Method for evaluating the effect of anti-inflammatory or analgesic drugs on the growth of rootlets of germinating plant seeds.

Abstract

The invention relates to a method for evaluating the effect of anti-inflammatory or analgesic drugs on the growth of rootlets of germinating plant seeds, said drugs being used clinically in humans and animals and for basic research.

Description

Title of the invention: Method for rapid and simple screening of anti-inflammatory or analgesic drugs and results obtained by this method.

[0001] The present invention relates to a new screening or rapid sorting method for anti-inflammatory drugs, whether used clinically or not in humans or animals, starting from the rootlet of germinating plant seeds, and the results obtained by this method. This is the first time this method has been used for the rapid screening of anti-inflammatory drugs used clinically.

[0002] Some definitions.

[0003] To better understand this text, it is advisable to define some of the words and terms used in it. The majority of the terms and words used are defined in the Garnier Delamare Illustrated Dictionary of Medical Terms, 28th edition, or in the latest Larousse French edition.

[0004] Analgesic: An analgesic product is a product that suppresses or reduces pain. Antipyretics act not only against pain, but also against fever and have anti-inflammatory effects. Antinarcotics, in addition to their analgesic action, have a sedative, euphoric, or anxiolytic effect that can lead to drug dependence (another term for narcotic). These are opium alkaloids and their synthetic derivatives. Central analgesics mainly include narcotics and tricyclic antidepressants. Peripheral analgesics are mainly paracetamol, aspirin, and non-steroidal anti-inflammatory drugs (NSAIDs). In this invention, the term analgesic will refer to analgesics, antipyretics, narcotics, and anti-inflammatories.

[0005] Pain reliever: in the context of this invention, pain relievers will refer to anti-inflammatories.

[0006] Anti-inflammatory: that which opposes inflammation. Anti-inflammatory drugs are classified as steroidal (cortisones and derivatives) and non-steroidal (divided into three main groups: aspirin, classic non-salicylic NSAIDs (carboxylic acid, indole, oxycodone, and pyrazolone derivatives), and more recently, coxibs). Finally, some pro-inflammatory cytokinin inhibitors are used only in exceptional circumstances.

[0007] Drug candidate: A substance or combination of substances still under development presented as potentially possessing curative or preventive properties with regard to human or animal diseases, as well as any product under development intended to be administered to humans or animals for the purpose of establishing a medical diagnosis, to restore, correct or modify their organic functions. In the context of this invention, the drug candidate refers to the anticancer or antibiotic, or the antifungal, or the antiparasitic.

[0008] 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 stop signal, malignant cells multiply to form tumors in organs and tissues or, in the case of blood cancers, displace normal cells from the bloodstream and bone marrow.

[0009] In a healthy body, cells grow and divide in a controlled and orderly manner to replace those that have aged or been damaged and that die by design in a process called apoptosis. Cancer occurs when these natural processes fail and malfunction.

[0010] When the genetic material of a cell—DNA—is damaged, mutations can occur, potentially disrupting normal growth and division. An accumulation of mutations can transform normal cells into precancerous cells, which sometimes multiply and develop into cancerous cells. Cancer is the result of the accumulation of these cells.

[0011] 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.

[0012] Cancers are classified according to the organ or cell type from which they originate. Carcinomas (skin cancers), sarcomas (cancers of the bones, cartilage, fat, muscles, blood vessels or any other connective or supporting tissue), leukemias (cancers of the blood cells and bone marrow), lymphomas (cancers of the cells of the immune system and which generally appear in the lymphatic system), cancers of the central nervous system are cancers that occur in the cells of the brain and spinal cord.

[0013] Host cell: Here, the host cell is defined as the cell that receives the anticancer drug, antibiotic, antifungal, or antiparasitic agent. In this context, the host cell is considered to be the cancer cell, fungus, parasite, or bacterium.

[0014] Chromosome: Rod-shaped structure of the cell nucleus, constituting the physical support of heredity.

[0015] IC50: Concentration that reduces the maximum effect by 50 percent: The concentration that reduces the normal growth of the rootlet by half.

[0016] Screening: In the Larousse dictionary, screening refers to the operation of sorting materials for the purpose of particle size classification. In the context of this document, screening refers to the rapid sorting of anti-inflammatory drugs.

[0017] Excipient: This is a vehicle for a medicinal product. It is a substance into which the active ingredients are incorporated to make them easily absorbed.

[0018] Aqueous extract: By aqueous extract we mean the extract using only water in liquid or vapor form as a solvent.

[0019] Genome: The complete set of genes on the chromosomes. The complete set of genetic material, i.e. the DNA molecules of a cell.

[0020] 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 active life state of a closed organ whose life has been greatly slowed until then (spore, egg, tuber).

[0021] Medicinal product: A substance or combination of substances presented as having curative or preventive properties with regard to human or animal diseases, as well as any product that can be administered to humans or animals for the purpose of establishing a medical diagnosis, restoring, correcting, or modifying their organic functions. In the context of this invention, the candidate medicinal product refers to an anti-inflammatory or analgesic.

[0022] Essential medicine: A medicine that meets the priority healthcare needs of the population. These medicines are generally used for priority conditions and are included on the national list of essential medicines.

[0023] Mitosis: cell division (indirect), characterized by complex duplication (series of modification of the chromatin of the nucleus) or splitting 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).

[0024] Rootlet: Fine branching of the roots. In this document, rootlet also refers to the single or multiple fine roots obtained at the time of seed germination in a plant. For example, the seed of garden cress (Lepidium sativum) or onion (Allium cepa) or any other plant.

[0025] Ribosome: Ribosomes are ribonucleoprotein complexes present in eukaryotic and prokaryotic cells. Extremely well conserved throughout evolution, their function is to synthesize proteins by decoding the information contained in messenger RNA.

[0026] Screening: In this document, screening refers to the rapid sorting of anticancer drugs, antibiotics, or antifungals. Screening may be replaced by screening.

[0027] Xenobiotic: A xenobiotic is a product that is foreign to biological chemistry and can behave as a toxin or allergen towards the body. These are primarily household and industrial chemicals. In the context of this invention, xenobiotics refer to drugs or drug candidates, chemicals, trace elements, and total or partial plant extracts intended for therapeutic use. Technical field of the invention.

[0028] The present invention relates to a method for the rapid screening of anti-inflammatory drugs or drug candidates whose target action is the blocking of protein synthesis (inhibition of genome replication) in host cells, starting from the rootlet of a germinating plant seed. The method must be feasible at very low cost.

[0029] The applicant fortuitously discovered that the rootlet of Cress (Lepidium sativum) can be used effectively as a host tissue for the rapid and inexpensive sorting of anti-inflammatory drugs used in clinical practice or for basic or applied research, with very good correlation when this method is compared to conventional methods known to those skilled in the art. Prior art.

[0030] There is no single method for rapid screening of anti-inflammatory drugs. The methods used are very expensive and the setup time is also very long compared to the process according to the invention presented. Screening of anti-inflammatory drugs.

[0031] Anti-inflammatory drug screening can be performed in several ways. The most commonly used screening tests employ techniques of varying complexity. They are carried out in vivo on the whole animal and / or in vitro on isolated organs or organ fragments, cell cultures, or cells, and may include receptor studies. The process aims to select molecules or biological targets from a collection called, respectively, a chemical library and a target library, based on pharmacological, biological, toxicological, and physicochemical properties. The following methods can be used:

[0032] Preclinical evaluation of anti-inflammatory drugs involves a series of in vitro (laboratory) and in vivo (animal) tests to assess the efficacy, safety, and mechanism of action of the drugs. The most common methods are: In vitro methods

[0033] 1. Enzyme tests: These tests evaluate the inhibition of cyclooxygenase enzymes (COX-1 and COX-2) by anti-inflammatory drugs. Enzymatic activity is measured in the presence and absence of the anti-inflammatory drug to determine its inhibitory efficacy.

[0034] 2. Cell culture: Inflammatory cells (such as macrophages or Synovial cells are cultured and exposed to pro-inflammatory agents to induce an inflammatory response. The anti-inflammatory agent is then added to observe its effect on the production of inflammatory mediators (such as cytokines, prostaglandins, and tumor necrosis factors).

[0035] 3. The anti-inflammatory activity of a xenobiotic (medicine, plant extract) etc..) can be assessed throughout the blood by monitoring inflammatory mediators of arachidonic acid catabolism and their by-products 12 rHHT and 13 HODE.

[0036] 4. Binding tests: These tests evaluate the ability of the anti-inflammatory to bind to its specific molecular targets (such as receptors or enzymes). In vivo methods

[0037] Models of acute inflammation:

[0038] 1. Rat paw edema test: An inflammatory agent (such as the X-carrageenan is injected into the paw of a rat to induce edema. The anti-inflammatory drug is administered, and the reduction in edema is measured (according to the method of Vinegar et al. (Vinegar et al., 1969)). The baseline volume of the right hind paw is determined before any drug administration. Fifty microliters of 2.5% X-carrageenan suspended in 0.9% saline are injected into the plantar surface of the right hind paw. The paw volume is then measured at 1, 2, 3, and 4 hours post-injection using a plethysmometer.

[0039] 2. Xylene-induced inflammation test: A xylene solution is applied to the ear of a mouse to induce inflammation. The anti-inflammatory drug is then administered and the reduction in inflammation is assessed.

[0040] 3. Evaluation of central analgesic activity by the withdrawal method Tail under the influence of heat. The central analgesic activity of a drug (or xenobiotic) can be studied by the tail-flick test, as described by D'Amour and Smith (D'Amour & Smith, 1941). Radiant heat was applied to the base of the tail using a tail-flick meter. The latency time for tail retraction from the stimulus was recorded. The intensity of the thermal stimulus was adjusted to induce tail movement within 10 to 12 seconds. A time limit of 20 seconds was used to avoid tissue damage in laboratory animals (rats, mice, etc.).

[0041] 4. Acetic acid-induced torsion reaction. The mouse torsion test is performed according to the method of Koster et al. (Koster et al., 1959). The twists are induced by an intraperitoneal injection of acetic acid at different doses. The number of muscle contractions is counted over a 5-minute period following the acetic acid injection. The data represents the total number of movements observed during a 10-minute period.

[0042] 5. Models of chronic inflammation: a) Adjuvant-induced arthritis in rats: A complete Freund adjuvant is injected to induce chronic arthritis. The anti-inflammatory drug is administered, and parameters of inflammation, such as joint thickness and cytokine levels, are measured. b) Collagen-induced arthritis in mice: A rheumatoid arthritis model is created by injecting IL-type collagen. The effects of the anti-inflammatory drug on arthritis symptoms are evaluated.

[0043] 6. Models of inflammatory pain: a) Formalin test: An injection of Formalin in a rodent's paw induces biphasic inflammatory pain. An anti-inflammatory drug is administered, and the reduction in the pain response is measured. b) Chronic nerve constriction test: A model of neuropathic pain is created by constricting a nerve. The effects of the anti-inflammatory drug on pain are assessed by measuring behavioral parameters. Parameters assessed

[0044] -Reduction of inflammation: Measured by the decrease in edema, redness and levels of pro-inflammatory cytokines.

[0045] - Pain reduction: Evaluated by behavioral tests in animals.

[0046] - Safety profile: Evaluated by toxicological studies to determine the effects potential secondary effects.

[0047] These methods make it possible to select promising anti-inflammatory candidates for clinical trials in humans, while ensuring that they are both effective and safe. Presentation of the invention.

[0048] The objectives of the invention are:

[0049] 1) to develop a rapid process that allows for the screening of anti- anti-inflammatory and other analgesic drugs.

[0050] 2) this procedure must be easy to carry out, in a short amount of time.

[0051] 3) the cost of this process must be as low as possible to allow the most deprived of access to anti-inflammatory screening, if possible in environments lacking comfort or energy.

[0052] 4) Ideally the cost should not exceed 5 US dollars for several tests. Summary of the invention.

[0053] Statement of the claimed elements with their justification.

[0054] Within the scope of the invention, the proposed screening process fulfills the following conditions: 1. The same process is used for screening anti-inflammatories, analgesics, from the rootlet of germinating plant seeds. 2. One of the targets of all these drugs and xenobiotics must be the inhibition of protein synthesis. 3. The process is easy to carry out as it only requires plant seeds. For example, a packet of common garden cress seeds, petri dishes, filter paper, and water. 4. The cost of the process must be low. 5. The time required to complete the process and obtain reliable results must not exceed 3 days. Description of the implementation methods.

[0055] The implementation of the invention can be done by measuring the action of xenobiotics (medicines, plant extracts or even xenobiotics) on seed germination by measuring the length of the rootlet. Operating procedure.

[0056] Tests to measure the growth of the germinating seed rootlet are carried out after seed germination in Petri dishes or suitable containers following an incubation period in the dark of 1 to 3 days. The rootlet is measured with a ruler, and the length is recorded. The germination of seeds in the presence of different concentrations of xenobiotics is compared with the control. In the control, germination occurs normally under the same conditions as the treated seeds but with water free of xenobiotics.

[0057] Preparation of plant seeds.

[0058] Plant seeds, 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 pre-germination process aims to eliminate seeds that do not show any signs of germination and have a closed cuticle. These seeds are unlikely to germinate and are therefore eliminated from the germination tests.

[0059] Preparation of the concentrations to be tested.

[0060] Different concentrations of the xenobiotic (drug) to be tested are preferably prepared at concentrations ranging from femtograms (fg / ml) to micrograms (pg / ml). The containers (in the case of Petri dishes) may contain volumes of 5 to 7 ml per container.

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

[0062] After pre-germination of the seeds in the presence of different concentrations of drugs (xenobiotics) for 2 to 3 days, the rootlet lengths are measured to obtain mean values ​​and standard deviations. It is preferable to have more than 8 measurements per drug concentration or control to make accurate comparisons of the effects of each product on rootlet growth. Brief description of the figures.

[0063] The list of figures.

[0064] [Fig. 1]: shows the effect of different concentrations of clinically used anti-inflammatory drugs on the germination of the rootlet of garden cress seeds. The different drugs and their IC50 values ​​are as follows: KETO (Ketoprofen, IC50: 542.2 pg / mL), INDO (Indomethacin, IC50: 32.51 pg / mL), DEXA (Dexamethasone, IC50: 3.15 pg / mL), IBUP (Ibuprofen, IC50: 364.4 pg / mL), DICL (Diclofenac, IC50: 98.10 pg / mL).

[0065] [Fig. 2]: shows the effect of different concentrations of clinically used anti-inflammatory drugs on the germination of the rootlet of garden cress seeds. The different drugs and their IC50 values ​​are as follows: AHA (acetohydroxamic acid, IC50: 259.5 pg / mL), TFP (tiaprofenic acid, IC50: 121.9 pg / mL). The advantages of the invention and applications.

[0066] The invention presented here has several advantages and possible applications:

[0067] 1) the use of a single process for screening not only the drugs used clinically in humans, but also in animals and even in plants for medical, scientific, applied and basic research.

[0068] 2) It is a simple process that can be carried out anywhere, including at home. in developing countries, in the most remote places and without energy or sophisticated equipment.

[0069] 3) It is a process that makes it possible to identify new anti-inflammatory drugs and analgesics during basic or applied scientific research.

[0070] 4) for a person skilled in the art, the cost of implementing the process 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.

[0071] 5) The method according to the invention can use any kind of easily germinating seed, but preferably the seeds of Lepidium sativum and Allium cepa.

[0072] 6) The method according to the invention can be used for fundamental studies or applied. Bibliographical references.

[0073] Vinegar R, Schreiber W & Hugo R. Biphasic development of carrageenan edema in rats. J Pharmacol Exp Ther, 166, 1969, 96-103.

[0074] Meddah Bouchra. et al. International Journal of Phytopharmacology, 4(2), 2013, 133-140.

[0075] D’Amour GE & Smith DL. A method for determining loss of pain sensation. J Pharmacol Exp Ther, 72, 1941, 74-78.

[0076] Koster R, Anderson M & and E. J. Beer. Acetic acid for analgésie screening. Fédération Proceeds, 18, 1959,412-416.

Claims

Demands

1. A method for evaluating the effect of anti-inflammatory or analgesic drugs on the growth of rootlets of germinating plant seeds, characterized in that said method comprises: - germination, in the dark for 1 to 3 days, of said plant seeds incubated in the presence of different concentrations of xenobiotics, namely anti-inflammatory or analgesic drugs, said germination being compared to a control incubated under the same conditions, but with water without the xenobiotics, - and the measurement of the growth of the rootlets of said germinating plant seeds.

2. Method according to claim 1, characterized in that the measurement of the growth of the rootlets of germinating plant seeds is carried out by measuring the length of said rootlets with a ruler.

3. A method according to any one of the preceding claims, characterized in that it comprises a pre-germination operation consisting of placing plant seeds in petri dishes on filter papers or blotting papers soaked in water, in total darkness overnight.

4. A method according to any one of the preceding claims, characterized in that the germinating plant seeds are Lepidium sativum seeds or Allium cepa seeds.

5. Use of the method according to any one of claims 1 to 4 for drug screening.

Images