Chemistry: analytical and immunological testing – Measurement of electrical or magnetic property or thermal... – Of a liquid
Reexamination Certificate
2001-09-25
2003-09-30
Witz, Jean C. (Department: 1651)
Chemistry: analytical and immunological testing
Measurement of electrical or magnetic property or thermal...
Of a liquid
C436S149000, C436S151000, C436S063000
Reexamination Certificate
active
06627452
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to medicine, in particular, to methods for screening drug preparations performed to choosing a drug and its optimal dosage for treatment of a particular patient.
DESCRIPTION OF THE PRIOR ART
In the current art, the conventional way to select a drug for a particular patient involves medical examination of the patient, comparison of the results of the examination with recommendations provided by pharmacopoeia, prescribing of a drug or drug combination, and correction of the prescription basing on observation of the patient's response (Methods of Experimental Chemotherapy: a Practical Guide. Ed. by N. G. Pershin, Moscow, 1971, 234 pp.—In Russian).
However, the time cost and unreliability of choosing a treatment methodology for a particular patient and a high degree of subjectivity made it expedient to search for novel, more objective methods for choosing drugs and their dosages for a particular patient suffering from a particular disease.
A method for screening of therapeutic drugs is disclosed in Russian Patent No 2 007 117 (IPC A 61 B 5/04, 1992), which involves positioning of investigator's hands over a patient and test substances and choosing the optimal drug basing on the temperature response of the investigator.
This method is sufficiently universal, however it is also somewhat subjective and insufficiently reliable.
Soviet and Russian Patents and Certificates of Authorship No 1 806 601 (IPC A 61 B 5/04476, 1990), No 1 540 804 (IPC A 61 B 10/00, 1987), No 2 000 739 (IPC A 61 B 5/02, 1991), and No 2 046 341 (IPC G 01 N 33/48, 1994) disclose methods for screening of therapeutic drugs for treatment of particular CNS disorders and viral, cancerous, and other diseases. The methods are based on administration of test drugs into patient's body or application of the drugs to tissue preparations or body fluids, distinguishing of significant parameters, measuring of these parameters for each test drug, and choosing the drug that causes the greatest effects on the chosen parameters as the optimal drug.
However, these methods are of a limited applicability and do not yield sufficiently reliable results where complex therapy is required.
A method for choosing a medicinal drug to treat cardiovascular deficiency has been disclosed that involves chemiluminescence analysis of venous blood after its incubation with the solutions of the drug at different concentrations and the choice of the dose of the drug that produces minimal chemiluminescence as the target dose. (Russian Patent No 2072100 CI of Jan. 20, 1997).
This method is also of a limited applicability.
SUMMARY OF THE INVENTION
The aim of the present invention is to provide a more rapid and universal method for screening of drug preparations.
To this end, it was suggested to perform screening basing on culturing of a sample of whole heparinized blood with aqueous solutions of test drugs at different doses, to determine the ratio of —SH and —SS groups in the cellular fraction of patient's blood after culturing, and to choose the drug at its dose that produces the greatest ratio of —SH and —SS groups as the optimal drug at the optimal dose. (In choosing the prescribed drug one should proceed from that the optimal drug and its optimal dose for treatment of a patient makes said ratio to be about 3.0, as experiments have shown.)
Blood culturing is typically performed for 1 h at a temperature maximally close to 37° C., the tested drugs being added at doses amounting to 1:5000 of their therapeutic doses.
The method is based on the hypothesis that blood sulfur-containing compounds are the most sensitive to factors of various etiologies associated with body diseases, so drugs capable of normalizing them are the most efficacious in treatment of these disease.
The major distinctions of the method provided by this invention are the use of whole blood for culturing, determination of a new parameter, i.e., the ratio of —SH groups and —SS groups in the cell fraction of blood, and the assumption of the optimal value of the parameter.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the method for screening drug preparations involves the following:
Each test drug at its in vitro dosage calculated to amount to 1:5000 of its therapeutic dosage is added to a test tube containing I ml of heparinized whole blood obtained from a peripheral vein, respective vehicles being added to control tubes, and the mixtures are incubated for 1 h at 37° C. The incubation time may be optionally protracted up to 2 h, which allows some increase in the accuracy of the method, however, this is reasonable only when test results for different drugs are close to each other, or the drugs render small effects on the ratio of —SH and —SS groups.
The amounts of —SH groups and —SS groups and their ratio in the cell fraction of blood are determined mainly using the methods of direct (—SH) and reverse (—SS) amperometric titration.
For direct amperometric titration, 10 ml of venous blood is added to a test tube containing 1-2 drops of 5000 IU/ml heparin as an anticoagulant. Then the blood sample is dispensed as 1-ml aliquots into the required number of test tubes (usually 7-9), which depends upon the number of drugs to be tested and their dosages. The test drugs are added to the test tubes at dosages required (each in vitro dosage is calculated to amount to 1:5000 of respective therapeutical dosage), whereas respective vehicles are added to control tubes. Then the mixtures are incubated in a thermostat for 1 h at 37° C. After incubation, the test and control tubes are centrifuged for 5-7 min at 800 rpm, and plasma is removed. Then each cell fraction is hemolyzed in a 0.1% Trylon B (Na-EDTA) solution (pH=7.0), for which purpose 1 part of blood cells is mixed with 19 parts of the solution, the mixture is placed into a refrigerator (+4° C.-+5° C.), and after 30 min the mixture is centrifuged for 15 min at 6000 rpm to sediment lysed cells. The supernatant (hemolysate) is used further. Into a titration vessel (a 30-40 ml beaker) 25-30 ml of ammonia buffer is added. The beaker is placed onto a magnetic stirrer, a platinum electrode and the free end of a salt bridge are immersed into the test solution and connected to an ammeter, and a capsule with a magnet is placed onto the bottom of the beaker and driven to rotation at such a rate as to avoid the sputtering of the solution. After the ammeter pointer has stabilized, titration is started by adding of 0.05-ml or 0.1-ml portions of a 10
−3
M AgNO
3
solution. After each consecutive portion, the pointer is allowed to stabilize, its position is recorded, and thereafter titration is continued. After the end point is achieved, each next portion of the titrating solution causes a sharp increase in electric current and the respective shift of pointer position toward greater values. After additional 4-5 check measurements, the titration procedure is stopped and its results are used to graphically determine the amount of silver nitrate required to titrate the solution, which makes the basis to calculate —SH group content.
For reverse amperometric titration, 25 ml of ammonia buffer is added to a titration vessel. The vessel is put onto the top of a magnetic stirrer, the indicator electrode and the end of the salt bridge connected to the reference electrode are immersed into the solution, the ammeter and magnetic stirrer are linked up to mains, the stirrer is switched on, and the following reagents are sequentially added to the buffer: 0.5 ml of 1×10
−4
M AgNO
3
solution, 0.2 ml of a hemolysate, and 200 mg of sodium sulfite.
The ammeter is switched on and, after its pointer has been stabilized (after about 3-5 min), titration of a test sample is started using a 5×10
−4
M unithiol solution added to the reaction mixture by 0.05 portions or 0.1 ml portions till the final volume of 0.5 ml is added. After titration is completed, the amount of unithiol required to titrate the sample is determined graphically, and the amount determin
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