MXPA99008488A - Rapid method of cancer diagnosis - Google Patents

Abstract

This invention refers to a rapid method of cancer diagnosis. In particular, the present invention relates to a rapid method for cancer diagnosis which is suitable to detect the presence of a carcinogenic process in a subject in the asymptomatic phase or the early phase. The present invention further relates to an activator compound of carbonic anhydrase II, i.e., a tumor marker, which is present in the serum of subjects having cancer, to a method for treating cancer which method comprises detecting cancer in the patient in the asymptomatic phase or in early stage by said method for cancer diagnosis;and to a kit for performing said method for cancer diagnosis.

Description

RAPID DIAGNOSTIC METHOD OF C NCER BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a rapid method of cancer diagnosis, which is suitable for detecting the presence of carcinogenic processes in a subject in the asymptomatic phase or early phase. The present invention also relates to a carbonic anhydrase activating compound II, i.e., an oral marker thereof, which is present in the serum of subjects having cancer; to a method for treating cancer which comprises detecting cancer in the patient in the asymptomatic phase or in the early stages by said method of cancer diagnosis; and a packet or it to execute said method of cancer diagnosis. The method for diagnosing cancer of the present invention is based on an effect of the serum of cancer patients on the activity of purified carbonic anhydrase II. It was found that the activity of purified carbonic anhydrase II is significantly increased by the addition of an adequate amount of serum from cancer patients. On the other hand, neither the serum of healthy volunteers nor the serum of patients who had other diseases, except cancer, showed a similar effect.

REF .: 31345 DESCRIPTION OF THE PREVIOUS TECHNIQUE The carbonic anhydrase is an enzyme with zinc described by Meldrum et al. , in 1932 (Meldrum N. U., Roughton J.: Carbonic Anhydrase: Its preparation and properties, J. Physiol. (London), 1933, 80, 113-142). This enzyme is located in erythrocytes and other cells of the body and participates in the maintenance of the acid-base balance (Maren TH: Carbonic Anhydrase, Physiological Reviews 1967, 47, 585-782, He ett-Em ett D., Hopkins PJ , Tashian RE, Czelusniak J.: Origins and molecular evolution of the carbonic anhydrase isozymes, Ann., NY Acad. Sci. 1984, 429, 338-358). Eight different isoenzymes of carbonic anhydrase are known in vertebrates (designated from I to VIII), which have different physiological functions and participate in: gastric secretion of acid, formation of aqueous humor and cerebrospinal fluid, secretion of pancreatic bicarbonate, intermediate metabolism, calcification, etc. Previous research by the applicants showed that specific inhibitors of carbonic anhydrase (acetazolamide, ethoxzolamide, benzothiazole-2-sulfonamide) administered orally at normal therapeutic doses, reduce the secretion of gastric acid and lead to the disappearance of ulcerative pain and the endoscopic healing of gastroduodenal ulcers in a very short period (I. Puscas et al.: Les inhibiteus de l'anhydrase carbonique dans le traitement de 1 'ulcere gastrique et duodenal, Archive Francaises des Maladies de l'Appareil Digestif, Paris, 1976 , 65: 577-583; I. Puscas: Treatment of gastroduodenal ulcers with carbonic anhydrase inhibitors, Ann. New York Academic of Sciences, 1984, 587-591; I. Puscas: Carbonic anhydrase inhibitors in the treatment of gastrie and duodenas ulcers , in: New Pharmacology of Ulcer Disease, S. Szabo, Gy. Mozsik (Editors) Elsevier Pubis, House, New York, USA, 164-178, 1987. I. Puscas: Clinical Pharmacology of Ulce Peptic, in: Aparelho Digestivo, Clínica e Cirurgia, Julio Coelho (Ed) MEDSI Publ. House, Rio de Janeiro, Ia Edition in 1990, 2nd Edition in 1996, 1704-1734; I. Puscas: Avaliagao do patient co doenga do stomago and duodeno, in: Aparelho Digestivo. Clínica e Cirurgia, Julio Coelho (Ed.), MEDSI Publ. House, Rio de Janeiro, Brazil, Edition in 1990, 2nd Edition in 1996, 173-179). Additional research by the Applicants has shown that histamine, gastrin and acetylcholine - which are major stimulators of gastric acid secretion - directly activate carbonic anhydrases II and IV in the gastric mucosa (I. Puscas: New concepts concerning the mechanism of stimulation of gastric acid secretion by hista ine VIth World congress of Gastroenterology, Madrid, 1978, 213; I. Puscas: Direct activation by histamine of the carbonic anhydrase in the human gastric mucosa, Rev. Roum. Biochim., 1979, 4: 317-320). Studies of the Applicants have also shown that isoenzyme I of carbonic anhydrase, which is located in erythrocytes and vascular walls, is involved in vascular modulation processes (I. Puscas et al .: Inhibition of carbonic anhydrase by nitric Arzneimittel - Forschung / Drug Research, 1995, 8: 846-848, I. Puscas et al .: Prostaglandins having vasodilating effects inhibit carbonic anhydrase while leukotrienes B4 and C4 increase carbonic anhydrase activity.

Int. J. Clin. Pharmacol Therapeutics, 1995, 32, 3: 176-181; I. Puscas et al. : Isosorbide nitrates, nitroglycerine and sodium nitroprusside induces vasodilation, conco itantly inhibiting down to abolish carbonic anhydrase I in erythrocytes. American Journal of Hypertension, 1997, 10 (1), 124-128). The isoenzyme II of carbonic anhydrase, which is located in erythrocytes and in secretory cells, modulates the secretory process in the body (I. Puscas et al .: World Congress of Gastroenterology, Los Angeles, oct. 1994: 1329-32; 1366-81; I. Puscas et al. : Nonsteroidal anti-inflammatory drugs activate carbonic anhydrase by a direct mechanism of action. J. Pharmacol. Exp. Therap., USA, 1996, 277, 3: 1146-1148).

The results of the Requesters led to the postulation of a new theory for the transmission of signals within the cell, the theory of pH. According to this theory, stimuli or primary messengers that produce vascular and secretory modifications, act by a dual mechanism: both in specific receptors of the cell membrane and directly in the carbonic anhydrase bound to the membrane. This enzyme, by its activation or inhibition, ensures an adequate pH for the formation of the receptor complex of stimuli in order to transmit the information to the cellular effector. In this way, through pH modifications initiated by carbonic anhydrase, this enzyme is involved in the modulation of physiological and pathological processes in the organism, as well as in carcinogenesis (I. Puscas et al .: Carbonic anhydrase and modulation of physiological and pathologic processes in the organism, I. Puscas - (Editor), Helicón Publishing House Timisoara, Romania, 1994, the Romanian version p 155-205 and 577-585, and the English version p. 551-559; I. Puscas: Carbonic anhydrase - a modulator of physiological and pathologic processes in the organism: The pH Theory, The 4th International Conference on the Carbonic Anhydrase, July 1995, University of Oxford, England, I. Puscas: Carbonic anhydrase Modulating Physiological and Pathological Processes in Organism, The Theory of Philosophy, The Medical Novelty (Noutatea Medicale), Bucharest, Romania, 1997, 3: 5-15). Recent years have witnessed remarkable progress in understanding the biological and biochemical basis of cancer. Cancer is a disease characterized by the uncontrolled growth of abnormal cells that spread from the anatomical site of origin to other tissues. This metastatic process involves a variety of cellular events that take place before any tumor cell gives rise to a metastatic colony. This spread, if left unchecked, is the primary cause of the fatal outcome of oncological diseases. However, many cancers can be cured if they are detected early and are treated quickly; others can be controlled for many years with a variety of treatments. In vitro and in vivo studies of the Applicants have shown that the carcinogenic substances reduce the activity of carbonic anhydrase II and of superoxide dismutase, and the anticarcinogenic substances increase the activity of these enzymes (I. Puscas et al.: Relation between carbonic anhydrase I, CA II, non-steroidal anti-inflammatory drugs and carcinogenic substances, Digestive Disease Week, San Diego, California, May 1995, 0643; I. Puscas et al .: The valúes of superoxid dismutase in patients with gastric cancer as compared to controls, Digestive Disease Week, San Francisco, California, May 1996, 0798; I. Puscas et al.: Anticancer chemotherapy increases red cell and gastric mucosa carbonic anhydrase activity In vivo studies Digestive Disease Week, San Francisco , California, May 1996, 0797). Other recent studies by the Requesters - carried out with patients who presented different forms of cancer (histologically and to oogrically confirmed) - have shown that the activity of carbonic anhydrase II and the superoxide dismutase of erythrocytes are weak in all patients, compared to the control group consisting of healthy volunteers (I. Puscas et al .: The effect of benzpyrene and cyclophosphamide on superoxid dismutase and carbonic anhydrase activity in gastric carcinoma patients as compared to controls.) The First Foru of Gastroenterology, "Banat Crisana ", Timisoara, Romania, Oct. 1996). Until now, in many cases the early detection of tumors and the careful surveillance of the disease are conceivable through the use of tumor markers that are biochemical indicators of the presence of a neoplastic proliferation and that can be detected in serum, plasma or other body fluids. The main utility of tumor markers such as carcinoembryonic antigen (ACE), the tumor necrosis factor TNF-a and ß), sialic acid, prostate specific antigen (PSA), etc., has been in the determination of the response to treatment, the detection of residual disease or relapses. To date, no tumor marker has been shown to be specific; virtually all of them are present at low levels in the normal physiological state or in non-neoplastic diseases. Thus, these markers are not applicable in a general screening test for cancer. In order to eliminate the main disadvantages of tumor markers, in the present invention a rapid method of cancer selection is established in its asymptotic stages, or its confirmation in the symptomatic phase. This early cancer detection allows an efficient surgical or pharmacological therapy, together with the other usual methods used to locate tumors. The Applicants have now found that the activity of purified carbonic anhydrase II is activated significantly by the serum of cancer patients and that such activation is not observed in the serum of healthy persons or in the serum of patients who have other diseases that are not Cancer. Based on these observations, the Applicants found that by testing the effect of blood serum on the activity of purified carbonic anhydrase II, it is possible to diagnose any type of cancer, even those in their early stages. BRIEF DESCRIPTION OF THE INVENTION Thus, the present invention provides (1) a method for the diagnosis of cancer comprising (a) preparing a first reaction mixture by combining a diluted blood serum sample of the subject to be tested with a purified carbonic anhydrase solution; (b) determining the activity of carbonic anhydrase II in the first reaction mixture; (c) determine the activity of carbon dioxide II in a second reaction mixture containing all the components of the first reaction mixture except blood serum; and (d) evaluating the degree of activation of carbonic anhydrase II from step (b) in relation to the passage of part (c); (2) a tumor marker present in the serum of subjects having cancer, wherein the tumor marker activates carbonic anhydrase at concentrations of 1 μM or less; (3) a method for treating cancer, which comprises detecting the cancer process in a patient in the asymptomatic phase or in the early stages by a method for the diagnosis of cancer as defined in subsection (1) above; and (4) a package or kit to perform the cancer diagnosis method as defined in subsection (1) above, wherein the package or kit comprises a composition containing carbonic anhydrase II. DETAILED DESCRIPTION OF THE INVENTION The method for diagnosing cancer of the present invention is an in vi tro method and the preferred subjects to be tested are human beings. In a preferred embodiment of the method for diagnosing cancer, the diluted blood serum contains from 0.1 to 50% by volume, preferably from 1 to 10% by volume of blood serum. The diluent for the diluted blood serum is selected from the group consisting of water and aqueous organic solvents, eg, aqueous mixtures containing from 0.1 to 50% by volume, preferably from 1 to 15% by volume of one or more organic solvents, wherein the diluents may optionally contain a buffer solution and / or organic or inorganic salts. Suitable organic solvents include lower alcohols such as methanol, ethanol, propanol, isopropanol and butanol, ketones such as acetone and butanone, diols and triols such as ethylene glycol, 1,3-propanediol and glycerin; and ethers such as diethyl ether diethylene glycol and 2-methoxyethanol. Suitable buffer solutions include HEPES, DIPSO, TAPSO, TES and MOPS solutions and suitable organic or inorganic salts include Na2S04 and K2S0. The concentration of carbonic anhydrase II in the purified carbonic anhydrase II solution varies from O.Ol M to O.l mM, preferably from 1.0 nM to 1.0 μM. Suitable solvents for the solution are selected from the group consisting of water and aqueous organic solvents, which may also contain a buffer solution, organic or inorganic salts and / or one or more indicators. With respect to the organic solvent, the buffer solutions and the organic or inorganic salts, these refer to the diluted blood serum sample defined above. Suitable indicators include p-nitrophenol, phenol red and tetrazolium nitro blue. In accordance with the present invention, it is preferred that the diluted blood serum sample and the purified carbonic anhydrase solution are combined in a ratio of 100: 1 to 1: 100, preferably in a ratio of 10: 1 to 1:10 and more preferably in a ratio of 1: 1.

In a preferred embodiment, the first reaction mixture contains 0.05 to 25% by volume, preferably 0.5 to 5% by volume of blood serum. It should be understood that an activation of 100% or greater in the first reaction mixture - wherein the first reaction mixture contains 25% by volume or less, preferably 5% by volume or less of serum - in relation to the activity of the carbonic anhydrase II of the second reaction mixture (viz, the activity of carbonic anhydrase in the first reaction mixture being at least twice as much as the second reaction mixture), indicates the presence of a carcinogenic process in the subject. In another preferred embodiment of the method of the present invention, the diluted blood serum sample is an aqueous solution containing 10% by volume of serum, the purified carbonic anhydrase II solution is an aqueous buffer containing 1.0 to 10 μM carbonic anhydrase II and the first reaction mixture contains the diluted blood serum sample and the purified carbonic anhydrase II solution in a ratio of 1: 1. The activity of carbonic anhydrase II can be determined using one of the known methods, e.g., as described in the specific scientific literature cited above. In a preferred embodiment of the method of the present invention, the activity of carbonic anhydrase is determined by the stop-flow method (Khalifah RG: The carbon dioxide hydration activity of carbonic anhydrase: stop-flow kinetic studies on the native human isozymes B and CJ Biol. Chem., 1971, 246: 2561-2573). This method involves measuring the enzymatic activity of CO2 hydration. The hydration of CO2 is followed by a colorimetric method based on the change in pH. The time in which the pH of the reactive mixture decreases from its initial value of 7.5 to its final value of 6.5 is measured. The reaction is followed spectrophotometrically at a wavelength of 400 nm, using a HI-TECH SF-51MX fast kinetic spectrophotometer (England), equipped with a mixing unit and a system of two syringes that supply the reagents. The reagent supply in the reaction cuvette is carried out by two separate routes: the syringe one introduces the reaction mixture made of enzyme-regulatory solution (ie, the first or second reaction mixture), while the second syringe introduces the solution of substrate, which is an aqueous CO2 solution having a CO2 content of 5 to 50 mM, preferably 15 nM. The reagents are mixed when entering the reaction cell and in this the reaction begins and its monitoring. The signal transmitted by the photomultiplier from the mixing chamber is received and visualized by a computer equipped with a mathematical coprocessor and a kinetic software RKBIN IS-1. The detection of carbonic anhydrase activity can also be tested by other methods such as: Methods based on pH changes (Philpot F. J. et al., A modified colorimetric estimation of carbonic anhydrase, Bioche. J., 1986; 30: 2192-94; Kernohan J. K. et al. : The activity of concentrated solutions of carbonic anhydrase: chemistry, physiology and inhibition. Physiol. Rev. 1967, 47: 595-781; Wilbur K. M. & Anderson N. 0.: Electrometric and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 1948, 176: 147-153). This method is based on the measurement of the production and consumption of H and can be carried out a) by pH indicators in which case the production of H is visualized by the color change of an indicator, or b) by the electrodes of pH, in which case the production and consumption of H is measured with pH electrodes. Methods based on changes in CO2 concentration or PCO2, which are based on the measurement of the production or consumption of C02 of the reversible reaction C? 2 <;?: > HC? 3 by a) the gas manometric technique (Meldrum N.

U., Roughton F.J.W., J. Physiol, London, 1933, 80: 113-142: Roughton F.J.W. et al. , Biochem J., 1946, 40: 319- 390) or by b) CÜ2 electrodes (Holland R. A. B. et al., J. Physiol., London, 1975, 228: 1589-1596; Klocke R. A., J. Appl. Physiol., 1976, 40: 707-714). Methods based on changes in the reaction temperature. These are based on the measurement of the rapid initial temperature increase caused by the reaction, where the measurement is carried out with an apparatus equipped with thermocouples (Kernohan J.C. et al., J. Physiol., London, 1968, 197: 345-361). Methods with labeled isotopes (tracers) such as O (Silverman D. N., Methods Enzymol, 1982, 87: 732-752). Methods with application of nuclear magnetic resonance (NMR) with 13C (Simonsson I. et al., Eur. J.

Biochem. 1979, 93: 409-417). Relaxation methods. In these the equilibrium in solution of HCC, 3, > C? 2 suddenly modifies its physical properties, for example its electric field, pressure or temperature, with the consequent modification of the chemical equilibrium (Eigen M. et al., Z. Physik. Chem. NF, 1961, 30: 130-136). Immunohistochemical methods are based on the cobalt trapping CO2 produced by carbonic anhydrase and which serves to localize carbonic anhydrase at a number of body sites (Hansson HPJ: Histochemical daya-slcaücn of carbonic anhydrase activity, Histochemie, 1967, 11: 112-128; Lonnerholm G.: Histochemical demonstration of carbonic anhydrase activity in the human kidney, Acta Physiol. Scand., 1973, 88: 455-468). Fluorometric methods (Shingles R., Moroney J.V.: Measurement of carbonic anhydrase activity using a fluoro etric assay, Analytical Biochemistry, 1997, 252, 1: 190-198). Methods that use other substances, these are methods that use other substances to measure carbonic anhydrase such as p-nitrophenyl acetate (Schneider F. et al.: Über die Reaktion von Carbonathydrolyase mit p-Nitrophenylacetat, Z. Physiol. Chem., 1963, 334: 279-282). As stated above, an activation value of 100% or higher in the first reaction mixture (which contains 25% by volume or less, preferably 5% by volume or less of serum) relative to the activity of the carbonic anhydrase II of the second reaction mixture, indicates the presence of a carcinogenic process. In addition, the Applicants found that a weaker activation of carbonic anhydrase II was obtained using the serum of cancer patients in the early stages (regardless of the location of the cancer). Thus, the method of the present invention is suitable for determining the stage of the cancer process in patients (by comparing the degree of activation of carbonic anhydrase II) in comparison with the activation produced by the serum of patients who are in stages more advanced However, in all cases of cancer patients in the early stages, the activation of carbonic anhydrase II (having the first reaction mixture 5% by volume of serum) exceeded 100% and the activation produced by the serum of patients They were in advanced stages, reached 400% or even more. The method of the present invention has the following advantages: 1. It allows the search of carcinomas in their asymptomatic phase, respectively in early stages in which the carcinomas are undetectable by conventional methods. 2. It is done using a small amount of blood serum. 3. It is highly sensitive and the tests are reproducible. 4. It is economically feasible due to the low prices of the required reagents. Starting from the previous results -referring to the activating effect of the serum of cancer patients on carbonic anhydrase II-, it was attempted to identify some responsible factors (ie, tumor markers) of the activation of carbonic anhydrase II in the serum of said patients with Cancer. So that, it was demonstrated that the tumor necrosis factors a and ß, carcinoembryonic antigen, sialic acid and cti-antiqui iotrypsin are potent activators of carbonic anhydrase II. Studies by the Requesters have also shown that serum gastrin levels are high in 80% of cancer patients, compared to healthy volunteers. Therefore, it could be found among the endogenous serum activators of carbonic anhydrase II. The tumor markers according to the present invention activate carbonic anhydrase II even at concentrations below 1 μM, in particular even at concentrations of 1 nM or lower, and even at concentrations of 1 pM or lower. Preferred tumor markers include tumor necrosis factors a and β, carcinoembryonic antigen (ACE), sialic acid, ai-antichymotrypsin and oncoprotein sp 185Her2. The tumor markers of the present invention can be used as standard solutions to quantitate the percentage of detected activation, in particular to determine the stage of the cancer process. The method of treating cancer in accordance with the present invention, which comprises detecting the cancer process in a patient in the asymptomatic phase or in the early stages as defined above, is suitable for treating any type of cancer in patients. The method for diagnosing cancer in accordance with the present invention, therefore, can be combined with any type of therapeutic treatment (e.g., surgical or chemotherapeutic). Detection in the early stages of the cancer process increases the chances of curing the cancer process. The package or kit for carrying out the method for diagnosing cancer according to the present invention comprises a composition containing carbonic anhydrase II. The composition may be in the dry state or it may be a solution. For information on other ingredients of the composition and the amount of carbonic anhydrase II in the composition, refer to the discussion of the compounds of the first reaction mixture described above. The package or kit may further comprise compositions containing a regulatory solution, eg, buffer solutions, compositions having an indicator substance and compositions containing one or more tumor markers, wherein the regulatory solution, the indicator and the tumor marker are as previously defined. The present invention will be described in more detail by means of the following non-limiting Examples. EXAMPLES Reagents: - p-nitrophenol -as a color indicator- was used at a concentration of 0.2 mM; pH = 7.5; temperature 20- 25 ° C = room temperature (hereinafter "t.a.");. A concentrated solution of purified carbonic anhydrase II (obtained from SIGMA Diesenhofen, Germany) was used at a concentration of 3.44 x 10 ~ M; pH = 7.5; t.a. A CO2 solution at a concentration of 15 mM (as a substrate) was obtained by bubbling CO2 into bidistilled water until saturation. Na2SO4 was used at a concentration of 0.1 M to keep the ionic strength constant. The purified carbonic anhydrase II activity assay was performed by measuring the time in which the hydrogenation of CO2 occurred in the presence of said enzyme. The measurement started at the time when the reaction started (when the absorbance of the reaction mixture was 3 units) until the end of the reaction (when the absorbance of the reaction mixture became zero). The absorbance values are illustrated in the reaction curve. The reaction cuvette comprised two syringes containing: Syringe I: 1 ml of a mixture of: 0.8 ml of buffer + indicator + 0.1 ml of carbonic anhydrase II solution (3.44 x 1GM) + 0.1 ml H20. Syringe II: 1 ml of a CO2 solution (15 mM). The reagent injection and carbonic anhydrase activity measurements were performed in the manner described above. The reaction time measured in this way was called T. The activity of carbonic anhydrase was obtained by the Formula: A = To-T [EU / ml] wherein o represents the reaction time without catalysis; and T represents the catalyzed reaction time (in the presence of carbonic anhydrase II).

The measurement of the activity of carbonic anhydrase II in the presence of serum according to the method of the present invention was carried out in the following manner: EXAMPLE 1 5 ml of venous blood was collected from a patient diagnosed with gastric cancer. The blood was kept for 30 minutes in a thermostat and then centrifuged for 5 minutes at 4,000 r.p.m. After centrifugation, 2 ml of serum were removed and three dilutions of 1/10, 1/50 and 1/100 were made in bidistilled water as follows: from 0.5 ml of serum + 4.5 ml of H2O a 1/10 dilution; taking 1 ml of serum from the 1/10 dilution and adding 4 ml of H2O, the 1/50 dilution was obtained; finally, taking 1 ml of serum from the 1/50 dilution + 4 ml of H2O, the 1/100 dilution was obtained. The first step was the measurement of the uncatalyzed reaction time, ie, the determination of the reaction time between: 0.8 ml of buffer + indicator and + CO2 solution- The time measured in this way was denoted as To and was 6.5 sec. The second step was the measurement of the reaction time of the CO2 solution with 0.1 ml of purified carbonic anhydrase II (3.44 x 10 M) in 0.8 ml of buffer + indicator and + 0.1 ml of H2O. The time measured in this way was denoted as T and was 3.25 sec. The activity of carbonic anhydrase II purified in the presence of H2O was obtained from the following Formula: A = T0 - T = 6. 5N - 3.25s = 1.00 [EU / ml] T 3.25s Third step: serum dilutions of 1/100, 1/50 and 1/10 were added in a volumetric ratio of 1.1 to 0.1 ml of a purified carbonic anhydrase II solution (3.44 x 10 ~ 6 M) in 0.8 ml of HEPES buffer (20 mM) + indicator (p-nitrophenol, 0.2 mM). Each reaction time of each mixture was denoted as Ti, T2, T3 and was measured in the presence of CO2. These values (Ti, T2, T3) represent the interval in which the absorbance of each reaction mixture fell to zero. The following values were obtained: Ti = 1.679 sec - for the 1/100 dilution; T2 = 1.264 sec - for the 1/50 dilution; T3 = 0.874 sec - for the 1/10 dilution. The activity of carbonic anhydrase II in the presence of serum was calculated as follows: Al = T2 = 6.5S - 1.679s = 2.871 [EU / ml] - in the dilution Ti 1.679s 1/100 serum A2 = or ~ T2 6.5S - 1.264s = 4.142 [EU / ml] - in the T2 dilution 1.264s 1/50 of serum A3r To ~ T3 6.5S - 0.874s ~ 6.435 [EU / ml] - in the dilution T3 0.874s 1/10 of serum The percentages of activation of the dilutions of serum were calculated as follows: j - ¿x 100 = 2.871-1.00 x 100 = 187% - in the dilution 1/100 At 1.00 of serum A2 - A x 100 = 4.142-1.00 x 100 = 314% - in the 1/50 dilution At 1.00 of serum A3 - A x 100 = 6.435-1.00 x 100 = 543% - in the 1/10 dilution At 1.00 of serum EXAMPLE 2 5 ml of venous blood was collected from a patient diagnosed with lung cancer and processed as in the first axis 1. First step: To = 6.40 sec. Second step: T = 3.20 sec; A = 1.00 [EU / ml]. Third step: T = 2.143 sec (dilution 1/100); Ai = 1986 [EU / ml]; 98% activation; T2 = 1449 sec (1/50 dilution); A2 = 3.415 [EU / ml]; 241% activation; T3 = 1.125 sec (1/10 dilution); A3 = 4,686 [EU / ml]; 368% activation; EXAMPLE 3 5 ml of venous blood was collected from a patient diagnosed with breast cancer and processed as in Example 1. First step: To = 6.35 sec. Second step: T = 3.175 sec; A = 1.00 [EU / ml]. Third step: i = 2,201 sec (1/100 dilution); i = 1.885 [EU / ml]; 88% activation; T2 = 1.545 sec (1/50 dilution); A2 = 3.108 [EU / ml]; 211% activation; T3 = 1170 sec (1/10 dilution); A3 = 4.425 [EU / ml]; 342% activation; EXAMPLE 4 5 ml of venous blood was collected from a patient diagnosed with ovarian cancer and processed as in Example 1. First step: o = 6.52 sec. Second step: T = 3.26 sec; A = 1.00 [EU / ml]. Third step: Ti = 2.330 sec (dilution 1/100); Ai = 1.798 [EU / ml]; 80% activation; T2 = 1664 sec (1/50 dilution); A2 = 2.917 [EU / ml]; 192% activation; T3 = 1338 sec (1/10 dilution); A3 = 3.870 [EU / ml]; 287% activation; EXAMPLE 5 5 ml of venous blood was collected from a patient diagnosed with testicular cancer and processed as in Example 1. First step: To = 6.24 sec. Second step: T = 3.12 sec; A = 1.00 [EU / ml]. Third step: Ti = 2.155 sec (1/100 dilution); Ai = 1895 [EU / ml]; 90% activation; T2 = 1553 sec (1/50 dilution); A2 = 3.018 [EU / ml]; 202% activation; T3 = 1.210 sec (1/10 dilution); A3 = 4.156 [EU / ml]; 316% activation; EXAMPLE 6 5 ml of venous blood was collected from a patient diagnosed with prostate cancer and processed as in Example 1. First step: To = 6.42 sec. Second step: T = 3.21 sec; A = 1.00 [EU / ml]. Third step: Ti = 2,407 sec (1/100 dilution); i = 1,708 [EU / ml]; 71% activation; T2 = 1.798 sec (1/50 dilution); A2 = 2626 [EU / ml]; 163% activation; T3 = 1.475 sec (1/10 dilution); A3 = 3.418 [EU / ml]; 242% activation; EXAMPLE 7 5 ml of venous blood was collected from a patient diagnosed with leukemia and processed as in Example 1. First step: o = 6.36 sec. Second step: T = 3.18 sec; A = 1.00 [EU / ml]. Third step: Ti = 2,108 sec (dilution 1/100); Ax = 2.016 [EU / ml]; 102% activation; T2 = 1.279 sec (1/50 dilution); A2 = 3.610 [EU / ml]; 261% activation; T3 = 1.046 sec (1/10 dilution); A3 = 5.080 [EU / ml]; 408% activation; EXAMPLE 8 5 ml of venous blood was collected from a patient diagnosed with healthy volunteer and processed as in Example 1. First step: To = 6.28 sec. Second step: T = 3.14 sec; A = 1.00 [EU / ml]. Third step: Ti = 3.14 sec (dilution 1/100); Ai = 1.0 [EU / ml]; 0% activation; T2 = 3.09 sec (1/50 dilution); A2 = 1.032 [EU / ml]; 3% activation; T3 = 3.06 sec (1/10 dilution); A3 = 1.051 [EU / ml]; 5% activation; EXAMPLE 9 The rapid method of diagnosing carcinoma according to the present invention was tested in two groups of patients in the following manner: Group 1 (N = 2320): patients suffering from different types of carcinoma, where the carcinomas were histologically confirmed. Group 2 (N = 2638): healthy volunteers and patients who had other diseases, but not cancer. In both groups venous blood was collected and the effect of the serum was determined at 1/100, 1/50 and 1/10 dilutions on the activity of the purified carbonic anhydrase II, according to Example 1. The results are presented in the Next tables, where the values are the average values of the activating effect of serum on purified carbonic anhydrase II.

The results show that the purified carbonic anhydrase II is activated exclusively by the serum of patients with carcinoma and that the activity of a carbonic anhydrase II is not altered by the serum of healthy volunteers or by the serum of patients without cancer. From the above results, it is clear that the activating effect is increased proportionally to the serum concentration, ie, in the Examples, the maximal activating effect was obtained at a 1/10 dilution (~ 5% by volume of serum in the first reaction mixture); and, on the other hand, by increasing the serum dilution (from 1/10 to 1/100) of ~ 5% by volume to 0.5% by volume, the activating effect of the serum is reduced. However, the activating effect is obvious even at a dilution of 1/100 (~ 0.5% by volume of serum in the first reaction mixture). UTILITY The method of the present invention provides a rapid test for the diagnosis of different types of cancer. The studies done by the Applicants to date include more than 3,000 patients, with different stages and locations of the tumor process, including: pharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, testicular cancer, prostate cancer, urinary bladder cancer, thyroid cancer, lip cancer, leukemia, malignant melanomas, Hodgkin's lymphoma, etc. The Applicants described numerous categories of carbonic anhydrase activators and inhibitors that can be administered as therapeutic agents in different cases and which could influence the test method described in the application. For this reason, any treatment with H2 receptor antagonists is recommended five days before carrying out the method for diagnosing cancer and also stopping any treatment with carbonic anhydrase inhibitors (acetazolamide, prostaglandins Ei, E2, I2) or any anti-inflammatory drug. no steroid 10 days before the test. Studies of the Requesters verify that the method of the present application can be used as a search test for all types of cancer. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (16)

1. REGVINDICATIONS Having described the invention as background, the content of the following claims is claimed as property: 1. A method for diagnosing cancer, characterized in that it comprises: (a) preparing a first reaction mixture by combining a diluted blood serum sample of the subject be tested, with a solution of purified carbonic anhydrase II; (b) determining the activity of carbonic anhydrase II in the first reaction mixture; (c) determining the activity of carbonic anhydrase II in a second reaction mixture containing all the components of the first reaction mixture except blood serum; and (d) evaluating the degree of activation of carbonic anhydrase II from step (b) in relation to the passage of part (c); 2. The method according to claim 1, characterized in that the diluted blood serum sample contains from 0.1 to 50% by volume, preferably from 1 to 10% by volume of blood serum. The method according to any of claims 1 or 2, characterized in that the diluent of the diluted blood serum sample is selected from the group consisting of water and aqueous organic solvents, wherein the diluent optionally can contain a buffer solution. The method according to claim 1, characterized in that the concentration of carbonic anhydrase II in the purified carbonic anhydrase II solution varies from 0.01 nM to 1.0 mM, preferably from 1.0 nM to 1.0 μM, where the solvent of the solution is selected from the group consisting of water and aqueous organic solvents and wherein the solvent may additionally contain a buffer solution and / or an indicator. The method according to claim 1, characterized in that the diluted blood serum sample and the purified carbonic anhydrase solution are combined in a ratio of 100: 1 to 1: 100, preferably in a ratio of 10: 1 to 1:10 and more preferably in a ratio of 1: 1. The method according to claim 1, characterized in that the first reaction mixture contains from 0.05 to 25% by volume, preferably from 0.1 to 10% by volume and more preferably from 0.5 to 5% by volume of blood serum. The method according to claim 6, characterized in that an activation of 100% or greater in the first reaction mixture, which contains 25% by volume or less, preferably 5% by volume or less of serum, in relation to with the activity of carbonic anhydrase II of the second reaction mixture, indicates the presence of a carcinogenic process in the subject. The method according to claim 1, characterized in that the diluted blood serum mixture is an aqueous solution containing 10% by volume of serum, the purified carbonic anhydrase II solution is an aqueous buffer solution containing from 1.0 to 10. nM carbonic anhydrase II and the first reaction mixture is prepared by combining the diluted blood serum sample with the purified carbonic anhydrase II solution in a ratio of 1: 1. The method according to any of claims 1 to 8, characterized in that the determination of carbonic anhydrase II is carried out by means of a kinetic stop-flow measurement of the hydrogenation of CO2 of said enzyme. The method according to claim 9, characterized in that the initial pH of the first and second reaction mixtures is 7.5 and wherein the determination of the activity of the enzyme comprises combining the first or second reaction mixture with a solution of substrate, which is in aqueous solution, containing 5 to 50 mM of CO2, preferably 10 to 25 mM of CO2 and more preferably 15 mM of CO2, and measuring the time until the final pH of 6.5 is reached. The method according to claim 1, characterized in that it is suitable for detecting a cancer process in a subject independently of the stage of the cancer process, particularly in the asymptomatic phase or in the early stages. The method according to claim 1, which is suitable for a diagnostic screening test for cancer at different stages and locations, including pharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, pancreatic cancer, cancer pulmonary, breast cancer, ovarian cancer, uterine cancer, testicular cancer, prostate cancer, urinary bladder cancer, thyroid cancer, lip cancer, leukemia, malignant melanomas and Hodgkin's lymphoma and any other form of cancer. 13. A method for the treatment of cancer, characterized in that it comprises detecting the cancer process in a patient in the asymptomatic phase or in the early stages by a method for diagnosing cancer comprising: (a) preparing a first reaction mixture by combining a sample of diluted blood serum of the subject to be tested, with a solution of purified carbonic anhydrase II; (b) determining the activity of carbonic anhydrase II in the first reaction mixture; (c) determining the activity of carbonic anhydrase II in a second reaction mixture containing all the components of the first reaction mixture except blood serum; and (d) evaluating the degree of activation of carbonic anhydrase II from step (b) in relation to the passage of part (c); 14. A package or kit for carrying out the method for diagnosing cancer according to any of claims 1 to 13, characterized in that it comprises a composition containing carbonic anhydrase II. The package or kit according to claim 14, characterized in that it further comprises a composition containing at least one tumor marker, wherein the tumor marker activates carbonic anhydrase II at concentrations of 1 μM or less. The package or kit according to claim 15, characterized in that the tumor marker is selected from the group consisting of tumor necrosis factors a and β, carcinoembryonic antigen, sialic acid, ai-antichymotrypsin and oncoprotein sp 185 Her2