CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug and preparation method of CRAC inhibitor freeze-dried powder preparation

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and discloses a CRAC inhibitor freeze-dried powder preparation based on a tromethamine phosphate prodrug and a preparation method of the CRAC inhibitor freeze-dried powder preparation. A CRAC inhibitor parent drug and tromethamine phosphate are combined to form a high-solubility prodrug, an ultrasonic-assisted freeze-drying technology is adopted, ultrasonic parameters are optimized in the stages of pre-freezing, primary drying and secondary drying, the freeze-drying time is remarkably shortened, the ice crystal size is controlled to be smaller than or equal to 50 microns, and the resolubility and stability are improved. The preparation is high in clinical safety, reduces local stimulation and systemic toxicity, is suitable for treating acute pancreatitis, and has high production efficiency and environmental protection property.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a CRAC inhibitor freeze-dried powder preparation based on a tromethamine phosphate salt prodrug and a preparation method thereof. Background Art

[0002] Currently, CRAC channel inhibitors (such as CM4620) have shown potential in the treatment of acute pancreatitis, but their dosage form development faces significant challenges: CM4620 has low solubility and needs to be formulated in an emulsion, which results in a complex production process, high cost, and limited dosage adjustment; emulsion preparations have poor stability and are easily affected by factors such as temperature and pH. Storage and transportation conditions are harsh and may cause drug degradation or precipitation; existing preparations are prone to strong local irritation at high concentrations, such as inflammation at the intravenous injection site, thrombosis and other adverse reactions, and their clinical safety is limited; emulsion preparations require complex preparation steps, which makes administration inconvenient, which is reflected in the extension of clinical administration time and reduced patient compliance.

[0003] At present, the recommended medications for acute pancreatitis are all for symptoms or complications, and there is no clear standard treatment plan, indicating that this market is not met. The above problems seriously limit the clinical application of CRAC inhibitors, and there is an urgent need to develop a dosage form with high stability, good solubility and excellent safety. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the present invention provides a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate salt prodrug and a preparation method thereof.

[0005] A method for preparing a CRAC inhibitor freeze-dried powder preparation based on a tromethamine phosphate prodrug comprises the following steps: The CRAC inhibitor parent drug is combined with tromethamine phosphate to form a prodrug; dissolving the prodrug and excipient in a buffer to form a base solution; Pre-freezing the base liquid to form a frozen base liquid; The frozen base liquid is subjected to primary drying and secondary drying in sequence to obtain a freeze-dried powder; Applying ultrasonic auxiliary treatment during the pre-freezing treatment, primary drying and secondary drying processes; The CRAC inhibitor parent drug is WXTJ0262-1.

[0006] Furthermore, the excipient is mannitol, and the buffer is Tris buffer.

[0007] Furthermore, the mass ratio of the excipient to the prodrug is 1:1 to 1:1.5.

[0008] Furthermore, the Tris buffer is Tris hydrochloric acid buffer or Tris phosphate buffer.

[0009] Further, the pre-freezing treatment is performed by cooling the temperature at a rate of 1°C / min to -40°C to -35°C and maintaining the temperature for 1-2 hours. Ultrasonic treatment of the base liquid at a power density of 1–2 W / cm² for 10–15 minutes in the range of 5 to -10°C promotes uniform nucleation of ice crystals, reduces the size of ice crystals, and ensures uniform distribution of ice crystals.

[0010] Furthermore, the primary drying temperature is -20°C and the vacuum degree is 5-10Pa. The end point is determined by the pressure rise test, and the criterion is that the pressure change is less than 1Pa / min. Ultrasonication was applied intermittently with a power density of 2–3 W / cm², with each ultrasonic treatment lasting 5 minutes and an interval of 25–30 minutes.

[0011] Furthermore, the temperature gradient of the secondary drying is from -20°C to 20-25°C, and the vacuum degree is controlled to be ≤5Pa. The drying end point was determined by Karl Fischer test, with the criterion being moisture ≤ 1.5%.

[0012] Furthermore, the power density of the intermittent application of ultrasound during secondary drying is 2–3 W / cm². During the heating stage, ultrasonic treatment was performed for 3-5 minutes each time with an interval of 10 minutes; During the incubation period, ultrasonic treatment was performed for 5 min each time with an interval of 20-30 min.

[0013] Furthermore, the ultrasonic frequency used in the pre-freezing process is 20kHz; The ultrasonic frequency used in the primary drying and secondary drying processes is 28kHz.

[0014] The reconstitution time of the CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug prepared according to the above preparation method is ≤1 minute.

[0015] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: 1. Reduce the incidence of preclinical adverse events Reduce local irritation: The concentration of the lyophilized powder after reconstitution is ≤2mg / mL (non-clinical data). Repeated administration tests in SD rats and Beagle dogs showed no vascular inflammation or thrombosis (compared with the high-dose group of the emulsion preparation); Improved systemic toxicity: The parent drug WXTJ0262-1 has extremely low off-target effects (only NET is inhibited among 42 safety targets, IC50=1.8μM), and all genetic toxicity tests are negative, which is significantly better than similar drugs.

[0016] 2. Improve the convenience of drug administration Rapid reconstitution: The reconstitution time of lyophilized powder is ≤ 1 minute, and no complicated preparation process is required, which is suitable for emergency scenarios; Excellent stability: No change in properties after 24 months storage at 2-8°C, loose transportation conditions, and no reliance on cold chain; Dosage flexibility: The specifications of lyophilized powder are standardized, supporting precise dosage adjustment to meet the needs of different patients.

[0017] 3. Preclinical data show high safety Phase I trial data: In the single-dose multiple ascending dose (SAD) regimen, the incidence of mild to moderate AEs was low, and there were no serious adverse reactions; Animal model support: The NOAEL for rats is 10 mg / kg, and the NOAEL for dogs is 20 mg / kg. The safety window is wide, which is significantly better than traditional dosage forms.

[0018] 4. Advantages of Ultrasonic-Assisted Freeze-Drying Technology Shorten freeze-drying time: Ultrasonic-assisted freeze-drying technology shortens the primary drying time from 48 hours to 36 hours, and the secondary drying time from 12 hours to 8 hours, thus improving production efficiency; Improve product quality: Ultrasonic treatment promotes uniform nucleation of ice crystals, reduces ice crystal size, and improves the solubility and stability of freeze-dried products; Energy saving and emission reduction: shorten freeze-drying time, reduce energy consumption, and meet green production requirements.

[0019] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is an XRD comparison diagram of an accelerated test in one embodiment of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0023] WXTJ0262 is a calcium release-activated calcium (CRAC) channel inhibitor. It inhibits the increase of calcium ions in pancreatic acinar cells, thereby preventing excessive activation of digestive enzymes and preventing acinar cell dysfunction, death or necrosis. It can also act on the CRAC channel of T cells, blocking T cell activation, reducing pancreatic and systemic inflammation, and achieving the purpose of treating acute pancreatitis.

[0024] The present application develops a CRAC inhibitor freeze-dried powder preparation and a preparation method thereof. The parent drug component of the freeze-dried powder preparation is WXTJ0262-1. In order to prepare the subsequent freeze-dried powder preparation, WXTJ0262-1 is first combined with trometamol phosphate to form a prodrug. The prodrug has high solubility and a solubility of >50 mg / mL. It has low water solubility in combination with other Tris bases, with a solubility of <10 mg / mL.

[0025] Preliminary tests were also conducted on the metabolism, active release efficiency and safety of the prodrug.

[0026] Under the action of alkaline phosphatase (ALP), the half-life of tromethamine phosphate salt is only 2 minutes, and the parent drug WXTJ0262-1 is released rapidly. After a single intravenous injection of 14C WXTJ0262 in SD rats, the total radioactivity was distributed in the pancreas, lungs, and kidneys, accounting for >70%, indicating efficient targeting; tromethamine combined with the parent drug relies on non-specific esterase or amidase hydrolysis, the half-life is significantly prolonged, and the release of the active ingredient is delayed.

[0027] The results of bacterial reverse mutation test and chromosome aberration test of tromethamine phosphate salt were both negative. The off-target risk was only for NET (IC50=1.8μM). NOAEL=10mg / kg for SD rats and NOAEL=20mg / kg for Beagle dogs. No serious organ damage was observed (pathological score ≤grade 2). Unphosphorylated Tris base accumulated in the kidneys, and some renal function indicators of SD rats showed abnormalities.

[0028] WXTJ0262-1 is a safe and effective active ingredient in the treatment of pancreatitis. In order to safely prepare a lyophilized powder that is easy to store, the prodrug WXTJ0262 formed by combining WXTJ0262-1 with trometamol phosphate is lyophilized to prepare a lyophilized powder preparation that meets the requirements of the drug.

[0029] The structural formula of WXTJ0262-1 is .

[0030] Example 1 (1) Solution preparation The base solution preparation process includes mixing WXTJ0262 API and mannitol in a ratio of 1:1.2 (w / w), dissolving in 10mM Tris buffer, and controlling the pH in the range of 7.6-7.8.

[0031] The Tris buffer can be selected from Tris hydrochloric acid buffer or Tris phosphate buffer. In this embodiment, Tris phosphate buffer is selected.

[0032] The operation of filtering and sterilizing the base liquid includes processing the base liquid through a 0.22μm PVDF filter membrane and filling it into a 7mL vial (filling volume 5mL).

[0033] The purpose of adding mannitol (5% w / v) and Tris buffer to the base solution is to ensure the stability of the drug structure during the freeze-drying process.

[0034] (2) Pre-freezing stage The temperature was decreased from 25°C to -40°C at a rate of 1°C / min and maintained for 2 hours.

[0035] After completing this step, the frozen base liquid was taken for scanning electron microscopy (SEM) observation, which showed that the ice crystal diameter was ≤80 μm and the drug molecules were not obviously embedded.

[0036] (3) Primary drying parameters: The temperature of the partition is -20°C and the vacuum degree is 10Pa. The drying is carried out for 48 hours. The end point is determined by the pressure rise test (PRT), and the criterion is that the pressure change is less than 1Pa / min.

[0037] Moisture removal target: residual moisture ≤5%.

[0038] (4) Secondary drying parameters: The temperature was raised to 25°C at a rate of 0.5°C / min and the vacuum degree was 5Pa.

[0039] The endpoint was determined by Karl Fischer test, with the criterion being moisture ≤ 1.5% and drying time being 12 hours.

[0040] Key control point: Pressure fluctuation inside the vial is ≤2Pa to avoid collapse.

[0041] (5) Packaging and stability testing The headspace nitrogen filling is controlled and adjusted to fill with 99.99% nitrogen during packaging, and the oxygen content is ≤0.5%.

[0042] Storage conditions: Store in a sealed container at 2-8℃ away from light. Valid for 24 months. Accelerated test shows no degradation at 40℃ for 6 months.

[0043] After testing, the freeze-dried powder preparation prepared in Example 1 can meet the national standard for injection, and the adverse reactions are significantly reduced. However, the freeze-drying process time is relatively long and the energy consumption is high. Therefore, in production practice, the process of introducing ultrasonic treatment during the freeze-drying stage of the active substance of the drug is further explored. The time and power of ultrasonic treatment need to be selected according to the active ingredient and.

[0044] Example 2 (1) Solution preparation WXTJ0262 API was mixed with mannitol in a ratio of 1:1.1 (w / w) and dissolved in 10 mM Tris buffer with a pH control range of 7.4-7.6.

[0045] Tris buffer: Tris phosphate buffer.

[0046] The rest is consistent with Example 1.

[0047] (2) Pre-freezing stage The temperature was lowered from 25°C to -40°C at a rate of 1°C / min and maintained for 2 hours. In the temperature range of 5 to -5°C, ultrasonic waves with a frequency of 20kHz and a power density of 1-2W / cm² were used for 10 minutes to promote uniform nucleation of ice crystals, reduce the size of ice crystals, and ensure uniform distribution of ice crystals.

[0048] Scanning electron microscopy (SEM) showed that the diameter of ice crystals was ≤50 μm and there was no obvious entrapment of drug molecules.

[0049] (3) Primary drying parameters Drying was carried out under the conditions of a partition temperature of -20°C and a vacuum degree of 10Pa.

[0050] Intermittently apply 28kHz ultrasound with a power density of 2–3W / cm², each ultrasound treatment lasts for 5 minutes with an interval of 30 minutes.

[0051] The drying time was shortened to 36 hours with ultrasonic assistance, and the endpoint was determined by the pressure rise test (PRT), with the criterion being a pressure change of <1 Pa / min.

[0052] (4) Secondary drying parameters The temperature was raised to 25°C at a rate of 0.5°C / min and the vacuum degree was 5Pa.

[0053] Intermittent application of 28kHz ultrasound with a power density of 2–3W / cm², During the warming stage from -20 to 25°C, ultrasonic waves were sonicated for 3 min each time with 10 min intervals; During the insulation stage after the temperature rises to 25°C, ultrasonic treatment is performed for 5 minutes each time with an interval of 30 minutes. The endpoint is determined by Karl Fischer test, with the criterion being moisture ≤ 1.5%, and the drying time is shortened to 8 hours.

[0054] (5) Packaging and stability testing The same as Example 1.

[0055] Storage conditions: Store in a sealed container at 2-8℃ away from light. Valid for 24 months. Accelerated test shows no degradation at 40℃ for 6 months.

[0056] Example 3 (1) Solution preparation WXTJ0262 API was mixed with mannitol at a ratio of 1:1.35 (w / w) and dissolved in 10 mM Tris buffer with a pH control range of 7.6-7.8.

[0057] Tris buffer: Select Tris hydrochloric acid buffer.

[0058] The rest is consistent with Example 1.

[0059] (2) Pre-freezing stage Decrease from 25℃ to -35℃ at a rate of 1℃ / min and maintain for 2 hours In the temperature range of 5 to -10°C, ultrasonic waves with a frequency of 20kHz and a power density of 1-2W / cm² were used for 15 minutes to promote uniform nucleation of ice crystals, reduce the size of ice crystals, and ensure uniform distribution of ice crystals.

[0060] Scanning electron microscopy (SEM) showed that the diameter of ice crystals was ≤50 μm, and the drug molecules had no obvious (3) Primary drying parameters Drying was carried out under the conditions of a partition temperature of -20°C and a vacuum degree of 10Pa.

[0061] Intermittent application of 28kHz ultrasound with a power density of 2–3W / cm², each ultrasound treatment lasts for 5 minutes with an interval of 25 minutes; The drying time was shortened to 30 hours with ultrasonic assistance, and the endpoint was determined by the pressure rise test (PRT), with the criterion being a pressure change of <1 Pa / min.

[0062] (4) Secondary drying parameters The temperature was raised to 20°C at a rate of 0.5°C / min and the vacuum degree was 5Pa.

[0063] Intermittent application of 28kHz ultrasound with a power density of 2–3W / cm², During the warming stage from -20 to 20 °C, ultrasonic waves were sonicated for 5 min each time with 10 min intervals; During the insulation stage after the temperature rises to 20°C, ultrasonic treatment is performed for 5 minutes each time with an interval of 20 minutes. The drying time was 8 hours, and the endpoint moisture test was determined by the Karl Fischer method, with the result being ≤1.5% moisture.

[0064] (5) Packaging and stability testing The same as Example 1.

[0065] Storage conditions: Store in a sealed container at 2-8℃ away from light. Valid for 24 months. Accelerated test shows no degradation at 40℃ for 6 months.

[0066] The applicant also conducted performance and pharmacological tests on the freeze-dried powders prepared in Examples 1-3. The test items and results are as follows.

[0067] (1) Redissolution test The finished product used is 50 mg / bottle of freeze-dried powder, and the reconstitution medium is 5 mL of water for injection. Shake for 10 seconds until completely dissolved, and the clarity of the solution meets the 0904 standard of the "Chinese Pharmacopoeia".

[0068] The freeze-dried powders prepared in Examples 1-3 can meet the requirements of the above standards, and the specific data are shown in Table 1.

[0069] Table 1 Time to reach the dissolution standard in the re-dissolution test (unit: seconds)

[0070] From the data table summary, we can see that the reconstitution performance of CRAC inhibitor freeze-dried powder preparations based on tromethamine phosphate prodrug can meet the 0904 standard of the Chinese Pharmacopoeia. The re-dissolution effects of Example 2 and Example 3 are better and more complete, and the dissolution time is shorter, which indicates that the ultrasonic treatment makes the particle size of the prodrug lower and more conducive to dissolution.

[0071] (2) Drug infusion test: Low dose (0.1-1.0 mg / kg): infuse at a constant rate (0.25 mL / min) over 4 hours.

[0072] High dose (1.6-3.0 mg / kg): 2-hour infusion (0.5 mL / min), Cmax controlled at ≤5000 ng / mL (based on Phase I SAD trial data).

[0073] The drip test after the freeze-dried and reconstituted products prepared in Example 1, Example 2 and Example 3 had no obvious adverse reaction feedback.

[0074] (3) Stability Accelerated test: 6 months at 40℃ / 75% RH.

[0075] The data of the rapid test of Example 2 are listed in Table 2.

[0076] Table 2

[0077] As can be seen from Table 2, the lyophilized powder preparation prepared by this scheme is consistent with the control group, the ultrasonic treatment does not change the active ingredients, and the amount of impurities generated in the accelerated test meets the mandatory regulations for medical safety.

[0078] The relevant substances in Table 2 are explained as follows: The structural formula of WXTJ0262-BP12 is ; The structural formula of WXTJ0262-BP11 is ; The structural formula of WXTJ0262-BP10 is ; The structural formula of WXTJ0262-BP7 is ; RRT0.56 and RRT0.67 are methods of expressing the relative retention time of impurities in drugs, which are used to describe the retention time relationship between impurities and main components in chromatographic analysis.

[0079] RRT0.56 means that the retention time of the impurity relative to the main component is 0.56, that is, the retention time of the impurity is 56% of the retention time of the main component.

[0080] RRT0.67 means that the retention time of the impurity relative to the main component is 0.67, that is, the retention time of the impurity is 67% of the retention time of the main component.

[0081] From the attached Figure 1 Comparison of the XRD pattern after 6 months of the medium accelerated test with the XRD pattern at 0 time shows that the properties of the freeze-dried powder are stable and the crystal consistency is good.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a CRAC inhibitor freeze-dried powder preparation based on a tromethamine phosphate prodrug, characterized in that: The following steps are involved: The CRAC inhibitor parent drug is combined with tromethamine phosphate to form a prodrug; dissolving the prodrug and excipient in a buffer to form a base solution; Pre-freezing the base liquid to form a frozen base liquid; The frozen base liquid is subjected to primary drying and secondary drying in sequence to obtain a freeze-dried powder; Applying ultrasonic auxiliary treatment during the pre-freezing treatment, primary drying and secondary drying processes; The CRAC inhibitor parent drug is WXTJ0262-1.

2. The method for preparing a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug according to claim 1, characterized in that: The excipient is mannitol, and the buffer is Tris buffer.

3. The method for preparing a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug according to claim 2, characterized in that: The mass ratio of the excipient to the prodrug is 1:1 to 1:1.

5.

4. The method for preparing a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug according to claim 2, characterized in that: Tris buffer is Tris hydrochloric acid buffer or Tris phosphate buffer.

5. The method for preparing a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug according to claim 1, characterized in that: Pre-freeze treatment: cool down to -40℃ to -35℃ at a rate of 1℃ / min and maintain for 1-2 hours. Ultrasonic treatment of the base liquid at a power density of 1–2 W / cm² for 10–15 minutes in the range of 5 to -10°C promotes uniform nucleation of ice crystals, reduces the size of ice crystals, and ensures uniform distribution of ice crystals.

6. The method for preparing a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug according to claim 1, characterized in that: The primary drying temperature is -20°C and the vacuum degree is 5-10Pa. The end point is determined by the pressure rise test, and the criterion is that the pressure change is less than 1Pa / min. Ultrasonication was applied intermittently with a power density of 2–3 W / cm², with each ultrasonic treatment lasting 5 minutes and an interval of 25–30 minutes.

7. The method for preparing a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug according to claim 1, characterized in that: The temperature gradient of secondary drying is from -20℃ to 20-25℃, and the vacuum degree is controlled to ≤5Pa. The drying end point was determined by Karl Fischer test, with the criterion being moisture ≤ 1.5%.

8. The method for preparing a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug according to claim 1, characterized in that: The power density of intermittent ultrasonic wave applied in secondary drying is 2–3W / cm². During the heating stage, ultrasonic treatment was performed for 3-5 minutes each time with an interval of 10 minutes; During the incubation period, ultrasonic treatment was performed for 5 min each time with an interval of 20-30 min.

9. The method for preparing a CRAC inhibitor freeze-dried powder preparation based on tromethamine phosphate prodrug according to claim 1, characterized in that: The ultrasonic frequency used in the pre-freezing process is 20kHz; The ultrasonic frequency used in the primary drying and secondary drying processes is 28kHz.

10. A CRAC inhibitor freeze-dried powder preparation based on a tromethamine phosphate prodrug prepared according to the method for preparing a CRAC inhibitor freeze-dried powder preparation based on a tromethamine phosphate prodrug according to any one of claims 1 to 9, characterized in that: The reconstitution time of the lyophilized powder preparation is ≤ 1 minute.

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