WO2015160284A1 - Pharmaceutical composition for treating endometriosis and method of producing same - Google Patents

Abstract

The group of inventions relates to pharmaceutical compositions and techniques for producing same from animal-derived raw materials, and can be used for the treatment and rehabilitation of various types of endometriosis and certain oncological diseases. A pharmaceutical composition for treating endometriosis is produced from the follicular fluid of cattle, from which serum albumin and a protein of the TGF-β family having the immune specificity of inhibin βA are separated, and NaCl salt is added, with the following ratio of components by mass percent: 0.0000015-0.3% TGF-β family protein having the immune specificity of inhibin βA; 3-5% serum albumin; the remainder NaCl salt. A method of producing a pharmaceutical composition for treating endometriosis is carried as follows: follicular fluid is collected from cattle, the fluid is cooled and then centrifuged to produce a supernatant, the supernatant is mixed with cooled acetone, the resulting mixture is left to sit and is then freeze-dried and subsequently purified by means of repeated centrifugation to produce a freeze-dried product, characterized in that the freeze-dried product is dissolved in a buffer, the resulting suspended matter is centrifuged at 8000-10000 revolutions per minute for 15-30 minutes in order to produce a supernatant, which is separated by means of ion exchange chromatography into DEAE-Sepharose, the eluate produced under an NaCl concentration of 0.11 M to 0.19 M in an eluting solvent is collected and dialyzed against water for no less than 8 hours, and NaCl salt is added to produce the preparation.

Description

 PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF ENDOMETRIOSIS

 AND METHOD FOR ITS PRODUCTION.

 Technical field

The group of inventions relates to pharmaceutical compositions and technologies for their preparation from raw materials of animal origin and can be used for the treatment and rehabilitation of various types of endometriosis and some oncological diseases.

State of the art

Currently, in medical practice, the conservative method is most widely used for the treatment of endometriosis, including the suppression of the clinically active process of endometriosis by the introduction of a hormonal drug (see A. Strizhakov, A. Davydov (Endometriosis: clinical and theoretical aspects. - M .: Medicine, 1996 .-- p.206).

Common disadvantages of the traditionally used methods are the weakly pronounced effectiveness of hormonal drugs in patients with moderate and severe endometriosis, the development of relapses shortly after the course of treatment, the need for long-term use of the drug, which leads to undesirable complications, as well as insufficient effectiveness associated with the tendency of endometriosis to the development of relapses leading to an increase in the frequency and extent of surgical interventions without a guarantee of complete cure.

A pharmaceutical composition is known for the treatment of endometriosis or infertility or for increasing the ability to reproduce offspring, comprising a therapeutically effective amount of a β-adrenergic agonist and a pharmaceutically acceptable bioadhesive carrier, in which the β-adrenergic agonist is terbutaline and the bioadhesive carrier includes a crosslinked, water-insoluble, but water insoluble, but water insoluble, a polycarboxylic acid polymer, while the concentration of terbutaline is in the range from less than 0.1 to about 0.4 wt.%, and polycarbophil is used as the polymer. A composition in which terbutaline is a β-adrenergic agonist is intended for administration in a dosage of from about 0.5 to 2.5 g and which delivers from less than 1 to about 8 mg of terbutaline (see RU 2310474, C2 04.10.2000).

As a result of the analysis of a well-known drug, it should be noted that it is not a direct-acting drug and all its effects are indirect. It has poorly expressed effectiveness in patients with moderate and severe endometriosis, requires long-term use of the drug, which leads to undesirable complications inherent in all adrenomimetics. The most common complications are tachycardia and tremors. Sometimes hyperglycemia, central nervous system agitation, lowering blood pressure.

A known method for the treatment of uterine endometriosis, including the suppression of the clinically active process of endometriosis with the introduction of a hormonal drug, moreover, in parallel with the hormonal drug, the patient is additionally injected with animal preparation Squaacan, intramuscularly daily, 2.0 ml for 75 days with a seven-day break every 30 days, and as a hormonal drug, for example, duphaston is used. (see RU 221 1700 C2, 07.23.2001).

This method will increase the degree of suppression of the prevalence and growth of foci of endometriosis, reduce treatment time, make it safer for patients with uterine endometriosis, because antitumor cellular immunity is activated, the antibacterial activity of neutrophils and the phagocytic activity of macrophages increase.

The drug "Squaacan" used for treatment is obtained as follows. Initially, the raw materials are homogenized, centrifuged with subsequent membrane filtration, where the catran shark liver is used as raw material, the liver after homogenization is degreased by cold sedimentation at 1 - 5 ° C for 20-24 hours, then the fat-free homogenate is sent to thermal and chemical protein coagulation at pH 1.0-6 , 0, stand at room temperature for 5-6 hours to obtain a stabilized extract, which, after centrifugation and membrane filtration, is amplified and autoclaved (see RU 2336884 C1, 07/11/2007).

Squaacan is a highly active bioorganic preparation from a stabilized extract of catran liver cells with multifaceted biological activity: antitoxic, antimetastatic, immunostimulating properties. It has a long shelf life (three years).

As a result of the analysis of the Squaacan preparation, it can be concluded that none of its listed activities is specific for endometriosis, and the immunostimulating effect can be attributed to contraindications, because endometriosis has an autoimmune component (that is, there is a danger of stimulating the disease). In addition, the effectiveness of the drug “Squaacan” is insufficient for its use in the treatment of endometriosis as an independent drug. It should also be noted that the Squaacan preparation obtained by the above method contains many biologically active substances, the quantitative ratio of which is not controlled. The quality of the raw material, and this is the Katran liver, will depend on the habitat, on the fishing season, on its age and condition. All these factors lead to instability of the medicinal properties of the drug from batch to batch.

Disclosure of invention

The technical result of the claimed group of inventions is to increase the effectiveness of treatment of endometriosis due to a significant reduction or complete absence of relapse after a course of treatment, the absence of significant side effects, normalization of hormonal status. The specified technical result is ensured by the fact that the pharmaceutical composition is obtained from the follicular fluid of cattle and contains NaCl salt, serum albumin and a protein of the TGF-β family, transforming growth factor having immunospecificity of βина inhibin, in the following ratio of their components, in wt.%:

 - a protein of the TGF-β family having the immunospecificity of β ингиб inhibin: 0.0000015-

0.3%;

 - serum albumin: 3-5%

- NaCl salt: the rest.

 To implement the method of obtaining the pharmaceutical composition, the cattle follicular fluid is selected, cooled, and centrifuged after cooling to obtain the supernatant, mixed with cooled acetone, the resulting mixture is defended, then it is freeze-dried, and purified by centrifugation to obtain a lyophilized product, which dissolved in buffer, the resulting suspension is centrifuged at 8000-10000 rpm for 15-30 minutes to obtain a supernatant that is separated by ion exchange chromatography on DEAE-Sepharose, the eluate is collected, leaving at a concentration of NaCl in an eluting solution from 0.1 1 M at 0.19 M, which is dialyzed against water for at least 8 hours, after which the protein concentration in the dialysate is determined by the Bradford method, NaCl is added from calculation of 9 mg of salt per 0.3 mg of protein, sterilizing filtration is carried out, it is poured into vials in an amount of 0.3 mg of protein and 9 mg of salt per vial and freeze-dried.

The use as a raw material for obtaining a composition of follicular fluid injected from cattle ovarian follicles is very significant, because: firstly, it provides an accessible source, and secondly, studies have shown that the use of other follicular fluid as raw materials species of animals, it is not possible to obtain a product with the necessary number of active components .. As shown by experiments, the use of follicular protein preparation, for example, follicular fluid of pigs (US patent N ° 5102867) does not allow to obtain a composition of a given protein composition.

 Cooling the liquid before primary centrifugation allows you to keep the proteins in their native state and increase their yield.

 Centrifugation (primary and secondary) purifies soluble proteins from cell debris.

 Carrying out the deposition in chilled acetone allows you to clean the drug from non-protein components.

 Ion exchange chromatography on DEAE-Sepharose results in a follicular protein preparation of a specific composition, consisting of a protein of the TGF-β family, having immunospecificity of βΑ inhibin and bovine serum albumin.

The term "βин inhibin" is commonly used. In the literature, there is also its identical designation - inhibin A.

Lyophilization of the follicular protein preparation, containing NaCl as a stabilizing agent, allows the drug to be stored for a sufficiently long time.

 It should be noted, and this is confirmed experimentally, that the claimed technical result can be achieved only with the unconditional fulfillment of all the actions reflected in the application materials with strict adherence to their declared sequence and modes, as well as the quantitative ratio of the components of the drug. Thus, the features set forth in the claims and the sequence of their implementation are essential.

 A method of obtaining a pharmaceutical composition is carried out in several stages.

Initially, follicular cattle fluid is removed from the ovaries. The selected fluid is centrifuged at 8000-10000 g for receiving supernatant. The supernatant obtained by centrifugation is poured into a clean container and cooled to 4-8 ° C to increase the amount of precipitated protein. Then, acetone (grades of special purity grade) was added to the supernatant, cooled to -18 ° C in a ratio of 1/1 to 2/3 by volume, stirred and centrifuged for 15-25 minutes at 8000-10000 g (Eppendorf 5804 centrifuge) to obtain a precipitate. The supernatant is discarded. The precipitate was transferred to a container, cooled to -18 ° C and dried on a freeze dryer (VirTis benchtop SLC) until it was completely dried. The result is a lyophilized product in the form of a granular powder from white to dark yellow, poorly soluble in water.

In the next step, the lyophilized product is dissolved in a pH 7.2 buffer with constant stirring for 15-30 minutes. The resulting suspension is centrifuged at 8000-10000 rpm for 15-30 minutes to obtain a supernatant. The resulting supernatant is discarded and applied to a DEAE-Sepharose column. Column filling time is 6-8 hours. Balance the column with a buffer of 12-24 hours to a pH of 7-7.2.

Options for implementing the proposal

Further isolation of the protein preparation can be done in two ways, getting the same result:

1. Apply the supernatant obtained from 3 g of lyophilized follicular fluid to the column and dispense 180 ml of buffer. Using a chromatographic system (BioRad BioLogic LP with a UV detector with a wavelength of λ = 280 nm), 1 12 ml of 0.1 1 M NaCl solution (approximately 2.6 column volumes) is supplied. Then, 120 ml of a 0.1-1- 0.19 M NaCl solution in a buffer are fed and at the same time, the eluate is collected in a container. After that, 40 ml of a 2M NaCl solution are fed onto the column to remove residues of the applied substances from the column. The collected eluate is frozen in a freezer (Sanyo MDF-436) at -18 ° C to 35 ° C, then freeze-dried. 2. The supernatant obtained from 3g lyophilized follicular fluid is applied to the column and eluted with a NaCl gradient from 0 to III in a pH 7.05-7.20 buffer on a chromatographic system (for example, BioRad). Select the eluate, washed with salt concentrations of 0.11 - 0.19 M NaCl. Control by the recorder. The collected eluate is frozen in a freezer (Sanyo MDF-436) at -18 ° C to 35 ° C, then freeze-dried.

As a result of using process 1 or process 2, the same eluate is obtained, therefore, the coherence of using one or the other process is determined by the instrument base and technological conditions.

As a result of the described manipulations, a protein preparation with a buffer is obtained - a light, airy white powder

To remove impurity buffer from the resulting preparation, the next step is the dialysis of the protein preparation with the buffer, for which:

- dissolve it in distilled water;

- dialysis through a membrane (ROTH) is carried out with an exception volume of 14 kDa against 2 liters of distilled water with a change in the distillate according to the scheme:

dialysis 3 hours (2 liters of distillate);

- change of distillate;

dialysis 3 hours (2 liters of distillate);

- change of distillate;

dialysis for 2 hours (2 liters of distillate).

The process is carried out at a temperature of 4 to 8 ° C. Get dialysate. The membrane is incised and the solution is poured into a glass beaker, the amount of total protein in the solution is determined, NaCl is added at the rate of 9 mg of salt per 0.3 mg of protein, sterilization filtration is carried out, 0.3 mg of protein and 9 mg of salt are poured into vials and freeze-dried.

The quantitative content of the components indicated in the claims is controlled by well-known enzyme-linked immunosorbent assay, and the activity of the protein preparation is controlled by the standard method for determining the biological activity in a VERO cell culture.

As a result of the implementation of this method, a follicular protein preparation containing NaCl, serum albumin and a protein of the TGF-β family having immunospecificity of β ингиб inhibin is obtained in the following ratio of their components, May. %:

- a protein of the TGF-β family having immunospecificity of β inhibin: 0.0000015-0.3%;

- serum albumin: 3-5%

- NaCl salt: the rest.

The essence of the claimed method will be more clear from the above example. Initially, the follicular fluid of cattle is taken, for which the ovaries in the area of the follicular vesicles are wiped with 96% alcohol, then they are punctured with a disposable sterile syringe and 10 ml of follicular fluid is taken into a centrifuge tube rinsed with alcohol, with a capacity of at least 20 ml. Follicular fluid is centrifuged for 25 min at 8000 g (Eppendorf 5804 centrifuge). The resulting supernatant is poured into a clean container and cooled to 6 ° C. Then acetone was added to the supernatant (grades of special purity grade), cooled to -18 ° C in a ratio of 1/1 by volume, stirred and centrifuged for 15 min at 8000g (Eppendorf 5804 centrifuge). The supernatant is discarded. The precipitate was transferred to a container, cooled to -18 ° C and dried on a freeze dryer (VirTis benchtop SLC) until it was completely dried. Next, 1.3 g of lyophilized follicular fluid is dissolved in 40 ml of a pH 7.2 buffer in a 50 ml glass flask using an ultrasonic stirrer USM 001 (Poland) with constant stirring for 30 minutes. The resulting suspension was centrifuged in an Eppendorf 5804 centrifuge at 8,000 rpm for 30 minutes. The supernatant (approx. 8 ml) is drained and applied to a column (Cat. 34 "57407-08-6 DFF100, Sigma, Diethylaminoethyl Sepharose with DEAE-Sepharose. Column: glass, dimensions 1.5 * 30 cm, column working volume - 42 ml, Column filling time is 6 hours, Column balancing time with buffer is 12 hours, to pH 7.2). The supernatant obtained from 1.3 g of lyophilized follicular fluid is then applied to the column and the column is washed with 180 ml of buffer. Using a chromatographic system (BioRad BioLogic LP with a UV detector with a wavelength of λ = 280 nm), 112 ml of a 0.11 M NaCl solution (approximately 2.6 column volumes) is supplied. Then, 120 ml of a 0.19 M NaCl solution are fed in the buffer and at the same time they begin to collect the eluate in a container (we obtain 120 ml of solution). After that, 40 ml of a 2M NaCl solution are fed onto the column to remove residues of the applied substances from the column. The collected eluate is frozen in a freezer (Sanyo MDF-436) at -35 ° C, then freeze-dried (VirTis benchtop SLC).

Then dialysis of the obtained lyophilisate is carried out for which

 - Dissolve 0.26 g of protein in 10 ml of distilled water.

- subjected to dialysis through a membrane (ROTH) with an exception volume of 14 kDa against 2 liters of distilled water with a change in the distillate according to the scheme:

dialysis 3 hours (2 liters of distillate);

 - change of distillate;

dialysis 3 hours (2 liters of distillate);

- change of distillate;

dialysis for 2 hours (2 liters of distillate).

 The process is carried out at a temperature of 6 ° C.

Receive dialysate in an amount of 12 ml. The membrane is incised and the solution is poured into a 50 ml glass beaker. Determine the concentration of protein according to the method of Bradford, NaCl is added at the rate of 9 mg of salt per 0.3 mg of protein, sterilization filtration is carried out, 0.3 mg of protein and 9 mg of salt are poured into vials and lyophilized.

 The quantitative content of the components indicated in the claims is monitored by enzyme-linked immunosorbent assay, and the activity of the protein preparation is monitored by determining the biological activity in a VERO cell culture.

 The result is 8.06 g of follicular protein preparation containing:

 - NaCl 7.8 g

- bovine serum albumin 0.254813 g

- a protein of the TGF-β family having immunospecificity of inhibin β ингиб 0.000013 g

- impurities of 0.005174 g, which do not significantly affect the achievement of the specified technical result.

 It should be noted that the indicated technical result is provided only within the limits of the follicular protein preparation components indicated in the claims.

At lower values of the protein of the TGF-β family, which has the immunospecificity of βина inhibin, the drug loses its activity; at higher values, the risk of pathological effects increases. The presence of serum albumin determines the stability of inhibins in the protein preparation.

 This drug was tested on a rat model of endometriosis. Intraperitoneal administration of the drug at a dose of 40 μg per animal daily in a single dose for 10 days in rats with surgically induced endometriosis led to a decrease in implant volume in rats by 7.9 times.

Obtained by this method and with the above composition, the drug was used in the treatment of endometriosis in women.

Examples. AND

 The treatment was carried out with a drug (dosage of 0.3 mg) of the following composition:

 - NaCl 9 mg

 - bovine serum albumin 0.299 mg

 - a protein of the TGF-β family having immunospecificity of inhibin βΑ 0.000013 mg

 Therapy scheme:

• first month - 10 injections of 0.3 mg from the fifth day of the monthly cycle every day.

• second month - 10 injections of 0.3 mg from the fifth day of the monthly cycle every day.

• the third month - 10 injections of 0.3 mg from the fifth day of the monthly cycle every day.

Patient U., 27l. Clinical diagnosis: uterine endometriosis, diffuse form, made in 2008. Complaints of heavy, painful menstruation.

Before treatment, clinical and biochemical parameters: urinalysis, clinical blood analysis, biochemistry, coagulation, tumor markers are within normal limits. Blood hormones: estradiol 0.618 nmol / L., Progesterone 7.6 nmol / L. Ultrasound of the organs of the m / pelvis: endometriosis of the uterus. Morphological conclusion: active adenomyosis.

On the background of treatment, positive dynamics: there are no pains, menstruation is moderate. Clinical and biochemical parameters are within normal limits. In the hormonal analysis of blood, an increase in the level of estradiol to 0.72 nmol / L, a decrease in the level of progesterone to 3.97 nmol / L was noted. Ultrasound of the organs of the m / pelvis: positive dynamics. Morphological conclusion: endometriosis was not detected (positive dynamics).

 Patient C, 39 L. Clinical diagnosis: uterine endometriosis, focal form, made in 2002. Complaints of heavy, painful menstruation.

Before treatment, clinical and biochemical parameters: urinalysis, biochemistry, coagulogram, tumor markers are within normal limits. Clinical blood test: HB- PO g / l. Oncomarkers CA 125 - 47.6 (normal - less than 35 U / ml). Blood hormones: estradiol 0.56 nmol / L., Progesterone 1.8 nmol / L. Ultrasound of the organs of the m / pelvis: endometriosis of the uterus. Morphological conclusion: moderate adenomyosis.

 Against the background of treatment, positive dynamics were noted: pain and hyperpolymenorrhea syndrome decreased. Clinical and morphological indicators within normal limits. Clinical blood test: HB-102g / l. In a hormonal blood test, an increase in estradiol levels to 0.8 nmol / L, a decrease in progesterone levels to 0.2 nmol / L was noted. Ultrasound of the organs of the m / pelvis: positive dynamics. Morphological studies: adenomyosis of weak activity (positive dynamics).

Patient K., 33. Clinical diagnosis: Endometriosis of the uterus, diffuse form, made in 2000. Complaints of heavy menstruation. The menstrual cycle is irregular.

Before treatment, clinical and biochemical parameters: urinalysis, clinical blood analysis, biochemistry, coagulation, tumor markers are within normal limits. Blood hormones: estradiol -0.538 nmol / l, progesterone - 3.7 nmol / l. Ultrasound of the organs of the m / pelvis: endometriosis of the uterus. Morphological conclusion: adenomyosis of weak activity.

 During treatment, a positive trend was noted: hyperpolymenorrhea syndrome decreased. Clinical and biochemical parameters are within normal limits. A hormonal blood test showed a decrease in estradiol to 0.218 nmol / L, an increase in progesterone to 36.7 nmol / L. Ultrasound of the organs of the m / pelvis - positive dynamics. Morphological study: adenomyosis was not detected (positive dynamics).

 Side effects during treatment in patients were not observed.

The use of the drug by patients suffering from endometriosis led to the disappearance of hyperpolymenorrhea - in 8% of cases, and its decrease - in 92% of cases; to the disappearance of algodismenorea in 36% of cases and its decrease in 64% of cases; When assessing the severity of the disease - to the disappearance of symptoms in 32% of cases and a decrease in the severity of the disease - in 68% of cases.

 Based on the conclusions of a comprehensive morphological (histological and immunomorphological) study, a positive effect of therapy was detected in 80% of cases, and in other cases, a tendency to a decrease in the activity of foci of adenomyosis was revealed.

The drug can also be used to treat certain cancers and in veterinary medicine.

Claims

CLAIM  1. A pharmaceutical composition for the treatment of endometriosis, comprising serum albumin and a protein of the TGF-β family having immunospecificity of inhibin βΑ and NaCl salt in the following ratio of their components, in wt.%:  - a protein of the TGF-β family having immunospecificity of β inhibin: 0.0000015-0.3%; - serum albumin: 3-5%; - NaCl salt: the rest. 2. A method of obtaining a pharmaceutical composition for the treatment of endometriosis, according to which the cattle follicular fluid is taken, cooled, and centrifuged after cooling to obtain the supernatant, mixed with chilled acetone, the resulting mixture is defended, then it is freeze-dried, and purified by centrifugation again, obtaining a lyophilized product, the lyophilized product being dissolved in a buffer, the resulting suspension was centrifuged at 8000-10000 rpm for 15-30 minutes to obtain super of the natant, which is separated by ion-exchange chromatography on DEAE-Sepharose, collect the eluate, leaving at a concentration of NaCl in an elution solution of 0.11 M in 0.19 M, which is dialysed against water for at least 8 hours, add NaCl salt, carry out sterilization filtration and lyophilic drying, getting the drug.