CN114748496B

CN114748496B - gemcitabine sensitizer

Info

Publication number: CN114748496B
Application number: CN202210215173.XA
Authority: CN
Inventors: 焦作义; 周辉年; 蒋祥彦; 马勇
Original assignee: Lanzhou University Second Hospital
Current assignee: Lanzhou University Second Hospital
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2023-11-14
Anticipated expiration: 2042-03-04
Also published as: CN114748496A

Abstract

The present invention provides a sensitizer for gemcitabine, i.e., 1,2,3,4, 6-O-galloylglucose (PGG).

Description

Gemcitabine sensitizer

Technical Field

The present invention relates to the field of cancer treatment. In particular, the present invention relates to sensitizers of gemcitabine, 1,2,3,4, 6-O-galloylglucose (PGG).

Background

Gemcitabine is a difluoro nucleoside antimetabolite anticancer agent that disrupts cell replication and is used for the treatment of breast cancer, uterine body cancer, ovarian cancer, prostate cancer, lung cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, head and neck squamous cell carcinoma, and other solid tumors. However, the role of gemcitabine in cancer therapy is limited by its resistance, and thus, solving the resistance of gemcitabine is currently a critical issue.

1,2,3,4, 6-O-galloylglucose (PGG), which is a polyphenol monomeric compound, has various biological and pharmacological activities such as antioxidation, anti-inflammatory, antagonism of intracellular toxins, and treatment of diabetes, but has not been reported as a sensitizer for chemotherapeutics.

Disclosure of Invention

The invention discovers that the 1,2,3,4, 6-O-galloylglucose and gemcitabine can be combined for treating cancers, and the treatment effect of the gemcitabine is improved. In particular, the present invention has found that 1,2,3,4, 6-O-galloylglucose can sensitize gemcitabine, thereby enhancing the effect of gemcitabine in the treatment of cancer. More specifically, the present invention finds that 1,2,3,4, 6-O-galloylglucose sensitizes gemcitabine by inhibiting UBE 2T. Without being limited by theory, it is believed that 1,2,3,4, 6-O-galloylglucose inhibits the degradation of gemcitabine by UBE2T by inhibiting UBE2T, thereby enhancing the therapeutic effect of gemcitabine.

Accordingly, in one aspect, the present invention relates to the use of 1,2,3,4, 6-O-galloylglucose and gemcitabine in the manufacture of a medicament for the treatment of cancer.

In particular embodiments, 1,2,3,4, 6-O-galloylglucose sensitizes gemcitabine.

In particular embodiments, 1,2,3,4, 6-O-galloylglucose sensitizes gemcitabine by inhibiting UBE 2T.

In a specific embodiment, the cancer is selected from breast cancer, endometrial cancer, ovarian cancer, prostate cancer, lung cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, squamous cell carcinoma of the head and neck, preferably pancreatic cancer.

In one aspect, the invention relates to a pharmaceutical composition comprising 1,2,3,4, 6-O-galloylglucose and gemcitabine. In particular embodiments, the pharmaceutical composition is for use in treating cancer.

In particular embodiments, 1,2,3,4, 6-O-galloylglucose sensitizes gemcitabine.

In particular embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes excipients, vehicles, adjuvants and diluents well known in the art and is available from commercial sources for use in Pharmaceutical formulations (see, e.g., gennaro (2003) Remington: the Science and Practice of Pharmacy with Facts and Comparisons: drugs Plus, 20 th edition, mack Publishing; anse et al (2004) Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th edition, lippencott Williams and Wilkins; kibbe et al (2000) Handbook of Pharmaceutical Excipients, 3 rd edition, pharmaceutical press.).

Suitable pharmaceutically acceptable carriers comprise substances that are relatively inert and may facilitate administration of the pharmaceutical composition or may assist in processing the active compound into a preparation that is pharmaceutically optimized for delivery to the site of action.

Such pharmaceutically acceptable carriers include agents that can alter the form, consistency, viscosity, pH, tonicity, stability, osmolarity, pharmacokinetics, protein aggregation or solubility of the formulation, and include buffers, wetting agents, emulsifying agents, diluents, encapsulating agents and skin permeation enhancers. Some non-limiting examples of carriers include saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.

As used herein, 1,2,3,4, 6-O-galloylglucose and gemcitabine include derivatives thereof. As used herein, the term "derivative" means a similar compound having a similar function derived from a given compound by a physical or chemical process. Derivatives may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and such as those described by j.march, "Advanced Organic Chemistry: reactions, mechanisms and Structure (higher organic chemistry: reactions, mechanisms and structures) ", 4 th edition (New York: wiley-Interscience, 1992). For example, base addition salts are prepared from the compounds using conventional means, including reacting one or more free hydroxyl groups of the compounds with a suitable base. Generally, the compound is dissolved in a polar organic solvent such as methanol or ethanol and a base is added thereto. The resulting salt precipitates or may be precipitated from solution by the addition of a less polar solvent. Salts of cystamine suitable for forming the base addition salts refer to cystamine salts derived from inorganic and organic acids and bases. Examples of suitable acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and oxalic acid. Salts derived from amino acids (e.g., L-arginine, L-lysine) are also included. Examples of suitable bases include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like. Those skilled in the art know how to obtain derivatives of compounds without substantially altering their function.

As used herein, the term "sensitize" gemcitabine refers to the enhancement of the effect of gemcitabine in the treatment of cancer.

As used herein, UBE2T refers to ubiquitin-binding enzyme E2T, which belongs to a ubiquitin-binding enzyme E2 family member, and was first found in related studies of fanconi anemia to be involved in DNA damage repair as an important member of the fanconi signaling pathway. The crystal structure of UBE2T is known to be located on chromosome chrl q32.1, its Open Reading Frame (ORF) is composed of 6 exons and 5 introns, spans 10.3kb on the chromosome, has a characteristically conserved domain, has a conserved cysteine residue at position 86 as its active site, can form a thioester bond with ubiquitin molecules, has an extension sequence at its C-terminus, and is associated with localization of UBE2T to the nucleus. The research reports that UBE2T plays an important role in the occurrence and development of various cancers such as lung cancer, breast cancer, bladder cancer, prostate cancer, liver cancer, myeloma, gastric cancer and the like.

As used herein, "inhibiting UBE2T" refers to PGG interacting with UBE2T to inhibit ubiquitination activity of UBE 2T.

Drawings

Figure 1 shows a comparison of tumor volumes in four groups of post-treatment humanized pancreatic cancer xenograft models.

Figure 2 shows a comparison of tumor volume curve changes in four groups of post-treatment humanized pancreatic cancer xenograft models.

FIG. 3 shows the knocking-out of UBE2T group (KO) in the control group (SgCtr)) Gemcitabine IC in the reverted wild type UBE2T group (RE-WT) and reverted C86A mutant UBE2T group (RE-C86A) ₅₀ And (5) detecting.

FIG. 4 shows a bicore experiment of PGG and UBE2T proteins.

FIG. 5 shows the effect of ubiquitination assay to detect PGG on UBE 2T-mediated ubiquitination of P53.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: sensitization of gemcitabine by PGG

1. Constructing a humanized pancreatic cancer xenograft model, transplanting tumor tissues of a pancreatic cancer patient into the subcutaneous of a mouse, and obtaining the humanized pancreatic cancer xenograft model after 3 passages;

2. the tumor length of the mice to be transplanted subcutaneously is 100cm ³ The administration is started at the moment;

3. dosing regimen and group:

(1) Control group: injecting physiological saline into the abdominal cavity;

(2) Gemcitabine dosing group: gemcitabine is injected intraperitoneally, 50 mg/kg/week;

(3) PGG dosing group: PGG was dissolved in physiological saline containing 2% TWEEN-20 and infused with stomach at 20 mg/kg/day;

(4) Gemcitabine group for PGG: gemcitabine is injected intraperitoneally, 50 mg/kg/week, PGG is dissolved in physiological saline containing 2% TWEEN-20, and the stomach is irrigated, 20 mg/kg/day;

4. mice were observed for changes in subcutaneous tumor volume.

The results showed that PGG combined with gemcitabine significantly reduced tumor volume growth in mice compared to control, gemcitabine and PGG single drug groups, thus PGG combined with gemcitabine significantly inhibited pancreatic cancer growth (fig. 1, 2).

Example 2: mechanism of PGG sensitization of gemcitabine

1. UBE2T is knocked out through CRISPR/Cas9 technology, and gemcitabine IC (integrated circuit) of pancreatic cancer cell lines before and after UBE2T knocking out is detected through MTT (methyl thiazolyl tetrazolium) method ₅₀ Is a variation of (c).

2. The binding force between UBE2T and PGG is studied through a bicore technology;

3. the blocking effect of PGG on UBE 2T-mediated P53 ubiquitination degradation is studied through a ubiquitination experiment;

293T cells were cultured to third generation at 5X 10 in the evening on the first day ⁶ The plates were plated, and the following day was followed by transfection of HA-ubi, flag-P53, non-tag-UBE 2T and non-tag-RING 1 by lip2000, and after 24h of transfection MG132 was added to a final concentration of 20. Mu.M, which was allowed to act for 4h, and samples were collected. After proteolytic cleavage, the beads were incubated with Flag gel for 2h at 4℃in a refrigerator and WB was detected after elution.

Through the above experiments, we discussed the mechanism of PGG sensitization gemcitabine, we knocked out UBE2T in pancreatic cancer cell line PANC-1 by CRISPR technology, and reverted to wild type and Cys86 site mutant, found out the IC of PANC-1 after knocking out UBE2T ₅₀ Significantly down-regulated, but reverted to IC of PANC-1 of wild-type UBE2T ₅₀ Significantly up-regulated, IC of PANC-1 reverting to C86A mutant UBE2T ₅₀ There was no change compared to the knockout strain (FIG. 3).

Based on the above data, we examined binding of UBE2T to PGG by the Bicore method and found that UBE2T was highly affinity to PGG (fig. 4). Furthermore, we have found that PGG can block ubiquitinated degradation of P53 by UBE2T (fig. 5).

Claims

1. Use of 1,2,3,4, 6-O-galloylglucose and gemcitabine of formula (i) below for the preparation of a pharmaceutical composition for the treatment of pancreatic cancer;

2. the use of claim 1, wherein 1,2,3,4, 6-O-galloylglucose sensitizes gemcitabine by inhibiting UBE 2T.

3. A pharmaceutical composition for treating pancreatic cancer, which comprises 1,2,3,4, 6-O-galloylglucose and gemcitabine represented by the following formula (i);

4. the pharmaceutical composition of claim 3, wherein 1,2,3,4, 6-O-galloylglucose sensitizes gemcitabine by inhibiting UBE 2T.