RU2604124C1 - Agent for treating osteomyelitis - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to an agent for treating osteomyelitis. It is suggested to use β-betulonic acid alanylamide of formula (I):

as an agent for treating osteomyelitis.

EFFECT: presented agent effectively prevents generalization of inflammatory process, formation of extensive zones of necrosis and infiltration, osteoclastic bone resorption and formation of sequestra, which in turn prevents the chronic inflammatory process and enables its elimination; promotes earlier and more expressed formation of loose fibrous connective and membrane reticulated bone in comparison to groups without using betamide, which indicates the positive effect of the preparation on the repair processes in bone; causes faster onset of functional adaptation phase, promoting faster completion of endochondral osteohistogenesis to form trabecular bone tissue.

1 cl, 59 dwg, 3 tbl

Description

The invention relates to medicine.

It is known that antibiotics are used to treat osteomyelitis (A. Trampuz, W. Zimmerli Diagnosis and treatment of infections associated with fracture-fixation devices // Injury, 37 (2006), pp. S59-S66; A. Trampuz, AF Widmer Infections associated with orthopedic implants // Curr Opin Infect Dis, 19 (2006), pp. 349-356; T. Miclau, AH Schmidt, JC Wenke, et al. Infection // Orthop Trauma, 24 (2010), pp. 583-586 ; L. Frommelt Principles of systemic antimicrobial therapy in foreign material associated infection in bone tissue, with special focus on periprosthetic infection // Injury, 37 (2006), pp. S87-S94; JA Forsberg, BK Potter, G Cierny 3rd, L Webb Diagnosis and management of chronic infection // J Am Acad Orthop Surg, 19 (Suppl. 1) (2011), pp. S8-S19; HG Schmidt, AH Tiemann, R. Braunschweig, et al. Arbeitsgemeinschaft septische Chirugie der DGOUC10 [Definition of the diagnosis osteomyelitis-osteomyelitis diagnosis scor e (ODS)] // Z Orthop Unfall, 149 (2011), pp. 449-460; R.O. Darouiche Treatment of infections associated with surgical implants // N Engl J Med, 350 (2004), pp. 1422-1429).

So, they use cefazolin, vancomycin, as well as various combinations with rifampicin: rifampicin + ciprofloxacin, rifampicin + levofloxacin (Korean Society for Infectious Diseases; Korean Orthopedic Association Clinical guidelines for the antimicrobial treatment of bone and joint joints in Korea / / Infect Chemother. 2014 Jun; 46 (2): 125-38).

However, these drugs have side effects:

cefazolin (http://www.tiensmed.ru/news/cefazolin-10x.html) - hepatotoxicity, it initiates pseudomembranous colitis, allergic reactions, disorders of the hematopoiesis system, has nephrotoxicity, promotes the development of superinfection;

vancomycin (https://health.mail.ru/drug/vancomycin/) - cardiotoxicity, nephrotoxicity, it causes disorders of the hematopoiesis system, allergic and dermatological reactions;

rifampicin (https://health.mail.ru/drug/rifampicin/) - hepatotoxicity, nephrotoxicity, it initiates pseudomembranous colitis, allergic reactions, thrombocytopenia, eosinophilia, leukopenia, hemolytic anemia, menstrual irregularities;

ciprofloxacin (https://health.mail.ru/drug/tsiprofloksatsin/) - hepatotoxicity, nephrotoxicity, cardiotoxicity, it initiates pseudomembranous colitis, sleep disturbances, hallucinations, visual disturbances, disorders of the blood system, phlebitis, vasculitis, allergic reactions;

levofloxacin (https://health.mail.ru/drug/levofloxacin_2/) - hepatotoxicity, cardiotoxicity, nephrotoxicity, causes sleep disorders, hallucinations, confusion, motor disorders, visual impairment, hearing impairment, hematopoiesis, arthralgia, myalgia, rupture tendons, tendonitis, allergic and dermatological reactions, contributes to the development of superinfection.

Of the above medicines, rifampicin is closest to the claimed drug, but it has a significant negative effect on the body, in addition, its use as a monotherapy for the treatment of osteomyelitis is impractical (W. Zimmerli, AF Widmer, M. Blatter, R. Frei, PE Ochsner , Foreign-Body Infection (FBI) Study Group Role of rifampin for treatment of orthopedic implant-related staphylococcal infections: a randomized controlled trial // JAMA, 279 (1998), pp. 1537-1541).

Betulonic acid β-alanilamide does not have hepatotoxicity, nephrotoxicity, does not cause allergic reactions and endocrine disruption, and can also be used as monotherapy.

Amides and peptides of betulonic acid having antitumor activity are known. So, in [B.B. Saxena, L. Zhu, M. Nao, E. Kisilis, M. Katdare, O. Oktem, A. Bomshteyn, P. Rathnam, Bioorg. Med. Chem. Lett., 2006, V. 14. 6349-6358.] Describes an amide of betulonic acid and N-Boc-lysine, which has specific activity in prostate cancer in vitro and in vivo. Amides of betulonic acid containing fragments of caprylic, pelargonic and undecanoic acids inhibit the growth of tumor cells MT-4, MOLT-4, CEM and Hep G2 [Pokrovsky AG, Shintyapina AB, N.V. Pronkina, V.S. Kozhevnikov, Plyasunova O.A., Schulz E.E., Tolstikov G.A. Reports of the Academy of Sciences. 2006.Vol. 407. No. 5. S. 698-701.]. The betulonic acid dipeptide (N ′ {N- [3-Oxolup-20 (29) -en-28-oil] -9-aminononanoyl} -3-amino-3-phenylpropionic acid) is patented as an antiviral agent (anti-HIV, anti-herpes) and immunostimulating activity [RF patent 2211843]. In the work [Shintyapina A.B., Shults E.E., Petrenko N.I., Uzenkova N.V., Tolstikov G.A., Pronkina N.V., Kozhevnikov B.C., Pokrovsky A.G. Bioorganic chemistry. 2007. T. 33. No. 6, 620-626.] It is shown that this compound inhibits the growth of tumor 3 types of cells - MT-4, MOLT-4, CEM and Hep G2. The dipeptide is an active inducer of apoptosis in leukemia cells and in vitro hepatocarcinoma cells [G. Avtandilov Medical morphometry: a guide. M .: Medicine, 1990. 384 p.].

It is known to use β-alanilamide of betulonic acid [3 oxo-20 (29) -lupen-28-oil] -3-amino-propionic acid, “betamide”) as a corrector for cytostatic polychemotherapy (RF patent No. 2353623). This derivative of betulonic acid is a low-toxic substance (LD ₅₀ over 5000 mg / kg). It has an inhibitory effect on tumor growth in vivo, enhances the antitumor effect of the complex of chemotherapeutic agents and has a pronounced organoprotective effect on the state of the liver, kidneys, heart, thymus, blood, showing a cytoprotective effect.

The cytoprotective properties of betamide were detected in experiments on mice and rats against the background of exposure to cytotoxic drugs simulating CHOP polychemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone) and monotherapy (cyclophosphamide, doxorubicin). Against the background of CHOP PCT, course administration of betamide (7 days at a dose of 50 mg / kg) to mice with transplantable malignant tumors reduces the severity of dystrophic and necrobiotic changes caused by polychemotherapy, improves the state of the microvasculature in the tissues of internal organs, and increases the plastic reserves of cells. In the kidneys of CBA / Lac mice with RLS lymphoma, the number of necrotic nephrocytes decreased by 77%, in connection with which the number of cells with mild hyaline droplet dystrophy increased by 53%, the lumen of the proximal tubule decreased by 43% compared with the PCT group [N. BUT. Zhukova, D.E. Semenov, I.V. Sorokina, T.G. Tolstikova S.V. Pozdnyakova, O.R. Greek. The effect of betulin derivatives on kidney structure in RLS lymphoma / Siberian Scientific Bulletin - IX. Novosibirsk, 2006. Pages 49-51]. In the liver, the volume density of necrosis zones decreased by 63%, and zones with dystrophically altered hepatocytes increased by 19%, while the degree of dystrophy decreased to a weakly expressed hyaline droplet, the volume density of sinusoids increased (by 28%), the intrahepatic cholestasis decreased, the number increased glycogen in all hepatocytes [N.A. Zhukova, D.E. Semenov, I.V. Sorokina, T.G. Tolstikova S.V. Pozdnyakova, O.R. Greek. The effect of betulonic acid and its derivative [3-oxo-20 (29) -lupen-28-oyl] -3-aminopropionic acid on the liver structure of mice with RLS lymphoma / Bulletin of experimental biology and medicine. 2005. Volume 140, No. 9, pp. 348-351].

In the liver of C57B1 / 6 mice with Lewis lung carcinoma, the administration of betamide after PCT caused a 53% decrease in monocellular necrosis and a decrease in the severity of dystrophy compared with the PCT group. In hepatocytes there were practically no zones of cytoplasm “devastation” and lipid infiltration, insignificant subplasmalemmal cytoplasm vacuolization was noted. The introduction of betamide in combination with PCT increased the bulk density of sinusoids by 33-40%) and reduced the number of metastases in them compared with the introduction of PCT alone [N.A. Zhukova, I.V. Sorokina, T.G. Tolstikova, E.L. Lushnikova, L.M. Nepomnyashchikh, D.E. Semenov. Liver structure of mice with transplanted Lewis lung carcinoma during polychemotherapy and correction with betulonic acid and its derivatives / Bulletin of Experimental Biology and Medicine. 2010. Volume 150, No. 7, pp. 108-112]. In the kidneys of these mice, under the influence of betamide, the bulk density of epithelial cells in the state of necrosis decreased by 80-98%, the proportion of cells with hydropic and balloon dystrophy decreased by 54-60%, and the volume of epithelial cells with signs of mild degeneration increased by 170-180%. A decrease in edema of epithelial cells and brush border on the background of the introduction of betamide was accompanied by an increase in the bulk density of the lumen of the proximal tubules (by 50% -75%) [N.A. Zhukova, I.V. Sorokina, T.G. Tolstikova, M.P. Dolgikh D.E. Semenov. The effect of betulonic acid and its derivatives on the kidney morphology of animals with transplanted Lewis lung carcinoma with and without chemotherapy chemotherapy / Chemistry for Sustainable Development. 2010. Volume 18, No. 4, pp. 483-488].

The administration of betulonic acid alanilamide to healthy Wistar rats for 14 days at a dose of 50 mg / kg against a background of a single administration of cytostatics according to the CHOP PCT regimen reduced the number of hepatocytes with dystrophic changes by 75-90% and decreased the number of necrotic cells by 35-45%. The bulk density of binuclear hepatocytes increased by 36-58%), which reflects an increase in the regenerative potential of the liver. In the kidneys, betulonic acid alanilamide significantly weakened the inflammatory effect of cytostatics, reduced the proportion of nephrocytes of proximal tubules with hydropic dystrophy by 65-72%, and reduced the proportion of necrotic changes by 60%. In the thymus, the protective effect of betulonic acid alanilamide was noted, which manifested itself in an increase in the cerebral-cerebral index and a decrease in the number of Gassal bodies in the brain [I.V. Sorokina, T.G. Tolstikova, N.A. Zhukova, N.I. Petrenko, E.E. Schulz, N.V. Uzenkova, O.R. Greek, S.V. Pozdnyakova, G.A. Tolstikov. Betulonic acid and its derivatives are a new group of agents that reduce the side effects of cytostatics // Doklady Akademii Nauk. 2004. Volume 399, No. 2, pp. 274-277; Grek O.R., Pozdnyakova S.V., Nadeev A.P., Pronin B.C., Zhukova N.A., Sorokina I.V., Volkova E.B., Tolstikova T.G. The effectiveness of betulonic acid and its alaninamide derivatives in the restoration of rat liver parenchyma in the post-cytostatic period // Exp. and wedge, pharmacology. 2005, T. 68, No. 6, p. 49-51; Pozdnyakova S.V., Grek O.R., Funtikov A.S., Zhukova N.A., Sorokina I.V. Tolstikova T.G. A comparative study of the nephroprotective activity of betulonic acid and its derivatives in experimental polychemotherapy // Zh. expert. wedge. honey. 2006, No. 1-2, p. 15-17]. The thymus protective effect of betulonic acid alanilamide was observed against a single dose of cyclophosphamide in healthy rats at a toxic dose of 125 mg / kg, which causes persistent inhibition of the blood-forming organs. The protective effect of betulonic acid alanilamide was expressed in maintaining the normal mass of the thymus during a 14-day administration of this drug at a dose of 50 mg / kg. With the combined introduction of betulonic acid alanilamide with doxorubicin, there was a tendency to more rapid restoration of the mass of the thymus [I.V. Sorokina. Ref. diss. Doct. biol. sciences. 2010].

The course administration of betulonic acid alanilamide against the background of a complex of cytostatics (cyclophosphamide, doxorubicin, vincristine, prednisone in doses of 1/20 of LD ₅₀ ) restored the activity of cytochrome P450IIIA4 6 days after exposure and increased the activity of P450IIE1 by 26.5 and 24.1%, respectively 7th and 14th day of observation. [S.V. Pozdnyakova, O.R. Greek, R.A. Zhogol, I.V. Sharapov, V.I. Sharapov, I.V. Sorokina, T.G. Tolstikova. Activity of liver monooxygenases and biological effects of betulonic acid and its amide derivatives // Bulletin of Novosibirsk State University. Series: Biology, Clinical Medicine. 2006. Volume 4, no. 3, p. 66-70].

In numerous in vitro experiments, the antiviral activity of betulonic acid and its amide has been identified, including against human immunodeficiency virus, influenza A viruses, herpes simplex type 1; antibacterial effect against Staphylococcus aureus, Bacillus antracoides, Eschericha coli and Salmonella typhimurium and antifungal activity against Candida albicans and Aspergillus niger, (Flekhter O.B., Boreko E.I., Nigmatullina L.R., Tretyakova E.V. , Pavlova N.I., Baltina L.A., Nikolaeva S.N., Savinova O.V., Eremin V.F., Galin F.Z., Tolstikov G.A. Synthesis and antiviral activity of amides and betulonic conjugates acids with amino acids // Bioorganic chemistry. - 2004. - T. 30. - No. 1. - S. 89-98).

The use of betulonic acid derivatives as a treatment for osteomyelitis has not been described.

Disclosure of invention

It is proposed to use betulonic acid β-alanylamide (name according to IUPAC nomenclature: N- [3-oxo-20 (29) lupen-28-oyl] -3-aminopropionic acid; the alleged commercial name is “betamide” as an agent for treating osteomyelitis; formula (I):

Technical result

Betulonic acid β-alanylamide:

1) prevents the generalization of the inflammatory process, the formation of extensive zones of necrosis and infiltration, osteoclastic bone resorption and the formation of sequesters, which in turn prevents the inflammatory process from being chronic and contributes to its resolution;

2) contributes to an earlier and more pronounced formation of loose fibrous connective and coarse fibrous bone tissue, compared with groups without the use of betamide, which indicates a positive effect of the drug on reparative processes in the bone;

3) causes a faster onset of the phase of functional adaptation, contributing to a more rapid completion of reparative endochondral osteohistogenesis with the formation of mature bone tissue.

A method for producing β-alanilamide of betulonic acid is described in RF patent No. 2353623.

List of figures

FIG. 1. The proximal end of the tibia of the rat 1 month after inoculation of Staphylococcus aureus. Bone deformity due to formed callus. Macrodrug.

FIG. 2. The proximal end of the tibia of the rat 1 month after inoculation of Staphylococcus aureus. In the area of the trepanation hole, inserted cotton threads are visible. The caseous contents of the trepanation hole, the rough structure of the surface of the tibia, the lack of translucent bone marrow through the plate of the diaphysis indicate the development of osteomyelitis. Macrodrug.

FIG. 3. The site of the tibia of the rat 1 month after inoculation of Staphylococcus aureus. Zone of necrosis, osteoclastic resorption of the tibia. Hematoxylin and eosin stain. SW one hundred.

FIG. 4. Plot of the tibia of the rat 1 month after inoculation of Staphylococcus aureus. Granulocyte-lymphocytic infiltration in the area of threads inserted into the trepanation hole with Staphylococcus aureus. Hematoxylin and eosin stain. SW 400.

FIG. 5. Plot of the tibia of the rat 1 month after inoculation of Staphylococcus aureus. A large number of macrophages in the area bordering the site of necrosis. Immunohistochemical reaction to an ED1-like antigen. SW 400.

FIG. 6. Plot of the tibia of the rat 1 month after inoculation of Staphylococcus aureus. The heterogeneity of the distribution of collagen fibers in relatively preserved bone beams associated with osteoclastic resorption of a portion of the bone located in close proximity to the zone of infiltration and necrosis. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 7. Plot of the tibia of the rat 1 month after inoculation of Staphylococcus aureus. The formation of thin beams of coarse fibrous bone tissue. Picrofuchsin stain according to van Gieson. SW 400.

FIG. 8. Plot of the tibia of the rat 1 month after inoculation of Staphylococcus aureus. Vascular ingrowth in hyaline cartilage tissue undergoing degeneration and destruction. The formation of thin beams of coarse-fibrous bone tissue. Stained with Alcian blue and Mayer karmalum. SW 400.

FIG. 9. A section of the tibia of the rat 1 month after inoculation of Staphylococcus aureus, an increased content of adipocytes and single mast cells in the bone marrow. Stained with Alcian blue (pH = 1.0) and Mayer karmalum. SW 400.

FIG. 10. The proximal end of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. Severe bone deformation due to the formed bone marrow. Macrodrug.

FIG. 11. The proximal end of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. Formed bone callus, inserted cotton threads and yellow caseous contents are visible in the area of the trepanation hole. Decrease in bone marrow transillumination through the diaphysis plate. Macrodrug.

FIG. 12. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. Zone of necrosis and fatty degeneration of bone marrow. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 13. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. Preservation of the bone marrow necrosis zone, limited by the lymphocyte-macrophage shaft, in close proximity to the introduced cotton threads. Hematoxylin and eosin stain. SW one hundred.

FIG. 14. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. The formation of a macrophage-fibroblast barrier that limits the area of necrosis. Immunohistochemical reaction to an ED1-like antigen followed by hematoxylin nuclei dubbing. SW 600.

FIG. 15. A plot of the tibia of a rat 2 months after inoculation of Staphylococcus aureus. Granulation tissue limiting foci of necrosis, osteoclastic resorption of the tibia. Hematoxylin and eosin stain. SW 400.

FIG. 16. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. Chondrocyte destruction and death, blood vessel ingrowth, reparative endochondral osteohistogenesis. Staining with Alcian blue at pH = 1.0 with Mayer karmalum dyeing. SW 400.

Fig.17. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. Active formation of intercellular substance of loose connective tissue containing sulfated (chondroitin-4 sulfate, chondroitin-6-sulfate) glycosaminoglycans, numerous blood vessels. Alcian blue stain at pH 1.0 with Mayer karmalum dyeing. SW 400.

FIG. 18. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. Active formation of intercellular substance of loose connective tissue containing collagen fibers. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 19. Plot of the tibia of a rat 2 months after inoculation of Staphylococcus aureus. The presence of bone fragments with dead osteocytes in the area of necrosis. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 20. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. The increased content of adipocytes and the absence of mast cells in the bone marrow. Stained with Alcian blue (pH = 1.0) and Mayer karmalum. SW 400.

FIG. 21. The proximal end of the tibia of the rat 3 months after inoculation of Staphylococcus aureus. Bone deformity due to formed callus. Macrodrug.

FIG. 22. The proximal end of the tibia of the rat 3 months after inoculation of Staphylococcus aureus. Closing the trepanation opening, the formation of bone protrusions surrounding the ends of the inserted thread. The boundary of decrease in bone marrow transillumination through the diaphysis plate is clearly visible. Macrodrug.

FIG. 23. Plot of the tibia of the rat 3 months after inoculation of Staphylococcus aureus. Preservation of the necrosis zone, limited by macrophage-fibrocytic shaft, in the immediate vicinity of the introduced cotton threads. Fragmentation of bone beams, the presence of zones with focal enlightenment, uneven distribution of osteocytes. Hematoxylin and eosin stain. SW one hundred.

FIG. 24. The tibia of the rat 3 months after inoculation of Staphylococcus aureus. A large number of macrophages next to the introduced thread. Immunohistochemical reaction to an ED1-like antigen. SW 400

Fig.25. Plot of the tibia of the rat 3 months after inoculation of Staphylococcus aureus. A giant multi-core Pirogov-Langhans cell containing numerous nuclei at the periphery of the cytoplasm in the form of a ring. Hematoxylin and eosin stain. SW 400.

FIG. 26. Plot of the tibia of the rat 3 months after inoculation of Staphylococcus aureus. Filling the spaces between the bone beams with fibrous tissue with a high content of collagen fibers, as well as an increased content of fat cells in the bone marrow. Immunohistochemical detection of type II collagen, followed by hematoxylin nuclei dyeing. SW one hundred.

FIG. 27. Plot of the tibia of a rat 3 months after inoculation of Staphylococcus aureus. The increased content of adipocytes in the borderline with the inflammatory infiltrate zone and single mast cells in the bone marrow. Stained with Alcian blue (pH = 1.0) and Mayer karmalum. SW 400.

FIG. 28. Plot of the tibia of the rat 3 months after inoculation of Staphylococcus aureus. The presence of bone fragments with dead osteocytes in the area of necrosis. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 29. The proximal end of the tibia of the rat 2 months after inoculation of Staphylococcus aureus and the introduction of an oil emulsion. Bone deformity due to formed callus. In the area of the trepanation hole there is a caseous yellow content. Macrodrug.

FIG. 30. Rat tibia 2 months after inoculation of Staphylococcus aureus and per os administration of an oil emulsion. Site of necrosis, zonal distribution of immunocompetent cell elements. Staining with Ehrlich hematoxylin and eosin. SW one hundred.

FIG. 31. Tibia of the rat 2 months after inoculation of Staphylococcus aureus and the introduction of per os oil emulsion. Zonal distribution of immunocompetent cell elements. Immunohistochemical reaction to an ED1-like antigen. SW 400.

FIG. 32. Rat tibia 2 months after inoculation of Staphylococcus aureus and per os administration of an oil emulsion. Osteoclastic bone resorption. Staining with Ehrlich hematoxylin and eosin. SW one hundred.

FIG. 33. Rat tibia 2 months after inoculation of Staphylococcus aureus and per os administration of an oil emulsion. The formation of thin beams of coarse fibrous bone tissue, the formation of coarse fibrous connective tissue, fatty degeneration of bone marrow. Picrofuchsin stain according to van Gieson. SW 400.

FIG. 34. Rat tibia 2 months after inoculation of Staphylococcus aureus and per os administration of an oil emulsion. The presence of a bone fragment with dead osteocytes in the area of necrosis. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 35. Rat tibia 2 months after inoculation of Staphylococcus aureus and per os administration of an oil emulsion. Foci of destruction and death of chondrocytes and reparative endochondral osteohistogenesis. Stained with Alcian Blue (pH 1.0) and Mayer Karmalum. SW one hundred.

FIG. 36. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus. Active formation of intercellular substance of loose connective tissue, numerous blood vessels. Picrofuchsin stain according to van Gieson. SW 400.

FIG. 37. Tibia of the rat 2 months after inoculation of Staphylococcus aureus and the introduction of per os oil emulsion. An increase in the number of mast cells in the border area of the bone marrow with inflammatory infiltrate. Stained with Alcian Blue (pH 1.0) and Mayer Karmalum. SW one hundred.

FIG. 38. The proximal end of the tibia of the rat 3 months after inoculation of Staphylococcus aureus and the introduction of an oil emulsion. Preservation of bone deformation due to the formed bone marrow. Macrodrug.

FIG. 39. Tibia of the rat 3 months after inoculation of Staphylococcus aureus and the introduction of per os oil emulsion. A site of necrosis, zonal distribution of immunocompetent cellular elements, fatty degeneration of bone marrow. Staining with Ehrlich hematoxylin and eosin. SW one hundred.

FIG. 40. Tibia of the rat 3 months after inoculation of Staphylococcus aureus and the introduction of per os oil emulsion. The formation of a macrophage-fibroblast barrier that limits the area of necrosis. Immunohistochemical reaction to an ED1-like antigen followed by hematoxylin nuclei dubbing. SW 600.

FIG. 41. Tibia of the rat 3 months after inoculation of Staphylococcus aureus and the introduction of per os oil emulsion. Fragmentation of bone beams containing few osteocytes. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 42. Tibia of the rat 3 months after inoculation of Staphylococcus aureus and the introduction of per os oil emulsion. Uneven distribution of collagen fibers in the bone beams, with the formation of areas of enlightenment. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 43. Rat tibia 3 months after inoculation of Staphylococcus aureus and per os administration of an oil emulsion. The formation of fibrous tissue with a high content of collagen fibers. Picrofuchsin stain according to van Gieson. SW 400.

FIG. 44. Rat tibia 3 months after inoculation of Staphylococcus aureus and per os administration of an oil emulsion. The formation of fibrous tissue with a high content of sulfated glycosaminoglycans. Stained with Alcian Blue (pH 1.0) and Mayer Karmalum. SW 400.

FIG. 45. The proximal end of the tibia of the rat 2 months after inoculation of Staphylococcus aureus and the introduction of an oil emulsion with betamide. Reduction of bone deformation due to formed bone callus. Macrodrug.

FIG. 46. The proximal end of the tibia of the rat 2 months after inoculation of Staphylococcus aureus and the introduction of an oil emulsion with betamide. In the area of the trepanation hole there is a yellowish caseous content. The surface of the tibia maintains a smooth structure, bone marrow transillumination through the diaphysis plate is noted. Macrodrug.

FIG. 47. A plot of the tibia of a rat 2 months after inoculation of Staphylococcus aureus and the per os administration of betamide oil emulsion. The absence of zones of necrosis and a significant decrease in zones of infiltration. Staining with Ehrlich hematoxylin and eosin. SW one hundred.

FIG. 48. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus and the per os administration of betamide oil emulsion. High macrophage content in inflammatory infiltrate. Immunohistochemical reaction to an ED1-like antigen followed by hematoxylin nuclei dubbing. SW 600.

FIG. 49. Plot of the tibia of a rat 2 months after inoculation of Staphylococcus aureus and per os administration of betamide oil emulsion. The absence of granulocytes in the focus of inflammatory infiltrates. Staining with Ehrlich hematoxylin and eosin. SW 400.

FIG. 50. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus and the per os administration of betamide oil emulsion. The formation of loose fibrous connective and coarse fibrous bone tissue in close proximity to the bacterial culture threads introduced into the trepanation opening. Picrofuchsin stain according to van Gieson. SW 400.

FIG. 51. Plot of the tibia of a rat 2 months after inoculation of Staphylococcus aureus and per os administration of betamide oil emulsion. The formation of loose fibrous connective tissue with a high content of sulfated glycosaminoglycans. Stained with Alcian Blue (pH 1.0) and Mayer Karmalum. SW 400.

FIG. 52. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus and the per os administration of betamide oil emulsion. Osteoclastic bone resorption. Staining with Ehrlich hematoxylin and eosin. SW 400.

FIG. 53. Plot of the tibia of the rat 2 months after inoculation of Staphylococcus aureus and the per os administration of betamide oil emulsion. Reparative endochondral osteohistogenesis, the inclusion of cotton thread inserted into the trepanation hole in newly formed bone tissue. Stained with Alcian Blue (pH 1.0) and Mayer Karmalum. SW 400.

FIG. 54. Plot of the tibia of a rat 2 months after inoculation of Staphylococcus aureus and per os administration of betamide oil emulsion. Fatty degeneration of bone marrow, marked increase in the number of mast cells. Stained with Alcian Blue (pH 1.0) and Mayer Karmalum. SW 400.

FIG. 55. The proximal end of the tibia of the rat 3 months after inoculation of Staphylococcus aureus and the introduction of an oil emulsion with betamide. Slight bone deformation due to the formed callus. Macrodrug.

FIG. 56. The proximal end of the tibia of the rat 3 months after inoculation of Staphylococcus aureus and the introduction of an oil emulsion with betamide. In the area of the trepanation hole there is a fibrous formation protruding above the surface of the bone. The surface of the tibia maintains a smooth structure, bone marrow transillumination through the diaphysis plate is noted. Macrodrug.

FIG. 57. Plot of the tibia of the rat 3 months after inoculation of Staphylococcus aureus and the per os administration of betamide oil emulsion. The formation of loose fibrous connective and coarse fibrous bone tissue around the threads inserted into the trepanation hole with a culture of microorganisms. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 58. Plot of the tibia of the rat 3 months after inoculation of Staphylococcus aureus and the per os administration of betamide oil emulsion. Delimitation from the surrounding bone marrow by the newly formed bone tissue of the introduced threads with the culture of microorganisms. Picrofuchsin stain according to van Gieson. SW one hundred.

FIG. 59. Plot of the tibia of a rat 3 months after inoculation of Staphylococcus aureus and per os administration of betamide oil emulsion. Delimitation from the surrounding bone marrow by the newly formed bone tissue of the introduced threads with the culture of microorganisms. Fatty bone marrow degradation, high mast cell count. Stained with Alcian blue (pH 1.0) and Mayer karmalum. SW one hundred.

Justification of the invention

A study was conducted, the purpose of which was to study the effect of betamide on the development of chronic osteomyelitis.

Material and research methods

The experiment was conducted on 48 sexually mature Wistar male rats with an initial weight of 180-220 g. In experimental animals, under a general inhalation anesthesia, tibia was treated, followed by plugging the hole with a cotton thread 30 minutes in the daily wash of Staphylococcus aureus culture (strain 20). A preliminary study made it possible to establish that with such processing of the thread it contains 1 × 10 ⁷ colony forming units. Subsequently, tibial osteomyelitis developed in rats.

Animals were removed from the experiment after 1, 2, and 3 months from the moment of inoculation of Staphylococcus aureus (groups “At 1 month”; “At 2 months”; “At 3 months”, the letter “B” means “inflammation”).

As a control, 6 intact animals were used (int group). Within 10 days before the expiration of a 2-month period after the inoculation of Staphylococcus aureus, 12 animals were injected with an oil-in-water emulsion (6 animals in the 2 months old group and 6 animals in the 3 months old group; the letter “BO” means “inflammation” + oleum "). In the group “IN 3 months”, the introduction of water-in-oil emulsion was continued for another 1 month before the expiration of the 3-month period from the moment of inoculation of Staphylococcus aureus.

Within 10 days before the expiration of a 2-month period after the inoculation of Staphylococcus aureus, 12 animals were injected with a water-fat emulsion with betamide (VOB 2 month group - 6 animals; VOB 3 month group - 6 animals; the letter “VOB” means “Inflammation + oleum + betamide”). In the group "PSA 3 months" the introduction of a water-oil emulsion with betamide continued for 1 month before the expiration of a 3-month period from the moment of inoculation of Staphylococcus aureus.

In total, in the experiment there were 7 groups with reproduction of osteomyelitis (6 animals each) and one group of intact animals (also 6 individuals).

The experiment was carried out in compliance with the principles of humanity set forth in the directives of the European Community (86/609 / EEC) and the Helsinki Declaration on the Protection of Vertebrate Animals used for laboratory and other purposes.

The tibia was fixed in 12% formalin. From paraffin-embedded objects, serial sections were made with a thickness of 7 μm. The tibial morphology was assessed by panoramic microscopy after staining the sections with Ehrlich hematoxylin and eosin. Collagen fibers were detected by van Gieson picrofuchsin, sulfated glycosaminoglycans were detected with Alcian blue at pH 1.0, mast cell populations were evaluated after staining with Alcian blue at pH 1.0, followed by Mayer karmalum staining with nuclei.

When preparing histological preparations for immunohistochemical analysis, the standard method of tissue fixation in neutral formalin was used, followed by dehydration and paraffin filling. An immunohistochemical study evaluating the expression level of a macrophage cell marker (ED1-like antigen) was performed using the indirect streptavidin-biotin peroxidase method. We used the Peroxidase Detection System (Novocastra) imaging system. After the unmasking procedure, the immunohistochemical staining of the preparations was carried out in accordance with generally accepted standards of the indicated technique (Ramos-Vara J.A., 2005; Bibeau F. et al., 2006) and in accordance with the recommendations of manufacturers.

The analysis of the intensity of staining of collagen fibers was carried out using a light-optical microscope and a morphometric complex based on a Micros MS 300A microscope, a CX 13 s digital camera (Baumer Electric GmbH, Germany) and ImageJ 1.42g software (National Institute of Health, USA). For each group, 48 images were evaluated.

Statistical processing of the results of the study was performed using the software package for statistical processing of SPSS v 13.0 for Windows. For comparison of independent groups, the Kruskal-Wallis test was used, followed by intergroup comparison using the Mann-Whitney test. Differences between the values of the compared parameters were regarded as statistically significant at p <0.05. The data obtained during the study are presented as the median (Me) and interquartile range (Q ₁ ; Q ₃ ).

Research results

Macroscopic examination of the tibia 1 month after inoculation of Staphylococcus aureus revealed bone deformation due to the formation of bone callus (Fig. 1). In the area of the trepanation hole, the introduced cotton threads and caseous contents were clearly visible (Fig. 2). The normal surface of the tibia in the proximal region has acquired a rough structure. The bone marrow did not show through the diaphysis plate.

Morphological examination of the tibia 1 month after trepanation and the introduction of a thread with a culture of Staphylococcus aureus revealed signs of osteomyelitis. So, in the place of the bone defect, extensive zones of necrosis and infiltration of granulo-, lympho- and histiocytic elements (Fig. 3) were found, which are located in close proximity to the staphylococcus aureus filaments introduced into the trepanation hole. At the same time, a fairly clear zoning of cellular elements was noted. As a rule, the focus of such infiltrates was clusters of neutrophils (Fig. 4), which were surrounded by a lymphocyte-macrophage shaft (Fig. 5). Attention was drawn to the absence at this time of signs of vasculogenesis inherent in an uninfected fracture and appearing, according to published data, already 10 days after an uninfected injury.

From the saved beams, osteoclastic bone resorption was observed (Fig. 6), as well as a productive and inflammatory reaction of the endosteum and periosteum.

Along with the signs of osteomyelitis described above, signs of regenerative osteohistogenesis were observed. So, in animals of this group, the formation of thin beams of coarse-fiber bone tissue (Fig. 7) along the edge of the rupture of bone plates of haversian systems, as well as an increase in the proportion of bone tissue due to its formation at the site of the cartilage (Fig. 8) was revealed. Vessels grew into the hyaline cartilage undergoing dystrophy and destruction, along with which cells with osteogenic potentials were introduced, providing the formation of reticulofibrous bone tissue.

A study of the bone marrow revealed an increased content of adipocytes in the area bordering the inflammatory infiltrate, and extremely rare mast cells (Fig. 9).

A macroscopic examination of the tibia 2 months after inoculation of Staphylococcus aureus showed marked bone deformation due to the formed callus (Fig. 10).

Introduced cotton threads and yellow caseous contents were clearly visible in the area of the trepanation hole. Translucent bone marrow through the plate of the diaphysis was reduced (Fig. 11).

Subsequently, 2 months after the inoculation of Staphylococcus aureus, zones of bone marrow necrosis (Fig. 12) remained in the immediate vicinity of the inserted cotton threads, bounded by a shaft of lymphocytic and macrophage elements (Fig. 13), passing into the macrophage-fibroblastic barrier (Fig. fourteen). Osteoclastic resorption (Fig. 15) and fragmentation of the spongy bone beams were observed (Fig. 13).

It should be noted that at the site of the bone defect a complex tissue regenerate was formed, in which there was a pronounced presence of hyaline cartilage tissue. Foci of calcification of hyaline cartilage, which led to the destruction and death of chondrocytes, were quite characteristic for this group of animals. (Fig. 16). It was noted that blood vessels grew in these areas, which contributed to reparative endochondral osteohistogenesis. In addition, intense angiogenesis was noted, as well as the active formation of intercellular substance of loose connective tissue (Fig. 17; Fig. 18).

Quite often, bone fragments with osteocytes that underwent destruction (Fig. 19) were found in the necrosis zone, which contributed to the chronicity of osteomyelitis.

A study of the bone marrow revealed an increased content of adipocytes in the area bordering the inflammatory infiltrate, and the absence of mast cells (Fig. 20).

A morphometric analysis of the intensity of staining of collagen fibers, reflecting the density of their distribution in the bone beams in animals of the groups “At 1 month”, “At 2 months”, “At 3 months” showed that, despite the signs of inflammation and osteoclastic bone resorption, the process reparative endochondral osteohistogenesis, accompanied by synthesis of collagen osteoblasts, significantly exceeds the same indicator in intact animals (Table 1).

Macroscopic examination of the tibia 3 months after inoculation of Staphylococcus aureus was observed preservation of bone deformation due to the formed bone callus (Fig. 21). Introduced cotton threads and yellow caseous contents were clearly visible in the area of the trepanation hole. In some cases, the closure of the trepanation hole was noted, however, bony protrusions surrounding the ends of the inserted thread were formed (Fig. 22). Translucent bone marrow through the plate of the diaphysis was reduced.

In a morphological study of the tibia 3 months after inoculation with Staphylococcus aureus, bone marrow necrosis zones limited by the macrophage-fibroblast barrier were observed (Fig. 23). A large number of macrophages were located in the zones of the introduced cotton threads (Fig. 24).

In addition, giant Pirogov-Langhans multinuclear cells were determined, containing numerous nuclei at the periphery of the cytoplasm in the form of a ring (Fig. 25).

Bone beams were fragmented, contained a few, unevenly located osteocytes, areas with focal enlightenment (Fig. 23). It was noted that the spaces between the bone beams were filled with fibrous tissue with a high content of collagen fibers (Fig. 26), as well as an increased content of fat cells in the border with infiltrate zone and single mast cells in the bone marrow (Fig. 27).

As in the previous group, quite often, in the area of necrosis there were bone fragments with osteocytes that underwent destruction (Fig. 28), which determined the chronic course of osteomyelitis.

The study of macropreparations in the group "IN 2 months" did not allow to reveal any significant differences with the group "In 2 months" (Fig. 29).

The dynamics and nature of morphological changes in the tibia in the “2 months” group did not differ from the “2 months” group. So, in the “2 months” group, extensive zones of necrosis and infiltration of granulo-, lympho-, and macrophage elements were noted with a preserved zonality of the distribution of immunocompetent cells (Fig. 30; 31); osteoclastic bone resorption (Fig. 32); fatty degeneration of bone marrow (Fig. 33), the presence of bone fragments with osteocytes that underwent destruction (Fig. 34), the presence of foci of destruction and death of chondrocytes and reparative endochondral osteohistogenesis (Fig. 35); the presence of intense angiogenesis (Fig. 36) and the active formation of intercellular substance of loose connective tissue (Fig. 33; 34). In contrast to the groups in which the oil emulsion was not administered, in this group there was a slight increase in mast cells in the border area of the bone marrow with inflammatory infiltrate (Fig. 37).

The study of macropreparations in the group "IN 3 months" did not allow to reveal any significant differences with the group "In 3 months" (Fig. 38).

In a morphological study of the tibia in the “3 months old” group, as in the animals of the “3 months old” group, the preservation of areas of bone marrow necrosis (Fig. 39), limited by the macrophage-fibroblastic barrier (Fig. 40), was noted. Bone beams were fragmented, contained a few, unevenly located osteocytes (Fig. 41), there was an uneven distribution of collagen fibers and areas with the formation of focal enlightenment of bone tissue (Fig. 42). It was noted that the spaces between the bone beams were filled with fibrous tissue with a high content of collagen fibers (Fig. 43) and sulfated glycosaminoglycans (Fig. 44), as well as an increased content of fat cells in the bone marrow (Fig. 39).

A morphometric analysis of the intensity of staining of collagen fibers, reflecting the density of their distribution in the bone beams in animals of the group "2 months" and group "2 months", animals of the group "3 months" and the group "3 months", showed that on similar the terms of the group of experimental animals with the introduction of an oil emulsion and without its introduction do not differ statistically significantly (Table 2).

A macroscopic examination of the tibia of experimental animals of the “PSA 2 months” group, in contrast to the “2 months” group, showed less pronounced bone deformation due to the formed bone callus (Fig. 45). In the area of the trepanation hole (group "PSA 2 months"), a yellowish caseous content was clearly visible; the surface of the tibia in the proximal section maintained a smooth structure, transmission of bone marrow through the plate of the diaphysis was noted (Fig. 46).

During the morphological study of the group "PSA 2 months" attention was drawn to the absence of zones of necrosis and a significant decrease in zones of infiltration (Fig. 47). Moreover, unlike the “2 months” group, it was noted that the very nature of the inflammatory infiltrates, which were represented by lymphocytic macrophage elements with a significant predominance of the latter (Fig. 48), as well as the almost complete absence of granulocytes (Fig. 49), changed . At the same time, marked formation of loose fibrous connective and coarse fibrous bone tissue was noted (Fig. 50). It should be noted that the content of sulfated glycosaminoglycans in the loose fibrous connective tissue was higher than in the “2 months” group (Fig. 51).

From the side of the bone beams, osteogenic bone resorption was observed (Fig. 52), which was combined with pronounced reparative endochondral osteohistogenesis. Moreover, quite often the newly formed bone tissue included fragments of the cotton thread inserted into the trepanation hole (Fig. 53).

In contrast to the “2 month old” group, rats of the “VOB 2 month old” group showed a higher mast cell count and more pronounced fatty degeneration of the bone marrow (Fig. 54), which is a response of the bone marrow to inflammation. Nevertheless, such a distinguishing feature in the absence of foci of necrosis and infiltrates did not lead to the generalization of the inflammatory process, since the presence of an increased number of mast cells contributed to a more pronounced bone remodeling already at this time, which can be explained by the stimulating role of mast cells in relation to the connective tissue differential (Gruber BL, 2003; Garbuzenko E., 2000; 2004).

In a macroscopic examination of the tibia of experimental animals of the “PSA 3 months” group, in contrast to the “3 months” group, a significantly less pronounced bone deformation was observed due to the formed callus (Fig. 55). In some cases, a fibrous mass protruding above the bone surface remained in the area of the trepanation hole. The surface of the tibia in the proximal region maintained a smooth structure, transmission of bone marrow through the plate of the diaphysis was noted (Fig. 56).

During a morphological study of the group “PSA 3 months”, the absence of zones of necrosis and infiltration was noted, the inflammatory reaction from the endosteum and periosteum was not detected. At the same time, pronounced formation of loose fibrous connective and coarse fibrous bone tissue was noted (Fig. 57). Quite interesting was the fact that in some cases, the newly formed bone tissue delimited the introduced filaments from the surrounding bone marrow (Fig. 58). Attention was drawn, as in the “PSA 2 months” group, to pronounced fatty degeneration of the bone marrow in the area bordering the inserted threads, as well as a higher content of mast cells (Fig. 59).

A morphometric analysis of the intensity of staining of collagen fibers, reflecting the density of their distribution in bone beams in animals of the “2 months” and “PSA 2 months” groups, showed that it did not differ statistically significantly (which indicated the same level of severity of collagen formation in bone beams) ( Table 3), but differed from the group of intact animals. The intensity of staining of collagen fibers in the “3 months” and “PSA 3 months” groups was statistically significantly different. Moreover, the intensity of staining of collagen fibers in the group "PSA 3 months" and in the group of intact animals did not significantly differ, which indicated the practical completion of endochondral osteohistogenesis.

The above data show that betulonic acid β-alanilamide has a therapeutic effect in osteomyelitis.

Claims (1)

An agent for treating osteomyelitis, characterized in that it is betulonic acid β-alanilamide of the formula (I):

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