("Lydasum") with polyoxidonium.

3, 5, 8 days using the method of limiting dilutions was determined by the number of mesenchymal (true) stem cells (MSCS) [12], as well as a method of cloning in poluvyazkie environment studied the content of hematopoietic stem cells (HSC) - granulocyte-Erythro-macrophage-megakaryocyte units (CFU-GEMM), commiteeman mesenchymal (CFU-f) and granulometry (CFU-GM) progenitor cells in the bone marrow [13], represents the modern view depot SC in the body [6].

Analysis was performed by the method of variation statistics using t-test, t-test and non-parametric U-test, Wilcoxon-Mann-Whitney. The frequency of occurrence of MSC in bone marrow and peripheral blood were determined using a generalized linear model for Poisson distribution. Compliance data limiting dilutions of one-dimensional Poisson model was estimated via a linear log-log regression. In this case, theoretical fraction of negative wells µidistributed as µi=exp(-fxi), where f is the frequency of MCK, xithe number of cells planted in the hole [12]. Was performed with the program Statistica 6.0.

The drug immobilizovannoi using electron-beam exposure on poliatilenglikole hyaluronidase (imgd) at a dose of 1000 U/kg, was administered to intact the animal once intraperitoneally and orally. Control mice at equivalent volume (0.2 ml) intraperitoneally and orally injected drug comparison "Longido" (NPO Petrovax Pharm. Russia) at a dose of 1000 IU/kg

Introduction animal study drug in all cases, except for oral administration of the drug comparisons resulted in a significant increase in the content of the analyzed progenitor elements in the bone marrow.

Intraperitoneal injection of longitute number MCK, SOME GEMM, CFU-f and CFU-GM was increased throughout the experiment and reached maximum values at the 5 day experience.

Use imgd led to significantly more pronounced and lasting changes from the studied pools of progenitor cells. Moreover, it should be noted that the maximum effectiveness of the proposed tool was shown in relation to the earliest precursor cells: MSC and CFU-GM. Moreover, practically there were no differences in the groups of animals receiving the drug in different ways (oral and parenteral) (table 1).

Example 4

The tool was obtained by immobilization of hyaluronidase on molecules hydroxyethyl-starch using different types of ionizing radiation: 1) gamma radiation (imgd-1), 2) ultraviolet radiation (imgd-2) and (3) laser radiation (imgd-3). Drugs immobilization the Oh DG (LLC "Scientific Future Management", Novosibirsk, Russia) at a dose of 50 IU/kg was administered to intact animals orally 1 time a day for 5 days.

Introduction imgd in all cases led to significant and virtually identical to the increase in the content of early (MSK, SOME GEM) and commiteeman progenitor cells (CFU-f) in hematopoietic tissue. The greatest value increase of indicators took place in relation to MSC and SOME HAM on the 8 days of the experiment (table 2).

Table 2
Dynamics of the content of stem cells and commiteeman precursor cells of haematopoiesis in the bone marrow of mice CBA/CaLac with the introduction of DG immobilizovannoi using gamma radiation (imgd-1), ultraviolet radiation (imgd-2) and laser (imgd-3), (X±m)
The time frame of the study (day)MSC 106the myelokaryocytesCFU-f, 250 thousand myelokaryocytesCCM (SOME GAMM), 105the myelokaryocytes
background18,0±2,46,3±0,515,67±0,57
3rdimgd-129,0±0,6*12,4±0,63*11,9±1,1*
imgd-232,0±1,3*11,7±0,21*12,3±0,9*
imgd-331,0±0,84*10,9±0,36*12,1±0,87*
8thimgd-143,0±0,7*17,3±1,2*9,87±0,4*
imgd-241,0±1,4*18,6±0,97*10,12±0,56*
imgd-342,0±0,9*19,3±0,56*9,67±0,39*
* - marked significance of differences with the background values at p<0,05

Example 5

Comparative study of the allergenic (immunogenic) properties of drugs hyaluronidase, immobilizovannoi on polyethylene glycol with ionizing radiation (imgd), and "Longido" (NPO Petrovax Pharm., Russia).

Preparations were examined in the same doses (defined as units actively the ti hyaluronidase), which was determined to be optimal in relation to the stimulation of stem cells, and 10 times its excess (contents hyaluronidase 100 and 1000 IU/kg, respectively). Assessment of the allergenic properties of the drug was performed using the following tests: total reaction anaphylaxis reactions specific agglomeration of cells, skin and conjunctival testing on Guinea pigs, the reaction of delayed-type hypersensitivity in mice [14].

For sensitization of Guinea pigs medicines were introduced: first, 1 times subcutaneously, then the same dose 2 times intramuscularly, every day, in the thigh. Anaphylactogenic properties of hyaluronidase preparations revealed by intracardiac injection on the 21st day after the last sensitizing injections. Allow testing intracardiac injection of the drug was carried out at a dose equal to the total sensitizing, ie 300 IU/kg served as Control desensibilisation Guinea pigs, which the drug was administered by intracardiac.

Accounting for the intensity of the anaphylactic reaction was performed using the formula

,

where AI - anaphylactic index;

n - number of animals, anaphylactic reaction which was fatal;

n1- the number of animals with significant manifestations of the Academy of Sciences of the vention reaction;

n2- the number of animals with secondary manifestations of the reaction;

n3- the number of animals with weak manifestations of reaction;

N is the total number of animals in the group.

For evaluation of the skin sensitizing properties of the drug in 20 days after the end of the sensitizing injections on the side surface of the body of Guinea pigs was vestigal wool, and each animal was injected intradermally drug imgd or longido in doses of 10 IU/animal (50 IU/kg) in 0.02 ml of physiological solution (dose not cause visual changes skin intact Guinea pigs).

Skin reaction to the drug was evaluated in points with regard to the severity of hyperemia and size of damaged area (average diameter in millimeters). Identified four degrees of hyperemia, which corresponded to a numeric indexes: strong (+++) - 1,0; moderate (++) - 0,66; weak (+) - 0,33; equivocal (+/-) is 0.17. Multiplying the diameter of the damaged area on the index of the corresponding degree of hyperemia, allowed in a single index (points) to Express both the characteristics of the response.

On the 20th day after the end of the cycle sensitizing injections were performed conjunctival testing. The test consisted of instillation into the left eye of each animal 0,02 ml of physiological solution containing 10 IU of the drug, in the right eye - physiological the practical solution in the same volume. Assessment of the conjunctiva of the eye was performed after 4 and 24 hours after exposure.

To confirm the results of the above tests, we used the reaction alergodiagnostiki in vitro of RSAL (reaction specific agglomeration of cells). The working dose of hyaluronidase preparations for this reaction was 5 IU per 0.05 ml of blood.

In mice sensitization was performed using a full adjuvant's adjuvant (PAF). Preparations of hyaluronidase was injected once subcutaneously at the base of the tail in 0.06 ml PAF, which was taken in the ratio 1:1 by volume solution of the drug. The dose was 1000 mg/kg Control animals were injected with PAF in the same amount.

Through 5 days after the injection of the test and control mice in the tip of one of the hind paw was injected drugs hyaluronidase at a dose of 30 IU/animal 0.04 ml of physiological solution, in other paw - saline. 24 hours after the second injection with engineering micrometer type MK measured the thickness of both legs. The degree of response was defined as the difference in the thickness of the experimental and control legs in millimeters.

During the experiment it was found that the reaction of Guinea pigs by intradermal injection of the drug imgd on such visual indicators, such as redness or swelling, did not differ from the corresponding CSP is the GDP in animals which was administered distilled water, while with the introduction of longitute skin reaction was significantly more pronounced. Conjunctival test and reaction alergodiagnostiki in vitro showed no signs of allergic action in imgd, and found those in the comparison drug. When testing intracardiac administration of drugs imgd and longitute was a statistically significant increase in anaphylactic index in the group of animals treated longido, and the AI was greater than one (table 3).

Table 3
Assessment anaphylactogenic properties imgd and longitute in the experiment on Guinea pigs
The group of animalsIndicators
The reaction of the skin, pointsConjunctive. test scoresThe coefficient of RSALAI
Control0,75±0,0301,6±0,10,38
Longdata0,9±0,03*0,7±0,01* 1,65±0,21,1
imgd0,76±0,0401,55±0,20,56
* - the reliability of differences in the rate from its value in the control at p<0,05

In addition, the study allergenic (immunogenic) properties showed that intradermal testing injection imgd, unlike longitute, did not lead to a change in the value of the reaction GST compared with a control level indicator after 4 and 24 hours after exposure (table 4).

Thus, a drug immobilizovannyi using ionizing radiation hyaluronidase, has no immunogenic effect, manifested allergization of an organism, while the usual conjugate of an enzyme with the carrier has allergenic properties.

Table 4
Assessment of the sensitizing properties of the drug RCG-CSF in the experiment on mice (X±m)
The conditions of the experimentThe reaction GST, mm
The group of animalsSensib the lytic dose, U/kgResolution Units µg/kg4 hours24 hours
Control-10000,068±0,010,071±0,01
Longdata100010000,094±0,005*0,09±0,004*
imgd100010000,071±0,020,79±0,01

Thus, a means of representing mobilisierung on low molecular weight media using ionizing radiation hyaluronidase, is able to significantly increase the content of stem cells of different classes in the body and has no immunogenicity. The effectiveness of this tool significantly higher than that in the known analogues. Moreover, the peculiarities of the technology of this tool leads to the possibility of its effective use of not only dropped, but inside.

Cited literature:

1. Metcalf D. Hemopoietic growth factors. 1. The Lancet. - 1989. - Vol.15. - P.825-827.

2. Goldberg ED, Digi A.M., Sushkov GN. and other the Role of hyaluronidase in reg the regulation functions of mesenchymal precursor cells. Cell technologies in biology and medicine. - 2007. No. 2. - S-119.

3. Sushkov G.N., Zhdanov V., Digi A.M., Goldberg ED the Role of hyaluronidase in the regulation of haematopoiesis. Bull. the experimental. Biol. and medicine. - 2007. No. 12. - S-695.

4. Anderson J.A. Allergic reactions to drugs and biologic agents. JAMA. - 1992. - Vol.268. - p.2845-2857.

5. De Swarte R.D., Drug allergy. In: Patterson R. e.a. Allergic Diseases Diagnosis and Management, 4th ed. Philadelphia, PA. JB Lippincott. - 1993. - P.396-551.

6. Goldberg ED, Digi A.M., Zhdanov B.B., Sushkov GN. and other Pharmacological aspects of regenerative medicine. Bull. the experimental. Biol. and medicine. - 2008. - Annex 2. - P.14-21.

7. Vial So, Descotes J. Clinical toxicity of cytokines used as haemopoietic growth factors. Drug Saf. - 1995. - Vol.13. No. 6. - P.371-406.

8. Vereschagin E.I., Khan Do-Hung, et al. Radiation Technology in the Preparation of Polyethylene Oxide Hydrophilic Gels and Immobilization of Proteases for Use in Medical Practice. Arch. Pharm. Res. - 2001. - V.24. - N 3. - P.229-233.

9. Dubnicka L.V. Nazarenko T.A. Possible drug Logidata® in adjuvant therapy of pathological changes of the endometrium. Russian medical journal. - 2008. - T.16. No. 19. - S-1251.

10. Seyfulla RD, Timofeev A.B., Ordzhonikidze SG and other Problems of the use of nanotechnology in pharmacology. Experimental and clinical pharmacology. - 2008. No. 1. - P.61-69.

11. Y.M. Yevdokimov Spatially ordered forms of DNA and its complexes is the basis for the creation of nanoconstructs for medicine and biotechnology. Russian nanotechnology. - 2006. - №1-2. - S-264.

12. In't Anker P.S. Noort W.A., Scherjon SA e.a. Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica. - 2003. - Vol.88. - P.845-852.

13. Goldberg ED, Digi A.M., Shah V.P. Methods of tissue culture in Hematology. - Tomsk: Publishing house of Tomsk state University, 1992. - 272 S.

14. Manual on experimental (preclinical) study of new pharmacological substances. Edited Rugarama. - 2 ed. - M.: JSC "Publishing house "Medicine", 2005. - P.54-69.

The means increasing the reserve stem cells in the body, representing mobilisierung on low molecular weight water-soluble polymer hyaluronidase, wherein the hyaluronidase immobilizovana on the polymer by introducing into the solution of low molecular weight water-soluble polymer, pre-exposed to ionizing radiation at a dose of 1-5 Mrad, to a final concentration of 70 IU per 1 ml



 

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