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Medicinal composition of recombinant carboxypeptidase G2

A technology of carboxypeptidase and composition, which is applied in the field of recombinant carboxypeptidase G2 pharmaceutical composition freeze-dried powder injection preparation and preparation thereof, can solve the problems of reduced purity, lack of availability, influence on efficacy and the like, and achieves low cost and increased Toxic side effects, easy to produce effects

Active Publication Date: 2012-07-11
重庆科润生物医药研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, recombinant carboxypeptidase G2 and lactose are commonly used to form a pharmaceutical composition, because lactose has the following defects as a freeze-dried preparation: BTG Company’s product manual records that lactose is used as a freeze-dried preparation, but during preservation and freeze-drying, protein and reduced Sexual lactose is prone to Maillard reaction (Maillard reaction), resulting in reduced purity and affecting drug efficacy (see Withdrawal assessment report for Voraxaze, EMEA / CHMP / 171907 / 2008)
So far, there is no preparation report suitable for stable storage and clinical application of carboxypeptidase G2

Method used

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  • Medicinal composition of recombinant carboxypeptidase G2
  • Medicinal composition of recombinant carboxypeptidase G2
  • Medicinal composition of recombinant carboxypeptidase G2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: The impact of excipient concentration on preparation molding, solubility

[0048] Two excipients, mannitol and sorbitol, were investigated on the formability and reconstitution solubility of lyophilized products under different concentration conditions. The specific experimental process is: to contain 0.2mM ZnCl 2 25mM Tris-HCl buffer solution with a pH of 7.3 to prepare 20% mannitol and sorbitol; respectively get 5ml carboxypeptidase G2 stock solution (2000U / ml) and a certain amount of mannitol or sorbitol mother solution (0ml, 0.5ml . Enzyme G2 solution, divided into 1.1ml / cartridge, freeze-dried; at 25°C, after standing for 20 days, each sample was reconstituted with 1ml water for injection, and the solubility was observed, and analyzed by RP-HPLC:

[0049] 1. The influence of mannitol on the stability of preparations

[0050]

[0051]

[0052] 2. The effect of sorbitol on the stability of the preparation

[0053]

[0054] It can be seen fr...

Embodiment 2

[0055] Embodiment 2: the impact of different carbohydrates on the stability of preparations

[0056] Investigate the influence of different carbohydrates on the stability of freeze-dried preparations according to the following conditions: respectively with 25mM, pH7.3Tris-HCl, 0.2mM ZnCl 2 Prepare the buffer solution with a concentration of 10% sucrose, lactose, glucose and trehalose; take 5ml of 2000U / ml carboxypeptidase G2 stock solution, add 3ml of the above sugar solution and 2ml of buffer solution to obtain: 1000U of carboxypeptidase G2 activity / ml, a solution with a sugar content of 3%. A total of 10ml of each sample was prepared, divided into vials at a rate of 1.1ml / branch, freeze-dried, placed at 25°C for 20 days, and then sampled for RP-HPLC analysis and activity determination.

[0057]

[0058] The above results show that the addition of sugar is beneficial to the maintenance of stability, among which sucrose has the best protective effect, and the activity and...

Embodiment 3

[0064] Embodiment 3: the impact of different pH buffering agents on formulation stability

[0065] The final purified sample (containing 25mM Tris-HCl pH7.3) was ultra-filtered with a membrane with a molecular weight cut-off of 10kD, respectively, with 25mmol / L disodium hydrogen phosphate-citric acid buffer solution with a pH of 4.0-6.0, pH7.0 ~pH9.0 25mmol / L Tris-HCl buffer solution for buffer solution exchange. Add 3% mannitol and 1% sucrose into the sample, prepare 10ml of each sample, put 1.1ml / branch into vials, freeze-dry, and take samples after 20 days at 25°C for RP-HPLC analysis and activity Determination.

[0066]

[0067] The above results show that the pH7.0-8.0 buffer system maintains the best drug activity. According to existing research reports, the optimal pH of the enzyme protein is 7.3, which is close to the physiological pH environment of the human body. Therefore, pH 7.3 is the best buffer pH for the preparation.

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Abstract

The invention relates to a medicinal composition of recombinant carboxypeptidase G2. The medicinal composition comprises a medicinal effective dosage of recombinant carboxypeptidase G2 proteins, a stabilizer, an excipient and a nonvolatile buffering agent for adjusting the pH value of a preparation to keeping optimal biological activity of the preparation. Every 1ml of recombinant carboxypeptidase G2 medicinal composition solution contains 500-2,000 U of recombinnt carboxypeptidase G2, 3-6 percent by weight volume of an excipient and 1-3 percent by weight volume of a stabilizer, a tri-methylolaminomethane-hydrochloric acid (Tris-HCl) buffering agent for adjusting the pH range to 6.8-7.8 and 0.1-0.5 mM of zinc chloride. A recombinant carboxypeptidase G2 lyophilized powder injection preparation has the beneficial effects that: under the pserving condition of 2-8 DEG C, the activity can be kept for 24 months, and the active structure of a natural dipolymer is kept. The preparation is aseptic, and can play a pharmacological action through intravenous injection.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant carboxypeptidase G2 pharmaceutical composition freeze-dried powder preparation capable of improving drug stability and a preparation method thereof. Background technique [0002] Carboxypeptidase G2 (Carboxypeptidase G2, referred to as CPG2) can hydrolyze folic acid C-terminal glutamic acid residues, folic acid polyglutamyl derivatives, folic acid analogs, such as methotrexate (MTX), and folic acid subfragments such as p-aminobenzene Formylglutamate (Chabner et al. 1972; Goldman 1975; Kalghatgl & Bertino 1981). CPG2 was originally a bacterial enzyme isolated from Pseudomonas strain RS-16 (Levy & Goldman 1967), and was cloned and purified in Escherichia coli in the 1980s (Minton, et al 1983). Carboxypeptidase G2 hydrolyzes MTX to nontoxic inactive metabolites 2,4-dimethyl-N10-methylpterinic acid (DAMPA) and glutamate, both of which can be metabolized by the liver, providi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61P39/02
Inventor 李树刚辛渝张伟于廷和但国平柴新娟龚会英曹莉君李晓丽张勇
Owner 重庆科润生物医药研发有限公司
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