WO2008150080A1

WO2008150080A1 - Injectable ready to use solutions comprising human chorionic gonadotropin

Info

Publication number: WO2008150080A1
Application number: PCT/KR2008/002971
Authority: WO
Inventors: Beom-Soo Park; Ho-Young Yi; Kyu-Heum Na; Sung-Hee Lee; Soo-Hyung Kang
Original assignee: Dong-A Pharm. Co., Ltd.
Priority date: 2007-06-04
Filing date: 2008-05-28
Publication date: 2008-12-11
Also published as: KR20080106636A; EP2152296A1

Abstract

There is provided an injectable ready-to-use solution comprising human chorionic gonadotropin (hCG) capable of stably storing hCG. The injectable ready-to-use solution comprising human chorionic gonadotropin (hCG) according to the present invention may be useful to stably store hCG without reconstitution from a freeze-dried materials since the injectable ready-to-use solution comprises pharmaceutical non-reducing sugars such as trehalose and methionine.

Description

INJECTABLE READY TO USE SOLUTIONS COMPRISING HUMAN CHORIONIC GONADOTROPIN

Technical Field

[1] The present invention relates to an injectable ready-to-use solution comprising human chorionic gonadotropin, and more particularly to an injectable ready-to-use solution comprising human chorionic gonadotropin (hCG) capable of stably storing the hCG.

[2]

Background Art

[3] Human chorionic gonadotropin (hereinafter, referred to as 'hCG'), which is one of compounds belonging to glyco-hormones that act at the beginning of pregnancy by stimulating the generation of steroid hormones in corpus luteum, is synthesized in placental tissues. hCG is composed of two different subunits α and β that bind to each other through noncovalent bonds. However, only heterodimeric hCG has a biological activity. The α subunit composed of 92 amino acids has carbohydrate complexes bound to 52° and 78 asparagine residues thereof, and is stabilized by 5 disulfide bonds. The β subunit composed of 145 amino acids has carbohydrate complexes bound to 13 and 30 asparagine residues and 4 serine residues in C-terminus thereof, respectively, and is stabilized by 6 disulfide bonds.

[4] The preparation of hCG injectable ready-to-use solutions has many problems caused by the reduction of active components due to the physical and chemical effects, and therefore the hCG injectable ready-to-use solutions are reconstituted from freeze-dried materials for the purpose of practical uses.

[5]

[6] International Publication No. WO 88Ol 0270 discloses a freeze-dried formulation in which urine-derived follicle stimulating hormones (FSH) are stabilized with lactose and human serum albumin, and European Patent No. EP 448146 discloses a freeze- dried gonadotropin formulation which is stabilized with dicarboxylic acid such as citric acid. Also, International Publication No. WO 93011788 discloses a method for stabilizing a freeze-dried gonadotropin formulation using sucrose andύr glycine. In particular, sucrose has been proposed as a desirable vehicle so as to maintain the stability of hCG, and a freeze-dried pharmaceutical composition including sucrose has a superior stability to a freeze-dried pharmaceutical composition including lactose or mannitol.

[7]

[8] However, when a freeze-dried formulation is administered, double handling of medicines is required. In this case, a freeze-dried cake is first formulated again and administered. Also, some formulations should be stirred for an extended time for the purpose of complete dissolution of powders since they are not easily solubilized.

[9]

[10] Also, a technology using human serum albumin (HSA) as an additive in an hCG injectable ready-to-use solution has been developed, but the use of HSA causes problems regarding the viral infection, the formation of aggregates, and the infection by prion that has been issued recently. Therefore, there has been a demand for developing a liquid formulation that is not freeze-dried but also free from HSA.

[H]

[12] As the prior-art literatures regarding the stability of an hCG injectable ready-to-use solution, International Publication No. WO 93022335 discloses a citric acid buffer (pH 5.0-5.5) so as to stabilize liquid polypeptide, and International Publication No. WO 96XE9095 discloses a liquid hCG composition comprising mannitol and acetic acid, succinic acid or phosphate (pH 7.0). In the patent applications, the stability of the hCG according to the kind of stabilizing agents was compared under the same pH buffer condition. In this case, it was revealed that the stability of a liquid formulation including mannitol is more excellent than that of a formulation including sucrose, glycine, glucose, lactose and sodium chloride. U.S. Patent Publication No. 5929028 discloses a liquid gonadotropin- stabilized formulation comprising 25-100 mM citric acid (pH 6.5-7.2), 1-10 mM methionine, 25-300 mM sucrose, and a non-ionic surfactant.

[13]

[14] The present inventors have studied on an injectable ready-to-use solution that is free from human serum albumin and may stably store hCG, and found that the storage stability of an hCG injectable ready-to-use solution containing trehalose and methionine is very excellent. Therefore, the present invention was completed on the basis of the facts.

[15]

Disclosure of Invention Technical Problem [16] Accordingly, the present invention is designed to solve such drawbacks of the prior art, and therefore an object of the present invention is to provide an injectable ready- to-use solution comprising human chorionic gonadotropin (hCG) capable of stably storing the hCG.

[17]

Technical Solution

[18] The present invention relates to an injectable ready-to-use solution comprising human chorionic gonadotropin (hCG) that is stabilized with trehalose and methionine.

[19]

[20] According to an aspect of the present invention, there is provided an injectable ready-to-use solution comprising human chorionic gonadotropin (hCG) that essentially includes trehalose and methionine, is not reconstituted from a freeze-dried material comprising hCG and a vehicle, has a pH from 6.0 to 8.0, is free from pyrogens, and stably store hCG.

[21]

[22] hCG may be isolated from natural sources, for example human urine, and also be expressed in Chinese Hamster Ovary(CHO) cell transfected with DAN using genetic engineering technologies. In this case, the recombinant hCG may be included at a content of 10 to 1000 ug/ml, and preferably 250 to 500 ug/ml.

[23]

[24] Trehalose may be present at a content of 10 to 100 mg/ml, and preferably 60 mg/ml, and methionine may be present at a content of 0.1 to 1 mg/ml, and preferably 0.5 mg/ ml. Also, pH adjusted by a phosphate buffer may be in a range from 6.0 to 8.0, and particularly preferably 7.0.

[25]

[26] The present invention provides a process for preparing a liquid pharmaceutical composition and the process includes dilution step of an hCG solution with a vehicle- containing buffer. In this case, the solution of the present invention is provided in a sealed container, for example, in a glass container. The solution of the present invention may also be provided in the form of a single dose, or multiple doses using the method according to the present invention.

[27]

Advantageous Effects

[28] As described above, the present invention provides the injectable ready-to-use solution comprising human chorionic gonadotropin (hCG) containing non-reducing sugar such as trehalose and methionine, and the solution which has not been reconstituted from a freeze-dried materials and not containing HSA as an additive may store stably the hCG for an extended time.

[29]

Best Mode for Carrying Out the Invention [30] Hereinafter, Examples and Experimental examples of the present invention will be described in more detail.

[31] [32] Example 1 : Preparation of vehicle-containing hCG injectable ready- to-use solution

[33] Injectable solutions stabilized with 4 different stabilizing agents were prepared in 1OmM phosphate buffer (pH 7.0) including 500 ug/ml of recombinant hCG. Trehalose, mannitol, sucrose and lactose were used as the stabilizing agent or vehicle. The injectable solution containing recombinant hCG was prepared according to the components and their contents as listed in the following Table 1. Trehalose was prepared by adding phosphoric acid (0.98g) and Tween 20 (0.2g) to injectable distilled water (600 ml). pH was adjusted into 7.0. Trehalose (6Og) was added, a recombinant hCG solution (500 mg) was added to a vehicle solution, and the resulting mixture solution was adjusted to pH 7.0+0.2 using IM sodium hydroxide when it is necessary to check pH of the mixture solution.

[34] [35] Table 1 [Table 1] Compositions of recombinant hCG solutions

[36] [37] Example 2 : Preparation of amino acid-containing hCG injectable ready-to-use solution [38] Compositions were prepared in the same manner as in Example 1, and amino acid such as methionine was added to a vehicle solution prior to adding a recombinant hCG solution (500 mg) to the vehicle solution. The compositions are listed in the following Table 2.

[39] [40] Table 2 [Table 2]

Compositions of recombinant hCG solutions

[41] [42] Experimental example 1 : Measurement of storage stability of hCG injectable ready-to-use solutions according to vehicles

[43] The injectable solution prepared in Example 1 was adjusted to IL with water for injection, and the injectable solutions were filtered with a sterile filter, 1 ml of samples of the compositions were put into sealed 3ml vials, and then kept at 4⁰C, 25⁰C and 4O⁰C, respectively. Then, the composition samples were subject to HPSEC (high-performance size-exclusion chromatography) and RP-HPLC (reverse-phase high-performance liquid chromatography) methods to perform stability tests. Here, the HPSEC results are listed in the following Table 3, and the RP-HPLC results are listed in the following Table 4.

[44] [45] As seen from the analysis results as listed in Table 3 and Table 4, it was revealed that trehalose has more excellent stability than the other stabilizing agents.

[46] [47] Table 3 [Table 3]

Change (%) in concentration of recombinant hCG according to stabilizing agents or vehicles: HPSEC analysis results

[48] [49] Table 4 [Table 4]

Change (%) in concentration of recombinant hCG according to stabilizing agents or vehicles: RP- HPLC analysis results

[30] [51] Experimental example 2 : Measurement of storage stability of hCG injectable ready-to-use solutions according to amino acids

[52] Samples of the recombinant hCG injectable solutions (500 mg) prepared in Example 2 were kept at 4⁰C, 25⁰C and 4O⁰C in the same manner as in the Experimental example 1, stability tests on the samples were performed using HPSEC and RP-HPLC. Here, the HPSEC results are listed in the following Table 5, and the RP-HPLC results are listed in the following Table 6.

[53] [54] As seen from the analysis results as listed in Table 5 and Table 6, it was revealed that the stability of the recombinant hCG were increased at the presence of methionine. Also, it was revealed that the stability of the liquid composition was more increased with increasing content of methionine.

[55] [56] Table 5 [Table 5]

Change (%) in concentration of recombinant hCϋ according to the content of methionine: HPSEC analysis results

[57] [58] Table 6 [Table 6]

Change (%) in concentration of recombinant hCG according to the content of methionine: RP-HPLC analysis results

Claims

[1] A sterile injectable ready-to-use solution comprising human chorionic gonadotropin (hCG) that is not re-prepared from a freeze-dried material comprising human chorionic gonadotropin and a vehicle, and is free from pyrogens, wherein hCG is present at a content of 10 to 1000 μg/ml, and stably stored by trehalose and methionine that are included as essential components.

[2] The sterile injectable ready-to-use solution according to claim 1, wherein the human chorionic gonadotropin is a recombinant human chorionic gonadotropin.

[3] The sterile injectable ready-to-use solution according to claim 1, wherein trehalose is present at a content of 10 to 100 mg/ml, and methionine is present at a content of 0.1 to 1 mg/ml.

[4] The sterile injectable ready-to-use solution according to claim 3, wherein the trehalose is present at a content of 60 mg/ml, and the methionine is present at a content of 0.5 mg/ml.