Novel crystal form of L-serine and preparation method thereof

Abstract

The invention relates to a novel crystal form of L-serine and a preparation method thereof. The novel crystalline form of L-serine has improved hygroscopicity and can be effectively used in the preparation of medicaments.

Description

Technical Field

[0001] The present invention relates to a new crystal form of L-serine and a method for preparing the same. More specifically, the present invention relates to a new crystal form of L-serine and a method for preparing the same, wherein the new crystal form has improved stability by improving hygroscopicity, thereby having improved storage and handling properties and improved dissolution, solubility and pharmacokinetic properties. Background Art

[0002] L-serine represented by the following formula 1 is a non-essential amino acid that participates in various metabolic systems and plays an important role in the normal growth and development of nerve cells, plays a vital role in cell proliferation by synthesizing bases that become components of DNA, and also plays an important role in anti-oxidation by serving as a precursor for glutathione synthesis.

[0003] In Korean Patent No. KR101948066B1, a composition containing L-serine as an active ingredient for preventing, improving or treating neurodevelopmental disorders is disclosed, wherein the L-serine required for effective treatment is 200 mg / kg to 400 mg / kg, twice a day. At the same time, Korean Patent Publication No. 10-2022-0131186 discloses a syrup form, which is an optimal pharmaceutical preparation that can be taken by children with autism spectrum disorders.

[0004] It is known that L-serine is a white solid that is highly soluble in water and decomposes at a melting point of 246°C.

[0005] [Formula 1]

[0006]

[0007] For the formulation of drugs and health functional foods containing active ingredients, it is important to deliver an effective amount of the active ingredient at an appropriate time and maintain the concentration in the body so that the effect can be achieved, regardless of the administration route of the drug or health functional food.

[0008] Most active ingredients of new therapeutic substances exist in solid form, which may be amorphous or crystalline. The solid particles of each drug have different hardness, shape, size and density, so their activity and stability also vary. The physical form of the active ingredient not only affects the difficulty of formulation, but also affects the stability before and after formulation and the biological activity of the drug in the human body after administration. Therefore, regardless of the structure and mechanism of action (MOA) of the drug, the optimal form of the active ingredient should be selected to ensure that the structure and composition of the drug are maintained when formulating the new drug.

[0009] In particular, as disclosed in the inventor's earlier invention, in the case where L-serine is an active ingredient in solid form, the dose required to show its efficacy is as high as 200 to 400 mg / kg; therefore, it is necessary to develop a crystalline form with optimized solid stability and biochemical activity. In addition, the inventor has confirmed the effect of L-serine for treating developmental disorders through Korean Patent No. KR101948066B1. When developing L-serine as a pharmaceutical preparation that is easy to administer to pediatric patients, various dosage forms that can provide ease of administration are crucial for children with developmental disorders who suffer from a variety of eating disorders; therefore, the dissolution, solubility and pharmacokinetics of the solid are more important factors.

[0010] Generally speaking, the amorphous form has high solubility, which contributes to improved drug efficacy and rapid efficacy, but it is unstable, which shortens the shelf life and also makes it difficult to release the drug and control blood concentration. Meanwhile, the crystalline form has low solubility, so the bioavailability per unit weight is low, but it has the advantage that stability can be ensured, thereby enabling the preparation of sustained controlled release preparations.

[0011] According to non-patent literature 1 (Science of The Total Environment, Vol. 11, 2020, 139318), it can be seen that the hygroscopicity of amino acids is closely related to water solubility. For L-serine, its water solubility is relatively high, at 50 g / kg; therefore, according to relative humidity, the increase in hygroscopicity is relatively higher than that of aspartic acid or glutamine as amino acids in the control group.

[0012] Non-patent document 2 (Physica B: Condensed Matter, Vol. 541, pp. 32-42, 2018) discloses a crystalline form of L-serine (hereinafter referred to as crystalline form A), which has Fig.15 X-ray diffraction pattern shown.

[0013] The present inventors have confirmed that there are the following problems in the process of preparing and formulating amorphous and crystalline forms of L-serine: since L-serine is highly hygroscopic, care must be taken to avoid contact with moisture during handling such as storage and transportation and when manufacturing finished drugs; it changes color to a yellow oxidized form when stored at a relatively low temperature of 40° C. for 5 to 7 days. Furthermore, in the case of L-serine, it has been confirmed that an increase in hygroscopicity not only has the disadvantage of hardening the surface of the powder in contact with moisture, which greatly reduces its solubility in water, but also affects the saturated solubility due to the influence of excipients such as thickeners and sweeteners that are mainly used in the preparation of liquid preparations such as syrups, thereby causing technical difficulties in controlling pharmacokinetic properties.

[0014] Under these circumstances, in order to solve the above-identified problems, the present inventors provide a new crystalline form of L-serine and a method for preparing the same, wherein the new crystalline form has improved stability by improving hygroscopicity, thereby having improved storage and handling properties as well as improved dissolution, solubility and pharmacokinetic properties. Summary of the invention

[0015] Technical issues

[0016] An object of the present invention is to provide a new crystalline form of L-serine having improved stability by improving hygroscopicity, thereby having improved storage and handling properties and improved dissolution, solubility and pharmacokinetic properties.

[0017] Another object of the present invention is to provide a method for preparing a new crystalline form of L-serine.

[0018] Solution to the problem

[0019] [1] In one aspect of the present invention, the present invention relates to a crystalline form of L-serine represented by the following formula 1, wherein the crystalline form has diffraction peaks at diffraction angles (2θ) of 19.06, 20.76, 22.86, 28.28 and 30.58 in X-ray powder diffraction analysis:

[0020] [Formula 1]

[0021]

[0022] [2] In the above [1], the crystalline form of L-serine may have an additional diffraction peak at one or more diffraction angles (2θ) selected from 18.49, 36.89, 42.13, 42.96 and 44.17.

[0023] [3] In the above [1], the crystalline form of L-serine can show an endothermic point of 218.36° C. in differential scanning calorimetry analysis.

[0024] [4] In the above [1], the crystal form of L-serine can be at 3457cm -1 、2940cm -1 、1572cm -1 、1500cm -1 、1466cm -1 、1408cm -1 、1123cm -1 、1082cm -1 and 1008cm -1 It has an absorption peak in the infrared spectrum.

[0025] [5] In one aspect of the present invention, the present invention relates to a method for preparing a crystalline form of L-serine according to the above-mentioned [1], which comprises: (a) mixing and stirring L-serine and a solvent; and (b) filtering the crystals produced after stirring.

[0026] [6] In the above [5], the solvent may be one or more selected from water, methanol and ethanol.

[0027] [7] In the above [5], when the solvent is a mixed solvent of water and methanol or a mixed solvent of water and ethanol, the mixing ratio may be 1:9 to 9:1.

[0028] [8] In the above [5], the filtration temperature may be 10°C or lower.

[0029] [9] In the above [5], the method may further include (c) drying after the filtration.

[0030]

[10] In the above [9], the drying temperature may be 40°C to 50°C.

[0031]

[11] In the above [9], the drying time may be 3 hours to 8 hours.

[0032]

[12] In the above [1], the crystalline form of L-serine may have Figure 3 X-ray powder diffraction values ​​are shown.

[0033]

[13] In the above [1], the crystalline form of L-serine may have Figure 6 Infrared spectra values ​​shown.

[0034] Advantageous Effects of the Invention

[0035] The new crystalline form of L-serine according to the present invention (hereinafter referred to as type 1 crystalline form) has improved stability by improving hygroscopicity, thereby having improved storage and handling properties and improved dissolution, solubility and pharmacokinetic properties, and can therefore be effectively used in the manufacture of pharmaceutical preparations.

[0036] In particular, the new crystal form of L-serine has almost no hardening phenomenon, so it can show rapid solubility in aqueous solution, and it has less thermal denaturation, so the generation or discoloration of related substances in the preparation containing the crystal form can be suppressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the X-ray powder diffraction pattern of amorphous L-serine.

[0038] Figure 2It is an X-ray powder diffraction pattern of type A crystalline form of L-serine.

[0039] Figure 3 This is an X-ray powder diffraction pattern of Type 1 crystal of L-serine.

[0040] Figure 4 This is the infrared analysis chart of the amorphous form of L-serine.

[0041] Figure 5 This is the infrared analysis diagram of the A-type crystal of L-serine.

[0042] Figure 6 This is an infrared analysis chart of Type 1 crystal of L-serine.

[0043] Figure 7 This is a dynamic water adsorption / desorption curve which is a result of dynamic water adsorption (DVS) analysis of an amorphous form of L-serine.

[0044] Figure 8 This is a dynamic water adsorption / desorption curve which is a result of dynamic water adsorption (DVS) analysis of the A-type crystal form of L-serine.

[0045] Fig. 9 This is a dynamic water adsorption / desorption curve which is a result of dynamic water adsorption (DVS) analysis of the Type 1 crystal form of L-serine.

[0046] Fig.10 It is a differential scanning calorimetry chart of Type 1 crystal of L-serine.

[0047] Fig.11 Differential scanning calorimetry analysis of the amorphous form of L-serine.

[0048] Fig.12 The pharmacokinetic results of amorphous L-serine, crystal form A of L-serine and crystal form 1 of L-serine in mice are shown.

[0049] Fig.13 The color change results of amorphous L-serine, type A crystal form of L-serine, and type 1 crystal form of L-serine after 8 weeks are shown.

[0050] Fig.14 The results of comparing the X-ray powder diffraction patterns of the L-serine type A crystal form and the L-serine type 1 crystal form by overlaying them are shown.

[0051] Fig.15It is an X-ray diffraction pattern of a crystalline form of L-serine disclosed in Non-Patent Document 2 (Physica B: Condensed Matter, Vol. 541, pp. 32-42, 2018). DETAILED DESCRIPTION

[0052] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Generally speaking, the terms used herein are well known and commonly used in the art.

[0053] In one aspect, the present invention relates to a crystalline form of L-serine represented by the following Formula 1 (Type 1 crystalline form).

[0054] [Formula 1]

[0055]

[0056] In the present invention, "L-serine" as one kind of amino acid constituting protein is a monoamide of glutamic acid, and is an L-amino acid having the structure of Formula 1 above.

[0057] In one embodiment of the present invention, the crystalline form of L-serine represented by Formula 1 has diffraction peaks at diffraction angles (2θ) of 19.06, 20.76, 22.86, 28.28, and 30.58 in X-ray powder diffraction analysis.

[0058] In another embodiment of the present invention, in the X-ray powder diffraction analysis, the diffraction peak at the diffraction angle (2θ), where I / I0 (I: intensity of the peak at each diffraction angle, I0: intensity of the maximum peak) is 10% or greater, may be at 19.06, 20.76, 22.86, 28.28 and 30.58.

[0059] In one embodiment of the present invention, the crystalline form of L-serine represented by Formula 1 may further have a diffraction peak at one or more diffraction angles (2θ) selected from 18.49, 36.89, 42.13, 42.96, and 44.17.

[0060] In one embodiment of the present invention, the crystalline form of L-serine represented by Formula 1 may have Figure 3 X-ray powder diffraction analysis value.

[0061] In another embodiment of the present invention, in X-ray powder diffraction analysis, diffraction peaks of diffraction angles (2θ), where I / I0 is less than 10%, may be at 18.49, 36.89, 42.13, 42.96, and 44.17.

[0062] In one embodiment of the present invention, the crystalline form of L-serine represented by Formula 1 may exhibit an endothermic point of about 218.36° C. in differential scanning calorimetry. Therefore, the crystalline form of L-serine represented by Formula 1 has excellent thermal stability with a thermal decomposition temperature of about 218.36° C., and the active ingredient can remain stable without decomposition or discoloration even when stored for a long time.

[0063] In one embodiment of the present invention, the crystalline form of L-serine represented by Formula 1 can be detected at 3457 cm in infrared spectrum (IR). -1 、2940cm -1 、1572cm -1 、1500cm -1 、1466cm -1 、1408cm -1 、1123cm -1 、1082cm -1 and 1008cm -1 There is an absorption peak at .

[0064] In one embodiment of the present invention, the crystalline form of L-serine represented by Formula 1 may have Figure 6 Infrared spectrum values.

[0065] In another aspect, the present invention relates to a method for preparing a crystalline form of L-serine represented by Formula 1, comprising: (a) mixing and stirring L-serine and a solvent; and (b) filtering the crystals produced after stirring.

[0066] In one embodiment of the present invention, the "L-serine" used in step (a) is a compound represented by Formula 1, and may be L-serine obtained by a known method. Traditionally, L-serine is mainly prepared by fermentation or by using mercury or the like to obtain a DL-serine mixture and then obtaining pure L-serine via optical resolution. Representatively, as a method for preparing serine in the document (Advances in Biochemical Engineering / Biotechnology 79 (2003) 1), a method for preparing 45 g / L of L-serine using 100 g / L of glycine and 88 g / L of methanol over 3 days using Hyphomicrobium sp. NCIB10099 is disclosed. In another document (U.S. Pat. No. 5,382,517), a preparation method is disclosed, which uses Escherichia coli MT-10350 to ferment an aqueous solution of glycine and formaldehyde (485 g / L) at 50°C for 35 hours to obtain a yield of 89%. As another preparation method, the document [Org. Synth. 1940, 20, 81] discloses a preparation method using acrylic acid ester as a starting material.

[0067] In one embodiment of the present invention, the form of L-serine used in step (a) is not limited, for example, L-serine can be oily L-serine obtained by concentrating the solvent after preparing L-serine, or an amorphous or A-type crystal form of L-serine.

[0068] In step (a), the "solvent" may be a polar solvent, specifically one or more solvents selected from water, methanol and ethanol.

[0069] In an embodiment of the present invention, when the solvent is a mixed solvent of water and methanol or a mixed solvent of water and ethanol, the mixing ratio may be 1:9 to 9:1.

[0070] Furthermore, considering process economic efficiency, the amount of the solvent may be about 3 to 20 times, preferably about 4 to 15 times, and most preferably about 5 to 10 times the amount of L-serine used in the production.

[0071] In one embodiment of the present invention, in the above-mentioned step (a), L-serine is dissolved when L-serine and a solvent are mixed; when dissolving, the temperature does not need to be raised from room temperature, but the temperature can be raised if necessary; and considering process economic efficiency and preventing discoloration, it is most desirable to dissolve at about 50° C. or lower.

[0072] In one embodiment of the invention, after stirring in the above-mentioned steps (a), it is further possible to include concentrating and removing the solvent and gradually lowering the temperature to carry out crystallization. When the solvent removed in the process of concentrating and removing the solvent is too little, the process yield can be reduced, and when the solvent removed is too much, not only the preparation time can be increased to reduce economy, but also the risk of collision during the process of concentrated solvent under reduced pressure can be caused. Therefore, it is most desirable to concentrate the solvent to about 2 / 5 to 1 / 2 of the remaining total solvent.

[0073] Meanwhile, the cooling temperature at the time of crystallization is preferably about -5°C to 30°C, more preferably about 0°C to 20°C, most preferably about 5°C to 15°C.

[0074] In one embodiment of the present invention, in step (b), any tool capable of performing a filtering function may be used. For example, on a relatively small scale, a Buchner funnel may be used, and from an industrial perspective, the production efficiency may be improved by performing the process under reduced pressure using a Nutsche filter, but the process is not limited thereto. In addition, the temperature for filtering may be about 10° C. or less.

[0075] In one embodiment of the present invention, it may further include (c) drying after filtration, and the drying temperature may be about 40°C to 50°C, but the drying temperature is not limited thereto, and considering economic efficiency, the drying time may be appropriately set to preferably about 12 hours or less, most preferably about 3 hours to 8 hours.

[0076] The new crystal form type 1 of L-serine according to the present invention has relatively low hygroscopicity and reduced hardening compared to the amorphous form of L-serine or the crystal form A of L-serine, and exhibits rapid dissolution in an aqueous solution, and exhibits higher solubility in a syrup solution; therefore, it can be effectively used in the manufacture of pharmaceutical preparations. In particular, the crystal form type 1 of L-serine can not only suppress the generation or discoloration of related substances in a preparation containing the crystal form due to its low sensitivity to thermal denaturation, but also allows the storage of raw materials and the manufacturing process to be carried out at room temperature and a relative humidity of 60% due to its low hygroscopicity, so it is convenient. In addition, the crystal form type 1 of L-serine exhibits excellent pharmacokinetic properties compared to the amorphous form of L-serine or the crystal form A of L-serine; therefore, it can be expected that a therapeutic effect can be achieved with a smaller amount of L-serine.

[0077] Hereinafter, in order to help understand the present invention, the present invention will be described in detail using embodiments. However, embodiments according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the present invention are provided to more fully illustrate the present invention to people with general knowledge of the field to which the present invention belongs.

[0078] Example

[0079] Comparative Example 1: Preparation of amorphous L-serine

[0080] The L-serine obtained by the following method was subjected to X-ray powder diffraction analysis, and the results are shown in Figure 1 Infrared spectroscopy (IR) data are shown in Figure 4 The differential scanning calorimetry data are shown in Fig.11 It was confirmed that L-serine is amorphous: 100 g of O-acetyl-L-serine (Aldrich, 99% purity) was dissolved in 500 mL of water, 3 mL of acetic acid was added, and then stirred at 70°C for 6 to 8 hours, the solvent was concentrated under reduced pressure, and freeze-dried.

[0081] Comparative Example 2: Preparation of Type A Crystalline of L-Serine

[0082] According to the production method in Non-Patent Document 2, crystal form A of L-serine was obtained with a yield of 70%.

[0083] 250 g of L-serine (purchased from Aldrich) was added and dissolved in 100 mL of water. The resultant was heated to 60° C. to 70° C. and stirred for 6 hours. After complete dissolution, the resultant was cooled to 5° C. to 10° C., stirred for 1 hour, and then filtered to obtain pure L-serine as a white solid.

[0084] The obtained L-serine was subjected to X-ray powder diffraction analysis, and the results are shown in Figure 2 Infrared spectroscopy (IR) data are shown in Figure 5 and the differential scanning calorimetry data are shown in Fig.10 middle.

[0085] Example 1: Preparation of crude L-serine

[0086] 355g of DL-serine (purchased from Aldrich) and 232g of L-(-) camphorsulfonic acid (purchased from EOS, China) are added and dissolved in 2200mL of ethyl acetate (purchased from Samchun Chemicals). The resulting mixture is heated to 80°C to 90°C and stirred for 3 hours. After the reaction is complete, the mixture is cooled to 5°C to 10°C, stirred for 1 hour, then filtered to obtain pure L-serine-camphorsulfonic acid as a white solid. The obtained solid is dissolved in 120L of anhydrous ethanol, and ammonia is added thereto to adjust the pH to between 6.0 and 7.0. The reactant is stirred at room temperature for 1.5 hours, cooled to 5°C to 10°C, stirred for another 3 hours, then filtered to obtain 150g of crude L-serine as an off-white solid.

[0087] Example 2: Preparation of a new crystal form of L-serine (1)

[0088] After 50 g of the amorphous form of L-serine obtained in the above-mentioned Comparative Example 1 was dissolved in 0.40 L of water and then stirred, about 1 / 2 of the solvent was removed by concentration, the resultant was cooled to 10° C. or lower, the obtained solid was filtered, and vacuum dried at 40° C. for 6 hours to obtain Type 1 crystal of L-serine with a yield of 74%.

[0089] The obtained L-serine crystal form was subjected to X-ray powder diffraction analysis, infrared analysis, differential scanning calorimetry and DVS analysis, and the results are shown in Figure 3 , Figure 6 , Fig.10 and Fig. 9 middle. Fig.14 The result of overlapping the X-ray powder diffraction patterns of the type A crystal form of L-serine obtained in Comparative Example 2 and the type 1 crystal form of L-serine obtained in Example 2 is shown. Fig.14 It can be seen that the A-type crystal form and the 1-type crystal form are different crystal types from each other.

[0090] Example 3: Preparation of a new crystal form of L-serine (2)

[0091] After 50 g of the crude L-serine from Example 1 was dissolved in 0.40 L of water and then stirred, about 1 / 2 of the solvent was removed by concentration, the resultant was cooled to 10° C. or lower, the resulting solid was filtered, and vacuum dried at 40° C. for 6 hours to obtain Type 1 crystal of L-serine with a yield of 85%.

[0092] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis on the obtained crystal form, it was confirmed that L-serine was the same crystal form as in Example 2.

[0093] Example 4: Preparation of a new crystal form of L-serine (3)

[0094] After dissolving 30 g of the Type A crystal of L-serine prepared in Comparative Example 2 in 0.50 L of water and then stirring, about 1 / 2 of the solvent was removed by concentration, the resultant was cooled to 10° C. or lower, the obtained solid was filtered, and vacuum dried at 40° C. for 7 hours to obtain Type 1 crystal of L-serine with a yield of 60%.

[0095] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis on the obtained crystal form, it was confirmed that L-serine was the same crystal form as in Example 2.

[0096] Example 5: Preparation of a new crystal form of L-serine (4)

[0097] After 50 g of the crude L-serine from Example 1 was dissolved in 0.40 L of water / methanol (9 / 1) and then stirred, about 1 / 2 of the solvent was removed by concentration, the resultant was cooled to 10° C. or lower, the resulting solid was filtered, and vacuum dried at 40° C. for 6 hours to obtain Type 1 crystal of L-serine with a yield of 77%.

[0098] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis on the obtained crystal form, it was confirmed that L-serine was the same crystal form as in Example 2.

[0099] Example 6: Preparation of a new crystal form of L-serine (5)

[0100] After 50 g of the crude L-serine from Example 1 was dissolved in 0.40 L of water / methanol (9 / 1) and then stirred, about 1 / 2 of the solvent was removed by concentration, the resultant was cooled to 10° C. or lower, the resulting solid was filtered, and vacuum dried at 40° C. for 6 hours to obtain Type 1 crystal of L-serine with a yield of 79%.

[0101] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis on the obtained crystal form, it was confirmed that L-serine was the same crystal form as in Example 2.

[0102] Example 7: Preparation of a new crystal form of L-serine (6)

[0103] After dissolving 200 g of O-acetyl-L-serine (Aldrich, 99% purity) in 100 mL of water, one drop of acetic acid was added thereto, the mixture was stirred at 70° C. for 6 hours, and then about 1 / 2 of the solvent was removed by concentration, the resultant was cooled to 10° C. or lower, the obtained solid was filtered, and vacuum dried at 40° C. for 8 hours to obtain Type 1 crystal of L-serine with a yield of 77%.

[0104] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis on the obtained crystal form, it was confirmed that L-serine was the same crystal form as in Example 2.

[0105] X-ray powder diffraction analysis

[0106] X-ray powder diffraction analysis (XRD) was performed using a powder X-ray diffraction detector to obtain a diffraction pattern in the 2θ range of 5° to 80°. The powder X-ray diffraction analysis conditions are as follows.

[0107] -Equipment: EMPYREAN Panalytical

[0108] -Time per step: 0.5s

[0109] - Scan method: Continuous PSD Fast

[0110] -X-ray tube; Cu 1.8KW

[0111] -Detector: PIXcel3D (1D mode)

[0112] Differential Scanning Calorimetry

[0113] Differential scanning calorimetry (DSC) was performed using a SCINCO DSC N-650 model. About 2 to 3 mg of the sample was placed in an aluminum pan and covered with a perforated lid to prepare the sample required for the DSC experiment. After recording the exact weight of the sample, it was heated to 30°C to 350°C at a rate of 10°C / min under nitrogen.

[0114] Based on saturation solubility test of raw materials

[0115] Each raw material was prepared and tested as a 2000 mg / mL L-serine solution (MW=105.09) according to the following test method, and the amount was measured three times using the following method.

[0116] a. 10.0 g of each raw material as L-serine was added to a 50 mL conical tube, and then about 5 mL of purified water (or syrup composition) was added thereto, and the mixture was vigorously mixed using a vortexer for 30 seconds.

[0117] b. The resulting solution was allowed to stand at room temperature for 5 minutes and then mixed vigorously using a vortexer for 30 seconds.

[0118] c. Repeat process b 6 times (about 30 minutes), and then let the result stand at room temperature until the floating matter settles.

[0119] d. The supernatant of the solution was placed in a 2.0 mL EP tube and centrifuged at 23°C and 4000 rpm for 20 minutes using a centrifuge.

[0120] e. Take 1 mL of the solution supernatant, place it in a 100 mL volumetric flask, and fill it to the mark with purified water. Then, take 5 mL (or 10 mL) of the solution, place it in a 50 mL volumetric flask, and fill it to the mark with purified water to use as the test solution.

[0121] f. Analyze the test solution according to the HPLC operating conditions for testing the L-serine content.

[0122] Experimental Example 1: Thermal Stability Test

[0123] 8 mg of each of the L-serine obtained in Comparative Example 1, Comparative Example 2 and Example 3 was placed in an opaque glass vial and stored at 40±2° C., 75±5% RH. After 8 weeks, each sample was taken out, the content was analyzed using high performance liquid chromatography (HPLC), and the presence or absence of discoloration was visually confirmed, such as Fig.13 shown.

[0124] HPLC conditions

[0125] - Equipment: Waters Alliance e2695

[0126] -Column: SIELC Primsep 100, 150mm×4.6mm×5μm

[0127] - Column temperature: 30℃

[0128] -Flow rate: 1.0mL / min

[0129] -Detection: 210nm, UV

[0130] -Injection volume: 10 μL

[0131] -Total analysis time: 40mins

[0132] - Mobile phase: Adjust pH to 2.2 ± 0.05 using phosphoric acid in 1000 mL of water.

[0133] As a result of the 8-week analysis, it was confirmed that the crude L-serine obtained in Comparative Examples 1 and 2 showed a decrease in L-serine content or discoloration compared to the standard product, and that the Type 1 crystal form of L-serine obtained in Example 3 showed excellent stability over a period of up to 8 weeks without any change in the content value or color of the powder. When the crystal form change of L-serine was confirmed by XRD after 8 weeks, some of the amorphous form was transformed into a crystal form, and the Type A crystal form of L-serine obtained in Comparative Example 2 and the Type 1 crystal form of L-serine obtained in Example 3 did not show a crystal form change. These results are shown in Table 1 below.

[0134] [Table 1]

[0135]

[0136] As shown in the above Table 1, it was confirmed that the Type 1 crystal form according to Example 3 was stable without any reduction in content due to hygroscopicity or discoloration due to oxidation.

[0137] Experimental Example 2: Moisture absorption test

[0138] The amorphous and A-type crystal forms of L-serine obtained in Comparative Examples 1 and 2, respectively, and the type 1 crystal form of L-serine obtained in Example 3 were each tested in the following manner: using a dynamic moisture adsorption (DVS) apparatus (VTI SGA-100, TA Instruments) as a dynamic moisture adsorption and desorption apparatus, adsorption and dehumidification were repeated once at intervals of 5% RH in a relative humidity range of 2% to 98% under isothermal conditions at 25° C. The moisture adsorption and desorption behaviors according to relative humidity are shown in Figures 7 to 9 middle.

[0139] As a result of DVS analysis, the amorphous form of L-serine obtained in Comparative Example 1 has begun to absorb moisture at a relative humidity of 40% (eg Figure 7 As shown), the A-type crystal form of L-serine obtained in Comparative Example 2 began to absorb moisture at a relative humidity of about 70% (as shown Figure 8 As shown), while the type 1 crystal form of L-serine obtained in Example 3 began to absorb moisture at a relative humidity of about 80% (as shown Fig. 9 Therefore, it is confirmed that the type 1 crystal form of L-serine exhibits relatively low hygroscopicity.

[0140] Experimental Example 3: Pharmacokinetic Comparison

[0141] After fasting for 12 hours, blood samples were collected at 6 hours, 4 hours, 2 hours and 0 hours before drug administration, each crystalline form of L-serine was administered once (400 mg / kg, n=5) using a probe for oral administration, and about 40 to 50 μL of blood was collected and measured at 0.25 hours, 0.5 hours, 1 hour, 3 hours, 6 hours, 9 hours and 12 hours after drug administration. The measurement results are shown in Fig.12 The results after baseline correction of L-serine are shown in Table 2.

[0142] As a result, as shown in Table 2 and Fig.12 As shown, it is confirmed that the maximum blood concentration (C 20 ) of the type 1 crystal of L-serine prepared in Example 3 is higher than that of the amorphous and type A crystals prepared in Comparative Examples 1 and 2, respectively. max ) showed no significant difference; however, the half-life of the drug (T 1 / 2 ) varies significantly depending on the crystalline form. In the case of Type 1 crystalline form of L-serine, the 0-12 hour concentration-time curve (AUC 0-12 ) was increased by about 2 times or more compared to the amorphous and type A crystalline forms of L-serine.

[0143] [Table 2] Comparison of baseline-corrected pharmacokinetics

[0144]

[0145]

[0146] Experimental Example 4. Measurement of the saturated solubility of each crystal form of L-serine in purified water

[0147] For each crystal form of L-serine, the saturated solubility was measured while observing the dissolution using purified water as a solvent, and the results are shown in Table 4.

[0148] a. 10.0 g of each of L-serine obtained in Comparative Example 1, Comparative Example 2 and Example 3 was added to a 50 mL conical tube, and then about 5 mL of purified water was added thereto, and the mixture was vigorously mixed using a vortexer for 30 seconds.

[0149] b. The resulting solution was allowed to stand at room temperature for 5 minutes and then mixed vigorously using a vortexer for 30 seconds.

[0150] c. Repeat process b 6 times (about 30 minutes), and then let the result stand at room temperature until the floating matter settles.

[0151] d. The supernatant of the solution was placed in a 2.0 mL Eppendorf tube and centrifuged at 23° C. and 4000 rpm for 20 minutes using a centrifuge.

[0152] e. Take 1 mL of the solution supernatant, place it in a 100 mL volumetric flask, and fill it to the mark with purified water. Then, take 5 mL (or 10 mL) of the solution, place it in a 50 mL volumetric flask, and fill it to the mark with purified water to use as the test solution.

[0153] f. Analyze the test solution according to the HPLC operating conditions for testing the L-serine content. Experimental Example 5. Measurement of the saturated solubility of each crystal form of L-serine in a syrup solution

[0154] For each crystal form of L-serine, the saturated solubility was measured while observing the dissolution using a solution of a syrup preparation as a solvent, and the results are shown in Table 4.

[0155] As shown in Table 3 below, a syrup composition was prepared using the method described in Korean Patent Application No. 10-2022-0033655, and the experimental method of saturated solubility was performed in the same manner as Experimental Example 4, except that the solution of the syrup composition in Table 3 was used instead of purified water in Experimental Example 4.

[0156] [Table 3]

[0157]

[0158]

[0159] As shown in Table 4 below, it was confirmed that the average solubility of the new crystal form Type 1 of L-serine in water was almost the same as that of the amorphous form and the crystal form A of L-serine, but the average solubility in the syrup composition was 5% to 10% higher than that of the amorphous form and the crystal form A of L-serine, and because there was no hardening phenomenon compared with the amorphous form and the crystal form of L-serine, the dissolution rate in terms of dissolution was fast and the dissolution process was carried out smoothly.

[0160] [Table 4]

[0161]

[0162] Therefore, the new crystal form type 1 of L-serine has relatively low hygroscopicity and reduced hardening compared to the amorphous or A-type crystal form of L-serine, and shows more rapid dissolution in aqueous solutions, so it can be effectively used for the manufacture of pharmaceutical preparations. In particular, since the crystal form type 1 of L-serine is less susceptible to thermal denaturation, it can not only suppress the generation of variable substances or discoloration in the preparation containing the crystal form, but also allow the storage of raw materials and the manufacturing process to be carried out at room temperature and 60% relative humidity due to low hygroscopicity, which is convenient. In addition, the new crystal form type 1 of L-serine shows improved pharmacodynamic properties compared to the amorphous or A-type crystal form of L-serine; therefore, it can be expected that a therapeutic effect can be achieved with a smaller amount of L-serine.

Claims

1. A crystalline form of L-serine represented by the following formula 1: [Formula 1] The crystal form has diffraction peaks at diffraction angles (2θ) of 19.06, 20.76, 22.86, 28.28 and 30.58 in X-ray powder diffraction analysis.

2. The crystalline form of L-serine according to claim 1, which has additional diffraction peaks at one or more diffraction angles (2θ) selected from 18.49, 36.89, 42.13, 42.96 and 44.

17.

3. The crystalline form of L-serine according to claim 1, which shows an endothermic point of 218.36°C in differential scanning calorimetry analysis.

4. The crystalline form of L-serine according to claim 1, which has a -1 、2940cm -1 、1572cm -1 、1500cm -1 、1466cm -1 、1408cm -1 、1123cm -1 、1082cm -1 and 1008cm -1 It has an absorption peak in the infrared spectrum.

5. A method for preparing the crystalline form of L-serine according to claim 1, comprising: (a) mixing L-serine and a solvent and stirring; as well as (b) Filter the crystals produced after stirring.

6. The method according to claim 5, wherein: The solvent is one or more solvents selected from water, methanol and ethanol.

7. The method according to claim 5, wherein: When the solvent is a mixed solvent of water and methanol or a mixed solvent of water and ethanol, the mixing ratio is 1:9 to 9:

1.

8. The method according to claim 5, wherein: The filtration temperature is 10°C or lower.

9. The method according to claim 5, further comprising (c) drying after filtering.

10. The method according to claim 9, wherein: The drying temperature is 40°C to 50°C.

11. The method according to claim 9, wherein: Drying time is 3 hours to 8 hours.

12. The crystalline form of L-serine according to claim 1, characterized in that: The X-ray powder diffraction values ​​are shown in FIG3 .

13. The crystalline form of L-serine according to claim 1, characterized in that: The infrared spectrum values ​​are shown in FIG6 .

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