CN107823204B

CN107823204B - New application of gemifloxacin

Info

Publication number: CN107823204B
Application number: CN201711029621.2A
Authority: CN
Inventors: 孔韧; 许晓双; 朴莲花
Original assignee: Jiangsu University of Technology
Current assignee: Jiangsu University of Technology
Priority date: 2017-10-30
Filing date: 2017-10-30
Publication date: 2020-05-05
Anticipated expiration: 2037-10-30
Also published as: CN107823204A

Abstract

The invention discloses an application of a compound in a medicine for treating diseases caused by bacterial glucuronidase, wherein the compound is Gemifloxacin (Gemifloxacin), a metabolite thereof or a pharmaceutically acceptable salt of the compound. The compounds can be used for treating cancers, and can also relieve diarrhea caused by irinotecan, gastrointestinal ulcer caused by non-steroidal anti-inflammatory drugs, or the curative effect and side effect of other drugs for regulating the metabolism of bacterial glucuronidase, and related diseases caused by the bacterial glucuronidase.

Description

New application of gemifloxacin

Technical Field

The invention belongs to the technical field of medicines, and relates to a new application of Gemifloxacin (Gemifloxacin), in particular to Gemifloxacin serving as a bacterium β glucuronidase inhibitor and an application thereof.

Background

By the bacterium β -glucuronidase, microorganisms in the gastrointestinal tract deprive sugar groups from glucuronidated compounds for energy supply, resulting in the conversion of various drugs or their metabolites from a non-toxic state to a toxic state, thereby affecting drug metabolism and toxic side effects^[1,2]The symptoms affected by bacterial β -glucuronidase include tardive diarrhea caused by irinotecan, gastrointestinal ulcer caused by non-steroidal anti-inflammatory drugs, and the like^[3,4]。

Irinotecan (Irinotican, CPT-11), which is called Kemptotar (Camptosar), is one of the clinically common antitumor chemotherapeutic drugs, and the main target of the irinotecan is type I DNA topoisomerase. Since its introduction into the market in 1996, irinotecan has been widely used in the treatment of colorectal cancer, lung cancer, brain tumors, and drug-resistant leukemia and lymphoma, and is listed as a first-line therapeutic drug for metastatic colorectal cancer. Years of clinical use have shown that enterotoxicity is one of the major dose-limiting side effects of irinotecan, manifested as late diarrhea, usually in patients who are on drug for 24 hoursLater, major features include unpredictability, high morbidity, and a degree of mortality. Statistics show that up to 88% of patients treated with irinotecan exhibit symptoms of diarrhea, with 20-30% of patients experiencing severe diarrhea of grade 3-4 (CTCAE grade), and about 3.5% of patients dying from complications due to severe diarrhea^[5,6]. A considerable number of patients have to reduce the dosage of the medicine and even stop the medicine due to diarrhea, and no effective treatment means exists so far.

Although a variety of "second generation irinotecan" have been developed in recent years, such as ONIVYDE (also known as MM-398, PEP02, or nal-IRI, Merrimack Pharmaceuticals, Inc.), an irinotecan liposome formulation capsule approved by the U.S. FDA for marketing in 2015; PEG-SN38 (polyethylene-linked SN38, BelrosePharma Inc.) was in the clinical phase III of the experiment. Although these "new irinotecan" have improved bioavailability and potency, diarrhea remains a dose-limiting side effect that is difficult to overcome.

The mechanism of diarrhea development has a close relationship with the metabolic pathway of irinotecan. The main metabolic site of CPT-11 is liver, CPT-11 firstly removes di-piperidine group by Carboxylesterase (CES) to generate active metabolite SN-38; subsequently, SN-38 is subjected to glycosylation modification under the catalysis of glucuronosyl-transferase (UGT) and further converted into an inactive metabolite SN-38G^[7]. SN-38 is the main active metabolite of irinotecan, has over one hundred times of the inhibition ability to DNA topoisomerase compared with the original drug CPT-11 and the inactive metabolite SN-38G, and has extremely strong lethality to rapidly-divided tumor cells^[8]It was found that symbiotic bacteria rich in the intestinal tract use the inactive metabolite SN-38G as one of the energy sources, and the bacterial β -glucuronidase (β -glucuronidase, β -GUS) removes the sugar group of SN-38G to supply its own energy and reactivates it to SN-38, resulting in a greatly increased concentration of toxic compound SN-38 in the intestinal lumen, killing the intestinal epithelial cells and causing irreversible damage to the intestinal mucosa, and finally resulting in the production of delayed diarrhea^[3,9]Therefore, the selective inhibition of the activity of β -GUS enzyme in intestinal tract can directly reduce the local concentration of SN-38 and protect intestinal tract tissues, thereby fundamentally preventing and treating the delayed diarrhea.

The bacterial β -GUS enzyme exists in a tetramer form, the small molecular compound is combined in a catalytic pocket of the enzyme, and has stronger interaction with a loop structure consisting of a section of 17 amino acid residues specific to the bacterial enzyme, because the sequence and the structure of the corresponding region of the loop region in human homologous protein β -GUS are obviously different, the combination with the specific loop structure of the bacterial enzyme ensures that the series of compounds can selectively inhibit β -GUS in bacteria, and has no obvious influence on β -GUS of mammals^[3]Animal experiment results show that the bacterium β -GUS inhibitor 2 can reduce the incidence rate of diarrhea, prolong the survival time of animals, and further verify the safety and the effectiveness of the target point^[3,10]. However, since the toxicological and pharmaceutical properties of the newly developed compound are not clear, the safety and efficacy of the compound after being used in combination with CPT-11 are more difficult to evaluate, and thus, a large gap still exists between the compound and clinical application.

The invention discovers that Gemifloxacin can selectively inhibit bacterial glucuronidase by adopting a method of integrating virtual screening and experimental determination. Since Gemifloxacin is a known drug, is used in a human body, has definite safety and pharmaceutical properties, can quickly enter clinical experiments, and is used for relieving diarrhea caused by irinotecan, gastrointestinal ulcer caused by non-steroidal anti-inflammatory drugs, or regulating curative effects and side effects of other drugs metabolized by bacterial glucuronidase and related diseases caused by the bacterial glucuronidase.

Disclosure of Invention

The invention discovers that Gemifloxacin can selectively inhibit bacterial glucuronidase by adopting a method of integrating virtual screening and experimental determination. Since Gemifloxacin is a known drug, is used in human bodies, has definite safety and pharmaceutical properties, can be rapidly introduced into clinical experiments for relieving diarrhea caused by irinotecan or regulating the curative effect and side effect of other drugs metabolized by bacterial glucuronidase and related diseases caused by bacterial glucuronidase.

The invention provides application of a compound A in preparing a medicament for treating diseases caused by bacterial glucuronidase, wherein the compound A is a compound shown in a formula I, a metabolite of the compound shown in the formula I or a pharmaceutically acceptable salt of the compound shown in the formula I.

The disease of the invention is preferably diarrhoea, gastrointestinal toxicity or other conditions caused by bacterial glucuronidase. The diarrhea includes, but is not limited to, being caused by a chemotherapeutic drug; such gastrointestinal toxicity includes, but is not limited to, that caused by non-steroidal anti-inflammatory drugs.

The chemotherapy drugs of the invention include, but are not limited to, irinotecan or its active metabolite 7-ethyl-10 hydroxycamptothecin, or other drugs taking camptothecin compounds and derivatives thereof as main active ingredients.

The compound of formula I and one or more pharmaceutically acceptable carriers form a pharmaceutical composition.

The invention also provides an application of a chemotherapeutic medicament and a compound A in preparing a combined medicament for treating tumors, wherein the compound A is a compound shown in the formula I, a metabolite of the compound shown in the formula I or a pharmaceutically acceptable salt of the compound shown in the formula I.

The chemotherapeutic drug of the invention includes, but is not limited to irinotecan or the active metabolite 7-ethyl-10 hydroxycamptothecin, or other drugs taking camptothecin compounds and derivatives thereof as main active ingredients. The compound A and one or more pharmaceutically acceptable carriers form a pharmaceutical composition, and the dosage form of the pharmaceutical composition is tablets, capsules, granules, pills or other dosage forms which can be prepared. The tumor comprises one or more of colorectal cancer, gastric cancer, liver cancer, breast cancer, brain tumor, drug-resistant leukemia, lymph cancer, prostate cancer, lung cancer or bladder cancer.

The invention takes the relocation of the drug as a basic strategy, adopts a computer simulation method to carry out early evaluation on the effectiveness of the drug molecules, and adopts a biological experiment method to test the activity, so that Gemifloxacin is found to be an effective selective inhibitor of the β -GUS enzyme of the bacterium, and the enzymology level IC of the Gemifloxacin is₅₀Values of 2.0900. + -. 1.2270. mu.M, respectively (FIG. 1); at the cellular level IC₅₀The value is 0.9157 +/-1.4830 mu M (figure 2), the compound has no obvious effect on β -GUS enzyme of mammal sources under the concentration of 100 mu M, and the compound has good selectivity (figure 3), and the compound has no obvious influence on the growth of escherichia coli, and the compound has no obvious cytotoxicity, and can inhibit the activity of β -GUS enzyme under the condition of not killing the escherichia coli (figure 4).

Gemifloxacin in proper dosage can be used for selectively inhibiting bacterial β -GUS enzyme, thereby influencing the efficacy and side effects of drugs metabolized by bacterial β -GUS enzyme and treating diseases caused by bacterial β -GUS enzyme.

Since Gemifloxacin is a known drug, is used in human bodies, has definite safety and pharmaceutical properties, can be rapidly introduced into clinical experiments for relieving diarrhea caused by irinotecan or regulating the curative effect and side effect of other drugs metabolized by bacterial glucuronidase and other related diseases caused by bacterial glucuronidase.

Drawings

FIG. 1 IC of Gemifloxacin on protein level for inhibition of bacterial β -GUS enzyme₅₀Gemifloxacin has good inhibitory effect on bacterial β -GUS enzyme at protein level, and IC thereof₅₀The value was 2.0900. + -. 1.2270. mu.M.

FIG. 2 IC of Gemifloxacin on the cell level for the inhibitory potency of the bacterium β -GUS enzyme₅₀Gemifloxacin has good bacterial β -GUS enzyme inhibition effect at cellular level, and IC thereof₅₀The values are 0.9157 + -1.4830 μM。

Gemifloxacin has no obvious inhibitory activity to mammal-derived Bovine taurus β aGUS enzyme at a concentration of 100 mu M Gemifloxacin is used for testing the activity of mammal-derived Bovine taurus β -GUS enzyme by using Gemifloxacin at a concentration of 100 mu M, and the compound is found to have no obvious inhibitory effect to mammal-derived β -GUS enzyme.

FIG. 4 shows the results of incubation of bacteria with Gemifloxacin in 1% DMSO solution, 100. mu.M aspartame and Gemifloxacin in 10. mu.M concentration in black, dark grey and light grey columns, respectively.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Test examples

Determination of inhibitory Activity of Gemifloxacin on bacterial glucuronidase

1. Experimental methods

1.1 virtual receptor-based screening

First adopt

The Protein Preparation module in the software package treats the crystal structure (PDB: 3LPF) of the β -GUS enzyme, including modifying the bond level, adding hydrogen atoms and partial charges, deleting all the crystal water molecules, optimizing the whole system based on OPLS-2005 force field, and when the RMSD value reaches

The optimization terminates. By using

9.0 Ligprep 2.5 Module preprocesses LOPAC and Microresource Spectrum electronic version molecule files of known drug molecule libraries, includingHeavy, free of salt ions and inorganic substances, under pH7.4 conditions to give compounds of possible ionization states and tautomers, for chiral compounds with undetermined chiral centers to give various possible chiral low energy compound structures, under default conditions, up to 32 stereoisomers per molecule, and finally about 5 ten thousand compound conformations. The Glide SP mode was first used for docking and scoring, with all small molecule binding modes retained. In literature reports, active molecules essentially comprise two distinct pharmacophore characteristics: hydrogen bonding with GLU413 and hydrophobic interaction with PHE 365. For the binding mode of the first 1 ten thousand small molecules generated by docking, the pharmacophore characteristics are taken as screening conditions, the binding mode which forms hydrogen bonds with GLU413 and hydrophobic interaction with PHE365 is selected, and finally, part of compounds are selected and purchased for experimental activity determination.

1.2 bacterial glucuronidase enzymatic level determination step

4mg/ml of bacterial β -GUS (300,000X 125pM) was diluted to 1X 125pM with 50mM HEPES buffer (pH7.4with 0.017% Triton x-100) and substrate 4MUG was dissolved and diluted to 312.5. mu.M with 50mM HEPES (pH 7.4). The specific procedures were 1) 20. mu.l of the compound to be tested was added to a 96-well plate (blackboard), 2) 40. mu.l of GUS enzyme (125pM) was added, 3) 40. mu.l of substrate (4MUG, 312.5. mu.M) was added, 4) incubation was carried out at room temperature for 30 minutes and 40. mu.l of 1M Na2CO3 was added to terminate the reaction, and 5) fluorescence was carried out on an EnVision (Perkin Elmer USA) multifunctional microplate reader with excitation wavelength of 335nm, emission wavelength of 460nm, with the plate without the compound added as a positive control and the plate without the enzyme as a negative control.

1.3 enzymatic Activity measurement procedure for glucuronidase from mammalian sources

The specific steps are the same as the activity test experiment of the enzymatic level of bacterial glucuronidase, wherein the enzyme is replaced by a mammalian source of Bovine taurus β -GUS enzyme, and the final concentration of the enzyme is 1 nM.

1.4 cellular level GUS enzyme Activity test experiment

Empty plasmid pGex-4T-1 was transformed into E.coli (DH5 α), cultured overnight in LB (100. mu.M ampicillin) at 37 ℃, then grown up to OD600 to 0.6 at 1/100, centrifuged at 8000rpm for 5 minutes, the pellet was washed twice with 50mM HEPES (100. mu.M ampicillin, pH7.4), the pellet OD600 was concentrated to 1.0, and this bacterial solution was used to replace GUS enzyme for experimental detection the reaction was carried out at 37 ℃ for 2 hours with 50mM HEPES (pH7.4) as buffer, and the other experimental steps and data processing were identical to those of the GUS enzyme experiment.

1.5 bacterial cytotoxicity assay

And performing cytotoxicity experiments by using escherichia coli liquid used in GUS cell experiments. The final concentrations of test compound, 100. mu.M and 10. mu.M (1% DMSO), were used for cytotoxicity assays. 20 mu L of the compound to be detected and 80 mu L of the bacterial solution are added into a 96-well plate, 1% DMSO is used as a control group, the reaction is carried out for 2h at 37 ℃, then 10 mu L of Cell Counting Kit-8(Dojindo, Japan) is added into each well, and the mixture is mixed evenly. The reaction was carried out at 37 ℃ for 5 minutes, 30 minutes and 60 minutes, followed by measurement of absorbance at 490nm using a multifunctional microplate reader (Thermo Scientific, USA).

2. Results of the experiment

Gemifloxacin was assayed for activity at the protein level against β -GUS, a bacterium, as well as β -GUS enzyme from mammalian sources, as well as activity of the compound at the cellular level against β -GUS enzyme, and cytotoxicity of the compound.

IC for inhibition of bacterial β -GUS activity at the protein level₅₀The curve is shown in FIG. 1, Gemifloxacin has good inhibitory effect on bacterial β -GUS enzyme at protein level, and IC thereof₅₀The value was 2.0900. + -. 1.2270. mu.M.

IC for inhibition of bacterial β -GUS enzyme at cellular level₅₀The curve is shown in FIG. 2, and the bacterial β -GUS enzyme inhibition effect is good at the cellular level, and the IC is₅₀The values were 0.9157. + -. 1.4830. mu.M, respectively.

Gemifloxacin was tested for β -GUS enzyme activity from mammalian sources using a concentration of 100. mu.M and was found to have no significant inhibitory effect on β -GUS enzyme from mammalian sources (FIG. 3).

Gemifloxacin incubated with E.coli at a concentration of 100. mu.M at 10. mu.M had no significant effect on E.coli growth, indicating that the compound was not significantly cytotoxic and inhibited the β -GUS enzyme activity without killing E.coli (FIG. 4).

Experiment for Gemifloxacin in relieving irinotecan-induced diarrhea in mice

The CT-26 cell line was used to construct a mouse tumor model. 18 female Balb/cJ mice were selected for 6-8 weeks and injected subcutaneously with PBS suspension of cells at the back sites of the mice. After about 10 days, the tumor of the mouse reaches about 500mm³(tumor volume is expressed by the formula π/6 × a²X b calculation, where a is the minor axis of the tumor and b is the major axis of the tumor). The mice were then randomized into three groups for administration: (1) control group, received equal volume of intraperitoneal distilled water, and gavage with 1% DMSO solution (total about 100 μ L, twice daily); (2) CPT-11 group, i.e., intraperitoneal injection of CPT-11 at a dose of 50mg/kg, and gavage with 1% DMSO solution (total about 100. mu.L, twice a day); (3) CPT-11 plus Gemifloxacin combination, was injected intraperitoneally with CPT-11 at a dose of 50mg/kg, and Gemifloxacin solution was gavaged (total of about 100. mu.L, twice daily) at a dose of 50 mg/kg. In order to obtain a mouse diarrhea model, CPT-11 is continuously injected for 9 days in the CPT-11 administration group and the combined drug group; the combination drug combination starts to take Gemifloxacin orally from the day before CPT-11 injection, continues to take the drug orally for two days after the CPT-11 injection is finished, and then finishes the drug administration. Diarrhea was observed in the control group, CPT-11 administration group, and CPT-11 plus Gemifloxacin combination group, respectively, and the body weight and tumor size of the mice were recorded.

The experimental result shows that the incidence rate of the diarrhea in the blood sample of the mice in the CPT-11 administration group is 83.33 percent, while the incidence rate of the diarrhea in the blood sample of the CPT-11 and Gemifloxacin combined group is 50 percent, and the combined drug has obvious effect of improving the incidence rate of the diarrhea compared with the single use of the CPT-11. In addition, the weight reduction of the combination group is also obviously improved compared with the single CPT-11 administration group (P is less than 0.05). Meanwhile, the combined medicine group and the single CPT-11 administration group have no obvious difference in the effect of inhibiting the size of the tumor.

Claims

1. Use of compound a in the manufacture of a medicament for reducing diarrhea caused by irinotecan, or its active metabolite 7-ethyl-10 hydroxycamptothecin, said compound a being a compound of formula I,

2. the use according to claim 1, wherein compound a and one or more pharmaceutically acceptable carriers comprise a pharmaceutical composition.

3. The use according to claim 2, wherein the pharmaceutical composition is in the form of tablets, capsules, granules, pills or other preparable dosage forms.