Method for Preserving P1, P4-di(uridine 5′)-tetraphosphate Crystals

Abstract

The present invention provides a method for easily and stably storing P1, P4-di(uridine 5')-tetraphosphate crystals for a long period of time; the method is a method for storing packaged P1, P4-di(uridine 5')-tetraphosphate crystals or pharmaceutically acceptable salts thereof, characterized by selecting any one of the following storage conditions (1) to (3) and storing the crystals under the conditions; (1) the storage temperature is above 0°C and below 25°C, (2) the storage temperature is above 25°C and below 40°C and the crystal pH is 4.5 to 8.0, and (3) the storage temperature is above 40°C and below 60°C and the crystal pH is 5.0 to 6.4.

Description

Technical Field

[0001] The present invention relates to a method for stably preserving P1, P4-di(uridine 5')-tetraphosphate crystals. Background Art

[0002] P1,P4-di(uridine 5')-tetraphosphate (commonly known as "UP4U") or a salt thereof, represented by the following formula (1), is used as a therapeutic agent for corneal epithelial disorders associated with dry eye. Furthermore, this compound has an expectorant effect and is expected to be developed as an expectorant or pneumonia treatment.

[0003] (Chemistry 1)

[0004]

[0005] In order to stably store P1,P4-di(uridine 5′)-tetraphosphate (hereinafter sometimes referred to as "UP4U"), crystals of this compound or its salts have been obtained (Patent Document 1). However, although previous literature indicates that crystals are more stable than freeze-dried products, the conditions under which crystals can be stably stored have not been studied.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2000 / 020430 Summary of the Invention

[0009] UP4U is a compound used as a pharmaceutical raw material. When used as a pharmaceutical raw material, extremely high-purity intermediates are required to minimize the formation of byproducts caused by impurities. These specifications are sometimes so stringent that even 0.1% impurities can be problematic. Therefore, storage conditions for manufactured intermediates must be set to minimize decomposition. However, it was previously unknown what conditions would promote decomposition when storing UP4U crystals, or what conditions would effectively suppress decomposition.

[0010] Therefore, an object of the present invention is to provide a method for easily and stably storing UP4U crystals for a long period of time.

[0011] The inventors of the present invention conducted intensive research to address the above-mentioned issues and discovered that the stability of UP4U varies significantly depending on the storage temperature and pH of the crystals. Further research revealed that the rate of change in HPLC purity after 4 weeks of storage of UP4U crystals under any of the following storage conditions (1) to (3) was suppressed to less than 0.15%, thereby reducing the rate of decrease in HPLC purity during storage. This led to the completion of the present invention. (1) Storage temperature: 0°C to 25°C; (2) Storage temperature: 25°C to 40°C, pH: 4.5 to 8.0; (3) Storage temperature: 40°C to 60°C, pH: 5.0 to 6.4.

[0012] Effects of the Invention

[0013] By using the storage method of the present invention, UP4U hardly decomposes and can be stored in a stable state for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The chart shows the HPLC purity change rate (%) when UP4U crystals exhibiting various crystal pH values ​​were packaged in containers and stored at 5°C to 60°C for 4 weeks.

[0015] As an example, * represents 5°C, x represents 25°C, △ represents 40°C, and ● represents 60°C, and their respective approximate curves are also shown. DETAILED DESCRIPTION

[0016] The storage method of the present invention relates to a method for storing UP4U crystals. The UP4U used in the crystals may be free or a pharmaceutically acceptable salt. Specific examples of salts include alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, and pharmaceutically acceptable salts such as ammonium salts. The number of metal substitutions in the salts may range from 1 to 4.

[0017] In the storage method of the present invention, UP4U crystals are stored under any of the following conditions (1) to (3) in order to suppress the change in HPLC purity of the crystals to less than 0.15% after 4 weeks of storage. (1) Storage temperature: 0°C to 25°C; (2) Storage temperature: 25°C to 40°C, pH of the crystals: 4.5 to 8.0; (3) Storage temperature: 40°C to 60°C, pH of the crystals: 5.0 to 6.4.

[0018] Furthermore, in the storage method of the present invention, in order to suppress the change in HPLC purity of the crystals after 4 weeks of storage to less than 0.10%, the UP4U crystals are stored under any of the following conditions (4) to (6): (4) Storage temperature is 0°C or higher and 25°C or lower; (2) Storage temperature is 25°C or higher and 40°C or lower, and the pH of the crystals is 4.5 to 7.4; (3) Storage temperature is 40°C or higher and 60°C or lower, and the pH of the crystals is 5.2 to 6.2.

[0019] Furthermore, in the storage method of the present invention, in order to suppress the change in HPLC purity of the crystals after 4 weeks of storage to less than 0.05%, the UP4U crystals are stored under any of the following conditions (7) and (8): (7) the storage temperature is 0°C or higher and 25°C or lower; (8) the storage temperature is 25°C or higher and 40°C or lower, and the pH of the crystals is 4.6 to 6.6.

[0020] In the present invention, the pH of crystals refers to the pH value measured when 1g of crystals is dissolved in 20ml of water. To adjust the pH of UP4U crystals to within a desired range, for example, the pH of the stock solution used for crystallization can be adjusted. Any acid or base used in the manufacture of pharmaceutical raw materials can be used to adjust the pH, as needed. Acids such as hydrochloric acid, nitric acid, and phosphoric acid can be used, while bases such as sodium hydroxide, sodium carbonate, and sodium bicarbonate can be used.

[0021] The UP4U used in the present invention can be synthesized and crystallized using known methods (e.g., Patent Document 1). In a specific example, crude UP4U synthesized by any method, such as chemical synthesis or enzymatic synthesis, is purified using anion exchange chromatography and activated carbon chromatography. A hydrophilic organic solvent is then added to the purified UP4U solution to produce UP4U crystals. Crystals produced by other methods are also acceptable, as long as they can produce crystals. Furthermore, the resulting crystals can be dried appropriately using conventional methods such as reduced pressure drying, ventilation drying, and heat drying.

[0022] Considering the pharmaceutical application of the UP4U crystals produced, a purity of 95% or higher is preferred, 97% or higher is more preferred, and 99% or higher is even more preferred. Furthermore, these crystals may adsorb water and form hydrates. More specifically, one molecule of UP4U may bind to or adsorb 3 to 8 molecules of water.

[0023] In the method of the present invention, the UP4U crystals are packaged in any container for storage. Containers of any shape, such as bags, bottles, cans, boxes, and small glass tubes, can be used as storage containers.

[0024] As the material of the container, unstretched films and uniaxial or biaxially stretched films of polyolefin resins such as low-density polyethylene, high-density polyethylene, and polypropylene, ethylene resins such as polystyrene and polyvinyl chloride, polyamide resins such as nylon 6 and nylon 66, and polyester resins such as polyethylene terephthalate and polybutylene terephthalate can be used.

[0025] These film materials can be used as a single layer or as a laminate of two or more films. Metal films such as aluminum, or vapor-deposited films of metals or metal oxides, can also be laminated. Furthermore, the container can be two or three layers or more. The container is preferably placed in a closed, airtight, or sealed state, preferably in a sealed or airtight state.

[0026] If UP4U is stored under conditions where the crystal pH or temperature is outside the above range, UP4U is likely to decompose, and there is a risk that it will be unsuitable for use as a pharmaceutical raw material.

[0027] The method of the present invention can control the decrease in UP4U purity by high performance liquid chromatography (HPLC) after 4-week storage to 0.15% or less, or 0.10% or less, or even 0.05% or less, which is extremely suitable for suppressing decomposition.

[0028] That is, when storing UP4U, for example, as a raw material for manufacturing a pharmaceutical product, and requiring a decrease in HPLC purity of 0.15% or less, it can be stored in a sealed, airtight, or airtight state under any of the following storage conditions: (1) a storage temperature of 0°C or higher and 25°C or lower; (2) a storage temperature of 25°C or higher and 40°C or lower, with a crystal pH of 4.5 to 8.0; or (3) a storage temperature of 40°C or higher and 60°C or lower, with a crystal pH of 5.0 to 6.4. If the storage environment can maintain a storage temperature of 0°C or higher and 25°C or lower, the crystal pH does not need to be adjusted. However, if the storage environment temperature is unstable and may reach 25°C or higher and 40°C or lower, the crystal pH needs to be adjusted to 4.5 to 7.4 to control the decrease in HPLC purity after 4 weeks of storage to less than 0.15%. If the temperature is 40°C or higher and 60°C or higher, the crystal pH should be adjusted to 5.2 to 6.2.

[0029] Similarly, in order to control the reduction in HPLC purity of UP4U to below 0.10%, it is necessary to store it under any one of the following storage conditions: (4) storage temperature is above 0°C and below 25°C; (5) storage temperature is above 25°C and below 40°C, and the crystal pH is between 4.5 and 7.4; (6) storage temperature is above 40°C and below 60°C, and the crystal pH is between 5.2 and 6.2.

[0030] Furthermore, in order to control the reduction in HPLC purity of UP4U to below 0.05%, it is necessary to store it under any one of the following storage conditions: (7) the storage temperature is above 0°C and below 25°C; (8) the storage temperature is above 25°C and below 40°C, and the crystal pH is between 4.6 and 6.6.

[0031] To summarize the above, (A) if the storage temperature can be maintained between 0°C and 25°C, the HPLC purity of UP4U crystals will hardly decrease, regardless of their pH, making them suitable for long-term storage. On the other hand, to store UP4U crystals in environments where temperature control is difficult, the pH of the crystal stock solution must be adjusted to maintain the pH within a specified range.

[0032] (B) When the storage temperature can reach 25°C or higher and 40°C or lower, it is appropriate to control the decrease in HPLC purity of the crystals after 4 weeks of storage to below 0.15%, 0.10%, and 0.05%, respectively, by setting the crystal pH to 4.5-8.0, preferably 4.5-7.4, and more preferably 4.6-6.6.

[0033] (C) When the storage temperature is likely to reach 40°C or higher and 60°C or lower, by setting the crystal pH to 5.0-6.4, preferably 5.2-6.2, the decrease in HPLC purity of the crystals after 4 weeks of storage can be controlled to less than 0.15% and 0.10%, respectively. When it is necessary to control the decrease in HPLC purity to less than 0.05%, a storage temperature exceeding 40°C is not preferred.

[0034] (D) Furthermore, if the storage temperature exceeds 60°C, the HPLC purity of the crystals may be significantly reduced regardless of the pH of the crystals, which is not preferred.

[0035] In addition, when UP4U crystals are stored for more than 4 weeks (2 to 3 months), in order to control the decrease in HPLC purity to below the desired value, the storage temperature can be set to above 0°C and below 25°C. The crystal pH can also be set within a range that is more preferred than the range of the necessary crystal pH described in the respective temperature ranges in the above (B) or (C).

[0036] Example

[0037] The following examples are given to explain the present invention in detail, but the present invention is not limited to these examples.

[0038] (Example 1) Preparation of UP4U crystals at different pH values

[0039] UP4U crystals prepared by a known method were dissolved in deionized water. A phosphoric acid solution or a sodium hydroxide solution was added to the solution containing UP4U to prepare solutions with different pH values ​​(hereinafter referred to as "stock solutions").

[0040] Ethanol was added to each stock solution until slightly cloudy, and a small amount of UP4U crystals was added. The mixture was stirred for more than ten hours. After confirming the precipitation of crystals, further ethanol was added, stirred, and then separated by centrifugation. The resulting crystals were dried in a ventilated dryer, and the UP4U crystals were adjusted to different pH values.

[0041] (pH Measurement) Prepare cooled distilled water after thorough boiling and attach a carbon dioxide absorption tube. Dissolve 1.0 g of the test sample in 20 ml of this distilled water. Measure the pH of this solution (crystallization pH). Table 1 below shows the correspondence between the pH of the stock solution prepared before crystallization and the pH of the crystals.

[0042] Table 1

[0043]

[0044] (Example 2) Approximately 20 mg of the test specimen for the stability test (test specimen packaging) was placed in a low-density polyethylene bag and sealed by heat sealing. This bag was then placed in a low-density polyethylene bag and sealed by heat sealing. This bag was then placed in a low-density polyethylene bag and sealed by heat sealing. This bag was then placed in an aluminum-coated film bag and sealed by heat sealing to prepare the stability test specimen.

[0045] (Stability Test) The packaged specimens were placed in thermostatic chambers at 5°C, 25°C, 40°C, and 60°C and stored for 4 weeks.

[0046] (Analysis) The sealed packages of the test specimens from the day 0 of the stability test and the test specimens after 4 weeks of storage at various temperatures were opened, and the test specimens were dissolved in deionized water. The solutions were analyzed by HPLC, and the stability was evaluated based on the results. The HPLC measurement conditions for this analysis are as follows.

[0047] Table 2

[0048]

[0049] In the evaluation, the HPLC purity (%) of the crystals on the 0th day of the stability test was subtracted from the HPLC purity (%) of the crystals after 4 weeks of storage as the change rate. The change rate (%) under each condition is shown in Table 3 and Figure 1 .

[0050] Table 3

[0051] pH 4.60 5.16 5.54 5.64 5.97 6.32 6.66 6.91 7.12 7.43 7.97 5℃ 0.01 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 0.00 0.01 0.03 25℃ -0.01 -0.02 -0.02 -0.01 -0.02 -0.01 -0.03 -0.02 -0.02 -0.01 -0.01 40℃ -0.05 -0.04 -0.03 -0.02 -0.03 -0.02 -0.04 -0.04 -0.08 -0.15 -0.12 60℃ -0.25 -0.08 -0.07 -0.07 -0.08 -0.14 -0.18 -0.24 -0.46 -0.50 -0.87

[0052] From Table 3 and Figure 1 It can be seen from the results that the storage conditions for UP4U crystals are as follows: (1) the storage temperature is above 0°C and below 25°C; (2) the storage temperature is above 25°C and below 40°C, and the crystal pH is between 4.5 and 8.0; (3) the storage temperature is above 40°C and below 60°C, and the crystal pH is between 5.0 and 6.4. The change rate of HPLC purity can be suppressed to below 0.15%.

[0053] In addition, as for the storage conditions, as long as any one of the following conditions is met: (4) the storage temperature is above 0°C and below 25°C; (2) the storage temperature is below 25°C and below 40°C, and the crystal pH is between 4.5 and 7.4; (6) the storage temperature is above 40°C and below 60°C, and the crystal pH is between 5.2 and 6.2, the change rate of HPLC purity can be suppressed to below 0.10%.

[0054] Furthermore, as for the storage conditions, as long as any one of the following conditions is met: (7) the storage temperature is above 0°C and below 25°C; (8) the storage temperature is above 25°C and below 40°C, and the crystal pH is between 4.6 and 6.6, the change rate of HPLC purity can be suppressed to below 0.05%.

Claims

1. A method for preserving P1, P4-di(uridine 5′)-tetraphosphate crystals, characterized in that: In order to suppress the decrease rate of HPLC purity of the packaged crystals to less than 0.15% when stored for 4 weeks, the crystals were stored under the following storage conditions: [1] Crystal pH is 5.16 at 60°C; [2] Crystal pH is 5.54 at 60°C; [3] Crystal pH is 5.64 at 60°C; [4] Crystal pH is 5.97 at 60°C; The crystal pH is the pH value measured when 1 g of crystals is dissolved in 20 ml of water.

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