JP3200173B2 - Determination of quinolones - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for determining a quinolone agent.

[0002]

BACKGROUND OF THE INVENTION In chemical structure, the 6-position of quinolone nucleus (or
Derivatives having fluorine at the corresponding position) and a piperazine ring and a pyrrolidine ring at the 7-position, so-called quinolones, are widely used as broad spectrum antibiotics. Of which
9-fluoro-3-methyl-10- (4-methyl-1-
Piperazinyl) -7-oxo-2,3-dihydro-7H
-Pyrido [1,2,3-de] [1,4] benzoxazine-6-carboxylic acid (JP-A-57-46986), ie, the oral quinolone agent ofloxacin (hereinafter abbreviated as OFLX), is almost completely metabolized in the human body. Since it is excreted in the urine in the unaltered form without undergoing treatment, it is recognized as a drug that is very easy to design for administration. Recently, the elderly,
There are also increasing opportunities to be prescribed, especially for patients with renal insufficiency.
However, it has been found that it is necessary to modify the administration design and monitor blood levels in patients with OFLX being renal excretory and having reduced renal function.

[0003]

SUMMARY OF THE INVENTION Monitoring of a quinolone agent does not require sophisticated equipment, and it is necessary to obtain results in a short time at the bedside.

[0004]

Most of the quinolone agents are 40
Since fluorescence is emitted around 0 to 500 nm, the fluorescence may be measured. Furthermore, when the sample is irradiated with excitation light,
When the fluorescence wavelength shifts to the longer wavelength side under certain conditions, it is considered that the lightness increases with respect to the naked eye, and detection sensitivity that allows fluorescence observation with the naked eye is obtained. By applying this method, blood, urine, and saliva concentrations can be measured sufficiently, and it is thought that the measurement can be performed by comparing with a standard sample with the naked eye instead of using a fluorometer.

OFLX has an asymmetric carbon at the 3-position in its structure, and can be obtained as a racemic form ((±) -body specific rotation [α] D 0 で は) by an ordinary production method. This optically active substance 3S
It is known that the body ((-) body) has about twice the antibacterial activity of the (±) body and is less toxic than the (+) body (Japanese Patent Application Laid-Open No. 62-252790). This optically active body,
S-(-)-9-fluoro-3-methyl-10- (4-
Methyl-1-piperazinyl) -7-oxo-2,3-dihydro-7H-pyrido [1,2,3-de] [1,4]
Benzoxazine-6-carboxylic acid (abbreviated as DR-3355) can be similarly quantified. Furthermore, one of quinolone agents having a pyrrolidine ring at the 7-position of the quinolone nucleus, 10
-(8- (S) -Amino-6-azaspiro [3,4] octan-6-yl) -9-fluoro-2,3-dihydro-3- (S) -methyl-7-oxo-7H-pyrido [ 1,2,3-de] [1,4] benzoxazine-6
-Carboxylic acid (abbreviated as DV-7751)
5176) can be similarly quantified.

That is, it is acidic, and more preferably has a pH of 3 to
4, the fluorescence wavelength of OFLX is on the longer wavelength side as compared with other quinolone agents (ciprofloxacin, norfloxacin, etc.), which contributes to increase the brightness that can be observed by fluorescence with the naked eye. Since it is considered that the one-pair electron of the oxygen atom bonded to the 8-position of the skeleton is involved in the resonance structure of the aromatic ring, the DR-
3355 and DV-7751 can also be quantified.

[0007] The salivary concentration of OFLX, one of the oral quinolones, is well correlated with the serum concentration (Ichihara Norikata et al., C
hemotherapy (Tokyo), 32 , 118
-149, (1984), detection of OFLX concentration in saliva can be a sufficient blood concentration monitoring method.

Here, as a result of repeated studies, the present inventors have found that a quinolone agent containing a quinolone agent is irradiated with excitation light using a fluorescent lamp or the like as an irradiation source under acidic conditions, and the resulting fluorescence is measured. The quantitative method was completed. At this time, it was found that if the maximum fluorescence wavelength of the fluorescence of the quinolone agent was in the vicinity of 490 nm to 550 nm in the acidic state, it could be quantified by visual observation. Furthermore, we have completed a method for quantifying quinolone agents in saliva by irradiating saliva with excitation light under acidic conditions and measuring the resulting fluorescence.

[0009]

SUMMARY OF THE INVENTION The present invention provides a method for quantifying a quinolone agent, which comprises irradiating a sample containing the quinolone agent with excitation light under acidic conditions and measuring the resulting fluorescence. At the time of this measurement, if the maximum fluorescence wavelength is around 490 nm to 550 nm at the time of acidity, it is a quantification method that can be quantified even by visual observation.

Samples containing a quinolone agent include blood, urine, saliva and the like. Among them, saliva is irradiated with excitation light under acidic conditions, and the resulting fluorescence is measured. The method for quantifying a quinolone in saliva provides a useful quantification method for monitoring a quinolone.

The assay of the present invention is useful for quinolone agents, especially those having a pyridobenzoxazine skeleton.
Examples of such a quinolone include those having a structure of the following general formula (although the configuration at the 3-position is represented by the RS configuration, but also includes the S configuration (β configuration). Is.)

[0012]

Embedded image

In the formula, R ¹ represents a lower alkyl group, particularly preferably a methyl group. As for the configuration at this site, it has been found that the S configuration is preferable in terms of pharmacological activity.

R ² represents a saturated nitrogen-containing heterocyclic substituent. The size of the ring is preferably a four-membered ring to a seven-membered ring,
Membered and six-membered rings are preferred. Further, it may contain an oxygen atom, a sulfur atom or a plurality of nitrogen atoms as ring constituent atoms, such as oxazolidine, morpholine, thiazolidine, thiomorpholine, imidazolidine, pyrazolidine and piperazine. As the saturated nitrogen-containing heterocyclic substituent, a pyrrolidinyl group and a piperazinyl group are particularly preferable, and these may further have a substituent. At this time, it is preferable that the substituent is stereoisomerically a single substituent.

Examples of such a substituent include an amino group which may have a substituent, an aminoalkyl group which may have a substituent, a 5-substituted-2-oxo-1,3-dioxol-4-ylmethyl group. And a polar group such as a hydroxyl group, and a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms. In the case of a polar group, it may be bonded to a saturated nitrogen-containing heterocyclic substituent via an alkylene group having 1 to 6 carbon atoms. Here, examples of the substituent of the amino group include an alkyl group, an acyl group, and an acyloxycarbonyl group.

As the above-mentioned polar group, an unsubstituted amino group, aminomethyl group, 1-aminoethyl group and hydroxyl group are particularly preferred.

The alkyl group on the saturated nitrogen-containing heterocyclic substituent includes a methyl group, an ethyl group, a propyl group and an isopropyl group, a gem ^- dimethyl group and
It is also preferable that a gem ^- diethyl group or the like, and furthermore, these be a saturated nitrogen-containing heterocyclic substituent which forms a cyclopropane ring or a cyclobutane ring to form a spiro ring system. Also,
The 4- to 7-membered saturated nitrogen-containing heterocyclic substituent may be cross-linked to form a bicyclocyclic saturated nitrogen-containing heterocyclic substituent.

Of these saturated nitrogen-containing heterocyclic substituents,
In particular, examples of those substituted with an amino group or those having a second nitrogen atom include those having the following structures.

[0019]

Embedded image (Wherein, R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ ,
R ¹⁰ , R ¹¹ , R ¹² and R ^{13 each} independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms;
¹² and R ¹³ combine to form a polymethylene chain,
A six-membered ring may be formed from a membered ring. )

Specific examples of these substituents include a 3-aminopyrrolidinyl group, a 3-methylaminopyrrolidinyl group, a 3-dimethylaminopyrrolidinyl group, a 3-ethylaminopyrrolidinyl group, Propylaminopyrrolidinyl, 3-isopropylaminopyrrolidinyl, 3-amino-4-methylpyrrolidinyl, 4-amino-2-methylpyrrolidinyl, 4-amino-2,3-dimethylpyrrolidinyl Dinyl group, 3-methylamino-4-methylpyrrolidinyl group, 4-methylamino-2-methylpyrrolidinyl group,
4-methylamino-2,3-dimethylpyrrolidinyl group,
3-dimethylamino-4-methylpyrrolidinyl group, 4-
Dimethylamino-2-methylpyrrolidinyl group, 4-dimethylamino-2,3-dimethylpyrrolidinyl group, 3-methylpiperazinyl group, 4-methylpiperazinyl group, 3,
4-dimethylpiperazinyl group, 3,5-dimethylpiperazinyl group, 3,4,5-trimethylpiperazinyl group, 4
-Ethyl-3,5-dimethylpiperazinyl group, 4-isopropyl-3,5-dimethylpiperazinyl group, 3-aminomethylpyrrolidinyl group, 3-methylaminomethylpyrrolidinyl group, 3- (1 -Amino) ethylpyrrolidinyl group, 3- (1-methylamino) ethylpyrrolidinyl group,
3- (1-ethylamino) ethylpyrrolidinyl group, 3-
(1-amino) propylpyrrolidinyl group, 3- (1-methylamino) propylpyrrolidinyl group, 3-aminopyrrolidinyl group, 4-amino-3,3-dimethylpyrrolidinyl group, 7-amino -5-azaspiro [2,4] heptane-5-yl group, 8-amino-6-azaspiro [3,4]
Octane-6-yl group, 1,4-diazabicyclo [3,2,1] octan-4-yl group, 3,8-diazabicyclo [3,2,1] octan-3-yl group, 8-methyl-3 , 8-diazabicyclo [3,2,1] octan-3-yl group, 8-ethyl-3,8-diazabicyclo [3,2,1] octan-3-yl group, and the like.

Examples of the saturated nitrogen-containing heterocyclic substituent having a substituent other than an amino group include, for example, a 3-hydroxypyrrolidinyl group, a 3-mercaptopyrrolidinyl group and a 3-hydroxy-4-methylpyrrolidinyl group. , 3-mercapto-4-methylpyrrolidinyl, morpholino, thiomorpholino, 2-methylmorpholino, 2-methylthiomorpholino, 2,6-dimethylmorpholino, 2,6
-Dimethylthiomorpholino group, 2,2-dimethylmorpholino group, 2,2-dimethylthiomorpholino group and the like.

The present invention provides a method for quantifying a quinolone agent, which comprises irradiating excitation light with saliva containing a quinolone agent having a pyridobenzoxazine skeleton under acidic conditions and measuring the resulting fluorescence. And a quinolone having a pyridobenzoxazine skeleton as 9-fluoro-3-methyl-10- (4-methyl-1-piperazinyl) -7-oxo-2,3-dihydro-7H-pyrido [1,2 , 3-de] [1,4] benzoxazine-6
-Carboxylic acid, S-(-)- 9-fluoro-3-methyl
-10- (4-Methyl-1-piperazinyl) -7 -oxo-2,3-dihydro-7H-pyrido [1,2,3-d
e] [1,4] benzoxazine-6-carboxylic acid, and 10- (8- (S) -amino-6-azaspiro [3,
4] Octane-6-yl) -9-fluoro-2,3-dihydro-3- (S) -methyl-7-oxo-7H-pyrido [1,2,3-de] [1,4] benzoxazine −
The present invention provides a method for quantifying a quinolone agent of 6-carboxylic acid.

Hereinafter, the present invention will be described in detail with reference to embodiments.

[0024]

Example 1 Saliva Collection Method A: A method in which a piece of absorbent cotton was torn off and lightly rounded into a sphere having a diameter of about 5 mm was contained in 2-3 mouths, and lightly chewed to sufficiently contain saliva. At this time, the case where candy, citric acid, and the like were included in the mouth together with the absorbent cotton was also examined. Take out absorbent cotton with tweezers,
A transparent sample tube (polystyrene, Assist, No. 17) in which gauze was previously set as shown in FIG.
452.722), covered with a lid so as to hold the gauze, and centrifuged for about 1 minute with a simple centrifuge (Tibitan, manufactured by Nippon Millipore).

Method B--For the purpose of measuring the recovery of OFLX, saliva was directly collected from a healthy adult male into a test tube.

The simplest method for collecting saliva is B
Although it is a law, it is painful for those who collect it, and it takes some skill to collect the required amount of saliva in a short time. Ichihara et al. Put a bioassay paper disk in their mouth to collect saliva (Norikata Ichihara et al., Ch.
emotherapy (Tokyo), 32 , 118-
149, (1984)). Although this method is relatively simple, the amount required for fluorescence detection cannot be collected, and it is difficult to recover it from filter paper. In the method A, about 0.3 ml could be easily obtained in a short time with little pain to the sampled person. When saliva was collected, candy, citric acid, and the like were also included in the mouth, which further promoted secretion of saliva and facilitated collection. The properties of saliva collected by this method were slightly different from those of saliva directly collected in a test tube, and there were relatively few saliva-specific high-viscosity proteins.

[0027]

Example 2 Examination of Fluorescence Observation Conditions OFLX was prepared by using various pH solutions (solvent 1: 0.1 M phosphate buffer (pH 7.0). 2: 0.05 M acetate buffer (pH 4.0). 3: 0.04M phosphate buffer (pH
3). 4: 5M sulfuric acid) at a concentration of 10 μg / ml, and a Hitachi 650-60 spectrofluorometer (Hitachi, Ltd.)
And the fluorescence spectrum was measured. At the time of measurement, a pre-scan was performed with a solution having a pH of 7 to standardize the sensitivity of the spectrofluorometer, and thereafter, the sample was measured at this sensitivity. Figure 2 shows the results.
Shown in

It is known that fluorescent compounds generally differ in fluorescence wavelength and intensity depending on the dissociation state. For OFLX, when the pH changes from 7 to 4,
The excitation spectrum did not change much, but the fluorescence spectrum shifted to longer wavelengths by as much as 40 nm. The fluorescence spectrum did not change much from the acidic side, and the fluorescence intensity became maximum at pH 3 and decreased on the acidic side.

Such a change in the fluorescence spectrum was observed to the naked eye as a change in the color tone of the fluorescence, and changed from deep blue at pH 7 to yellow-green in the acidic region. With this change, the brightness perceived by the naked eye increased significantly on the acidic side. Therefore, the saliva sample was adjusted to pH 3 and a fluorescent lamp (UVGL-25) was used.
Mold, UVP, Inc. (CA, USA), the color changes from deep blue to yellowish green which can be clearly distinguished due to the reflection of the irradiation light of the fluorescent lamp, and when observed with the naked eye, the sensitivity of quinolone agent detection was increased and the reliability was increased. .

[0030]

Example 3 Preparation of Standard Addition Sample 0.2 ml of saliva collected by the method B in Example 2 was placed in a transparent sample tube, and an aqueous OFLX solution (concentration: 4, 1)
0, 20, 40, 100 μg / ml), mix well, and add OFLX concentrations of 0.2, 0.5, 1.0,
Each sample was 2.0 and 5.0 μg / ml. To this, 0.2 ml of 0.04 M phosphate buffer (pH 3) was added and mixed, and the mixture was centrifuged with a simple centrifuge for about 10 minutes to precipitate the protein. Fluorescent lamp (UVGL-25 type, UVP, In) with sample tube against black Kentrasha paper as background
c. (CA, USA) and the fluorescence was observed with the naked eye.
FIG. 3 shows the results. From FIG. 3, the concentration of OFLX was quantitatively detected with the naked eye, and was detected even at a saliva concentration of 0.2 μg / ml. The detection limit with the naked eye is considered to vary from individual to individual, but was about 0.2 μg / ml.

In a single oral administration of 100 mg of OFLX to a human, the concentration in saliva is 0.4-0.
7 μg / ml (Norikata Ichihara et al., Chemot
herpy (Tokyo), 32 , 118-149,
(1984). When 200 mg was continuously administered every 12 hours, the plasma concentration immediately before the second administration was 0.53 μg / m 2.
l, the concentration immediately before the fifth administration was reported to be 1.19 μg / ml (Couraud, L. et al., Drug).
s, 34 , (Suppl. 1), 37-38, (198
7)). At this time, the concentration in saliva is expected to be approximately the same as the concentration in plasma, and therefore, can be monitored with the above detection sensitivity. When the dose is increased, it is expected that the salivary concentration will be higher than this.
It is sensitive enough to be used for the purpose of preventing the accumulation of X.

In 1 ml of saliva collected by the method B in Example 2,
The sample was prepared by adding an OFLX solution to 0.5 to 0.004 μg / ml and further adding 4 ml of 0.04 M phosphate buffer (pH 3) to prepare a UV-clade methacrylate cuvette (Aldrich Catalog No. Z1880).
1-8) and measured with a JASCO FP777 spectrofluorometer. Table 1 shows the results when 303 nm, 330 nm and 360 nm were used as the excitation wavelength and 500 nm was used as the fluorescence wavelength.

[0033]

[Table 1]

[0034]

Example 4 Measurement of Recovery Rate OFLX was added to saliva collected by Method B in Example 1 in an amount of 5 μg.
/ Ml. This solution was mixed with absorbent cotton, placed in an inner tube of a transparent sample tube, and centrifuged to obtain a filtrate.
The OFLX concentration in this filtrate and the original saliva was determined by HPLC (Matsubayashi K. et al., Journal).
of Chromatography, 495 , 35
4-357 (1989)), and the recovery rate of the operation by absorption and recovery by absorbent cotton was determined.

The excitation spectrum was measured at the maximum fluorescence wavelength, and the fluorescence spectrum was measured at the maximum excitation wavelength.

As shown in Table 2 , the recovery of OFLX was 93.5
It was ± 5. 4%. That is, in the method in which saliva is absorbed by absorbent cotton and collected by centrifugation (method A in Example 1), OFLX was quantitatively recovered.

[0037]

[Table 2]

[0038]

Example 5 Quantification of DR-3355 DR-3355 may be quantified as follows.

The saliva is separated according to the method A of Example 1.
0.1ml 0.04M phosphate buffer 1 in 0.2ml saliva
The solution (pH 3) was added to make the solution acidic. On the other hand, a standard addition sample is prepared according to Example 3. The sample tube is irradiated with a fluorescent lamp against black Kentrasha paper as a background, and the fluorescence is observed with the naked eye. The saliva concentration of DR-3355 is determined relative to the fluorescence of the standard spiked sample.

In addition to the first embodiment, saliva can be collected using a capillary tube, a dropper or the like. Final dozen μl
If there is, it can be placed on a Saran wrap or the like as a water drop and observed with the naked eye. Therefore, saliva can be quantified if it is about several tens μl. If the amount of observation under acidic conditions is small, water drops are formed on the Saran wrap or a sample is inserted into the narrow slit between the two glass plates. Black lash paper is placed under the Saran wrap or the glass plate and irradiated with a fluorescent lamp. Thus, it is possible to determine the amount of quinolone by observing the fluorescence of the quinolone in saliva and comparing it with a standard sample.

[0041]

Example 6 Examination of Fluorescence Observation Conditions for Other Oral Quinolone Agents-1 As in Example 2, 10- (4-amino-3,3-dimethyl-1-pyrrolidinyl) -9-fluoro-2,3 -Dihydro-3- (S) -3-methyl-7-oxo-7H-
Solutions of pyrido [1,2,3-de] [1,4] -benzoxazine-6-carboxylic acid (abbreviated as DV-7777) at two pHs, 1. 0.1 M phosphate buffer (pH 7.
0) or 2. 0.04M phosphate buffer (pH3)
Was dissolved to a concentration of 2 μg / ml, and the fluorescence spectrum was measured with a Hitachi 650 spectrofluorometer. A prescan was performed at each measurement to standardize the sensitivity of the spectrofluorometer. FIG. 4 shows the results.

DV-7777 has p as in OFLX.
When H changed from 7 to 3, the excitation spectrum did not change much, but the fluorescence spectrum shifted to longer wavelengths by as much as 40 nm.

Such a change in the fluorescence spectrum was observed to the naked eye as a change in the color tone of the fluorescence, and changed from deep blue at pH 7 to yellow-green in the acidic region. With this change, the brightness perceived by the naked eye increased significantly on the acidic side.

Further, the sample was irradiated with a fluorescent lamp under acidic conditions, and the detection limit with the naked eye was measured.
/ Ml. This was slightly better or equivalent to the sensitivity of OFLX.

[0045]

Example 7 Examination of Fluorescence Observation Conditions for Other Oral Quinolone Agents-2 As in Example 2, 10- (8- (S) -amino-6-
Azaspiro [3,4] octan-6-yl) -9-fluoro-2,3-dihydro-3- (S) -methyl-7-oxo-7H-pyrido [1,2,3-de] [1, 4] Benzoxazine-6-carboxylic acid (DV-7751) in two pH solutions, 0.1 M phosphate buffer (pH 7.
0) or 1μ in 0.04M phosphate buffer (pH3)
g / ml, and the fluorescence spectrum was measured with a Hitachi 650 spectrofluorometer. A prescan was performed at each measurement to standardize the sensitivity of the spectrofluorometer. The results are shown in FIG.

As in the case of DV-7777, when the pH changed from 7 to 3, the excitation spectrum did not change much, but the fluorescence spectrum shifted to a longer wavelength by 45 nm. This change in the fluorescence spectrum was also caused by DV-77.
Same as 77. Irradiation with a fluorescent lamp under acidic conditions, the detection limit was measured with the naked eye, 0.2μ
g / ml.

[0047]

According to this method, the result can be known in about 5 minutes in a short time, and the demand can be quickly met at the bedside. In the operation, the patient simply puts cotton wool in his mouth, takes it out, adds a buffer solution, makes it acidic, and measures the fluorescence intensity. When performing measurements by visual observation, only a fluorescent lamp is sufficient in the equipment. Therefore, it is possible to quickly measure at the bedside of the clinic, and to consider and adjust the dose.

[0048]

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a method for separating a saliva sample.

FIG. 2 is a diagram of an excitation spectrum and a fluorescence spectrum of OFLX.

FIG. 3 is a diagram showing the fluorescence intensity of an OFLX standard sample in saliva.

FIG. 4 is a diagram of an excitation spectrum and a fluorescence spectrum of DV-7777.

FIG. 5 is a diagram of an excitation spectrum and a fluorescence spectrum of DV-7751.

──────────────────────────────────────────────────続 き Continued on the front page (56) References Antimicrobial Agents and Chemothera py, Vol. 33, No. 1, P. 27-29 (1989) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 21/78 G01N 21/64 G01N 33/50 G01N 33/52 BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A quinolone agent having a maximum fluorescence wavelength in the range of 490 to 550 nm at pH 3 to 4, wherein the saliva is irradiated with excitation light at pH 3 to 4, and the resulting fluorescence is measured by comparison with a standard added sample. Quantitation method. 2. A method for quantifying a quinolone having a pyridobenzoxazine skeleton, which comprises irradiating saliva with excitation light at pH 3 to 4, and measuring the resulting fluorescence by comparison with a standard added sample. 3. The method of claim 1, wherein the quinolone having a pyridobenzoxazine skeleton is 9-fluoro-3-methyl-10- (4-
Methyl-1-piperazinyl) -7-oxo-2,3-dihydro-7H-pyrido [1,2,3-de] [1,4]
3. The method according to claim 2, which is benzoxazine-6-carboxylic acid. 4. The method of claim 1, wherein the quinolone having a pyridobenzoxazine skeleton is S-(−)-9-fluoro-3-methyl-.
10- (4-methyl-1-piperazinyl) -7-oxo-2,3-dihydro-7H-pyrido [1,2,3-d
e) The method according to claim 2, which is [1,4] benzoxazine-6-carboxylic acid. 5. A quinolone having a pyridobenzoxazine skeleton which is 10- (8- (S) -amino-6-azaspiro [3,4] octan-6-yl) -9-fluoro-
2,3-dihydro-3- (S) -methyl-7-oxo-
3. The method according to claim 2, which is 7H-pyrido [1,2,3-de] [1,4] benzoxazine-6-carboxylic acid.