CN116196415B - Mixed preparation for sensitizing PD-1 antibody and method of use thereof - Google Patents

Abstract

The invention discloses a mixed preparation for sensitizing PD‑1 antibodies and a method for using the same, and relates to the field of medical technology. The pharmaceutical composition comprises PD-1 antibody, PRMT5 inhibitor and PARP inhibitor and one or more pharmaceutically acceptable excipients, diluents or carriers. The two-drug combination therapy of PD-1 antibody combined with PRMT5 or the three-drug combination therapy of PD-1 antibody combined with PRMT5 and RAPR inhibitor provided by the present invention enables some patients who are ineffective against PD-1 antibody to benefit from the combination therapy Benefit, can effectively enhance the effect of PD‑1 antibody.

Description

Mixed preparation for sensitizing PD-1 antibody and method of use thereof

technical field

The invention belongs to the technical field of medicine, and in particular relates to a mixed preparation for sensitizing PD-1 antibodies and a method of use thereof.

Background technique

Immunotherapy has made some progress in the treatment of various solid tumors. However, there are still a considerable number of tumor patients who are not sensitive to immunotherapy. Studies have shown that the degree of immune cell infiltration in tumor tissue can reflect the effect of immunotherapy to a certain extent. Tumors with more immune cell infiltration in the tumor microenvironment are called "hot tumors", and vice versa are called "cold tumors". In patients with cold tumors, the infiltration of immune cells in the tumor does not increase significantly after treatment with PD-1 antibodies, which is an important reason for the failure of immunotherapy. Therefore, how to convert "cold tumor" into "hot tumor" is an urgent problem in this field. The purpose of the present invention is to provide a combined treatment plan for tumors that are not sensitive to PD-1 antibody immunotherapy, specifically for patients with various solid tumors with unstable chromatin, the two-drug combination therapy using PD-1 antibody combined with PRMT5 Some patients who are ineffective against PD-1 antibodies can benefit from the combination therapy.

Contents of the invention

The purpose of the present invention is to provide a mixed preparation for sensitizing PD-1 antibody and its use method, and propose a two-drug combination treatment plan of PD-1 antibody combined with PRMT5 or a three-drug combination of PD-1 antibody combined with PRMT5 and RAPR inhibitor The combination therapy allows some patients who are ineffective against PD-1 antibodies to benefit from the combination therapy and can effectively enhance the effect of PD-1 antibodies.

The technical scheme that the present invention takes for realizing the above object is:

A pharmaceutical composition, comprising a PD-1 antibody, a PRMT5 inhibitor and a PARP inhibitor, and one or more pharmaceutically acceptable excipients, diluents or carriers. The present invention provides a two-drug combination scheme of PD-1 antibody combined with PRMT5 or a three-drug combination scheme of PD-1 antibody combined with PRMT5 and RAPR inhibitors. After analyzing the biological activity, it is found that the combination of LLY-283 and PD-1 has a significant effect on mice. The source cell line 4T1 has a stronger inhibitory effect on subcutaneous tumor bearing, and the effect is significantly better than that of single drug, with additive or synergistic enhancement effects. At the same time, through the analysis of biological activity, it was found that the combination of LLY-283, Olaparib and PD-1 had a stronger inhibitory effect on the subcutaneous tumor bearing of the mouse cell line EMT6, and the effect was significantly better than that of the single drug, with additive or synergistic effects. Enhanced effect; Compared with traditional monotherapy with Olaparib, it can effectively reduce the concentration of Olaparib, and can significantly inhibit tumor proliferation, and avoid or reduce the occurrence of relapse and acquired drug resistance events due to monotherapy alone. The antitumor pharmaceutical composition provided by the invention has clear ingredients and simple preparation process, and can be used alone for tumor treatment, and can also be used in conjunction with radiotherapy and surgery. The antitumor pharmaceutical composition of the invention provides a new idea for antitumor treatment or prevention, and has great application prospects.

Preferably, the PARP inhibitor is selected from one of Olaparib, Veliparib, Talazoparib, Niraparib and Rucaparib.

Preferably, the PRMT5 inhibitors are selected from GSK3326595, AMG-193, MRTX1719, TNG-908, PF-069399999, PRT543, PRT811, SH-3765, onametostat, SCR-6920, SKL27969, SYHX2001, AGXX, AGXX 323, BRD0639, C220, DS- One of 437, DW14800, GSK3203591, GSK3235025, JBI-778, LLY-283, MRTX9768, MS4322, MS4369, MS4370, PF-06855800.

Preferably, the PD-1 antibody includes but not limited to toripalimab, and various inhibitors targeting PD-1 should also fall within the protection scope of the present invention.

Preferably, the dose range of the PD-1 antibody is selected from 10-100 mg in vivo; further, it can be 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg.

Preferably, the dose range of the PRMT5 inhibitor is selected from 10-100 mg in vivo; further, it can be 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg.

Preferably, the dosage range of the PAPR inhibitor is selected from 10-100 mg in vivo; further, it can be 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg.

Preferably, the combined administration route is selected from oral administration, parenteral administration, and transdermal administration, and said parenteral administration includes but not limited to intravenous injection, subcutaneous injection, and intramuscular injection.

Preferably, the administration frequency of the PRMT5 inhibitor can be once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks or once a month; the administration frequency of the PARP inhibitor can be one Once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks or once a month.

Another object of the present invention is to provide the use of PD-1 antibody in combination with PRMT5 in the preparation of a drug for preventing or treating solid tumors.

Another object of the present invention is to provide the use of PD-1 antibody in combination with PRMT5 and RAPR inhibitors in the preparation of drugs for preventing or treating solid tumors.

Preferably, a solid tumor refers to a solid tumor with unstable chromatin.

Specifically, solid tumors include benign solid tumors and malignant solid tumors.

Further, benign solid tumors mainly include hamartoma, leiomyoma, hemangioma, lymphangioma, and various adenomas and adenomatous polyps; malignant solid tumors include Hodgkin's lymphoma, non-Hodgkin's lymphoma, Lymphoma, lung cancer, breast cancer, ovarian cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, as well as head and neck malignancies, urinary system malignancies, endometrial cancer, cervical cancer, osteosarcoma, cartilage Sarcoma, Ewing's sarcoma, thyroid cancer, hepatoblastoma, Wilms tumor, etc.

It should be noted that the pharmaceutical composition of the present invention can be administered alone or in combination with one or more therapeutic agents.

The "combination" in the present invention is a mode of administration, which refers to the administration of at least one dose of PRMT5 inhibitor and at least one dose of PD-1 antibody within a certain period of time, or the administration of at least one dose of PARP inhibitor agent and at least one dose of PD-1 antibody, or at least one dose of PRMT5 inhibitor and at least one dose of PARP inhibitor and at least one dose of PD-1 antibody. The time period can be within one administration cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours, more preferably within 12 hours. This term includes treatments in which the PRMT5 inhibitor, the PARP inhibitor and the PD-1 antibody are administered by the same route of administration or by different routes of administration.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a pharmaceutical composition for the prevention or treatment of solid tumors, specifically the two-drug combination of PD-1 antibody combined with PRMT5 or the three-drug combination of PD-1 antibody combined with PRMT5 and RAPR inhibitor, which is a tumor drug Treatment offers new drug options. The present invention finds that PRMT5 inhibitors can effectively increase the curative effect of PD-1 antibodies after biological activity analysis. At the same time, through the analysis of biological activity, it was also found that the combination of PRMT5 inhibitor and PARP inhibitor can effectively increase the efficacy of PD-1 antibody; compared with traditional single drug therapy, it can effectively reduce the concentration of single drug and significantly inhibit tumor proliferation , to avoid or reduce the occurrence of relapse and acquired drug resistance events due to the use of only single drug therapy; this combination therapy also provides new ideas for the treatment of some patients who are ineffective against PD-1 antibodies, and provides a basis for the design and development of drugs It provides new theoretical support and has great value in the research and treatment of tumors.

Therefore, the present invention provides a mixed preparation for sensitizing PD-1 antibody and its use method, and proposes a two-drug combination treatment plan of PD-1 antibody combined with PRMT5 or a three-drug combination of PD-1 antibody combined with PRMT5 and RAPR inhibitors The combination therapy allows some patients who are ineffective against PD-1 antibodies to benefit from the combination therapy and can effectively enhance the effect of PD-1 antibodies.

Description of drawings

Figure 1 shows the effect of PRMT5 inhibitors on increasing PD-1 antibodies in the BALB/C mouse model bearing subcutaneous tumors of the murine cell line 4T1;

Figure 2 shows the effect of PRMT5 inhibitor combined with PARP inhibitor on increasing PD-1 antibody in the subcutaneous tumor-bearing BALB/C mouse model of murine cell line EMT6;

Figure 3 shows the effect of shikonin derivatives on increasing PD-1 antibody in the subcutaneous tumor-bearing BALB/C mouse model of the murine cell line EMT6.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, various implementation modes of the present invention will be described in detail below in conjunction with the embodiments. However, those of ordinary skill in the art can understand that, in each implementation manner of the present invention, many technical details are provided for readers to better understand the present application. However, even without these technical details and various changes and modifications based on the following implementation modes, the technical solution claimed in this application can also be realized.

Example 1:

In vivo pharmacodynamic study of the combination of PRMT5 inhibitor and PD-1 antibody in the BALB/C mouse model bearing subcutaneous tumors of the murine cell line 4T1:

1. Test drug

Drug name: PRMT5 inhibitor LLY-283, PD-1 antibody.

2. Experimental animals

BALB/c mice, 6-8 weeks old, female, feeding environment: SPF grade.

Breeding environment: temperature: control temperature 20-26°C; relative humidity: control relative humidity 40%-70%; light: automatic light, alternating light and dark every 12 hours.

3. Experimental procedure

4T1 cells were resuspended in 1:1 PBS and Matrigel (2×10 ⁵ ) (RPMI1640, 10% fetal bovine serum, 1% P/S (PB180120), cultured at 37°C, 5% CO ₂ ), subcutaneously The tumors were located subcutaneously in the right flank of 24 BALB/c nude mice. When the average tumor volume of the mice reached 100-150 mm ³ , they were randomly divided into 4 groups with 6 mice in each group. After grouping, 0.5% carboxymethylcellulose sodium water/normal saline (group 1), LLY-283 (group 2), PD-1 antibody (group 3), LLY-283+PD-1 antibody (group 3) were orally administered according to the protocol. 4); experimental groups and dosing regimens are shown in Table 1. Tumor volume was measured every two days, body weight was weighed, and the data were recorded. The formula for calculating tumor volume (V) is:

V=0.52×a×b ² , where a and b represent length and width, respectively.

Table 1 Experimental grouping and dosing regimen

Note: QDx15: 3 days on and 4 days off, 15 times of administration; QDx10: start of administration after 5 days, administration every two days, 10 times of administration; po: oral administration; i.p.: intraperitoneal injection.

The test results are shown in Figure 1. From the analysis in the figure, it can be seen that the tumor volume of the PRMT5 inhibitor and PD-1 antibody combined administration group was significantly lower than that of the respective administration alone, indicating that the PRMT5 inhibitor can effectively increase the efficacy of the PD-1 antibody. The murine cell line 4T1 has a better in vivo pharmacodynamic effect in the subcutaneous tumor-bearing BALB/C mouse model.

4. Compatibility calculation method

Calculate the q value according to King's formula, q=E _A+B /(E _A +E _B -E _A ×E _B ), which is used to judge whether the effect of the two drugs in combination is better than that of single administration. If q<0.55, the effects of the two drugs are significantly antagonistic; if 0.55≤q<0.85, the effects of the two drugs are antagonistic; if 0.85≤q<1.15, the effects of the two drugs are purely additive; if 1.15≤q<20, the effects of the two drugs are synergistic enhancement ; q≥20, the effect of the two drugs is significantly enhanced.

From the data analysis in Figure 1, it can be seen that after 35 days of administration, compared with the control group, the tumor volume reduction rates in groups 2, 3, and 4 were 45.45%, 34.06%, and 74.33%, respectively. According to King’s formula, q= 74.33%/(45.45%+34.06%-45.45%×34.06%)=1.16, in line with 1.15≤q<20, indicating that the combination of LLY-283 and PD-1 antibody has a synergistic enhancement effect on tumor inhibition.

Example 2:

In vivo pharmacodynamic study of PRMT5 inhibitor combined with PARP inhibitor and PD-1 antibody three-drug combination administration and each administration alone on the subcutaneous tumor-bearing BALB/C mouse model of the murine cell line EMT6:

1. Test drug

Drug name: PRMT5 inhibitor LLY-283, PARP inhibitor Olaparib, PD-1 antibody.

2. Experimental animals

BALB/c mice, 6-8 weeks old, female, feeding environment: SPF grade.

Breeding environment: temperature: control temperature 20-26°C; relative humidity: control relative humidity 40%-70%; light: automatic light, alternating light and dark every 12 hours.

3. Experimental procedure

EMT6 cells were resuspended in PBS (4×10 ⁵ ) (high glucose DMEM medium, 10% fetal bovine serum, 1% P/S (PB180120), cultured at 37°C, 5% CO ₂ ), subcutaneously bearing tumors at 64 Subcutaneously in the right flank of BALB/c nude mice. When the average tumor volume of the mice reached 100-150 mm ³ , they were randomly divided into 8 groups with 8 mice in each group. After grouping, 0.5% carboxymethylcellulose sodium water/normal saline (group 1), LLY-283 (group 2), PD-1 antibody (group 3), Olaparib (group 4), LLY-283+ were orally administered according to the protocol PD-1 antibody (group 5), Olaparib+PD-1 antibody (group 6), LLY-283+Olaparib (group 7), LLY-283+Olaparib+PD-1 antibody (group 8); experimental grouping and administration The program is shown in Table 1. Tumor volume was measured every two days, body weight was weighed, and data was recorded. The formula for calculating tumor volume (V) is:

V=0.52×a×b ² , where a and b represent length and width, respectively.

Table 2 Experimental grouping and dosing regimen

Note: QDx15: once a day, 15 times of administration; QDx7: 3 days on and 4 days off, 7 times of administration; QDx5: administration every two days, 5 times of administration; po: oral administration; i.p.: intraperitoneal injection.

The test results are shown in Figure 2. From the analysis in the figure, it can be seen that the tumor volume of PRMT5 inhibitor combined with PARP inhibitor and PD-1 antibody three-drug combination administration group was significantly lower than that of each administration alone and two-two combination administration, indicating that PRMT5 inhibitors and PARP inhibit The combination of the two agents can effectively increase the efficacy of the PD-1 antibody, and the combined use of the two has a better in vivo pharmacological effect in the BALB/C mouse model of the murine cell line 4T1 subcutaneously bearing tumors.

4. Compatibility calculation method

Calculate the q value according to King's formula, q=E _A+B /(E _A +E _B -E _A ×E _B ), which is used to judge whether the effect of the two drugs in combination is better than that of single administration. If q<0.55, the effects of the two drugs are significantly antagonistic; if 0.55≤q<0.85, the effects of the two drugs are antagonistic; if 0.85≤q<1.15, the effects of the two drugs are purely additive; if 1.15≤q<20, the effects of the two drugs are synergistic enhancement ; q≥20, the effect of the two drugs is significantly enhanced.

From the data analysis in Figure 2, it can be seen that after 21 days of administration, compared with the control group, the tumor volume reduction rates in groups 2, 3, 4, 5, 6, 7, and 8 were 29.67% and 19.36%, respectively. %, 48.51%, 82.20%.

Compared with group 5, group 6, and group 8, according to King's formula, q=82.20%/(29.67%+19.36%-29.67%×19.36%)=1.90, which meets 1.15≤q<20;

Compared with group 5, group 7, and group 8, according to King's formula, q=82.20%/(29.67%+48.51%-29.67%×48.51%)=1.29, which is consistent with 1.15≤q<20;

Compared with group 6, group 7, and group 8, according to King's formula, q=82.20%/(19.36%+48.51%-19.36%×48.51%)=1.41, which is consistent with 1.15≤q<20;

The above results indicate that the combination of LLY-283, Olaparib and PD-1 antibody has a synergistic effect on tumor inhibition.

Example 3:

The chemical structure of shikonin derivatives is shown in formula I:

I.

Another object of the present invention is to provide the use of the PD-1 antibody combined with the shikonin derivative shown in formula I in the preparation of a drug for preventing or treating solid tumors.

The preparation method of the above-mentioned shikonin derivatives comprises: using chromene card to chemically modify the shikonin through esterification to prepare the shikonin derivatives. The present invention uses chromene to chemically modify the natural plant extract shikonin to further improve the biological activity of shikonin derivatives, and combine it with PD-1 antibody to provide a pharmaceutical composition that is compatible with single drug or some Compared with other combined drugs, it has a significant synergistic effect in inhibiting solid tumors such as benign or malignant tumors. Shikonin derivatives can effectively increase the efficacy of PD-1 antibodies. Better in vivo pharmacodynamics in the tumor-bearing BALB/C mouse model; concomitantly improved resistance to monotherapy.

Specifically, the preparation method of the above-mentioned shikonin derivatives, the steps are:

Dissolve chromene, DCC and DMAP in anhydrous dichloromethane, stir for 10-15 minutes, then add shikonin, place the reaction system in an ice bath, stir and react for 10-15 hours, and monitor by TLC to confirm whether the reaction is complete; After the reaction was completed, the reaction system was placed in a -20°C refrigerator, frozen overnight, filtered quickly, and the filtrate was concentrated at low temperature using a rotary evaporator, and the mixed solvent of ethyl acetate/petroleum ether (v/v, 1:6~8) was used as Developing solvent, thin-layer chromatography separation to obtain shikonin derivatives.

Preferably, the molar ratio of chromene card, DCC and DMAP is 1:1.5~2:0.2~0.3; the solid-liquid ratio of chromene card and anhydrous dichloromethane is 2~3g:1mL; shikonin and chromene The molar ratio of card is 0.8~0.9:1.

In this embodiment, the preparation of shikonin derivatives is as follows:

Take chromene, DCC and DMAP (the molar ratio of the three is 1:1.8:0.24) and dissolve it in anhydrous dichloromethane (the solid-liquid ratio of chromene and anhydrous dichloromethane is 2.6g:1mL), Stir for 12 minutes, then add shikonin (the molar ratio to chromene card is 0.85:1), put the reaction system in an ice bath, stir and react for 12 hours, TLC monitors to confirm whether the reaction is complete; after the reaction, place the reaction system in In the -20℃ refrigerator, freeze overnight, filter quickly, and concentrate the filtrate at low temperature with a rotary evaporator, and use a mixed solvent of ethyl acetate/petroleum ether (v/v, 1:7.5) as a developing solvent, and obtain purple Grass Ning Derivatives. ¹ H NMR (500 MHz, CDCl ₃ ) δ 12.62 (s, 1H, -OH), 12.49 (s, 1H, -OH), 8.09 – 7.40 (4H,Ar-H), 7.61 (dd, 2H,Ar- H), 7.82, 7.43, 5.21 (3H, CH=C), 5.45 (m, 1H, -CH), 2.21, 2.40(2H, -CH ₂ ), 1.77 (s, 3H, -CH ₃ ), 1.65 ( s, 3H, -CH ₃ ).

In vivo pharmacodynamic study of shikoning derivatives combined with PD-1 antibody combined administration and each administration alone on the murine cell line EMT6 subcutaneously bearing tumor-bearing BALB/C mouse model:

1. Test drug

Drug name: Shikonin derivative prepared in this example, PD-1 antibody.

2. Experimental animals

BALB/c mice, 6-8 weeks old, female, feeding environment: SPF grade.

Breeding environment: temperature: control temperature 20-26°C; relative humidity: control relative humidity 40%-70%; light: automatic light, alternating light and dark every 12 hours.

3. Experimental procedure

EMT6 cells were resuspended in PBS (4×10 ⁵ ) (high glucose DMEM medium, 10% fetal bovine serum, 1% P/S (PB180120), cultured at 37°C, 5% CO ₂ ), subcutaneously bearing tumors at 32 Subcutaneously in the right flank of BALB/c nude mice. When the average tumor volume of the mice reached 100-150 mm ³ , they were randomly divided into 4 groups with 8 mice in each group. After grouping, 0.5% carboxymethylcellulose sodium water/normal saline (group 1), PD-1 antibody (group 2), shikonin derivatives (group 3), shikonin derivatives + PD- 1 antibody (group 4); the experimental grouping and dosing regimen are shown in Table 3. Tumor volume was measured every two days, body weight was weighed, and the data were recorded. The formula for calculating tumor volume (V) is:

V=0.52×a×b ² , where a and b represent length and width, respectively.

Table 3 Experimental grouping and dosing regimen

Note: QDx15: 3 days on 4 days off, 15 times of administration; QDx7: 3 days on 4 days off, 7 times of administration; po: oral administration; i.p.: intraperitoneal injection.

The test results are shown in Figure 3. From the analysis in the figure, it can be seen that the tumor volume in the combination administration group of the shikoning derivative and the PD-1 antibody was significantly lower than that in the single administration group, indicating that the shikoning derivative can effectively increase the curative effect of the PD-1 antibody. The combined use of the two has better pharmacological effects in vivo in the BALB/C mouse model bearing subcutaneous tumors of the murine cell line 4T1.

4. Compatibility calculation method

Calculate the q value according to King's formula, q=E _A+B /(E _A +E _B -E _A ×E _B ), which is used to judge whether the effect of the two drugs in combination is better than that of single administration. If q<0.55, the effects of the two drugs are significantly antagonistic; if 0.55≤q<0.85, the effects of the two drugs are antagonistic; if 0.85≤q<1.15, the effects of the two drugs are purely additive; if 1.15≤q<20, the effects of the two drugs are synergistic enhancement ; q≥20, the effect of the two drugs is significantly enhanced.

From the data analysis in Figure 3, it can be seen that after 21 days of administration, compared with the control group, the tumor volume reduction rates in groups 2, 3, and 4 were 19.47%, 18.54%, and 77.22%, respectively. According to King’s formula, q= 77.22%/(19.47%+18.54%-19.47%×18.54%)=2.24, in line with 1.15≤q<20, indicating that the combination of shikonin derivatives and PD-1 antibody has a synergistic effect on tumor inhibition.

The conventional technologies in the above embodiments are known to those skilled in the art, so they will not be described in detail here.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (4)

1. A mixed formulation for sensitizing a PD-1 antibody comprising a PD-1 antibody and shikonin derivative represented by formula I:

I。

2. the cocktail for sensitizing PD-1 antibodies according to claim 1, wherein said PD-1 antibodies comprise terlipressin Li Shan antibodies.

3. Use of a PD-1 antibody in combination with shikonin derivatives of formula I for the manufacture of a medicament for the prevention or treatment of solid tumors:

I。

4. the use of claim 3, wherein the solid tumor comprises a benign solid tumor or a malignant solid tumor.