Drug testing method for the treatment of cancer cell treatment

Abstract

The invention relates to a drug testing method that enables the testing of inhibitor drugs against the heparanase enzyme, which plays a crucial role in the formation and proliferation of cancer cells, in a more cost-effective and faster manner. It involves the use of the DNS (3,5-Dinitrosalicylic acid) molecule.

Description

[0001] DESCRIPTION

[0002] Drug Testing Method for Cancer Cell Treatment

[0003] Field of the Invention

[0004] The invention is generally related to methods that enable the testing of drugs for cancer treatment.

[0005] The invention specifically relates to a drug testing method used for testing inhibitor drugs against the heparanase enzyme, which plays a crucial role in the formation and proliferation of cancer cells, enabling the testing process to be conducted in a more cost-effective and faster manner.

[0006] State of the Art

[0007] Currently, research continues on the treatment and prevention of cancer. As part of this research, it is necessary to test inhibitor drugs against the heparanase enzyme, which plays a crucial role in the formation and proliferation of cancer cells. Therefore, the heparanase enzyme has become a target for the development of drugs that exhibit inhibitory effects on angiogenesis and metastasis.

[0008] Although heparanase research has been targeted for cancer treatment for over twenty years in the current state of the art, the drug development process has been slow due to the lack of a simple, optimized test capable of demonstrating the enzyme's activity. Furthermore, the high cost of heparanase, its heterogeneity, the variety of its compounds, and the diversity and difficulty of heparanase cleavage have also contributed to the slow progress of the process. Fondaparinux (FPX), as identified by scientists, has been used as a substrate in place of heparin, and activity assays have been conducted. Fondaparinux is a completely synthetic, sulfated heparin sulfate mimic that can be degraded by heparanase into disaccharides and trisaccharides. The degradation of fondaparinux by heparanase can be measured colorimetrically by detecting the reduced disaccharide units using tetrazolium salts (WST-1 ). With this method, it is possible to determine the enzyme's activity, and it is an application already present in the literature. The testability of drugs can be carried out colorimetrically using this method. However, the tetrazolium salts (WST-1 ) used in this application are very expensive, and the WST-1 chemical binds to enzyme products, with a temperature for color change at 60°C and a color change duration of 60 minutes. Since the color change duration is quite long, obtaining test results also takes an extended period.

[0009] In the prior art related to the subject, several documents have been encountered. The document numbered CN101310770A pertains to antibodies developed against cancer. This document discusses an antigenic and separating combination conjugate present in Cripto-1 albumin, Pim-1 albumin, or in a colon cancer cell lysis agent. As seen in the summary of the mentioned document, the use of DNS molecules in cancer treatment is discussed. However, it does not mention that DNS is used as a method in heparanase enzyme inhibitor studies, nor does it explain exactly how it is used.

[0010] The document numbered WO2018177857A1 pertains to symmetric 2- aminophenyl-benzazolinyl-5-acetate compounds and their use as anti- heparanase agents. This document discusses the use of symmetric 2- aminophenyl-benzazolinyl-5-acetate compounds as a drug, particularly in the treatment of diseases and disorders associated with heparanase activity, as well as pharmaceutical compositions containing these compounds. In this document, tetrazolium salts are used as inhibitors. However, there is no information in this document regarding the use of DNS chemicals as a method in heparanase enzyme inhibitor studies.

[0011] As a result, advancements are being made in the treatment of cancer, and therefore, there is a need for new developments that will eliminate the aforementioned disadvantages and provide solutions to the existing systems. Aim of the Invention

[0012] The present invention relates to a drug testing method that meets the requirements mentioned above, eliminates all disadvantages, and offers additional advantages.

[0013] The main objective of the invention is to enable the testing of drugs that will be used in the treatment of cancer cells.

[0014] One objective of the invention is to enable the testing of inhibitor drugs against the heparanase enzyme, which plays a crucial role in the formation and proliferation of cancer cells.

[0015] Another objective of the invention is to enable the testing of drugs used in the treatment of cancer cells through a more cost-effective and faster method.

[0016] The present invention, as outlined above and in the detailed description below, provides all the advantages necessary to achieve the objectives. It is a drug testing method that enables the testing of inhibitor drugs against the heparanase enzyme, which plays a crucial role in the formation and proliferation of cancer cells, in a more cost-effective and faster manner. This method involves the use of the DNS (3,5-Dinitrosalicylic acid) molecule.

[0017] The structural and characteristic features of the invention, along with all its advantages, will be more clearly understood through the figures provided below and the detailed description made with references to these figures. Therefore, the evaluation should be conducted with these figures and the detailed description in mind.

[0018] Detailed Description of the Invention

[0019] In this detailed description, the preferred embodiments of the drug testing method of the invention are explained solely for the purpose of better understanding the subject and without creating any limiting effect. The invention is a drug testing method that enables the testing of inhibitor drugs against the heparanase enzyme, which plays a crucial role in the formation and proliferation of cancer cells, in a more cost-effective and faster manner; it involves the use of the DNS (3,5-Dinitrosalicylic acid) molecule.

[0020] The chemical used in the test method of the invention includes DNS, fondaparinux, and heparanase. In this chemical, the content consists of 40-60% DNS, 10-18% fondaparinux, and 10-18% heparanase. Additionally, the chemical preferably contains 3-8% sodium potassium tartrate, 3-8% sodium hydroxide, 3-8% water, 3-8% sodium acetate, and 3-8% acetic acid. In the preferred embodiment of the invention, the mentioned chemical contains 50% DNS, 5% sodium potassium tartrate, 5% sodium hydroxide, 5% water, 15% fondaparinux, 10% heparanase, 5% sodium acetate, and 5% acetic acid.

[0021] The drug testing method of the invention includes the following steps:

[0022] • Preparation of the DNS solution,

[0023] • The heparanase enzyme reacting with the fondaparinux substrate at 37°C for 24 hours,

[0024] • The products formed reacting with DNS and undergoing a color change,

[0025] • Reading the results at a wavelength of 540 nm.

[0026] DNS is a chemical molecule that can react with reducing sugars. The heparanase enzyme must react with the fondaparinux substrate and break it down, indicating the enzyme's active function. At this stage, the commercial heparanase enzyme is dissolved in a pH 5, 40mM acetate buffer, and a 10-9dilution is made. The enzyme substrate, the fondaparinux molecule, is also prepared in the same buffer. The enzyme (100 pl) and substrate (100 pl) are incubated in an incubator at 37°C for 24 hours. Afterward, 400 pl of DNS is added, and the mixture is boiled at 95°C for 5 minutes. At this stage, the drug molecule added to the system can reduce or stop the activity of the heparanase enzyme, potentially helping to partially control the cancer. As a result, the products formed react with DNS, and a color change is observed. After cooling, 200 pl of the test material is transferred to a 96-well plate. The resulting outcomes are read at 540 nm wavelength using a spectrophotometer and converted into numerical data.

Claims

CLAIMS1. It is a drug testing method that enables the testing of inhibitor drugs against the heparanase enzyme, which plays a crucial role in the formation and proliferation of cancer cells, in a more cost-effective and faster manner. Its distinguishing feature is the use of the DNS (3,5-Dinitrosalicylic acid) molecule.

2. The drug testing method according to claim 1 , characterized by the following steps:• Preparation of the DNS solution, • The heparanase enzyme reacting with the fondaparinux substrate at37°C for 24 hours,• The products formed reacting with DNS and undergoing a color change,• Reading the results at a wavelength of 540 nm.