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WO2004091650A1 - Utilisation de la troponine i cardiaque pour preparer des medicaments antitumoraux - Google Patents

Utilisation de la troponine i cardiaque pour preparer des medicaments antitumoraux Download PDF

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Publication number
WO2004091650A1
WO2004091650A1 PCT/CN2004/000339 CN2004000339W WO2004091650A1 WO 2004091650 A1 WO2004091650 A1 WO 2004091650A1 CN 2004000339 W CN2004000339 W CN 2004000339W WO 2004091650 A1 WO2004091650 A1 WO 2004091650A1
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WO
WIPO (PCT)
Prior art keywords
cardiac troponin
seq
tumor
expression
cell line
Prior art date
Application number
PCT/CN2004/000339
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English (en)
Chinese (zh)
Inventor
Fengming Liu
Original Assignee
Fengming Liu
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fengming Liu filed Critical Fengming Liu
Publication of WO2004091650A1 publication Critical patent/WO2004091650A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin

Definitions

  • the present invention relates to new uses of compounds, and particularly to new uses of cardiac troponin I.
  • Malignant tumors are one of the most serious diseases that endanger human health.
  • the common methods for treating malignant tumors are: surgical treatment, chemotherapy, radiation therapy, biological therapy, and traditional Chinese medicine.
  • surgical treatment, chemotherapy, and radiation therapy are the most commonly used.
  • Surgical treatment as the first choice and main program for most cancer treatments, has enabled countless cancer patients to recover, prolong life or improve quality of life.
  • surgical treatment is only applicable to patients with certain conditions and ranges, and because of tumor recurrence and metastasis, surgical resection cannot always achieve the goal of radical cure.
  • the use of chemotherapy and radiation therapy also plays a very important and indispensable role in the treatment of tumors, and is also constantly developing.
  • angiogenesis is very active, and blood is provided for tumor tissue growth in a timely manner.
  • tumor tissues in order to adapt to the rapid growth of tumor tissues, angiogenesis is very active, and blood is provided for tumor tissue growth in a timely manner.
  • vascular endothelial growth factor produced by tumor cells, which induces tumors to generate microvessels; the other is that the host blood vessels remaining in the tumors gradually become tumor blood vessels.
  • Tumorization Unlike normal blood vessels, the vascular endothelial cells of tumor blood vessels are incomplete, the basement membrane is sparse and leaks easily. After tumor angiogenesis, it will not be further differentiated into arteries and veins, without smooth muscles and nerve endings. It is a passive blood vessel. Its blood flow is completely dependent on the systemic circulation.
  • the new microvessels are the first stop for tumor invasion and metastasis. Tumor cells enter the blood circulation through the tumor microvessel wall and transfer to distant places.
  • Solid tumor growth requires a large blood supply. Many experimental studies have confirmed that inhibiting neovascularization in animals can effectively cut off the blood supply that provides nutrients for tumor growth, causing the tumor to shrink and even disappear.
  • Cardiac troponin I is one of the three subunits of cardiac troponin. It is often used as a marker for the diagnosis of myocardial infarction. It has the sequence of SEQ ID N 2: 1 amino acid residue in the sequence listing and consists of 210 amino acids. Residue composition.
  • E. coli and Pichia pastoris expression systems are one of the most successful expression systems for the expression of foreign proteins. They have developed rapidly in recent years, and hundreds of proteins have now been expressed in the expression system.
  • Pichia yeast is a methanol nutritional yeast, which grows rapidly, has simple culture conditions, and can be continuously cultured at high density. The genetic operation is similar to that of Saccharomyces cerevisiae, and the technology is quite mature. It is a high-expressing strain commonly used in industrial production.
  • the object of the present invention is to provide a new use of cardiac troponin I.
  • cardiac troponin I has the effect of inhibiting tumor proliferation. Therefore, the technical solution of the present invention is:
  • one or more pharmaceutically acceptable carriers may be added to the medicine using cardiac troponin I as an active ingredient.
  • the carrier includes a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, and a lubricant that are conventional in the pharmaceutical field. Wait.
  • the effective dose of cardiac troponin I is 1 microgram to 1 milligram per kilogram of body weight per day, and can be made into injections, lyophilized powder injections and other forms.
  • the aforementioned various dosage forms can be prepared according to a conventional method in the field of pharmacology.
  • Another object of the present invention is to provide a cardiac troponin I-encoding gene that is highly expressed in Pichia and E. coli expression systems.
  • cardiac troponin I encoding genes There are two types of cardiac troponin I encoding genes provided by the present invention. The first is a cardiac troponin I encoding gene that is highly expressed in the Pichia expression system and is one of the following nucleotide sequences:
  • the DNA sequence defined by SEQ ID No : 2 in the sequence listing has more than 90% homology, and a DNA sequence encoding the same functional protein.
  • the DNA sequence of Sequence 2 in the Sequence Listing is composed of 633 nucleotides and encodes the protein of Sequence 1 in the Sequence Listing.
  • PPIC9K-AAF vector (physical map shown in Figure 1) and GS115 Pichia yeast cell line.
  • the second cardiac troponin I encoding gene provided by the present invention is a cardiac troponin I encoding gene that is highly expressed in an E. coli expression system, and is one of the following nucleotide sequences:
  • the DNA sequence of Sequence 3 in the Sequence Listing is composed of 633 nucleotides and encodes the protein of Sequence 1 in the Sequence Listing.
  • Both expression vectors and cell lines containing the above genes belong to the protection scope of the present invention, such as the pET-AAF vector (the physical map is shown in Figure 4) and the BL21-DE3 E. coli cell line.
  • Figure 1 is a physical map of the expression plasmid pPIC9K-AAF
  • Figure 2 shows the digestion electrophoresis map of plasmid PPIC9K-AAF
  • Figure 3 shows the electrophoresis of Pichia GS115 culture medium after methanol induction.
  • Figure 4 is a physical map of the expression plasmid pET-AAF
  • Figure 5 is a restriction enzyme electrophoresis map of the expression plasmid pET-MF
  • Figure 6 shows the electrophoresis profile of BL21-DE3 E. coli culture medium after IPTG induction.
  • the high-efficiency expression plasmid pPIC9K purchased from Invitrogen, USA was used to digest the pGEM-AAFl plasmid and pPIC9K plasmid with Ndel and Notl, respectively.
  • the target fragments were collected by gel electrophoresis, and ligated with T 4 ligase at 16 ° C overnight. Transformation, picking bacteria, and amplification to obtain expression plasmids by conventional methods
  • the plasmid PPIC9K- AAF was linearized with Sacl, and the linearized pPIC9K-AAF was introduced into Pichia pastoris GS115 (purchased from Invitrogen) using Zhejiang Xinzhi Electric Gene Introducer, which was cultured, selected, and screened. In the process, a monoclonal bacterium resistant to G418 mg / ml was obtained.
  • mice Twenty-four Kunming mice were divided into two groups: the methanol-induced supernatant and the methanol-induced supernatant.
  • the mice were subcutaneously inoculated with the S180 ascites cell line. Three days later, the mice were injected subcutaneously with the methanol-induced supernatant and methanol-induced supernatant. 5ml / head, injection every 12 hours, continuous injection for 7 days.
  • the tumor group was observed and excised and weighed. As a result, the tumor weight of the supernatant before methanol induction and the supernatant after methanol induction was 0.82 + 0.22 g and 0.1 11 + 0.05 g, respectively, and the tumor inhibition rate was 86. . 6%.
  • Example 2 E.coli expression of cardiac troponin I in vitro
  • the synthetic gene sequence 3 has a full length of 1-210 bases, respectively, with a Snabl digestion site at the 5 end and a Notl digestion site at the T end to obtain the sequence AFF2.
  • the artificially synthesized full sequence is loaded into the pGEM-T-EASY plasmid to construct the pGEM-AAF2 cloning vector.
  • the high-efficiency expression plasmid pET21b purchased from Novagen Corporation was used to cut the PGEM-AAF2 plasmid and pET21b plasmid with Snabl and Notl, respectively.
  • the target fragments were collected by gel electrophoresis and ligated with T 4 ligase 16 ⁇ overnight. After transformation by conventional methods, picking bacteria, and amplification to obtain the expression plasmid pET-AAF, the structure of which is shown in Figure 4.
  • the results of enzyme digestion identification, as shown in Figure 5, prove that the structure of the expression plasmid is correct.
  • the plasmid pET-AAF was introduced into BL21-DE3 E. coli by transformation method, and after culturing, selection, screening and other processes, monoclonal bacteria were obtained.
  • the tumor inhibition rate was 85.3%.
  • mice Twenty-four Kunming mice were divided into phosphate buffer solution and expression purification group, and the phosphate buffer solution and expression purification solution were injected subcutaneously at 2.0 mg / kg protein, 8 hours after injection, blood was collected from the fundus vein, and the serum was separated. Serum cardiac troponin I concentration was measured by conventional enzyme-linked immunosorbent assay. Results in phosphate buffer and The concentration of serum cardiac troponin I in the purified solution group was 0.52 + 0.08ng / ml,
  • the present invention creatively discloses the antitumor activity of cardiac troponin I.
  • Drugs using cardiac troponin I as an active ingredient do not directly affect tumor cells. Inhibiting tumors has a broad spectrum without producing drug resistance, which has far-reaching practical significance.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des méthodes d'utilisation de la troponine I cardiaque pour préparer des médicaments antitumoraux. Cette invention se rapporte également à la dose efficace de troponine I cardiaque qui se situe entre 1 νg et 1 mg/kg de poids corporel du patient par jour. Les médicaments antitumoraux selon l'invention renferment un ou plusieurs vecteurs pharmaceutiquement acceptables. Cette invention concerne également des gènes codant la troponine I cardiaque qui s'expriment fortement dans le système d'expression de Escherichia coli et de Pichia pastoris.
PCT/CN2004/000339 2003-04-15 2004-04-12 Utilisation de la troponine i cardiaque pour preparer des medicaments antitumoraux WO2004091650A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN03109861.4 2003-04-15
CNB031098614A CN1294987C (zh) 2003-04-15 2003-04-15 心肌肌钙蛋白i在制备抑制肿瘤药物中的应用

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CN1312173C (zh) * 2004-12-23 2007-04-25 复旦大学 一种人心肌肌钙蛋白i合成肽段s28－42及其用途
CN102146372B (zh) * 2011-01-24 2012-09-05 四川农业大学 一种克隆山羊tnnc2基因编码区全序列的方法
CN107245102A (zh) * 2016-12-30 2017-10-13 广西壮族自治区药用植物园 抗肿瘤重组蛋白gpti及其编码基因与应用
CN107245103A (zh) * 2016-12-30 2017-10-13 广西壮族自治区药用植物园 抗肿瘤重组蛋白ifti及其编码基因与应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030085A1 (fr) * 1996-02-16 1997-08-21 Children's Medical Center Corporation Sous-unites et fragments de troponine utiles comme inhibiteurs de l'angiogenese

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030085A1 (fr) * 1996-02-16 1997-08-21 Children's Medical Center Corporation Sous-unites et fragments de troponine utiles comme inhibiteurs de l'angiogenese

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOSE M.A. ET AL: "Troponin I is present in human cartilage and inhibits angiogenesis", PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 2645 - 2650, XP002131230, DOI: doi:10.1073/pnas.96.6.2645 *
YU WANG ET AL: "Cloning and expression of Troponin and its inhibitory effects on human overian carcinoma SKOV3", PROG OBSTET GYNECOL, vol. 11, no. 3, 2002, pages 168 - 170 *

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