JP3453522B2 - Pyrmenol derivative and anti-pirmenol antibody - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention is useful for quantifying pirmenol in biological samples such as serum or urine,
The present invention relates to a labeled antigen or an immunogen, a pirmenol derivative, and an anti-pirmenol antibody.

[0002]

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION Pirmenol hydrochloride [(±) -4- (cis-2,6-dimethylpiperidino) -1-phenyl-1- (2-pyridyl)]
Butanol monohydrochloride monohydrate] is an antiarrhythmic agent represented by the following formula.

[0003]

[Chemical 2]

Due to its electrophysiological properties, the drug is Vaug
It has been reported that it belongs to the class Ia group antiarrhythmic drugs of the Han Williams classification and exhibits an excellent inhibitory effect on various arrhythmias such as ventricular premature contraction and supraventricular premature contraction. The blood levels of pirmenol needed to maintain these effects are
It is said to be about 400 to 1000 ng / mL.

On the other hand, side effects of this drug include dysuria,
Dry mouth, blurred vision, syncope, dizziness, light-headedness, numbness of limbs, etc. have been reported, and if the blood concentration of pirmenol in a patient is higher than necessary, the possibility of these side effects increasing.

Even if the same amount of drug per body weight is administered to each patient, the same blood concentration cannot be obtained. This is because there are individual differences in drug absorption, distribution, metabolism, excretion, etc. For example, in a person with impaired renal function, the drug may be easily accumulated in the body, and when there is a concomitant drug, the interaction with the drug may affect the blood concentration.

Therefore, in the field of prevention or treatment of arrhythmia, it is necessary to measure the blood concentration of pirmenol in each patient as a proof of the administration design for maintaining the optimum blood concentration of pirmenol.

Conventionally, high performance liquid chromatography (HP
LC) is used to measure blood pirumenol [eg Kato, R. et al., Jpn. J. Clin. Pharma.
col. Ther., 23 , 495-505 (1992)].

However, the HPLC method requires special equipment and its operation is complicated, and the precision of the measurement is easily influenced by the skill of the measurer. Moreover, in this method, a biological sample such as serum cannot be directly measured, and a pretreatment for extracting pirmenol is required.

Therefore, in the HPLC method, a large number of samples cannot be processed at one time, and the measurement of pyrmenol by this method is not a very preferable method for daily clinical examination.

Under such circumstances, it is desired to provide a reagent capable of measuring pyrmenol rapidly, accurately and simply.

[0012]

In order to solve the above-mentioned problems, the present inventors have earnestly studied for a reagent capable of measuring pyrmenol rapidly, accurately and easily, and as a result,
By performing an immunoassay of pirmenol using a pirmenol-labeled antigen bound to a label in the pyridine ring portion of pirmenol, and a highly specific anti-pirmenol antibody prepared by an immunogen bound to a carrier substance in the part, It was found that the intended purpose could be achieved, and further research was conducted to complete the present invention.

The present invention relates to a pyrmenol derivative represented by the following general formula (I).

[0014]

[Chemical 3]

(In the formula, L is a binding group, and X means a residue of a labeling substance or a residue of a carrier substance.) Furthermore, the present invention relates to an anti-pyrumenol antibody.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION When X of the compound (I) of the present invention is a carrier substance residue, it is an immunogen for producing an anti-pirmenol antibody (hereinafter sometimes simply referred to as immunogen), and X is When it is a labeled residue, it is a pyrmenol-labeled antigen (hereinafter sometimes simply referred to as a labeled antigen).

The carrier substance in the definition of X of the compound (I) of the present invention may be any as long as it can bind to the compound (I) and the resulting conjugate can serve as an immunogen, for example, proteins and polypeptides. Is mentioned. Specific examples include albumin, globulin, cycloglobulin, shell hemocyanin, edestin and the like. Preferred is albumin or shell hemocyanin.

Examples of labeling substances in the definition of X include enzymes, fluorescent substances, radioactive substances and the like. Specific examples of the enzyme include peroxidase, β-D-galactosidase, lipase, alkaline phosphatase, glucose-6-phosphate dehydrogenase and the like. Further, examples of the fluorescent substance include fluorescein and its derivatives, and examples of the radioactive substance include
¹²⁵ I and the like can be mentioned.

The linking groups in the definition of L are known in the art. The binding group is appropriately selected depending on the specific pair of the pyrmenol derivative represented by the general formula (II) or a salt thereof and the carrier substance or the labeled substance.

These linking groups include a binder, for example, a homobifunctional binder such as glutaraldehyde or m-maleimidobenzoyl-N-hydroxysuccinimide (MB).
It also includes a linking group formed as a result of binding with a heterobifunctional binder such as S).

When X is a carrier substance residue or an enzyme residue, that is, when X is a protein or polypeptide, specific examples of the linking group L include those represented by the following formula.

[0022]

[Chemical 4]

[0023]

[Chemical 5]

(In the formula, Q means a phenylene group or a lower alkylene group).

Specific examples of the linking group L in the case where X is a radioactive substance such as ¹²⁵ I include the following.

[0026]

[Chemical 6]

When X is a fluorescent substance such as fluorescein or a derivative thereof, examples of the bonding group include a bonding group formed as a result of a bonding reaction (described later) between fluorescein and compound (II) described later.

Further, these linking groups are used to provide an additional space between the portion corresponding to pyrmenol in the above formula (I) (hereinafter referred to as hapten portion) and the carrier substance or the labeled substance. A spacer may be included. For example, as a spacer, C ₁ -C ₂₀ which may contain a hetero atom such as O, S or N
And a straight-chain or branched alkylene group.

A preferable example of the bonding group containing a spacer is --Z-(CH ₂ ) n--CONH-- (wherein Z represents an oxygen atom or a sulfur atom, and n is 1 to 6).
Include groups represented by the an integer), where the definitive -Z- (CH ₂₎ n-corresponds to a spacer moiety.

Preferred compound (I) of the present invention is -L-X.
Is a compound in which the group represented by is bonded to the 3-position of the 2-pyridyl group.

The bond ratio between X and the hapten moiety is not shown in the formula (I), but varies greatly depending on the type of X. For example, when X is a carrier substance residue, the binding ratio of both is 1 to 50 mol of the hapten moiety to 1 mol of the carrier substance residue. In this case, the bonding ratio of the both can be adjusted by changing the kind of the substituent R or the binder in the compound (II) described later, or by changing the used ratio or the reaction condition of them in the bonding reaction.

When X is an enzyme residue, the proportion of the hapten moiety is 1 to 1 for 1 mol of the enzyme residue.
The amount is 0 mol, and preferably 1 to 5 mol. This binding ratio can be adjusted in the same manner as in the case of the above-mentioned carrier substance.

When X is a radioactive substance residue or a fluorescent substance residue, the binding ratio of the radioactive substance residue or the fluorescent substance residue is 1 to 1 mol of the hapten moiety.
It is equivalent to 10 moles. This binding ratio can also be adjusted in the same way as for the carrier substance.

The pyrmenol derivative of the present invention can be obtained, for example, by the following general formula (II)

[0035]

[Chemical 7]

(In the formula, R means a group capable of being bonded to the carrier substance or the labeled substance directly or by a binder)
It can be produced by binding the compound represented by or a salt thereof with a carrier substance or a labeled substance according to a conventional method in this field.

For example, the pyrmenol derivative of the present invention in which X is a residue of the carrier substance converts the carboxyl group contained in the substituent R of the compound (II) into an active ester, which is condensed with the amino group of the carrier substance. Alternatively, it can be produced by binding the amino group contained in R and the amino group of the carrier substance with the aid of a binder, preferably a binder such as glutaraldehyde, toluene diisocyanate or dihalogenated nitrobenzene. It is also possible to produce the pyrmenol derivative of the present invention in which X is a carrier substance residue by diazotizing the amino group and then coupling it with a tyrosine residue or a histidine residue of the carrier substance.

In the pyrmenol derivative of the present invention in which X is a residue of the carrier substance, the thiol group contained in the substituent R of the compound (II) and the amino group of the carrier substance are bound to each other using a binder. Can be manufactured by Examples of the binder that crosslinks between the thiol group and the amino group include N- (m
-Maleimidobenzoyloxy) maleimide succinimide derivatives such as succinimide and the like.

The pyrmenol derivative of the present invention in which X is an enzyme as a labeling substance, that is, an enzyme-labeled antigen can be produced in the same manner as in the case of the immunogen described above. In this case, it is produced under mild conditions so that the enzyme activity is not inactivated.

X is a radioactive substance as a label, for example, ¹²⁵
The pirmenol derivative of the present invention which is I, that is, the radioactive substance-labeled antigen is a method using a Bolton Hunter reagent,
It can be produced by any known method such as the chloramine T method and the lactoperoxidase method.

Further, X is a fluorescent substance as a labeling substance, for example,
The pirumenol derivative of the present invention which is fluorescein or its derivative, that is, the fluorescence-labeled antigen is, for example, a compound (II) containing a carboxylated fluorescein and a substituent R containing an amino group according to the method disclosed in JP-A-57-150680. Can be condensed, preferably in the presence of a condensing agent, or according to the method disclosed in JP-A-57-58695.
It can be produced by reacting dichlorotriazinylaminofluorescein which is a reaction product of aminofluorescein and cyanuric chloride with a compound (II) having a substituent R containing an amino group.

The present invention also relates to anti-pirumenol antibodies, and not only polyclonal antibodies but also monoclonal antibodies are included in the antibodies of the present invention.

The polyclonal antibody of the present invention uses the pyrmenol derivative (I) in which X is a carrier substance as an antigen,
It is produced by immunizing non-human animals. More specifically, the polyclonal antibody of the present invention is parenterally administered (immunized) to a nonhuman animal such as rabbit, guinea pig, sheep and goat by mixing the pirmenol derivative (I) with a suitable adjuvant. It can be produced by collecting serum and performing a known treatment.

The monoclonal antibody can be produced by collecting antibody-producing cells of the animal thus immunized from the spleen, and then performing operations such as fusion with myeloma cells and screening of clonal cells according to a conventional method.

The antibody of the present invention can be used in an immunoassay method for pyrmenol. In this case, the antibody of the present invention is usually used in the form of an insolubilized antibody in order to advantageously perform the B / F separation described later. The antibody can be insolubilized by binding the antibody and the insoluble carrier in the same manner as in the method for producing the pyrmenol derivative (I), or by physically adsorbing it to the insoluble carrier. Examples of the insoluble carrier include bacterial cell wall, natural insoluble polysaccharide, chemically treated dextran gel, agar gel, plastic beads, acrylamide gel, glass beads, fine metal powder, synthetic rubber tube, metal chip and the like. The antibody can also be insolubilized by adsorbing the antibody to the microplate well.

For the determination of pirmenol, the pirmenol in the sample and the labeled antigen are allowed to undergo an antigen-antibody reaction competitively with the anti-pirumenol antibody, and then the labeled antigen bound to the antibody and the free labeled antigen are separated ( B / F separation), and the labeling activity of either of them is measured. B
The / F separation can be advantageously performed by using the above-mentioned insolubilized antibody, an antibody against the immunoglobulin of the animal used for antibody preparation (second antibody), or the like.

Further, using fluorescein or a derivative thereof as a labeling substance, the fluorescence polarization immunoassay method (FPIA; Fluorescence Polarization) disclosed in JP-A-57-58695.
Immunoassay) can be used to quantify pirmenol. This method allows quantification of pirmenol without performing B / F separation.

The compound (II) or a salt thereof used in the production of the pyrmenol derivative of the present invention is a novel compound and can be produced, for example, by the method described in Reference Example below or a method analogous thereto.

[0049]

EXAMPLES The present invention will be described more specifically with reference to the following Reference Examples and Examples.

Reference Example 1- (±) 4- (cis-2,6-dimethylpiperidino) -1
Synthesis of-(3-hydroxypyridin-2-yl) phenylbutanol

2-Bromo-3-methoxymethoxypyridine (8.8 g) was dissolved in diethyl ether (300 mL) at -60 ° C.
N-Butyllithium (1.6M hexane solution) with stirring
28 mL was added dropwise. After 10 minutes, (±) 4- (cis-2,6
-Dimethylpiperidino) -1-phenylbutanone 10.4 g
Was added to 100 mL of diethyl ether, and added to -40
The temperature was raised to ℃ and stirred for 10 minutes. The reaction mixture was added to ice water, saturated aqueous sodium hydrogen carbonate and then ethyl acetate were added, and the organic layer was separated. After the solvent was distilled off, the residue was purified by basic silica gel column chromatography (hexane: ethyl acetate = 2: 1) to obtain 10.1 g of a yellow oily substance. 3.0 g of this to 50 m of ethanol
It was dissolved in L, 20 mL of 20% aqueous sulfuric acid was further added, and the mixture was refluxed with stirring for 1 hour and a half. Ethyl acetate was added, the mixture was made alkaline with saturated aqueous sodium hydrogen carbonate, and the organic layer was separated. After distilling off the solvent, the residue was subjected to basic silica gel column chromatography (hexane: ethyl acetate =
It was purified by 1: 2) and recrystallized from isopropyl ether to obtain 2.0 g of the desired title compound (colorless crystal). Melting point: 117-119 ° C

Reference Example 2- (±) 4- (cis-2,6-dimethylpiperidino) -1
-[3- (3-Carboxypropoxy) pyridine-2-
Synthesis of phenyl] phenylbutanol dihydrochloride

(±) 4- (cis-obtained in Reference Example 1)
3.5 g of 2,6-dimethylpiperidino) -1- (3-hydroxypyridin-2-yl) phenylbutanol were dissolved in 30 mL of dimethylformamide and then 0.4 g of 60% sodium hydride was added. Further, 1.95 g of ethyl 4-bromobutanoate was added, and the mixture was stirred at room temperature for 1 hour. Water was added to the reaction mixture, the mixture was extracted with ethyl acetate, the solvent was evaporated, 50 mL of 10% aqueous hydrochloric acid was added to the residual oily substance, and the mixture was stirred under reflux for 1 hr. After the water was distilled off, diethyl ether was added for crystallization to obtain 3.1 g of the title compound. Mass spectrum: MH ⁺ 441

Example 1-Preparation of bovine serum albumin conjugate (immunogen)

1.215 mmol / 6 mL of the compound obtained in Reference Example 2
Distilled aqueous solution, N-hydroxysuccinimide 1.215 mmol /
A mixture of 2 mL distilled aqueous solution and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 1.337 mmol / 2 mL distilled aqueous solution was allowed to stand at room temperature for 25 minutes to give an N-hydroxysuccinimide ester aqueous solution of the compound.
Prepared mL. 5 ml of this ester solution and 200 mg of bovine serum albumin (BSA, Fraction V; Bayer) 50 mm
The solution dissolved in 100 mL of ol / L borate buffer (pH 9.5) was mixed, and the mixture was left for 1 hour with stirring under ice cooling. This reaction solution was placed in a dialysis tube (manufactured by Visking) and dialyzed against 5 L of distilled water eight times in total. This was freeze-dried to obtain 200 mg of the title conjugate as a dried product.

Example 2-Preparation of keyhole limpet hemocyanin conjugate (immunogen)

200 mg of a dried product of the title conjugate was obtained in the same manner as in Example 1 except that keyhole limpet hemocyanin (KLH, manufactured by CALBIOCHEM) was used instead of bovine serum albumin.

Example 3-Preparation of horseradish-derived peroxidase conjugate (labeled antigen)

Peroxidase derived from horseradish (HRP
O (manufactured by Toyobo Co., Ltd.) 5 mg (as protein) was dissolved in 5 mL of 50 mmol / L borate buffer (pH 9.5). Then, 0.5 mL of an aqueous solution of the N-hydroxysuccinimide ester of the compound obtained in Reference Example 2 was prepared in the same manner as in Example 1, and this was added to the above enzyme solution and stirred under ice-cooling to prepare 1 Left for hours. This solution was ultrafiltered using Omega Cell (manufactured by Filtron), the residue remaining on the membrane was washed with 50 mL of 20 mmol / L phosphate buffer (pH 7.0), and then 20 mmol / L phosphate buffer ( It was washed with 3 mL of pH 7.0) to make the final volume 5 mL. This was used as a labeled antigen solution and stored in a refrigerator.

Example 4 -Preparation of anti-Pirmenol antibody

The bovine serum albumin conjugate prepared in Example 1 was dissolved in 0.9% NaCl to a concentration of 0.2%,
Emulsified by adding an equal amount of Freund's complete adjuvant was injected at 0.25 mL at 1 foot pad and 7 subcutaneous sites at the back of the rabbit for the first immunization, and 0.25 mL at 8 subcutaneous sites on the back for the second and subsequent immunizations. Each mL was injected. After that, immunization was performed 7 times in total every 2 weeks, and 10 days after the final immunization, whole blood was collected from the carotid artery to obtain anti-pirmenol serum.

Reference Example 3- Production of enzyme-linked immunosorbent assay reagent for pirmenol in serum

A title reagent consisting of the following components (1) to (4) was prepared.

Standard solution : Pirmenol was dissolved in a small amount of purified water and then appropriately diluted with human serum to prepare a standard solution (10
0,250,500,1000,2000 ng / mL) was prepared. The standard solution with a concentration of pirmenol of 0 is the human serum.

Sample diluent : 0.1% BSA-0.9% Na
12 mL of Cl-0.04 mol / L phosphate buffer (pH 7.0) was filled in a 15 mL brown bottle.

Anti-pirmenol antibody : 8 mL of the antiserum prepared in Example 4 diluted 1.5 million times with the above-mentioned sample diluent.
Was packed in a 12 mL amber bottle.

Enzyme-labeled antigen : 20% of the labeled antigen solution prepared in Example 3 was added with 1.0% BSA-0.9% NaCl-0.04mo.
7.98 mL of l / L phosphate buffer (pH 7.0) was added for dilution.
This was packed in a 15 mL amber bottle.

Insolubilized secondary antibody binding well : anti-rabbit Ig
G To 20 mL of goat serum (manufactured by Scantibodies), 20 mL of 0.2 mol / L phosphate buffer (pH 6.5) was added, and 40 mL of saturated ammonium sulfate solution was added to it under ice cooling, stirred for 20 minutes, and then centrifuged at 16000 rpm for 30 minutes. The precipitate was collected. This precipitate was dissolved in 40 mL of 0.02 mol / L phosphate buffer (pH 6.5), and this solution was ultrafiltered using Omega Cell (manufactured by Filtron). The residue remaining on the membrane was washed with 20 mmol / L phosphate buffer (pH 6.5) 50 mL x 3 times, and then the DEAE cellulose anion exchange column (φ
2.5cm x 14cm: Whatman) purified and anti-rabbit
About 40 mL of IgG fraction of IgG goat serum was obtained. About 40 mL of the IgG fraction of anti-rabbit IgG goat serum was dissolved in 50 mmol / L citrate buffer and solidified in a microplate well (96 wells), and the well was packed in an aluminum bag.

Stock solution for washing : 0.2 mol containing 1.0% polyoxyethylene sorbitan monolaurate and 9.0% NaCl
/ L phosphate buffer (pH 7.0) was prepared and 30 mL was packed in a 30 mL plastic bottle. This washing stock solution should be purified water before use.
It was diluted 10-fold and used as a washing solution.

Substrate : Tetramethylbenzidine (TM
14 mL of a substrate solution (manufactured by DAKO) containing B) was packed in a 15 mL light-shielding plastic bottle.

Stop solution : 14 mL of 1.6 N sulfuric acid solution was filled in a 30 mL plastic bottle.

Reference Example 4- Quantification of pirmenol by enzyme immunoassay (preparation of standard curve)

Using the reagents produced in Reference Example 3, a standard curve for the determination of pyrmenol was prepared according to the following procedure.

Pre-prepare the sample diluent () on the preplate.
Then, 30 μL of the standard solution () was dispensed, and the mixture was stirred for 1 minute and diluted 4-fold. Insolubilize this standard solution
25 μL each was dispensed to the antibody-binding well (), and then 50 μL of labeled antigen solution () and 50 μL of antiserum solution () were dispensed. After stirring for 30 seconds, the mixture was incubated at room temperature for 30 minutes. After that, wash with the washing solution () three times to remove the substrate solution ()
Was dispensed in 100 µL aliquots and incubated at room temperature for 30 minutes. The reaction stop solution () was dispensed in 100 µL aliquots, stirred for 10 seconds, and then the absorbance (450 nm / 620 nm) was measured to prepare a standard curve (Fig. 1).

Reference Example 5- Pirmenol addition recovery test by enzyme immunoassay

A prescribed amount of pirmenol was added to each of the serum samples A, B and C of patients taking pirmenol, and a test for addition and recovery of pirmenol was conducted using the reagents prepared in Reference Example 3 and the standard curve prepared in Reference Example 4. went.

The results are shown in Table 1. The average recovery rate of each sample exceeded 95%, and it was confirmed that the amount of pirmenol in serum can be accurately measured by the enzyme immunoassay using the labeled antigen and antibody of the present invention.

[0078]

[Table 1]

[0079]

By using the labeled antigen and anti-pirumenol antibody of the present invention, the concentration of pirmenol in a biological sample such as a serum sample can be measured quickly, accurately and simply. The immunogen of the present invention is also useful for obtaining the antibody.

[Brief description of drawings]

FIG. 1 shows a standard curve obtained in Reference Example 4.

Front page continued (72) Inventor Noriyuki Sunahara 86-2 Kawashima Arisugawa-cho, Nishikyo-ku, Kyoto City, Kyoto Prefecture (72) Inventor Shozo Ueda 3-26 Hattoridai, Kamimaki-cho, Kitakatsuki-gun, Nara (56) References Special Kaihei 2-245661 (JP, A) JP 59-132362 (JP, A) Pharmacology and Treatment, (1991), 19 (12), P. 4767-76 (58) Fields investigated (Int.Cl. ⁷ , DB name) C07D 401/06 CA (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] 1. A pyrmenol derivative represented by the following formula: (In the formula, L is a bonding group, X is a labeling residue or immuno
Means a proteinogenic or immunogenic polypeptide residue). 2. The pyrmenol derivative according to claim 1, wherein a group represented by -L-X is bonded to the 3-position of the 2-pyridyl group. Wherein L is -Z- (CH ₂₎ n-CONH-
The pyrmenol derivative according to claim 1, which is a group represented by (wherein Z represents an oxygen atom or a sulfur atom, and n represents an integer of 1 to 6). 4. X is an immunogenic protein or immunogenic poly
The pyrmenol derivative according to any one of claims 1 to 3, which is a peptide residue. 5. The pyrmenol derivative according to any one of claims 1 to 3, wherein X is an enzyme residue. 6. produced by immunizing an animal other than human with at Pirumenoru derivative according to claim 4, Pirumeno
Antibody that can recognize