WO2023177325A2

WO2023177325A2 - Parenteral pharmaceutical composition for regenerating bone tissues

Info

Publication number: WO2023177325A2
Application number: PCT/RU2023/050044
Authority: WO
Inventors: Владимир Леонидович ИВАНОВ
Original assignee: КОЛЫШ, Александр Львович; НЕГОРЕЛОВ, Сергей Владимирович
Priority date: 2022-03-18
Filing date: 2023-03-03
Publication date: 2023-09-21
Also published as: WO2023177325A3

Abstract

The invention relates to the field of medicine, and more particularly to drugs for parenteral administration for regenerating bone tissues. The present parenteral pharmaceutical composition for regenerating bone tissues contains 0.04-0.5 wt% calcium 1-hydroxyethylidene dihydrogen phosphate, 2·10-5 - 5·10-5 wt% silver, and water up to 100 wt%. The proposed composition is effective in regenerating bone tissues, does not cause sterile inflammation in the region of a bone defect, and improves the extent and dynamics of the reparative processes.

Description

NAME OF INVENTION:

Pharmaceutical parenteral composition for bone tissue restoration

TECHNICAL FIELD

The invention relates to the field of medicine, namely to preparations for parenteral use to restore bone tissue.

Currently, the problem of post-traumatic restoration of bone tissue (reparative regeneration) is of particular importance due to the increase in local armed conflicts and man-made disasters, and also has socio-economic relevance associated with the need for long-term and expensive treatment.

BACKGROUND ART

Currently, several of the most common methods of stimulating reparative regeneration are known (Von Wersen R., 1993):

1. transplantation of determined osteogenic prodromal cells (DOPC), which have their own potential for bone formation - osteoblastic osteogenesis (Fridenstein A.Ya., Lalykina K.S., 1973; Matti N., 1932);

2. a method of passive stimulation of DOPC using allogeneic bone grafts, synthetic or semi-synthetic bone substitutes, osteoconductive osteogenesis or osteoconduction (Gerbart T.N. et al, 1993; Mitteimeir H, Katthagen B.D., 1983). Implants of artificial or biological origin in this case serve as a framework (conductor) for the germination of blood vessels, after which the ingrowth of cells (osteoblasts) from the bone bed occurs;

3. exposure to specific substances to which bone morphogenetic protein (BMP - bone morphogenetic protein) belongs, more precisely, some representatives of the family of morphogenetic proteins that induce the phenotypic transformation of pluripotent connective tissue stem cells or inducible osteoprodromal cells (Von Wersen R., 1993) into osteoblasts, - osteoinductive osteogenesis or osteoinduction (Urist M.R., 1965);

4. influence on osteogenesis by factors stimulating new bone formation (TGF-p, IGF-I, IGF-II, PDGF, bFGF. aFGF, BMPs) - stimulated osteogenesis. These factors are constantly present in native bone tissue, being mediators of cell proliferation and differentiation, angiogenesis and mineralization, both during physiological and reparative regeneration of bone tissue (Solheim E., 1998).

Currently, BMPs and growth factors are commercially available and used in clinical practice in some countries. However, their small amount in bone tissue, the difficulty of isolating and purifying, and the impossibility of synthesizing some of them using genetic engineering methods (for example, BMPs due to their unclear chemical structure) significantly limit the use of these factors in experimental and clinical traumatology and orthopedics.

Another way to influence reparative regeneration is the transplantation of determinate osteogenic prodromal cells (DOPC), which have their own potential for osteogenesis (Fridenstein A.Ya., Lalykina K.S., 1973).

However, due to the complexity and high cost of culturing osteoblast precursor cells and the short lifespan of cells outside the nutrient medium, the proposed method of influencing osteogenesis has not yet been widely implemented in experimental and clinical traumatology and orthopedics.

There are known experimental and clinical data on the use of grafts from demineralized bone matrix, coral hydroxyapatite, porous polymer materials saturated with osteogenic host cells (Jean J.L., Wang S.J., Ai M.K., 1997; Bruder S.P., Kraus K.N., Goldberg V.M. , Kadiyala S., 1998, Rosenberg E., Rose L.E., 1998).

Currently promising, according to research (Paralkar V.M., Hammonds R.G., Reddi A.N., 1991; Solheim E., Pinholt E.M., Andersen R., Bang G., Sudmann E., 1992; Solheim E., Pinholt E.M., Bang G., Sudmann E. 1992; Solheim E., Pinholt E.M., Bang G., Sudmann E. 1992; Sudmann B., Anfinsen O.G., Bang G. et al., 1993), is a resorbable synthetic implant (system delivery of growth factors), made from polyorthoester. However, the long-term results of using this material are unknown, which so far limits the use of polyorthoester in the clinic.

An injection solution for bone tissue regeneration is known (RF patent No. 2061402), containing 1-hydroxyethylidene diphosphonic acid, calcium chloride, gadolinium nitrate hexahydrate and water in the following ratio of ingredients, wt. %: 1-hydroxyethylidene diphosphonic acid 1.8-2.0; anhydrous calcium chloride 1.4-2.2; gadolinium nitrate hexahydrate 0.3-0.4; water up to 1000, with pH 7.5-8.0. The known solution shortens the duration of the process of bone tissue regeneration at the site of its damage or defect, however, the use of different anions in the composition of its constituent salts (chloride and nitrate ions) does not allow obtaining a pharmaceutically active complex of 1-hydroxyethylidene diphosphonic acid of a constant composition, which makes the drug ineffective.

An injection solution for bone tissue regeneration is known (RF patent No. 2521344), containing 1-hydroxyethylidene diphosphonic acid in an amount of 1.80-2.06 g/l, anhydrous calcium chloride in an amount of 1.44-2.22 g/l, gadolinium nitrate (III) hexahydrate in an amount of 0.30-0.40 g/l, dysprosium chloride (III) hexahydrate in an amount of 0.038-0.076 g/l, with a pH of 7.3-7.8. The specified solution before its introduction into the fracture zone bring to a temperature of 30-100 °C, maintain it at this temperature for 1-48 hours and cool again to room temperature. The known solution reduces the duration of the process of regeneration of bone tissue at the site of its damage or defect and the time of restoration of the normal physiological function of the injured bone, however, the use of different anions in the composition of its salts (chloride and nitrate ions) does not allow obtaining a pharmaceutically active complex of 1-hydroxyethylidene diphosphonic acid permanent composition. In addition, the use of a known composition requires preliminary preparation, which limits its use for operational actions.

A parenteral composition is known that includes bisphosphonic acid or its pharmaceutically acceptable salt (bisphosphonate) as an active component, a pharmaceutically acceptable chelating agent, namely EDTA and DTPA and their pharmaceutically acceptable salts, and pharmaceutically acceptable excipients (RF patent No. 2238736). The known composition is used in the treatment and prevention of diseases including bone resorption, especially osteoporosis, Paget's disease, hypercalcemia in malignant tumors and metabolic disorders of bone tissue. The composition is particularly useful for improving the local tolerance of the active component when administered parenterally, especially by the subcutaneous route. However, the known composition does not affect post-traumatic restoration of bone tissue.

It is known to use an N-bisphosphonate or a pharmaceutically acceptable salt thereof or any hydrate thereof for the preparation of a medicament intended for the treatment of conditions characterized by abnormally increased bone turnover, wherein the medicament is adapted for parenteral administration in the form of a unit dosage form containing: approximately 1 to approximately 10 mg of N-bisphosphonate or a pharmaceutically acceptable salt thereof or any hydrate thereof (RF application No. 2003100503). The known drug is used for the preventive treatment of osteoporosis, but does not affect the post-traumatic restoration of bone tissue.

A known composition for parenteral administration for the treatment of osteonecrosis and related disorders, such as osteochondritis dissecans, containing as an active ingredient a pharmaceutically acceptable bisphosphonate salt [RF patent No. 2284821, publ. 10.10.2006]. However, this drug is not effective in post-traumatic bone tissue restoration.

IMPLEMENTATION OF THE INVENTION

The problem solved by the proposed invention is the development of a pharmaceutical parenteral composition for the restoration of bone tissue, combining restorative and bacteriostatic effects with high pharmacokinetic parameters and a constant composition.

The declared technical result is achieved by the proposed pharmaceutical parenteral composition for the restoration of bone tissue, containing 1-hydroxyethylidene dihydrogen phosphate of calcium, silver and water, with the following ratio of components, mass. %: 1-hydroxyethylidene dihydrogen phosphate calcium 0.04-0.5 silver 2- 10' ⁵ - 5 - 10' ⁵ water up to 100.

The technical result from the use of the invention is to increase the effectiveness of the drug due to its stability and, as a consequence, increase the rate of diffusion of active substances into the bone tissue area, and expand the arsenal of means for restoring bone tissue through the use of a bacteriostatic component in the form of colloidal silver, which in combination with calcium 1-hydroxyethylidene dihydrogen phosphate exhibits a synergistic effect.

Due to the synergistic interaction of the components, the proposed composition has a complex effect in the area of the bone defect. The 1-hydroxyethylidene dihydrogen calcium phosphate included in the proposed composition is a stable chelate complex of constant composition and accelerates the regeneration process by enriching the bone defect area with chelated calcium. The colloidal silver present, on the one hand, acts on the cell membrane, being a conductor of calcium 1-hydroxyethylidene dihydrogen phosphate, and on the other, has bacteriostatic effect, due to which there is no resistance of microorganisms, which enhances the positive effect on the degree and dynamics of reparative processes.

It was experimentally established that the indicated ranges of concentrations of active ingredients are optimally acceptable, pharmacologically significant and accepted in medicinal practice for similar injection solutions of this pharmacological group.

OPTION FOR IMPLEMENTATION OF THE INVENTION

The production of the proposed pharmaceutical composition is carried out in the following way.

The required amount of distilled water and 1-hydroxyethylidene diphosphonic acid is placed in a glass container. Stir until the acid is completely dissolved and place silver electrodes of arbitrary shape into the solution. A direct current is passed through the electrodes for a time calculated to achieve the required silver concentration in accordance with Faraday's law of electrolysis. After this, the calculated amount of calcium hydroxide or oxide is added to the solution, mixed and homogenized on a submersible ultrasonic laboratory homogenizer at a power of 180 watts for 15 minutes at a temperature not exceeding 90 °C. If overheating occurs, a technological pause is made. The hot solution is packaged in 10 ml ampoules using standard pharmaceutical equipment. The finished product does not require preliminary preparation, is sterile and has a shelf life of at least a year.

Monitoring the quantitative content of active ingredients in the composition and determining the sterility of the resulting solution is carried out according to known methods.

INDUSTRIAL APPLICABILITY

Examples of obtaining the proposed pharmaceutical composition.

Example 1.

4.224 g of 1-hydroxyethylidene diphosphonic acid are dissolved in 995 ml of distilled water. Silver electrodes are immersed in the solution and a direct current of 180 mA is passed for 10 seconds. After this, 1.517 g of calcium hydroxide is added to the solution, mixed and homogenized using a submersible ultrasonic laboratory homogenizer at a power of 180 watts for 15 minutes at a temperature of 80 °C. The solution is transparent with slight opalescence. The resulting solution contains 1-hydroxyethylidene dihydrogen calcium phosphate 0.5 wt. %, silver 2x10-5 wt. %.

Example 2.

0.845 g of 1-hydroxyethylidene diphosphonic acid is dissolved in 999 ml of distilled water. Silver electrodes are immersed in the solution and a direct current of 180 mA is passed for 25 seconds. After this, 0.23 g of calcium oxide is added to the solution, mixed and homogenized using a submersible ultrasonic laboratory homogenizer at a power of 180 watts for 15 minutes at a temperature of 80 °C. The solution is transparent with slight opalescence.

The resulting solution contains 1-hydroxyethylidene dihydrogen calcium phosphate 0.1 wt. %, silver 5x10-5 wt. %.

Example 3.

0.535 g of 1-hydroxyethylidene diphosphonic acid is dissolved in 998 ml of distilled water. Silver electrodes are immersed in the solution and a direct current of 180 mA is passed for 15 seconds. After this, 0.689 g of calcium oxide is added to the solution, mixed and homogenized using a submersible ultrasonic laboratory homogenizer at a power of 180 watts for 15 minutes at a temperature of 80 °C. The solution is transparent with slight opalescence.

The resulting solution contains 1-hydroxyethylidene dihydrogen calcium phosphate 0.3 wt. %, silver 3x10-5 wt. %.

Example 4.

0.34 g of 1-hydroxyethylidene diphosphonic acid is dissolved in 1000 ml of distilled water. Silver electrodes are immersed in the solution and a direct current of 100 mA is passed for 27 seconds. After this, 0.121 g of calcium hydroxide is added to the solution, mixed and homogenized using a submersible ultrasonic laboratory homogenizer at a power of 180 watts for 15 minutes at a temperature of 80 °C. The solution is transparent with slight opalescence.

The resulting solution contains 1-hydroxyethylidene dihydrogen calcium phosphate 0.04 wt. %, silver 3x10-5 wt. %.

Example 5.

0.59 g of 1-hydroxyethylidene diphosphonic acid is dissolved in 999 ml of distilled water. Silver electrodes are immersed in the solution and a direct current of 100 mA is passed for 36 seconds. After this, 0.161 g of calcium oxide is added to the solution, mixed and homogenized on a submersible ultrasonic laboratory homogenizer at a power of 180 watts for 15 minutes at a temperature of 80 °C. The solution is transparent with slight opalescence.

The resulting solution contains 1-hydroxyethylidene dihydrogen calcium phosphate 0.07 wt. %, silver 4x10-5 wt. %.

Example 6.

3.38 g of 1-hydroxyethylidene diphosphonic acid is dissolved in 996 ml of distilled water. Silver electrodes are immersed in the solution and a direct current of 250 mA is passed for 18 seconds. After this, 0.95 g of calcium oxide is added to the solution, mixed and homogenized using a submersible ultrasonic laboratory homogenizer at a power of 180 watts for 15 minutes at a temperature of 80 °C. The solution is transparent with slight opalescence.

The resulting solution contains 1-hydroxyethylidene dihydrogen calcium phosphate 0.4 wt. %, silver 5x10-5 wt. %.

The pharmaceutical parenteral composition for bone tissue restoration, obtained in accordance with the examples given, in all cases confirmed the achievement of the stated technical result.

In order to determine the general toxic effect and prove the effectiveness of the proposed composition, its preclinical tests were carried out at the central research laboratory of the Volga Research Medical University.

The experiment was carried out on male rabbits of the "Soviet Chinchila" line weighing 3.5-5 kg, which were divided into three groups: control - no administration of the drug in the injury, experimental - administration of the proposed composition in the injury, comparison - administration of the reference drug "Bioplast-dent" in injury. In animals of the control and experimental groups, a trephine with a diameter of 3.0 mm was used to produce a defect in the upper jaw and ilium to a depth of 1-3 mm. In rabbits of the control group, the bone defect was left free; In rabbits of the first experimental group, bone tissue defects were filled with an experimental drug. In rabbits of the second experimental group, defects in the bone tissue of the upper jaw and ilium were filled with a comparison drug.

The surgical intervention was carried out under 3-component anesthesia, which included intramuscular administration of drugs such as Zoletil 5 mg/kg mg intramuscularly (Virbac France), Xylanit 0.1 mg intramuscularly (ZAO NITA-PHARM, Russia, . Saratov) and inhalation - with the drug Isoflurane (Carizoo Laboratory, Spain). Innings The respiratory mixture during inhalation anesthesia was carried out using an ArMed 7F-1L oxygen concentrator and an anesthesia system for small laboratory animals, isoflurane inhalation, Biosthesia (“Vilber Lourmat”, France). Monitoring of the physiological state of the animals was carried out throughout the operation and during the postoperative period. In fig. Figure 1 shows the skeleton of a rabbit, indicating the zones, which are highlighted by rectangles, surgical intervention and collection of morphological material.

All animals underwent surgery satisfactorily. Rabbits from each group were removed from the experiment on days 3, 7, 14, 28 and 90. All manipulations on animals were carried out under combined three-component anesthesia in compliance with the rules of asepsis and antisepsis, as well as in accordance with the international principles of the Declaration of Helsinki on the humane treatment of animals. To carry out hematological and biochemical analysis of the blood of laboratory animals, blood was taken intravenously from the medial ear vein of rabbits.

To carry out morphological analysis, a fragment of bone tissue of the upper jaw and ilium, including intact areas and the regenerated area, was explanted in animals of all groups after they were removed from the experiment.

Morphological assessment and photographic recording of the experimental material was performed using a Leica DMLS light microscope and a small-format color camera (CCD) in the working magnification range from 40 to 400. A total of 172 histological preparations of the ilium and 134 of the maxillary bone were analyzed (Tables 1, 2).

Table 1. Distribution of animals by experimental groups and timing of experimental material collection (ilium)

Table 2. Distribution of animals by experimental groups and timing of experimental material collection (maxillary bone)

Results and discussion

In rabbits of all groups after 3 days. postoperative period, the area of the formed defect was clearly determined at the light-optical level. In bone tissue samples from animals in the control group, the defect was filled with tissue detritus. Along the entire perimeter of the wound, fibrin separated the ends of the cuts of the upper jaw and ilium from necrotic masses and blood cells. In bone tissue samples from animals of the first experimental group, on the 3rd day of the postoperative period, the entire volume of the bone defect was filled with the experimental preparation. The preparations show that the material adheres tightly to the bone fragments. During this observation period, the surface of the bone wound of the upper jaw is covered with loose fibrous connective tissue, in which the formation of blood vessels is noticeable. Among the components of the intercellular substance of connective tissue, numerous poorly differentiated cells are identified.

In bone tissue samples from animals of the second experimental group, after 3 days. after damage and filling the wound with the reference drug, the edges of the bone along the perimeter are delimited from the drug by a leukocyte shaft. Fibrin deposits and small particles of the reference drug are visible between the blood cells. The surface of the defect is covered with a layer of fibrin, among which blood cells and necrotic masses are visible. Morphological characteristics of the acute phase of inflammation in a bone wound in rabbits of all groups after 3 days. postoperative period have pronounced differences. The intensity of the inflammatory reaction in the tissue upon administration of the comparison drug is lower than in animals of the control group. However, the lowest level of inflammatory reaction was observed when using the experimental drug. Obviously, this fact is due to different mechanisms of action of the substances included in the preparations.

On the 7th day, differences in the process of reparative regeneration in the bones of the upper jaw and ilium become more pronounced. In the second case, the healing process is more intense.

Morphology of the regenerate on the 14th day. observations has pronounced differences in animals of experimental groups. In samples obtained from rabbits of the first experimental group, the degradation of the experimental drug is associated with its replacement by intensively vascularized coarse-fiber bone tissue. In rabbits of the second experimental group, signs of inflammation remain in the regenerate tissues, and osteoblasts have signs of low functional activity. After 28 days, in the bone tissue samples of rabbits from the control group, the defect was completely filled with regenerate and the surface was covered with a thickened periosteum. In the area of the bone defect bed, the regenerate is represented by young bone tissue. Functionally active osteoblasts are identified on the surface of the developing bone trabeculae. In the area of the ends of the cuts, the regenerate is made of fibrous tissue with signs of intense vascularization. On histological preparations of bone tissue of animals of the first experimental group by the 28th day. observations, the defect is represented by developing lamellar bone tissue. On the surface between the periosteum and the regenerate there are small areas filled with an experimental preparation, which has signs of resorption and replacement with bone tissue. It should be noted that along the entire perimeter of the defect, the regenerate is integrated with the bone tissue of the upper jaw and ilium. In similar samples of bone tissue from rabbits of the second experimental group, the defect along the entire perimeter of the edges of the cuts of the upper jaw and the bed was made of developing coarse-fiber bone tissue.

It should be especially noted that sections of the ilium, starting from days 3 to 28 of observation in the operated animals, revealed components of hyaline cartilaginous tissue, chondroblasts and chondrocytes of varying degrees of differentiation, tightly adjacent to the bone defect (Fig. 2). The presence of chondrocytes in the early stages of observation and their persistence 28 days after surgery in the area of the bone defect serves as a springboard for the development of enchondral osteogenesis.

In animals of the control group, 3 months after bone damage, the defect was completely filled with regenerate, represented by coarse-fiber bone tissue (Fig. 3). In fragment A, black arrows indicate the boundary between the regenerate and the bone defect; fragment B is an enlarged part of fragment A (staining according to Van Gieson, magnification Ax 100; B x200). In the histological preparations of the experimental groups, the defect zone is not identified.

The morphological study showed that bone tissue regeneration using the experimental drug is superior to that when using the comparison drug. The data obtained indicate the feasibility of using the proposed drug to optimize osteogenesis in case of bone tissue damage. Probably, the proposed drug indirectly affects the development of inflammation in the wound and stimulates proliferation osteogenic cells. The results obtained allow us to conclude that the proposed drug is a promising material for further morphofunctional studies as a stimulator of reparative bone tissue regeneration.

Analysis of hematological parameters in the experimental group after 3 months (number of red blood cells, leukocyte content, ratio of band neutrophils, basophils, monocytes in the leukocyte formula) did not reveal statistically significant differences relative to the control. There was a statistically significant decrease in the content of platelets, segmented neutrophils and eosinophils in the leukocyte formula, an increase in hemoglobin and lymphocytes relative to the control. In the comparison group, as in the experimental group, there was a statistically significant increase in hemoglobin levels compared to the control. The statistically significant changes recorded were within the physiological norm of this animal species.

As studies have shown, the proposed pharmaceutical parenteral composition is effective for the restoration of bone tissue, does not cause aseptic inflammation in the area of the bone defect, and increases the degree and dynamics of reparative processes.

Claims

Claim

Pharmaceutical parenteral composition for the restoration of bone tissue, obtained by dissolving 1-hydroxyethylidene diphosphonic acid in distilled water, placing silver electrodes in the resulting solution and passing a direct current of 180 mA through them for 10, 15 or 25 seconds, 100 mA for 27 or 36 seconds or 250 mA for 180 seconds, then add calcium hydroxide or calcium oxide, mix and homogenize at 180 watts for 15 minutes at a temperature not exceeding 90°C.