WO2006012791A1

WO2006012791A1 - Composition containing active prourokinase, its freeze-drying process and lyophilized preparation

Info

Publication number: WO2006012791A1
Application number: PCT/CN2005/001149
Authority: WO
Inventors: Yan Jiang; Tieqiang Hu; Chengzu Xiao
Original assignee: Shanghai Tasly Pharmaceutical Co., Ltd.
Priority date: 2004-08-06
Filing date: 2005-07-28
Publication date: 2006-02-09
Also published as: CN100420485C; HK1088243A1; CN1730098A

Abstract

The present invention discloses a pharmaceutical composition containing active prourokinase, its freeze-drying process and lyophilized preparation.

Description

TECHNICAL FIELD The present invention relates to the field of medicine, and in particular to a pharmaceutical composition containing active prourokinase, a lyophilization method thereof, and a lyophilized preparation. BACKGROUND OF THE INVENTION Cardiovascular diseases caused by blood clots are a serious threat to human health, and there is an urgent need for a drug having a good thrombolytic effect and a small side effect. Pro-ukogenase (Pro-uk) specifically activates plasminogen on the surface of the thrombus, which specifically dissolves the thrombus, while not destroying the fibrinolytic system. The side effects of systemic bleeding are small; preclinical animal experiments And clinical trials verify that Pro-uk also has the advantages of small dosage, safe use, good thrombolytic effect, low recombination rate, no allergic reaction, etc. It is a safe and non-toxic, ideal biological thrombolytic preparation with Broad market pre-study

Due to the low content of Pro-uk in natural materials, it is difficult to prepare in large quantities. Since the 1980s, genetic engineering technology has been used to produce recombinant Pro-uk, which is currently in E. coli (Homes WE, et al. Cloning and expression of the gene for pro-urokinase in Escherichia coli. Biotechnology, 1985; 3:923; Winkler ME, et al. Purification and characterization of recombinant single-chain

Urokinase produced in Escherichea coli. Biochemistry, 1986; 25:4041), mammalian cells such as mouse IC9 cells, CHO cells, Namalwa cells (Nolli ML, et al. Production and charaterisation of human recombinant single chain

Urobinase-type plasminogen activator from mouse cells. Fibnrinolysis, 1989 3 : 101; Nelles L, et al. Characterization of recombinant human single chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of Lysine. J Biol Chem, 1987; :5682; Li Fengzhi et al. Stable expression of human prourokinase full-length cDNA in Chinese hamster ovary cells. Chinese Journal of Biotechnology, 1991; 7 : 1 14; Satoh M, et al. Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the espressed-protein stability. Cytotechnology, 1993; 13:79; Satoh M, et al. Efficient expression of pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells using Moloney retroviral promoter. Cytotechnology, 1996;

18: 167) and insect cells (Xu Yang et al. High expression of human prourokinase cDNA in the eukaryotic expression system of insect baculovirus. J. Biol. Chem., 1993; 6:709), myeloma cells (Weaver WD, et Al. New recombinant glycosylated prourokinase for treatment of patients with acute myocardial infarction. J Am Coll Cardiol, 1994;

24: 1242) Both were highly expressed and purified.

The Pro-uk preparation is extremely unstable and is easily cleaved by various proteases into urokinase (UK). At the same time, it is automatically decomposed even in the liquid state even after cryopreservation (Yu Fang et al., Stability observation of recombinant semi-finished recombinant urokinase, Proceedings of the Academy of Military Medical Sciences, 2000, 24 (1): 70); In addition, due to laboratory measurements, clinical use, and daily transportation and storage requirements, it is often necessary to use a dry method to stabilize its activity to prevent degradation. Summary of the invention

In order to solve the problem of instability of the prourokinase preparation in the prior art, the inventors of the present invention have obtained a composition containing active prourokinase by long-term research, and the present invention is compared with the existing prourokinase preparation. The composition can be stably stored for a long time.

Another aspect of the invention provides a method of preparing the prourokinase composition.

The pharmaceutical compositions of the present invention contain appropriate amounts of Pro-uk, excipients, and buffer solutions and salts. Wherein, the excipient may be a saccharide and/or a polyhydric alcohol, and one or more selected from the group consisting of sucrose, trehalose, mannitol, lactose, glucose, sorbitol, maltose, etc., wherein mannitol is preferred because mannitol Freezing products are superior to other forms in appearance and lyophilization time, and mannitol has been used in clinical practice for many years, and the price is also cheaper. Therefore, the choice of mannitol can shorten the freeze-drying time of the product and increase the freezing. Dry efficiency, reduce costs. The preferred range of mannitol content is 6% to 12% (weight/volume ratio), and most preferably 6% (weight/volume ratio) from the economical point of view; the buffer may be phosphate buffer and/or Tris-HCl, Wherein the content of the phosphate buffer is preferably in the range of 8 to 25 mmol/L, and the content of the Tris-HCl is preferably in the range of 0.005 to 0.015 mol/L; in the composition, the salt thereof may be sodium chloride, chlorinated The preferred range of sodium content is from 0.03 to 0.20 mol/L; Pro-uk can be any possible concentration, but for ease of lyophilization and medical use, a preferred concentration range is from 1.5 to 5.5 mg/ml.

In the above composition, the protective agent can be added if lyophilized to give better results, the protective agent may be a protein, preferably albumin, the albumin content is preferably in the range of the composition is 5-7.5% ₀ · 1 (weight/volume ratio) It should be noted that Pro-uk in the above pharmaceutical composition can be prepared according to the prior art method, but is not limited to these methods; the solution can be prepared according to the general solution preparation method, but in order to prevent degradation of Pro-uk, it is best Formulated at low temperatures, the action should be mild during formulation.

The invention also provides a freeze-drying method for a solution containing a composition of active Pro-uk, the method comprising the steps of: placing the packaged Pro-uk product in a pre-cooling to -35 ° C to - 45 ° C Dry machine, pre-freeze for 2-4h, then start vacuuming, first temperature rise to -25 °C ~ - 30 °C, when the water in the bottle is basically sublimated, the second temperature is raised, and the temperature rises to 25 T:~ At 35 °C, after standing for 0.5h~1.5h, the lyophilization can be ended.

There are many methods for drying biological products, but most of them can denature proteins. The freeze-drying technique uses a frozen solution to remove dryness from a frozen state without direct liquid sublimation under low temperature and low pressure conditions. Freezing and drying technology can maintain the original physical and chemical properties and physiological activity of the drug, and the loss of active ingredients is minimal. In addition, the loose porous structure unique to the lyophilized preparation can make the drug easy to rehydrate and restore activity, and the lyophilized preparation has low water content and is easy to store for a long time. Therefore, freeze drying is one of the best methods for clinical and laboratory preservation of protein products.

The active Pro-uk-containing pharmaceutical composition of the present invention was freeze-dried by the above method to obtain a frozen Pro-uk preparation. The lyophilized preparation has a good appearance, and has no change in storage for 3 years under storage conditions of -20 ° C and 4 ° C; the long-term stability of the lyophilized preparation under different conditions is observed by an accelerated storage test, and the storage life is performed. Estimated, the results are as follows: Pro-uk lyophilized preparation can be stored for 173 days at 37 ° C, 2.78 years at 25 ° C, 70.8 years at 4 ° C, and stored at 0 ° C for 1 18 cases with Pro-uk activity loss of 50%. Year: When the activity loss is 10%, Pro-uk lyophilized preparation can be stored for 20 days at 37 °C, stored for 100 days at 25 °C, stored for 7.6 years at 4 °C, and stored for 1 year at 0 °C, which is sufficient for Pro-uk laboratory. And the need for clinical storage. DRAWINGS

Figure 1 : Pro-uk activity degradation rate curve.

Figure 2: Storage life curve of Pro-uk. detailed description

The following are examples of specific embodiments for carrying out the invention. The examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Embodiment 1 Preparation of prourokinase

(See Chinese patent: Preparation method of recombinant human glycosylated prourokinase, bulletin number

CN1062016C, Announcement Date: February 2001, 14曰)

(1) Cloning of prourokinase gene and construction of expression vector

Detroit 562 cells were induced by myristyl ester (PMA), total RNA was extracted, and a cDNA library was constructed. A positive clone containing a urokinase gene fragment was obtained by screening, and a human prourokinase full-length cDNA gene was obtained. The gene recombination technique operates to place two transcription units expressing the prourokinase and the dihydrofolate reductase gene in the same vector, which are respectively controlled by metallothionein (MT) and the SV40 early promoter.

( 2 ) Transfection and screening of highly expressed CHO engineered cells

20-40 μg of pMTSV-du plasmid DNA was transfected into CHO-dhfr-cells by phospho-facial coprecipitation, first screened with HAT selection medium, and replaced with l-3xl (T ⁸ M MTX selective culture after 10 days). The dhfr and MTX dual screening were performed, and the positive transformed cells expressing prourokinase activity were subjected to multiple subcloning and MTX pressure amplification genes, induced by zinc ions, and finally a cell line capable of efficiently expressing prourokinase was screened.

(3) CHO engineering cell culture and amplification

The engineered cell strain expressing prourokinase was cultured at a high density using a microcarrier (Cytopore) perfusion culture technique and a low serum medium to obtain a prourokinase-containing material.

(4) Purification of recombinant human prourokinase

Purified prourokinase is obtained by a four-step method, namely CM cation chromatography, MPG adsorption chromatography, Sephacryl S-200 gel filtration and p-aminobenzidine chromatography.

Embodiment 2

Vacuum freeze drying of Pro-uk composition

Purified Pro-uk pure product 2mg/ml, mannitol to 6% (weight/volume ratio), albumin to 3%. (weight/volume ratio), concentration of p-acid buffer to 10mmol/L, sodium chloride concentration to 0.05mol/L, dispense into 1 ml ampoules, and freeze-dried to -40 °C beforehand. Machine, pre-freezing for 3h, then start vacuuming, first temperature rise to -30 °C, when the water in the bottle is basically sublimated, the second temperature is raised, the temperature is raised to 30 Ό, and after stabilization for 1 h, the lyophilization is completed. .

Embodiment 3

Vacuum freeze drying of Pro-uk composition

Purified Pro-uk pure product 4mg/ml, add mannitol to 6% (weight/volume ratio), Albumin to 3%. (weight/volume ratio), phosphate buffer concentration to 10mmol/L, sodium chloride concentration to 0.10mol/L, dispense into 1 ml ampoules, and put into freeze dryer with pre-cooling to -40 °C , pre-freezing for 3h, then start vacuuming, for the first time to warm up to -30 ° C, when the water in the bottle is basically sublimated, the second temperature is raised, the temperature is raised to 30 ° C, and after stabilization for 1 h, the freezing can be ended. dry.

Embodiment 4

Vacuum freeze drying of Pro-uk composition

Purified Pro-uk pure product 5mg/ml, mannitol to 10% (weight/volume ratio), albumin to 7%. (weight/volume ratio), phosphate buffer concentration to 20mmol/L, sodium chloride concentration to 0.20mol/L, dispensed into 1 ml ampoules, and placed in a lyophilizer pre-cooled to -40 °C , pre-freezing for 4h, then start vacuuming, for the first time to warm up to -30 ° C, when the water in the bottle is basically sublimated, the second temperature is raised, the temperature is raised to 35 ° C, and after stabilization for 1 h, the freezing can be ended. dry.

Embodiment 5

Vacuum freeze drying of Pro-uk composition

Purified Pro-uk pure 2 mg/ml, mannitol to 6% (weight/volume ratio), albumin to 3%. (weight/volume ratio), phosphate buffer concentration to 20mmol/L, sodium chloride concentration to 0.20mol/L, dispensed into 1ml ampoules, and placed in a lyophilizer pre-cooled to -40 °C, Pre-freezing for 3h, then start vacuuming, for the first time to warm up to -30 °C, after the water in the bottle is basically sublimated, the second temperature is raised, the temperature is raised to 30 ° C, and after stabilization for 1 h, the freeze-drying is completed. .

Embodiment 6

Vacuum freeze drying of Pro-uk composition

Purified Pro-uk pure 2 mg/ml, mannitol to 6% (weight/volume ratio), albumin to 3%. (weight/volume ratio), Tris-HCl buffer concentration to 0.10mol/L, sodium chloride concentration to 0.05mol/L, dispense into 1ml ampoules, and freeze-dried to -40 °C beforehand. Machine, pre-freezing for 3h, then start vacuuming, first temperature rise to -30 °C, when the water in the bottle is basically sublimated, the second temperature is raised, the temperature is raised to 30 °C, and it is kept for 1h after stabilization, then it can be finished. Freeze dried.

Example 7

Effects of different concentrations of excipients and protective agents on the morphology and activity of Pro-uk frozen preparations. Purified Pro-uk pure 2mg/ml, plus phosphate buffer concentration to 10 mmol/L, sodium chloride concentration to 0.05 mol/L, and different concentrations of excipients and protectants were added to evaluate their effects on the morphology and activity of the pro-uk lyophilized formulation. The activity of Pro-uk was determined using a modified agarose-fibrinolytic method (see Xiao C, Huang Z, Lin F, et al. High density cultivation of genetically-engineered CHO cell line with microcarrier culture system. Chin Med Sci J , 1994, 9(2): 71-74). Unless otherwise specified below, the same method was employed for the activity measurement. Table 1: Effect of different concentrations of excipients and protective agents on the morphology and activity of pro-uk lyophilized preparations Types of excipients and protective agents (%) Protection ratio

1 2 3 -1 5 6 7 8 9 ( % ) Mannitol One + One + + 33 Lactose One ― ++ + + + + + 28

3 %. Albumin + mannitol - one + + + + 80

+ + + +

Ν=3 _: · + + good appearance, low loss of activity; + good appearance; no effect of forming the buffer type, buffer concentration and sodium chloride concentration on the shape of Pro-uk lyophilized preparation For Pro-uk products with different buffer and sodium chloride concentrations, add 7% mannitol, 5%. The human albumin is completely dissolved and placed in a vial, 1 ml per bottle, and placed in a freeze-drying box for freeze-drying. After drying, the product is taken out for visual inspection, and it can be seen that the phosphate buffer (PB) is contained. The Pro-uk preparation of Tris-HCl has a loose appearance and good morphology (see Table 2). After adding water, the solution dissolves quickly and completely, and the transparency is good, and the original trait is immediately restored.

Table 2: Effect of different buffer concentrations on the appearance of pro-uk frozen product. Buffer type Sodium chloride (mol/L)

0.50 0 0 025 O20 0Λ 5 0J 0 0 Λ5 l OmM PB + ― + + +■ + + + + +

20mM PB + + ― ― + + + + + +

9.2mM NaAc ― ― ― ― ― ―

5mM NaAc ― 一― ― ― ―

0.01 M Tris-HCl + + + + + +

N=4 : + + Appearance is good; + Appearance is good; One is not formed. Stability of Pm-uk lyophilized preparation is observed. Purified Pro-uk pure 2 mg/ml, and mannitol is added to 6 % (weight/volume ratio), albumin to 3%. (weight/volume ratio), the phosphate buffer concentration was 10 mmol/L, the sodium chloride concentration was 0.05 mol/L, and the mixture was dispensed into a 1 ml ampoule and lyophilized as in Example 2. Pro-uk lyophilized preparations are stored at -20 ° C, 4 ° C, 25 ° C and 37 ° C, and regularly (January, March, June, September, December, December) The stability of the activity was observed. It can be seen that the activity is unchanged at -20 and 4, and the activity is decreased by 37% and 81% at 25 °C and 37 °C, respectively (see Table 3).

Table 3: Pro-uk activity at -20 ° C, 4 ° C, 25 ° C and 37 ° C (IU/ml) Time (month) -20 °C 4 °C 25 °C 37 °C

1 26704 27627 27627 26704

3 25630 25632 21471 20669

6 23000 21200 1 8023 12500

9 27253 26786 21429 15000

12 25522 26055 18593 6678

1 5 25070 25000 1 7500 5000

Accelerated storage test of Pro-uk lyophilized product was purified and purified Pro-uk pure 2 mg/ml, mannitol to 6% (weight/volume ratio), albumin to 3%. (weight/volume ratio), phosphate buffer concentration to 10 mmol/L, sodium chloride concentration to 0.05 mol/L, dispensed into a 1 ml ampoule, and lyophilized as in Example 2. Plus The multi-point isothermal stability test (MIS) reported by Greiff et al. (Griff D, et al, An accelerated storage test for predicting the stability of suspension of measles virus dried by sublimation in vacuo. J Immunol, 1965; 94: 395) ), the Pro-uk vials after freezing are placed in a water bath of 60 ° C (20 bottles), 70 ° C (12 bottles), 80 ° C (5 bottles), and water baths from 3 different temperatures at intervals of 24 hours. Take a bottle and measure the activity of Pro-uk (Table 5).

Table 5: MIS test results of prourokinase determination

60 °C 70 °C 80 °C

Time active time . activity time activity

(d) LogIU/ml (d) LogIU/ml (d) LogIU/ml

0 5.1 03 0 5.1 03 0 5.103

1 5.048 1 5.005 1 4.557

2 5.022 2 4.862 2 3.90

3 4.954 3 4.752 3 3.53

4 4.935 4 4.580 4 3.24

5 4.927 5 4.291 5 2.88

6 4.883 6 4.229

7 4.81 8 7 4.015

8 4.699 8 3.799

9 4.622 9 3.398

10 4.589 10 3.286

1 1 4.426 1 1 3.081

12 4.380 12 2.970

13 4.3 19

14 4.163

1 5 4.097

16 4.000

17 3.999

21 3.875

28 3.476

3 1 3.327 The data processing is roughly the same as that described by Greiff et al. The logarithm of the Pro-uk titer measured at different temperatures after storage and the logarithm of the storage time and the corresponding Pro-uk activity. The degradation logarithm was input into the computer, and the Pro-uk activity degradation rate (K1) was determined to be -0.068, -0.161 and -0.443, respectively. The degradation rate curve is shown in Fig. 1. From this curve, the degradation rates of Pro-uk lyophilized preparations at 37° (:, 25 V, 4 ° 〇, and 0 ° C were -0.00608, -0.00169, -0.00016, and -0.00011, respectively. According to the degradation rate K1 Pro-uk's storage life estimation curve was plotted against the time required for Pro-uk activity to degrade 10% or 50% corresponding to each K1 value, and the degradation rate of Pro-uk lyophilized preparation activity was based on the above two curves. Estimation of shelf life: Calculated according to the MIS test, the Pro-uk lyophilized preparations were stored at 60 ° C, 70 ° C and 80 ° C, and the time required for loss of activity was 50%, 6.27d, 1.92d and 0.48. The time required for loss of activity of 10% is 0.8d, 0.2d and 0.04d, and thus the storage life curve of Pro-uk can be plotted (Fig. 2). This curve can estimate the loss of Pro-uk activity by 50%. In the case of Pro-uk, the frozen preparation can be stored for 173 days at 37 °C, 2.78 years at 25 °C, 70.8 years at 4 °C, and 118 years at 0 °C. Also, according to this method, it can be inferred that when the activity loss is 10%, The Pro-uk lyophilized preparation can be stored at 37 ° C for 20 days, stored at 25 ° C for 100 days, stored at 4 ° C for 7.6 years, and stored at 0 ° C for 11 years.

Claims

Rights request

A prourokinase composition comprising an appropriate amount of prourokinase, an excipient, a buffer solution and a salt.

2. The composition of claim 1 further comprising a protective agent.

3. The composition of claim 1 or 2, wherein the prourokinase content is from 1.5 to 5.5 mg/ml.

4. The composition of claim 2 wherein said protective agent is at a concentration of 1.5%. -7.5%.

(weight/volume ratio) of albumin.

5. The composition of claim 1 or 2 wherein the excipient is a saccharide and/or a polyol.

6. The composition of claim 6 wherein the saccharide and/or polyol is selected from the group consisting of mannitol, sucrose, trehalose, mannitol, lactose, glucose, sorbitol, maltose.

7. The composition of claim 1 or 2 wherein the buffer solution is a diacid buffer or a Tris-HCl buffer.

The composition according to claim 1 or 2, wherein the salt is sodium chloride having a concentration of 0.03 to 0.20 mol/L.

9. A method of freeze-drying any of claims 1 to 8, the method comprising the steps of: i) taking any of the compositions of claims 1 to 8 and pre-cooling to -35 ° C to - 45

°C lyophilizer, pre-freezing 2-4h;

Ii) Then start vacuuming and heat up to -25 °C ~ - 35 °C;

Iii) When the water in the container is substantially sublimated, the temperature is raised for the second time, and the temperature is raised to -25 °C to -35 °C, and is maintained for 0.5h~1.5h after stabilization.

10. A prourokinase procalculin prepared by the method of claim 9.