CN108659104B

CN108659104B - Preparation method of terlipressin and pharmaceutical composition thereof

Info

Publication number: CN108659104B
Application number: CN201810707079.XA
Authority: CN
Inventors: 宋雪萍
Original assignee: Beijing Xinli Pharmaceutical Technology Co Ltd
Current assignee: Beijing Xinli Pharmaceutical Technology Co., Ltd
Priority date: 2018-07-03
Filing date: 2018-07-03
Publication date: 2020-06-09
Anticipated expiration: 2038-07-03
Also published as: CN108659104A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of terlipressin and a pharmaceutical composition thereof; the preparation method adopts two different preparation liquid phases to obtain terlipressin; the method for purifying terlipressin provided by the invention is simple and easy to operate, the purity of the obtained terlipressin can reach more than 99.7%, and the yield can reach more than 60%. The freeze-dried powder injection prepared by the composition has good solubility, good stability and good redissolution property; the freeze-dried powder injection prepared by the composition has the advantages of convenient use, easy storage and transportation, simple preparation method, easy industrial production and low production cost.

Description

Preparation method of terlipressin and pharmaceutical composition thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of terlipressin and a pharmaceutical composition containing the terlipressin.

Background

Terlipressin (Terlipessin, also called thymosin acetate α 1) with chemical name of N- α -triglycyl-8-lysine-vasopressin and structural formula as follows, and molecular formula as C₅₂H₇₄N16O₁₅S₂The relative molecular mass was 1227.37, and the powder was white powder.

The terlipressin is a novel artificially synthesized long-acting vasopressin preparation, belongs to a prodrug, has no activity, and slowly releases active lysine vasopressin after 3 glycyl residues at the N terminal of the terlipressin are removed by the action of aminopeptidase in vivo. It is this "slow release" mechanism that maintains smooth muscle contraction for up to 10 hours after a single administration, whereas vasopressin activity is maintained for only 20-40 minutes at equivalent doses. On the other hand, because the enzymolysis speed is slow, the circulating lysine vasopressin can not reach the toxic level, so the use of the terlipressin is safer.

The pharmacological action of terlipressin is to shrink the smooth muscle of the blood vessel of the internal organ, reduce the blood flow of the internal organ (such as reducing the blood flow of mesentery, spleen, uterus and the like), thereby reducing the blood flow of portal vein and reducing the portal vein pressure, and on the other hand, the terlipressin also has the action of reducing the concentration of plasma renin, thereby increasing the blood flow of the kidney of the patients with hepatorenal syndrome, improving the kidney function and increasing the urine volume. Terlipressin is the only drug capable of improving the survival rate of patients with esophageal variceal bleeding at present, is mainly used for treating variceal bleeding, and is widely used for treating hepatorenal syndrome, ascites due to cirrhosis, septic shock, burns, acute liver failure, sudden cardiac arrest and the like clinically. Compared with vasopressin, it has lasting effect, no dangerous complications including fibrinolysis and serious complications in the cardiovascular system, simple use method (intravenous injection is available), and is more suitable for the rescue and treatment of critically ill patients.

The terlipressin is a polypeptide, is easy to degrade under the conditions of acid, alkali, illumination, oxidation and high temperature, has low purity and yield of the terlipressin prepared at present, can not meet the requirement of industrial production, particularly can not purify the terlipressin on a large scale, and restricts the application of the terlipressin. Therefore, it is of great significance to provide a method for purifying terlipressin.

Disclosure of Invention

In order to solve the problems in the prior art, a great deal of experimental research is carried out, and a novel method for purifying terlipressin acetate is found, so that the terlipressin acetate obtained by the method has higher purity. Another object of the present invention is to provide a terlipressin acetate pharmaceutical composition and a method for preparing the same, wherein the preparation obtained by the method has more excellent product quality.

The invention is realized by the following technical scheme:

a preparation method of terlipressin comprises the following steps:

step 1, dissolving a terlipressin acetate crude product by using an acetic acid aqueous solution with the mass concentration of 1-5%, filtering and collecting filtrate, and then diluting acetic acid in the terlipressin crude product solution by using water to the mass concentration of below 0.5%;

step 2, taking octadecylsilane as a stationary phase, taking a terlipressin crude product solution obtained in the step 1 as a sample, taking a phosphate buffer solution containing 35-45% of methanol by mass concentration as a phase A, taking a methanol water solution with 80-95% of mass concentration as a phase B, carrying out linear gradient elution, collecting a characteristic peak eluent, filtering the eluent by using a filter membrane, and concentrating to a relative density of 1.05-1.08 at 25 ℃;

and 3, taking the octaalkylsilane bonded silica as a stationary phase, after the concentrated solution obtained in the step 2 is sampled, firstly washing the concentrated solution with 80% methanol aqueous solution of ammonium acetate with the mass concentration of 0.5-1.6% for 30-45 min, then eluting the concentrated solution with 70% acetonitrile aqueous solution of ammonium acetate with the mass concentration of 0.05-0.1%, collecting characteristic peak eluent, concentrating and drying the characteristic peak eluent to obtain the terlipressin acetate.

In the step 1, the crude terlipressin is dissolved by using an acetic acid aqueous solution with the mass concentration of 1-5%, and 1L of acetic acid aqueous solution with the mass concentration of 1-5% is added into each 100g of the crude terlipressin.

The pH value of the phosphate buffer solution in the step 2 is 5.0-7.0.

4. A pharmaceutical composition containing terlipressin comprises terlipressin acetate, excipient, sodium acetate and glacial acetic acid, and is characterized in that the excipient is a mixture consisting of polydextrose, mannitol and trisodium citrate.

The composition comprises the following components in parts by weight: 0.8-1 part by weight of terlipressin acetate, 30-50 parts by weight of excipient, sodium acetate and glacial acetic acid.

The polydextrose in the vehicle is spray dried polydextrose.

Polydextrose in the vehicle: mannitol: the weight ratio of trisodium citrate is (3-5): 1: (0.1-0.3).

The pH value of the composition is 4.5-6.0.

A method for preparing a freeze-dried powder injection from a pharmaceutical composition containing terlipressin comprises the following steps:

1) dissolving excipient in 2/3 prescription amount of water for injection, and stirring;

2) preparing acetic acid-sodium acetate buffer solution by using glacial acetic acid and sodium acetate for later use;

3) and (3) cooling the prepared solution to room temperature, adding terlipressin acetate, measuring the pH value, adjusting the pH value to 4.5-6.0 by using an acetic acid-sodium acetate buffer solution, adding water for injection to full volume, finely filtering by using a 0.22 mu m microporous membrane, and freeze-drying to obtain the freeze-dried powder injection.

Compared with the prior art, the invention has the following advantages and positive effects:

1. the method for purifying terlipressin provided by the invention is simple and easy to operate, the purity of the obtained terlipressin can reach more than 99.7%, and the yield can reach more than 60%.

2. The freeze-dried powder injection prepared by the composition has good stability and good redissolution property; the freeze-dried powder injection prepared by the composition has the advantages of convenient use, easy storage and transportation, simple preparation method, easy industrial production and low production cost.

Detailed Description

The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.

The crude terlipressin acetate is purchased from Jianyuan medicine science and technology Limited company in Shenzhen, and the HPLC purity of the crude terlipressin acetate is detected to be 92.02%.

The preparation method of the acetic acid-sodium acetate buffer solution comprises the following steps: according to the formula pH 4.75+ lg [ C ]_(NaAc)/C_(HAc)](C_(HAc)Means the concentration of acetic acid, C_(NaAc)The concentration of sodium acetate) to calculate the usage amount of acetic acid and sodium acetate, then adding the weighed sodium acetate and glacial acetic acid into 1000mL of water to dissolve, and stirring uniformly. For example, if a buffer with a pH of 5.5 is pre-configured, it will beSubstitution thereof gives C_(NaAc)/C_(HAc)5.6. Such as C_(HAc)0.1mol/L, then C_(NaAc)Sodium acetate solid mass 82 × 0.56 × 46 g was weighed 0.56mol/L, glacial acetic acid volume 0.1 × 1000/17.5 × 5.7mL was measured.

Example 1

Step 1: dissolving 1000g of a terlipressin acetate crude product in 10L of an acetic acid aqueous solution with the mass concentration of 5%, filtering and collecting filtrate, and then diluting the acetic acid in the crude product solution to 0.45% by mass concentration with water;

step 2: taking octadecylsilane as a stationary phase, sampling the crude product solution obtained in the step 1, taking disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH6.9) containing 35% methanol by mass as a phase A, taking 60% methanol aqueous solution by mass as a phase B, eluting at the flow rate of 105ml/min for 350min and the gradient of 40% B → 70% B, collecting eluent with the purity of more than 95%, filtering the eluent by using a 0.22 mu m filter membrane, and concentrating to the relative density of 1.05-1.08 at the temperature of 25 ℃;

and step 3: and (2) taking octaalkylsilane bonded silica as a stationary phase, after the solution obtained in the step (2) is loaded, firstly washing for 45min by using 80% methanol aqueous solution of ammonium acetate with the mass concentration of 1%, then eluting by using 70% acetonitrile aqueous solution of ammonium acetate with the mass concentration of 0.1%, collecting eluent with the purity of more than 99%, concentrating and drying to obtain 643.2g of terlipressin acetate with the purity of 99.93%, wherein the total purification yield is 64.32%.

Example 2

Step 1: dissolving 100g of a terlipressin peak crude product by 1000ml of acetic acid aqueous solution with the mass concentration of 3%, filtering and collecting filtrate, and then diluting the acetic acid in the crude product solution to the mass concentration of 0.45% by using water;

step 2: taking octadecylsilane as a stationary phase, sampling the crude product solution obtained in the step 1, taking a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH5.5) containing 40% by mass of methanol as a phase A, taking a 90% by mass of methanol aqueous solution as a phase B, eluting at the flow rate of 90ml/min for 40min and the gradient of 40% B → 70% B, collecting an eluent with the purity of more than 95%, filtering the eluent by using a 0.22 mu m filter membrane, and concentrating to the relative density of 1.05-1.08 at the temperature of 25 ℃;

and step 3: and (2) taking octaalkylsilane bonded silica as a stationary phase, after the solution obtained in the step (2) is loaded, firstly washing the solution for 30min by using 80% methanol aqueous solution of ammonium acetate with the mass concentration of 1.6%, then eluting the solution by using 70% acetonitrile aqueous solution of ammonium acetate with the mass concentration of 0.05%, collecting eluent with the purity of more than 99%, concentrating and drying the eluent to obtain 63.21g of terlipressin acetate with the purity of 99.81%, wherein the total purification yield is 63.21%.

Example 3

Step 1: dissolving 50g of a terlipressin acetate crude product by 500ml of acetic acid aqueous solution with the mass concentration of 1%, filtering and collecting filtrate, and then diluting the acetic acid in the crude product solution to 0.5% of mass concentration by using water;

step 2: taking octadecylsilane as a stationary phase, sampling the crude product solution obtained in the step 1, taking disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH6.5) containing 45% methanol by mass as a phase A, taking 80% methanol aqueous solution by mass as a phase B, eluting at the flow rate of 75ml/min for 25min and the gradient of 40% B → 70% B, collecting eluent with the purity of more than 95%, filtering the eluent by using a 0.22-micrometer filter membrane, and concentrating;

and step 3: and (2) taking octaalkylsilane bonded silica as a stationary phase, after the solution obtained in the step (2) is loaded, firstly washing the solution for 30min by using an 80% methanol aqueous solution of ammonium acetate with the mass concentration of 1.6%, then eluting the solution by using a 70% acetonitrile aqueous solution of ammonium acetate with the mass concentration of 0.05%, collecting an eluent with the purity of more than 99%, concentrating and drying the eluent to obtain 34.52g of terlipressin acetate with the purity of 99.81%, wherein the total purification yield is 69.04%.

The method of the above example is chromatography, and the detection wavelength is 220 nm.

Test example 1:

taking appropriate amount of related substances, and preparing into solution containing about 0.2mg per 1ml with water as test solution; 1ml of the solution was measured precisely, placed in a 100ml measuring flask, diluted to the mark with water, and shaken up to give a control solution. Precisely measuring 1ml of the control solution, placing the control solution in a 50ml measuring flask, diluting the control solution to a scale with the mobile phase, and shaking up to obtain the sensitivity solution. According to the chromatographic condition under the content determination item, 20 mul of the sensitive solution is injected into a liquid chromatograph, and the signal-to-noise ratio of the terlipressin peak is more than 10. Precisely measuring 20 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording chromatogram.

The content measurement is performed by high performance liquid chromatography (2015 version of Chinese pharmacopoeia). Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability tests; the mobile phase A is 0.067mol/L sodium dihydrogen phosphate (pH value is adjusted to 3.5 by phosphoric acid), the mobile phase B is acetonitrile, and gradient elution is carried out according to the table 1; the detection wavelength is 210 nm; the column temperature was 28 ℃. Taking a proper amount of terlipressin acetate reference substance and desglycine terlipressin reference substance, preparing a mixed solution containing about 0.1mg of terlipressin and desglycine terlipressin in each 1ml by using water, taking 20 mu l of the mixed solution as a system applicable solution, injecting the mixed solution into a liquid chromatograph, wherein the number of theoretical plates is not less than 2000 calculated according to a terlipressin peak, and the separation degree of the terlipressin peak and the desglycine terlipressin peak is not less than 1.0.

The determination method comprises precisely weighing appropriate amount of the product, dissolving in water, diluting to obtain solution containing terlipressin 0.2mg per 1ml, precisely weighing 20 μ l, injecting into liquid chromatograph, and recording chromatogram; and taking a proper amount of terlipressin acetate reference substance, and measuring by the same method. Calculating according to the peak area by an external standard method to obtain the product. Results are shown in Table 2.

TABLE 1 gradient elution

Time (minutes)	Mobile phase A	Mobile phase B (%)
			0	92	8
4	92	8
			18	85	15
30	80	20
			30.1	92	8
40	92	8

Table 2 sample impurity comparison

Sample (I)	Total related substances (%)	Single maximum impurity (%)	Number of impurities
				Example 1	0.07	0.03	3
Example 2	0.08	0.03	3
				Example 3	0.09	0.04	4
Raw materials	6.45	0.98	14

And (4) conclusion: the results show that the total related substances of the terlipressin acetate prepared by the invention are less than 0.1 percent, and the single maximum impurity is less than 0.05 percent, which are all superior to the raw materials.

Example 4

Under clean conditions, 41g of polydextrose, 8.2g of mannitol and 0.82g of trisodium citrate were put into a liquid preparation apparatus, 700ml of water for injection was added and stirred to dissolve the components, 1g of terlipressin acetate (prepared by the method described in example 1) was added after the above preparation liquid was cooled to room temperature, the pH was measured, the pH was adjusted to 5.2 with an acetic acid-sodium acetate buffer (0.1M, pH 4.5), water for injection was added to the full volume, and the filtrate was filtered through a 0.22 μ M microfiltration membrane to obtain terlipressin acetate filtrate. Subpackaging 3ml in an ampere bottle, and freeze-drying for 72 hours to prepare the sterile freeze-dried powder injection.

Example 5

Under clean conditions, 34.8g of polydextrose, 11.6g of mannitol and 3.48g of trisodium citrate were put into a preparation apparatus, 700ml of water for injection was added and dissolved by stirring, and after the above preparation liquid was cooled to room temperature, 1g of terlipressin acetate (prepared by the method described in example 3) was added, the pH was measured, the pH was adjusted to 5.1 with an acetic acid-sodium acetate buffer (0.1M, pH 4.5), water for injection was added to the full volume, and the filtrate was filtered through a 0.22 μ M microfiltration membrane to obtain terlipressin acetate filtrate. Subpackaging 3ml in an ampere bottle, and freeze-drying for 72 hours to prepare the sterile freeze-dried powder injection.

Example 6

30.8g of polydextrose, 7.7g of mannitol and 1.54g of trisodium citrate are put into a liquid preparation device under clean conditions, 700ml of water for injection is added and stirred to dissolve the mixture, 1g of terlipressin acetate (prepared by the method described in example 2) is added after the above preparation liquid is cooled to room temperature, the pH value is measured, acetic acid-sodium acetate buffer (0.1M, pH 4.5) is added to 5.0, water for injection is added to the total amount, and the filtrate is filtered through a 0.22 mu M microfiltration membrane to obtain terlipressin acetate filtrate. Subpackaging 3ml in an ampere bottle, and freeze-drying for 72 hours to prepare the sterile freeze-dried powder injection.

Example 7

28.5g of polydextrose, 7.5g of mannitol and 1.50g of trisodium citrate were put into a preparation device under clean conditions, 700ml of water for injection was added and dissolved by stirring, and after the preparation liquid was cooled to room temperature, 1.0g of terlipressin acetate (prepared by the method described in example 1) was added, the pH was measured, the pH was adjusted to 4.9 with an acetic acid-sodium acetate buffer (0.1M, pH 4.5), water for injection was added to the full volume, and the filtrate was filtered through a 0.22 μ M microfiltration membrane to obtain terlipressin acetate filtrate. Subpackaging 3ml in an ampere bottle, and freeze-drying for 72 hours to prepare the sterile freeze-dried powder injection.

Prescription screening test

1. Screening of formulation buffer systems

Acetic acid-sodium acetate buffer (0.1M, pH 4.5), citric acid-sodium citrate buffer (0.1M, pH 4.8), disodium hydrogen phosphate-citric acid buffer (0.1M, pH 7.0) were prepared. The solubility of terlipressin in each buffer solution is determined by the following steps: weighing a proper amount of sample, placing the sample in a certain amount of solution at 25 +/-1 ℃, strongly oscillating the solution for 30 seconds every 5 minutes, observing the dissolution condition within 30 minutes, regarding the sample as dissolved when solute particles are not visible, and selecting water, acetonitrile, DMSO and normal saline as solvents. The test results are shown in Table 3, and the preparation solution is preferably acetic acid-sodium acetate buffer solution, judged by the degree of solubility.

TABLE 3 solubility of terlipressin acetate in buffer

Buffer solution	Solubility (mg/ml)
		Acetic acid-sodium acetate buffer	＞50
Citric acid-sodium citrate buffer	～0
		Disodium hydrogen phosphate-citric acid buffer	～10

2. Stability analysis in acetate-sodium acetate buffer

Preparing acetic acid-sodium acetate buffer solution (0.1M, pH 4.5), weighing a certain amount of terlipressin acetate to be dissolved into the buffer solution to form a solution with the concentration of 1mg/ml, placing the solution in an incubator at 37 ℃ for incubation, and carrying out HPLC (high performance liquid chromatography) detection on the purity of the terlipressin in the solution for 0, 1, 2, 4, 8 and 12 hours, wherein the test result is shown in Table 4. The preparation solution is preferably an acetic acid-sodium acetate buffer as a buffer solution from the viewpoint of stability.

Table 4 purity of terlipressin in buffer (%)

Time points (hours)	Acetic acid-sodium acetate buffer
		0	99.82
1	99.82
		2	99.76
4	99.68
		8	99.67
12	99.63

3. Screening of excipients in formulation solutions

An acetic acid-sodium acetate buffer (0.1M, pH 4.5) containing terlipressin acetate at 1mg/ml was prepared. And contains 4% (W/V) of mannitol, polydextrose and glycine (weight ratio 1:1), sorbitol, and lactose as excipients, respectively. Placing the solution in an incubator at 37 ℃ for incubation, and detecting the stability of the terlipressin acetate in the solution at each time point. And freeze-drying (freeze-drying procedure: pre-freezing at-40 ℃ for 4 hours, vacuumizing to 300mT, setting temperature-raising procedure of 1 ℃/min to-20 ℃, freeze-drying for 12 hours, setting temperature-raising procedure of 1 ℃/min to 30 ℃, drying for 4 hours, and plugging and capping) to observe the appearance of the freeze-dried preparation, wherein the test results are shown in Table 5. The formulation solution is preferably a combination of mannitol or polydextrose with glycine as an excipient, as judged from the stability and lyophilized appearance.

TABLE 5 purity (%) and lyophilized appearance of terlipressin in buffer containing excipient formulation solution

4. Screening of excipients in formulation solutions

An acetic acid-sodium acetate buffer (0.1M, pH 4.5) containing terlipressin acetate 0, 0.1, 0.25, 0.5, 1.0, 1.6, 3.2, or 6.0mg/ml was prepared. And each contained 4% (W/V) glycine, a composition of polydextrose and mannitol, sorbitol, lactose as excipients. At the same time, a preparation solution without excipients is prepared. And (4) taking a glass bottle, subpackaging the preparation solution according to 1 ml/bottle, and freeze-drying. Reconstitution of the lyophilized form of the pharmaceutical preparation was examined. The test results are shown in Table 6.

TABLE 6 clarity of lyophilized formulations when different excipients are included in the formulation solution, after reconstitution

Note: <1 indicates that the turbidity is less than that of the No. 1 standard solution; standard solution with turbidity greater than No. 1

The test result shows that solute molecules such as excipients such as glycine, polydextrose or sorbitol are introduced, the concentration range of the terlipressin acetate in the drug preparation solution for preparing the soluble and unfrozen dry preparation is only increased from 0-0.1 mg/ml to 0-0.25 mg/ml, and the concentration of the terlipressin acetate in the lyophilized re-soluble drug preparation is slightly increased; the introduction of some excipients (e.g. lactose) has little effect on increasing the concentration of terlipressin in a reconstituted pharmaceutical formulation after lyophilization.

5. Screening of excipients in formulation solutions

Preparing preparation solution, wherein acetic acid-sodium acetate buffer solution (0.1M, pH 4.5), polydextrose, glycine, trisodium citrate composition, and terlipressin acetate 1mg/ml in different weight ratio are mixed with water. The pH value was measured by using a PB-10 type pH meter (Sartorius, USA) according to the requirement of pH value measurement in pharmacopoeia 2015 edition of the people's republic of China. The solubility of terlipressin in the formulation solution was examined. The formulation solution was filled into 2ml glass freeze-drying bottles, 1 ml/bottle, and transferred to a cryo-freeze dryer (Supermodulyo type, 0.4m2, E-CApparatus Co.) product cabinet for freeze-drying. The lyophilized preparation was reconstituted with 1ml of water for injection, and the reconstitution of the lyophilized form of the pharmaceutical preparation was examined. And (3) putting the freeze-dried preparation into an incubator at 37 ℃, re-dissolving the freeze-dried preparation with 1ml of injection water in each bottle after 8 weeks of incubation bath, and detecting the purity of the terlipressin acetate in the preparation solution. The pH of solutions of polydextrose and mannitol, trisodium citrate in various weight ratios, the appearance and reconstitution of the lyophilized formulations, and the purity of terlipressin acetate after storage at 37 ℃ for 8 weeks in the lyophilized formulations are shown in table 7, and the purity of terlipressin acetate after lyophilization of each formulation solution was found to be 99.86%. Comprehensively judging the pH value of the preparation solution, the appearance of the freeze-dried preparation and the stability of the freeze-dried preparation, wherein the weight ratio of polydextrose to mannitol to trisodium citrate is (3-5): 1: (0.1-0.3). The lyophilized pharmaceutical preparations can be reconstituted with 1ml to 5ml of water for injection and reconstituted into liquid pharmaceutical preparations. TABLE 7 pH, lyophilized appearance, reconstitution time and

stability for 8 weeks

Note: <1 indicates that the turbidity is less than that of the No. 1 standard solution; and the turbidity is larger than that of the No. 1 standard solution by 1.

And (4) test conclusion: the tests show that the lyophilized preparation of terlipressin has more excellent product quality by adopting a certain proportion of polydextrose, mannitol and trisodium citrate as excipients.

Test example of terlipressin for injection

1. Apparatus and medicine

High performance liquid chromatograph (model HP1100, hewlett packard, usa), DAD detector, AgilentChemstation chromatography workstation: a clarity tester type YB-2 (precision instruments works of Tianjin university); DU640 type ultraviolet spectrophotometer (beckmann, usa); pHS-3C type digital acidimeters (Shanghai Lei magnetic Instrument works); WS/08-0l type temperature and humidity regulating box (Hangzhou blue sky instrument production Co., Ltd.); analytical balance model mayler.ae200 (switzerland); terlipressin (sample prepared as described in example 5) was injected.

2. Method of producing a composite material

Taking 5 pieces of the product, adding 3ml of water into each bottle to dissolve, mixing uniformly, and determining according to the method (Chinese pharmacopoeia 2015 year) that the pH value should be 4.5-6.0.

Taking the product, dissolving in water, and diluting to obtain solution containing terlipressin 0.4mg per 1ml as test solution; precisely measuring 1ml, placing in a 100ml measuring flask, diluting with water to scale, and shaking up to obtain a control solution; 1ml of the control solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale with the mobile phase and shaken up to be used as a sensitive solution. According to the chromatographic condition under the content determination item, 10 mul of the sensitive solution is injected into a liquid chromatograph, and the signal-to-noise ratio of the terlipressin peak is more than 10. Precisely measuring 10 μ l of each of the reference solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatogram.

Measuring content by adding 2ml water into 10 bottles, shaking to dissolve the content, and precisely measuring 10 μ l as sample solution according to the content measurement method of terlipressin acetate; taking a proper amount of terlipressin acetate reference substance, adding water to dissolve, quantitatively diluting to prepare a solution containing about 0.4mg of terlipressin in each 1ml, and measuring by the same method to obtain the average content of 10 bottles.

2.3 influencing factor experiments

Under the packaging conditions of the marketed drugs, three batches of the samples of example 5 (batch numbers 160505, 160507, 160601) were examined under high temperature (60 ℃), high light (4500lx) and high humidity conditions for 5 days and 10 days, and the acidity, content and related substance indexes were examined, and all indexes were in accordance with the regulations. The results are shown in Table 8.

TABLE 8 influence factor test results

A. B, C represents three batches of samples from example 5, batch numbers 160505, 160507, 160601.

2.6 accelerated test

Under the packaging conditions of the marketed drugs, three batches of the samples of example 5 (batch numbers 160505, 160507, 160601) were stored at 40. + -. 2 ℃ and 75% relative humidity at 5% soil, and sampled at the end of 0, 1, 2, 3, 6 months, respectively, to determine the respective indices. All samples were white loose blocks in character. The results of the acidity, related substances and content measurement are shown in Table 9.

2.7 Long term experiments

Under the packaging conditions of the marketed drugs, three batches of the samples of example 5 (batch numbers 160505, 160507, 160601) were stored at 25. + -. 2 ℃ and 60. + -. 10% relative humidity, and samples were taken at the end of 0, 3, 6, 12, 18, 24 months, respectively, to determine the respective indices. All the samples are white loose blocks, and the results of measuring acidity, related substances and content are shown in Table 9.

TABLE 9 accelerated test and Long term test investigation results

And (4) conclusion: influence factor tests and accelerated test results show that various measurement indexes of the terlipressin for injection have no obvious change and the stability is good; the quality indexes of the injection Teli for 24 months under the condition of long-term room temperature have no obvious change, and the product has good stability.

Stability determination test after reconstitution of terlipressin for injection

The samples 160505, 160507, 160601 were reconstituted with 250ml of water for injection, placed in a refrigerator at 4 ℃ and measured once a day for 14 days, and then subjected to linear regression analysis with respect to time (X) as measured value (Y). The results are shown in Table 10.

TABLE 10 stability of terlipressin for injection after reconstitution

P >0.05 indicates that the terlipressin concentration is substantially stable over the time measured.

And (4) conclusion: as can be seen from Table 10, the terlipressin content remained stable after the three batches of the product of the present invention were reconstituted and continuously measured for 14 days. The injection terlipressin prepared by the method disclosed by the invention is stable in property after redissolution.

Claims

1. A preparation method of a terlipressin freeze-dried powder injection is characterized by comprising the following steps:

under clean conditions, 34.8g of polydextrose, 11.6g of mannitol and 3.48g of trisodium citrate are put into a liquid preparation device, 700ml of water for injection is added and stirred to be dissolved, 1g of terlipressin acetate is added after the prepared liquid is cooled to room temperature, the pH value is measured, the pH value is adjusted to 5.1 by 0.1M, pH-4.5 acetic acid-sodium acetate buffer solution, the water for injection is added to the full 1050ml, and the filtrate is filtered by a 0.22 mu m microporous membrane to obtain terlipressin acetate filtrate; subpackaging 3ml in an ampere bottle, and freeze-drying for 72 hours to prepare sterile freeze-dried powder injection;

the preparation method of terlipressin acetate comprises the following steps:

step 2: taking octadecylsilane as a stationary phase, sampling the crude product solution obtained in the step 1, taking a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the pH value of 6.5 and the mass concentration of 45% methanol as an A phase, taking an 80% methanol aqueous solution as a B phase, eluting at the flow rate of 75ml/min for 25min and the gradient of 40% B → 70% B, collecting eluent with the purity of more than 95%, filtering the eluent by using a 0.22 mu m filter membrane, and concentrating;