JP3290914B2 - Apparatus for the determination of quinolone antibacterial agents in ocular tissues - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to levofloxacin,
The present invention relates to a device for non-invasively quantifying the concentration of a quinolone antibacterial agent such as ofloxacin in an eye tissue.

2. Description of the Related Art Quinolone-based antibacterial agents such as ofloxacin are agents having a broad antibacterial spectrum and are widely used in ophthalmology. It is known that a quinolone-based antibacterial agent emits fluorescence when irradiated with ultraviolet excitation light, and a method for quantifying the quinolone-based antibacterial agent in various biological tissues using this property has been studied. However, as these quantification methods, a method of isolating a living tissue and performing a chemical treatment followed by measurement is generally used, and the concentration of the quinolone antibacterial agent in the living tissue is directly measured without isolating the living tissue. Little is known about the method for quantification, and particularly no method is known for directly quantifying the concentration in eye tissue.

On the other hand, as a method for measuring the function of eye tissue, particularly the barrier function of corneal epithelium, a method using fluorescein (fluorophotometry) is actually used. This method, after fluorescein is administered by eye,
This method measures the barrier function of the corneal epithelium by measuring the intensity of the fluorescence to be excited after a certain period of application of the excitation light and measuring the rate of decrease in the concentration of fluorescein. As an apparatus for this fluorophotometry, there has been reported an apparatus capable of measuring excitation light having a wavelength of 460 to 520 nm by an exciter filter using a halogen lamp as a light source, and measuring the excited fluorescence as a bandwidth of 520 to 620 nm by a barrier filter. (Journal of the Japanese Society of Ophthalmology, 98, 641-647).

[0003]

The effect of an antimicrobial agent depends on the concentration of the antimicrobial agent in a target tissue. The effect expression concentration is generally represented by a minimum growth inhibitory concentration (MIC) of the bacterium, and it is necessary that the MIC be present in the target tissue at a concentration higher than the MIC in order to exhibit the effect as an antibacterial agent. Therefore, concentration measurement in the target tissue becomes very important. In experiments with animals, after isolating the tissue,
It is easy to measure the concentration in the tissue, but in the case of humans, it becomes very difficult to isolate the tissue depending on the target tissue. Measurement of blood concentration, etc. can be easily performed by collecting blood from a patient, but it is extremely difficult to measure the concentration of a drug in a tissue, such as eye tissue, which cannot be separated from the living body in practice. Become. For a drug such as an antibacterial agent, which has a very high correlation between the concentration in the tissue and the effect, if a method for non-invasively measuring the concentration in the tissue without damaging the tissue can be found, drug development research and eventually medical treatment It would be very useful, and research on such a method was desired. Accordingly, an object of the present invention is to provide a quantitative device for quantitatively measuring the concentration of an administered quinolone antibacterial agent in eye tissue without damaging the tissue.

[0004]

SUMMARY OF THE INVENTION The present invention is based on the finding that the quinolone antibacterial agent in the tissue emits fluorescence when irradiated with ultraviolet excitation light, and the above-mentioned problem can be efficiently achieved by measuring the intensity. It was based on knowledge. That is, the present invention relates to a device for quantifying the concentration of a quinolone antibacterial agent in eye tissue, a means for converting light emitted from a light source into excitation light in the ultraviolet region, a means for irradiating the eye with the excitation light, Provided is a quantitative device as described above, characterized by comprising means for converging fluorescence in the visible region excited and excited by the quinolone antibacterial agent in the optometry tissue, and means for measuring the fluorescence intensity of the converged fluorescence. Here, a method for quantitatively measuring the concentration of the quinolone antibacterial agent in the eye tissue is performed by irradiating the subject's eye with ultraviolet excitation light from the outside to excite the administered quinolone antibacterial agent when excited. Measuring the fluorescence intensity of the area,
It is preferable to use the above-described quantification method, wherein the concentration is calculated from the intensity.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Quinolone-based antibacterial agents emit fluorescence upon irradiation with ultraviolet rays, but the present invention is a product of this property and can be widely applied to quinolone-based antibacterial agents. The quinolone antibacterials that can be detected by the method of the present invention include 6-fluoroquinoline-4-oxo-4H-1,4.
-Dihydro-3-carboxylic acid or 9-fluoro-2,3-
Dihydro-7-oxo-7H-pyrido [1,2,3-d
e] those having a skeleton of [1,4] benzoxazine-6-carboxylic acid and further having various substituents. These compounds are described, for example, in
141286, JP-A-58-74667,
JP-A-59-67279, JP-A-60-6497
No. 9, JP-A-60-174786, JP-A-60-174786
JP-A-228479, JP-A-2-231475, JP-A-3-95176, JP-A-7-3098
No. 64, etc., Internal Medicine [Vol. 62, No. 1, pp. 28-33 (1988)], Pharmacia [Vol. 25, No. 5, 434]
To 440], and the production thereof can be carried out according to the methods disclosed in these publications. Of these, ofloxacin (O
FLX), levofloxacin (LVFX: S (-)
-9-Fluoro-2,3-dihydro-3-methyl-10
-(4-methyl-1-piperazinyl) -7-oxo-7
H-pyrido [1,2,3-de] [1,4] benzoxazine-6-carboxylic acid hemihydrate), norfloxacin (NFLX: 1-ethyl-6-fluoro-1,
4-dihydro-4-oxo-7- (1-piperazinyl)
-3-quinolinecarboxylic acid, ciprofloxacin hydrochloride (CPFX: 1-cyclopropyl-6-fluoro-1,
4-dihydro-4-oxo-7- (1-piperazinyl)
-3-quinolinecarboxylic acid hydrochloride, lomefloxacin (LFFLX: (±) -1-ethyl-6,8-difluoro-1,4-dihydro-7- (3-methyl-1-piperazinyl) -4-oxo- 3-quinoline carboxylic acid / hydrochloride). The method of the present invention is suitably used in the case of ophthalmic administration as a dosage form of a quinolone antibacterial agent, but can also be used in the case of systemic administration such as oral administration. The measurement may be performed at a desired time after administration of the quinolone antibacterial agent. The measurement method of the present invention is a means for converting light emitted from a light source into excitation light in an ultraviolet region, a means for irradiating the eye with the excitation light,
Use of a device for quantifying the concentration of a quinolone antibacterial agent in eye tissue having means for converging fluorescence in the visible region generated by excitation of the quinolone antibacterial agent in the eye tissue to be examined and means for measuring the fluorescence intensity of the converged fluorescence Thus, the measurement can be performed efficiently.

A quantitative device used in the present invention will be described with reference to FIG. In the figure, reference numeral 20 denotes a means for converting light emitted from a light source into excitation light in an ultraviolet region, and the light emitted from an ultraviolet light source (preferably a mercury xenon lamp) 1 passes through a quartz fiber 2 and passes through an illumination optical system 21 of a measuring apparatus. After the emitted light is converged by the lens 3, the exciter filter 4
Through which the light is in the wavelength band of 260 to 390 nm, and is used as excitation light. Note that an alignment filter 16 that transmits visible light coaxial with the ultraviolet light may be used for positioning the eye to be exposed to the ultraviolet light. In the figure, 30
Is a means for irradiating the eye with the excitation light. The excitation light passes through the opening of the slit 5, and forms an image of the slit opening on the measurement site of the eye 15, such as the cornea and the aqueous humor, by the imaging lens 6. Let it. As a result, only the measurement site of the eye to be examined can be locally irradiated, and the quinolone antibacterial agent present at the measurement site emits fluorescence in the visible region.

In the figure, reference numeral 40 denotes a means for converging fluorescence in the visible region emitted by excitation of the quinolone antibacterial agent in the tissue of the eye to be examined, which has a converging lens 7, and further has a barrier filter 8 and a mask 9. Is good. The barrier filter is preferably designed to have a transmission wavelength band in the visible range of 430 to 630 nm and at the same time to have a characteristic of not transmitting the excitation light wavelength of 260 to 390 nm.
Only the fluorescence (430-630 nm) emitted by the quinolone antibacterial agent does not reach the means for measuring the fluorescence intensity.
The mask 9 is optically conjugate with the measurement site, and the measurement size is determined by adjusting the size of the opening of the mask and the observation optical system.

In the figure, reference numeral 50 denotes a means for measuring the fluorescence intensity of the converged fluorescence, and the electric output signal output from the photomultiplier tube 10 through the mask 9 is amplified by the multiplier 11 and the control unit 12 controls the signal. Perform analysis. The measured value is display 13
Alternatively, the output is displayed on the printer 14. At the time of actual quantification, the fluorescence intensity of a standard solution having a constant concentration is measured in advance to prepare a calibration curve, and calculation is performed based on the calibration curve. It is preferable to measure the fluorescence intensity (background) of the eye tissue before administration, and to subtract the background from the fluorescence intensity measured after administration of the antibacterial agent.

The method of the present invention makes it possible to quantitatively measure the concentration of a quinolone antibacterial agent in ocular tissue without damaging ocular tissue. It can be measured at a desired time after administration. The excitation light may be in the ultraviolet region,
If the wavelength is too short, it may damage the eye tissue,
As a result of studying a more suitable wavelength, 260-39 was obtained.
It has also been found that a wavelength of about 0 nm is preferred. Also,
The fluorescence to be measured may be in the visible range, but as a result of research on a more suitable wavelength, 430-630 was obtained.
It has also been found that wavelengths on the order of nm are preferred. In the present invention, it is not strictly limited to the range of the preferable wavelengths of the excitation light and the fluorescence, but should be interpreted with a certain width. The wavelengths of the excitation light and the fluorescence to be measured can be appropriately selected according to the type of the quinolone antibacterial agent to be used. The irradiation time of the excitation light is 0.
It may be an instantaneous one of about 2 seconds.

The method of the present invention is particularly suitable for quantification of the concentration in the cornea or aqueous humor. For quantitative measurement of the corneal concentration, the measurement angle (the angle between the incident excitation light and the fluorescence to be measured) is 30.
It was also found that a measurement angle of 90 ° was suitable for the determination of ° and aqueous humor. The method of the present invention is a method capable of correctly measuring the concentration of a quinolone antibacterial agent in eye tissue,
It was revealed in an experiment using rabbits. An experiment using this rabbit will be described in detail in Examples.

[0011]

【Example】

Example 1 Levofloxacin was used as a representative example of a quinolone antibacterial agent, and after instillation in rabbits, the concentration in eye tissues was quantified over time. As the quantification method, the method of the present invention and a conventional high-performance liquid chromatography method were used, and the quantitative values of both methods were compared and examined. Quantification by the method (FPM) of the present invention 1) Preparation of a calibration curve Levofloxacin was used at 0, 0.05, 0.1, 0.25, 1, 2.
An aqueous solution containing 5, 10 or 20 μg / ml is prepared, and the fluorescence (430) emitting excitation light (260-390 nm) is applied.
-630 nm) The intensity was measured. The measurement was performed by setting the angles of the excitation light and the fluorescence at 30 ° and 90 °. A calibration curve was created based on the measured fluorescence intensity.

2) Measuring device The device shown in FIG. 1 was used. In the figure, reference numeral 20 denotes a means for converting light emitted from a light source into excitation light in an ultraviolet region, guides light emitted from a mercury xenon lamp 1 through a quartz fiber 2 to an illumination optical system of a measuring apparatus, and converts emitted light into a lens. After the convergence at 3, it passes through the exciter filter 4 and
Light of a wavelength band of -390 nm is used as excitation light. 30
Is a means for irradiating the eye to be examined with excitation light. The excitation light passes through the opening of the slit 5 and forms an image of the slit opening on the measurement site of the eye to be inspected by the imaging lens 6. Numeral 40 is a means for converging the visible-range fluorescence emitted when the quinolone antibacterial agent in the eye tissue to be examined is excited, and has a converging lens 7, a barrier filter 8 and a mask 9, and the barrier filter 8 has a wavelength of 430-630 nm. It is designed to have a characteristic of having a transmission wavelength band in the visible region and not transmitting the excitation light at a wavelength of 260 to 390 nm. 50 is a means for measuring the fluorescence intensity of the converged fluorescence, and amplifies the electric output signal output from the photomultiplier tube 10 through the mask 9 by the multiplier tube 11;
The control unit 12 performs signal analysis. The measured values are output and displayed on the display 13 or the printer 14.

3) Measurement of concentration in ocular tissue after instillation of levofloxacin 50 μl of 0.5% levofloxacin ophthalmic solution was instilled into both eyes of male Dutch rabbits, and 5, 15, 30 after instillation.
After 1, 2, 4, and 6 hours, the fluorescence intensity in the cornea and aqueous humor was measured using the apparatus shown in FIG. In the case of the cornea, the angle between the excitation light and the fluorescence was set to 30 °, and in the case of the aqueous humor, the angle was set to 90 °. The obtained fluorescence intensity was converted based on the calibration curve prepared in 1) to obtain the concentration. The fluorescence in the cornea and the aqueous humor immediately before the instillation was measured (background), and subtracted from the fluorescence intensity after the instillation.

Determination by high performance liquid chromatography (HPLC)
The amount of 0.5% Levofloxacin ophthalmic solution (dissolved at a concentration of 0.5% in saline levofloxacin prepared, hereinafter) 50
μl was instilled into both eyes of male Dutch breed rabbits, respectively, and euthanized by Nembutal administration at 5, 15, 30 minutes, 1, 2, 4 and 6 hours after instillation. The cornea was harvested. The collected sample was processed as follows, and measured by HPLC. (Processing method) The collected cornea was homogenized with methanol and centrifuged to obtain a supernatant, and the aqueous humor was used as a sample. Phosphate buffer (pH 7.0) was added to these samples and extracted with chloroform. After the organic layer was evaporated to dryness under reduced pressure, the HPLC mobile phase (1
0 mM potassium phosphate buffer (pH 3.0 / acetonitrile (85/15)) and HPLC (column: TSK
gel ODS-80Ts). A fluorescence detector (excitation wavelength 294 nm, fluorescence wavelength 51
0 nm).

Results The results determined by the FPM method of the present invention and the conventional HPLC method are shown in FIGS. The concentrations in the aqueous humor were almost the same in both methods. Corneal concentrations were generally lower with the FPM method. This is presumed to be due to the difference in the refractive index between the standard solution used for the calibration curve and the cornea, but if corrected by multiplying by a certain constant (1.7), the FPM method and the HPLC method can also be used for the cornea. It can be seen that the quantified values of agree substantially (see FIG. 4). The above results show that the method of the present invention can correctly quantify the concentration of levofloxacin in cornea and aqueous humor.

Reference Example 1 To determine whether or not the irradiation of 260-390 nm excitation light (ultraviolet light) affects the eye tissue, a change in the eye tissue (cornea, iris, lens, retina) after irradiation was measured using a microscope. Examined. As a result, it was found that 260-390 nm excitation light (ultraviolet light) did not affect ocular tissues. The effect on the retina potential was also examined, but no abnormality was found.

[Brief description of the drawings]

FIG. 1 shows an outline of a quantitative device of the present invention.

FIG. 2 shows levofloxacin concentrations in aqueous humor when the quantification method (FPM method) of the present invention is used and when a conventional method (HPLC method) is used.

FIG. 3 shows levofloxacin concentrations in the cornea when the quantification method (FPM method) of the present invention is used and when a conventional method (HPLC method) is used.

4 is a quantification method of the present invention in FIG. 3 (FPM method)
The value obtained by multiplying the levofloxacin concentration in the cornea by using a constant (1.7) and correcting it using the conventional method (HPLC method)
Are shown together with the levofloxacin concentration in the cornea when. In the figure, 1 is a light source, 3 is a lens, 4 is a filter,
6 is an imaging lens, 7 is a converging lens, 8 is a filter,
10 is a photomultiplier tube, 11 is a multiplier tube, 20 is a unit for converting light emitted from a light source into excitation light in an ultraviolet region, 30 is a unit for irradiating excitation light to the eye to be examined, and 40 is a unit in the tissue of the eye to be examined. Means for converging the visible-range fluorescence emitted when the quinolone antibacterial agent is excited, 50 means for measuring the fluorescence intensity of the converged fluorescence,
It is.

──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Tatsuo Ikei 8916-16 Takayamacho, Ikoma City, Nara Prefecture Santen Pharmaceutical Co., Ltd. (72) Inventor Takayoshi Suzuki 1-3-1 Shintoda, Hamamatsu City, Shizuoka Prefecture (56) References JP-A-4-211615 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61B 3/00-3/16 JICST file ( JOIS)

Claims (2)

(57) [Claims] An apparatus for quantifying the concentration of a quinolone antibacterial agent in eye tissue, comprising: means for converting light emitted from a light source into excitation light in an ultraviolet region; means for irradiating the excitation light to an eye to be examined; The above-described quantification device, comprising: means for converging fluorescence in the visible region emitted when a quinolone antibacterial agent in the tissue is excited, and means for measuring the fluorescence intensity of the converged fluorescence. 2. The means for generating excitation light in the ultraviolet region has a wavelength of 26.
2. The apparatus according to claim 1, wherein the means for obtaining the excitation light of 0-390 nm and the means for converging the fluorescence in the visible region are means for converging the fluorescence wavelength of 430-630 nm but not transmitting the wavelength of the excitation light.