阅读说明：本技术 包含Fab I抑制剂的口服给药的药物组合物及制备其的方法 (Orally administered pharmaceutical compositions comprising Fab I inhibitors and methods of making the same ) 是由 曹在平 曹重明 于 2019-05-29 设计创作，主要内容包括：本发明涉及包含Fab I抑制剂的口服给药的药物组合物,并且涉及制备其的方法。本发明可以有效地应用于对抗生素等具有抗药性的细菌感染。更具体地,本发明可以通过提高溶解和洗脱速率而更快速地发挥疗效。此外,本发明可以通过调节粒径来改善制剂的混合均匀性和含量均匀性。(The present invention relates to orally administered pharmaceutical compositions comprising Fab I inhibitors, and to methods of making the same. The present invention can be effectively applied to bacterial infections having resistance to antibiotics and the like. More specifically, the present invention can exert therapeutic effects more rapidly by increasing the dissolution and elution rates. In addition, the present invention can improve the mixing uniformity and content uniformity of the preparation by adjusting the particle size.)

1. A pharmaceutical composition for oral administration comprising 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one or a salt thereof.

2. The orally administered pharmaceutical composition of claim 1, wherein the 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one or salt thereof has a particle size distribution D of 0.1 μ ι η to 500 μ ι η₉₀。

3. The orally administered pharmaceutical composition of claim 1, wherein the composition is provided in the form of a tablet or capsule.

4. The orally administered pharmaceutical composition of claim 1, wherein the 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one, or a salt thereof, or both, are present in an amount of 10% to 60% by weight, based on the total weight of the composition.

5. A method for preparing a pharmaceutical composition for oral administration, the method comprising:

adding 4-benzyloxy-1H-pyridone, 2-methyl-3-nitrobenzyl chloride and potassium tert-butoxide to dimethylformamide, and mixing them to conduct a heating reaction;

adding purified water and heating to dry to obtain a dried product;

dissolving the dried product in an organic solvent and adding purified water for layering;

recovering the organic layer and filtering and concentrating the organic layer to obtain a concentrate;

re-concentrating the concentrate and adding hexane to obtain a precipitate;

dissolving the resulting precipitate and cooling, filtering and drying the resulting precipitate to obtain a dried product; and

the resulting dried product is dissolved in an organic solvent, ferric chloride hexahydrate, activated carbon and hydrazine monohydrate are added to the organic solvent, they are cooled and filtered to obtain a resulting precipitate and then the resulting precipitate is dried and pulverized.

6. The method for preparing a pharmaceutical composition for oral administration of claim 5, wherein the method further comprises adding an acidic substance.

7. The method for preparing a pharmaceutical composition for oral administration of claim 6, wherein said acidic substance comprises hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, oxalic acid, fumaric acid, malonic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, EDTA, and combinations thereof.

8. The process for preparing a pharmaceutical composition for oral administration of claim 5, wherein the precipitate is formulated as a tablet containing nanoparticles or a capsule containing solid dispersed particles.

9. The orally administered pharmaceutical composition of claim 1, wherein the composition is for the treatment of a bacterial infection.

Technical Field

The present invention relates to orally administered pharmaceutical compositions comprising Fab I inhibitors, and to methods of making the same.

Background

Infectious diseases caused by bacteria are diseases that afflict humans up to human history and have had a great impact on human history, such as black death. In order to overcome these threats of bacteria, continuous efforts have been made by humans, which have resulted in rapid development of medicine and drugs. The development of the modern concept of antibiotics began with the first discovery of penicillin by alexanda fleming in 1928. Since then, the development of antibiotics to treat bacterial infections has taken a leap forward. However, resistance of bacteria to antibiotics has begun to be known, and the use of antibiotics has been limited.

Since then, with the development of new antibiotics and the continued emergence of drug-resistant bacteria against the new antibiotics, the development of new antibiotics has become an essential task for the treatment of bacterial infections. In addition, research strategies are also being changed to overcome drug resistant bacteria. It is of interest to develop antibiotics with new mechanisms of action, since even the development of new antibiotics with better efficacy using previously established mechanisms of bacterial inhibition cannot overcome the already expressed resistance. In addition, large pharmaceutical companies around the world, such as Bayer (Bayer), Bristol-Myers Squibb, Merck (Merck), puerarin Smith Kline (Glaxo Smith Kline), and Astrazeneca (Astrazeneca) are striving to develop new antibiotic concepts that can overcome resistance through a completely different mechanism of action than conventional antibiotics. Among these resistant strains, one of the most difficult to treat is MRSA (methicillin-resistant staphylococcus aureus). MRSA is a staphylococcus aureus resistant to the penicillin antibiotic methicillin. MRSA is not only resistant to methicillin, but also to most antibiotics, and is therefore a pathogen that can only be treated with very limited antibiotics. Outbreaks of MRSA infection are on the rise worldwide. This strain is of concern not only because it is resistant to existing antibiotics, but also because it is the most common pathogenic organism among the pathogens that induce nosocomial infections, and it can be fatal to immunocompromised patients or elderly, infirm people. In recent years, not only healthcare-and community-acquired MRSA infections have increased significantly, indicating that exposure of MRSA is likely to occur in daily life. Vancomycin has been used for its treatment. However, vancomycin resistant strains have been reported. Other therapeutic agents include linezolid and daptomycin, but the choice of antibiotics for therapeutic purposes is very limited. Therefore, there is an urgent need to develop a novel antibiotic that can be used for antibiotic-resistant bacterial infections.

Drawings

Fig. 1 is a graph showing the results of particle size measurement of nanoparticles.

Fig. 2 and 3 are diagrams showing respective preparation examples and dissolution patterns of the examples in distilled water.

Fig. 4 is a graph showing the dissolution pattern of the pH 1.2 aqueous solution of each example.

FIG. 5 is a graph showing the antibacterial effect according to T/MIC values.

Fig. 6 is a graph showing the concentration of drug in serum as a function of time.

Best mode for carrying out the invention

Since various modifications and changes can be made in the present invention, specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. It should be understood, however, that the intention is not to limit the invention to the particular embodiments, but to include all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. In the following description of the present invention, if it is determined that the gist of the present invention may be obscured, a detailed description of known functions will be omitted.

Hereinafter, the pharmaceutical composition according to an embodiment of the present invention will be described in more detail.

The term "pharmaceutical composition" as used herein is described interchangeably with "pharmacological composition" and "pharmaceutically acceptable composition" and refers to any composition that may be relatively non-toxic and have an innocuous effective effect on a subject to be administered. In addition, it may refer to any organic or inorganic compound preparation, since the side effects produced by the composition do not impair the efficacy of the drug, and do not cause severe irritation to the subject to which the compound is administered, and do not impair the biological activity and properties of the compound.

As used herein, the term "subject to be administered" is used interchangeably with "individual to be administered" and "organism to be administered" and may refer to any animal, including humans, in which acute or chronic pain is or may be caused.

In addition, the term "bacterial infection" is used interchangeably with "bacterial-related disease" and refers to a condition or disease caused by a bacterial infection. The condition or disease may include, for example, urinary tract, respiratory tract or skin tissue infection, sepsis, but is not limited thereto.

The present invention provides orally administered pharmaceutical compositions comprising a Fab I inhibitor. In particular, in the present invention, the selective Fab I inhibitor may comprise 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one, a salt thereof, or a combination thereof. 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one is represented by the following formula 1.

[ formula 1]

Chemical name: 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one

For example, a selective Fab I inhibitor having the structure of formula 1, which exhibits antibiotic efficacy by inhibiting the action of Fab I, an enzyme essential for protein synthesis in bacteria, is a compound having a completely different mechanism of action from existing antibiotics such as β -lactam antibiotics (penicillins, cephalosporins, etc.), glycopeptides (vancomycin, etc.), tetracyclines, aminoglycosides, glycylcyclines (glycocyclines), macrolides, chloramphenicol, quinolones, sulfonamides, and oxazolines.

Fatty acids are not only the energy source of living organisms, but also the major components of cell membranes and play a crucial role in maintaining life phenomena. Thus, the biosynthesis of fatty acids in cells is an essential biochemical process present in all living cells. The genes involved in these processes are among the essential genes from bacteria to humans.

Among the four enzymes involved in bacterial fatty acid biosynthesis, Fab I, which is an enoyl-ACP reductase in the last step of the cycle, is reported to play a role in converting enoyl-ACP to the corresponding acyl-ACP by 1, 4-reduction (Payne et al, Drug Discovery Today 6,2001, 537-544). Fab I is the most important protein in fatty acid synthesis and participates in reactions that determine the rate of the overall synthetic process. However, in mammals such as humans, unlike bacteria, a large group of enzymes called fatty acid synthases is used to synthesize such fatty acids. Furthermore, their structures are completely different from proteins in the bacterial fatty acid synthesis pathway. Therefore, since selective Fab I inhibitors have little toxicity and are inhibitors against novel target proteins that have not been targeted with any antibiotics so far, the development of drugs acting on the target proteins can improve the success rate of treatment against bacteria with drug resistance, in particular multi-drug resistance.

According to an embodiment, the compound of formula 1 may be provided in amorphous form, crystalline form, or a mixture thereof, and for example the compound of formula 1, a salt thereof, or a combination thereof may be included in an amount of 10% to 60% by weight, such as 20% to 40% by weight, such as 10% to 30% by weight, such as 30% to 60% by weight.

According to a further aspect of the invention, there is provided a process for the preparation of an orally administered pharmaceutical composition comprising a Fab I inhibitor, the process comprising:

adding 4-benzyloxy-1H-pyridone, 2-methyl-3-nitrobenzyl chloride and potassium tert-butoxide to dimethylformamide, and mixing them to conduct a heating reaction;

adding purified water and heating to dry to obtain a dried product;

dissolving the dried product in an organic solvent and adding purified water for layering;

recovering the organic layer and filtering and concentrating the organic layer to obtain a concentrate;

re-concentrating the concentrate and adding hexane to obtain a precipitate;

dissolving the resulting precipitate and cooling, filtering and drying the resulting precipitate to obtain a dried product; and

the resulting dried product is dissolved in an organic solvent, ferric chloride hexahydrate, activated carbon and hydrazine monohydrate are added to the organic solvent, they are cooled and filtered to obtain a resulting precipitate and then the resulting precipitate is dried and pulverized.

According to one embodiment, the pharmaceutically acceptable salt of the compound of formula 1 may be included in a pharmaceutical composition for oral administration. The pharmaceutically acceptable salt may be an acid addition salt formed using an acid. Examples of acids include, but are not limited to, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, oxalic acid, fumaric acid, malonic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and EDTA.

According to one embodiment, the present invention can improve the dissolution rate and uniformity of the formulation by adjusting the particle size of the compound of formula 1 or a salt thereof. Specifically, the particle size distribution D of the compound of formula 1 or a salt thereof₉₀May be from 0.1 μm to 500 μm, such as from 0.1 μm to 300 μm, such as from 0.1 μm to 50 μm, such as from 0.1 μm to 25 μm.

According to one embodiment, the pulverization of the compound of formula 1 or a salt thereof may be carried out by a wet method or a dry method, but the present invention is not particularly limited thereto, and may be appropriately selected if it is a general pulverization method. For example, an air jet mill, a fluid energy mill, a micro mill, or the like may be used, and the present invention is not limited thereto.

According to one embodiment, the present invention may be provided in oral dosage forms and may be formulated in solid or liquid form. In particular, the compositions according to the invention may be provided in liquid or solid form, and may be provided in any convenient form, such as in the form of tablets, pills, granules, capsules, suspensions, emulsions or powders, suitable for reconstitution with water or other suitable liquid medium.

According to one embodiment, the composition of the present invention may be provided in the form of a capsule or tablet. Capsules or tablets may be prepared by dissolving or mixing them in non-toxic pharmaceutically acceptable excipients suitable for oral administration.

According to a particular embodiment, the composition according to the invention may be provided in finished form by: the manner is to additionally add an excipient and a solubilizer to completely dissolve it, spray-dry to prepare a powder, and then fill the resulting powder into hard capsules.

For example, excipients include water soluble polymers such as dextrins, polydextrose, dextrans, pectins and pectin derivatives, alginates, starches, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose acetate succinate, hydroxyethyl methylcellulose, guar gum, locust bean gum, tragacanth gum, carrageenan, acacia gum, gellan gum, xanthan gum, gelatin, casein, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene acetaldehyde diethylamide acetate, polybutylmethacrylate, (2-dimethylaminoethyl) methacrylate, methyl methacrylate copolymers, polyethylene glycol, polyethylene oxide, carbomers, and the like. More specific examples of the excipient include polyvinylpyrrolidone. The excipient may be present in an amount of 10% to 60% by weight, for example 30% to 55% by weight, for example 40% to 50% by weight, based on the total weight of the composition.

For example, solubilizing agents include, but are not limited to, polyols, surfactants, and the like, such as propylene glycol, polyethylene glycol, dipropylene glycol, diethylene glycol monoethyl ether, glycerol, tween 80, cremophor, carbitol, and the like. More specifically, the solubilizing agent includes tween 80. The solubilizer may be present in an amount of 0.5% to 20%, 1% to 10%, e.g., 2% to 5% by weight, based on the total weight of the composition.

According to one embodiment, the compound of formula 1, a salt thereof, or a combination thereof may be present in an amount of 10% to 60%, such as 30% to 55%, such as 40% to 50%, by weight, based on the total weight of the composition.

According to a specific embodiment, the composition according to the present invention may be provided in the form of a tablet by adding excipients, disintegrants and lubricants to form wet granules and combining, drying, sieving and mixing the wet granules.

For example, the solvent that may be used for wet granulation may include at least one selected from the group consisting of water, methanol, ethanol, and dichloromethane. Specifically, the solvent that may be used for wet granulation may include ethanol or an aqueous ethanol solution, but is not limited thereto.

For example, excipients may include microcrystalline cellulose, silicified microcrystalline cellulose, mannitol, lactose, silicon dioxide, and the like, but are not limited thereto.

For example, the disintegrant may include croscarmellose sodium, starch, and the like, but is not limited thereto.

For example, the lubricant may include corn starch, talc, magnesium stearate, calcium stearate, polyethylene glycol, sodium lauryl sulfate, and the like, but is not limited thereto.

According to an embodiment, in the preparation of the tablet form of the present invention, the composition may further include a binder, but is not limited thereto, and for example, includes gelatin, starch, glucose, povidone, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like.

According to one embodiment, the present invention may provide a tablet for oral administration, the tablet comprising: microcrystalline cellulose, D-mannitol, and the like as an excipient which may be used alone or in combination of two or more; croscarmellose sodium as a disintegrant, and the like; and magnesium stearate as a lubricant.

As a specific example, the tablet according to the present invention comprises: 10 to 1000 parts by weight (such as 100 to 300 parts by weight) of an excipient, 1 to 30 parts by weight (such as 10 to 20 parts by weight) of a disintegrant, 0.1 to 20 parts by weight (such as 5 to 15 parts by weight) of a binder, and 0.1 to 10 parts by weight (such as 3 to 5 parts by weight) of a lubricant, based on 100 parts by weight of the compound of formula 1, a salt thereof, or a combination thereof.

According to one embodiment, a pharmaceutically acceptable solvent may be used to dissolve the compound of formula 1 or a salt thereof, and the pharmaceutically acceptable solvent may include an aqueous solution containing an acid having a pH of 1 to 3 (e.g., pH 1.2), water, methanol, ethanol, dichloromethane, and the like, but is not limited thereto.

According to one embodiment, the present invention may be used to treat gram-positive bacterial infections such as MRSA (methicillin-resistant staphylococcus aureus) or various infectious diseases caused thereby. Gram-positive bacteria include, for example: staphylococci (Staphylococcus), such as Staphylococcus aureus (Staphylococcus aureus) and Staphylococcus epidermidis (Staphylococcus epidermidis); and Streptococci (Streptococcus) such as Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus thermophilus (Streptococcus pyrogenes), group C/F/G Streptococcus (group C/F/G Streptococcus), and group chlorella Streptococcus (viridans group Streptococcus).

According to one embodiment, the composition of the invention may further comprise pharmaceutically acceptable and physiologically suitable additives.

For example, any one may be used as an additive as long as it is pharmaceutically acceptable and generally used in each formulation, such as a filler, a bulking agent, a binder, a disintegrant, a glidant, a preservative, a buffer, a coating agent, a sweetener, a solubilizer, a suspending agent, a colorant, a water-soluble additive, an excipient, a carrier, a filler, a lubricant, a desiccant, and the like. For example, the additive may be present in an amount of 5% to 90% by weight, for example 40% to 90% by weight, based on the total weight of the composition.

The pharmacological or pharmaceutical compositions according to the invention may be prepared in any form suitable for administration to humans, including infants, children, adults and animals, by standard methods known to those skilled in the art.

Hereinafter, embodiments of the present invention will be described in detail in order to enable those skilled in the art to easily practice the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Preparation example 1: preparation of the Compound of formula 1

To prepare the compound of formula 1, 0.9mol of 4-benzyloxy-1H-pyridone is added to 10L of Dimethylformamide (DMF), and then 0.9mol of potassium tert-butoxide is added thereto with stirring. It was warmed to 55 ℃ for 30 minutes with stirring. 0.9mol of 2-methyl-3-nitrobenzyl chloride is slowly added and reacted while mixing is continued for a further 2 hours. After the reaction was completed, 4L of purified water was added, heated to 60 ℃ and evaporated in a rotary evaporator (C.), (R-220, BUCHI). 14L of dimethyl chloride was added to dissolve the dried product, and 7L of distilled water was added to conduct layer separation. After taking the supernatant, 0.7kg of each of magnesium sulfate and activated carbon was added thereto and stirred for 1 hour, filtered through celite, and dried using a rotary evaporator (yield: 60%).

After about 2kg of the resulting dried product was dissolved in ethanol, 100g of ferric chloride hexahydrate, 600g of activated carbon and 10kg of hydrazine monohydrate were added to cool the reaction solution. Thereafter, it was filtered to obtain a white precipitate, which was dried in a vacuum oven at 40 ℃ overnight to yield 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one. The yield was 85%.

In order to remove the relevant substances produced during the synthesis, purification may be performed as necessary. Purification was performed as follows: 2kg of the starting material was dissolved in 30L of methylene chloride, and then layer separation was carried out by adding purified water. After taking the organic layer, 0.7kg of sodium sulfate was added, and 0.7kg of activated carbon was additionally added thereto and stirred for 1 hour, filtered and concentrated under reduced pressure to remove dichloromethane. Further, 10L of ethyl acetate was added to dissolve, concentrate, and recrystallize by adding 20L of hexane, followed by drying at 40 ℃. The purification operation may be repeated as necessary.

Preparation example 2: preparation of salt of compound of formula 1

The compound of formula 1 has very low solubility in distilled water of 2 μ g/mL, resulting in decreased bioavailability due to low solubility after administration to the body. In addition, the process of preparing polymer dispersed particles by spray drying, which can be used to prepare pharmaceutical compositions, is not only very complicated, but also has the disadvantage of relatively large loss of raw materials during the manufacturing process. To ameliorate these problems, salts of the compounds of formula 1 are prepared.

6g of the compound of formula 1 (molecular weight 340.45) according to preparation example 1 was added to 100mL of solvent dichloromethane, and then stirred at room temperature for 30 minutes to completely dissolve it (0.176M). When the acidic substance is a liquid, it is used as it is, and when the acidic substance is a solid, it is dissolved in water for use. The acidic substance was slowly added while stirring the solution of the compound of formula 1 dissolved in the solvent at a speed of 100 rpm. The kind of the acidic substance and the properties according to the added acidic substance are shown in table 1.

[ Table 1]

From the results of salt formation using various types of acidic substances shown in table 1, addition of hydrochloric acid and sulfuric acid immediately resulted in precipitation. However, in the case of sulfuric acid, the color of the precipitate gradually turns brown over time. In addition, the other acidic substances remained in their phase separated state or in liquid form, as shown in the table. When a precipitate is produced, the precipitate is filtered, diluted with dichloromethane, and filtered again to remove any acidic species that may remain. After repeating these processes twice, the obtained precipitate was dried in a vacuum oven preheated to 40 ℃ for 12 hours. The yield was 95% to 98%, and the dried product was pulverized to measure solubility. Samples dried using a rotary evaporator (Tokyo Rikakai Co., Ltd./Eyela laboratory evaporator N-1000) were used in the case of liquid phase or phase separation. The solubility of each sample obtained was measured. The results are shown in table 1. The water solubility of phosphate in neutral water is 452 times higher than that of the free base, and the water solubility of sulfate and hydrochloride is improved by more than 330 times. In addition, the solubility of citrate and tartrate is improved by more than 150 times, the solubility of ascorbate and fumarate is improved by about 40 times and about 100 times, respectively, and finally the solubility of acetate and EDTA salts is improved by about 20 times. In view of ease of preparation, properties after drying and solubility, the hydrochloride salt can be applied to the salt preparation of the compound of formula 1.

Example 1: preparation of capsules

The compound of formula 1 has very low solubility in water. It is known that the lower the solubility, the lower the bioavailability. To overcome this problem, the finished form is prepared by preparing solid dispersed particles based on polymers and filling them into capsules. More specifically, first, about 80g of 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one, a salt thereof, or a mixture thereof was dissolved in 1.6L of dichloromethane, and then 80g of hydrophilic polymer polyvinylpyrrolidone and 3.26g of solubilizer tween 80 were added, followed by stirring at room temperature for 30 minutes to completely dissolve them. Performing a spray drying process (GEA/Niro SDMICRO)^TM) To form granules. The spray drying process was performed as shown in table 2 below, collecting the particles and then drying overnight using a vacuum oven preheated to 40 ℃ to remove possible residual dichloromethane. The prepared powder was filled into a hard gelatin capsule to prepare a capsule.

[ Table 2]

Setting parameters

Example 2: preparation of tablets containing salt.

Preparation of a catalyst composition comprisingTablets of the salt of the compound of formula 1 according to preparation example 2. First, on a per tablet basis, to a compound of formula 1, 42.4% (w/w) hydrochloride, 27.1% (w/w) microcrystalline cellulose, 20.3% (w/w) D-mannitol, 3.4% (w/w) croscarmellose sodium, and 1.4% (w/w) Aerosil (Tomita Pharmaceuticals) (Japan),) To the mixture of (a) was slowly added 60 μ L of a binder solution having hydroxypropylcellulose dissolved in ethanol (10% (w/v)) to form granules, and then dried in a hot air dryer preheated to 60 ℃ for 3 to 4 hours. The moisture content of the dried product was set to 3% or less. The dried granules were sieved through an 18 mesh sieve. After 2% (w/w) croscarmellose sodium as a disintegrant and 1.4% (w/w) magnesium stearate as a lubricant were added, mixing and tableting were performed to prepare tablets.

Example 3: preparation of tablets containing nanoparticles

In order to improve the water solubility of the compound of formula 1 according to preparation example 1, the particle size of the compound of formula 1 is set to be nano-sized, thereby increasing the surface area of the raw material to improve the solubility. For this, 1g of the compound of formula 1 was stirred in 10mL of dimethyl sulfoxide (DMSO) and completely dissolved to prepare a transparent solution. After dissolving 0.5g of poloxamer 407 in 20mL of myristyl alcohol at 70 ℃, a solution of the compound of formula 1 is slowly added thereto, and by using a homogenizer (R) ((R))-Werke GmbH&Co.kg,T25 digital LR) was emulsified by mixing at 15,000rpm for 5 minutes. The temperature during emulsification was 80 ℃. After the emulsification process, it was stored at room temperature for a period of time to rapidly reduce the temperature, and the resulting solid was pulverized to an average particle size of about 290nm using a tory Hills T65 three roll mill. Subjecting the obtained pulverized material to supercritical treatmentIn the extraction system (ILSHIN autoclave/SC-CO 2 extraction system) carbon dioxide was injected simultaneously. At this time, the pressure in the reactor was maintained at about 70atm to remove myristyl alcohol and dimethyl sulfoxide, and only solid particles remained. The weight of the obtained solid was 1.48g, and the yield was 98.7%. The particle size of the obtained solid was measured by a zeta potential measuring system (ELS-2000Z, Otsuka Electronics Korea Co., Ltd.). As a result, the average particle diameter was 318nm, and the results are shown in FIG. 1 and Table 3.

TABLE 3

To 45.5% (w/w) nanoparticles, 22.7% (w/w) microcrystalline cellulose, 22.7% (w/w) D-mannitol, 3.0% (w/w) croscarmellose sodium and 1.4% (w/w) Aerosil (Futian pharmaceutical (Japan) containing the compound of formula 1 prepared as described above as the main component,) To the mixture of (a) was slowly added 60 μ L of a binder solution having Hydroxypropylmethylcellulose (HPMC) dissolved in ethanol (10% (w/v)) to form granules, and then dried in a hot air dryer preheated to 60 ℃ for 3 to 4 hours. The moisture content of the dried product was set to 3% or less. The dried granules were sieved through an 18 mesh sieve. After adding 1.8% (w/w) croscarmellose sodium as a disintegrant and 1.2% (w/w) magnesium stearate as a lubricant, mixing and tableting were performed to prepare tablets.

Example 4 to example 8: preparation of tablets

In order to solve the decrease in productivity and economic efficiency that may occur due to the loss of the raw material, polymer dispersed particles, and nano-sized particles in the salt introduction, the compound of formula 1 as the raw material is pulverized with a Jet Mill (Komachine, Micro Jet Mill), and by changing the particle size (D) of the raw material₉₀) Values were prepared as shown in table 4. To the compound of formula 1 having different particle sizes, 24.0% (w/w) microcrystalline cellulose (MCC), 21.7% mannitol (D-mannitol) and 0.8% (w/w) croscarmellose sodium, which are sieved to remove lumps that may be contained, and then they are homogeneously mixed. After mixing, 150 μ L of a binder solution having hydroxypropylmethylcellulose dissolved in ethanol (6% (w/v)) was added to form granules, and drying was performed until the moisture content was 2% or less. After sieving, 0.8% (w/w) magnesium stearate as a lubricant was added on a per tablet basis, mixed and subjected to tableting to prepare tablets.

[ Table 4]

	D90(μm)
		Example 4	10.1
Example 5	25.6
		Example 6	60.1
Example 7	124.3
		Example 8	253.1

Experimental example 1: evaluation of inhibition of MRSA strains

To verify the inhibitory effect of the compound of formula 1 on antibiotic-resistant strains, the compounds were prepared byThe isolated staphylococcus aureus phenotype of each patient was treated with the compound of formula 1 to evaluate drug susceptibility. In vitro tests for antibiotic development, the most important result may be the MIC₉₀(minimum inhibitory concentration required to inhibit growth by 90% of the total bacterial population). Table 5 shows MIC using representative drugs currently on the market as control drugs for about 100 methicillin-sensitive strains and about 100 currently socially problematic MRSA strains isolated in the laboratory of Peter c.appelbaum doctor (approved by its authority in the field of anti-infection all over the world) at hershesey hospital₉₀And (5) testing results.

[ Table 5]

As shown in Table 5, MIC was found₉₀The value was 0.25. mu.g/mL regardless of the sensitive strain and the non-sensitive strain, i.e., the MRSA strain, which indicates two to several tens times superior results compared to the control drug. In particular, these strains include vancomycin-resistant intermediate staphylococcus aureus (VISA) strain and vancomycin-resistant staphylococcus aureus (VRSA) strain as a superbacteria (vancomycin MIC)>64. mu.g/mL). From these results, it can be seen that the compound of formula 1 can be used as an effective therapeutic agent for diseases or disorders caused by bacterial infection, compared to conventional drugs such as vancomycin, teicoplanin, linezolid, amoxicillin-clavulanic acid, daptomycin and the like. In particular, although not limited thereto, it can be effectively used as a therapeutic agent for bacterial infection associated with diseases including urinary tract, respiratory tract, skin tissue infection, sepsis, and the like.

Experimental example 2: analysis of solubility of Compound of formula 1

Prior to preparing the salt of the compound of formula 1, the compound of formula 1 is analyzed to determine solubility in various solvents. First, the compound of formula 1 was supersaturated in a pharmaceutically acceptable solvent as shown in table 5, and stirred at room temperature for 12 hours in a dark room. It was first centrifuged to take the supernatant, and then filtered again with a 0.25 μm PVDF filter to remove insoluble substances remaining in the solution. The filtrate was diluted with methanol and then subjected to HPLC analysis to quantify the solubility. The results are shown in table 6. In addition, the HPLC analysis conditions were as follows.

Each 20 μ L of the test solution and the standard solution was tested under the following conditions according to a liquid chromatography method (HPLC) of a general test method of korean pharmacopoeia, and the peak area of the main component in each solution was measured.

Operating conditions and calculations

[ operating conditions ]

A detector: UV spectrophotometer (measuring wavelength 286nm)

Column: aegispak C18-L (4.6 mm. times.250 mm, 5 μm) column

Column temperature: 25 deg.C

Mobile phase: acetonitrile: water 3:2

Flow rate: 1.0mL/min

[ calculation ]

A_T: peak area of main component in test solution

A_S: peak area of main component in standard solution

D_T: dilution factor of test solution

D_S: dilution factor of standard solution

P: purity of main component in Standard solution (%)

[ Table 6]

As shown in table 6, it was found that the solubility increased as the pH of the compound of formula 1 decreased. For example, it has a low solubility of 2.5. mu.g/mL in water (neutral), but has a solubility of about 0.9mg/mL under strongly acidic conditions at pH 1.2. The solubility in ethanol, methanol and glycerol is very low, i.e. below 10mg/mL, while the solubility in dichloromethane and DMSO is high, i.e. above 50 mg/mL. However, DMSO is expected to be difficult to dry due to its low volatility from high boiling point, and thus is judged to be unsuitable for salt production. Thus, in the salt production process, for example, methylene chloride, which has high volatility and relatively high solubility, may be used.

Experimental example 3: capsule stability analysis

The stability of the capsule of example 1 according to storage conditions was verified. The storage conditions are long-term conditions and accelerated conditions, specifically, the long-term conditions and accelerated conditions are a temperature of 25 + -2 deg.C and a humidity of 60 + -5%, and a temperature of 40 + -2 deg.C and a humidity of 75 + -5%, respectively. The results are shown in table 7.

[ Table 7]

As shown in table 7, the dissolution rates, contents and related substances were found to meet the standards for at least 12 months under long-term storage conditions and 6 months under accelerated conditions. In this regard, the shelf life of the product may be determined to be, for example, 24 months.

Experimental example 4: stability analysis of tablets containing the hydrochloride salt of the Compound of formula 1

The stability of the tablet of example 2 according to storage conditions was verified. The storage conditions were long-term conditions and accelerated conditions, and the dissolution rate, the content and the related substances were evaluated as test items. The long-term storage conditions and the accelerated storage conditions were 25. + -. 2 ℃ and 60. + -. 5% humidity, and 40. + -. 2 ℃ and 75. + -. 5% humidity, respectively. The results are shown in table 8.

[ Table 8]

As shown in table 8, the result values showed that the stability remained within the set range in all the test items under both conditions, and no significant change was observed in 6 months for each item. It can be seen that the tablets containing the hydrochloride salt of the compound of formula 1 have excellent physical and chemical stability.

Experimental example 5: stability analysis of tablets containing nanoparticles

The stability of the tablets containing the nanoparticles of example 3 according to storage conditions was verified. The storage conditions were long-term conditions and accelerated conditions, and the dissolution rate, the content and the related substances were evaluated as test items. The long-term storage conditions and the accelerated storage conditions were 25. + -. 2 ℃ and 60. + -. 5% humidity, and 40. + -. 2 ℃ and 75. + -. 5% humidity, respectively. The results are shown in table 9.

[ Table 9]

As shown in table 9, no significant change was observed in all tested items over 6 months under both conditions. It can be seen that the tablets containing the compound of formula 1 have excellent stability.

Experimental example 6: solubility and dissolution tests

The solubility of the compound of formula 1 and its salt, and the powders of polymer dispersed particles and nanoparticles prepared with the compound of formula 1 in preparation example 1, preparation example 2, example 1, and example 3, respectively, in distilled water was verified. First, an excess of each substance was added to distilled water and mixed at room temperature for 12 hours. After standing for 2 hours, the supernatant was collected and filtered through a filter having a pore size of 0.25. mu.m. After it was diluted again in methanol, HPLC analysis was performed. The HPLC conditions were the same as the analysis conditions in the solubility test of experimental example 2. The results are shown in table 10.

[ Table 10]

Item	Water solubility (μ g/mL)	pH
			Preparation of example 1	2.5	6.8
Preparation example 2	927	2.1
			Example 1	154	6.7
Example 3	168	6.9

As shown in table 10, it was found that the solubility was improved in the case of salt introduction, polymer dispersed particles and nanoparticles, compared to the free base.

In addition, each of the compound of formula 1 according to production example 1, the hydrochloride of the compound of formula 1 according to production example 2, the polymer dispersed particle powder according to example 1, and the nanoparticle powder according to example 3 as described above was filled into a gelatin hard capsule. Then, the dissolution test was performed in distilled water by a dissolution test method 2 (paddle method) of the general test methods in korean pharmacopoeia. The results are shown in FIG. 2. Samples obtained during the experiment were analyzed by HPLC to quantify the drug content per time period. The HPLC conditions were as follows.

[ dissolution test conditions ]

-test method: dissolution testing method of Korean pharmacopoeia 2 (Paddle method)

-test solution: distilled water

-test temperature: 37 +/-0.5 DEG C

-rotational speed: 50rpm

According to liquid chromatography (HPLC) of a general test method of korean pharmacopoeia, peak areas of main components of each solution were measured under the following conditions.

Operating conditions and calculations

[ operating conditions ]

The operating conditions and the calculation equation were the same as the content test used in the solubility test of experimental example 2. As shown in fig. 2, it was found that the dissolution pattern in water was significantly improved when the techniques of preparation example 2 (salt), example 1 (polymer dispersed particles) and example 2 (nanoparticles) were applied, as compared to preparation example 1.

Experimental example 7: analysis of dissolution Pattern according to particle size

Prior to dissolution testing of the tablets according to examples 4 to 8, testing of dosage form uniformity was performed. In the testing of dosage form uniformity, 10 samples of each example were prepared and the content per tablet of each sample was quantified. The acceptance value of the uniformity of the dosage form was determined according to the content uniformity test of the korean pharmacopoeia general test method. Generally, the acceptance value is inversely proportional to the dosage form uniformity. The acceptance values of examples 4 to 8 are shown in table 11.

[ Table 11]

Examples of the invention	Uniformity of dosage form (average:%)
		Example 4	2.5
Example 5	2.8
		Example 6	4.8
Example 7	5.7
		Example 8	6.8

As shown in table 11, the acceptance value tends to increase with an increase in the particle size of the raw material used. That is, as the particle size increases, the uniformity of the dosage form decreases. In particular, when the particle size distribution (D)₉₀) At about 50 μm, the acceptance of dosage form uniformity increases rapidly, which is believed to represent a decrease in dosage form uniformity.

In order to determine the difference in dissolution rate according to the particle size of the raw materials, an experiment was performed according to the dissolution test method 2 (paddle method) of the general test method of the korean pharmacopoeia. The experiment was performed using 900mL of distilled water and a pH 1.2 aqueous solution as a dissolution medium at a rotation speed of 50rpm and a temperature of 37. + -. 0.5 ℃. The results using distilled water as dissolution medium are shown in figure 3 and the results using aqueous pH 1.2 as dissolution medium are shown in figure 4.

The dissolution medium was removed at regular intervals, filtered through a 0.45 μm filter and then subjected to HPLC analysis to assess the extent of dissolution. HPLC analysis conditions were the same as the dissolution test method used in experimental example 6. As shown in fig. 3 and 4, the dissolution rates in the distilled water and the dissolution medium at pH 1.2 increased with the decrease in the particle size of the raw material. In particular, example 4 showed a similar dissolution rate in distilled water as in the case of applying the techniques of preparation example 2 (salt introduction), example 1 (polymer dispersed particles) and example 2 (nanoparticles).

In addition, the difference in dissolution rate of each sample was also gradually decreased. Specifically, the deviation of the dissolution test was within 1% to 3% in the case of examples 4 and 5, and it was shown to be 3% to 10% in the case of examples 6 to 8. From the above, it was found that as the particle size becomes finer, the uniformity of the dose unit improves, the dissolution rate improves, and the dissolution deviation decreases. Therefore, a faster and more consistent pattern of body absorption is expected to be exhibited when orally administered, thereby expecting to ensure a fast and constant therapeutic effect.

Experimental example 8: stability analysis of tablets according to example 4

After storing the tablets according to example 4 in plastic bottles, it was observed whether the physical and chemical properties of the products changed significantly under long-term conditions of 25 ± 2 ℃ and 60 ± 5% RH and accelerated conditions of 40 ± 2 ℃ and 75 ± 5% RH. The results are shown in table 12.

[ Table 12]

As shown in table 12, no significant changes were observed in all test items over 6 months under long term and accelerated conditions. From this it can be seen that the tablets have stability. In examples 5 to 8, they were expected to be very stable physically and chemically under the same conditions, although the results were not shown.

Experimental example 9: pharmacokinetic and pharmacodynamic analysis in mouse models

For the compound of formula 1, pharmacokinetic/pharmacodynamic experiments were performed using a mouse infection model. For this purpose, experiments were carried out with Staphylococcus aureus ATCC 29213(MSSA, standard strain) and 13B-382(MRSA, clinical strain). Mueller-Hinton broth or cation-adjusted Mueller-Hinton broth was used as the culture medium. For sensitivity testing (minimum inhibitory concentration (MIC)), the compound of formula 1 is used.

For sterile (specific pathogen free, SPF) females, 6-week-old (23-27 g) ICR mice (Orient Bio Inc, Gapyeong, Korea), experiments were performed according to the regulations and procedures, according to the animal protection and laboratory animal protocols, with the permission of the animal experimental ethics committee. Subcutaneous injections of cyclophosphamide (Bexter, Frankfurt, Germany) to induce neutropenia: (<100/mm³). Prior to the experiment, the test strains were incubated in Muller Hinton II broth for 24 hours at 37 ℃ to obtain 10⁸CFU/mL concentration. Then, they were diluted with physiological saline. 0.1mL of the solution was inoculated into the thigh of a mouse (inoculum size 1.0X 10)⁵CFU/mL). After 2 hours, oral administration of the compound of formula 1 was started. The drug is administered at a dose of 7.5 mg/kg/day to 240 mg/kg/day every 3 hours, 6 hours, 12 hours, and 24 hours.

After 24 hours of administration, mice were euthanized with carbon dioxide gas to isolate their thighs, placed in physiological saline, and homogenized with a homogenizer (Kinematica)) And (5) fine cutting. This was diluted 10-fold, spread on Muller Hinton II broth, and incubated at 37 ℃ for 24 hours. Viable cell numbers were counted and recorded. The results are expressed as log10 CFU/thigh and the limit of viable cell count in the laboratory is 1X 10²CFU/thigh.

The T/MIC is evaluated as an index for determining the effect of the antibiotic as well as the antimicrobial effect and pharmacokinetic results, depending on the antimicrobial dose and mode of administration. The T/MIC values represent the percentage of dose intervals at which serum levels exceeded MIC. The results are shown in fig. 5. As shown in FIG. 5, it was found that the effect of eradicating bacteria rapidly increased with an increase in T/MIC value. When the T/MIC value is about 20% or higher,over 99.9% of the bacteria were eradicated. In addition, it was found that when AUC_0-24hMIC and C_maxThe effect of eradicating bacteria such as MRSA strains increases rapidly when the value of/MIC increases.

As shown in FIG. 5, it can be seen that the MIC values of Staphylococcus aureus ATCC 29213 and 13B-382 of the compound of formula 1 were 0.25. mu.g/mL regardless of the strain. This value is lower than that of oxacillin (0.25. mu.g/mL and 16. mu.g/mL) and vancomycin (0.5. mu.g/mL and 1. mu.g/mL).

Experimental example 10: pharmacokinetic evaluation in beagle model

For the formulations according to example 4 and example 8, a pharmacokinetic evaluation of beagle dogs (n ═ 5) was performed. Prior to pharmacokinetic testing, beagle dogs were fed at 30 g/head/day for about 30 minutes prior to administration of the test substance, and the test substance was administered without fasting. The dose was about 5mg/kg, and blood samples were obtained from the jugular vein at 9 time points of 0 min, 10 min, 30 min, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours per body. After processing the blood samples, the concentration of the drug in the plasma was determined to obtain the pharmacokinetic profile as shown in figure 6.

The particle size of the starting material used in the manufacture of the tablet greatly influences the pharmacokinetic profile. As shown in FIG. 6, AUC_0-24hAnd C_maxThe value tends to increase as the particle size decreases. In addition, the concentration of antibiotics in plasma is higher than the MIC (T)>MIC) also tends to increase with smaller particle size. Based on the results according to experimental example 9, it can be seen that AUC_0-24h、C_maxChanges in the values and the time period during which the concentration of the antibiotic in the plasma is above the MIC have a large effect on the antibacterial effect of the antibiotic. Following AUC_0-24h、C_maxThree indicators of value and MIC value increased, with a significant increase in antimicrobial activity.

As described above, it can be seen that the present invention can be effectively applied to the treatment of multi-drug resistant bacterial infections.

The above description is merely illustrative of the technical idea of the present invention, and various modifications and changes can be made by those skilled in the art without departing from the essential characteristics of the present invention. The embodiments disclosed herein are not intended to limit the technical spirit of the present invention, but to explain the technical spirit, and the scope of the technical spirit of the present invention is not limited to the embodiments. The scope of the present invention should be construed as defined in the appended claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Detailed description of the invention

Technical problem

In order to solve the above problems, the present invention provides a pharmaceutical composition for oral administration comprising a Fab I inhibitor and a salt thereof as an active ingredient.

In addition, the present invention provides a method for preparing a pharmaceutical composition for oral administration comprising a Fab I inhibitor and a salt thereof as an active ingredient.

Solution to the problem

The present invention provides orally administered pharmaceutical compositions comprising a Fab I inhibitor, comprising 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one, a salt thereof, or a combination thereof.

According to one embodiment, the composition may have a particle size distribution D of 0.1 μm to 500 μm₉₀。

According to one embodiment, the composition may be provided in the form of a tablet or capsule.

According to one embodiment, 1- (3-amino-2-methylbenzyl) -4- (2-thiophen-2-yl-ethoxy) -1H-pyridin-2-one, a salt thereof, or a combination thereof may be present in an amount of 10% to 60% by weight, based on the total weight of the composition.

According to another aspect of the present invention, there is provided a process for the preparation of an orally administered pharmaceutical composition comprising a Fab I inhibitor, the process comprising:

adding 4-benzyloxy-1H-pyridone, 2-methyl-3-nitrobenzyl chloride and potassium tert-butoxide to dimethylformamide, and mixing them to conduct a heating reaction;

adding purified water and heating to dry to obtain a dried product;

dissolving the dried product in an organic solvent and adding purified water for layering;

recovering the organic layer and filtering and concentrating the organic layer to obtain a concentrate;

re-concentrating the concentrate and adding hexane to obtain a precipitate;

dissolving the resulting precipitate and cooling, filtering and drying the resulting precipitate to obtain a dried product; and

the resulting dried product is dissolved in an organic solvent, ferric chloride hexahydrate, activated carbon and hydrazine monohydrate are added to the organic solvent, they are cooled and filtered to obtain a resulting precipitate and then the resulting precipitate is dried and pulverized.

According to one embodiment, the method may further comprise adding an acidic substance.

According to one embodiment, the acidic substance may include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, oxalic acid, fumaric acid, malonic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, EDTA, and combinations thereof.

According to one embodiment, the precipitate may be formulated as a tablet containing nanoparticles or a capsule containing solid dispersed particles.

According to one embodiment, the method may comprise filling the resulting precipitate into capsules in the form of solid dispersed particles comprising a hydrophilic polymer.

According to one embodiment, the composition of the invention may be used for the treatment of bacterial infections.

Additional details of embodiments of the invention are included in the detailed description below.

Effects of the invention

The orally administered pharmaceutical composition comprising a Fab I inhibitor according to the present invention can be effectively applied to bacterial infections resistant to antibiotics and the like. More specifically, the present invention can exert therapeutic effects more rapidly by increasing the dissolution and elution rates. In addition, the present invention can improve the mixing uniformity and content uniformity of the preparation by adjusting the particle size.