Means for stimulation of normal and reparative osteogenesis
The invention relates to the stimulation of osteogenesis and may find application in experimental medicine and veterinary medicine. Developed the drug naloxone hydrochloride effectively stimulated normal (physiological) and reparative osteogenesis. The invention expands the Arsenal of the declared destination. 3 tab., 27 ill.
The invention relates to stimulating osteogenesis, and may find application in experimental medicine and veterinary medicine.
The problem of stimulation of osteogenesis, along with finding ways of controlling the regeneration of bone tissue is one of the urgent (in particular, orthopedics/traumatology and veterinary) (U. I. Bogdanovich, D. L. Akberdina, 1976, R. I. Kai, C. D. Charles, 1981, G. I. Lavrishcheva, G. A. Onoprienko, 1996, I. C. Derevyanko, 2001). This is due to the large number of patients with orthopedic-traumatological injuries occurring to the oppression of reparative osteogenesis, as well as diseases of the musculoskeletal system, the pathogenesis of which is based on an imbalance of normal bone formation (e.g., osteoporosis).
It is known that General biological stimulation of reparative processes (including bone tissue is s, the use of pyrimidine and purine derivatives (pentoksil, methyluracil, potassium orotate) (C. I. Rusakov, 1976, S. M. Borovecki et al., 1981). With regard to bone tissue, in addition to General biological stimulation of reparative processes in it, used drugs, complementary deficiency of calcium in the body, which stimulate phase of calcification of bone matrix without affecting the rate of formation of the protein matrix of the bone - formation rate of which determines the rate of osteogenesis in General (Slutsky, 1969).
As a prototype, you can use the drug “Osteogene” (Osteogenon), which is used for the treatment of various forms of osteoporosis, as well as to accelerate healing of bone fractures (RLS - encyclopedia of medicine”, 2001, page 656).
Osteogene in systemic osteoporosis is applied inside of 2-4 tablets (one tablet is covered with a sheath, 830 mg), and to accelerate the healing of fractures 1-2 tablets per day (RLS - encyclopedia of medicine”, 2001, page 656). According to the creators of the drug, osteogene derived from bone tissue of animals, he stops (reduces bone resorption (inhibits osteoclasts and stimulates bone formation (activates osteoblasts) (RLS-encyclopedia of medicine”, 2001, months to years) application, the possibility of exacerbation in patients with urolithiasis, control the level of calcium and phosphorus in the urine, the high cost of the drug.
The novelty of the invention and its difference is the ligand of opiate receptors naloxone hydrochloride - for stimulation of normal and reparative osteogenesis.
Naloxone hydrochloride powder photosensitive substance of white color with the molecular formula C19H21NO4·HCl molecular weight 363,8, is an antagonist of mu, Sigma, Kappa - opioid receptor. The solubility in water of 50 mg/ml, in ethanol 3.4 mg/ml
As a stimulator of normal and reparative osteogenesis offers the use of a solution of naloxone hydrochloride in a 1/15M phosphate buffer, Sorensen pH was 7.36 as a solvent of the following composition:
- naloxone hydrochloride, 150 mg/kg;
phosphate buffer, Sorensen pH of 7.36, 1/15 M - 0,1 ml
Because naloxone hydrochloride (experiment used the drug, manufactured by Sigma) is a powdered substance, for parenteral administration it is necessary to prepare a solution, and in the literature (as well as in the documentation Mock solvent, we decided to use isotonic sterile blood 1/15 M phosphate buffer, Serensen with a pH of 7.36 as solvent production company “Sigma”.
Currently naloxone hydrochloride is used only for the diagnosis and treatment of acute poisoning with narcotic analgesics and hypotension in septic shock.
Study of the effect of naloxone hydrochloride on osteogenesis was performed on two models: the normal osteogenesis was studied on the model of embryonic osteogenesis chick embryos, and reparative osteogenesis was studied on the model of a closed fracture of both bones of the right leg mice weighing 25-30 g
Study of the effect of naloxone hydrochloride in normal osteogenesis
Incubated fertilized chicken eggs cross “Smena-2” at a temperature of 38.5° C humidity - 85% in the incubator. Eggs turned over daily in offline mode. Chicken embryo (egg) on the 11th day of incubation were weighed, placed in the instrument for x-ray and determined the localization of the large veins near the edge of the air chamber by the following features:
1. smooth contours;
2. branching in the direction of the air chambers;
3. the blood flow in the direction of the air chamber.
The shell on top of vein SC is the outline of a rectangle has penetrated the shell. The cut section of the shell with the outer part of podkorkovoi shell shot eye with tweezers, making sure that the eruption of the shell does not cause damage chorioallantoic membrane. The resulting eggs were put on the stand for eggs and performed an injection of naloxone hydrochloride in a dose of 150 mg/kg in a volume of 0.1 ml in the series “A” or an injection of 0.1 ml of 1/15 M phosphate buffer, Sorensen pH of 7.36 in the series “b” in the direction of blood flow, i.e. in the direction of the air chamber. The needle tip was slightly bent upwards in order to Vienna to come under the most obtuse angle. In the shell over the air cell was drilled using a drill a small hole to compensate for the pressure rise in the Bud. After injection hole was sealed with tape and incubated egg in the usual way in the incubator.
As described above, the injection of naloxone hydrochloride in a dose of 150 mg/kg, dissolved in 0.1 ml of 1/15 M phosphate buffer, Sorensen pH of 7.36 and injection of 0.1 ml of 1/15 M phosphate buffer, Sorensen pH of 7.36 performed on 11, 13, 15 or 14, 16, 18 day incubation to assess the degree of influence of naloxone hydrochloride on growth of bone tissue at the second and third week of embryonic development. Injection of phosphate buffer was performed only for isklucheniya was taken 21 days after 2 h after hatching Chicks. Histological preparations of bone tissue of chicken embryos were prepared according to standard methods (C. I. Merkulov et al., 1973) with the subsequent colouring obtained paraffin sections were hematoxylin-eosin and by the method of van Gizona.
Prepared histological bone tissue of chicken embryos were subjected to morphometric analysis method Avtandilov 5 series of experiments on the following criteria:
1. Determination of the total number of cells in series (the results of the study are presented in Fig.9)
2. Determining the average number of mitoses per 100 cells (Fig.8; Fig 1-5)
3. Determining the average number of osteoblasts (Fig.10) in compared series
4. Determining the average number of osteocytes (Fig.11)
5. Determination of the average thickness of the woven bone cuff (TPCM) in µm (Fig.12; Fig.6-7)
Feature comparison series of experiments, the results of which are reflected in Fig.8-12, are also reflected in table 1.
Study of the effect of a solution of naloxone hydrochloride in 1/15 M phosphate buffer, Serensen with a pH of 7.36 (hereinafter the product) on normal osteogenesis conducted on 150 chicken embryos showed that the drug, administered at 3 h the increase in the number of mitoses in 6,56 times compared with intact embryos (p<0,01) (Fig.2, Fig.8). The number of mitoses per 100 cells with the drug embryos averages of 13.3, a y intact embryos of 2.5. This trend continues when comparing mitotic activity in the growth zone of the femur in embryos with the introduction of the 3 week of embryogenesis of the drug and in embryos exposed to phosphate buffer. The number of mitoses in this case with the drug increased 5.3-fold (p<0.01) (Fig.1, 3 and 8). The number of mitoses per 100 cells with the drug was 13.3 and 2.0 with the introduction of phosphate buffer. It should be noted that the drug, administered at 2 week of embryogenesis, does not change the number of mitoses (Fig.4) (no effect on their expression) compared with intact embryos and embryos exposed to phosphate buffer (Fig.5 and 8).
To exclude the effect of phosphate buffer on normal osteogenesis, we performed morphometric analysis of histological preparations of chicken embryos, which were introduced phosphate buffer for 2 and 3 week of embryogenesis.
It was found that phosphate buffer, input on the 2 and 3 week development of the chicken embryo does not encourage mitoses (Fig.8) in the growth zone of the femur, does not affect the density of the cells (Fig.9) bone (citedeleau square 4 mm2bone compared with intact embryos.
Method of morphometric analysis us a study of the effect of the drug on the cellular composition of the growth zone of the femur chicken embryo. It was found that the introduction of the drug for 2-3 week development of the chicken embryo does not affect the density of bone cells (Fig.9) (number of cells per field of view), nor on the average number of osteoblasts (Fig.10) defined on the space of 4 mm2bone compared with intact embryos and embryos exposed to phosphate buffer.
We also studied the integral indicator of embryonic osteogenesis - thickness woven bone cuff (TPCM). It was found that the drug, administered at 2 week of embryogenesis, caused increase in TPKM compared with intact embryos and embryos exposed to phosphate buffer to 100% (p<0,01) (Fig.6; 7 and 12).
The introduction of the drug on the 3rd week of embryogenesis is accompanied by a statistically significant increase TPKM compared with intact embryos (39,9%) and embryos exposed to phosphate buffer (37,3%) (Fig.12).
Thus, the introduction of the drug in week 2 of the development of chicken EMW is manifested by increasing the integral index - TPCM. Against this background, the lack of increase in the number of mitoses to 21 days, is a logical continuation of the completion of proliferation occurring in earlier periods.
The introduction of the drug on the 3rd week of embryogenesis also leads to stimulation of embryonic osteogenesis, which is manifested by an increase in the number of mitoses. Against this background, the decrease in the number of osteocytes reflects not a lack of education, and characterized by incomplete formation of bone cuff, i.e., the process of differentiation of bone cells. The latter, probably due to small term effect of the drug, to check what we raised an additional series of experiments (the results of which are shown in Fig.13 and 14) with the introduction of the drug for 3 weeks incubation (14, 16, 18 days) and analysis of its influence on osteogenesis after 6 days of life birds. Characteristic series of experiments is presented in table 2. In this mode of experience data were obtained, identical to the drug for 2 week incubation with analysis on the 21st day of incubation.
We found no changes in other organs and tissues (brain, liver, heart), in addition to bone, under the influence of a solution of naloxone hydrochloride in a dose of 150 μg/kg, diluted 1/15 M phosphate buffer of Zeron hydrochloride on reparative osteogenesis was performed on 120 mice With2L57That have under ether anesthesia between the branches of the tweezers inflicted closed fracture of both bones of the right leg. All mice were divided into 3 groups: intact - group, in which applied fracture and without injection of the intervention, the control group it was applied fracture and intraperitoneally three times (2, 48 and 96 hours after injury) were administered 0.1 ml of phosphate buffer, Serensen 1/15 m, pH 7.36, which was the solvent for naloxone hydrochloride and experienced group - it was applied fracture and intraperitoneally three times (2, 48 and 96 hours after injury) was administered naloxone hydrochloride in a dose of 150 μg/kg, dissolved in 0.1 ml of phosphate buffer, Serensen 1/15 m, pH 7.36.
Influence of solution of naloxone hydrochloride during bone repair were studied using x-ray, histological methods, and test mechanical strength of callus by A. C. Cuchicheo. What on 7, 11, 14, 21 days after injury in 15 animals from each series were taken from experiment by transcervical dislocation, 10 of which were produced preparative isolation of both bones of the lower leg, followed by x-rays and test mechanical tensile strength, and the remaining 5 makepossible manufacturing histological preparations according to the above method.
Research has shown that under the influence of naloxone hydrochloride was significantly increased strength fusion of fragments of the Shin bone as compared with both control groups. The results of the comparison test, the mechanical strength of callus on gap control and experimental groups are presented in table 3 and Fig.15-18. We discovered that at 7 days after injury, the amount of force needed to rupture a non-Union of bone fragments in mice recipient, naloxone hydrochloride increased 2-fold (p<0.01) as compared with both control groups (table 3; Fig.15). Then at 11, 14, 21 days after fracture was observed a tendency of reduction of the difference of the amount of force to break a bone spur in the experimental and control groups due to the consolidation of fractures. But the strength of callus in the experimental group was always significantly higher (p<0.001) compared to both control groups (table 3; Fig.16, 17, 18).
The study of micropreparative found that abnormal mobility of fragments from injured mice recipient of a solution of naloxone hydrochloride was significantly decreased on day 7 after injury compared with thinking of all experimental mice to 7 days after fracture, while in the control group, only 20% of the animals. When this bone spur in the control groups was always less pronounced than in the experimental group of animals. In the course we conducted histological examination of the process of fusion of the fracture in the experimental and control groups found that, already on day 7 after injury there are significant differences in the histological picture of the fracture site of the above groups of animals. Thus, in the experimental group on day 7 after injury is determined by the histological pattern (Fig.19) pretty active bone formation. In megaloschemoi cracks are sporadic remnants of a blood clot, is determined immature fibrous tissue residues of granulation tissue. A significant part of immature fibrous tissue is composed of fibroblastic cells in the presence of sparse and thin collagen and reticulin fibers on the background concentrations of lymphocytes, macrophages and erythrocytes.
Differences in the histological picture of the place Paloma experimental and control groups of animals are saved and 14 days after injury. Thus, in the experimental group there is a marked pattern of bone formation (Fig.21). The diastasis between the fragments filled Mature, differentiated roughly wondertales surface is defined by a dense network of Mature bone beams.
At the same time, the histological pattern of the fracture site of the control group animals at 14 days after injury (Fig.22) presents only the initial elements of bone formation in megaloschemoi cracks observed differentiated connective tissue network of capillaries and sinusoids. There is also a slight increase in network bone beams, endofline bone formation (moderately Mature and immature), a moderate increase in periosteal bone formation. Remain differences in the histological picture of the place of fracture control and experimental groups and at 21 days after injury. Thus, the histological pattern of callus from the experimental group (Fig.23) in these terms after the fracture is characterized by the fact that in place of diastasis (megaloschemoi gap) observed Mature bone beams characterizing the fracture fracture. Megalomania gap, as such, are not differentiated (not VisualRoute). Bone spur of the control group animals at 21 days after fracture (Fig.24) presents coarse-fibered connective tissue with foci of hyaline cartilage, moderate bone network of beams.
Using x-ray method, we carried out a study of the fracture site on 7, 14, 21 days after rsnake slaboraschlenennoe corn (Fig.25S). At the same time study the fracture site in animals of the control group signs of callus was not detected (Fig.25A, B).
Remain differences in x-ray picture of the fractured bone in animals from the control and experimental groups, and 14 days after injury. On the radiograph of the fractured bone of animals from the experimental group (Fig.26C shows a well-pronounced bone spur. The edges of the bone fragments are not differentiated, indicating that the fusion of the fracture. At the same time on the x-ray fractured bone in animals of the control group is only visible laboratoriekontrolleret corn (Fig.26A, B).
At 21 days after fracture of the experimental animals (Fig.27C shows a well-pronounced callus events recanalization of bone-marrow channel. In the control group of animals x-ray picture of the fracture site presents moderate callus, less intense than in the experimental group, with no signs of recanalization (Fig.27A, B).
Thus, naloxone hydrochloride is a stimulant reparative osteogenesis.
The study shows high biological activity of naloxone hydrochloride in relation to the processes of bone repair in animals with ohii.
The use of naloxone hydrochloride as a means to stimulate normal and reparative osteogenesis.
where the values of A, B, K, T, W, X, Y, U, V, Z, R1specified in paragraph 1 of the claims
where R1and R3designate one or more identical or different substituents selected from the group consisting of halogen, (C1-C3)-alkyl, (C1-C3)-alkoxy; provided that, if R1denotes one Deputy, he is in the ortho-position, and if R1refers to several substituents, at least one substituent R1located in the ortho-position; and R2denotes one substituent in the ortho-position, and this Deputy is selected from the group consisting of halogen, (C1-C3)-alkoxy; and R3can additionally denote hydrogen; R4represents hydrogen; X represents oxygen; Q represents -(CO)- or a bond; Y represents (C5-C15)alkyl, (C2-C15)olefinic group; and any of these groups may be optionally substituted by one or more identical or different substituents selected from the group consisting of substituents of formula R5defined below, except that when Q represents a bond, then Y appears lcil, substituted by one or more substituents selected from the group R5; or a group of formula - (Z-O)n- Z, where Z is a (C1-C3)alkyl, n is an integer >1, and the number of atoms in a continuous linear sequence of atoms in the group Y does not exceed 15; R5denotes halogen, hydroxy, amino, (C1-C6)-alkylamino, (C1-C3)alkoxycarbonyl, -COOH, -CONHR' or-COONR'R' R' means (C1-C3)alkyl; or its pharmaceutically acceptable salt