Novel crystal form of l-serine and method for preparing same

Abstract

The present invention relates to a novel crystal form of L-serine and a method for preparing same. The novel crystal form of L-serine according to the present invention has improved hygroscopicity, and thus can be effectively used in the preparation of pharmaceutical preparations.

Description

Novel crystalline form of L-serine and method for preparing the same

[0001] The present invention relates to a novel crystalline form of L-serine and a method for preparing the same. More specifically, the present invention relates to a novel crystalline form of L-serine and a method for preparing the same, which improves stability through improved hygroscopicity, thereby improving ease of storage and handling, and has improved dissolution rate, solubility, and pharmacokinetic properties.

[0002] L-Serine, represented by the following chemical formula 1, is a non-essential amino acid that is involved in many metabolic systems and plays an important role in the normal growth and development of nerve cells. It plays an essential role in cell proliferation by synthesizing bases that become the materials of DNA, and also plays an important role in antioxidant activity by acting as a precursor for glutathione synthesis.

[0003] Patent No. KR101948066B1 discloses a composition containing L-serine as an active ingredient for the prevention, improvement, or treatment of neurodevelopmental disorders. The dosage of L-serine required for effective treatment is 200-400 mg / kg twice daily. Meanwhile, Korean Publication No. 10-2022-0131186 reports a syrup formulation, the optimal drug formulation for children with autism spectrum disorder.

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

[0005] [Chemical Formula 1]

[0006]

[0007] When formulating medicines 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 its concentration in the body so that it can be effective, regardless of the route through which the medicine or health functional food is administered.

[0008] Most active ingredients in new therapeutic substances exist in solid form, which can be amorphous or crystalline. Each drug's solid particles have different hardness, shape, size, and density, and thus, their activity and stability. The physical form of the active ingredient not only affects the difficulty of formulation, but also its stability before and after formulation, and its biological activity in the human body after administration. Therefore, regardless of the drug's structure or mechanism of action (MOA), when formulating a new drug, the optimal active ingredient form must be selected to maintain the drug's structure and composition.

[0009] In particular, as revealed through the prior invention of the present inventor, in the case of L-serine, an active ingredient in solid form, the dosage required to exhibit its efficacy is a large amount, such as 200-400 mg / kg, so it is absolutely necessary to develop a crystalline form with optimized stability and biochemical activity of the solid. In addition, the present inventor has clarified the effect of L-serine for treating developmental disorders through patent KR101948066B1. In developing L-serine into a pharmaceutical preparation that is easy for pediatric patients to take, various formulations that can provide convenience of taking for pediatric developmental disorder patients with many eating disorders are absolutely necessary, so the dissolution rate, solubility, and pharmacokinetics of the solid are even more important factors.

[0010] Amorphous forms generally have high solubility, which helps enhance efficacy and provide rapid onset of action. However, they are unstable, resulting in a shorter shelf life and making it difficult to control drug release and blood concentration. Crystalline forms, on the other hand, have low solubility, resulting in lower bioavailability per unit weight. However, they offer stability and the advantage of creating sustained-release controlled-release formulations.

[0011] According to non-patent literature 1 (Science of The Total Environment, vol 11, 2020, 139318), the hygroscopicity of amino acids is closely related to their water solubility. L-serine has a relatively high water solubility of 50 g / kg, and thus exhibits a relatively higher increase in hygroscopicity than the comparative amino acids aspartic acid and glutamine, depending on relative humidity.

[0012] Non-patent literature 2 (Physica B: Physics of Condensed Matter, 2018) discloses a crystalline form of L-serine (hereinafter referred to as crystalline form A) having an X-ray diffraction pattern as shown in Figure 1 below.

[0013] [Figure 1]

[0014]

[0015] The present inventors have confirmed that during the process of manufacturing and formulating amorphous and crystalline forms A of L-serine, care must be taken to avoid contact with moisture during handling such as storage and transportation and during the manufacture of finished pharmaceutical products due to their high hygroscopicity, and that there is a problem of discoloration into a yellow oxidized form when stored for 5-7 days at a relatively low temperature of 40℃. In addition, in the case of L-serine, the increase in hygroscopicity not only has the disadvantage of hardening the surface of the powder in contact with moisture, significantly reducing the dissolution rate in water, but also affects the saturated solubility depending on the influence of excipients that are essential in the manufacture of liquid formulations such as syrups, and thus there are technical difficulties in controlling the pharmacokinetic characteristics.

[0016] Accordingly, in order to solve the above problem, the inventors of the present invention provide a novel crystalline form of L-serine and a method for producing the same, which improves stability through improvement of hygroscopicity, thereby improving ease of storage and handling, and has improved dissolution rate, solubility, and pharmacokinetic properties.

[0017] The purpose of the present invention is to provide a novel crystalline form of L-serine having improved stability through improved hygroscopicity, thereby improving ease of storage and handling, and improved dissolution rate, solubility, and pharmacokinetic properties.

[0018] Another object of the present invention is to provide a method for producing a novel crystalline form of L-serine.

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

[0020] [Chemical Formula 1]

[0021]

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

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

[0024] [4] In the above [1], the crystalline L-serine has wavelengths of 3457, 2940, 1572, 1500, 1466, 1408, 1123, 1082, 1008 cm -1 It may have an absorption peak in the infrared spectral spectrum.

[0025] [5] In another aspect of the present invention, the present invention relates to a method for producing crystalline L-serine according to [1], comprising the steps of (a) mixing and stirring L-serine and a solvent; and (b) filtering the crystals produced after the stirring.

[0026] [6] In the above [5], the solvent may be at least one selected from the group consisting of 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 filtering temperature may be 10°C or lower.

[0029] [9] In the above [5], (c) a drying step after the filtration may be additionally included.

[0030]

[0010] In the above [9], the drying temperature may be 40 to 50°C.

[0031]

[0011] In the above [9], the drying time may be 3 to 8 hours.

[0032]

[0012] In the above [1], the crystalline L-serine may have the X-ray powder diffraction analysis values ​​of Figure 3 below.

[0033] [Figure 3]

[0034]

[0035]

[0013] In the above [1], the crystalline L-serine may have the infrared spectral spectrum value of FIG. 6 below.

[0036] [Figure 6]

[0037]

[0038] The novel crystalline form of L-serine according to the present invention (hereinafter, crystalline form 1) can improve stability by improving hygroscopicity, thereby improving ease of storage and handling, and improving solubility and pharmacokinetic properties, and thus can be usefully used in the manufacture of pharmaceutical preparations.

[0039] In particular, the novel crystalline form of L-serine exhibits little hardening, so it can exhibit a fast dissolution rate in an aqueous solution, and has little denaturation due to heat, so it can suppress the generation of flexible substances or discoloration in a preparation containing it.

[0040] Figure 1 is an X-ray powder diffraction diagram of amorphous L-serine.

[0041] Figure 2 is an X-ray powder diffraction diagram of L-serine crystalline form A.

[0042] Figure 3 is an X-ray powder diffraction diagram of L-serine crystalline form 1.

[0043] Figure 4 is an IR analysis diagram of amorphous L-serine.

[0044] Figure 5 is an IR analysis diagram of L-serine crystalline form A.

[0045] Figure 6 is an IR analysis diagram of L-serine crystalline form 1.

[0046] Figure 7 is a dynamic vapor absorption (DVS) analysis result of L-serine amorphous form, showing a dynamic vapor adsorption / desorption curve.

[0047] Figure 8 is a dynamic vapor absorption (DVS) analysis result of L-serine crystalline form A, showing a dynamic vapor adsorption / desorption curve.

[0048] Figure 9 is a dynamic vapor absorption (DVS) analysis result of L-serine crystalline form 1, showing a dynamic vapor adsorption / desorption curve.

[0049] Figure 10 is a differential scanning calorimetry analysis diagram of L-serine crystalline form 1.

[0050] Figure 11 is a differential scanning calorimetry analysis diagram of amorphous L-serine.

[0051] Figure 12 shows the pharmacokinetic results of L-serine amorphous, crystalline A, and crystalline 1 in mice.

[0052] Figure 13 shows the color change results of L-serine amorphous, crystalline A, and crystalline 1 after 8 weeks.

[0053] Figure 14 is a comparison result of overlapping X-ray powder diffraction patterns of L-serine crystalline form A and crystalline form 1.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0055] In one aspect, the present invention relates to crystalline L-serine (crystalline form 1) represented by the following chemical formula 1.

[0056] [Chemical Formula 1]

[0057]

[0058] In the present invention, “L-serine” is a type of amino acid that constitutes proteins, is a monoamide of glutamic acid, and is an L-amino acid having the structure of the chemical formula 1.

[0059] In one embodiment of the present invention, the crystalline L-serine represented by the chemical formula 1 has diffraction peaks at diffraction angles (2θ) of 19.06, 20.76, 22.86, 28.28, and 30.58 when analyzed by X-ray powder diffraction.

[0060] In another embodiment of the present invention, in X-ray powder diffraction analysis, diffraction peaks at diffraction angles (2θ) at which I / I0 (I: intensity of peak at each diffraction angle, I0: intensity of the largest peak) is 10% or more may be 19.06, 20.76, 22.86, 28.28, and 30.58.

[0061] In one embodiment of the present invention, the crystalline L-serine represented by the above chemical formula 1 may additionally have a diffraction peak at one or more diffraction angles (2θ) selected from the group consisting of 18.49, 36.89, 42.13, 42.96, and 44.17.

[0062] In one embodiment of the present invention, the crystalline L-serine represented by the chemical formula 1 may have the X-ray powder diffraction analysis value of FIG. 3 below.

[0063] [Figure 3]

[0064]

[0065] In another embodiment of the present invention, the diffraction peaks at diffraction angles (2θ) at which I / I0 is less than 10% in X-ray powder diffraction analysis may be 18.49, 36.89, 42.13, 42.96, and 44.17.

[0066] In one embodiment of the present invention, the crystalline L-serine represented by the chemical formula 1 may exhibit an endothermic point of about 218.36°C in differential scanning calorimetry analysis. Therefore, the crystalline L-serine represented by the chemical formula 1 has excellent thermal stability with a thermal decomposition temperature of about 218.36°C, and the active ingredient can be stably maintained without decomposition or discoloration even when stored for a long period of time.

[0067] In one embodiment of the present invention, the crystalline L-serine represented by the above chemical formula 1 has infrared spectroscopy (IR) spectrum at 3457, 2940, 1572, 1500, 1466, 1408, 1123, 1082, 1008 cm -1 The absorption peak value can be expressed in .

[0068] In one embodiment of the present invention, the crystalline L-serine represented by the chemical formula 1 may have the infrared spectral value of FIG. 6 below.

[0069] [Figure 6]

[0070]

[0071] In another aspect, the present invention relates to a method for producing crystalline L-serine represented by the above chemical formula 1, comprising the steps of (a) mixing and stirring L-serine and a solvent; and (b) filtering the crystals produced after the stirring.

[0072] In one embodiment of the present invention, the "L-serine" used in the step (a) is a compound represented by the above chemical formula 1, and may be L-serine prepared by a known method. Conventionally, L-serine has been prepared mostly by fermentation or by preparing a DL-serine mixture using mercury or the like and then using a method of obtaining pure L-serine through optical resolution. A representative method for preparing serine in the literature (Advances in Biochemical Engineering / Biotechnology 79 (2003) 1) discloses a method for preparing 45 g / L L-serine from 100 g / L glycine using Hyphomicrobium sp. NCIB10099 and 88 g / L methanol over 3 days. Another document (US 5382517) describes a manufacturing method using Escherichia coli MT-10350 to ferment glycine and an aqueous formaldehyde solution (485 g / L) at 50°C for 35 hours, yielding 89%. Other manufacturing methods include a method using acrylate as a starting material described in [Org. Synth. 1940, 20, 81].

[0073] In one embodiment of the present invention, the form of L-serine used in the step (a) is not limited, and for example, the L-serine may be oil-phase L-serine obtained by concentrating the solvent after producing L-serine, or amorphous or crystalline form A of L-serine.

[0074] In the above step (a), the “solvent” may be a polar solvent, and specifically, may be at least one selected from the group consisting of water, methanol, and ethanol.

[0075] In one 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.

[0076] Additionally, the amount of the solvent may be about 3 to 20 times the amount of L-serine used in the production, preferably about 4 to 15 times, and most preferably about 5 to 10 times, taking into account the economic feasibility of the process.

[0077] In one embodiment of the present invention, when L-serine and the solvent are mixed in step (a), L-serine is dissolved, and when dissolving, the temperature does not need to be raised from room temperature, but the temperature can be raised when necessary, and considering the economic feasibility of the process and prevention of discoloration, it is most preferable to proceed at about 50°C or lower.

[0078] In one embodiment of the present invention, after stirring in step (a), a step of concentrating and removing the solvent and a step of gradually lowering the temperature to crystallize may be additionally included. In the process of concentrating and removing the solvent, if too little solvent is removed, the process yield decreases, and if too much solvent is removed, the manufacturing time increases, which not only reduces economic efficiency but also carries the risk of bumping during the process of concentrating the solvent under reduced pressure. Therefore, it is most preferable to concentrate the solvent so that it remains between about 2 / 5 and 1 / 2 of the total solvent used.

[0079] Meanwhile, the cooling temperature of the crystallization step is preferably about -5 to 30°C, more preferably about 0 to 20°C, and most preferably about 5 to 15°C.

[0080] In one embodiment of the present invention, filtration in step (b) may be performed using any tool capable of performing a filtration function. For example, a Buchner funnel may be used on a relatively small scale, and industrially, a Nutsche filter may be used under reduced pressure to increase production efficiency, but this is not limited thereto. In addition, the filtration temperature may be approximately 10°C or lower.

[0081] In one embodiment of the present invention, (c) a drying step after the filtration may be additionally included, and the drying temperature may be about 40 to 50°C, but is not limited thereto, and the drying time may be appropriately set in consideration of economy, and is preferably about 12 hours or less, and is most preferably about 3 to 8 hours.

[0082] The novel crystalline form 1 of L-serine according to the present invention exhibits relatively less hygroscopicity and less hardening than the amorphous or crystalline form A, and thus exhibits a rapid dissolution rate in an aqueous solution and higher solubility in a syrup solution, and thus can be usefully used in the manufacture of pharmaceutical preparations. In particular, crystalline form 1 of L-serine is less denatured by heat, and therefore not only can the generation of reactive substances or discoloration be suppressed in preparations containing it, but also, due to its low hygroscopicity, the storage of raw materials and the manufacturing process can be conveniently performed at room temperature with a relative humidity of 60%. In addition, crystalline form 1 exhibits superior pharmacokinetic properties than the amorphous or crystalline form A, and thus a therapeutic effect can be expected with a smaller amount of L-serine.

[0083] Hereinafter, the present invention will be described in detail, using examples and the like, to aid understanding. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0084] Example

[0085] Comparative Example 1: Preparation of amorphous L-serine

[0086] 100g of O-Acetyl-L-serine from Aldrich (purity 99%) was dissolved in 500ml of water, 3ml of acetic acid was added, and the mixture was stirred at 70℃ for 6-8 hours, the solvent was concentrated under reduced pressure, and freeze-dried. X-ray powder diffraction analysis of L-serine was performed, and the results are shown in Fig. 1, infrared spectroscopy (IR) data is shown in Fig. 4, and differential scanning calorimetry data is shown in Fig. 11. From this, it was confirmed that it was amorphous.

[0087]

[0088] Comparative Example 2: Preparation of L-serine crystalline form A

[0089] L-serine crystalline form A was manufactured with a yield of 70% according to the manufacturing method of non-patent literature 2.

[0090] 250 g of L-serine (purchased from Aldrich) was dissolved in 100 ml of water. The solution was heated to 60 to 70°C and stirred for 6 hours. After complete dissolution, the solution was cooled to 5 to 10°C, stirred for 1 hour, and filtered to obtain pure L-serine as a white solid.

[0091] X-ray powder diffraction analysis of the obtained L-serine was performed, and the results are shown in Fig. 2, infrared spectroscopy (IR) data in Fig. 5, and differential scanning calorimetry data in Fig. 10.

[0092]

[0093] Example 1: Preparation of crude L-serine

[0094] 355 g of DL-serine (purchased from Aldrich) and 232 g of L-(-)camphorsulfonic acid (purchased from China EOS) were dissolved in 2200 ml of ethyl acetate (purchased from Samcheon Pure Chemicals). The mixture was heated to 80 to 90°C and stirred for 3 hours. After the reaction was completed, the mixture was cooled to 5 to 10°C, stirred for 1 hour, and filtered to obtain L-serine-camphorsulfonic acid salt as a white solid. The obtained solid was dissolved in 120 L of absolute ethanol, and ammonia water was added to adjust the pH to 6.0 to 7.0. The reaction mixture was stirred at room temperature for 1.5 hours, lowered to 5 to 10°C, stirred for an additional 3 hours, and filtered to obtain 150 g of crude L-serine as a white solid.

[0095]

[0096] Example 2: Preparation of a novel crystalline form of L-serine-1

[0097] 50 g of amorphous L-serine prepared in the above comparative example 1 was dissolved in 0.40 L of water, stirred, and then about 1 / 2 of the solvent was concentrated and removed, the temperature was lowered to 10°C or lower, and the resulting solid was filtered and vacuum dried at 40°C for 6 hours to obtain L-serine crystalline form 1 with a yield of 74%.

[0098] X-ray powder diffraction analysis, IR analysis, differential scanning calorimetry analysis, and DVS analysis of the obtained crystal forms were performed, and the results are shown in FIGS. 3, 6, 10, and 9, respectively. FIG. 14 is a result of overlapping the X-ray powder diffraction patterns of crystal form A obtained in Comparative Example 2 and crystal form 1 obtained in Example 2. It can be seen from FIG. 14 that crystal form A and crystal form 1 are different crystal forms.

[0099]

[0100] Example 3: Preparation of a novel crystalline form of L-serine-2

[0101] 50 g of crude L-serine of Example 1 was dissolved in 0.40 L of water, stirred, and then about half of the solvent was concentrated and removed. The temperature was lowered to 10°C or lower, and the resulting solid was filtered and vacuum-dried at 40°C for 6 hours to obtain L-serine crystalline form 1 with a yield of 85%.

[0102] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis of the obtained crystal form, it was confirmed that it was the same crystal as in Example 2.

[0103]

[0104] Example 4: Preparation of a novel crystalline form of L-serine-3

[0105] 30 g of crystalline form A manufactured in Comparative Example 2 was dissolved in 0.50 L of water, stirred, and then about half of the solvent was concentrated and removed, the temperature was lowered to 10°C or lower, and the resulting solid was filtered and vacuum-dried at 40°C for 7 hours to obtain L-serine crystalline form 1 with a yield of 60%.

[0106] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis of the obtained crystal form, it was confirmed that it was the same crystal as in Example 2.

[0107]

[0108] Example 5: Preparation of a novel crystalline form of L-serine-4

[0109] Example 1: 50 g of crude L-serine was dissolved in 0.40 L of water / methanol (9 / 1), stirred, and then about half of the solvent was concentrated to remove it. The temperature was lowered to 10°C or lower, and the resulting solid was filtered and vacuum-dried at 40°C for 6 hours to obtain L-serine crystalline form 1 with a yield of 77%.

[0110] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis of the obtained crystal form, it was confirmed that it was the same crystal as in Example 2.

[0111]

[0112] Example 6: Preparation of a novel crystalline form of L-serine - 5

[0113] 50 g of crude L-serine of Example 1 was dissolved in 0.40 L of water / ethanol (9 / 1), stirred, and then about half of the solvent was concentrated to remove it, the temperature was lowered to 10°C or lower, and the resulting solid was filtered and vacuum-dried at 40°C for 6 hours to obtain L-serine crystalline form 1 with a yield of 79%.

[0114] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis of the obtained crystal form, it was confirmed that it was the same crystal as in Example 2.

[0115]

[0116] Example 7: Preparation of a novel crystalline form of L-serine-6

[0117] After dissolving 200 g of O-Acetyl-L-serine (Aldrich, purity 99%) in 100 ml of water, 1 drop of acetic acid was added, and the mixture was stirred at 70°C for 6 hours. After concentrating and removing about half of the solvent, the temperature was lowered to 10°C or lower, and the resulting solid was filtered and dried at 40°C for 8 hours to obtain L-serine crystalline form 1 with a yield of 77%.

[0118] As a result of performing X-ray powder diffraction analysis and differential scanning calorimetry analysis of the obtained crystal form, it was confirmed that it was the same crystal as in Example 2.

[0119]

[0120] X-ray powder diffraction analysis

[0121] The above X-ray powder diffraction (XRD) analysis was performed using a powder X-ray diffractometer to obtain a diffraction pattern in the range of 5 to 80° 2θ. The powder X-ray diffraction analysis conditions are as follows.

[0122] - Device: EMPYREAN Panalytical

[0123] - Time per step: 0.5 s

[0124] - Injection method: Continuous PSD fast

[0125] - X-ray tube; Cu 1.8KW

[0126] - Detector: PIXcel3D (1D mode)

[0127]

[0128] Differential scanning calorimetry

[0129] Differential scanning calorimetry (DSC) was performed using a SCINCO DSC N-650 model. Approximately 2–3 mg of sample was placed in an aluminum pan and covered with a perforated lid to prepare the sample for DSC experiments. After recording the exact weight, the sample was heated to 30–350°C at a rate of 10°C / min under nitrogen.

[0130]

[0131] Saturation solubility test according to raw material

[0132] Each raw material was prepared and tested as a 2,000 mg / mL solution of L-serine (MW=105.09) according to the following test method, and the measured amount was measured three times using the following method.

[0133] a. Take 10.0 g of each raw material as L-serine, place it in a 50 ml conical tube, add about 5 ml of purified water (or syrup composition), and mix vigorously for 30 seconds using a vortexer.

[0134] b. Leave the above liquid at room temperature for 5 minutes and then mix vigorously for 30 seconds using a vortexer.

[0135] c. Repeat the above step b 6 times (approximately 30 minutes) and leave at room temperature until the floating matter settles.

[0136] d. Take the supernatant of the above solution, place it in a 2.0 ml EP tube, and centrifuge it at 23°C and 4000 rpm for 20 minutes.

[0137] e. Take 1 ml of the supernatant of the above solution and place it in a 100 ml volumetric flask, fill to the mark with purified water, then take 5 ml (or 10 ml) of this solution and place it in a 50 ml volumetric flask, fill to the mark with purified water, and use it as the test solution.

[0138] f. L-serine content test is analyzed according to HPLC operating conditions.

[0139]

[0140] Experimental Example 1: Thermal Stability Test

[0141] Each of 8 mg of L-serine obtained in Comparative Examples 1, 2, and Example 3 was placed in an opaque glass vial and stored at 40±2°C and 75±5% RH. After 8 weeks, each sample was taken out and the content was analyzed using high-performance liquid chromatography (HPLC), and the presence of discoloration was visually confirmed as shown in Figure 13.

[0142] High-performance liquid chromatography conditions

[0143] - Device: Waters Alliance e2695

[0144] - Column: SIELC Primsep 100, 150 mm x 4.6 mm x 5 μm

[0145] - Column temperature: 30℃

[0146] - Flow rate: 1.0 mL / min

[0147] - Detection: 210 nm, UV

[0148] - Injection volume: 10 μL

[0149] - Total analysis time: 40 minutes

[0150] - Mobile phase: Adjust the pH to 2.2 ± 0.05 using phosphoric acid in 1000 mL of water.

[0151]

[0152] As a result of the 8-week analysis, it was confirmed that the L-serine content of Comparative Examples 1 and 2 decreased or the color changed compared to the standard product, and the L-serine crystalline form 1 obtained in Example 3 showed excellent stability for up to 8 weeks without any change in the content value or color of the powder. When the presence or absence of change in the crystalline form was confirmed through XRD after 8 weeks, some of the amorphous cases changed into crystalline forms, and the crystalline form A obtained in Comparative Example 2 and the crystalline form 1 obtained in Example 3 showed no change in the crystalline form. These results are shown in Table 1 below.

[0153] Storage conditions 40℃ / 75% RH Comparative example 1 (amorphous) Comparative example 2 (crystalline form A) Example 3 (crystalline form 1) L-serine content after 0→8 weeks 99.0→90.4% 99.1→93.7% 99.3→99.2% Discoloration / change in crystalline form Change to off-white Change to some crystalline form Change to off-white Maintain crystalline form No color change Maintain crystalline form

[0154]

[0155] As shown in Table 1 above, it was confirmed that the crystalline form 1 according to Example 3 was stable without any decrease in content due to hygroscopicity or discoloration due to oxidation.

[0156]

[0157] Experimental Example 2: Hygroscopicity Test

[0158] The amorphous and crystalline forms A obtained in Comparative Examples 1 and 2 and the crystalline form 1 obtained in Example 3 were tested using a dynamic vapor absorption and desorption device, DVS (dynamic vapor absorption, VTI SGA-100, TA Instruments), under isothermal conditions at 25°C and relative humidity ranging from 2 to 98% at 5% RH intervals, repeating moisture absorption and dehumidification once. The moisture adsorption and desorption behavior according to relative humidity is shown in Figures 7 to 9, respectively.

[0159] As a result of DVS analysis, the amorphous form of L-serine obtained in Comparative Example 1 started absorbing moisture already at a relative humidity of 40%, as shown in FIG. 7, and the crystalline form A of L-serine obtained in Comparative Example 2 started absorbing moisture at around 70% relative humidity, as shown in FIG. 8, whereas the crystalline form 1 of Example 3 started absorbing moisture at around 80% relative humidity, as shown in FIG. 9, and thus it was confirmed that the crystalline form 1 showed relatively low moisture absorption.

[0160]

[0161] Experimental Example 3: Pharmacokinetic Comparison

[0162] After fasting for 12 hours, blood samples were collected at -6, -4, -2, and 0 hours before drug administration. Then, each crystalline form of L-serine was administered as a single dose (400 mg / kg, n = 5) using an oral gavage. Approximately 40-50 μl of blood was collected and measured at 0.25, 0.5, 1, 3, 6, 9, and 12 hours after drug administration. The measurement results are shown in Fig. 12, and the results after baseline correction of L-serine are shown in Table 2.

[0163] As a result, as shown in Table 2 and Figure 12, the L-serine of crystalline form 1 manufactured in Example 3 had a higher maximum blood concentration (C) after drug administration compared to the amorphous and crystalline forms A of L-serine manufactured in Comparative Examples 1 and 2. max ) showed no significant difference between drugs, but the half-life (T 1 / 2 ) showed significant differences depending on the crystal type, and the 0-12 hour concentration-time curve (AUC) 0-12 ) It was confirmed that the drug concentration increased by about 2 times or more in crystalline form 1 compared to amorphous and crystalline forms A.

[0164] Pharmacokinetic comparison table after baseline correction Parameter Unit Comparative Example 1 Comparative Example 2 Example 3 t 1 / 2 h4.15±0.992.56±0.763.56±1.36T max h0.250.500.50C maxμg / ml67.5±25.9277.69±8.4975.48±10.70AUC 0-12 μg / ml*h100.53±20.15165.81±40.99248.89±37.34AUC 0-inf μg / ml*h121.45±17.20177.36±39.9296.22±69.63MRT 0-inf h5.11±0.83.44 ±1.495.29±2.10Vz / F(mg / kg) / (μg / ml)19.87±4.8911.69±4.259.91±1.97Cl / F(mg / kg) / (μg / ml) / h3.35±0.433.19±0.672.11±0.49

[0165]

[0166] Experimental Example 4. Measurement of saturated solubility of each crystal form in purified water

[0167] For each crystal type, the dissolution rate was observed using purified water as a solvent, and the saturated solubility was measured, and the results are shown in Table 4.

[0168] a. Take 10.0 g of each L-serine obtained in Comparative Example 1, Comparative Example 2, and Example 3, place it in a 50 ml conical tube, add about 5 ml of purified water, and mix vigorously for 30 seconds using a vortexer.

[0169] b. Leave the above liquid at room temperature for 5 minutes and then mix vigorously for 30 seconds using a vortexer.

[0170] c. Repeat the above step b 6 times (approximately 30 minutes) and leave at room temperature until the floating matter settles.

[0171] d. Take the supernatant of the above solution, place it in a 2.0 ml Eppendorf tube, and centrifuge it using a centrifuge at 23°C and 4000 rpm for 20 minutes.

[0172] e. Take 1 ml of the supernatant of the above solution and place it in a 100 ml volumetric flask, fill to the mark with purified water, then take 5 ml (or 10 ml) of this solution and place it in a 50 ml volumetric flask, fill to the mark with purified water, and use it as the test solution.

[0173] f. L-serine content test is analyzed according to HPLC operating conditions.

[0174]

[0175] Experimental Example 5. Measurement of saturated solubility of each crystal form in syrup solution

[0176] For each crystal type, the dissolution rate was observed using a syrup-type solution as a solvent, and the saturated solubility was measured, and the results are shown in Table 4.

[0177] The composition of the syrup was prepared using the method specified in Korean Patent Application No. 10-2022-0033655 as shown in Table 3 below, and the saturated solubility test method was conducted in the same manner as Experimental Example 4 above, but the syrup composition solution in Table 3 was used instead of the purified water of Experimental Example 4.

[0178] Content (Amount in 1 mL (mg)) Diluent D-sorbitol solution 30.0 Thickener Carbomer 971P4.0 Flavoring Apple mint flavor SJ-G (22005221) 2.0 Preservative Methyl parahydroxybenzoate 0.05 Propyl parahydroxybenzoate 0.05 Sweetener Tablet White sugar 80.0 Acidifier Citric acid monohydrate 0.3 Buffer Potassium citrate monohydrate 1.5 Solvent Tablet water qs Final pH 5.4

[0179]

[0180] As shown in Table 4 below, the average solubility of the new crystalline form 1 L-serine in water is almost the same as that of the amorphous and crystalline forms A, but the average solubility in the syrup composition is about 5-10% higher than that of the amorphous and crystalline forms A. In addition, it was confirmed that the dissolution rate is fast and the dissolution process is smooth because there is no solidification phenomenon compared to the amorphous and crystalline forms in terms of the dissolution rate in water.

[0181] Sample name Average solubility (mg / ml) Syrup composition Average solubility (mg / ml) Note 1 Comparative example 1 300.4 207.7 When the first purified water was added, the crystals hardened, so the vortex time was increased to 2 minutes and the standing time was increased to 8 minutes. 2 Comparative example 2 299.0 195.9 When the first purified water was added, the crystals hardened, so the vortex time was increased to 3 minutes and the standing time was increased to 10 minutes. 3 Example 3 300.6 220.1 When dissolving, it proceeded smoothly within the time without any hardening phenomenon.

[0182]

[0183] Therefore, the novel crystalline form 1 of L-serine has relatively less hygroscopicity and less hardening than the amorphous or crystalline form A, so it exhibits a fast dissolution rate in an aqueous solution and higher solubility in a syrup solution, and can be usefully used in the manufacture of pharmaceutical preparations. In particular, since crystalline form 1 of L-serine is less denatured by heat, not only can it suppress the generation of reactive substances or discoloration in preparations containing it, but also its low hygroscopicity allows the storage of raw materials and the manufacturing process to be conveniently performed at room temperature with a relative humidity of 60%. In addition, it exhibits superior pharmacokinetic properties than the amorphous or crystalline form A, so that a therapeutic effect can be expected with a smaller amount of L-serine.

Claims

1. Crystalline L-serine represented by the following chemical formula 1: [Chemical Formula 1] Here, the crystalline L-serine is characterized in that the crystalline form has diffraction peaks at diffraction angles (2θ) of 19.06, 20.76, 22.86, 28.28, and 30.58 when analyzed by X-ray powder diffraction.

2. In the first paragraph, crystalline L-serine additionally having a diffraction peak at one or more diffraction angles (2θ) selected from the group consisting of 18.49, 36.89, 42.13, 42.96, and 44.

17.

3. In the first paragraph, crystalline L-serine exhibiting an endothermic point of 218.36℃ in differential scanning calorimetry analysis.

4. In paragraph 1, 3457, 2940, 1572, 1500, 1466, 1408, 1123, 1082, 1008 cm -1 Crystalline L-serine having an absorption peak in the infrared spectroscopy spectrum. 5.(a) a step of mixing and stirring L-serine and a solvent; and (b) A method for producing crystalline L-serine according to claim 1, comprising the step of filtering the crystals produced after the stirring.

6. A manufacturing method in paragraph 5, wherein the solvent is at least one selected from the group consisting of water, methanol, and ethanol.

7. A manufacturing method in which, in the fifth paragraph, 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. A manufacturing method in which the filtering temperature in paragraph 5 is 10°C or lower.

9. A manufacturing method in accordance with paragraph 5, further comprising the step of drying after the filtration (c).

10. A manufacturing method according to claim 9, wherein the drying temperature is 40 to 50°C.

11. A manufacturing method according to claim 9, wherein the drying time is 3 to 8 hours.

12. Crystalline L-serine characterized by the X-ray powder diffraction analysis values ​​of FIG. 3 below, in the first paragraph. [Figure 3] 13. Crystalline L-serine characterized by the infrared spectral spectrum values ​​of FIG. 6 in the first paragraph. [Figure 6]

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