CN118252945A

CN118252945A - Tumor microenvironment activated drug conjugate and antibody drug conjugate

Info

Publication number: CN118252945A
Application number: CN202410140476.9A
Authority: CN
Inventors: 刘源; 陈涛; 丁成立; 张蕊; 刘辰
Original assignee: Yafei Shanghai Biolog Medicine Science & Technology Co ltd
Current assignee: Yafei Shanghai Biolog Medicine Science & Technology Co ltd
Priority date: 2023-08-15
Filing date: 2023-12-22
Publication date: 2024-06-28

Abstract

The invention relates to a tumor microenvironment activated drug conjugate structure and an antibody drug conjugate. Specifically, the present invention provides a drug conjugate represented by the following formula, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The invention also provides a pharmaceutical composition of the drug conjugate and application thereof.

Description

Tumor microenvironment activated drug conjugate and antibody drug conjugate

The application is a divisional application of an application patent application with the application number 202311791063.9, the application date 2023, 12 months and 22 days, and the application name of a drug conjugate for activating tumor microenvironment and an antibody drug conjugate.

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a tumor microenvironment activated drug conjugate and an antibody drug conjugate.

Background

In order to improve the therapeutic window of antibody or protein conjugated drugs, efforts have been made to improve the tumor specificity and release efficiency of conjugated drugs. In recent years, the research and development of tumor-targeted therapeutic drugs has been greatly progressed in many aspects, driven by the development of tumor molecular biology and other fundamental subjects.

The method of linking effector molecules to antibodies using native cysteines in the antibody sequences has been widely used, such as cytotoxins, immune agonists, immune antagonists, etc., further extending the use of antibodies. In general, the drug effect of antibody-coupled drugs needs to enter tumor cells through endocytosis, and effector molecules with cell killing effect are released in the cells through degradation, so that the inhibition effect on tumors is realized. However, antibody-conjugated drugs have been developed for many years, and only two types of linkers activatable by cathepsin B have been successful and must enter tumor cells by endocytosis, which is a major contributor to the limitation of various antibody patent drugs.

Tetrapeptides Gly-Gly-Phe-Gly (GGFG) are cathepsin B activatable linkers that are used by ADC drugs Enhertu. The first three-common Enhertu is a plasma stable ADC, DAR 7.7, with proteolytic degradation in lysosomes, releasing DX-8951f derivative, a potent topoisomerase I inhibitor, derived from irinotecan Exatecan. Because of the GGFG linker specificity, achieving such high DAR is very considerable, which also suggests that the structure-activity relationship of the linker to the toxin molecule is critical for development into ADC drugs. However, GGFG linker compatible DX-8951f derivatives can bring about toxicity of interstitial pneumonia, including clinical occurrence of interstitial pneumonia of Enhertu(T-Dxd),Datopotamab deruxtecan(Dato-DXd),Raludotatug deruxtecan(R-DXd),Ifinatamab deruxtecan(I-DXd). Comprehensive analysis of T-DXd single drug treatment experiments: 879 patients with different tumor types, 139 (15.8%) patients were judged to have developed ILD.108 have had level 1 or level 2 events and 21 have had level 5 events. This is probably because the expression of cathepsin B in lung epithelial cells, GGFG, which activates Linker for cathepsin B, will produce a certain release in the lung. Comprehensive analysis of 23 PD-1 monotherapy trials: 3284 patients with different tumor types, 4% of whom developed ILD. In the future, ILD incidence may be further increased if T-DXd is combined with Pd-1 treatment.

We find that the rickettsia is highly expressed in tumor specificity, and develop a novel drug of rickettsia activated by small molecules coupled with rickettsia, and clinically find that hundreds of patients adopt novel chemotherapy without interstitial pneumonia, thereby indicating that the rickettsia activation has more tumor tissue specificity than cathepsin B. DX-8951f derivatives, and immune agonist T785, are far more toxic than conventional chemotherapeutic drugs such as doxorubicin and paclitaxel, and development of broad-spectrum small molecule coupled drugs is more difficult. Through various connection and enzyme cleavage activation of the Leucaezyme cleavable linker and toxin, AAN-toxin compatibility suitable for development is screened, and through R1 and R2 compatibility linker and toxin screening, small molecule coupling drugs with extremely high pre-clinical therapeutic indexes which can be considered to be developed at present are obtained, and the small molecule compounds can be independently formed into drugs or can be coupled to antibodies through EMC or other coupling modes to form antibody coupling drugs.

Disclosure of Invention

The invention aims to provide a drug conjugate and an antibody drug conjugate with strong specificity and high stability, and application thereof in treating and/or preventing cancers and/or inflammations. The drug conjugates and antibody drug conjugates of the invention are activated only in pathological microenvironments (e.g., in tumor microenvironments or inflammatory sites) to release active molecules, overcome drug resistance of the drug, and reduce toxicity.

Specifically, the present invention provides a drug conjugate represented by the following formula:

The present invention also provides a pharmaceutical composition comprising: (i) A drug conjugate according to any of the embodiments herein, a stereoisomer or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

The invention also provides the use of a drug conjugate, stereoisomer or pharmaceutically acceptable salt thereof as described in any one of the embodiments herein in the manufacture of a medicament for the treatment and/or prophylaxis of tumors and/or inflammation.

Detailed Description

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.

It must be noted that, as used herein and in the appended claims, the singular forms "a," an, "" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and reference to "the antibody" includes reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

The previously developed small molecular drug of Legulase (Legumain) activation, le Gu Bi star, contains AANL-DOX, and Le Gu Shachun contains AAN-PABC-taxel and paclitaxel, which are currently patented in China and are tested in critical clinical phase II/III and clinical phase I respectively. However, the toxicity of doxorubicin and paclitaxel cannot meet the more severe toxicant requirements of ADC, and therefore, innovative generation of conjugate structures with novel structure-activity relationship is required. The research shows that the complex formed by the levetisole and integrin can activate specific substrate structure on the surface of tumor cell, so that the inventor uses levetisole as activating enzyme, combines various toxin and immune agonist chemical structures, auxiliary structure and connecting structure to make a great amount of structure screening, and obtains the limited special conjugate structure through the action mechanism of organism and in-vitro and in-vivo curative effect. The conjugate structures are highly stable in human plasma, and can be activated and released to compatible chemical molecules in tumor microenvironment (outside tumor cells) with high efficiency. Therefore, compared with the traditional ADC, the novel release mode can better exert bystander effect and activate immune cells of accessories when the compatible load medicament is an immune agonist, and has different proprietary mechanism and expansibility compared with the traditional ADC.

Drug conjugates

Provided herein are compounds of the formula:

Pharmaceutical composition

The pharmaceutical composition of the present invention comprises: (i) A drug conjugate or a stereoisomer or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier. The carrier may be any pharmaceutically acceptable carrier or excipient, which may vary with the dosage form and mode of administration. The pharmaceutically acceptable carrier is generally safe and non-toxic and may comprise any known substance used in the pharmaceutical industry in formulating pharmaceutical compositions, including fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, disintegrants, and the like. Pharmaceutically acceptable suitable carriers include sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; starches, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate; and/or polyethylene glycol, and the like. When selecting pharmaceutically acceptable carriers, the mode of administration of the pharmaceutical dosage form is mainly considered. This is well known in the art.

The pharmaceutical composition may comprise a therapeutically or prophylactically effective amount of a drug conjugate or a stereoisomer or a pharmaceutically acceptable salt thereof. By "effective amount" is meant an amount of the ingredient sufficient to produce the desired reaction. The specific effective amount will depend on various factors such as the particular disease to be treated, the physical condition of the patient, such as weight, age and sex, the duration of the treatment, the co-administered treatment (if any), and the particular formulation used. In general, an "effective amount" as described herein is a conventional amount of a biomolecule. However, in some embodiments, the therapeutically or prophylactically effective amount of the conjugate included in the pharmaceutical compositions of the invention may be less than conventional amounts of the biomolecule but may result in better therapeutic or prophylactic effects, as the biomolecule is protected by a protecting group before reaching the pathological microenvironment for binding to its ligand or receptor.

The pharmaceutical compositions of the present invention may be formulated into a variety of suitable dosage forms including, but not limited to, tablets, capsules, injectables, and the like, and may be administered by any suitable route to achieve the intended purpose. For example, it may be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally, or externally. The dosage of the drug may depend on the age, health and weight of the patient, concurrent treatment, frequency of treatment, and the like. The pharmaceutical compositions of the invention may be administered to any subject in need thereof, e.g., as a mammal, particularly a human.

Use of the same

The drug conjugates disclosed in the present invention or stereoisomers or pharmaceutically acceptable salts thereof can be used for treating and/or preventing tumors or inflammations, or can be used as active ingredients for preparing drugs for treating tumors or inflammations.

The invention discloses a drug conjugate or stereoisomer or pharmaceutically acceptable salt thereof, which can treat diseases related to active ingredients contained in the conjugate. The indications for these active ingredients are well known in the art.

In some embodiments, tumors described herein may include hematological tumors and solid tumors, including, but not limited to, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, uterine, ovarian, testicular, blood, and the like.

The invention also includes a method for treating or preventing a tumor or inflammation comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a drug conjugate as described herein, or a stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein. The method may be used in combination with any known radiation therapy or immunotherapy.

The invention also provides the use of a drug conjugate as described herein or a stereoisomer or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of a tumor. Preferably, the tumor is selected from the group consisting of hematological tumors and solid tumors. Preferably, the tumor includes, but is not limited to, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and blood cancer.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

It should be understood that the terms "comprising" and "including" or similar expressions as used in this invention also mean "consisting of … …" and the like. The sum of all weight percentages or volume percentages should be equal to 100%. Unless otherwise indicated, the various reagents and products used in the examples were commercial products. Unless otherwise indicated, the methods mentioned in the examples are carried out according to conventional techniques. The following examples are not intended to limit the scope of the invention.

Example 1: preparation of drug conjugate S43

Typical preparation methods of the compounds are as follows:

Synthetic route general formula (III)

1) Synthesis of Fmoc-Ala-Ala-Asn-AM-OAc

Fmoc-Ala-Ala-Asn (Trt) -Gly-OH (1.0 eq) was dissolved in dichloromethane/tetrahydrofuran (15/5 by volume), cooled to 0deg.C, acOH (1.0 eq), lead acetate (1.2 eq) and copper acetate (0.2 eq) were added under nitrogen protection and stirred for 2-3 hours under nitrogen protection at 40-50deg.C. TLC monitored reaction was complete. The reaction solution was diluted with dichloromethane, washed with water, separated, the aqueous phase was extracted with dichloromethane, the organic phases were combined and dried over anhydrous sodium sulfate, spin-dried, and column chromatographed to give Fmoc-Ala-Ala-Asn-AM-OAc.

2) Synthesis of Fmoc-Ala-Ala-Asn (Trt) -AM-Ac-OBn

The compound Fmoc-Ala-Ala-Asn-AM-OAc (1.0 eq) was added to a three-necked flask, benzyl hydroxy acetate (2.0 eq) was added, and trifluoroacetic acid/dichloromethane (TFA/DCM volume ratio=1/5) was added under nitrogen and ice bath, and stirred at room temperature for 2-3h. After the reaction is completed, the reaction solution is concentrated, and Fmoc-Ala-Ala-Asn-AM-Ac-OBn white solid is obtained by high-pressure chromatography separation.

3) Synthesis of Fmoc-Ala-Ala-Asn-AM-Ac-OH

The compound Fmoc-Ala-Ala-Asn-AM-Ac-OBn (1.0 eq) was dissolved in an appropriate amount of MeOH, pd/C (10 wt%,0.1 eq) was slowly added and stirred under hydrogen for 2-3h. The compound Fmoc-Ala-Ala-Asn-AM-Ac-OH is obtained by high pressure preparation and is a pale yellow product.

4) Synthesis of Fmoc-Ala-Ala-Asn-AM-DXd

The compound Fmoc-Ala-Ala-Asn-AM-Ac-OH (1.0 eq), eptification Kang Jiawan sulfonate (1.0 eq) was dissolved in DMF, cooled to 0℃and the condensing agent DEPBT (1.2 eq) and organic base DIPEA (2.0 eq) were added in sequence under nitrogen protection and stirred at room temperature for 2-3h. TLC monitored completion of the original reaction. The reaction solution is concentrated in vacuum, DMF is redissolved, filtered, and a high-pressure liquid phase is prepared to obtain a compound Fmoc-Ala-Ala-Asn-AM-DXd which is a pale yellow product.

5) Synthesis of Compound H-Ala-Ala-Asn-AM-DXd

The compound Fmoc-Ala-Ala-Asn-AM-DXd (1.0 eq) was dissolved in DMF and piperidine (2.0 eq) was added and stirred at room temperature under nitrogen for 2-3h. TLC monitored completion of the original reaction. The reaction solution is redissolved, the solution is filtered, and then the compound H-Ala-Ala-Asn-AM-DXd is obtained by high-pressure liquid phase preparation.

6) Synthesis of Compound R ₁-R₂ -Ala-Ala-Asn-AM-DXd

Compound R ₁-R₂ -OH (1.0 eq) was dissolved in an appropriate amount of DMF, H-Ala-Ala-Asn-AM-DXd (1.0 eq) was added, HBTU (1.2 eq) and DIPEA (2.0 eq) were added under nitrogen and stirred at room temperature for 1-2H. TLC monitored completion of the original reaction. The crude reaction solution is filtered, and then the compound R ₁-R₂ -Ala-Ala-Asn-AM-DXd is obtained by high-pressure liquid phase preparation.

Using the corresponding starting materials, the S43 compounds shown in Table 1 were obtained according to the above-described methods, and the properties of S43 are shown in Table 2.

TABLE 1

TABLE 2

Numbering of compounds	Traits (3)	Yield (mg)	Molecular weight	Mass spectrometric detection [ M+H ]
					S43	Pale yellow powder	27	1770.90	1770.83

Example 2: cleavage assay for S43 drug conjugates

The stability and cleavage efficiency of the drug conjugate compound are measured as follows, and the test results are shown in Table 3.

Accurately weighing the drug conjugate compounds respectively, adding a certain amount of DMSO solution to make the concentration of stock solution be 10 mu mol/mL, then taking 10 mu L, adding 80 mu L of DMSO to dilute to 1 mu mol/mL, and then according to the following steps of 1:4 ratio of pure water was added to dilute to a sample solution concentration of 0.2. Mu. Mol/mL. After the sample was clarified, it was placed in a 25deg.C/37deg.C water bath, and after 0h, samples were taken and tested by HPLC (Agilent 1260, column: eclipse Plus C18,4.6 x 250mm,5 μm, mobile phase A:0.1% TFA+H ₂ O, mobile phase B: CH3CN, flow rate: 1.0ml/min, column temperature: 30deg.C, test wavelength: 214 nm) for a relative 0h content, so that solution stability data for the different compounds could be obtained.

Weighing a proper amount of sample, preparing into a 1mM solution by using DMSO, adding human plasma according to the volume ratio of 1:9, placing in a constant temperature box at 37 ℃ for incubation, sampling after 0,2,4,6 and 24 hours, adding DMSO/MeOH (1:1) according to the volume ratio of 1:3 for removing protein, centrifuging at 12000rpm for 5 minutes, taking supernatant, and adopting an HPLC method as the above (Agilent 1260, chromatographic column: eclipse Plus C18,4.6 mM 250mM,5 μm, mobile phase A:0.1% TFA+H2O, mobile phase B: CH ₃ CN, flow rate: 1.0ml/min, column temperature: 30 ℃ and detection wavelength: 214 nm).

An amount of drug conjugates was weighed, dissolved and diluted ten times to a concentration of 0.1 mM/ml. The drug conjugate was added to 100. Mu.g of acidified CT-26 tumor tissue homogenate (pH 5.0) at 37℃at a concentration of 1 mg/ml. Enzymes in the tumor tissue homogenates can be released and detected by HPLC to compare the activation efficiency of the tumor tissue to the linker.

TABLE 3 Table 3

Numbering of compounds	Stability of aqueous solution (24 h)	Plasma stability% (24 h)	Enzyme cutting efficiency% (2 h)
				Control Compound C	80	70	80
S43	95	95	100

As can be seen from Table 3, the stability of the compound S43 of the present invention is higher, wherein the stability of the aqueous solution for 24 hours is higher than 85%, the stability of the plasma for 24 hours is higher than 75%, and the digestion efficiency for 2 hours is higher than 90%. The stability of the control compound C (compound S1 disclosed in CN 104262455B) in 24h in water, plasma and 2h cleavage efficiency were all lower than that of the compound S43 of the present invention, because the stability of the amide bond and urethane bond between the linker and the load in the present invention was much higher than that of the carbonate bond in the control compound C, and the ester bond was more easily hydrolyzed.

The results show that the compound S43 provided by the invention has higher stability in aqueous solution and plasma, the corresponding compound is not easy to degrade or deteriorate into other compounds after entering the body, and the targeted therapeutic effect is stable after entering tumor cells or tumor tissues. In addition, after the compound S43 provided by the invention enters tumor cells and is cut by a specific enzyme for 2 hours, the toxic drug is released, and the killing rate of the toxic drug to the tumor cells is higher.

Example 3

1. Animals: nude mice, 6-8 weeks old, were all female (Shanghai Ling Biotechnology Co., ltd.).

2. And (3) cells: corresponding cells were purchased from American type culture Collection (AMERICAN TYPE culture collection, ATCC) and identified according to the instructions provided by ATCC, and the cells were cultured using modified eagle's medium (DMEM medium for short) containing 10% fetal bovine serum Dalberg's) at 37℃under 5% carbon dioxide. Cells were used within 15 passages once every 3 days.

3. Tumor production, 1.5X10 ⁶ HT1080 cells were subcutaneously injected into the back of nude mice, and when tumors grew to around 100mm3, the treatment was started at random, with the day of treatment being the first day.

4. Treatment process

The dose (5. Mu. Mol/Kg) was administered once a week for 3 weeks.

The grouping and result measurements are shown in table 4.

Table 4: effect of corresponding compound and control on tumor inhibition

5. Results and discussion: compared with the treatment effect of the irinotecan derivative, the compound S43 in the invention has greatly improved treatment effect and can almost achieve the effect of curing tumor.

Example 4

1. Test purpose: the acute toxicity of the drug in the organism is known by determining the MTD experiment of intravenous administration of the mice.

2. Test drug: the irinotecan derivative, compound S43, was dissolved in DMSO and diluted to the corresponding dose with physiological saline at the time of assay.

3. Animals: primary BaLB/C mice (purchased from Shanghai Laek laboratory animal Co., ltd.) weigh 19-21g, all females.

4. Method and results: the BALB/C mice tested were 48, 19-21g in weight, all females, and randomly divided into 7 groups of 6 according to body weight. As shown in table 5, the irinotecan derivative, compound S43, was administered intravenously at once, respectively. And a saline group control test was performed, and each mouse was dosed at a volume of 0.2ml. The animals were continuously observed for 17 days, and whether or not they were standing, matted, sleeping, bending over, hunchback, overdriving, etc., were observed daily, and weight and death were recorded. Whole blood counts were performed on day 3,5, and 14 blood samples, and on day 14 animals were dissected and observed on heart, liver, kidney, lung, spleen, pancreas HE staining.

Table 5: mortality rate results of test mice receiving different doses of compound injection and physiological saline respectively are compared with each other

Group of		Dosage (micro-mole/kg)	Animals (only)	Number of deaths (only)
					1	Physiological saline	0	6	0
2	Eptification control derivatives	20	6	3
					3	S43	20	6	0

5. Results and discussion: compared with the irinotecan derivative, when the invention is injected into S43, the animal does not have the conditions of standing hair, messy and mattness, sleeping, bending over and humpback, overdriving reaction and death, which indicates that the toxicity of the small molecule coupled medicine is obviously reduced compared with that of the unconjugated medicine.

In conclusion, the antibody-coupled drug provided by the invention can be targeted to identify and aggregate around tumor cells, can activate the drug only on the surfaces of the tumor cells, can kill tumors efficiently, and has very good application prospects, and the toxicity of the drug is reduced to a certain extent.

Claims

1. A drug conjugate of the formula:

2. A pharmaceutical composition, the pharmaceutical composition comprising: (i) The drug conjugate of claim 1, a stereoisomer or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

3. Use of a drug conjugate according to claim 1, a stereoisomer or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prophylaxis of tumors and/or inflammation.

4. The use of claim 3, wherein the tumor is a hematological tumor or a solid tumor.

5. The use of claim 3, wherein the tumor is selected from the group consisting of: bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and blood cancer.