Optics: measuring and testing – Plural test
Reexamination Certificate
1998-07-15
2001-06-12
Font, Frank G. (Department: 2877)
Optics: measuring and testing
Plural test
C356S364000, C356S367000
Reexamination Certificate
active
06246470
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns to the medical engineering and the pharmaceutical industry, and more particularly—to the way of determination in an analyzed liquid of a biologically active substance and the device for its realization.
The offered invention can be used in medical and clinical biochemistry and also in the molecular pharmacology at research of pharmaco-kinetics of biologically active substances, in pharmaceutical industry and in ecology. The most effective use of the present invention is in clinical biochemistry.
DESCRIPTION OF THE RELATED ART
The basic problem at rational pharmaco-kinetics of biologically active substances (BAS), in particular, synthetic and half-synthetic antitumor substances, influencing a possibilities of effective therapy, is reduced to speed and accuracy of determination of concentration of these substances in biological liquids (blood, plasma of blood, urea, etc.) after treatment of patients with certain amount of an antitumor substance.
Two basic groups of methods are known for determination of the presence and the efficiency of “action” of BAS, basic “target” of which are molecules of double-stranded nucleic acids.
The first group is formed by the biological methods [Modern methods in biochemistry. Ed. by V. N. Orekhovich. M.: Medicine. 1968, p.372; Methods of practical biochemistry. Ed. by B. Williams, K. Wilson. M.: World, 1978, p. 256]. They are based on the investigation of well-registered changes in well-described genetic systems (bacteria, bacteriophages, cultures of cells) after their treatment with BAS. These methods are realized by means of the standard microscopes. However, on a way of BAS penetration, the modification of their structures can However, on a way of BAS penetration, the modification of their structures can take place influencing the accuracy of the establishment of correlation between the structure of BAS, its concentration and the biological activity. In addition, the specified methods differ by duration of realization of the experiment (from days till weeks).
The second group includes the physico-chemicalical methods or their different combinations. Since 1980, a few methods of determination of BAS, in particular, derivatives of the anthraquinone group have been offered. Among them there are various versions of the radioimmune analysis [Nicolau G., Szucs-Myers V., McWilliams W., Morrison J., Lanzilotti A. (1985). Investigational New Drugs, 3: pp.51-56 ], the high-pressure thin-layer chromatography [Avramis V. (1982), Abstract. Pharmacologist, 24, p.241; Ehninger G., Proksch B., Hartmann F., Gartner H. V., Wilms K. (1984). Cancer Chemotherapy and Pharmacology, 12, pp.50-52 ], the method of replacement of DNA bound ethydiumbromide [Horvath J. J., Gueguetchkeri M., Gupta A., Penumatchu D., Weetall H. H., (1995), Biosensor and Chemical Sensor Technology. Ed. by Rogers K. R. et al., Washington, ASC Symp.Ser. 613, pp. 45-60]. The greatest application has been gained by the columnar high-pressure-liquid-chromatography (HPLC) [Chiccarelli F. S., Morrison J. A., Cosulich D. B., Perkinson N. A., Ridge D. N. (1986), Cancer Research, 46, pp. 4858-4861; Lin K. T., Rivard G. E., Leclerc J. M. (1989). J. Chromatography, 465, pp.75-86], carried out by the standard chromatographic devices.
The application of the HPLC method for determination of one of important anthraquinone—mithoxantrone (MX) has been described rather recently [Nicoletto M. O., Padrini R., Ferrazi E., Nascimben O., Visona E., Tumolo S., Palumbo M., Cossta L., Vinante O., Monfardini S., Fiorentino M. V. (1993). Eur. J. Cancer. 29A, pp.1242-1248]. According to this method, MX and products of its methabolism are extracted from blood of patients, then they are concentrated, and the chromatographic allocation of MX is carried out with a subsequent spectrophotometric determination of its concentration. Despite rather high accuracy of MX determination (a few tens of ng/ml), the specified method is characterized by:
duration of the whole determination process reaching two days, that is caused by the necessity of a special pretreatment of samples (processing of patients blood, extraction and concentration of MX, etc.),
application of rather expensive equipment, namely, the high-pressure chromatographs or similar devices,
necessity to use the high skilled personnel for realization of the whole cycle of the analysis.
A laboratory method for determination of colored BAS, those “targets” are the double-stranded DNA molecules, that takes into account the interaction of BAS with DNA molecules forming liquid crystals immobilyzed in films (gels) biosensor is known as well [Skuridin S. G., Pozdnyakov V. N., Tokareva L. G., Yevdokimov Yu. M. (1991), Patent of the Russian Federation N 2016888].
This method includes:
formation of the lyotropic liquid-crystalline dispersion of DNA in an aqueous-salt solution containing a neutral polymer,
addition the special monomers capable to be polymerized, and polymerization of the obtained mixture,
reception of a film (gel) with the form and the size that are convenient for the experimentator,
immersing of the film (gel) into an analyzed laboratory solution containing BAS, and exposure of the film in this solution during time that is sufficient for diffusion of BAS into the film and interaction with DNA molecules,
registration of a spectrum of circular dichroism (abnormal optical activity) in the region of BAS absorption,
determination of the BAS presence by the shape of a band in the spectrum of circular dichroism.
However, the exact determination of BAS concentration and, consequently, the practical opportunities of the specified method for BAS determination are limited by the following factors:
difficulty of creation of films (gels) that are adequate to the certain physico-chemical requirements (neutral in relation to DNA character of a film, its transparency, optical isotropy, etc.),
difficulty in the maintenance of constant properties of DNA liquid crystals in a film structure, even during time for diffusion of BAS,
significant interval of time, after which the registration becomes possible of an appreciable value of the optical signal arising as a result of diffusion of BAS molecules into a polymeric film and their subsequent interaction with nucleic acid moleculs forming the liquid crystalline dispersion,
impossibility for exact determination of the value of the abnormal optical signal in the UV-region of the spectrum (i.e., in the DNA absorption region), that is caused by an unsufficient transparency of films (gels) in this part of the spectrum. Therefore, though the presence of the substance under analisis can be registered, the exact determination of its concentration is extremely difficult,
application for registration of the abnormal optical signal of the expensive stationary dichrographs (Jobin-Yvon, Mark III or Mark V; Jasco, Model 710/720) being available, as a rule, only in specialized scientific laboratories. A lack of these devices is not only their high cost but also low speed of registration of the optical signal.
A well known dichrograph of “Jasco” firm (Model J-710/720 Spectropolarimeter, Instruction Manual: Jasco Corporation, 2967-5, Ishikwa-Cho, Hachioji City, Tokyo, Japan (June, 1990)), which can be applied as a device for determination of BAS in an analyzed liquid, comprises installed consistently:
a source of light radiation,
a selector forming a light flow of a certain wavelength;
a polarizer forming a linearly polarized light of the specified light flow;
a modulator of polarization transforming the linearly polarized light flow into a circularly polarized light flow with a periodically varied direction of rotation of its polarization vector;
a cell for an analyzed sample;
a photodetector transforming an optical signal generated by components of the sample to be analyzed into the proportional electrical signal;
a synchronous amplifier of the specified electrical signal;
a processing unit for processing the received electrical
Chernukha Boris Alexandrovich
Evdokimov Jury Mikhailovich
Kolosov Vladimir Vasilievich
Kompanets Oleg Nikolaevich
Mikhailov Evgeny Leonidovich
Font Frank G.
Institut Molekulyarnoi Biologi Imeni V.A. Engelgardita Rossiisko
Ostrolenk Faber Gerb & Soffen, LLP
Punnoose Roy M.
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