JP2830139B2 - Blood separation agent - Google Patents

Description

The present invention relates to a blood separating agent. More specifically, when blood is separated by using a specific gravity difference between a blood cell component and a serum component, the separation operation of these two components is performed by forming a partition wall between the two components with an intermediate specific gravity between the two components. The present invention relates to a blood separating agent used for the purpose of facilitating the blood separation.

[Conventional technology]

Conventionally, various compounds have been known as a separating agent used in such a separating operation. For example, silicone oil (JP-A-54-2120, etc.), chlorinated polybutene oil (JP-A-57
No.-9718) or α-olefin-maleic acid diester copolymer (Japanese Patent Application Laid-Open No. 62-199644) and the like, and silica gel for adjusting specific gravity and viscosity or maintaining shape. What added inorganic fine particles, such as clay and clay, or an organic gelling agent is known. On the other hand, in recent years, drug therapy has been actively performed in clinical settings, and thus it is necessary to accurately and rapidly measure the blood concentration of a drug. Since separation treatment of blood and blood can be performed simply and quickly, a separation agent is increasingly used.

[Problems to be solved by the invention]

However, when a conventional separating agent is used, for example, Hospital Pharmacy vol. 12, No. 6,401 (1986), Kyushu Pharmaceutical Society No. 40
No. 39 (1986), etc., there is a problem that the drug component contained in serum decreases with time due to the effect of the separating agent used. This is because the drug component to be measured is absorbed by the separating agent during the blood separating operation, and the blood concentration thereof decreases over time. Therefore, it is inappropriate to use a conventional separating agent for the purpose of measuring the blood concentration of a certain drug, and there has been a strong demand for the development of a separating agent that improves these points.

We have already found that dialkyl esters of α-olefins and maleic acid, or copolymers thereof of a specific composition of chlorinated dialkyl esters, are suitable for this purpose.

However, in this method, there is a point to be further improved in industrial practice, such as difficulty in adjusting the liquid properties as a separating agent.

[Means for solving the problem]

In view of such circumstances, the present inventors have conducted further intensive studies to provide a high-performance blood separating agent such that the component to be measured does not decrease over time during the separation operation,
Even when a conventionally known compound having drug adsorption is used as a blood separating agent, by laminating compounds having no drug adsorption, the time-dependent decrease of the component to be measured can be remarkably reduced, and the blood can be suitably separated. The present invention has been found.

That is, the gist of the present invention is that a liquid compound which has a specific gravity of 1.02 to 1.08 and which may be a gelled liquid compound having substantially no drug-adsorbing property with respect to a layer made of the liquid compound (A) which may be gelled. A blood separating agent obtained by laminating a layer comprising (B), wherein the liquid compound (A) is a copolymer of a maleic acid and / or fumaric acid diester represented by the following general formula (I) and a vinyl compound. At least one compound selected from the group consisting of polymers, polyesters obtained from dibasic acids and diols, low polymers of (meth) acrylates and chlorinated polybutenes, and the liquid compound (B) is ,
At least one compound selected from the group consisting of a copolymer of a maleic acid and / or fumaric acid diester represented by the following general formula (II) and a vinyl compound, polybutene, liquid paraffin and an α-olefin oligomer: Blood separating agent characterized by having (Where R ₁ is a C ₄ -C ₁₈ alkyl group or a phenyl group which may have a substituent, and R ₂ , R ₃ , R ₄ and R ₅ are each independently
It represents an alkyl group of C ₁ -C _14. l is a positive number, m and n
Represents 0 or a positive number independently, provided that 1 ≦ m + n / l ≦
1.4. ) (Where R ₆ represents a C _{4 to} C ₁₈ alkyl group or a phenyl group which may have a substituent, and R ₇ , R ₈ , R ₉ and R ₁₀ each independently represent a C _{1 to} C ₁₄ halogen atom. P represents a positive number, q and r each independently represent 0 or a positive number, and 1.5 ≦ q + r / p ≦ 5, and R ₇ , R ₈ , R _In the case where at least one of ₉ and R ₁₀ is an alkyl group substituted with halogen, 1 ≦ q + r / p ≦ 5).

 Hereinafter, the present invention will be described in detail.

The liquid compound (A) which may be gelled and has a specific gravity of 1.02 to 1.08 used in the present invention is a maleic acid and / or fumaric acid diester represented by the following general formula (I) and a vinyl compound And polyesters obtained from dibasic acids and diols, low polymers of (meth) acrylates, and one or more compounds selected from chlorinated polybutenes, all of which are liquids. It is known as a base material for a separating agent.

(Where R ₁ is a C ₄ -C ₁₈ alkyl group or a phenyl group which may have a substituent, and R ₂ , R ₃ , R ₄ and R ₅ are each independently
It represents an alkyl group of C ₁ -C _14. l is a positive number, m and n
Represents 0 or a positive number independently, provided that 1 ≦ m + n / l ≦
1.4. The vinyl compound as a raw material of the copolymer represented by the general formula (I) is an α-olefin or styrene which may have a substituent.

The α-olefin used in the present invention has 6 to 20 carbon atoms and is a straight-chain or low-branching hydrocarbon having a carbon-carbon double bond at the terminal. Specifically, α-olefins obtained by low-polymerization of ethylene are preferably used, but α-olefins obtained by low-polymerization of propylene, isobutylene and the like can also be used. In the case of these low-polymerized products, they are separated by means such as distillation and used as having a predetermined carbon number. In the present invention, each α-olefin having 6 to 20 carbon atoms may be used alone or as a mixture thereof.

If the α-olefin has more than 20 carbon atoms, the copolymer represented by the general formula (I) becomes a solid at room temperature, which is not preferable. A complicated operation such as a reaction is required, which is not preferable.

Examples of the styrene substituent include a C ₁ -C ₂ alkyl group.

On the other hand, as the diester of maleic acid or fumaric acid, which is another raw material of the copolymer represented by the general formula (I), the alkyl group represented by R _{2 to} R ₅ constituting the ester is a methyl group, Ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, t-butyl group, n
-Hexyl group, n-octyl group, n-decyl group, n-dodecyl group and the like, and straight-chain or branched alkyl groups having 1 to 14 carbon atoms. If the number of carbon atoms is larger than 14, the maleic acid or fumaric acid diester itself becomes a solid, and the copolymer with an α-olefin also becomes a solid, which is not preferable.

Incidentally, the copolymer composition of the vinyl compound and maleic acid and / or fumaric acid diester is preferably in the range of 1 ≦ m + n / l ≦ 1.4 from the viewpoint that the viscosity and / or specific gravity can be adjusted relatively easily. Polyesters synthesized from dibasic acids and diols include dibasic acids having an alkyl chain having 4 to 10 carbon atoms such as adipic acid and azelaic acid, and carbons such as 1,4-butanediol and 1,3-propanediol. Condensates of diols of formulas 2 to 8 can be mentioned (see US Pat. No. 4,350,593).

Further, as the low polymer of (meth) acrylic acid ester, methyl acrylate, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl,
Esters such as t-butyl, n-amyl, isoamyl, hexyl, 2ethylhexyl, heptyl, octyl, capryl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, heptadecyl, stearyl and the like. (See JP-B-59-17385).

Chlorinated polybutene can be obtained by reacting liquid polybutene obtained by low-polymerization of butene with chlorine gas to chlorinate it (see JP-A-57-9718).

The specific gravity of these liquid compounds is adjusted between 1.02 and 1.08 to separate blood components. Known gelling agents such as hydrophobic silica fine powder, hydrophobic clay mineral fine powder (eg, alkylamine-treated bentonite), dibenzylidene sorbitol, hydrogenated castor oil, glutamic acid amide and the like to impart thixotropic properties to the liquid compound. Can be added.

The present invention is characterized in that a layer made of a liquid compound (B) which may be gelled and has substantially no drug-adsorbing property is laminated on a layer made of a known liquid compound (A).

In the present invention, the liquid compound (B) which does not substantially have a drug-adsorbing property and may be gelled includes a maleic acid and / or fumaric acid diester represented by the following general formula (II) and a vinyl compound: , Polybutene, liquid paraffin, and oligomers of α-olefins.

(Where R ₆ represents a C _{4 to} C ₁₈ alkyl group or a phenyl group which may have a substituent, and R ₇ , R ₈ , R ₉ and R ₁₀ each independently represent a C _{1 to} C ₁₄ halogen atom. And p represents a positive number, q and r each independently represent 0 or a positive number, and 1.5 ≦ q + r / p ≦ 5, but R ₇ , R
_When at least one of ₈ , R ₉ and R ₁₀ is an alkyl group substituted with halogen, 1 ≦ q + r / p ≦ 5. The vinyl compound which is a raw material of the copolymer represented by the general formula (II) is the same as the vinyl compound which is a raw material of the copolymer represented by the general formula (I).

On the other hand, as a diester of maleic acid or fumaric acid, which is another raw material of the copolymer represented by the general formula (II), maleic acid or fumaric acid which is a raw material of the copolymer represented by the general formula (I) is used. And the ester moiety may be further substituted with a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. As the halogen atom, a chlorine atom is particularly preferable.

The diester of maleic acid or fumaric acid is obtained by converting maleic anhydride or the like and an alcohol having 1 to 14 carbon atoms and / or an alcohol having 1 to 14 carbon atoms having at least one halogen atom to an acid catalyst such as p-toluenesulfonic acid. Or by a method such as dehydration-condensation in the presence of an alkali catalyst, or transesterification of a diester of maleic acid or fumaric acid with an alcohol having a predetermined number of carbon atoms in the presence of a similar acid or alkali catalyst. You. Also,
The maleic acid or fumaric acid diester containing a halogen atom has at least a portion containing a halogen atom in at least one of its ester chains, and therefore has 1 to 14 carbon atoms having at least one halogen atom as an alcohol.
And alcohols having 1 to 14 carbon atoms in combination.

The maleic acid or fumaric acid diester only needs to have at least a part thereof containing a halogen atom in at least one of its ester chains, for example, containing a halogen atom only in one of the ester chains of the maleic acid or fumaric acid diester. Alternatively, both ester chains may contain a halogen atom, or one or both ester chains may contain a plurality of halogen atoms. Also, only a part of the maleic acid or fumaric acid diester may contain a halogen atom in its ester chain, or all of the maleic acid or fumaric acid diester may contain a halogen atom in both ester chains.

In the general formula (II), when the alkyl group represented by R _{7 to} R ₁₀ does not contain halogen, 1.5 ≦ q + r / p ≦ 5
Is preferred. If it is less than 1.5, it will not be easy to laminate on the layer composed of the compound (A), or it will not be possible to use the component to be measured while storing the blood in the separation tube containing the blood separation agent prior to the blood separation operation. The concentration of a drug in a certain blood decreases significantly with time, which is not preferable.
On the other hand, if the above molar ratio is too large, the specific gravity of the copolymer becomes too large, which is not preferable.

When 1.7 ≦ q + r / p ≦ 4.0, the change of the acidic, neutral and basic drug concentrations over time during the separation operation is suppressed, which is more preferable, and the range of 2.0 ≦ q + r / p ≦ 3.0 is particularly preferable.

On the other hand, when at least one of R _{7 to} R ₁₀ is an alkyl group substituted with halogen, the range of 1 ≦ q + r / p ≦ 5 is preferable, and the content of the halogen atom is one molecule of the copolymer. Among them, an average of 2.5 to 60% by weight is preferred. More preferably, it is 10 to 30% by weight.

The polybutene used in the present invention is obtained by low polymerization in the presence of a radical initiator or an ionic initiator alone or as a mixture of isobutylene or n-butene, has a viscosity at 38 ° C of 5 to 50000 cP and a molecular weight of 200. A thing of about 4000 is preferable. Liquid paraffin specified in JIS K2231 can be used.

Oligomers of α-olefins are α-olefins having 6 to 18 carbon atoms.
Olefin oligomer having a molecular weight of 500 to 15,000
About 0 is preferable.

The above liquid compound (B) may be a known gelling agent such as hydrophobic silica fine powder, hydrophobic clay mineral fine powder (eg, bentonite treated with alkylamine), dibenzylidene sorbitol, hydrogenated castor oil, and glutamic acid amide, if necessary. May be added to impart thixotropic properties.

The liquid compounds (A) and (B) are used by being injected into a glass or plastic bottomed test tube.

The blood separating agent of the present invention is formed by laminating a layer composed of the liquid compound (B) on a layer composed of the liquid compound (A). As a method of laminating a layer made of the liquid compound (B) on a layer made of the liquid compound (A), first, the liquid compound (A) is injected into a glass or plastic bottomed tube, and then the liquid compound (A) is added. (B) as it is,
Alternatively, if necessary, the solution is injected after being diluted with a common organic solvent such as alcohol, acetone, benzene, and toluene.

When a solvent is used, the layers can be laminated by evaporating the solvent after injection. The amounts of compound (A) and compound (B) used are (A) :( B) = 95: 5 to 50: 5 by volume ratio.
The range of 0 is preferable, and the ratio is more preferably 95: 5 to 70:30. If the amount of (B) is less than the above range, a sufficient blocking effect will not be exhibited, and if it is more than the above range, the performance of the base compound (A) will be impaired or the cost will be too high. The blood separating agent of the present invention prepared as described above is pale yellow, transparent, and odorless.

When the blood separating agent of the present invention is used for separating blood, it floats by a separating operation to form a partition and separates into a serum layer and a clot layer. The specific gravity of compounds (A) and (B) Thus, the partition wall is formed as a mixture of the compounds (A) and (B) or as a partial mixture, or the layer substantially composed of the compound (A) forms the partition wall, and the layer composed of the compound (B) is formed. It takes various forms, such as when present on the clot layer side.

(Operation)

The blood separating agent of the present invention has a structure in which a layer made of the liquid compound (B) having substantially no drug-adsorbing property is laminated on a layer made of the liquid compound (A). Therefore,
When the blood separating agent is used for separating blood, prior to the blood separation, even if the collected blood is injected into a bottomed tube containing the blood separating agent at the bottom and stored for a long period of time, the blood can be stored. Since it is in contact with the liquid compound (B) having substantially no drug-adsorbing property, there is no fear of preventing the drug from being adsorbed and causing a phenomenon such as elution of blood.

Therefore, it is possible to suppress the blood concentration of the drug component from changing over time. In addition, the blood separating agent exhibits favorable fluidity during centrifugation, and forms a partition wall between blood serum and other components, so that it is suitably used for blood separation.

〔Example〕

Next, specific embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples unless it exceeds the gist thereof.

Production Example 1 A mixture of dodecene and tetradecene (trade name dialen
124: manufactured by Mitsubishi Kasei Corporation) 182 g (1 mol) and dimethyl maleate 144 g (1.0 mol) are charged into a four-necked flask, and heated to 160 ° C. under a nitrogen atmosphere while stirring with di-t-butyl peroxide (product). Name Kayabutyl D: manufactured by Kayaku Nury)
11 g (0.08 mol) were added over about 4 hours. Then about 1
After stirring at 65 ° C. for 1 hour, the mixture was heated under reduced pressure to remove light boiling components.

The yield of the obtained copolymer was 323 g (99% yield), and the specific gravity was
1.03, molecular weight (measured by gel permeation chromatography and converted to polystyrene) about 3,50
At 0, the copolymerization molar ratio was α-olefin: maleic acid diester = 1: 1.

Dibenzylidene sorbitol 0.25 in 100 g of this copolymer
g, and the mixture was heated and stirred at 140 ° C. for 1 hour, and then allowed to cool to give thixotropic properties.
(Specific gravity 1.03).

Production Example 2 100 g of liquid polybutene having a specific gravity of 0.860 and a viscosity of 400 cP was charged into a glass reactor having a stirrer and a high-pressure mercury lamp, and chlorine gas was blown at a rate of 70 ml / min. A time reaction was performed. Then, the temperature was raised to 80 ° C., and dechlorination under reduced pressure was carried out for 6 hours.
A reaction product (chlorinated polybutene) of 00 centipoise was obtained.

To 100 g of the reaction product, 2 g of smectite clay treated with a quaternary ammonium salt was added and kneaded with a three-roll mill to obtain a liquid compound A-II (specific gravity 1.06).

Production Example 3 A four-necked flask was charged with 188.2 g of azelaic acid, 78.1 g of neopentyl alcohol, and 27.0 g of 1,3-butanediol,
The esterification reaction was performed at 225 ° C. for about 4 hours in a stirred nitrogen stream. When about 36 g of water was distilled off, nitrogen was stopped, the pressure was reduced to 10 mmHg by a vacuum pump, and the reaction was continued for another 4 hours. Then, 0.1 g of dibutyltin was added, and the reaction was continued for 6 hours while reducing the pressure to 4.5 mmHg to obtain 255.1 g of a copolymer (yield 99.1%). The specific gravity was 1.05 and the viscosity was 60,000 cP. 2 g of fumed silica was added to 100 g of this copolymer to obtain a liquid compound A-III (specific gravity 1.07) in the same manner as in Production Example 2.

Example 4 A four-necked flask was charged with 240 g of toluene and kept at 105 ° C. Under stirring, a mixture of 144 g of 2-ethylhexyl acrylate, 216 g of butyl acrylate, and 3.6 g of t-butyl per-oxide was continuously dropped in a nitrogen stream over about 3 hours, and after maintaining at about 104 ° C. for 2 hours, The solvent, unreacted substances and the like were removed by distillation under reduced pressure. The yield of the obtained copolymer was 356.9 g (99.1% yield), the specific gravity was 1.03, and the viscosity was 52,000 cP.

1.3 g of dibenzylitene sorbitol in 100 g of this copolymer
And the liquid compound A-IV (specific gravity) in the same manner as in Production Example 1.
1.03).

Production Example 5 Dibutyl fumarate 16 was added to the four-necked flask of 1.
0 g (0.7 mol) and 102 g (0.3 mol) of di-2-ethylhexyl fumarate were charged at 160 ° C. under a nitrogen atmosphere.
Then, under stirring, a mixture of 104 g (1 mol) of styrene and 8.76 g (0.06 mol) of di-tert-butyl peroxide was added dropwise continuously over 5 hours. After the addition, the mixture was further added at the same temperature for 1 hour. The reaction was allowed to proceed. After the reaction is over, 1
Under reduced pressure of 33 mmHg, light boiling components and unreacted monomers contained in the reaction mixture were removed by distillation to obtain a liquid compound.

The yield of the obtained copolymer was 329 g (99% yield),
The specific gravity was 1.04 and the viscosity was 90,000 cP.

100 g of this copolymer and 0.25 of dibenzylidene sorbitol
Using g, a liquid compound AV (specific gravity 1.04) was obtained in the same manner as in Production Example 1.

Production Example 6 122 g (1 mol) of octene and 288 g of dimethyl maleate
(2 mol) was charged into a four-necked flask, and 20.0 g (0.14 mol) of di-t-butyl peroxide was added under the same conditions as in Example 1. Then, after stirring at about 165 ° C for 1 hour,
By heating under reduced pressure to remove light boiling components, the liquid compound BI
I got

The yield of the obtained copolymer was 402 g (98% yield), and the specific gravity was
1.08, the molecular weight was about 3,500, and the copolymerization molar ratio was α-olefin: maleic acid diester = 1: 2.04.

Production Example 7 98 g (1 mol) of maleic anhydride and 180 g (2.2 mol) of 2-chloroethanol were dissolved in xylene 1, 1 g of p-toluenesulfonic acid was added, and the mixture was distilled while heating and stirring at about 110 ° C. for 10 hours. I drew out the water to make. After allowing to cool, neutralize with an aqueous solution of sodium hydrogen carbonate and wash with water,
Magnesium sulfate was added, shaken for a while, filtered off, and the filtrate was concentrated. The filtrate was heated under reduced pressure to obtain bis (2-chloroethyl) maleate. 230g yield
(94% yield).

Then, 56.0 g (0.33 mol) of dodecene and 97.0 g (0.40 mol) of bis (2-chloroethyl) maleate were charged into a four-necked flask, and 4.3 g of t-butylperoxyoctoate was charged.
(0.02 mol) was added under the same conditions as in Production Example 1. Thereafter, the mixture was stirred at about 110 ° C. for 1 hour, and then heated under reduced pressure to remove light-boiling components to obtain a liquid compound B-II.

The yield of the obtained copolymer was 100 g (yield 97%), and the specific gravity was
1.18, copolymerization molar ratio: dodecene: bis (2-maleate)
Chloroethyl) = 1: 1.2.

Examples 1 to 8 and Comparative Examples 1 to 6 Liquid compounds (AI) to (AV) described in Table 1 were synthesized in a test tube having a constant diameter, and then the liquid compounds (B- I) to (B-II) were added at the ratios shown in Table 1 so that the total amount became about 1.5 g, and 1 cc of bovine serum containing a certain concentration of the drug was added thereto, and the mixture was added at 20 ° C. At rest. After 72 hours, only bovine serum was collected, and the amount of drug contained therein was measured by the immunofluorescent immunoassay. The ratio of the drug concentration in the bovine serum after standing for 72 hours relative to the drug concentration in the original bovine serum was determined and defined as the drug residual ratio.

Evaluation: Drug residual rate 85% or more: ○, 75-85% △, 75%
The following was evaluated as x.

[Effect of the Invention] A liquid compound (B) having substantially no adsorptivity to the drug is laminated on a conventionally used blood separating agent (A) having an adsorptivity to a specific drug. The blood separating agent according to the present invention does not impair the separation performance of the conventional blood separating agent, does not significantly control the liquidity of the liquid compound (B), and furthermore, the blood separating agent during the blood separating operation. It is possible to suppress the change with time and is extremely useful in practical use.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C07B 63/00 G01N 33/48

Claims (2)

(57) [Claims] 1. A liquid compound (B) which has substantially no drug-adsorbing property with respect to a layer comprising the liquid compound (A) which may be gelled and has a specific gravity of 1.02 to 1.08. A blood separating agent obtained by laminating layers consisting of
The liquid compound (A) is a copolymer of a maleic acid and / or fumaric acid diester represented by the following general formula (I) and a vinyl compound, a polyester obtained from a dibasic acid and a diol, (meth) acrylic acid At least one compound selected from the group consisting of a low polymer of ester and chlorinated polybutene, and the liquid compound (B) is a compound of maleic acid and / or fumaric acid represented by the following general formula (II): A blood separating agent, which is at least one compound selected from the group consisting of a copolymer of a diester and a vinyl compound, polybutene, liquid paraffin, and an oligomer of α-olefin. (Where R ₁ represents a C ₄ -C ₁₈ alkyl group or a phenyl group which may have a substituent, and R ₂ , R ₃ , R ₄ and R ₅ each independently represent a C ₁ -C ₁₄ alkyl group. 1 represents a positive number, m and n each independently represent 0 or a positive number, provided that 1 ≦ m + n / l
≦ 1.4. ) (Where R ₆ represents a C _{4 to} C ₁₈ alkyl group or a phenyl group which may have a substituent, and R ₇ , R ₈ , R ₉ and R ₁₀ each independently represent a C _{1 to} C ₁₄ halogen atom. And p represents a positive number, q and r each independently represent 0 or a positive number, and 1.5 ≦ q + r / p ≦ 5, provided that R ₇ , R ₈ ,
When at least one of R ₉ and R ₁₀ is an alkyl group substituted with halogen, 1 ≦ q + r / p ≦ 5. ) 2. The method according to claim 1, wherein the volume ratio of the liquid compounds (A) and (B) is in the range of (A) :( B) = 95: 5 to 50:50. Blood separation agent.