CN115869312B - PDC anti-tumor medicine and preparation method and application thereof - Google Patents

Abstract

The invention discloses a PDC anti-tumor drug, a preparation method and application thereof, and belongs to the technical field of anti-tumor drugs. In order to solve the technical problems of poor water solubility, no targeting property, low bioavailability and a plurality of side reactions of the camptothecin drug. The invention provides a PDC anti-tumor drug which is obtained by connecting camptothecin with a targeting peptide through a connecting bond. The PDC antitumor drug provided by the invention has the advantages of improving the water solubility of the camptothecine drug and improving the solubility of the compound; the targeting polypeptide is coupled with camptothecine, so that the targeting problem of the medicine is solved, and the bioavailability of the medicine is improved; designing a connecting arm with a cleavable disulfide bond structure to connect the polypeptide and the drug, so as to solve the transmission of the drug; reduce toxicity of camptothecine and side effects.

Description

PDC anti-tumor medicine and preparation method and application thereof

Technical Field

The invention belongs to the technical field of antitumor drugs, and particularly relates to a PDC antitumor drug as well as a preparation method and application thereof.

Background

In recent years, different peptides have been used notably as carriers to deliver various antitumor agents and as one of the most promising therapeutic ligands in nanotechnology to treat cancer. Short peptides (e.g., dipeptides and tripeptides) play an important role in this regard because their structures are very small, biocompatible, and easy to modify. Peptides such as cyclic, alpha-helical, linear and amphiphilic may self-assemble to form different nanostructures, e.g., nanorods, nanospheres, nanotubes and nanofibers. Preclinical and clinical studies of these self-assembled peptides containing several antineoplastic agents, such as curcumin, paclitaxel and doxorubicin, have been successfully conducted. Physiologically stable dipeptides are advantageous over other peptides because they are less toxic and biodegradable. Peptide-drug conjugates, because of their ease of preparation, economy and wide range of uses, play an important role as drug delivery vehicles in targeting anticancer drugs.

Most of conventional tumor drugs have the problems of poor water solubility, untimely metabolism and the like, and after targeting polypeptide is linked, the water solubility of a linked product can be improved, the patent drug property is provided, the addition of a cosolvent harmful or irritant to human bodies is avoided, and the bioavailability in vitro and in vivo is also improved. With the increase of the using times of the traditional tumor drugs, tumor cell membranes can generate proteins which block the drugs from entering cells, so that the absorption and utilization rate of the drugs are reduced, and the drug effect is reduced. The drug linked with the targeting peptide can be combined with a tumor cell surface specific receptor to effectively bring the drug into tumor cells, so that the problem of drug resistance is avoided.

Camptothecin (CPT) is an indole alkaloid extracted from camptotheca acuminata of the Chinese Davidiaceae, has good anti-tumor activity on a plurality of solid tumors, but has low water solubility, no targeting property and low bioavailability, and can generate a plurality of side reactions.

Disclosure of Invention

The invention aims to solve the technical problems that the camptothecin medicament has poor water solubility, no targeting property and low bioavailability and can generate a plurality of side reactions.

The invention provides a PDC anti-tumor drug which is obtained by connecting camptothecin with a targeting peptide through a connecting bond.

Further defined, the camptothecin has the formula:

further defined, the linkage is a cleavable disulfide linkage.

Further defined, the cleavable disulfide bond has the structure ofFurther defined, the targeting peptide is [ Arg-Gly-Pro-Asp]n, n is a positive integer greater than or equal to 1.

Further defined, the targeting peptide has the structure of

Further defined, the structure of PDC antitumor drugs is:

the invention provides application of the PDC anti-tumor medicament in preparing an anti-cancer medicament.

Further defined, the anti-cancer is anti-lung cancer, anti-pancreatic tail cancer, pancreatic head cancer and anti-brain glioma.

The invention provides a preparation method of the PDC anti-tumor medicament, which is characterized by comprising the following steps:

step 1: modifying the target peptide by using a connecting compound SPDP-COOH to obtain a modified target peptide;

step 2: modifying the camptothecin by using a connecting compound SPDP-COOH to obtain modified camptothecin;

step 3: and (3) connecting the substances obtained in the step (1) and the step (2) through disulfide bonds to obtain the PDC antitumor drug.

The beneficial effects are that: the PDC antitumor drug provided by the invention has the advantages of improving the water solubility of the camptothecine drug and improving the solubility of the compound; the targeting polypeptide is coupled with camptothecine, so that the targeting problem of the medicine is solved, and the bioavailability of the medicine is improved; designing a connecting arm with a cleavable disulfide bond structure to connect the polypeptide and the drug, so as to solve the transmission of the drug; reduce toxicity of camptothecine and side effects.

The invention provides application of camptothecine PDC in antitumor drugs. The polypeptide coupling medicine can obviously improve the water solubility of the medicine without adding an organic solvent for assisting dissolution. Provides a reference for improving the aqueous solution of the medicine by modifying the polypeptide sequence in the follow-up process.

The invention provides a preparation method of RGPD targeting polypeptide, and also comprises a connection process of the targeting polypeptide, a connecting arm and camptothecine.

The invention provides a preparation method of a connecting arm Linker containing disulfide bonds, which breaks disulfide bonds in cells to release medicines, solves the problem of medicine transmission, improves the bioavailability of medicines, and provides a thinking for the development of connecting arms with other disulfide bond structures.

Drawings

FIG. 1 is a synthetic technology roadmap;

FIG. 2 is a graph showing HPLC detection after SPDP-COOH synthesis;

FIG. 3 is a HPLC detection pattern after the synthesis of Mpa (Trt) -CPT;

FIG. 4 is a HPLC detection pattern after deprotection of Mpa (Trt) -CPT;

FIG. 5 is a mass spectrum detection profile of the synthetic end Product (PDC);

FIG. 6 is a bar graph of JP-001-C0 acting at high, medium and low concentrations for three days in cell lines PANC-1 and A549;

FIG. 7 is a bar graph of JP-001-C3 effect at high, medium and low concentrations for three days in cell lines PANC-1 and A549;

FIG. 8 is a bar graph of data for three days of action of JP-001-C0 at high, medium and low concentrations in cell line MIA;

FIG. 9 is a bar graph of data for three days of action of JP-001-C3 at high, medium and low concentrations in cell line MIA;

FIG. 10 is a bar graph of data for three days of action of JP-001-C0 at high, medium and low concentrations in cell line U251;

FIG. 11 is a bar graph of JP-001-C3 effect at high, medium and low concentrations for three days in cell line U251;

FIG. 12 is a bar graph of JP-001-C3 action at high, medium and low concentrations for three days in cell lines PANC1 and A549.

Detailed Description

Synthesis of SPDP-COOH (3- (2-pyridinedithio) propionic acid)

33.75g of 2, 2-dithiodipyridine is weighed into a round bottom flask, 270ml of ethanol is added for stirring and dissolution, 3.6ml of glacial acetic acid is added, 8.11g of mercaptopropionic acid is weighed, diluted with 180ml of ethanol and added dropwise into the reaction solution, and after the dropwise addition is completed within 30min, the reaction is carried out for 5h at room temperature. The solvent was removed by vacuum concentration, separated and purified by column chromatography, mobile phase dichloromethane: ethanol=3:2, (3% glacial acetic acid) to give the target collection. Concentrated in vacuo to remove glacial acetic acid by extraction and concentrated in vacuo to give 12.52g SPDP-COOH. The results are shown in FIG. 2.

Synthesis of SPDP-RGPD-OH

CTC resin with substitution degree of 1.14mmol/g is weighed into a reaction vessel, added with DMF to swell for 1h and then filtered by suction. The amino acids Fmoc-Asp (OtBu) -OH and DIEA were weighed to prepare Fmoc-Asp (OtBu) -CTC resin, and the substitution rate was 0.60mmol/g.

Fmoc-Asp (OtBu) -CTC resin 10g was weighed, 6mmol of synthesis scale was obtained, HOBT/DIC was used as condensing agent, amino acid was calculated as 3 times the amount of the mole of synthesis scale, fmoc-Pro-OH 6.07g, HOBt 2.43g, DIC1.2ml were weighed, 50ml of DMF was added to dissolve, and then the mixture was poured into resin for 2 hours of reaction

And (3) repeating the subsequent operation, and sequentially connecting Fmoc-Gly-OH, fmoc-Arg (pbf) -OH and SPDP-COOH according to the peptide sequence to obtain the peptide resin. Cleavage of the peptide resin at the ratio TFA: tis: water=95:2.5:2.5, precipitation of methyl tert-butyl ether, washing, drying yielded the crude peptide. Purification by HPLC and lyophilization yielded 3.5g SPDP-RGPD-OH.

Synthesis of Mpa-CPT

3.01g CPT (camptothecine), 6.02g 3- (3-phenylmethylthio) propionic acid and 0.21g DMAP are weighed, 100ml methylene chloride is added, stirring and dissolving are carried out, 5.4ml DIC is added after cooling to 5 ℃, after 0.5h reaction, the reaction is transferred to room temperature condition, no raw material remains by TLC detection, and the reaction is finished. The reaction was quenched with water, extracted with dichloromethane, washed with 5% aqueous sodium bicarbonate, washed with 0.1M aqueous hydrochloric acid, washed with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was collected. The liquid was concentrated to give a solid, which was slurried with a mixed solution of methylene chloride/petroleum ether, suction-filtered and dried to give a yellow solid of 5.21g Mpa (Trt) -CPT. The results are shown in FIG. 3.

20ml of 50% TFA/DCM (2% Tis) was added to the Mpa (Trt) -CPT solid, the reaction was carried out at room temperature for 1h, no starting material remained by TLC, and the reaction was completed. The reaction solution was concentrated in vacuo to give a solid, which was slurried with diethyl ether, suction filtered and dried to give a yellow solid, 3.23g of MPa-CPT. The results are shown in FIG. 4.

Synthesis of RGPD-Mpa-S-S-Mpa-CPT

0.32g of SPDP-RGPD-OH was dissolved in 20ml of DMF, 0.17ml of DIEA was added, and a DMF solution of 0.25g of MPa-CPT was added to the reaction solution under stirring, and the reaction was terminated by liquid phase monitoring. The reaction solution was diluted with water, purified by using acetonitrile and 0.1% aqueous trifluoroacetic acid as a preparative high-performance liquid phase, and lyophilized to give 0.38g RGPD-MPa-S-S-MPa-CPT (code JP-001-C3). The results are shown in FIG. 5.

English shorthand corresponds to Chinese name

EXAMPLE 1 preparation of PDC antitumor drug

1. The structure is as follows:(abbreviated as JP-001-C3).

The whole technical route is shown in figure 1:

2. the preparation method comprises the following steps:

(1) The mercaptopropionic acid is chemically modified to obtain a connecting compound SPDP-COOH:

disulfide bond connecting arm Linker structure is a cleavable disulfide bond, disulfide bond is broken in cells, and medicine is released

The structure is as follows:

(2) The targeting peptide RGPD or the resin containing RGPD peptide sequence is synthesized through full solid phase, and is modified by a connecting compound (SPDP-COOH), and the modified targeting peptide has the function of forming disulfide bond with free radical mercapto.

(3) The antitumor drug is camptothecine antitumor drug, and the hydroxy camptothecine is modified chemically with one connecting compound mercaptopropionic acid, and the modified compound has exposed free radical mercapto.

(4) And obtaining the PDC medicament with disulfide bond Linker through disulfide bond exchange by using the modified camptothecine and the modified targeting peptide.

The shorthand structure: RGPD-Mpa-S-S-Mpa-CPT.

The specific steps are as follows:

1. the preparation technical scheme of the targeting polypeptide RGPD is as follows:

the polypeptide RGPD is synthesized by adopting a full solid phase synthesis method, the solid phase carrier selects the king resin or CTC resin with the substitution degree of 0.2-1.5mmol/g, and the Fmoc-strategy is adopted to synthesize the polypeptide synthetic peptide resin. The condensing agent is HOBT/DIC or TBTU/DIEA, wherein the condensing agent HOBt/DIC is preferred

2. The mercaptopropionic acid reconstruction technical scheme is as follows:

the method comprises the steps of dissolving the mercaptopropionic acid and the dithio-pyridine in an organic solvent, reacting under the catalysis of acetic acid, purifying to obtain a compound (b) SPDP-COOH with active disulfide bond sites, and performing disulfide bond exchange reaction on the compound (b) SPDP-COOH, wherein the organic solvent comprises methanol, ethanol and dichloromethane, and ethanol is selected as preferable choice.

3. The modification technical scheme of the targeting peptide is as follows

The compound (b) was attached to the polypeptide resin intermediate (a) by a solid phase method, cleaved by TFA cleavage, and purified by HPLC to give (c) SPDP-RGPD-OH.

4. The chemical modification technology of camptothecin is used for preparing the Mpa-CPT as follows:

the method for manufacturing the Mpa-CPT comprises the following steps:

(1) Dissolving camptothecine and 3- (tritylthio) propionic acid in a solvent under the catalysis of DMAP and the condition of condensing agent to obtain a compound (d), wherein the solvent comprises one or two mixed solutions of DMF, pyridine, dichloromethane, NMP and DMSO, and preferably dichloromethane is selected.

The condensing agent comprises one of DCC, EDC, DIC, EEDQ, preferably DIC.

(2) Dissolving the compound (c) in an organic solvent, and then adding trifluoroacetic acid with a certain concentration and removing Trt protecting groups under the condition of Tis to obtain the compound (e), wherein the organic solvent comprises dichloromethane, ethyl acetate and 1, 4-dioxane, preferably, dichloromethane is selected, the concentration of the trifluoroacetic acid is between 10% and 90%, preferably, the concentration of 50% Tis is between 1% and 10%, preferably, 2%.

PDC pharmaceutical Compounds through disulfide exchange

Compound C and compound e are subjected to disulfide bond exchange under alkaline conditions to prepare compound f, and compound f (RGPD-Mpa-S-S-Mpa-CPT, code JP-001-C3) is obtained through preparation and liquid phase purification. The alkaline condition comprises one or a mixed solution of DMF, DIEA, et, 3 and N, NMM and sodium bicarbonate, and preferably, a mixed solution of DMF and DIEA is selected.

The following experiment was used to verify the experimental effect:

1. JP-001-C3 acute toxicity test

Experiment 1:

1. test purpose: confirmation of toxicity of JP-001-C3 and camptothecin

2. Test principle: in half Lethal Dose (LD) ₅₀ ) The weight change, movement, respiratory state and death of the experimental animal within 1 week are observed by intravenous injection or intraperitoneal injection of the experimental material or the leaching liquid of the experimental material as evaluation indexes, and the acute toxic effect of the experimental material is judged. Half of the deaths of mice intraperitoneally injected with camptothecinsThe amount is 68.4-83.6mg/kg, and the JP-001-C3 medicine is converted into camptothecin for administration. The dose was calculated as 20mg of the body weight of the mice. The doses administered are shown in table 1:

TABLE 1

Sample name	Molecular weight (g/mol)	Molar mass (mmol)	Weight (mg)
				Camptothecins (JP-001-C0)	348.34	0.00861	3
JP-001-C3	966.05	0.00861	8.32

JP-001-C3 (batch No. D20210916) 30.60mg was formulated into 20mg/ml with physiological saline.

3. The test steps are as follows:

1. healthy, non-otherwise tested mice after weighing were randomly divided into experimental and control groups of 3 mice each.

2. Test solutions of different concentrations were intraperitoneally injected into mice of the experimental group.

3. The body weight of each group of mice was recorded 7 days after injection, and various biological reactions were observed.

4. Evaluation method

The animal reaction observations after injection are shown in table 2:

TABLE 2

The experimental results are shown in table 3:

TABLE 3 Table 3

Conclusion: camptothecins (JP-001-C0) were comparable to the toxicity recorded in the literature, and mice had a recovery period of at least 4 days with a single administration. JP-001-C3 has significantly less toxicity than Yu Xi trealine (JP-001-C0). 285mg/kg was not dead, the mice were affected at the early stage, the body weight was slowly increased, and recovered after one week.

The doses administered are shown in table 4:

TABLE 4 Table 4

Sample name	Molecular weight (g/mol)	Molar mass (mmol)	Dosage of 20mg mice
				Camptothecins (JP-001-C0)	348.34	0.0048	1.67mg
JP-001-C3	966.05	0.0048	4.64mg

The results are shown in Table 5:

TABLE 5

Results: the mice were in good condition when the amount of JP-001-C3 administered was 300 mg/kg. The toxicity of JP-001-C3 is obviously reduced, and mice die when the composition is administered according to the upper limit of the half lethal dose of camptothecine.

2. Efficacy experiment:

1. cell lines used in the assay:

a549: human non-small cell lung cancer cells;

BxPC-3: human in situ pancreatic adenocarcinoma cells (pancreatic somatic carcinoma cells);

MIA PaCa-2: human pancreatic cancer cells (pancreatic tail cancer cells);

PANC-1: human pancreatic cancer cells (pancreatic head cancer cells);

u251: human brain glioma cells;

2. the test method comprises the following steps: three concentrations, H, M, L respectively, are set as shown in table 6.

TABLE 6

Concentration of	JP-001-C3	Camptothecins (JP-001-C0)
			Low concentrationDegree (ug/ml)	36.2	13.1
Middle concentration (ug/ml)	72.4	26.1
			High concentration (ug/ml)	144.8	52.5

Test control group: cells cultured normally without drug.

3. The test steps are as follows:

(1) Cell suspensions (100. Mu.L/well, 5000 cells/well) were inoculated in 96-well plates and the plates were pre-incubated in an incubator (37 ℃,5% CO) ₂ )；

(2) OD on day 0 after overnight incubation;

(3) The normal culture medium of the dosing group is discarded, the culture medium with the drug to be tested is replaced, and the culture plate is placed in an incubator for culture (37 ℃,5% CO) ₂ )；

(4) OD values were measured sequentially from day one to day three (per well of 10. Mu.L CCK8 solution)

(5) The plates were incubated in the incubator for 3 hours.

(6) Measurement of absorbance at 450nm with a microplate reader

Results: the effect of the same drug on different cells for three days was as follows (a) test cells: PANC-1 and A549

JP-001-C0 test data are shown in Table 7 and FIG. 6:

TABLE 7

JP-001-C3 test data are shown in Table 8 and FIG. 7:

TABLE 8

The results prove that the three JP-001-C3 samples with different concentrations have inhibition effects on two cells, and the inhibition effects under different concentrations are not greatly different.

(b) Test cells: MIA-PaCa-2

JP-001-C0 test data are shown in Table 9 and FIG. 8:

TABLE 9

JP-001-C3 experimental data are shown in Table 10 and FIG. 9:

table 10

The results prove that: JP-001-C3 showed remarkable inhibitory effect on MIA cell line, and inhibitory effect at three concentrations was similar.

(c) Test cells: u251

JP-001-C0 test data are shown in Table 11 and FIG. 10:

TABLE 11

JP-001-C3 experimental data are shown in Table 12 and FIG. 11:

table 12

The results prove that: JP-001-C3 has remarkable inhibitory effect on U251 at all three concentrations, and the inhibitory effect is slightly weakened with the decrease of the concentration.

(d) Test cells: PANC-1, A549

JP-001-C3 experimental data are shown in Table 13 and FIG. 12:

TABLE 13

The results prove that: JP-001-C3 has an inhibitory effect on both cell lines at a concentration of H, M, L, and the inhibitory effect decreases with decreasing concentration. (higher absorbance means more viable cells)

Comparative example 1.

Compounds obtained for different types of targeting peptides linked by links of camptothecins are shown in table 12:

table 12

Sequence number	Numbering device	Peptide sequence/Structure
			1	C2	RGPD-S-S-CPT
2	C3	RGPD-Mpa-S-S-Mpa-CPT
			3	C4	c[CDGRRGDC]-Mpa-S-S-Mpa-CPT

1. C2 acute toxicity test

The experimental object: a mouse

Administration of substances such as C3

Results: first, the patients are eating less and moving less, and are loose, and then the patients are not eating, moving still, eyelid sagging, weight loss, death on 3 rd day, 1 th and all death on 6 th day.

Conclusion: has high toxicity and poor safety, and can not be used as a medicine. Failure of the structure

Description: the test, due to the severe response of the mice, was followed by death, and no weight data was recorded.

2. C3, C4 efficacy contrast test

Cell lines used in this test: a549, PANC-1

The experimental steps are as follows:

2.1. cell suspensions (100. Mu.L/well, 5000 cells/well) were inoculated in 96-well plates and the plates were pre-incubated in an incubator (37 ℃,5% CO) ₂ )；

2.2. OD on day 0 after overnight incubation;

2.3. the normal culture medium of the dosing group is discarded, the culture medium with the drug to be tested is replaced, and the culture plate is placed in an incubator for culture (37 ℃,5% CO) ₂ )；

2.4. OD values were measured sequentially from day one to day three (10 μl of CCK8 solution was spiked into each well) as a function of time;

2.5. incubating the culture plates in an incubator for 3 hours;

2.6. the absorbance at 450nm was measured with a microplate reader as shown in Table 13.

TABLE 13

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Conclusion: compared with C3, the C4 structurally replaces the original targeting peptide with the self-designed annular targeting peptide, and the action effect is similar to that of C3, but the advantages are not reflected, and the inhibition effect is slightly lower than that of C3.

Claims (4)

1. The PDC anti-tumor drug is characterized by comprising the following structure:

。

2. the use of the PDC antitumor drug of claim 1 for preparing an anticancer drug.

3. The use according to claim 2, wherein the anti-lung cancer, anti-pancreatic tail cancer, anti-pancreatic head cancer and anti-brain glioma.

4. The method for preparing the PDC anti-tumor medicament of claim 1, which is characterized by comprising the following steps:

step 1: modifying the target peptide by using a connecting compound 3- (2-pyridine dithio) propionic acid to obtain a modified target peptide;

step 2: modifying the camptothecin by using a connecting compound 3- (2-pyridine dithio) propionic acid to obtain modified camptothecin;

step 3: and (3) connecting the substances obtained in the step (1) and the step (2) through disulfide bonds to obtain the PDC antitumor drug.