Rapid and simple screening process for anticancer, antibiotic, antifungal and antiparasitic drugs and results obtained by this process.

Abstract

The invention relates to a rapid screening method for anticancer drugs, antibiotics, antifungals, or antiparasitics used clinically in humans and animals, and for basic and applied research, starting from the rootlet of germinating seeds. The screening method according to the invention is the same for screening anticancer drugs, antibiotics, antifungals, antiparasitics, and food preservatives. The cost of implementing this method is very low.

Description

Title of the invention: Method for rapid and simple screening of anticancer, antibiotic, antifungal and antiparasitic drugs and results obtained by this method.

[0001] The present invention relates to a new method for screening or tri-rapid screening of anticancer or antibiotic or antifungal or antiparasitic drugs used clinically or not in humans or animals, from the rootlet of a germinating plant seed and the results obtained by this method. This is the first time that this method has been used for rapid screening of drugs used clinically. Some definitions.

[0002] To better understand this text it is desirable to define some of the words and terms used in it. The majority of all the terms and terms used are defined in the Illustrated Dictionary of Medical Terms by Garnier Delamare, 28th edition, or in the latest Larousse edition in French.

[0003] Antibacterial: which opposes bacteria. Substance with this property. Sometimes we speak of antimicrobial (which opposes microbes)

[0004] Antibiotic: Opposing life. Refers to substances (such as penicillin and sulfonamides) which prevent the development of certain microorganisms into which they penetrate and whose metabolism they disrupt or which destroy them.

[0005] Anticancer: Drug used in the chemotherapy treatment of certain cancers. They are also called antineoplastic, antitumor (which opposes cancer).

[0006] Antifungals or antifungal: Synonym antimycotic. Which opposes the development of fungi (yeasts). Antifungals can destroy (fungicides) or stop the growth (fungistatics) of fungi.

[0007] Antimitotic: Which opposes cell multiplication and therefore cell proliferation. It is a product which prevents mitosis. It is used to treat cancer. Antimitotics are products which can block mitosis in metaphase.

[0008] RNA: ribonucleic acid.

[0009] cRNA: complementary ribonucleic acid.

[0010] mRNA: messenger ribonucleic acid.

[0011] rRNA: ribosomal ribonucleic acid.

[0012] tRNA: transfer ribonucleic acid.

[0013] Drug candidate: Substance or combination of substances still under development presented as potentially possessing curative or preventive properties. preventives against human or animal diseases, as well as any product under development intended to be administered to humans or animals with a view to establishing a medical diagnosis, restoring, correcting or modifying their organic functions. In the context of this invention, the drug candidate designates the anticancer or antibiotic, or the antifungal, or the antiparasitic.

[0014] Cancer: According to the American Cancer Society, cancer is not a single disease. The term cancer encompasses more than 200 diseases, all characterized by the uncontrolled proliferation of cells. Ignoring the body's signal to stop, malignant cells multiply to form tumors in organs and tissues or, in the case of blood cancers, crowd out normal cells from the bloodstream and bone marrow.

[0015] In a healthy body, cells grow and divide in a controlled and orderly manner to replace those that have aged or been damaged and die by design in a process called apoptosis. Cancer occurs when these natural processes fail and malfunction.

[0016] When a cell's genetic material—DNA—is damaged, mutations can occur, potentially disrupting normal growth and division. An accumulation of mutations can turn normal cells into precancerous cells, which sometimes multiply and develop into cancerous cells. Cancer is the result of the accumulation of these cells.

[0017] Cancer is not an event, but a process that takes time, often years, to develop. The duration varies considerably and depends on the identity, order, and rate at which mutations accumulate. The stage of cancer describes the progression of the disease in the body from pre-cancer to cancer that has spread to distant organs or tissues - metastatic cancer.

[0018] Cancers are classified according to the organ or cell type from which they originate. Carcinomas (skin cancers), sarcomas (cancers of bones, cartilage, fat, muscles, blood vessels, or any other connective or supporting tissue), leukemias (cancers of blood cells and bone marrow), lymphomas (cancers of immune system cells and usually appear in the lymphatic system), central nervous system cancers are cancers that arise in the cells of the brain and spinal cord.

[0019] Host cell: By host cell we mean here the cell that receives either the anticancer drug, the antibiotic, or the antifungal or antiparasitic drug. The host cell is then considered in this context as the cancer cell, or the fungus, or the parasite or the bacterium.

[0020] Chromosome: Rod-shaped structure of the cell nucleus, constituting the physical support of heredity.

[0021] Minimum inhibitory concentration: The minimum inhibitory concentration (MIC) of an extract (or product) relative to the rootlet of the plant, corresponds to the concentration which inhibited the growth of the rootlet of this plant.

[0022] Screening: In the Larousse dictionary, screening refers to the operation of sorting materials, with a view to granulometric classification. In the context of this document, screening refers to the rapid sorting of anticancer, antibiotic and antifungal drugs.

[0023] Excipient: It is a vehicle for a drug. It is a substance into which the active ingredients are incorporated to make them easily absorbable.

[0024] Aqueous extract: We mean by aqueous extract, the extract using as solvent only water in liquid or vapor form.

[0025] Genome: Set of genes in chromosomes. Set of genetic material, i.e., DNA molecules in a cell.

[0026] Germination: The beginning of the development of a new plant individual, from a seed placed in favorable conditions. Synonym: hatching. It is the transition to the state of active life of a closed organ whose life has been very slow until then (spore, egg, tuber).

[0027] Drug: Substance or combination of substances presented as having curative or preventive properties with regard to human or animal diseases, as well as any product which can be administered to humans or animals with a view to establishing a medical diagnosis, restoring, correcting or modifying their organic functions. In the context of this invention, the drug candidate designates the anticancer or antibiotic, or the antifungal, or the antiparasitic.

[0028] Essential medicine: A medicine that meets the priority health care needs of the population. These medicines are generally used for priority conditions and are included on the national list of essential medicines.

[0029] Mitosis: division of the cell (indirect), characterized by complex duplication (series of modifications of the chromatin of the nucleus) or even doubling of all the chromosomes of the mother cell, which allows the two daughter cells to receive the same number of chromosomes as the mother cell (equal distribution in the two daughter cells).

[0030] Rootlet: Fine branching of the roots. In this document, rootlet also refers to the fine single or multiple root obtained at the time of germination of the seed of a plant. Example: the seed of garden cress (Lepidium sativum) or onion (Allium cepa) or any other plant.

[0031] Ribosome: Ribosomes are ribonucleoprotein complexes present in eukaryotic and prokaryotic cells. Extremely well conserved during evolution, their function is to synthesize proteins by decoding the information contained in message RNA.

[0032] Screening: in this document, screening refers to the rapid sorting of anticancer drugs or antibiotics, or antifungals. Screening can be replaced by screening.

[0033] Xenobiotic: A xenobiotic is a product that is foreign to biological chemistry and can behave as a toxic or allergenic product towards the body. These are mainly household and industrial chemical products. In the context of this invention, xenobiotics designate drugs or drug candidates, chemical products, trace elements, total or partial extracts of plants for therapeutic purposes. Technical field of the invention.

[0034] The present invention relates to a method for rapid screening of anticancer, antibacterial, antifungal and antiparasitic drugs or drug candidates having as their target of action the blocking of protein synthesis (inhibition of genome multiplication) of host cells, and this from the rootlet of the seed of a germinating plant. The method must be feasible at very low cost.

[0035] The applicant has accidentally found that the rootlet of Cresson (Lepidium sativum) can be used effectively as host tissue to quickly and inexpensively screen anticancer, antibiotic, antifungal and antiparasitic drugs used in the clinic or for basic or applied research purposes with a very good correlation when this method is compared to conventional methods known to those skilled in the art such as cancer cells in culture, monoclonal or polyclonal antibodies, bacterial, fungal or parasitic cultures. State of the prior art.

[0036] There is no single method for rapid screening of anticancer drugs, antibiotics, antifungals and antiparasitics. Each product group has its own methods. Each of these methods is very expensive and the start-up time is also very long compared to the method according to the invention presented. Screening for anticancer drugs.

[0037] Screening for anticancer drugs is done in several ways. The most widely used screening tests use more or less complex techniques. They are carried out in vivo on the whole animal and / or in vitro on an isolated organ or organ fragment, and can understand receptor studies. The operation aimed at selecting, on the basis of pharmacological, biological, toxicological, physicochemical properties, molecules or biological targets within a collection called, respectively, chemical library and target library. We can thus use:

[0038] 1. Human or animal cancer cells. The use of stem cells human or animal cancer is by far the most used in laboratories.

[0039] 2. immunoglobulins [IG], for example monoclonal or polyclonal antibodies constructed by phage libraries as described in WO1992001047A1, C07K16 / 2803 (CA3059542A1) Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies directed against animal or human material against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily.

[0040] 3. the nanostructures containing a pilin protein used in the patent EP1485129B1.

[0041] 4. synthetic polypeptide libraries and methods for creating variants. A method for producing a collection of nucleic acids, each nucleic acid encoding a human immunoglobulin variable domain comprising a plurality of complementarity determining region 3 (CDR3) sequences separately isolated from the variable domain repertoire, a method cited in patent EP2432878B1.

[0042] To ensure patient safety during clinical studies or the marketing of a drug, toxicology has benefited in recent years from the growth of new knowledge, whether scientific, technical or bioinformatic. These have enabled the development of in silico, in vitro and in omic experimental models which, without replacing the evaluation carried out largely on laboratory animals, make it possible to eliminate molecules with prohibitive toxicity very early on, thus contributing to the reduction in the number of animals used.

[0043] In silico methods: These methods, also called SAR or (Q)SAR for (Quantitative) Structure-Activity Relationship or expert systems, use computer tools, hence their name in silico.

[0044] In vitro methods: The term in vitro is opposed to the term in vivo, these methods being able to be complementary or alternative to animal experimentation. In vitro methods are increasingly used not only during the screening stages, but also during drug development. Their ease of implementation, their isolation from any physiological context which allows the study of a toxic action mechanism, and above all the possibility of using human cells which make it possible to overcome inter-species differences, make them an essential tool.

[0045] In silico methods: Screening can be carried out in silico (virtual screening using molecular modeling computer tools).

[0046] High throughput screening (HTS) involves a large number of molecules or targets. It is carried out in a short period of time. However, the preparation and development time for the targets is very long. Performed in silico, high throughput screening uses intensive computing resources (computer science, bioinformatics, chemoinformatics).

[0047] SCREENING OF ANTIBIOTICS, ANTIFUNGALS AND ANTIPARASTICS.

[0048] The screening of antibiotics, antifungals and antiparasitics is carried out by measuring the inhibition of microbiological or fungal or antiparasitic activity with antibiotic or antifungal or antiparasitic candidates. The method consists of cultivating bacteria, fungi, parasites in the laboratory. The inhibition of microbial or fungal or parasitic growth in vitro is studied, for example for antibiotics and antifungals, the agar dilution method. The activity is then estimated visually after incubation in an oven at 37°C for 24 hours. Presentation of the invention.

[0049] The objectives of the invention are:

[0050] 1) develop a unique process which allows both screening of anti cancer drugs, antibiotics, antifungals, antiparasitics.

[0051] 2) The target(s) on the host cells must be perfectly identified.

[0052] 3) this procedure must be easy to carry out, in a short time.

[0053] 3) the cost of this process must be as low as possible to allow the most lacking access to screening for anticancer, antibiotic, antifungal, and antiparasitic drugs. If possible, in environments without comfort or energy.

[0054] 4) Ideally the cost should not exceed 10 US dollars. Summary of the invention.

[0055] Statement of the claimed elements with their justification.

[0056] Within the scope of the invention, the screening method which is proposed meets the following conditions: 1. The same method is used for screening anticancer, antibiotic, antifungal, antiparasitic drugs from the germinating plant seed rootlet. 2. The target of all these xenobiotics must be the inhibition of protein synthesis. 3. The process is easy to carry out because it only requires plant seeds. For example, a bag of common garden cress seeds, boxes of Petri dish, filter paper and water. 4. The cost of the process is low. For example, a sachet of garden cress seeds capable of performing more than 50 tests costs €1.99 (euro). 5. The time to complete the process and obtain reliable results is 3 days. Description of the embodiments.

[0057] The implementation of the invention can be done by measuring the action of xenobiotics (medicines, plant extracts or even xenobiotics) on the germination of the seed by measuring the length of the rootlet. Operating mode.

[0058] The tests for measuring the growth of the germinating seed rootlet are carried out after the germination of the seed in petri dishes or in suitable containers after an incubation period in the dark of 1 to 3 days. The measurement of the rootlet is carried out with a ruler and the value of the length of the rootlet is noted. The germinations of the seeds in the presence of the different concentrations of xenobiotics are compared with the control. In the control, germination normally takes place under the same conditions as the treated seeds but with water without xenobiotic. Preparation of plant seeds.

[0059] Seeds of plants, for example, garden cress (Lepidium sativum), are pre-germinated in petri dishes on filter paper or blotting paper soaked in water, in total darkness overnight. This seed pre-germination operation aims to eliminate seeds that do not show the beginning of germination with a closed cuticle. These seeds are not at risk of germination and are therefore eliminated from germination tests. Preparations of the concentrations to be tested.

[0060] Different concentrations per xenobiotic (drugs) to be tested are preferably prepared at concentrations ranging from picogram (pg / ml) to microgram (pg / ml). The containers (in the case of petri dishes) can contain volumes of 5 to 7 ml per container.

[0061] Results obtained by measuring the length of the rootlet of germinating seeds.

[0062] After pregermination of the seeds in the presence of different concentrations of drugs (xenobiotics), for 2 to 3 days, the lengths of the rootlets are measured to have average values ​​and standard deviations. It is preferable to have more than 8 measurements per drug concentration or controls to make good comparisons of the effects of each product on the growth of the rootlet. Brief description of the figures. The list of figures.

[0063] [Fig.l]: shows the effect of different concentrations of anticancer drugs on the germination of the rootlet of the seeds of Garden cress.

[0064] [Fig.2]: shows the effect of different concentrations of antiparasitics on the ger mination of the rootlet of the seeds of Garden cress.

[0065] [Fig.3]: shows the effect of different concentrations of antibiotics and antifungals on the germination of the rootlet of the seeds of Garden cress.

[0066] [Fig.4]: shows the perfect linear correlation that exists between the process described according to the invention and the most widely used classical method which uses cancer cells in culture. The list of tables.

[0067] Table 1: shows the IC50 values ​​obtained with the different anticancer drugs when using the method of inhibition of the garden cress rootlet.

[0068] Table 2: shows the IC50 values ​​obtained with two antiparasitics (hydroxychloroquine and ivermectin) when using the method of inhibition of the garden cress rootlet.

[0069] Table 3: shows the IC50 values ​​obtained with antibiotics and antifungals when using the garden cress rootlet inhibition method.

[0070] Table 4: shows the IC50 values ​​obtained with the different anticancer drugs on the cultures of cancer cells and the IC50 obtained according to the method of the invention, namely the inhibition of the rootlet of garden cress. Advantages of the invention and applications.

[0071] The invention presented here has several advantages and possible applications:

[0072] 1) the use of a unique method for screening not only the me drugs used clinically in humans, but also in animals and even in plants for medical, applied and fundamental scientific research.

[0073] 2) it is a simple process that can be carried out anywhere including at home, in developing countries, in the most remote locations and without energy or sophisticated equipment.

[0074] 3) it is a process which makes it possible to identify new anticancer drugs, antibiotics, antifungal and antiparasitic drugs during basic or applied scientific research.

[0075] 4) for a person skilled in the art the cost of carrying out the method according to the invention is very low (less than €10) with very significant results for applications in the field of human and animal health, see nutrition.

[0076] 5) The method according to the invention also makes it possible to determine, study and prepare food preservatives (antifungals, antibacterials, food antiparasitics).

[0077] 6) Method for rapid screening of anticancer drugs or antibiotics or anti fungal or antiparasitic drugs from the germinating seed rootlet, characterized in that: 1) this screening method is the same for anticancer drugs, antibiotics, antifungals, antiparasitic drugs. 2) the target on host cells is the blocking of protein synthesis. 3) the cost of screening around ten xenobiotics is less than 10 US dollars or 10 euros.

[0078] 7) Method according to the invention can use any kind of seed that is easy to germinate, but preferably the seeds of Lepidium sativum and Allium cepa.

[0079] 8) Method according to the invention can be used for fundamental or ap studies folded. Bibliographic references.

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J Agric Food Chem 2018 Jun 20;66(24): 6031-6041. Table 1 Anticancer Drugs No. Designation EC50 (pg / mL) 1 Methotrexate 0.043 2 Etoposide 7.11 3 Berberine 38.33 4 Doxorubicin 85.75 5 Rubidium Salt 251.90 6 Cesium Salt 592.10 7 Metformin 694.10 8 Biodefensin 205 9 Biodefensin Vit.C 202.30

[0082] [Tables2] Table 2 Antiparasitic drugs No. Designations IC50 [pg / mL] 1 Hydroxychloroquine 359.50 2 Ivermectin 3366 Table 3 Antibiotics and Antifungals No. Designation IC50 (pg / mL) 1 Isoniazid 135.90 2 Enoxolone 342.00 3 Amikacin 25.94 4 Erythromycin 133.70 5 Pyrazinamide 105.60 6 Doxycycline 3.355 7 Cotrimoxazole 2.388 8 Monensin 2.576 9 Hydroxychloroquine 343.40 Table 4 : Anticancer plants Plant ic50 Root and ic50 Cancer cell Cells Tissue Ref. Agératum conyzoides 10.65 0.84 MDA-MB 231 Sein 9 0.78 P-388 Sang 9 Alstonia boonei (Wild) 100.00 73.10 A431 Peau 10 72.80 MDA-MB231 Sein 10 50, 90 UNCPC-LNGC-A 10 Annona muricata 33.97 24.94 NCI-H292 Lung 6 49.00 HL-60 Sang 3 14.00 HL-60 Sang 3 Artemisia vulgaris 19.50 18.07 TCCSUP Urogenital 1 45.87 TCCSUP Urogenital Bracelet 1 22.16 58.96 HEp-2 Urogenital 17 61.20 Vero81 Digestive 17 Carica papaya 11.11 12.02 MCF-7 Breast 13 39.22 25.60 MCF-7 Breast 8 Crocus sativus 12 Curcuma longa 199.53 150.80 HT-29 Digestif 14 Trigonella foenum-graecum 25.97 26.55 MOLT-4 Lymphoblastic and 2 16.67 HaCaT Skin 2 17.43 Urogenital HeLa 16.26-3 Urogenital SK-2 Fragaria x ananas sa 80.27 81.51 KB Tube digestif 18 62.00 HCT116 Digestif 18 102.00 CAL-27 Tube digestif 18 114.20 HT-29 Digestif 18 Imperata cynlindrica 100.00 57.30 HL60 Sang 11 Ipomoea puniculata sp 14.99 52.27 MCF-7 Sein 22 Lantana camara 79.46 112.20 MCF-7 Sein 5 46.62 MCF-7 Sein 4 Lippia multiflora 52.24 53.43 MCF-7 Sein 19 56.43 C32 Peau 21 65.34 COR-L23 Poumon 20 Piper longum 88.95 120.50 MCF-7 Sein 7 Punie a granatum 38.94 12.85 MCF-7 Sein 16 Sonchus Oleraceus 67.20 33.10 MCF-7 Sein 21 20.68 NCI-H460 Poumon 21 20.32 SF268 Gliome 21 Tetrapleura tetraptera 316.23 294.37 Hela Urogénital 16 340.37 Jurkat Sang 15

Claims

Claims

1. A method of screening for anticancer drugs, antibiotics, antifungal drugs or antiparasitics, characterized in that said method comprises germinating the plant seed and measuring the growth of the germinating plant seed rootlet.

2. Method according to claim 1, characterized in that the germination is carried out by incubating the plant seed in water having different concentrations of anticancer, antibiotic, antifungal or antiparasitic to be screened, in a container, in particular a Petri dish, in the dark for 1 to 3 days, said germination being compared to a control incubated under the same conditions in water without anticancer, antibiotic, antifungal or antiparasitic.

3. A method according to claim 1 or 2, characterized in that it comprises measuring the length of the germinating plant seed rootlet.

4. Method according to one of claims 1 to 3, characterized in that it comprises a pre-germination operation consisting of placing the plant seed overnight in the dark.

5. Method according to one of claims 1 to 4, characterized in that the germinating plant seed is Lepidium sativum or Allium cepa.

6. Use of the method according to one of claims 1 to 5 for screening drugs.

7. Use of the method according to one of claims 1 to 5 for screening food preservatives.

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