CN100336557C - Somatostation a aqua prepn and its prepn process and application - Google Patents

Abstract

The present invention relates to a water solution preparation of somatostatin, a preparation process and the application of preparing medicine for preventing and treating diseases, such as serious and acute esophageal varix hemorrhage, acute gastric or duodenal ulcer hemorrhage, acute erosive gastritis or hemorrhagic gastritis, postoperative complications of pancreas surgery, etc. The preparation is composed of somatostatin, medicinal auxiliary materials and water, wherein the specification of the somatostatin can be from 0.1 to 1000 mg/pipe; the volume of a unit preparation can be from 0.1 to 500 ml; the auxiliary materials are selected from and are not limited by carbohydrates, such as mannitol, or polyethylene glycol, etc., twenty kinds of human amino acid, such as all kinds of cyclodextrin, glycine, etc., low molecular dextran, poloxamer, buffering salts, such as phosphate, or acetate, etc. The preparation process of the preparation comprises the steps of solution compounding, disinfection, separated packing, tamponage, packing, etc. The water solution preparation has the characteristics of good stability and convenient preparation, and better keeps the structure and the activity of the somatostatin; the water solution preparation also has the characteristic of wide application prospect in preparing medicine for preventing and treating the diseases, such as serious and acute esophageal varix hemorrhage, serious and acute gastric or duodenal ulcer hemorrhage, acute erosive gastritis or hemorrhagic gastritis, the postoperative complications of pancreas surgery, gallbladder or intestinal fistula, diabetes ketoacidosis, etc.

Description

An aqueous solution preparation of somatostatin, its preparation method and application

technical field

The invention relates to the field of medicines and preparations thereof. The core of the invention is to provide a new preparation of somatostatin, its preparation method and its use in the preparation for the prevention and treatment of severe acute esophageal variceal bleeding, severe acute gastric or duodenal ulcer bleeding, acute erosive gastritis or Application in drugs for hemorrhagic gastritis, postoperative complications of pancreatic surgery, biliary or intestinal fistula, diabetic ketoacidosis and other diseases.

Background technique

Somatostatin is a fourteen peptide, its amino acid sequence is as follows:

The molecular formula of somatostatin is C ₇₆ H ₁₀₄ N ₁₈ O ₁₉ S ₂ , and its molecular weight is 1638.

Somatostatin is a cyclic polypeptide hormone secreted by D cells, mainly distributed in the gastrointestinal tract and the central nervous system, of which the gastrointestinal tract accounts for 70%, and its main function is to inhibit the proliferation, secretion and absorption of the gastrointestinal mucosa , Inhibit the release of gastrin, secretin, glucagon and growth hormone, known as the master switch of endocrine hormones. Itztu et al. found D cells in the mucosa of colorectal cancer by immunohistochemistry, and believed that D cells in the colon mucosa may have a local regulatory effect on the secretion of other hormones, such as inhibiting the transcription of gastrin mRNA in G cells, thereby inhibiting gastrin synthesis and release.

Somatostatin can inhibit the secretion of gastric and pancreatic juice, stimulate mucus secretion, reduce portal pressure, relax the biliary sphincter (Oddi sphincter), stimulate the mononuclear macrophage system to reduce endotoxemia, inhibit the release of platelet activating factor, directly Or indirectly regulate cytokine chains to produce cytoprotective effects, etc., and can be used for the treatment of gastrointestinal bleeding, esophageal variceal bleeding, acute pancreatitis and postoperative complications of pancreas, pancreatic, biliary and intestinal fistula. Since somatostatin has a wide range of biological effects, its clinical prospects as a therapeutic drug are very broad.

Somatostatin can inhibit the secretion of gastrin, gastric acid and pepsin, thereby treating upper gastrointestinal bleeding, and can significantly reduce the blood flow of internal organs without causing significant changes in systemic arterial blood pressure. It has certain clinical value.

Somatostatin can also reduce the endocrine and exocrine of the pancreas to prevent and treat complications after pancreatic surgery.

As a polypeptide hormone with multiple physiological functions, somatostatin has been successfully developed into adult drugs, and its indications include: 1. Severe acute esophageal variceal bleeding; 2. Severe acute gastric or duodenal bleeding Intestinal ulcer bleeding, or complicated by acute erosive gastritis or hemorrhagic gastritis; 3. Prevention and treatment of complications after pancreatic surgery; 4. Adjuvant treatment of pancreatic, biliary and intestinal fistula; 5. Adjuvant treatment of diabetic ketoacidosis .

In addition, Itztu et al. used immunohistochemistry to find D cells in the mucosa of colorectal cancer, and believed that it might be related to the occurrence and development of tumors. The mechanism of anti-tumor effect of somatostatin is: somatostatin binds to the specific somatostatin receptor on the cancer cell membrane to produce a direct anti-proliferation effect, including activation of phosphotyrosine phosphatase, inhibition of cAMP and gene transcription, It has an indirect anti-proliferation effect by inhibiting the synthesis, release and action of gastrin, bombesin, epidermal growth factor, etc., and can also inhibit the formation of blood vessels related to tumors. Many literatures have reported the use of somatostatin in the treatment of liver cancer and neuroendocrine tumors such as pancreatic islet tumors.

In summary, somatostatin has very broad application prospects.

Like other polypeptide substances, the preparation of somatostatin is limited by factors such as its stability in aqueous solution. At present, there is only freeze-dried powder injection of somatostatin on the market. This dosage form needs to be dissolved when it is used. While increasing the operation steps, it also increases the chance of drug contamination. The present invention just aims at the current single dosage form of somatostatin, creates the aqueous solution preparation of somatostatin, solves the stability problem of somatostatin in the aqueous solution, and this is the main purpose of the present invention.

Contents of the invention

Design thinking, research content and technical scheme of the present invention:

The main content of the invention includes three parts: preparation process development, stability investigation and pharmacology research. Among them, the stability of somatostatin in the aqueous solution is a problem that needs to be solved emphatically.

According to the requirements of research on aqueous preparations for human use, the solution-type preparation should be a clear, safe, harmless and stable solution. The solvent can be aqueous or non-aqueous, and the most commonly used aqueous solvent is water for injection. According to the characteristics of active drugs, various pharmaceutical excipients can be selected, such as osmotic pressure regulators, solubilizers, antioxidants, cosolvents, pH value regulators, etc. Combining the characteristics of somatostatin, we mainly considered the following auxiliary materials: osmotic pressure regulator, pH value regulator, solubilizer, antioxidant and preservative.

Optional osmotic pressure regulators can be but not limited to inorganic salt compounds such as sodium chloride; optional pH regulators can be but not limited to phosphate buffer, acetate buffer, citric acid buffer, etc.; optional Solubilizers or co-solvents can be, but not limited to, 20 kinds of human amino acids such as glycine and their salts (such as cysteine, leucine, methionine, etc.), various cyclodextrins, glycerin (or sorbitol, polyethylene glycol, etc.) , propylene glycol) and other carbohydrates, various cyclodextrins, low molecular dextran, poloxamers, etc.; optional antioxidants can be but not limited to ethylenediaminetetraacetic acid and its salts, ascorbic acid, citric acid, cysteine amino acid, glutathione, methionine, etc.; optional preservatives can be but not limited to cinnamyl alcohol, formic acid, etc.

A large number of orthogonal tests have shown that the above-mentioned excipients have a good stabilizing effect on somatostatin. Among them, the preferred pH regulator is phosphate buffer, the preferred solubilizer is cyclodextrin compound, the preferred osmotic pressure regulator is sodium chloride, and the preferred antioxidant is disodium EDTA.

In terms of dosage composition of somatostatin and auxiliary materials in the prescription, somatostatin can be 0.1-1000 mg/bottle, such as 0.5, 1, 2, 5, 10, 20, 50, 100, 250, 500 mg/bottle. The volume of the unit preparation may be 0.1-500ml.

A preferred prescription is: 0.25-250mg somatostatin, 5-50mmol/L phosphate buffer, 0.5-10mmol/L disodium ethylenediaminetetraacetic acid (EDTA), 0.5-5% sodium chloride. A more preferred solution is: 0.25-10 mg somatostatin, 10-30 mmol/L phosphate buffer, 0.5-5 mmol/L disodium ethylenediaminetetraacetic acid (EDTA), 0.5-2% sodium chloride. The most preferred scheme is: 3mg somatostatin, 20mmol/L phosphate buffer, 2mmol/L disodium ethylenediaminetetraacetic acid (EDTA), 0.7% sodium chloride, and the preparation specification is 3mg: 1ml/branch.

Another preferred prescription scheme is: 0.25-250mg somatostatin, 5-50mmol/L phosphate buffer, 0.5-10mmol/L disodium ethylenediaminetetraacetic acid (EDTA), 0.5-5% sodium chloride, 0.1-10% Hydroxypropyl Beta Cyclodextrin. On this basis, the more preferred component scheme is: 0.25-10mg somatostatin, 10-30mmol/L phosphate buffer, 0.5-5mmol/L disodium ethylenediaminetetraacetic acid (EDTA), 0.5-2% sodium chloride, 0.5-5% hydroxypropyl beta cyclodextrin. The most preferred component scheme is: 3mg somatostatin, 20mmol/L phosphate buffer, 2mmol/L disodium ethylenediamine tetraacetate (EDTA), 0.7% sodium chloride, 2% hydroxypropyl beta cycloglutamate Essence, preparation specification is 3mg: 1ml/branch.

Above-mentioned preparation prescription can be prepared by following process:

(1) Take the required sodium chloride, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and disodium ethylenediaminetetraacetate by weighing the required amount of feed under 100-level laminar flow, and pack into a suitable container, Add calculated amount of sterile water for injection to dissolve and use immediately.

(2) Add the prescribed amount of somatostatin into the buffer, dissolve and shake well, remove the pyrogen by ultrafiltration through the ultrafiltration column, and then filter and sterilize with a high-pressure steam sterilized filter (0.22 μm, 0.45 μm double-layer filter membrane) , sealed, ready to pack.

(3) Use sterile water for injection to adjust the dosage by 1ml per bottle before subpackaging. After full stoppering, the medicine bottle is sent to the upper bottle track for capping.

(4) Start the capping machine, the medicine bottle enters the track with the turntable, the aluminum cap is added to the medicine bottle through the track, and is locked by the machine head. After capping, the aluminum cap should be tight and the edges should be neat.

So far, our invention has completed the design and research of the preparation process of the preparation, and began to investigate the stability of the preparation. The sample used in the experiment is from the product prepared in Example 1.

According to the corresponding research requirements, we mainly investigated the appearance, pH value, clarity and color of the solution, related substances, content, clarity inspection, bacterial endotoxin and sterility in the stability study. Influencing factor tests, accelerated tests and long-term tests were mainly carried out.

The main factors to be investigated in the influence factor test are high temperature and strong light irradiation.

Take a batch of this product and place it under the conditions of light (4000Lx) and high temperature (40°C) respectively, and take samples for analysis on 5 and 10 days to investigate the properties, related peptides, and content. The results showed that the pH value of this product decreased significantly after being exposed to light, the related substances increased, and the content decreased significantly, so this product should be stored in shading. The test results at high temperature of 40°C show that after 10 days at high temperature of 40°C, the pH value of this product decreases, the related substances increase, and the content decreases. The results are shown in Table 1 and Table 2.

Table 1 Lighting Test Stability Investigation Results batch number time (days) investigation Appearance traits pH value Clarity Clarity and Color relative substance(%) content(%) 01 0 Colorless and clear 7.01 Compliance clear colorless 0.25 100.0 5 Colorless and clear 5.65 Compliance clear colorless 0.59 85.33 10 Colorless and clear 4.89 Compliance clear colorless 1.36 77.65

Table 2 High Temperature Test Stability Investigation Results (40°C) batch number time (days) investigation Appearance traits pH value Clarity Clarity and color of the solution relative substance(%) content(%) 01 0 Colorless and clear 7.01 Compliance clear colorless 0.27 100.0 5 Colorless and clear 6.54 Compliance clear colorless 0.66 89.67 10 Colorless and clear 5.68 Compliance clear colorless 1.91 78.65

Accelerated test: Place the product in an environment with a temperature of 25°C±2°C and a RH of 60±10% for 6 months, and take samples at the end of the first month, second month, third month, and sixth month, according to Stability key inspection items are tested. The test results are shown in Table 3.

Table 3 Accelerated test results (25°C±2°C, RH60±10%) batch number moon investigation Appearance traits pH value Clarity relative substance(%) endotoxin sterile content(%) 01 0 Colorless and clear 7.00 Compliance 0.28 Compliance Compliance 100.0 1 Colorless and clear 6.36 Compliance 5.69 - - 92.49 2 cloudy solution 5.57 cloudy solution 10.65 - - 85.68 3 cloudy solution 5.16 cloudy solution 14.67 - - 79.66 6 cloudy solution 4.87 cloudy solution 20.63 - - 71.25

Accelerated test results show that the somatostatin aqueous solution is placed under these conditions for 6 months, the pH value decreases, the related substances increase, and the content decreases, indicating that this product is a preparation that is particularly sensitive to temperature and needs to be stored in a cold place.

Long-term test: place the product at a temperature of 2°C-10°C, take a sample every 3 months, and test according to the key stability inspection items at 0 month, 3 months, 6 months, and 9 months respectively. The test results are shown in Table 4.

Table 4 Long-term test investigation results (2°C-10°C) batch number moon investigation Appearance traits Clarity and Color pH value Clarity relative substance(%) endotoxin sterile content(%) 02 0 Colorless and clear clear colorless 7.02 Compliance 0.23 Compliance Compliance 100.0 3 Colorless and clear clear colorless 7.02 Compliance 0.24 Compliance Compliance 100.0 6 Colorless and clear clear colorless 7.03 Compliance 0.31 Compliance Compliance 100.0 9 Colorless and clear clear colorless 7.03 Compliance 0.30 Compliance Compliance 100.0 03 0 Colorless and clear clear colorless 7.01 Compliance 0.25 Compliance Compliance 100.0 3 Colorless and clear clear colorless 7.01 Compliance 0.26 Compliance Compliance 100.0 6 Colorless and clear clear colorless 7.05 Compliance 0.33 Compliance Compliance 100.0 9 Colorless and clear clear colorless 7.04 Compliance 0.32 Compliance Compliance 100.0

The results showed that the somatostatin aqueous solution was placed under the long-term test conditions for 6 months, and all the indicators did not change significantly, indicating that the product has good stability.

Stability test conclusion: The test results of influencing factors show that the pH value of the somatostatin aqueous solution drops after being placed under the condition of light or high temperature of 40°C, the content drops, and the related substances increase; under the accelerated test conditions (25°C±2°C, RH 60± 10%) for 6 months, the pH value decreased, the content decreased, and the related substances increased; under the long-term test conditions (2°C-10°C,) for 6 months, all the indicators had no obvious change. It is determined that this product should be stored in a shading and cool place (2-10°C). The content detection liquid chromatogram of the 02 batch samples at 9 months is shown in the accompanying drawing 1 of the description.

Another part of the present invention is to investigate the pharmacology of said aqueous injection formulations. The main purpose of pharmacological research is to compare the biological activity of this dosage form with the existing somatostatin freeze-dried powder injection dosage form. By comparing the activity of the preparation described in the invention with the commercially available product, the equivalence between the preparation and the existing preparation in the treatment of somatostatin indications can be deduced, and the practicability of the preparation can be determined accordingly. For this we used the Pancreatitis Treatment Trial.

Therapeutic effect of somatostatin on acute pancreatitis:

Apparatus, Animals and Reagents:

40 SD rats, TNFα radioimmunoassay kit, micro-infusion pump, bombesin, etc.

Drug and serial number:

The No. 01 sample prepared in the embodiment of the present invention, somatostatin freeze-dried powder (trade name Stanning).

Test group:

40 healthy male SD rats were provided by the Animal Center of the Academy of Military Medical Sciences, with a body mass of (250±20) g. They were randomly divided into 4 groups, 10 rats in each group: negative control group, model group, No. 01 sample treatment group, and stanine treatment group.

Experimental steps:

The animals were kept in the laboratory for 1 week to adapt to the environment. Fasting 12h before the experiment, free to drink water. All experimental animals were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg), and subcutaneously injected with bombesin 10 μg/kg.h, 5 times in total. The normal control group was subcutaneously injected with the same amount of normal saline. The two treatment groups were intubated through the external jugular vein, and at the same time as acute pancreatitis was induced, somatostatin was infused intravenously. The first dose was 10 μg/kg, followed by continuous intravenous infusion of 10 μg/kg.h for a total of 7 hours. Animals were sacrificed 10 hours after the start of the experiment, blood was collected from the heart, serum was separated, and stored in a -70°C refrigerator to be tested for TNFα. The abdominal cavity was opened, the pancreas was taken out, the wet mass was weighed, and the pancreas/body mass value (MP/MB) was calculated. TNFα was performed according to the operating instructions of the radioimmunoassay kit. The experimental results were expressed as mean ± standard deviation, and the significant difference between groups was tested by T test. , the results are shown in Table 5.

Table 5. Therapeutic effect of somatostatin on acute pancreatitis (mean ± standard deviation, n=6) Pancreas (g)/body mass (kg) ratio TNFα(ρB/ng ml ^-1 ) negative control 2.44±0.45 ^** 1.27±0.28 ^** model group 5.42±0.73 2.36±0.19 Sample No. 01 3.67±0.32 ^** 1.75±0.14 ^** Stanning 3.86±0.75 ^** 1.74±0.17 ^**

Note: ^** means p<0.01 compared with the model group.

The results show that the somatostatin aqueous solution provided by the invention has the same therapeutic effect as the traditional freeze-dried powder injection, and there is no statistical difference between the two treatment groups.

In summary, the present invention provides a new dosage form of somatostatin. The production process of this dosage form is simpler than the existing freeze-dried powder injection production process, and it is easy to scale up in production, has obvious advantages, and is convenient for the realization of industrialization; at the same time, In terms of biological activity and preparation stability, the dosage form described in the claims and instructions has good stability and can be stored for a long time, and its activity is the same as that of the existing freeze-dried powder injection. Therefore, the present invention well embodies the patent elements of novelty, creativity and practicality.

Detailed ways

In order to better illustrate the implementation process of this patent, we list the following examples. It should be noted that the examples listed are one of the concrete manifestations of the idea and spirit of the present invention, and are not intended to limit the scope of the content of the invention patent description, nor to refine the claims. Under the thinking of the present invention, any aqueous solution product (injection, spray, drop, etc.) of somatostatin described in this patent should be included in the scope of this patent.

The preparation of the pharmaceutical preparation described in embodiment 1, claim 7

Accurately weighed sodium chloride 7.0g, disodium hydrogen phosphate (containing 12 crystal water) 4.37g, sodium dihydrogen phosphate (containing 2 crystal water) 1.22g, edetate disodium 1.28g, somatostatin 3.0g , dissolved in water for injection, finally dissolved to 1000ml, and divided into 1000 vials with 2ml. The final specification is 3mg/support. According to this prescription, a total of 3 batches of samples were produced, numbered 01, 02, and 03 respectively. The operating procedures are carried out as described in the manual.

The preparation of the pharmaceutical preparation described in embodiment 2, claim 10

Accurately weighed sodium chloride 7.0g, disodium hydrogen phosphate (containing 12 crystal water) 4.37g, sodium dihydrogen phosphate (containing 2 crystal water) 1.22g, edetate disodium 1.28g, somatostatin 3.0g 1. Hydroxypropyl β-cyclodextrin 20g, dissolved in water for injection, finally dissolved to 1000ml, subpacked into 1000 sticks with 2ml vials. The final specification is 3mg/support. According to this prescription, a total of 1 batch of samples was produced, numbered 04. The operating procedures are carried out as described in the manual.

Claims (5)

1, a kind of aqueous solution preparation of somatostatin, it is mainly formed: the disodiumedetate of 0.25-10mg somatostatin, 10-30mmol/L phosphate buffer, 0.5-5mmol/L, the sodium chloride of 0.5-2% are prepared into the preparation that unit volume is 0.1-500ml.

2, preparation as claimed in claim 1, it is mainly formed and is: the disodiumedetate of 3mg somatostatin, 20mmol/L phosphate buffer, 2mmol/L, 0.7% sodium chloride, being prepared into specification is the preparation that 3mg: 1ml/ props up.

3, preparation as claimed in claim 1, it is mainly formed: the disodiumedetate of 0.25-10mg somatostatin, 10-30mmol/L phosphate buffer, 0.5-5mmol/L, the sodium chloride of 0.5-2%, 0.5-5% hydroxypropyl beta cyclodextrin are prepared into the preparation that unit volume is 0.1-500ml.

4, preparation as claimed in claim 3, it is mainly formed: the disodiumedetate of 3mg somatostatin, 20mmol/L phosphate buffer, 2mmol/L, 0.7% sodium chloride, 2% hydroxypropyl beta cyclodextrin, being prepared into specification is the preparation that 3mg: 1ml/ props up.

5, as any described preparation of claim 1-4 preparation be used for preventing and treat that the serious acute esophageal varix is hemorrhage, the application of the medicine of serious acute stomach or duodenal ulcer and hemorrhage, acute erosive gastritis or hemorrhagic gastritis, pancreas surgical postoperative complication, gallbladder or intestinal fistula, diabetic ketoacidosis disease.

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