JPH06254392A - Separating agent - Google Patents

Abstract

PURPOSE:To provide the separating agent consisting of a fixed phase formed by bonding plasma protein to a carrier and further to provide the method for easily measuring the interaction of the compd. and the plasma protein by this separating agent. CONSTITUTION:The separating agent is obtd. by chemically bonding the plasma protein to the carrier, such as silica gel or synthetic polymer and a column for chromatography is obtd. by packing this separating agent therein. The interaction of the compd. and the plasma protein is measured by using this column. As a result, the interaction of the compd. of medical supplies, etc., and the plasma protein is easily and rapidly measured and the interaction of the medical supplies is easily known.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a separating agent characterized in that a plasma protein bound to a carrier is used.
INDUSTRIAL APPLICABILITY The present invention is used in the field of chemistry, in which the measurement of protein binding has been raised as a technical problem, particularly in the field of pharmaceuticals.

[0002]

2. Description of the Related Art Considering the efficacy and safety of pharmaceuticals, it is strongly demanded to clarify the drug interaction due to multiple drug administration and provide information on appropriate administration method. Has been done. When two or more drugs are administered, pharmacological and physicochemical interactions occur, and the phenomenon in which the enhancement or diminution of drug efficacy is observed is generically called drug interaction. The phenomenon in which a plurality of pharmaceutical agents compete with each other for binding to plasma proteins, including the case of a blood coagulant and an acidic anti-inflammatory agent, has been particularly noted because of the strong action that results.

A number of laboratory techniques have been reported for protein binding of chemical substances. For example, as shown in the following documents 1) to 9).

1) Yuichi Sugiyama: Protein binding experimental method “Pharmacokinetics experimental method for drug development” Manabu Hanano, Koshiro Umemura, Ryuji Iga, Soft Science, Tokyo, 33
7-381 2) Shigeru Goto et al .: Pharmacology, Vol. 31, (1971) p. 24 3) M. T. Ryan et al .: Analytical Biochemistry 40: 364 19
71 (Analytical Biochemistry 40 (1971) 364) 4) J. P. Hummmel et al: Biochemica et Biophysica Acta 63 (196)
2) 532) 5) H. H. Zia et al: Journal of Pharmaceutical Science 64 Vol. 1177, 1975 (Journal of Pharmaceutical Science 64
(1975) 1177) 6) C.I. J. CJ Heftmann et al: Biochemistry 11: 3493 1972 (Bioch
emistry 11 (1972) 3493) 7) M. Yuki Odagiri et al. (M. Otagiri et al): Pharmaceutical Research, Vol. 6, 156, 1989 (Ph
armaceutical Research 6 (1989) 156) 8) O. O. Jardetzky et al: Molecular Pharmacology Vol. 1 pp. 214 1965
Year (Molecular Pharmacology 1 (1965) 214) 9) P. P. Coassolo et al: Biochemical Pharmacology 27: 2787, 1978 (Biochemical Pharmacology 27 (1978) 2787)

Among the above documents, 1) discloses a method for measuring protein binding by equilibrium dialysis, and 2) discloses a method using a dynamic equilibrium method. 3) and 4) disclose a protein binding assay method using filtration as a principle, that is, an ultrafiltration method and a gel filtration method, respectively. However, in the techniques of 1) to 4), a series of operations must be complicated because a compound that is not involved in protein binding must be separated and a quantitative operation must be performed. 5), 6), 7) and 8) disclose a method for measuring protein binding by measuring the spectral change of a complex of compound and protein.
5) is the absorption spectrum method, 6) is the fluorescence spectrum method, 7)
Discloses a circular dichroism spectrum method, and 8) discloses a nuclear magnetic absorption spectrum method. Further, 9) discloses a protein binding measurement method by a microcalorimetric method. Although the method according to 5) to 9) is relatively simple, it is required that a single protein is used. In order to estimate a compound-protein interaction in a system containing two or more kinds of proteins such as plasma, it is necessary to carry out two or more experiments and obtain a plurality of binding constants. Could not be a practical way to do it.

[0006]

[Means for solving the problem] Considering the problems in the previous term,
The present inventors have conducted various studies for the purpose of providing a technique capable of measuring a drug interaction caused by protein binding, which is difficult to evaluate by conventional techniques, using a protein contained in plasma. As a result, they have found that the problems can be solved by the following configurations, and completed the present invention. That is, the present invention is a separating agent comprising a stationary phase in which plasma proteins are bound to a carrier.

The present invention is also a column packed with a stationary phase in which plasma proteins are bound to a carrier. Furthermore, the present invention is a method for measuring the interaction between a compound and plasma protein in high performance liquid chromatography or column chromatography, using a column packed with a stationary phase in which plasma protein is bound to a carrier. According to the present invention, the interaction between a compound and plasma protein can be measured quickly and easily, which is the object of the present invention.

The plasma protein in the present invention may be a plasma protein obtained by a simple method or a commercially available plasma protein and is not particularly limited. Serum contains many kinds of proteins. As a method of classifying them, that is, plasma proteins are classified into albumin fractions, globulin fractions, etc., depending on which region is electrophoretically migrated, and are classified as glycoproteins if they contain sugar. .

The plasma protein used in the present invention is a mixture of such proteins and does not need to be particularly purified. The plasma protein mixture used in the present invention can be obtained, for example, by adding a low molecular weight anticoagulant to blood and then separating blood cells by gel filtration to remove low molecular weight substances. However, it can be easily prepared by using other anticoagulants or removing low-molecular substances by ultrafiltration or the like. The carrier used in the present invention is not particularly limited as long as it can bind to a plasma protein and form a stationary phase, but for example, silica gel, cellulose, synthetic polymer and the like are preferably used.

The method of binding plasma proteins to a carrier is as follows:
It may be carried out according to a method generally used for forming a stationary phase. Thus, for example, aminopropyl silica gel is used as a carrier and plasma protein is bound using N, N-disuccinimidyl carbonate as a cross-linking agent, or silica gel is used as a carrier and 3-glycidoxypropyltrimethoxysilane is used as a cross-linking agent in plasma. To bind proteins,
Alternatively, there may be mentioned a method in which cellulose is used as a carrier and activated with bromocyan, and then a plasma protein is bound thereto.

The present invention is not particularly limited by the method of binding to a carrier, since the main point of the present invention is that a plasma protein mixture is used for measuring drug interaction. As described above, the separating agent according to the present invention is characterized by comprising a stationary phase obtained by binding a plasma protein to a carrier. Therefore, the stationary phase is contained in the separating agent of the present invention as an essential constituent component, but at the same time, other components such as silica gel and cellulose can be arbitrarily selected and added to the separating agent, and the separation efficiency can be increased. Can be appropriately performed for the improvement of

[0012]

[Effect] In the present invention, the phenomenon that drug interaction is observed due to protein binding means that when multiple drugs are administered,
It means a phenomenon that the pharmacological activity of one of the drugs strongly appears because the drugs bind competitively to the drug binding site of the blood protein. Specifically, for example, when phenylbutazone is administered to a patient who has been administered warfarin, the anticoagulant effect of warfarin appears strongly and a bleeding tendency occurs, or when multiple β-lactam antibiotics are administered, single agent administration There is a phenomenon in which the change in blood concentration is different from that in. The separating agent of the present invention is particularly effective for confirming whether the protein binding rate of each drug changes when multidrug administration is performed.

The separating agent of the present invention is mainly used in liquid chromatography. Therefore, the method of use may be carried out by a usual operation in liquid chromatography. For example, the separating agent of the present invention is packed in a column, a drug mixture for drug interaction measurement is charged, and then a buffer solution, an aqueous ethanol solution, etc. The drug interaction may be estimated by circulating the mobile phase and determining the rate of change in retention time when the drug alone is similarly operated.

[0014]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 3 g of aminopropyl silica gel and 2 g of N, N-disuccinimidyl carbonate were mixed with 0.1 M sodium hydrogen carbonate buffer solution.
The mixture was added to 100 ml of (pH 6.8), stirred overnight, taken on a glass filter and washed with water to prepare a suspension of activated aminopropyl silica gel. Separately, prepare a solution prepared by dissolving 30 ml of a 0.1 M sodium hydrogen carbonate buffer (pH 6.8) in which the amount of plasma protein mixture equivalent to that contained in 2 ml of plasma was added to the suspension, and The inventive separating agent was obtained. The obtained separating agent was packed in a steel column to prepare a plasma protein column for measuring drug interaction.

Example 2 10 g of silica gel was dried at 140 ° C. for 24 hours, cooled, suspended in 140 ml of toluene, and 15 ml of 3-glycidoxypropyltrimethoxysilane was added, and the mixture was heated under reflux for 5 hours. After removing the lower boiling fraction from the top, take it on a glass filter,
It was washed successively with toluene, tetrahydrofuran and methanol and dried at 60 ° C for 2 hours to obtain an epoxy activated silica gel. 5 g of this was suspended in 50 ml of borate buffer solution of pH 8.5, a plasma protein mixture in an amount corresponding to that contained in 4 ml of plasma was added, and the mixture was reacted at room temperature for 24 hours. It was put on a glass filter and washed with water to obtain the separating agent of the present invention.
The obtained separating agent was suspended in 20 mM phosphate buffer (pH 7.0) and packed in a column to give a plasma protein column for measuring drug interaction.

Example 3 Commercially available Bromcianum-activated Sepharose 4B was added to 0.1 M sodium hydrogen carbonate buffer (pH 8.3) to swell it, and a plasma protein mixture was added thereto and mixed to obtain the separating agent of the present invention. .

The effects of the present invention will be shown in more detail by the following experimental examples. Experimental Example 1 Using the plasma protein column for drug interaction measurement prepared in Example 1, co-chromatography of phenylbutazone, warfarin and indomethacin was performed. The results are shown in Table 1. The retention coefficient in the table was calculated by the equation 1.

[0019]

[Table 1]

[0020]

[Formula 1]

From Table 1, it was found that the warfarin retention coefficient was remarkably reduced in the presence of phenylbutazone, and it was found that these two drugs exhibit drug interaction via protein binding.

Experimental Example 2 Tolubutamide, sulfamethizole and clofibrate were co-chromatographed using the plasma protein column for measuring drug interaction prepared in Example 2. The results are shown in Table 2.

[0023]

[Table 2]

From Table 2, it was found that the retention coefficient of tolbramide was significantly reduced in the presence of sulfamethizole, and it was revealed that these two kinds of drugs exhibit drug interaction through protein binding. From the above, it is clear that the drug interaction via protein binding can be easily measured by the method for measuring the drug interaction between the separating agent and the plasma protein according to the present invention.

Claims (4)

[Claims] 1. A separating agent comprising a stationary phase in which plasma proteins are bound to a carrier. 2. The separating agent according to claim 1, wherein the carrier is silica gel, cellulose or a synthetic polymer. 3. A column packed with a stationary phase in which a plasma protein is bound to a carrier. 4. A method for measuring the interaction between a compound and plasma protein in a high performance liquid chromatography or column chromatography using a column packed with a stationary phase in which a plasma protein is bound to a carrier.