Kit for early diagnosis and monitoring of tumors

Abstract

The present invention relates to a ready-to-use kit for the early diagnosis of tumors by analyzing a sample of a biological liquid and its use for the early diagnosis of tumors.

Description

Background Art

[0001] The authors of the report "Global Cancer Statistics 2020", jointly produced by the American Cancer Society (ACS) and the International Agency for Research on Cancer (IARC), reported that one in every five people globally will develop a tumor during their lifetime.

[0002] According to the report in the manuscript of the CA: A Cancer Journal for Clinicians, in which the document was published, in 2020, there were approximately 19.3 million newly diagnosed cancer cases globally, and the disease caused approximately 10 million deaths.

[0003] There are prevention programs for different types of cancer. Screening programs are examinations performed on a subset of the population with the aim of detecting the disease or its precursors before symptoms or a disorder are suspected to exist.

[0004] So far, not all cancer screening tests are available. In fact, tests that are useful for determining the presence of the disease in symptomatic cases are not always effective tools in screening programs, which aim to identify a small number of individuals in need of further investigation among many without a disorder.

[0005] EP3521443 describes a method for diagnosing cancer, which includes preparing a temporary solution from a substrate of iodine and potassium iodide in water and alcohols, and mixing it with a saliva sample from the subject to be analyzed. Molecular iodine (I 2 ) reacts with L-fucose optionally present in saliva. L-fucose (6-deoxy-L-galactose) (C 6 H 12 O 5 ) is the only levorotatory sugar used by the mammalian system, and it increases in the body under conditions of persistent hypoxia when it activates anaerobic glycolysis and other energy metabolism pathways. An increase in L-fucose in the body indicates a change in the oxidative metabolism of aerobic organisms under various stress conditions, characterized by low oxygen levels (persistent hypoxia), which favors the survival of undifferentiated anaerobic cells.

[0006] High levels of serum fucose have been reported in breast cancer, ovarian cancer, lung cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, and others.

[0007] The method described in EP3521443 has several drawbacks. First, the reagents used for the preparation of the solution are not easily accessible to the general population. In addition, the solution must be stored in the dark and at low temperature because it is not stable at all. For these reasons, the method described in EP3521443 is not an effective method for performing early diagnosis of tumors and cannot be put into practice unless by individuals with appropriate laboratories and chemical reagents.

[0008] Therefore, there is a need for useful tools for the early diagnosis of various types of tumors, which can be put into practice by anyone willing to do so in order to take the necessary treatment measures in a timely manner. Summary of the Invention

[0009] An object of the present invention is to provide a ready-to-use, non-invasive and easy-to-use kit that allows for the early diagnosis of tumors, particularly but not limited to breast cancer, ovarian cancer, lung cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, and other tumors.

[0010] Another object of the present invention is to provide a method for the early diagnosis of tumors, which includes using the kit of the present invention.

[0011] A further object of the present invention is to provide the use of the kit of the present invention for the early diagnosis of tumors.

[0012] Finally, a further object of the present invention is to provide the use of the kit of the present invention for monitoring the evolution of cancer diseases. Brief Description of the Drawings

[0013] Figure 1 An exemplary embodiment of the kit of the present invention is shown. Detailed Description

[0014] Starting from the concept proposed by the method described in EP3521443, the applicant has developed a kit that makes the non-invasive diagnosis of tumors more immediate and easier for anyone, not just for healthcare professionals or experts in analytical laboratories.

[0015] According to one of its aspects, the subject matter of the present invention is a kit for the early diagnosis of tumors, comprising:

[0016] - an optionally graduated container (1) provided with means (2) for its closure and one or more stirring means (3);

[0017] - an optionally graduated device (4) for collecting a sample of a biological fluid;

[0018] - a device (5) for transferring a metered amount of the sample;

[0019] - A pre-measured amount of molecular iodine (I 2 )(6);

[0020] - A pre-measured amount of an aqueous solution of an alkali metal iodide (7), where the alkali metal is preferably potassium iodide;

[0021] - A glass slide (8), preferably provided with a cavity;

[0022] - An optional colorimetric scale (9); and

[0023] - An optional information sheet (10).

[0024] The kit may further include an additional container (11) for the initial collection of a biological fluid. Alternatively, any clean container can be used to collect the biological fluid.

[0025] The container (1) can be, for example, a test tube equipped with a lid (2), where the lid is made of a suitable material compatible with iodine, such as plastic, and is preferably made of dark glass or in any case a material that does not interact with molecular iodine and iodide.

[0026] The container preferably has a capacity of 5 ml to 20 ml, more preferably 10 ml to 15 ml, and a suitable thickness to resist the impact on its walls caused by one or more stirring devices (3).

[0027] One or more stirring devices (3) can be, for example, small balls made of glass or other materials that do not interact (i.e., do not react) with molecular iodine and potassium iodide.

[0028] Preferably, one or more stirring devices are glass balls, more preferably two or more small glass balls with a diameter of 2 mm - 5 mm. The presence of the stirring devices not only helps in the mixing of the reagents with the biological sample but can also cause cell lysis and thus promote the binding of iodine to optionally present L-fucose, thereby increasing the sensitivity of the test. This technical solution is not provided in EP3521443.

[0029] The device (4) for collecting a sample of the biological fluid can be graduated, such as a disposable graduated pipette for saliva samples or a graduated collector, preferably a graduated pipette that can hold 1 ml to 15 ml, more preferably 1 ml to 10 ml of the biological fluid. Alternatively, the test tube or other container (1) is graduated from 1 ml to 15 ml or from 1 ml to 10 ml; in this case, the device for collecting the sample, such as a pipette, can collect the sample without measuring it and release it into the test tube or other container until the desired volume is reached.

[0030] Alternatively, if the device (4) for collecting a sample of biological fluid is not graduated and the container (1) is not graduated either, there is in the kit a device (5) for transferring a metered amount of the sample into the container (1).

[0031] The biological fluid according to the invention is any biological fluid, such as a sample of saliva, urine, serum or semen, preferably a saliva sample.

[0032] A pre-metered amount of molecular iodine (I 2 )(6) can be in the range from 1 mg to 20 mg, preferably 1 mg to 10 mg, such as 2 mg, 3 mg, 4 mg or 5 mg, and is contained in a suitable closed vessel, preferably made of glass or a material that in any case does not interact with molecular iodine. Preferably, the molecular iodine is bisublimated iodine.

[0033] The iodide solution (7) is preferably potassium iodide. The potassium iodide solution is preferably an aqueous solution of 0.5% (w / v), and the pre-metered amount can be in the range from 0.1 ml to 2 ml, preferably from 0.1 ml to 1 ml, such as 0.2 ml, 0.3 ml, 0.4 ml or 0.5 ml, and is contained in a suitable closed vessel, which is preferably made of glass or a material that in any case does not interact with potassium iodide. However, other concentrations of potassium iodide solution can be used in the kit of the invention, taking into account the factors described below.

[0034] Preferably, the solution only includes water and potassium iodide and does not include alcohols, such as ethanol.

[0035] The amounts and concentrations of both the molecular iodine and the potassium iodide solution are related. For every 2 mg (milligrams) of molecular iodine, preferably 0.5 mg of potassium iodide is present in the solution.

[0036] Thus, if the kit contains 4 mg of molecular iodine, the solution may contain 1 mg of potassium iodide.

[0037] In other words, the molecular weight ratio of molecular iodine / potassium iodide is about 4 / 1.

[0038] Based on the amount of molecular iodine given herein, a person skilled in the art is fully able to calculate the amount of iodide salt to be used in the case of using an iodide of another alkali metal, such as sodium iodide.

[0039] Similarly, the amounts of the two reagents (6) and (7) are related to the amount of biological fluid to be analyzed. For every milliliter of biological fluid, such as saliva, preferably there are 2 mg of molecular iodine and 0.5 mg of potassium iodide in the aqueous solution in the kit.

[0040] Representative examples of the ratio of reagent to biological sample are as follows:

[0041] - 2 mg of iodine, 0.5 mg of potassium iodide, and 1 ml of biological sample;

[0042] - 4 mg of iodine, 1 mg of potassium iodide, and 2 ml of biological sample;

[0043] - 5 mg of iodine, 1.25 mg of potassium iodide, and 2.5 ml of biological sample;

[0044] - 6 mg of iodine, 1.5 mg of potassium iodide, and 3 ml of biological sample.

[0045] The container (1) and the receptacles containing the reagents (6) and (7) are preferably dark-colored, such as dark glass ampoules, to prevent light from penetrating to the interior.

[0046] The examples described in the experimental section of this specification illustrate representative embodiments of the kit of the present invention, as well as the relationship between the biological liquid and the reagent.

[0047] The kit of the present invention further includes a slide (8), which preferably has a cavity into which the sample to be analyzed can be poured after adding the reagents (6) and (7) and subsequent stirring. The slide is preferably made of glass or a material that does not interact with the reagent in any way.

[0048] The slide may have a colorimetric scale or a stain (spot block) of one or more reference colors. Alternatively, a colorimetric scale (9) ranging from white to dark brown can be found separately in the kit.

[0049] Preferably, the kit may further include an information sheet (10) that explains the procedure for performing the test.

[0050] The order of addition of the reagent and the biological liquid is not important. The molecular iodine reagent (6) and the iodide solution (7) can be added first, followed by the biological liquid, or vice versa.

[0051] For example, according to the embodiment of the reference Figure 1 , molecular iodine (6) and an iodide solution, preferably a potassium iodide solution (7), are added to a sample of biological liquid collected from the container (11) by means of a device (4) for collecting biological liquid, in this case a graduated pipette, and poured into the container (1). The color of the sample inside the container turns brown. The container must be closed and stirred for a few seconds, such as 2 to 10 seconds. When the stirring is complete, after 1.5 - 3 minutes, a few drops of the treated sample are poured into the slide cavity and verified visually, preferably by comparison with a colorimetric scale placed on or separate from the slide.

[0052] If the initially brown color in a container containing a sample and a reagent turns white, which is a sign that the iodine in the reagent has bound to L-fucose, then the test result is positive (presence of a tumor). In the presence of an intermediate color, it is useful to retest after one week or several weeks.

[0053] A color scale from white to dark brown can help in evaluating the result, but visual observation alone is also sufficient.

[0054] Compared to the method described in EP3521443, the kit of the present invention is a practical, fast, and easy-to-use solution for anyone.

[0055] In fact, the perfect pre-measurement of the reagents in the kit and their simple addition to the container where the sample to be tested is placed are significant technical advantages compared to the method of EP3521443.

[0056] As described above, the presence of a tumor causes an increase in the level of L-fucose in the body, which includes body fluids such as saliva, urine, serum, and semen. The reaction between the iodinating reagent and L-fucose causes the color inside the container containing the treated sample according to the present invention to change from brown to white.

[0057] According to another aspect thereof, the subject matter of the present invention is the use of the kit for the early diagnosis of tumors, in particular but not limited to breast cancer, ovarian cancer, lung cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, and other tumors.

[0058] According to another aspect thereof, the present invention includes an in vitro method for the early diagnosis of tumors, in particular but not limited to breast cancer, ovarian cancer, lung cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, and other tumors, the method comprising using the kit of the present invention to test a sample of a biological fluid selected from saliva, urine, serum, and semen, the biological fluid preferably being saliva.

[0059] The level of L-fucose gradually increases as the tumor disease worsens, so the kit of the present invention is also useful for monitoring tumor diseases, i.e., checking for their improvement or deterioration.

[0060] Therefore, according to another aspect thereof, the subject matter of the present invention is the use of the kit for monitoring tumor diseases, in particular but not limited to breast cancer, ovarian cancer, lung cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, and other tumors.

[0061] According to another aspect thereof, the present invention includes an in vitro method for monitoring tumor diseases, especially but not limited to breast cancer, ovarian cancer, lung cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer, and other tumors. The method includes using the kit of the present invention to test a sample of a biological fluid selected from saliva, urine, serum, and semen, and the biological fluid is preferably saliva.

[0062] The following experimental section describes representative embodiments of the present invention for illustrative purposes and is not limiting.

[0063] Experimental section

[0064] Example 1

[0065] The kit according to the present invention includes:

[0066] - A 10 ml dark glass container equipped with a hard plastic lid and two small glass beads;

[0067] - A dispensing pipette for collecting a sample of the biological fluid;

[0068] - 4 mg of double-sublimated molecular iodine in a sealed glass container;

[0069] - 0.2 ml of a 0.5% (weight / volume (w / v)) aqueous potassium iodide solution, equivalent to 1 mg of potassium iodide;

[0070] - A colorimetric scale from white to dark brown; and

[0071] - An information sheet.

[0072] Example 2

[0073] The kit according to the present invention includes:

[0074] - A 5 ml amber glass container equipped with a plastic lid and two small glass beads;

[0075] - A dispensing pipette for collecting a sample of the biological fluid;

[0076] - 2 mg of molecular iodine (I 2 ) in a sealed glass container;

[0077] - 0.1 ml of a 0.5% (weight / volume (w / v)) aqueous potassium iodide solution, equivalent to 0.5 mg of potassium iodide;

[0078] - A glass slide provided with a cavity and a colorimetric scale from white to dark brown on the side; and

[0079] - An information sheet.

[0080] Example 3

[0081] The kit according to the present invention comprises:

[0082] - A 10 ml dark glass container equipped with a plastic lid and two small glass beads;

[0083] - A dispensing pipette for collecting samples of biological liquids;

[0084] - 6 mg of molecular iodine (I 2 ) in a sealed glass container;

[0085] - 0.3 ml of a 0.5% (weight / volume (w / v)) aqueous potassium iodide solution, equivalent to 1.5 mg of potassium iodide;

[0086] - A glass slide provided with a cavity;

[0087] - A colorimetric scale from white to dark brown on the side; and

[0088] - An information leaflet.

Claims

1. A kit for the early diagnosis of tumors, said kit comprising: - An optionally graduated container (1), said container being provided with means (2) for its closure and one or more stirring means (3); - An optionally graduated device (4) for collecting a sample of a biological fluid; - A device (5) for transferring a metered amount of the sample; - A pre-metered amount of molecular iodine (6); - A pre-metered amount of an aqueous solution of an alkali metal iodide (7), said alkali metal preferably being potassium iodide; - A glass slide (8), preferably provided with a cavity; - An optional colorimetric scale (9); and - An optional information sheet (10).

2. The kit according to claim 1, characterized in that the container is a glass test tube provided with a lid and one or more glass beads.

3. The kit according to claim 1 or 2, characterized in that the biological fluid is selected from saliva, urine, serum and semen, preferably from saliva.

4. The kit according to any one of claims 1 to 3, characterized in that the weight ratio of molecular iodine / potassium iodide is 4 / 1.

5. The kit according to any one of claims 1 to 4, characterized in that the kit comprises a glass slide provided with a cavity.

6. The kit according to any one of claims 1 to 5, characterized in that the kit comprises a colorimetric scale ranging from white to brown.

7. Use of a kit according to any one of claims 1 to 6 in an in vitro method for the early diagnosis of tumors.

8. Use of the kit according to claim 7, characterized in that the tumors are selected from breast cancer, ovarian cancer, lung cancer, liver cancer, thyroid cancer, gastric cancer, pancreatic cancer, head and neck cancer.

9. Use of the kit according to claim 7 or 8, characterized in that the sample of the biological fluid is selected from saliva, urine, serum and semen, preferably from saliva.

10. An in vitro method for the early diagnosis of tumors, said tumors being in particular but not exclusively breast cancer, ovarian cancer, lung cancer and liver cancer, characterized in that the in vitro method comprises using a kit according to any one of claims 1 to 6 to test a sample of a biological fluid, preferably a sample of saliva.

11. An in vitro method for monitoring tumor diseases, said tumor diseases being in particular but not exclusively breast cancer, ovarian cancer, lung cancer and liver cancer, said in vitro method comprising: using a kit according to any one of claims 1 to 6 to test a sample of a biological fluid, said biological fluid preferably being saliva.

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