JP2010091398A - Stable natriuretic peptide composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new stable natriuretic peptide composition preventing adsorption onto a container, and moreover, preventing decomposition due to serine protease. <P>SOLUTION: The natriuretic peptide is provided stabilized by making a benzamidine derivative coexist. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a stabilized natriuretic peptide composition and a method for producing a stabilized natriuretic peptide composition. More particularly, the present invention relates to a composition of a natriuretic peptide stabilized by the coexistence of a benzamidine derivative and a method for producing a composition of a stabilized natriuretic peptide comprising the coexistence of a benzamidine derivative. is there.

  Diagnostic agents for measuring B-type natriuretic peptide (BNP), A-type natriuretic peptide (ANP), and C-type natriuretic peptide (CNP) belonging to the natriuretic peptide have been developed and used. Among these, BNP is extremely useful as a diagnostic agent for heart failure and is widely used.

  In the measurement of the above-mentioned natriuretic peptide, an immunoassay using an antibody that recognizes a part of the amino acid sequence of the peptide has been developed and is generally used. In an immunoassay, it is necessary to measure the peptide itself as a measurement target as a standard substance for quantification of the measurement target. Specifically, in the measurement of natriuretic peptide such as BNP, a composition of natriuretic peptide that is a measurement target is used as a standard substance. However, natriuretic peptides represented by BNP are easily degraded by serine proteases when compared to other peptides to be measured, and further, the amino acid primary sequence contains many basic amino acids. It is easy to generate. Therefore, it is difficult to store stably for a long period of time, that is, to store without changing the value detected by the immunoassay. Such difficulty is not limited to the case of providing a natriuretic peptide such as BNP as a standard substance, but until just before measuring the natriuretic peptide such as BNP present in a blood sample obtained from a subject by an actual immunoassay. Also exists in the case of stable storage.

  For methods other than natriuretic peptides, methods for solving the problems of adsorption and degradation include a method for preventing adsorption of the peptide to the holding container in the presence of mannitol, sucrose, etc., and serine protease inhibitors aprotinin, EDTA ( (Ethylenediaminetetraacetic acid) and benzamidine have been proposed to suppress degradation of the peptide (Patent Document 1). However, this method is not sufficient as a method for preserving natriuretic peptides such as BNP without changing the values detected by immunoassay over a long period of time, and there remains room for further improvement.

  As one improvement, a method for preserving natriuretic peptides such as BNP using a resin container has been proposed (Patent Document 2), avoiding a glass container that tends to cause adsorption.

Japanese Patent No. 3457324 Japanese Patent No. 3302376

  In Patent Document 2, adsorption of natriuretic peptide such as BNP to a holding container is prevented by using a plastic container such as polystyrene or polypropylene. However, in addition to the premise that plastic containers are used for immunoassay products and blood samples, there is a problem that degradation by serine protease cannot be addressed. In Patent Document 1, the degradation of a natriuretic peptide by a serine protease is prevented by the coexistence of a serine protease inhibitor. However, it is unclear whether or not the adsorption of natriuretic peptide to the holding container can be dealt with. Accordingly, when the natriuretic peptide is prevented from being adsorbed and decomposed by a conventionally known method, it is necessary to carry out the above two countermeasures separately.

  Therefore, an object of the present invention is to provide a novel stable natriuretic peptide composition that prevents the adsorption of natriuretic peptides such as BNP in both plastic and glass containers, and also prevents degradation by serine proteases. To provide things.

  The present invention made to achieve the above object is a natriuretic peptide stabilized by the coexistence of a benzamidine derivative. The present invention is also a method for producing a stabilized natriuretic peptide, which comprises coexisting a benzamidine derivative. Hereinafter, the present invention will be described in detail.

  Benzamidine is a compound disclosed in Patent Document 1 as a serine protease inhibitor. However, Patent Document 2 discloses nothing about the effect that benzamidine has on natriuretic peptides such as BNP, that is, the effect of preventing adsorption to the container. The present invention was made on the basis of a completely new finding that a benzamidine derivative also prevents adsorption of natriuretic peptides such as BNP to glass containers, as will be described later. It provides a natriuretic peptide that can be stored for a long period of time without fluctuation in the value detected by an immunoassay by the extremely simple operation of coexisting a benzamidine derivative with a natriuretic peptide such as BNP. .

  The stabilized natriuretic peptide provided by the present invention is used, for example, as a standard or control in an immunoassay, and is, for example, a blood sample obtained from a subject before being subjected to an immunoassay. . The benzamidine derivative used in the present invention is represented by Formula 1, and for example, benzamidine hydrochloride can be exemplified as a specific benzamidine derivative.

ベンズアミジン誘導体は、ＢＮＰ等のナトリウム利尿ペプチドと共存させることによって当該ナトリウム利尿ペプチドの保持容器への吸着を防止すると同時に、セリンプロテアーゼによる分解をも防止するものである。保持容器への吸着の防止に関しては、保持容器の材質がナトリウム利尿ペプチドの吸着し易いとされるガラス製容器であるか、ガラス製容器と比較してその吸着が少ないとされるポリスチレンやポリプロピレン製であるかを問わず、十分な効果を奏する。容器の形状はチューブ状、バイアル状等、いかなるなる形状であっても良く、ベンズアミジン誘導体を共存させた状態で凍結、凍結乾燥してもその効果が失われることはない。

The benzamidine derivative coexists with a natriuretic peptide such as BNP to prevent adsorption of the natriuretic peptide to the holding container and at the same time prevent degradation by serine protease. Regarding the prevention of adsorption to the holding container, the material of the holding container is a glass container that is said to be easy to adsorb natriuretic peptides, or is made of polystyrene or polypropylene that is said to be less adsorbed than a glass container Regardless of whether it is, it has a sufficient effect. The shape of the container may be any shape such as a tube shape or a vial shape, and the effect is not lost even if it is frozen and lyophilized in the state where the benzamidine derivative is present.

  Examples of natriuretic peptides that can be stabilized according to the present invention include ANP and CNP in which the content ratio of each amino acid residue in the structure and primary structure is similar to BNP specifically shown in the examples. These natriuretic peptides do not necessarily have to be derived from humans. For example, they can be derived from rats, pigs, dogs, eels, or derivatives having a ring site formed by -SS-bonding. good. Furthermore, the natriuretic peptide may be a chemical synthetic product, a product produced by a gene recombination technique, or the like in addition to an extracted and purified product derived from a living tissue.

  The amount of the benzamidine derivative to be coexisted is preferably set to a certain ratio with respect to the concentration of the natriuretic peptide to be stabilized. However, there are cases where the concentration of natriuretic peptide is unknown, and the actual usage amount is mostly pg / mL, so that the final concentration of the solution is 1 to 100 mM, such as BNP. The effect can be sufficiently obtained by coexisting in an aqueous peptide solution.

  In the present invention, in addition to a benzamidine derivative, a preservative (eg, bovine serum albumin), an antioxidant (eg, ascorbic acid or vitamin E), a binder (eg, carboxymethylcellulose), a wetting agent (eg, cellulose) depending on the purpose. , Polyethylene glycol, etc.), colorants (eg, synthetic food coloring), suspending agents (eg, polyvinyl pyrrolidone, etc.), emulsifiers (eg, alkyl sulfonic acid, etc.), solubilizers (eg, glycerin, etc.), buffering agents (eg, phosphorus) Acid, trishydroxylamine hydrochloride, etc.), isotonic agents (eg, D-sorbitol, sodium chloride, etc.), surfactants (eg, Triton X-100, Tween 20, etc.), etc. There is no hindrance.

  According to the present invention, by coexisting with a benzamidine derivative, it is possible to stably store a natriuretic peptide such as BNP, that is, without changing the value detected by an immunoassay. The effect of allowing the benzamidine derivative to coexist exceeds the effect as a serine protease inhibitor already known for benzamidine, and also has the effect of preventing the adsorption of natriuretic peptide to a glass container. Therefore, beyond the knowledge of conventional benzamidine, according to the present invention, adsorption to a storage container and serine protease can be achieved only by adding a benzamidine derivative without special attention to the material of the container for storing the natriuretic peptide. It is possible to prevent both of the decomposition at the same time.

  Specific embodiments of the present invention will be described by way of examples. However, the present invention is not limited to these examples. In the examples, the natriuretic peptide solution provided by the present invention was measured as a sample for enzyme immunoassay (EIA), but radioimmunoassay (RIA), fluorescent immunoassay (FIA) Alternatively, it can be applied to all methods such as luminescence immunoassay (LIA), and can be applied not only to a sample but also to a control containing a stabilized natriuretic peptide.

  A BNP solution (solution A) was prepared by adding BNP (manufactured by Peptide Institute) to a final concentration of 600 pg / mL in 20 mM Tris-HCl buffer (pH 7.5) supplemented with 1 wt% bovine serum albumin. BNP solutions (Solution B, Solution C, Solution D, Solution E, Solution F, respectively) with benzamidine hydrochloride added to this solution A so that the final concentrations are 5, 10, 20, 50, and 100 mM, respectively. did. The above operation was performed in a polypropylene container.

  After the above adjustment, 1 mL of each solution was dispensed into a glass container. For comparison, 1 mL of each of solutions A and F was dispensed into a polypropylene container to obtain solutions G and H.

  Thereafter, solutions A to F dispensed in glass containers and solutions G and H dispensed in polypropylene containers were freeze-dried, dissolved by adding 1 mL of distilled water, and stored at 4 ° C. BNP concentration was measured at each time point on day 7 and 30. A commercially available BNP measuring reagent (Shionoria BNP kit, trade name, manufactured by Shionogi & Co., Ltd.) was used for the measurement of the BNP concentration. The results are shown in Table 1 (Day 7) and Table 2 (Day 30).

“Concentration percentage” in Tables 1 and 2 is calculated based on the following formula.
Concentration percentage = ((BNP concentration when stored at 4 ° C. for 7 or 30 days) / (BNP concentration immediately after dissolution)) × 100
As is clear from Tables 1 and 2, in solution A not coexisting with benzamidine hydrochloride, the concentration percentage was significantly reduced by storage for 7 days or 30 days, but in solutions B to F, benzamidine hydrochloride Due to the coexistence of the salt, the concentration percentage was 93% or more even after 30 days. These results are for a glass container that is said to be easily adsorbed to the holding container, and the results for the polypropylene container (solutions G and H) showed 96% or more.

  From the above results, it was shown that the coexistence of the benzamidine derivative prevents the adsorption of BNP to the holding container and the degradation by the serine protease, and can provide a natriuretic peptide in which the value detected by the immunoassay does not vary over a long period of time.

Claims (3)

A natriuretic peptide stabilized by the coexistence of a benzamidine derivative. The natriuretic peptide according to claim 1, which is held in a glass or resin container. A method for producing a stabilized natriuretic peptide, comprising coexisting a benzamidine derivative.