CN114146181A

CN114146181A - Combination containing Pan-HDAC and immune checkpoint inhibitor and application

Info

Publication number: CN114146181A
Application number: CN202111273090.8A
Authority: CN
Inventors: 吴文涛; 徐英霖
Original assignee: Xunuo Pharmaceutical Nanjing Co ltd
Current assignee: Xunuo Pharmaceutical Nanjing Co ltd
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2022-03-08
Anticipated expiration: 2041-10-29
Also published as: CN114146181B

Abstract

The application provides the use of a combination of a Pan-HDAC inhibitor N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide and an immune checkpoint inhibitor, such as a PD-1 antibody, in the manufacture of a medicament for the prevention of recurrent lymphoma; the N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide salt of the invention is preferably p-toluenesulfonate, the immune checkpoint inhibitor is preferably PD-1, and the content ratio of the two is preferably 5-10: 1-5. Experiments have shown that a combination of a Pan-HDAC inhibitor N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide and an immune checkpoint inhibitor, such as a PD-1 antibody, is effective in preventing recurrent lymphoma.

Description

Combination containing Pan-HDAC and immune checkpoint inhibitor and application

Technical Field

The present application is in the field of cancer therapy, in particular, the present invention relates to the use of a combination comprising a Pan-HDAC inhibitor and an immune checkpoint inhibitor for the manufacture of a medicament for the prevention of recurrent lymphoma.

Background

Epigenetics is an emerging discipline for studying the heritable modifications of gene expression and regulation that do not involve changes in DNA sequence, i.e., exploring the processes and mechanisms that evolve from genes into phenotypes. Epigenetic modifications and their genetic mechanisms include DNA methylation, histone modifications, non-coding RNA, genomic imprinting, and the like. The most extensively and extensively studied histone modification at present is nucleosome histone lysine acetylation; plays an important role in chromatin remodeling and gene expression regulation, etc., and this modification is highly dynamically regulated by histone acetylases and deacetylases (HDACs) in vivo. HDACs play a very important role in multiple links of tumorigenesis and progression; the expression change and mutation of HDAC gene can induce abnormal transcription of key gene, so as to affect the silencing of tumor cancer suppressor gene, cell differentiation, angiogenesis, cell migration, cell cycle abnormality, signal transduction, cell attachment, etc. Pan-HDAC inhibitors regulate histone acetylation by targeting HDAC inhibition, and further regulate key tumor suppressor proteins and protooncogenes, thus becoming one of the alternative approaches for cancer treatment. N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide belongs to a Pan-HDAC inhibitor and can act on HDAC1,

subtypes

2, 3, 6 and 10, and experiments show that the inhibitor can generate an inhibiting effect on tumors through various ways.

Immune check points are protein small molecules capable of regulating autoimmune functions, the immune check points are one of main reasons of immune tolerance in tumor occurrence and progression, tumor treatment can be realized by inhibiting and stimulating proper immune check points, and currently, most of immune check point inhibitor drugs which are researched and put on the market are mainly PD-1, PD-L1 and CTLA-4 antibody drugs, such as ipilimumab, pammumab, nivolumab, alemtuzumab, Devolumab, avizumab and the like. Although immune checkpoint inhibitor drugs have met with great clinical success, there are some drawbacks, such as poor overall population efficiency and limited use due to drug resistance problems. Based on our deep knowledge of the mechanism of action of Pan-HDAC inhibitors, we studied N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide in improving the efficiency of immune checkpoint inhibitors and overcoming and delaying secondary resistance, and obtained significant and substantial results. Our studies indicate that N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide not only can significantly improve the effective rate of immune checkpoint inhibitors and prolong the life span of tumor-bearing animals, but also find that immune memory cells are generated in vivo for animals with complete tumor regression in treatment, and can play a long-term protective role when the same tumor cells are re-excited. The research result perfects the individual tumor treatment scheme and the application of different treatment strategies, and further promotes the development of accurate tumor treatment.

There is currently no precedent for drugs or therapies that combine Pan-HDAC with immune checkpoint inhibitors.

Disclosure of Invention

In one aspect, the present invention provides the use of a pharmaceutical combination comprising a Pan-HDAC inhibitor and an immune checkpoint inhibitor for the preparation of a medicament for the prevention of recurrent lymphoma; the Pan-HDAC inhibitor is preferably N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide or a salt thereof; the immune checkpoint inhibitor is preferably a PD-1 or CTLA-4 antibody.

The N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide salt is preferably p-toluenesulfonate, hydrochloride, naphthalene-1, 5-disulfonate, naphthalene-2-sulfonate, oxalate, benzenesulfonate, sulfate or phosphate.

Further, the ratio of the Pan-HDAC inhibitor to the immune checkpoint inhibitor is preferably 5-10: 1-5. Further, the Pan-HDAC inhibitor is p-toluenesulfonate salt of N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide, and the immune checkpoint inhibitor is PD-1; the ratio of Pan-HDAC inhibitor to immune checkpoint inhibitor content was 8: 1.

Further, the medicine composition is an oral preparation or an injection.

Abexinostat, abebestat, XP101, PCI-24781, N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide are used interchangeably herein and refer to compounds of the formula:

the pharmaceutical combination forms described herein include, but are not limited to, formulating Pan-HDAC inhibitors and immune checkpoint inhibitors into compositions that provide Pan-HDAC inhibitors and immune checkpoint inhibitors, respectively, in the same package and Pan-HDAC inhibitors and immune checkpoint inhibitors, respectively, in different packages but in combination.

By "preventing" in this application is meant preventing the reoccurrence of the cancer or associated symptoms after healing.

In addition to lymphoma, the cancers treated or prevented herein include cancers known and under investigation to be associated with Pan-HDAC or immune checkpoints, including but not limited to lymphoma, lung cancer, liver cancer, pancreatic cancer, melanoma, head and neck cancer, brain cancer, nasopharyngeal cancer, bone cancer, intestinal cancer, gastric cancer, neural tumor, blood cancer, and the like; lymphoma is preferred. The cancer described herein may be a primary, recurrent or metastatic cancer, preferably a recurrent cancer.

The compositions of the present application can be used in a variety of animals including, but not limited to, primate mammals, murine, bovine, equine, canine, chicken, duck, goose species, and the like, preferably primate mammals, and particularly preferably human families.

The administration characteristics of Pan-HDAC inhibitors and immune checkpoint inhibitors herein will vary depending on the type of salt, type of inhibitor, dosage form, subject condition, and the like, and one skilled in the art or physician may vary the dosage, frequency, duration, etc., of administration as desired.

The dosage form of the present application may be selected from a variety of dosage forms known and studied in the art, including but not limited to oral dosage forms such as tablets, capsules, oral liquid formulations, pills, granules, powders; injection such as water injection, oil injection, emulsion injection, and powder injection; and other dosage forms, such as topical dosage forms, e.g., ointments, patches, sprays, and the like; the methods and adjuvants required for the preparation of these dosage forms are available in the relevant books and literature of pharmacy,

those skilled in the art can select suitable adjuvants for the drugs referred to herein according to the general knowledge in the pharmaceutical field, and the types of adjuvants that can be used include, but are not limited to, solvents, cosolvents, stabilizers, dispersants, viscosity modifiers, antioxidants, sweeteners, binders, gas generants, and the like; the preferred injection of this application contains water and HP-beta-CD as adjuvants.

The pharmaceutical compositions and methods of the present application may be used in combination with other known and studied drugs or methods of treatment or adjuvant treatment of cancer, including, but not limited to, chemotherapy and corresponding chemotherapeutic agents, radiotherapy, immunotherapy methods and corresponding drugs, herbal medicines, and the like.

Drawings

FIG. 1 is a graph showing the average body weight change of each group.

FIG. 2 is a graph showing the change in the average body weight loss (%) of each group.

Figure 3 is a plot of mean tumor volume growth for each group.

FIG. 4 is a graph of mean relative tumor volume change for each group.

FIG. 5 is a graph of birth weight.

FIG. 6 shows the Re-Challenge tumor growth curve.

Detailed Description

Abbreviations and alternatives in this application are indicated:

technical solution studied in this application

The present application provides a pharmaceutical combination comprising a Pan-HDAC inhibitor and an immune checkpoint inhibitor.

Further, the Pan-HDAC inhibitor is N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide.

Further, the Pan-HDAC inhibitor is a salt of N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide.

Further, the Pan-HDAC inhibitor is p-toluenesulfonate, hydrochloride, naphthalene-1, 5-disulfonate, naphthalene-2-sulfonate, oxalate, benzenesulfonate, sulfate or phosphate of N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide.

Further, the Pan-HDAC inhibitor is p-toluenesulfonate salt of N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide.

Further, the immune checkpoint is PD-1, PD-L1 or CTLA-4.

Further, the immune checkpoint is PD-1.

Further, the immune checkpoint inhibitor is an antibody.

Further, the immune checkpoint inhibitor is a monoclonal antibody.

Further, the pharmaceutical combination is for use in the treatment or prevention of cancer.

Further, the cancer is lymphoma.

Further, the cancer is a relapsed cancer.

In another aspect, the present application provides the use of the above pharmaceutical combination for the preparation of a medicament for the treatment or prevention of cancer.

Further, the cancer is lymphoma.

Further, the cancer is a relapsed cancer.

Further, the medicine composition is an oral preparation or an injection.

Further, the medicine composition is an injection.

The present application also provides methods of treating or preventing cancer using the above pharmaceutical combinations comprising administering to a subject having cancer a Pan-HDAC inhibitor and an immune checkpoint inhibitor.

Further, the immune checkpoint is PD-1, PD-L1 or CTLA-4.

Further, the immune checkpoint is PD-1.

Further, the immune checkpoint inhibitor is an antibody.

Further, the immune checkpoint inhibitor is a monoclonal antibody.

Further, the cancer is lymphoma.

Further, the cancer is a relapsed cancer.

Further, the Pan-HDAC inhibitor and the immune checkpoint inhibitor are administered simultaneously.

Further, the Pan-HDAC inhibitor and the immune checkpoint inhibitor are administered in the same pharmaceutical composition.

Further, Pan-HDAC inhibitors and immune checkpoint inhibitors are administered separately.

Further, the Pan-HDAC inhibitor is administered at a dose of 40-100mg per administration.

Further, the Pan-HDAC inhibitor is administered at a dose of 80mg each.

Further, the Pan-HDAC inhibitor is administered 1 time per 2 days to 3 times per day, 1-7 times per week.

Further, the Pan-HDAC inhibitor is administered 2 times daily, 4 hours apart between 2 times weekly, 7 times weekly, for a week followed by 7 days off, 28 days as one treatment cycle; or 2 times daily every 2 hours between 2 times on days 1-4, 8-11 and 15-18 of a 28-day treatment cycle.

Further, the administration dose of the immune checkpoint inhibitor is 1-50 mg/kg.

Further, the immune checkpoint inhibitor is administered at a dose of 10 mg/kg.

Further, the frequency of administration of the immune checkpoint inhibitor is 1-6 times per week.

Further, the frequency of administration of the immune checkpoint inhibitor is 2 times per week.

Further, the immune checkpoint inhibitor is administered for a period of 1-6 weeks.

Further, the administration of the immune checkpoint inhibitor is for a period of 3 weeks.

Example 1 Experimental drugs, animals, instruments and general methods

Experimental drugs and main reagents:

the experimental drug XP101 is provided by the applicant, and the basic information is as follows:

the molecular formula is as follows: c₂₁H₂₃N₃O₅，C₇H₈O₃S

Molecular weight: 569.6(397.431+172.205)

Chiral/stereochemistry: non-asymmetric carbon

Base conversion factor: 0.698.

experimental drug anti-mouse CTLA4 was supplied by Bioxcell.

The experimental drug Anti-mouse PD-1 was supplied by Bioxcell.

Solvent 20% HP-beta-CD was supplied by Miss of quality, Guangzhou, Inc.

Cell lines for experiments: a20 mouse B cell lymphoma cell line was derived from ATCC.

Other reagents:

experimental animals and feeding:

animal species and strains: BALB/c mice

Dosing record: no history of drug administration

Sex, age, body weight: female, 5-6 weeks old, 18-20 g

Breeder/supplier: shanghai Ling Biotech Co., Ltd

An adaptation period: 7 days

Room: SPF area room

Indoor temperature: 20-26 deg.C

Indoor relative humidity: 40-70 percent

Lighting: fluorescent lamp illumination, 12-hour illumination (08:00-20:00) and 12-hour no-illumination

Animal feeding: each cage is 2-6 (same administration group)

Food: infinite amount of feed (sterilized by irradiation, Jiangsu province, cooperative medical bioengineering Co., Ltd., China)

Water: unlimited access to drinking water (tap water treated by reverse osmosis or autoclaving and acidified)

A total of 68 was purchased from the supplier for this experiment only. The animals have no specific pathogen and arrive at the animal house for 4-5 weeks. Upon arrival, animal house personnel transfer the animals from shipping packages to squirrel cages and examine each animal. The examination range includes appearance, limbs, cavities and the like, and whether the animal is abnormally represented when the animal is static or in motion. The adaptation period is 7 days.

The main apparatus comprises:

instrument for measuring the position of a moving object	Company(s)	Model number
			Centrifugal machine	Saimer Feishale	Thermo Fisher legend MACH 1.6/
Inverted microscope	Nikang medicine	TS100
			Biological safety cabinet (cell culture)	Saimer Feishale	1379
CO₂Culture box	Saimer Feishale	Thermo HERA cell 150
			Water bath pot	Shanghai essence macro	DK-8B
Liquid nitrogen tank	Saimer Feishale	Thermo LOCATOR				4
			Pipettor (electric) (cell culture)	Capp	PA-100
Pipettor (1ml) (cell culture)	Aibende	L20989H
			Pipettor (100. mu.l) (cell culture)	Aibende	L38752H
Pipettor (20. mu.l) (cell culture)	Aibende	N22990H
			Anesthesia machine	MATRX	MATRX VIP3000
Biological safety cabinet (cell inoculation)	Saimer Feishale	Thermo MSC-Advantage
			Biological safety cabinet (dispensing)	Saimer Feishale	Thermo MSC-Advantage
Balance (animal weight)	Denver, USA	DENVER INSTRUMENT TP-602
			Vernier caliper	Japan Sanfeng	mitutoyo CD-15cX

EXAMPLE 2 cell culture

The mouse B-cell lymphoma cell line A20 used in this experiment was cultured in RPMI-1640 medium supplemented with 10% FBS in 5% CO₂37 ℃ incubator. After two successive passages of cell culture, 68 BALB/c mice were inoculated subcutaneously. Each mouse was inoculated with 1X 10⁶A20 cells, which were resuspended in PBS, were inoculated into mice by subcutaneous injection in a volume of 100. mu.L. Mice were anesthetized with 2-5% isoflurane prior to inoculation.

Example 3 animal grouping and dosing regimens

When the tumor grows to 67mm³At that time, 48 tumor-bearing mice were randomly divided into 6 groups of 8 mice each according to tumor volume and body weight. The day of group administration was defined as day 0. The grouping and dosing schedule are shown in table 1:

TABLE 1 grouping and dosing regimens

N number of animals per group

Dose volume the dose volume of animals was adjusted to 10. mu.L/g

Example 4 pharmaceutical formulation:

TABLE 2 drug compounding procedure

Example 5 Experimental procedures

Cage-side observation

Each mouse was observed daily for appearance and behavior, with successive observations beginning from the group. All abnormal appearance morphology and behavioral activities were recorded in the laboratory clinical observation table.

Measurement of

Cage-side observation: the appearance and behavior of each mouse was observed daily from the start of dosing. Continuous observation starts from the packet.

All abnormal appearance and behavior activities were recorded in the expanded biological laboratory clinical observation table.

Tumor volume: tumor volumes were measured twice weekly after grouping until the end of the experiment. The tumor volume (V) was calculated as follows: v ═ length x width²)/2. The Relative Tumor Volume (RTV) per mouse was calculated as: RTV-Vt/V0, where Vt is the measured volume per day and V0 is the volume at the start of treatment.

Animal body weight: mice body weights were measured and recorded twice weekly after grouping.

Experimental discontinuation and recovery dosing criteria

During the experiment, the mice were stopped and re-dosed following the following principles: stopping administration when the weight loss is more than or equal to 10%, wherein the administration stopping period is long enough to recover the weight of the mouse; only one mouse is stopped taking the medicine, and the other mice are normally taken; dosing was resumed with a weight loss of < 10%. No discontinuation of the mice occurred in this experiment.

Euthanasia and material selection

During the experiment, animals should be euthanized if any one or more of the following occurs:

1) when the tumor-bearing volume of a single animal exceeds 2000mm³；

2) Ulceration, necrosis or infection of the tumor;

3) abnormal movement or paralysis of the animal;

4) the animal's body weight decreased by more than 20% of the body weight at the time of starting the drug treatment for 3 consecutive days.

At the end of the in vivo experiment, mice were treated with CO₂Suffocating, and then taking off the cervical spine for sacrifice. Tumors were collected, weighed, photographed and discarded. Dead animals were not collected before the end of the in vivo experiment.

Statistical analysis

The results will be presented as mean ± s.e.m. The mean comparison between groups will be checked with Dunnett' smulti-compare test; for the survival analysis, the differences between groups were counted in Log-Rank (Mantel-Cox). Statistically significant differences were considered if p < 0.05.

Example 6 Experimental results and analysis

Average body weight, average body weight loss rate and animal tolerance

The Anti-PD-1 and Anti-CTLA4 single administration groups (G2 and G3) show no weight loss and are well tolerated; 80mg/kg XP101, either administered alone or in combination with anti-PD-1 or anti-CTLA4 (G4, G5 and G6), showed slight weight loss (< 5% average weight loss), indicating that the animals were well tolerated. The body weight statistics for each group of animals are shown in table 3 and fig. 1, and the body weight loss rate statistics are shown in table 4 and fig. 2.

Tumor volume and relative tumor volume

Tumor volumes were measured twice weekly after grouping until the end of the experiment. The Relative Tumor Volume (RTV) per mouse was calculated as: RTV-Vt/V0, where Vt is the measured volume per day and V0 is the volume at the start of treatment.

Compared with the G1 control group, the average tumor volume and the average relative tumor volume of the animals in the G2 group are obviously reduced at Day 11-18; the average tumor volume and the average relative tumor volume of the animals in the G3 group are not significantly different from those in the Day0-18, and the average relative tumor volume is significantly reduced from that in the Day 7; the average tumor volume of the animals in the G4 group is remarkably reduced in Day11-18, and the average relative tumor volume is remarkably reduced in Day 7-18; the average tumor volume and the average relative tumor volume of the animals in the G5 group are obviously reduced in Day 4-18; the average tumor volume and the average relative tumor volume of the animals in the G6 group were significantly reduced in Day 7-18. The statistics of tumor volumes for each group of animals are shown in table 5 and fig. 3, and the statistics of relative tumor volumes are shown in table 6 and fig. 4.

Compared with the single drug group (G2) of anti-PD-1, the average relative tumor volume of the animals of the XP101+ anti-PD-1 group (G5) is remarkably reduced in the Day11-18 (p is 0.032,0.046, 0.049; Table 6);

compared with the single drug group of XP101 (G4), the average relative tumor volume of the XP101+ anti-PD-1 group (G5) animals is obviously reduced in Day7-18 (p is 0.014,0.002,0.001, 0.001; Table 6);

compared with the single drug group of anti-CTLA-4(G3), the average relative tumor volume of the animals of the XP101+ anti-CTLA-4(G6) group is remarkably reduced in Day11-18 (p is 0.015,0.014, 0.007; Table 6);

compared with the single drug group of XP101 (G4), the average relative tumor volume of the animals of the XP101+ anti-CTLA-4(G6) group has no significant difference in Day0-18 (p > 0.05; Table 6).

Relative tumor inhibition (%)

Relative Tumor inhibition rate (TGI, Tumor growth inhibition): TGI% (1-T/C) × 100%. The evaluation standard of the curative effect is as follows: TGI% > 60% is effective, and TGI% < 40% is ineffective.

Day18 timeTGI of the anti-PD-1 single drug group, the XP101 and anti-PD-1 combined drug group and the XP101 and anti-CTLA-4 combined drug group is more than 60 percent, which proves that the anti-PD-1 combined drug group has obvious inhibition effect on tumors (Table 7).

Day18 timeCompared with an anti-PD-1 single drug group (G2) or an XP101 single drug group (G4), the XP101+ anti-PD-1 group (G5) has the relative tumor inhibition rate TGI improved to 99.52 percent from 65.83 percent or 77.42 percent; compared with the anti-CTLA-4 single drug group (G3) or the XP101 single drug group (G4), the XP101+ anti-CTLA-4 group (G6) has the relative tumor inhibition ratio TGI improved from 42.70% or 77.42% to 91.32% (Table 7).

Complete tumor Regression rate (CR%; Complete Regression)

The complete tumor regression rates for each group until Day63 are shown in table 8 below. Wherein, compared with the single anti-PD-1 medicine group, the CR% of the XP101 combined anti-PD-1 group is improved to 100% from 37.5%; compared with the XP101 single drug group, the CR% is improved from 0 to 100%.

Survival analysis

In this experiment, the tumor volume of a single animal reaches 2000mm³As endpoints, animal survival statistics were performed (results see figure 5 and table 9).

By Day63, the median survival of G1-G6 was 18, 52.5, 23, 25, >56, and 33.5 days, respectively, and the survival of groups G2-G6 was significantly prolonged compared to the G1 control group.

G5 test drug XP101+ anti-PD-1 combination group:at Day63, all 8 animals survived significantly longer than the single group of G2 Anti-PD-1 (3/8 animals survived) for survival (p ═ 0.008); compared with the single drug group of the G4 test drug XP101, the survival time of the group which is combined with the G5 Anti-PD-1 test drug XP101 is obviously prolonged (p)<0.001); shows that the combined administration shows the synergy of the antitumor effect.

G6 test drug XP101+ anti-PD-L1 combination group:at Day63, survival was extended from 23 to 33.5 days compared to the G3 Anti-CTLA-4 monotherapy group, but showed no statistical difference (p ═ 0.100); compared with the single drug group of the test drug XP101 of G4, the survival time is also prolonged to a certain extent, and the significance is not shown (p is 0.095).

Re-challenge experiment and results

In this experiment, it was observed that 4 and 8 animals of groups G2 and G5 survived when Day 56, respectively, and 3 of the groups G2 and all 8 animals of group G5 were in a state of complete tumor regression; the 11 animals were selected and re-inoculated subcutaneously at 1X 10 on the left side of each animal⁶A20 cells, seeded at a volume of 100. mu.L, in 5 females aged approximately one week

BALB/c mice were inoculated at the same location with 1X 10⁶A20 cells, seeded at a volume of 100. mu.L, and observed tumor growth to Day 98 to end the experiment.

Re-challenge test results: 5 are

The tumors of the animals can grow normally until Day 84, 5 animals

All animals reached 2000mm due to tumor volume³Occasionally euthanize. During this period, none of the 3 animals in group G2 and 7 animals in group G5 found any growth of the left tumor, while the other animal in group G5 was euthanized by regrowth of the right tumor and reached 2000mm3 at Day 77; no tumor growth was observed in either side of the experimental endpoint Day 98 in 3 animals in group G2 and 7 animals in group G5, indicating that these 10 animals developed an immunological memory in vivo due to prior treatment, thereby achieving complete immunization of Re-challenge A20 cells. The tumor growth curve is shown in FIG. 5.

Conclusion

In the experiment, a mouse B cell lymphoma A20 subcutaneous transplantation tumor model is established on a BALB/c mouse, the tumor volume of a Vehicle control group (G1) is stably increased, and the average tumor volume of D0 before administration is 69.99 +/-1.31 mm³。

Compared with the G1 control group, the tumor volume and the relative tumor volume of the animals in the G2 group are obviously reduced in Day 11-18; the tumor volume and the relative tumor volume of the animals in the G3 group are not obviously different from those of the animals in the Day0-18, and the relative tumor volume is obviously reduced in the Day 7; the tumor volume of the animals in the G4 group is remarkably reduced in Day11-18, and is remarkably reduced in relative tumor volume in Day 7-18; the tumor volume and the relative tumor volume of the animals in the G5 group are obviously reduced in Day 4-18; the tumor volume and relative tumor volume of the animals in the G6 group were significantly reduced in the Day7-18 group.

Compared with a Vehicle control group (G1), the tumor volume and relative tumor volume of animals in the G2 Vehicle + Anti-PD-1 group, 10mg/kg and QD 4/wks x 3wks + BIW x 6time group are remarkably reduced in Day11-18, and the TGI of Day18 is 65.83%;

the tumor volume and the relative tumor volume of animals in G3 Vehicle + Anti-CTLA-4,10mg/kg, QD 4/wks 3wks + BIW 6time groups have no significant difference in Day0-18, and the TGI of Day18 is 42.70%;

the tumor volume of the animals in the G4 XP101+ PBS 80mg/kg and QD 4/wks 3wks groups is remarkably reduced in Day11-21, the relative tumor volume is remarkably reduced in Day7-18, and the TGI of Day18 is 77.42%;

the tumor volume and the relative tumor volume of the animals in the G5 XP101+ Anti-PD-180 +10mg/kg group, QD 4/wks 3wks + BIW 6time group are obviously reduced in Day4-18, and the TGI of Day18 is 99.52%; compared with the XP101 single drug group or the anti-PD-1 single drug group, the average tumor volume is obviously different; and compared with the anti-PD-1 single drug group, by Day63, the significance provides the survival rate of animals (p ═ 0.008).

The tumor volume and the relative tumor volume of the animals in G6 XP101+ Anti-CTLA-480 +10mg/kg, QD 4/wks 3wks + BIW 6time groups are obviously reduced in Day7-18, and the TGI of Day18 is 91.32%; compared with the anti-CTLA-4 single drug group, the average tumor volume and the average survival time of animals are significantly different; however, compared with the single drug group of XP101, the average tumor volume and the average survival time of animals have no significant difference (p is 0.095).

Subsequent Re-challenge experiments showed that: in the case of animals with complete regression of the tumor under treatment, immune memory cells are generated in vivo and can play a long-term protective role when re-stimulated by the same tumor cells.

TABLE 7 relative tumor inhibition Rate

TABLE 8 complete tumor regression Rate CR%

G1 Vehicle+PBS	0(0/8)
		G2 Vehicle+Anti-PD-1	37.5％(3/8)
G3 Vehicle+Anti-CTLA-4	0(0/8)
		G4 XP101+PBS	0(0/8)
G5 XP101+Anti-PD-1	100％(8/8)
		G6 XP101+Anti-CTLA-4	0(0/8)

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And such obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the present invention.

Claims

1. Use of a pharmaceutical combination for the manufacture of a medicament for the prevention of recurrent lymphoma, wherein said pharmaceutical combination comprises a Pan-HDAC inhibitor and an immune checkpoint inhibitor; wherein the Pan-HDAC inhibitor is N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide or a salt thereof; the immune checkpoint inhibitor is a PD-1 or CTLA-4 antibody.

2. Use of a pharmaceutical combination according to claim 1 for the preparation of a medicament for the prevention of recurrent lymphoma, wherein said N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide salt is p-toluenesulfonate, hydrochloride, naphthalene-1, 5-disulfonate, naphthalene-2-sulfonate, oxalate, benzenesulfonate, sulfate or phosphate.

3. Use of a pharmaceutical combination according to claim 1 for the preparation of a medicament for the prevention of recurrent lymphoma, wherein the Pan-HDAC inhibitor and immune checkpoint inhibitor are present in a ratio of 5-10: 1-5.

4. Use of a pharmaceutical combination according to claim 1 for the preparation of a medicament for the prevention of recurrent lymphoma, wherein said Pan-HDAC inhibitor is the p-toluenesulfonate salt of N-hydroxy-4- {2- [3- (N, N-dimethylaminomethyl) benzofuran-2-ylcarbonylamino ] ethoxy } -benzamide; the immune checkpoint inhibitor is PD-1; the ratio of Pan-HDAC inhibitor to immune checkpoint inhibitor content was 8: 1.

5. Use of a pharmaceutical combination according to any one of claims 1 to 4 in the manufacture of a medicament for the prevention of recurrent lymphoma, wherein said pharmaceutical combination is in an oral formulation or an injectable formulation.