A gonadotropin freeze-dried preparation and preparation method thereof

Abstract

The present invention provides a gonadotropin freeze-dried preparation and a preparation method thereof, and relates to the technical field of pharmaceutical preparations. The gonadotropin freeze-dried preparation of the present invention comprises the following components in parts by weight: 0.01-0.15 parts of gonadotropin, 10-20 parts of buffer, 0.1-1 parts of surfactant, 200-300 parts of trehalose, 100-350 parts of mannitol, and 50-100 parts of glycine. The present invention adopts a specific formula to prepare a gonadotropin freeze-dried preparation, which has high biological activity, good stability during freeze-drying and storage, and short re-dissolution time during use. The freeze-dried preparation of the present invention can have good stability and re-solubility at the same time. The present invention is particularly suitable for preparing freeze-dried preparations of luteinizing hormone (LH) and its variants, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a gonadotropin freeze-dried preparation and a preparation method thereof. Background Art

[0002] Gonadotropins (Gn) are glycoprotein hormones that regulate the development of vertebrate gonads and promote the production and secretion of sex hormones, such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secreted by the anterior pituitary gland, and human chorionic gonadotropin (HCG) secreted by the human placenta. If the human body lacks gonadotropins, it may cause a variety of diseases. For example, low gonadotropin levels in women may affect their fertility. For example, LH, as a glycoprotein gonadotropin, can promote the conversion of cholesterol into sex hormones in gonadal cells. If LH is deficient, various diseases including reproductive diseases may occur, such as irregular menstruation and infertility in women, and decreased testicular function in men. Luteinizing hormone α, also known as recombinant human luteinizing hormone (rh-LH), has the same structure and physiological effects as the naturally occurring luteinizing hormone (LH) in the human body. It is a glycoprotein hormone prepared by genetic engineering and cell culture technology, and can bind to the luteinizing hormone / chorionic gonadotropin (LH / CG) receptors on the ovarian membrane (and granulosa layer) and testicular interstitial cell membrane. For women who lack LH and FSH and do not ovulate, the use of rh-LH can increase the level of estradiol secreted by the follicles and stimulate follicle development.

[0003] It can be seen that gonadotropin plays an important role in regulating the normal physiological functions of the human body, and the lack of gonadotropin can lead to the occurrence of many diseases. Clinically, patients with gonadotropin deficiency are often supplemented by injection of corresponding gonadotropin. For example, when LH is deficient, LH is formulated into intramuscular (IM) or subcutaneous (SC) injection drugs for injection supplementation, or injection supplementation of rh-LH. At present, the medicinal injection preparations of gonadotropin are mainly freeze-dried preparations, and the freeze-dried preparations are reconstituted for injection when used. However, gonadotropin has poor stability during the preparation of freeze-dried preparations and the later storage process, and is easy to denature, resulting in unqualified products. For example, the commercially available human luteinizing hormone (LH) antigen is dissolved in 0.02MpH7.4 PBS solution and freeze-dried into freeze-dried powder. The freeze-dried powder can only be stored for a short period of time at 2-8°C, and long-term storage needs to be stored below -20°C.

[0004] In order to improve the stability of gonadotropin freeze-dried preparations during preparation and long-term storage. Patent applications with publication numbers WO2004087213A8 and WO2004112826A1 both disclose freeze-dried preparations containing follicle stimulating hormone (FSH) or its variants, and / or luteinizing hormone (LH) or its variants. In both patent applications, sucrose, dextrose, lactose, mannitol and / or glycerol are selected as stabilizers and tension regulators to ensure the stability of the freeze-dried preparations. Patent publication number EP0448146B1 discloses a stabilized freeze-dried powder containing gonadotropin, which comprises gonadotropin and a stable amount of dicarboxylate. The preparation will contain a sufficient amount of dicarboxylate, dicarboxylate and added sugars as stabilizers to ensure the stability of the freeze-dried preparation during the freeze-drying process and storage process. However, in the above patent applications or patents, only the stability of the preparation is considered when preparing the freeze-dried preparation, and the solubility of the preparation is not considered.

[0005] However, during the actual use of the lyophilized preparation, its resolubility is the key quality attribute of the lyophilized preparation. The resolubility time of gonadotropin lyophilized preparations such as LH or its variants and FSH or its variants generally needs to be controlled within 2 minutes, especially rh-FSH in clinical use and related regulations. The resolubility time is further controlled within 60 seconds. If the lyophilized preparation is insoluble or incompletely dissolved within a certain period of time, it may lead to a reduction in active ingredients during clinical use, affecting the effectiveness of the drug. Therefore, when preparing gonadotropin lyophilized preparations, both the stability of the lyophilized preparation and its resolubility should be considered. The patent application with publication number CN103432085A discloses the use of amino acids as solubilizers to prepare temozolomide lyophilized preparations, which increases the solubility of temozolomide and the stability of the solution, and the resolubility time is relatively fast, wherein the amino acids are arginine or lysine, etc. However, for different drugs, the effect of amino acid solubilization may be different. It is still unknown whether amino acids can be used for gonadotropin solubilization and how the solubilization effect is. How to provide a gonadotropin lyophilized preparation with good stability and good resolubility needs further study. Summary of the invention

[0006] The invention provides a gonadotropin freeze-dried preparation and a preparation method thereof.

[0007] The present invention provides a gonadotropin freeze-dried preparation, which comprises the following components in parts by weight:

[0008] 0.01-0.15 parts of gonadotropin, 10-20 parts of buffer, 0.1-1 parts of surfactant, 200-300 parts of trehalose, 100-350 parts of mannitol, and 50-100 parts of glycine.

[0009] Furthermore, the aforementioned gonadotropin freeze-dried preparation comprises the following components in parts by weight:

[0010] 0.04 parts of gonadotropin, 10-15 parts of buffer, 0.5-1 parts of surfactant, 200-300 parts of trehalose, 200-300 parts of mannitol, and 50-100 parts of glycine.

[0011] Furthermore, the aforementioned gonadotropin freeze-dried preparation comprises the following components in parts by weight:

[0012] 0.04 parts of gonadotropin, 12-13 parts of buffer, 0.5 parts of surfactant, 200-300 parts of trehalose, 200-300 parts of mannitol, and 50-100 parts of glycine.

[0013] Furthermore, the weight ratio of mannitol to glycine is 2 to 4:1.

[0014] Further,

[0015] The gonadotropin is luteinizing hormone or its variant, follicle stimulating hormone or its variant, chorionic gonadotropin secreted by human placenta or its variant;

[0016] and / or, the buffer is a phosphate buffer;

[0017] And / or, the surfactant is selected from polysorbate, Tween 20, Tween 40, Tween 80;

[0018] Preferably,

[0019] The phosphate buffer consists of disodium hydrogen phosphate and sodium dihydrogen phosphate;

[0020] And / or, the surfactant is Tween 20.

[0021] Preferably, the mass ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is (2-3):1.

[0022] In the present invention, the gonadotropin is preferably LH or an LH variant, which can be produced by a suitable method, such as a recombinant method, if possible, isolated or purified from a natural source, or chemically synthesized, or any combination thereof. "LH variant" refers to a molecule that has a different amino acid sequence, glycosylation pattern or inter-subunit linkage than human LH, but exhibits LH activity.

[0023] The term "recombinant" is used to refer to the preparation of LH or LH variants obtained by the use of recombinant DNA technology (see for example patent application published as WO85 / 01958). An example of a method for expressing LH using recombinant technology is by transfecting eukaryotic cells with DNA sequences encoding the alpha and beta subunits of LH (whether provided on one vector or two vectors, each subunit having a separate promoter), as described in patent applications published as EP0211894 and EP0487512.

[0024] LH is preferably produced by recombinant methods, particularly preferably in Chinese hamster ovary (CHO) cells transfected with an expression vector or vectors comprising DNA encoding human glycoprotein α-subunit and LH β-subunit. The DNA encoding α- and β-subunits may be present on the same or different vectors.

[0025] Another example of using recombinant technology to produce LH is to use homologous recombination technology to insert a heterologous regulatory segment operably linked to the endogenous sequence encoding the LH subunit, as described in patent application publication number EP0505500.

[0026] Furthermore, the preparation of LH or LH variant comprises 1-15 μg of LH or LH variant, 10-30 mg of trehalose, 10-30 mg of mannitol, and 5-10 mg of glycine.

[0027] Furthermore, the formulation of LH or LH variants further comprises 0.01 mg to or about 1 mg of a surfactant.

[0028] The formulation of the LH or LH variant also includes a phosphate buffer. Preferably a phosphate buffer, preferably a counter ion is sodium or potassium ion. Phosphate saline buffer is well known in the art, such as Dulbecco's phosphate buffered saline. The buffer concentration in the total solution may vary between 5mM, 9.5mM, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM and 500mM. Preferably, the buffer concentration is or is about 10mM. Particularly preferred is a 10M phosphate ion buffer having a pH of 7.0.

[0029] The buffer is preferably adjusted in such a manner that the pH of the reconstituted formulation of the lyophilized formulation of the present invention is about 6.0 or about 6.0 to about 8.0, more preferably about 6.8 or about 7.8 to about 7.8, including about pH 7.0, pH 7.2 and 7.4.

[0030] Furthermore, the luteinizing hormone (LH) is human luteinizing hormone (LH).

[0031] The luteinizing hormone (LH) is a human urinary luteinizing hormone (LH).

[0032] The luteinizing hormone (LH) is recombinant human luteinizing hormone (rh-LH).

[0033] Furthermore, the freeze-dried preparation is prepared by taking each component, adding it to water for injection, dissolving it, and then freeze-drying it.

[0034] Furthermore, after the components are dissolved, the pH value is adjusted to 6.0 to 8.0; preferably 6.8 to 7.8; more preferably 7.2.

[0035] Furthermore, the freeze-drying process comprises the following steps:

[0036] 1) Prefreezing: temperature is -40℃~-50℃, time is 5-15h, vacuum degree is 1-5atm;

[0037] 2) Sublimation: temperature is -25℃~-35℃, time is 12-18h, vacuum degree is 8-16pa;

[0038] 3) Heating: temperature is 10℃~35℃, time is 1.5-2.5h, vacuum degree is 8-16pa;

[0039] 4) Analytical drying: temperature is 20℃~35℃, time is 1-3h, vacuum degree is 8-16pa.

[0040] The present invention also provides a method for preparing the aforementioned gonadotropin freeze-dried preparation, which comprises the following steps:

[0041] (1) Weigh each component according to weight;

[0042] (2) Dissolve each component in water for injection and adjust the pH value to 6.0-8.0;

[0043] (3) freeze drying, the freeze drying process comprising the following steps: 1) pre-freezing: temperature -40°C to -50°C, time 5-15h, vacuum degree 1-5atm; 2) sublimation: temperature -25°C to -35°C, time 12-18h, vacuum degree 8-16Pa; 3) heating: temperature 10°C to 35°C, time 1.5-2.5h, vacuum degree 8-16Pa; 4) analytical drying: temperature 20°C to 35°C, time 1-3h, vacuum degree 8-16Pa.

[0044] The present invention also provides use of the aforementioned gonadotropin freeze-dried preparation in preparing a drug for treating female infertility and / or male infertility.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention provides a gonadotropin freeze-dried preparation. The present invention adopts a specific formula to prepare a gonadotropin freeze-dried preparation. The freeze-dried preparation has high biological activity, good stability during freeze-drying and storage, and short re-dissolution time during use. The freeze-dried preparation of the present invention can have good stability and re-dissolution. The present invention is particularly suitable for preparing a freeze-dried preparation of luteinizing hormone (LH) and its variants, and has good application prospects.

[0047] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0048] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0049] Source of raw materials: Unless otherwise specified, the raw materials of the present invention can be purchased commercially.

[0050] In the present invention, 4 μg of rh-LH has an activity of 99.4 IU.

[0051] Examples 1 to 4: Preparation of rh-LH lyophilized preparations

[0052] The formulas of the rh-LH freeze-dried preparations prepared in Examples 1 to 4 of the present invention are shown in Table 1 below.

[0053] Table 1. Composition of each rh-LH lyophilized preparation

[0054]

[0055] The preparation method of the freeze-dried preparation of formula 1-4 is as follows:

[0056] rh-LH、Na 2 HPO 4 ·2H 2 O、NaH 2 PO 4 ·H 2 O, Tween 20, trehalose, mannitol and glycine and water for injection were added to the container and mixed, stirred to completely dissolve, adjusted to pH 7.2, filtered, and the filtered solution was filled into the glass container containing the lyophilized preparation, the stopper was put in place, and the filled glass container was placed in a stainless steel tray. The tray was loaded into the freeze dryer and the product was freeze-dried using the following freeze drying cycle.

[0057] The freeze-drying process can be carried out according to the following steps:

[0058] Step 1: Pre-freezing: temperature is -40℃~-50℃, time is 5-15h, vacuum degree is 1atm;

[0059] Step 2: Sublimation: temperature is -25℃~-35℃, time is 12-18h, vacuum degree is 8-16pa;

[0060] Step 3: Heating: temperature 10℃~35℃, time 1.5-2.5h, vacuum degree 8-16pa;

[0061] Step 4: Drying: temperature is 20℃~35℃, time is 1-3h, vacuum degree is 8-16pa;

[0062] Freeze drying is complete.

[0063] The lyophilized preparations prepared in Examples 1-4 were stored at a temperature of 25±2°C, and the stability and resolubility of the lyophilized preparations were analyzed and tested immediately after preparation (storage time 0), after storage for 1 month, 3 months, and 6 months. The analytical test results related to the stability and resolubility time of the lyophilized preparations of Examples 1-4 are shown in Tables 2 and 3.

[0064] Stability indicators include:

[0065] 1. Bioactive rh-LH: SE-HPLC determination

[0066] 2. Percentage of oxidation products: measured by reverse phase HPLC (RP-HPLC) method

[0067] 3. Dimer / aggregate percentage: determined by SDS-PAGE

[0068] 4. Percentage of free subunits: determination by non-reduced SDS-PAGE

[0069] High biological activity, few oxidation products, few dimers / aggregates, and few free subunits indicate good stability of the lyophilized preparation. When testing stability, add water for injection to reconstitute the injection solution.

[0070] Resolubility indicators include:

[0071] 1. Redissolution time test: Take 5 bottles of test sample, add 1.0ml sterile water for injection along the bottle wall, start timing immediately and observe the dissolution of the test sample, and gently shake to help dissolution. When the test sample (lyophilized block or powder) is completely dissolved, record the redissolution time (in seconds as the time unit, rounded to the integer). If it exceeds 2 minutes, and there are still substances in the solution that cannot be completely dissolved, it is counted as "insoluble / incompletely dissolved".

[0072] Table 2. Stability results of lyophilized formulations of Examples 1-4

[0073]

[0074] Table 3. Reconstitution time of lyophilized formulations of Examples 1-4

[0075]

[0076] The results in Table 2 and Table 3 illustrate that the rh-LH freeze-dried preparations prepared by Formulas 1-4 of the present invention all have good stability and resolubility. Even after being kept at room temperature for 6 months, the various indicators remain stable and can be quickly resolubilized when used.

[0077] The present invention illustrates the beneficial effects of the preparation of the present invention in detail through the following experiments.

[0078] Experimental Example 1: Screening of Lyoprotectants

[0079] 1. Formulas 5-10 in Table 4 were designed respectively for preparing rh-LH freeze-dried preparations, and the freeze-dried preparations were prepared according to the method of the embodiment of the present invention. The protective effect of trehalose, sucrose, glycine, sorbitol, lysine and mannitol on the product during the freeze-drying process was studied.

[0080] Table 4. Composition of each formulation

[0081]

[0082]

[0083] The lyophilized preparations prepared by formula 5-10 were reconstituted into injection solutions by adding water for injection. The stability of the lyophilized preparations was tested according to the method described in the examples. The results are shown in Table 5.

[0084] Table 5. Stability results of freeze-dried formulations of formula 5-10

[0085]

[0086] From the data in Table 5 above, it can be seen that among trehalose, sucrose, glycine, sorbitol, lysine and mannitol, trehalose and mannitol as protective agents have better protective effects on freeze-dried preparations than sucrose, glycine, sorbitol and lysine, and the freeze-dried preparations prepared have better stability. Therefore, trehalose and mannitol were initially selected as protective agents.

[0087] 2. In the present invention, formulas 11-15 in Table 4 were designed to prepare rh-LH lyophilized preparations, and the lyophilized preparations were prepared according to the method of the embodiments of the present invention. The stability of each lyophilized preparation was tested, the protective effect of the protective agent compound on the lyophilized preparation was investigated, and the reconstitution time of the lyophilized preparation was tested. The lyophilized preparations prepared by formulas 11-15 were reconstituted into injection solutions by adding injection water, and the stability and resolubility of the lyophilized preparations were tested according to the method described in the embodiments, and the results are shown in Table 6.

[0088] Table 6. Stability and reconstitution results of freeze-dried formulations of formulas 11-15

[0089]

[0090] As can be seen from the results in Table 6 above, formula 12 trehalose compounded with glycine has the best stabilizing protection effect. During its storage process, it has high biological activity, low percentage of oxidation products and dimer / aggregate percentages, but it can be seen that its solubility is not good, and it is still not completely dissolved after 2 minutes of re-dissolution. Although the lyophilized preparation prepared by trehalose compounded with mannitol can be re-dissolved, the re-dissolution time is 38-40s, but the stability of the lyophilized preparation is poor. At this time, the lyophilized preparation prepared cannot have both good stability and solubility, and the effect is not good.

[0091] 3. Based on the results of the above-mentioned formula 12 and formula 15, the present invention compounded trehalose, glycine and mannitol as freeze-dried excipients, and screened the compounding ratio of each excipient, and the formula design is shown in Table 7. Rh-LH freeze-dried preparations were prepared according to formulas 16-21 in Table 7, and the freeze-dried preparations were prepared according to the method of the embodiment of the present invention. The freeze-dried preparations prepared by formulas 16-21 were tested for stability and solubility of the freeze-dried preparations according to the method described in the embodiment, and the results are shown in Table 8.

[0092] Table 7. Composition of each formulation

[0093]

[0094] Table 8. Stability and reconstitution results of freeze-dried formulations of formulations 16-21

[0095]

[0096] From the results in Table 8 above, it can be seen that the specific amount of trehalose, mannitol and glycine can ensure both the stability and rehydration of the lyophilized preparation. The ratio of mannitol to glycine is preferably 2-4:1. If the amount of glycine is too much, the resolubility of the lyophilized preparation is poor; if the amount of mannitol is too much, the stability of the lyophilized preparation is poor.

[0097] 4. Control example: 4μgh-LH, 1.11mg Na 2 HPO 4 ·2H2 O, 0.45 mg NaH 2 PO 4 ·H 2 The above formula was prepared into a lyophilized preparation according to the method described in the example, and the stability and solubility of the lyophilized preparation were tested according to the method described in the example. The results are shown in Table 9.

[0098] Table 9. Stability and reconstitution results of the freeze-dried formulations of the control examples

[0099]

[0100] In the control example, lysine was used to replace the glycine of the present invention, and it was found that the stability and resolubility of the prepared lyophilized preparation were both deteriorated.

[0101] 5. The present invention uses the specific formulas in the patents with publication numbers WO2004087213A8, WO2004112826A1 and EP0448146B1, prepares the lyophilized preparation according to the methods in the documents, adds injection water for reconstitution and detects its reconstitution time.

[0102] WO2004087213A8: 65.5 μg recombinant FSH, 18.0 μg recombinant LH, 30.0 mg sucrose, 0.104 mg NaH 2 PO 4 ·H 2 O, 1.65 mg Na 2 HPO 4 ·2H 2 O, 0.10 mg Pluronic F68 and 0.10 mg L-methionine.

[0103] WO2004112826A1: 12.0 μg (165 IU) FSH, 3.7 μg (92 IU) LH, 30.0 mg sucrose, 0.45 mg NaH 2 PO 4 ·H 2 O, 1.11 mg Na 2 HPO 4 ·2H 2 O, 0.05 mg Tween 20, 0.1 mg L-methionine.

[0104] EP0448146B1: 75 IU rFSH, 75 IU LH, 15 mg sodium citrate, 50 mg sucrose and 0.2 mg polysorbate 20.

[0105] The solubility of the lyophilized preparation was tested according to the method described in the examples of the present invention. The results are shown in Table 10.

[0106] Table 10. Reconstitution of similar lyophilized formulations in other patents

[0107]

[0108] It can be seen from Table 10 that the resolubility of the gonadotropin freeze-dried preparation prepared in the prior art is poor. The freeze-dried preparation prepared in the present invention overcomes this problem existing in the prior art.

[0109] In summary, the present invention provides a lyophilized preparation of gonadotropin. The present invention adopts a specific formula to prepare a lyophilized preparation of gonadotropin. The lyophilized preparation has high biological activity, good stability during the lyophilization process and storage process, and short re-dissolution time during use. The lyophilized preparation of the present invention can have good stability and re-dissolution. The present invention is particularly suitable for preparing lyophilized preparations of luteinizing hormone (LH) and its variants, and has good application prospects.

Claims

1. A gonadotropin freeze-dried preparation, characterized in that: It is composed of the following components in parts by weight: 0.04 parts of luteinizing hormone, 11.1 parts of disodium hydrogen phosphate, 4.5 parts of sodium dihydrogen phosphate, 0.5 parts of Tween 20, 200-300 parts of trehalose, 200-300 parts of mannitol, and 50-100 parts of glycine, wherein the weight ratio of mannitol to glycine is 2-4:1; after the components are dissolved, the pH value is adjusted to 7.

2.

2. The lyophilized preparation of gonadotropin according to claim 1, characterized in that: The freeze-drying process comprises the following steps: 1) Prefreezing: temperature is -40℃~-50℃, time is 5-15h, vacuum degree is 1-5atm; 2) Sublimation: temperature is -25℃~-35℃, time is 12-18h, vacuum degree is 8-16pa; 3) Heating: temperature is 10℃~35℃, time is 1.5-2.5h, vacuum degree is 8-16pa; 4) Analytical drying: temperature is 20℃~35℃, time is 1-3h, vacuum degree is 8-16pa.

3. The method for preparing the lyophilized gonadotropin preparation according to claim 1 or 2, characterized in that: It includes the following steps: (1) Weigh each component according to weight; (2) Dissolve each component in water for injection, adjust the pH to 7.2, and filter; (3) freeze drying, wherein the freeze drying process comprises the following steps: 1) pre-freezing: the temperature is -40°C to -50°C, The time is 5-15h, and the vacuum degree is 1-5atm; 2) Sublimation: temperature is -25℃~-35℃, time is 12-18h, and vacuum degree is 8-16pa; 3) Heating: temperature is 10℃~35℃, time is 1.5-2.5h, and vacuum degree is 8-16pa; 4) Desorption drying: temperature is 20℃~35℃, time is 1-3h, and vacuum degree is 8-16pa.

4. Use of the lyophilized preparation of gonadotropin according to claim 1 or 2 in the preparation of a medicament for treating female infertility and / or male infertility.

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