CN109999053B - Medical application of trifluridine or trifluridine tepridigidine composition - Google Patents
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- pirimidine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
- A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
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Abstract
The invention relates to the technical field of medicines, relates to medical application of a trifluridine or trifluridine pirimidyl composition, in particular to application of the trifluridine or trifluridine pirimidyl composition in preparation of medicines for treating blood diseases, and in particular relates to application of the trifluridine or trifluridine pirimidyl composition in preparation of medicines for treating blood diseases caused by beta hemoglobin gene defect or gene mutation. The trifluridine thiouracil composition consists of trifluridine and thiouracil in a weight ratio of: 1:1-4:1. The trifluridine or trifluridine tepipimidine composition can be further prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier for preparing a medicine for treating blood diseases.
Description
Technical Field
The invention relates to the technical field of medicines, relates to medical application of a trifluridine or trifluridine pirimidyl composition, in particular to application of the trifluridine or trifluridine pirimidyl composition in preparing a medicine for treating blood diseases, and especially relates to application of the trifluridine or trifluridine pirimidyl composition in preparing a medicine for treating blood diseases caused by beta hemoglobin gene defect or gene mutation.
Background
Infant and adult human hemoglobin is a tetramer consisting of 2 alpha chains and 2 gamma chains (infant) or beta chains (adult). They carry oxygen and carbon dioxide within the human body. The molecular structure and synthesis of hemoglobin are determined by genes, wherein due to deletion or point mutation of beta hemoglobin genes, beta chain synthesis disorder causes insufficient hemoglobin production or structural abnormality, so that oxygen cannot be effectively carried, and clinical symptoms of various degrees finally appear, thereby obviously shortening the life span of patients and reducing the quality of life. Blood diseases caused by defects or mutations of beta hemoglobin genes mainly comprise beta-type thalassemia, sickle-type anemia and the like. The severity of the disease caused by the abnormality of β -hemoglobin is closely related to the fetal hemoglobin (HbF) content of the patient. If the HbF production pathway in the patient is not completely closed, the clinical symptoms are mild and the survival time is significantly prolonged. The HbF level of the thalassemia patient is improved to be more than 20%, the defect of insufficient HbA can be obviously relieved, and the purpose of treating insufficient hemoglobin generation of the patient is achieved. Theoretically, the HbF production pathway in humans is essentially closed after one year of birth and no drug development mechanisms have found any effective drug to reactivate HbF production. Currently, the only medicine which can be used for clinically relieving the symptoms of beta-type thalassemia is Hydroxyurea (Hydroxyurea) all over the world, but the treatment mechanism of the medicine is unknown, the clinical treatment dosage is high, the effect is not obvious, and after the medicine is taken, a patient has the risk of serious side reaction which influences reproduction.
Through years of research, the HbF generation pathway of a human body is mainly regulated by the expression level of a gene KLF 1. Therefore, as long as a medicine capable of effectively reducing the expression of the KLF1 gene is found, HbF generation can be reactivated, and the aim of treating blood diseases caused by beta hemoglobin gene defect or mutation is fulfilled. Based on the theoretical basis, the inventor develops and perfects a set of high-throughput screening system which takes the in-vitro directional differentiation of human hematopoietic stem cells into erythrocytes as a model and aims at inducing the expression of HbF.
Trifluridine thiouracil (TAS-102) was prepared from trifluridine and thymine phosphorylase inhibitor thiouracil in a weight ratio of 2: 1, is an oral fluorouracil drug. FDA approval in the united states was obtained at 22 days 9/2015 for treatment of refractory metastatic colorectal cancer (mCRC) patients who no longer responded to other therapies (chemotherapy and biotherapy) and was approved by the european drug administration at 4/2016. Up to now, 21 countries and regions such as japan, usa, uk, canada, european union, etc. have been licensed for the treatment of metastatic colorectal cancer (mCRC). And, due to high level of evidence for evidence of evidence and good clinical experience, has been listed in "clinical practice guidelines for NCCN colorectal cancer", "consensus on management of ESMO metastatic colorectal cancer patients", and "guidelines for treatment of colorectal cancer" by JSCR (society of colorectal cancer, Japan), and it is recommended that trifluridine primidine be used in metastatic colorectal cancer patients (mCRC) who have previously received chemotherapy and targeted therapy with fluoropyrimidine, oxaliplatin and irinotecan. The traditional Chinese medicine composition is only used for treating cancers clinically, and related reports of application in preparation of medicines for treating blood diseases are completely absent.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to solve the technical problem of screening chemical components capable of effectively activating HbF generation in human erythrocytes for preparing a medicine for treating blood diseases caused by beta hemoglobin gene defect or mutation, so as to establish a brand-new method for treating the blood diseases caused by the beta hemoglobin gene defect or mutation by using the medicine. The application of the medicine can completely take the application of an iron-removing agent, reduce and even replace blood transfusion treatment, reduce the dependence degree of patients on blood transfusion, and relieve the current situation of shortage of medical blood sources in China.
The invention is realized by the following technical scheme:
the invention provides medical application of a trifluridine or trifluridine tepirimidine composition.
Further, the invention provides the application of the trifluridine or the trifluridine tepipimidine composition in preparing the medicines for treating the blood diseases.
Furthermore, the invention provides the application of the trifluridine or trifluridine pirimidine in preparing the medicines for treating blood diseases caused by beta hemoglobin gene deletion or point mutation.
Preferably, the blood disease is thalassemia or sickle's disease.
In the trifluridine tipepidine composition, the weight ratio of trifluridine to tipepidine is as follows: 1:1-4:1.
The invention provides a drug screening system which takes human hematopoietic stem cells to be directionally differentiated into erythrocytes in vitro as a model and takes KLF1 and HbF gene expression quantity as indexes. The small molecular drug trifluridine or trifluridine tipidine composition capable of reducing KLF1 gene expression in erythrocytes and remarkably improving HbF expression is screened out through the system.
Test results show that trifluridine can reduce KLF1 gene expression in erythrocytes, remarkably improve HbF expression, and that tipepidine mainly can reduce the degradation speed of trifluridine in a human body, wherein the weight composition ratio of trifluridine to tipepidine is as follows: the compositions of 1:1 to 4:1 are all effective in increasing HbF expression.
Drawings
FIG. 1 is PCR method for detecting the expression of KLF1 and BCL11A gene in human erythrocyte under the action of medicine; (trifluridine: tipipridine ═ 2: 1)
FIG. 2 is a PCR method for detecting the expression level of HbF gene in human erythrocytes under the action of a drug; (trifluridine: tipipridine ═ 2: 1)
FIG. 3 is a flow cytometry method for detecting the protein expression amount of HbF in human erythrocytes under the action of a medicament; (trifluridine: tipipridine ═ 2: 1)
FIG. 4Western immunoblotting to detect the protein expression of HbF in human erythrocytes under the action of drugs; (trifluridine: tipipridine ═ 2: 1)
FIG. 5PCR method for detecting the expression level of HbF gene in human erythrocytes under the action of drugs.
Detailed Description
Example 1PCR method for detecting the expression level of target gene in human red blood cell under the action of drug
Mix 5x105An individual CD34+Cells were cultured in StemBan SFEM medium (StemShell Technologies Inc.) containing the cytokines SCF (Stem cell factor), IL-3(Interleukin 3), TPO (Thrombopoetin) and Flt-3L (Fms-like tyrosine kinase 3 ligand). After 6 days, 1X105Individual cells were distributed to each well (24-well plates) and grown in SFEM medium containing 2% cyan/streptomycin, 20ng/ml SCF, 1U/ml Epo, 5ng/ml IL-3,2uM dexamethasone, and 1uM β -estradiol. Small molecule drugs were added to each well from the first day, the culture medium was changed every two days and new small molecule drugs were added. After 9 days of cell culture, mRNA in cells was extracted using an mRNA extraction kit (Invitrogen), a cDNA library was constructed using a reverse transcription kit (Applied Biosystem), and finally the expression amount of the target gene in human erythrocytes in each sample was analyzed by the PCR method, and the results are shown in FIG. 1, FIG. 2, and FIG. 5.
The primer information of the PCR method is as follows
Human GAPDH (internal reference): Forward 5'-GTGAAGGTCGGAGTCAACG-3' Reverse 5-
TGAGGTCAATGAAGGGGTC-3’
Human KLF1:Forward 5’-CCACAGCCGAGACCGCCTTGACC-3’Reverse 5’-
CTCTCATCGTCCTCTTCCTCCC-3’
Human BCL11A:Forward 5’-CGAGCACAAACGGAAACAATG-3’Reverse 5’-
GATTAGAGCTCCATGTGCAGAACG-3’
Humanγ-globin(HbF):Forward 5’-GATGCCATAAAGCACCTGGATG-3’Reverse 5’-
TTGCAGAATAAAGCCTATCCTTGA-3’
Humanβ-globin(HbA):Forward 5’-AACTGTGTTCACTAGCAACCTCAA-3’Reverse 5’-
GAGTGGACAGATCCCCAAAGGA-3’。
As shown in FIG. 1, trifluridine pirimidyl reduced the gene expression of KLF1 and BCL11A in vivo, while hydroxyurea, the control drug, did not reduce the gene expression of interest.
As shown in FIG. 2, trifluridine thiopirimid significantly increased the expression of the HbF gene in human erythrocytes, whereas hydroxyurea only slightly increased the expression of the HbF gene. The amount of hydroxyurea was 10. mu.M, whereas the amount of trifluridine primidine was 0.15. mu.M, which was only 1.5% of the amount of hydroxyurea. At this amount, trifluridine thiouracil increased HbF gene expression three times as much as hydroxyurea.
As shown in FIG. 5, the expression of HbF gene in human erythrocytes was significantly improved by using trifluridine alone or by combining trifluridine and tipepidine at different ratios (1: 1-4: 1), and there was no significant difference in drug effects, indicating that trifluridine is the main component for improving HbF expression in human erythrocytes.
Example 2: flow cytometry method for detecting protein expression amount of HbF in human red blood cell under action of drug
The cell culture method was as described in example 1. Cells were cultured for 14 days, washed with pBS containing 0.1% BSA, and cells were fixed with 0.05% glutaraldehyde (Sigma, G5882). The fixed cells were washed 3 times with pBS containing 0.1% BSA, then permeabilized in 1% Triton X-100(Life Technologies, HFH-10), followed by immunostaining with antibodies against HbF (Invitrogen, MHFH 04). The immunostained cells were then analyzed by flow cytometry, and the results are shown in FIG. 3.
As shown in FIG. 3, the HbF signal peak was significantly shifted to the right by trifluridine thiopirimid, indicating that the HbF protein expression level in human erythrocytes was significantly increased.
Example 3: western immunoblotting method for detecting protein expression quantity of HbF in human red blood cell under action of drug
The cell culture method was as described in example 1. Cells were cultured for 14 days, washed with pBS containing 0.1% BSA, then lysed in 1% Triton X-100(Life Technologies, HFH-10), and the supernatant was centrifuged. The protein lysate is quantified, protein loading buffer is added, SDS polyacrylamide gel electrophoresis separation is carried out, then protein is transferred to a vinylidene fluoride membrane (PVDF) by electrophoresis, the protein is labeled by an anti-HbF antibody (Abcam) and an anti-beta-actin (cellsignaling) antibody, and electrochemiluminescence immuno-chromogenic reaction is carried out after HRP coupling secondary antibody labeling (cellsingalling), and the result is shown in figure 4.
As shown in FIG. 4, HbF protein levels were significantly elevated under the effect of trifluridine compared to hydroxyurea, with β -acitn protein levels being the protein loading internal control.
In conclusion, trifluridine pirimidyl increased HbF expression in human erythrocytes more effectively than hydroxyurea, and it was used in an amount of only 1.5% of the amount of hydroxyurea.
Trifluridine thiouracil (TAS-102) was prepared from trifluridine and thymine phosphorylase inhibitor thiouracil in a weight ratio of 2: 1. Tests show that the trifluridine thiouracil (TAS-102) can obviously improve the expression of HbF in human red blood cells, and the weight composition of the trifluridine thiouracil is changed to obtain the compound: 1, substituted pyrimidine: 1-4:1, can improve the expression of HbF in human erythrocytes, and can ensure the efficacy of the composition in treating blood diseases caused by beta hemoglobin gene defect or mutation.
In conclusion, trifluridine or floxuridinopipirimid can be used for preparing the medicine for effectively treating blood diseases caused by beta hemoglobin gene defect or mutation.
Claims (11)
1. Application of trifluridine in preparing medicines for treating blood diseases caused by beta hemoglobin gene deletion or point mutation.
2. The application of the trifluridine tipyrimidine composition in preparing the medicines for treating blood diseases caused by beta hemoglobin gene deletion or point mutation is characterized in that the trifluridine tipyrimidine composition consists of trifluridine and tipyrimidine in the weight ratio of: 1:1-4:1.
3. The use according to claim 1 or 2, wherein the blood disorder caused by the deletion or point mutation in the β -hemoglobin gene is thalassemia or sickle's anemia.
4. The use of claim 1 or 2, wherein the trifluridine or trifluridine pirimidine composition improves fetal hemoglobin expression by decreasing expression of gene KLF1 in red blood cells, thereby ameliorating or curing a blood disorder caused by β -hemoglobin abnormality.
5. The use of claim 1 or 2, wherein the trifluridine or trifluridine pirimidine composition is prepared as a pharmaceutical composition together with a pharmaceutically acceptable carrier.
6. The use of claim 3, wherein the trifluridine or trifluridine pirimidine composition is formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier.
7. The use of claim 4, wherein the trifluridine or trifluridine pirimidine composition is formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier.
8. The use of claim 1 or 2, wherein the trifluridine or trifluridine pirimidine composition is formulated in a clinically acceptable formulation with a pharmaceutically acceptable carrier.
9. The use of claim 3, wherein the trifluridine or trifluridine pirimidine composition is formulated in a clinically acceptable formulation with a pharmaceutically acceptable carrier.
10. The use of claim 4, wherein the trifluridine or trifluridine pirimidine composition is formulated in a clinically acceptable formulation with a pharmaceutically acceptable carrier.
11. The use according to claim 9 or 10, wherein the formulation is a tablet, capsule, granule or injectable solution.
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| PCT/CN2020/082054 WO2020215999A1 (en) | 2019-04-26 | 2020-03-30 | Medical use of trifluridine or trifluridine tipiracil composition |
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| CN113967197B (en) * | 2020-07-24 | 2024-06-18 | 鲁南制药集团股份有限公司 | Compound tablet of trofloxuridine and tepirimidine hydrochloride and preparation method thereof |
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