RU2666148C1 - Method for preparing water-soluble lyophilized salt form bis(2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl)-(4-nitrophenyl)methane - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: invention relates to a novel water-soluble lyophilized salt form bis(2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl)-(4-nitrophenyl)methane of formula 2a, which has hepatoprotective activity and is intended for the liver treatment, in particular hepatitis. Method for preparing a water-soluble lyophilized salt form bis(2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl)-(4-nitrophenyl)methane of formula 2acomprises: preparing a solution of bis(2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl)-(4-nitrophenyl)methane, preparing a solution of tris-hydroxymethylammonium, combining the resultant solutions to form a solution of tris-hydroxymethylammonium salt of formula 2a, followed by lyophilization of the resultant saline. Salt solution 2a used for lyophilization usually contains from 1.5 to 5 wt.% of bis(2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl)-(4-nitrophenyl)methane. Solution of tris-oxy methyl ammonium salt of bis(2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl)-(4-nitrophenyl)methane used for lyophilization has a concentration from 2.5 to 8.2 wt.% and pH from 6.9 to 7.2. Lyophilization is carried out at a pressure of 0.3–0.5 bar for 15–16 hours, followed by drying for 2–4 hours at a pressure of 0.05 mbar. Resultant lyophilized water-soluble salt form is generally used in a pharmaceutical composition suitable for parenteral administration, in a therapeutically or prophylactically effective amount as a single dose. Typically, a lyophilized water-soluble salt form is used to prepare a medicament packaged in a vessel suitable for parenteral administration. Vessel is a multi-dose vial, ampoule, pre-filled syringe, flexible perfusion bag. For treating or preventing hepatitis, the compound of formula 2a is also administered by infusion, perfusion or injection or orally, for example as a suspension in an aqueous medium.EFFECT: resultant lyophilized water-soluble salt form has increased storage stability and reduced toxicity while maintaining high hepatoprotective activity.13 cl, 5 tbl

Description

Technical field

The invention relates to chemistry and pharmacology, specifically to a method for preparing a water-soluble lyophilized form (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl ) methane), which has hepatoprotective activity and is intended for use in medical practice for the treatment of liver diseases, as well as to the resulting water-soluble lyophilized form of this compound.

State of the art

A compound of the formula (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane) is known (structural formula 1) having a hepatoprotective activity and proposed as a means for treating liver diseases.

In its free form, compound 1 is an acid that is poorly soluble in water and therefore in this form is inconvenient for parenteral use. Obtaining a water-soluble form (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane) (1) is possible by converting it to salt, as this substance gives suitable for parenteral administration water-soluble salts with alkali metal cations, nitrogenous bases and other pharmacologically acceptable cations. However, aqueous solutions of salts 1 have low stability and are unsuitable for long-term storage, which limits the possibility of using the drug.

A common way to stabilize such drugs is to lyophilize them, that is, to obtain dry instant forms (lyophilisates) by low-temperature evaporation of frozen water-salt solutions [Kunal A. Gaidhani, Mallinath Harwalkar, Deepak Bhambere 1, Pallavi S. Nirgude World Journal of Pharmaceutical Research 2017 Vol 4, Issue 8, 516-543. LYOPHILIZATION / FREEZE DRYING - A REVIEW].

As part of the well-known lyophilized preparations, various pharmacologically acceptable cations are used; most common is sodium. With this in mind, and since compound 1 is a derivative of barbituric acid, barbiturates are also usually lyophilized in the form of sodium salts, for example sodium hexenal, thiopental sodium [5 - EUROPEAN PHARMACOPOEIA - 8th Ed, 2013, V. 1, 2.] , as a technical solution closest to the present invention, a general lyophilization method using sodium salt is considered [US Pat. USA 4138486, A61K 81/00, 1980] for (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane). To increase the stability of lyophilisates, various additives are introduced into their composition, in particular sugars, amino acids, and salts [US Patent B 4,138,486, A61K 81/00, 1980]. For example, in WO 2005/053745, publ. 06.06.16, Stefan A. et al. An example of the introduction of Tris-hydroxymethylaminomethane, known as a stabilizer of biochemical systems, into the lyophilisate is described.

It should be noted that in the known method, tris-hydroxymethylaminomethane is used as a component of the buffer system, and not as a salt-forming counterion. Due to the complexity of the chemical processes and the lack of published materials, it is not possible to predict the composition of the salt form necessary to obtain a lyophilisate (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl ) - (4-nitrophenyl) methane), which would be characterized by sufficient storage stability.

Another objective arising from the existing level of technology is the need to obtain such a pharmaceutical form (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4 -nitrophenyl) methane), which would have a reduced toxicity of the active substance while maintaining the target pharmacological activity.

Disclosure of invention

The problem is solved by obtaining a lyophilized Tris-hydroxymethylammonium salt of the compound (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane) formula (2a) having hepatoprotective activity and having an advantage over the known lyophilized salts of this compound, for example, compared with the compound of formula (2b) due to greater storage stability and lower toxicity.

More specifically, according to the invention, the problem is solved by obtaining a lyophilized salt of the formula 2a, where X ⁺ is a tris-hydroxymethylaminomethane cation:

The applicant is not aware of any sources of information that would contain information about identical technical solutions, which allows us to conclude that the claimed invention meets the criterion of "novelty." The applicant has not identified any sources of information containing information about the impact of the claimed distinctive features on the technical result achieved as a result of their implementation. This, according to the applicant, indicates that this technical solution meets the criterion of "inventive step". Thus, according to the applicant, the lyophilized form of the compound (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) proposed in the present invention ) methane) and the described method for its preparation are not obvious and do not follow from the existing level of technology.

The invention is illustrated by the following examples of obtaining the claimed substance and comparison samples, a table of characteristics of the obtained substances, experiments on the study of biological properties and tables of experimental results to determine the biological properties of the obtained substances, where:

Example 1 - General procedure for the preparation of lyophilized salts (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane) having the formula (2a-c);

Example 2 - a method for determining hepatoprotective activity;

Example 3 - methodology for the determination of acute toxicity;

Table 1 - characteristics of lyophilisates (2a-c);

Table 2 - the results of determining the hepatoprotective activity of lyophilisates (2a-c);

Table 3 - Results of determination of acute toxicity of lyophilisates (2a-c).

The invention is further explained in the detailed description of examples of its implementation.

The best embodiment of the invention

The general scheme for the preparation of lyophilisates (2a-c) is shown in Scheme 1. The procedure was carried out in accordance with the general procedure described in Example 1.

Example 1. General procedure for the preparation of lyophilized salts (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane) (2- from).

25 g (59 mmol) (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) are placed in a 1 L conical flask methane) 1 with a basic substance content of at least 99% and 500 ml of water are added. Then, with stirring, add a 10% aqueous solution of the appropriate base (NaOH, KOH or Tris-hydroxymethylaminomethane with a purity of 99.9%) in an amount sufficient to completely dissolve the substance 1 (about 120 mmol). After complete dissolution of substance 1, the solution is quantitatively transferred to a 1-liter volumetric flask and the volume is adjusted to the mark with water. The resulting solution with a drug content of 2.5% is filtered through a sterile cellulose acetate membrane filter with a pore diameter of 0.4 μm. The filtrate is poured into glass vials and frozen at a temperature (-20 ° C). The frozen product is placed in the drying chamber of the sublimation unit and kept at a pressure of 0.3-0.5 mbar for 15-16 hours. After this, drying is carried out for 4 hours, keeping the product at a pressure of 0.05 mbar.

After drying is completed, the chamber is filled with nitrogen and the vials with lyophilisate are closed with rubber stoppers.

The pH of a 2.5% aqueous solution of the claimed substance is 7.15 when using 120 mmol of tris-hydroxymethylaminomethane, 6.90 when using 115 mmol of tris-hydroxymethylaminomethane and 7.20 when using 122 mmol of tris-hydroxymethylaminomethane per 25 g of substance 1.

The results of the evaluation of the properties of lyophilisates 2a-c after 60 days of storage at room temperature are shown in table 1.

Thus, according to the results of evaluating the properties of lyophilisates 2a-c after 60 days of storage, the target product 2a has a clear advantage over the prototype (2b) and its analogue (2c). Product 2a is less colored, contains less impurities and has an almost neutral pH close to physiological.

Example 2. Obtaining dosage forms of the composition

A. The resulting solution with a drug content of 1.5% is filtered through a sterile cellulose acetate membrane filter with a pore diameter of 0.4 μm. The filtrate is poured into glass vials and frozen at a temperature (-30 ° C). The frozen product is placed in the drying chamber of the sublimation unit and kept at a pressure of 0.3-0.5 mbar for 20-24 hours. After this, drying is carried out for 2 hours, keeping the product at a pressure of 0.08 mbar. After drying is completed, the chamber is filled with nitrogen and the vials with lyophilisate are closed with rubber stoppers.

B. Sterilized filtration of the resulting solution is carried out, having a concentration of compound 2a 5% by weight, and under aseptic conditions, it is bottled under nitrogen in ampoules with a capacity of 2 ml of 1 ml in an ampoule. Filled ampoules are sealed under nitrogen.

In another embodiment, the ampoule cassettes are placed in a sublimation chamber, where they are frozen to a temperature of minus 40 ° C with a freezing rate of 5-10 ° C / h, and then subjected to drying in vacuum. Ampoules with the obtained dry lyophilized form of the drug are sealed under nitrogen.

Compositions suitable for oral administration may contain (a) lipid solutions, such as, for example, an effective amount of a compound dissolved in diluents, such as, for example, water, saline, (b) capsules, (c) suspensions in a suitable liquid, ( d) suitable emulsions and (e) ampoules. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic substances and solvents that make the composition compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solvents, thickeners, stabilizers and preservatives. The compositions can be presented in single dose doses or in sealed multiple dose doses, such as ampoules and vials, and can be stored in a cold (lyophilized) condition, requiring only the addition of a sterile liquid excipient, for example water for injection, immediately before use. Injectable solutions and suspensions prepared for immediate administration can be prepared from sterile powders, granules and tablets from the previously described.

In some embodiments, the composition is formulated to have a pH in the range of from about 6.8 to about 7.30, including, for example, a pH in any range of from about 6.8 to about 7.3, from about 6.9 to about 7.2 and from about 7.0 to about 7.1. In some embodiments, the pH of the composition is at least about 7.15. The composition can also be made of isotonic blood by adding an appropriate tonicity modifier, such as glycerol.

Also described are products containing the compositions described herein in their respective packaging. Suitable packages for the compositions described herein are known in the art and include, for example, vials (such as hermetic bottles), reservoirs, ampoules, vessels, containers, flexible packaging (such as hermetic lavsan or plastic bags) and the like. Such products may additionally be sterilized and / or sealed. Also provided are standard dosage forms containing the compositions described herein. These unit dosage forms can be stored in suitable packaging in single or multiple doses and can additionally be sterilized and hermetically sealed.

The present invention also describes kits containing compositions (or unit dosage forms and / or products of a manufacturer) described herein, and may further include instructions (s) on how to use the composition, such as, for example, the use described herein. In some embodiments, a kit of the invention comprises a package as described herein. In other embodiments, a kit of the invention comprises a package described herein and a second package containing a buffering agent.

The kits described herein may further include other materials described herein, preferred by the commercial and consumer parties, including other buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for implementing any of the methods described herein.

Kits may also be provided that contain sufficient dosages to achieve effective treatment of the subject over a long period of time, for example from a week to 6 months or more. Kits may also include a combination of dosages for a single administration of the pharmaceutical composition and instructions for use, packaged in quantities suitable for storage and use in pharmacies, such as hospital pharmacies and drugstores finished dosage forms.

Preferably, the lyophilized product of the invention is packaged in a vessel that can be hermetically sealed, or a sealed vessel adapted for parenteral administration. Such a vessel may be, for example, a vial called a "multi-dose", a syringe called a "pre-filled", a flexible bag for perfusion or ampoule. A multi-dose vial is usually provided with a septum stopper to allow the needle to pass through the syringe, and contains an amount of the preparation of the invention that can be removed in any desired amount sufficient to permit a certain number of perfusions or injections.

Industrial applicability

To implement the invention, well-known materials and equipment are used, which determines, according to the applicant, the invention meets the criterion of "Industrial Applicability" (IA).

Hepatoprotective activity

The hepatoprotective activity of the derivatives was studied on Wistar rats (males and females, 180-200 g, Rappolovo, St. Petersburg).

Acute toxic hepatitis in rats was caused by intragastric administration of acetaminophen (2.5 g / kg for 2 days) or CCl4 (0.5 ml / kg). The test drugs were administered orally in the form of a suspension in olive oil 1 time per day after the formation of a model pathology or intravenously at a dose of 25 mg / kg. The dynamics of the weight of rats was determined on the scales of the company "Sartorius".

Lyophilized products were recovered by adding a liquid diluent in less than 2 minutes. The recovery time of lyophilized formulations was 10-15 minutes.

The reconstituted formulations were administered, respectively, intraperitoneally (IP) to groups of ten male CD-1 (Crl.) Line mice weighing 24 +/- 2 g.

Industrial applicability

To implement the invention, known materials and equipment are used, which determines, according to the applicant, the invention meets the criterion of "Industrial applicability".

Claims (18)

1. A method of obtaining a water-soluble salt form (bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane) of formula 2a

including: - preparing a solution of bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane, - preparation of a solution of tris-hydroxymethylammonium, - combining the resulting solutions to obtain a solution of Tris-oxymethylammonium salt of the formula 2A, followed by lyophilization of the resulting saline. 2. The method according to p. 1, characterized in that the solution of salt 2a used for lyophilization contains from 1.5 to 5% wt. bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane. 3. The method according to p. 1, characterized in that used for lyophilization solution of tris-hydroxymethylammonium salt of bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane has a concentration of from 2.5% wt. up to 8.2% wt. 4. The method according to p. 1, characterized in that the resulting solution of the Tris-hydroxymethylammonium salt of the compounds of formula 2A has a pH from 6.9 to 7.2. 5. The method according to p. 1, characterized in that the lyophilization is carried out at a pressure of 0.3-0.5 bar for 15-16 hours, followed by drying for 2-4 hours at a pressure of 0.05 mbar. 6. Lyophilized water-soluble form of tris-hydroxymethylammonium salt of bis (2-thio-4,6-dioxo-1,2,3,4,5,6-hexahydropyrimidin-5-yl) - (4-nitrophenyl) methane of formula 2a for pharmaceutical use in the treatment of hepatitis. 7. The lyophilized water-soluble salt form according to claim 6 for use in a pharmaceutical composition suitable for parenteral administration. 8. The lyophilized water-soluble salt form according to claim 6 for use in a therapeutically or prophylactically effective amount in a pharmaceutical composition in unit dose form. 9. The lyophilized water-soluble salt form according to claim 6 for the manufacture of a medicament packaged in a vessel suitable for parenteral administration. 10. The lyophilized water-soluble salt form according to claim 9, wherein the vessel is a multi-dose vial, ampoule, pre-filled syringe, flexible bag for perfusion. 11. A method of treating or preventing hepatitis, comprising administering to a subject in need of such treatment a lyophilized, water-soluble salt form of formula 2a in an effective amount. 12. The method of treatment or prevention of hepatitis according to claim 11, characterized in that the compound of formula 2a is administered by infusion, perfusion or injection. 13. A method for the treatment or prevention of hepatitis according to claim 11, characterized in that the compound of formula 2a is administered orally, for example, as a suspension in an aqueous medium.