WO2022021786A1 - Tsl-1502 compound pharmaceutical composition - Google Patents

Abstract

A TSL-1502 compound pharmaceutical composition comprising temozolomide, cisplatin, carboplatin and irinotecan. The method for preparing the pharmaceutical composition comprises: using a desired amount of TSL-1502 and another antitumor drug together or separately as pharmaceutically active ingredients to prepare, according to conventional techniques of pharmaceutics, the pharmaceutical composition for administration. Dosage forms include injections. A study on the combined use of TSL-1502 and other drugs finds synergy when TSL-1502 is used in combination with temozolomide, cisplatin, carboplatin and irinotecan.

Description

TSL-1502 Compound Drug Combination technical field

The invention relates to a drug combination for treating tumors, particularly a compound drug combination consisting of the compound TSL-1502, temozolomide, cisplatin or carboplatin, and irinotecan.

Background technique

TSL-1502, chemical name is (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-((3-methyl-1-((S)-1-propyl-pyrrole) Alk-3-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-5-yl)oxy)tetrahydro-2H-pyran-2 - Carboxylic acid pentahydrate. TSL-1502 first appeared in the 0177 paragraph compound of Chinese patent 201180002886.8 (publication number CN102510863A, authorized announcement number CN102510863B). Its structural formula is shown in formula I.

The above patent discloses specific tumor types in claim 16, such as head cancer, thyroid cancer, neck cancer, eye cancer, skin cancer, oral cancer, throat cancer, esophagus cancer, breast cancer, bone cancer, blood cancer, bone marrow cancer, lung cancer , colon cancer, sigmoid colon cancer, rectal cancer, stomach cancer, prostate cancer, breast cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, brain cancer, colon cancer, heart cancer, adrenal cancer, subcutaneous tissue cancer, lymph node cancer, pigment cancer , malignant glioma, etc.

TSL-1502 is a novel oral poly(ADP-ribose) polymerase [PARP] inhibitor, which can inhibit PARP1, the two most important members of the PARP protein family, at very low doses It is intended to be used for the treatment of advanced solid tumors; in vitro anti-tumor mechanism studies have shown that the anti-tumor mechanism of TSL-1502 includes induction of tumor cell DNA damage, cell cycle arrest and apoptosis; The combined use of toxic and antitumor drugs can inhibit the repair of DNA damage after chemotherapy, thereby increasing the efficacy of chemotherapy drugs. In vitro and in vivo antitumor profiling studies have confirmed that TSL-1502 is more sensitive to a variety of DNA repair-deficient tumors (such as BRCA1/2 gene deficiency or mutant tumors), and is a specific antitumor and chemotherapeutic drug.

There is no report on the combined application of TSL-1502 and other drugs. The present invention studies the combined application of TSL-1502 and other drugs. It is unexpectedly found that the combined use of TSL-1502 and temozolomide, cisplatin or carboplatin and irinotecan has a synergistic effect. effect. The present invention has carried out the following experimental research for this purpose:

TSL-1502 + temozolomide combination for the treatment of melanoma; TSL-1502 + cisplatin combination for the treatment of breast cancer; TSL-1502 + carboplatin combination for the treatment of breast cancer; TSL-1502 + irisin hydrochloride Rinotecan in combination therapy for colorectal cancer.

SUMMARY OF THE INVENTION

The present invention provides a compound drug combination containing TSL-1502 and another anti-tumor drug, wherein the other anti-tumor drug is selected from temozolomide, cisplatin, carboplatin and irinotecan.

The present invention includes a pharmaceutical preparation composition containing two active ingredients of TSL-1502 and another anti-tumor drug, or a separate pharmaceutical preparation containing TSL-1502 and another anti-tumor drug, and the combination of the two preparations packaged together .

The preparation of the pharmaceutical preparation composition of the present invention includes the step of mixing TSL-1502 and another antitumor drug.

Or, the preparations containing TSL-1502 and another antitumor drug are separately prepared into drug preparations, and the two preparations are combined and packaged together, and the two drugs can be conveniently used in combination. As described in the combination packaging, TSL-1502 can be prepared into injections, and another anti-tumor drug can be prepared into injections. The two unit doses of injections are packaged together in the same packaging box, and can be injected separately or together when used.

The combination of TSL-1502 and another anti-tumor drug compound drug of the present invention, wherein the dose of each drug adopts a drug effective amount, and the "effective amount" means that each drug can be used alone or in combination. An amount that is clinically effective in preventing or treating a disease. If it is prepared as an injection, it is in the form of a unit dose. For example, it is divided into sterile vials of different colors and sizes, each containing 0.1-2000 mg of active pharmaceutical ingredients. After the vials are sealed, they are placed in the packaging box; Comes in boxes that can hold 2-100 bottles for easy storage and transportation.

The combination of the present invention comprises TSL-1502 and another antitumor drug, and the weight ratio of the two can be formulated according to their respective effective amounts, such as 1:1000 to 1000:1.

Further, the combination of the present invention comprises TSL-1502 and temozolomide in a ratio of effective amount (5-60):(10-100). Preferred (10-40): (40-60); most preferred (10-30): 50. The composition is used for the treatment of melanoma.

Further, the combination of the present invention comprises TSL-1502 and cisplatin in an effective amount (5-50):(2-10) in a ratio. Preferred (5-40): (2-8); most preferred (6.25-25): 6. The composition is used for the treatment of breast cancer, especially human breast cancer type MX-1.

Further, the combination of the present invention comprises TSL-1502 and carboplatin in an effective amount (10-90):(5-100) in a ratio. Preferably (20-70):(50-70), most preferably 25-50:60. The combination is used to treat breast cancer, particularly human breast cancer type MX-1.

Further, the combination according to the present invention comprises TSL-1502, irinotecan hydrochloride (Kipto) according to the ratio of effective amount (5-100): (5-30) 10, preferably (5-60): (5- 20), most preferably (5-50): 10. This combination is used to treat colorectal cancer.

The preparation method of the pharmaceutical combination of the present invention comprises the steps of preparing a pharmaceutical composition that can be taken according to the conventional technology of preparation, containing the required amount of TSL-1502 and another anti-tumor drug together or separately as active pharmaceutical ingredients. , and its dosage forms include injections, preferably dry powder injections, particularly preferably freeze-dried injections.

The injection of the present invention can be prepared without adding auxiliary materials or adding one or more pharmaceutical auxiliary materials, such as: glucose, lactose, mannitol, sodium chloride, hydroxypropyl-B-cyclodextrin, etc., and then using an appropriate method injection.

The use method of the present invention includes preparing an injection containing a required amount of TSL-1502 and another antitumor drug together as two active pharmaceutical ingredients for injection together. The required amount of TSL-1502 and another antitumor drug can also be made into injections and used separately.

The pharmaceutical preparation composition of the present invention can be in any ingestible pharmaceutical form: such as: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral Liquids, buccal preparations, granules, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches .

The pharmaceutical preparation composition of the present invention is preferably in the form of a unit dose pharmaceutical preparation. For example, when a pharmaceutical preparation is prepared, the unit dose of the pharmaceutical preparation may contain the TSL-1502 of the present invention, and another antitumor drug 0.1-1000 mg, and the rest It is a pharmaceutically acceptable excipient. The pharmaceutically acceptable adjuvants can be 0.01-99.99% by weight of the total weight of the formulation.

The dosage of the pharmaceutical preparation composition of the present invention is determined according to the patient's condition, such as 1-3 times a day. 1-20 tablets at a time, etc.

Preferably, the pharmaceutical preparation composition of the present invention is an oral preparation or an injection. Wherein, the oral preparation is selected from capsules, tablets, dropping pills, granules, concentrated pills, and oral liquids. Wherein, the injection is selected from liquid, semi-solid, solid, powder form, preferably injection, powder injection.

The pharmaceutical preparation composition of the present invention, its preparation for oral administration may contain adjuvants such as binders, fillers, diluents, tableting agents, lubricants, disintegrating agents, coloring agents, flavoring agents and wetting agents, as necessary Tablets may be coated.

Suitable fillers include cellulose, mannitol, lactose and other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable humectants include sodium lauryl sulfate.

The pharmaceutical preparation of the present invention can be prepared as a solid oral composition by mixing, filling, tableting and other common methods. Repeated mixing allows the active to be distributed throughout those compositions where large amounts of fillers are used.

Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, Emulsifiers such as lecithin, sorbitan monooleate or acacia; non-aqueous carriers (which may include edible oils) such as almond oil, fractionated coconut oil, oily esters such as glycerol esters, propylene glycol or ethanol; preservatives agents such as methylparaben or propylparaben or sorbic acid and, if desired, conventional flavoring or coloring agents.

For injections, liquid unit dosage forms are prepared containing the active substance of the present invention and a sterile carrier. Depending on the carrier and concentration, the compound can be suspended or dissolved. Solutions are generally prepared by dissolving the active material in a carrier, filter sterilizing before filling into a suitable vial or ampoule, and sealing. Adjuvants such as a local anesthetic, preservatives and buffering agents can also be dissolved in the carrier. To improve its stability, the composition can be frozen after filling into a vial and the water removed under vacuum.

The present invention further provides the application of the composition of the present invention in the preparation of anti-tumor drugs. In the application, the dosage of TSL-1502 and another anti-tumor drug can be 0.01 mg-2000 mg per day. The tumor is selected from breast cancer, melanoma, colorectal cancer.

The application of the present invention has achieved unexpected technical effects, and the relevant application examples are shown in the experimental examples of the present invention.

Description of drawings

Figure 1. Efficacy of TSL-1502 and AZD2281 alone or in combination with carboplatin on subcutaneous xenografts of human breast cancer MX-1 nude mice;

Figure 2. The effect of TSL-1502, AZD2281 alone or in combination with carboplatin on the body weight of tumor-bearing nude mice, mean ± SD; solvent, n=12, treatment group, n=6;

Figure 3. Efficacy of TSL-1502 and AZD2281 alone or in combination with carboplatin on the subcutaneous xenograft of human breast cancer MX-1 nude mice (tumor photos);

Figure 4. TSL-1502, AZD2281 pair

The synergistic effect of treating human colorectal cancer SW620 nude mice subcutaneously transplanted tumor;

Figure 5. TSL-1502, AZD2281 alone or with

The effect of combined use on the body weight of tumor-bearing nude mice, mean±standard deviation; solvent group, n=12, treatment group, n=6;

Figure 6. TSL-1502, AZD2281 pair

The synergistic effect of treating human colorectal cancer SW620 nude mice subcutaneously transplanted tumor (tumor photo);

Figure 7. The synergistic effect of HD199, veraparib and cisplatin in the treatment of human breast cancer MX-1 nude mice subcutaneously transplanted tumor-mean tumor volume (mm ³ );

Figure 8. The synergistic effect of HD199, veraparib and cisplatin in the treatment of human breast cancer MX-1 nude mice subcutaneously transplanted tumor-relative tumor volume RTV (%);

Figure 9. Effects of HD199, veraparib and cisplatin on body weight (BW, g) of human breast cancer MX-1 nude mice;

Figure 10. The synergistic effect of HD199, veraparib and temozolomide in the treatment of human melanoma B16F10 subcutaneously transplanted tumor in nude mice-mean tumor volume (mm ³ );

Figure 11. The synergistic effect of HD199, veraparib and temozolomide in the treatment of human melanoma B16F10 nude mice subcutaneously transplanted tumor-relative tumor volume RTV (%);

Figure 12. The synergistic effect of HD199, veraparib and temozolomide on the treatment of human melanoma B16F10 subcutaneously transplanted tumor in nude mice-relative tumor proliferation rate T/C (%).

detailed description

The following examples further illustrate the present invention, but are not intended to limit the present invention.

Example 1

The invention provides a compound drug freeze-dried injection composed of TSL-1502 and another anti-tumor drug, temozolomide, cisplatin, carboplatin and irinotecan.

Preparation example:

Take 20 g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and 10 g of irinotecan, and dissolve them in 1000 ml of aqueous solution containing 100 g of mannitol, respectively, and fill them into 2 g vials. A total of 500 bottles were placed in a freeze-drying box before capping, vacuum-dried for 24 hours, capped, and 1 bottle of each was combined and packaged.

Example 2

Take 5g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and irinotecan 10g, dissolve together in 1000ml of aqueous solution containing 100g of mannitol, mix well, and canned in a 2g vial , a total of 500 bottles, before capping, placed in a freeze-drying box, vacuum-dried for 24 hours, capped, and packaged.

or

Take 5g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, 20g of irinotecan, and dissolve them together in 1000ml of aqueous solution containing 100g of mannitol, mix them evenly, and put them in a 2g vial. , a total of 500 bottles, before capping, placed in a freeze-drying box, vacuum-dried for 24 hours, capped, and packaged.

or

Take 5g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, 50g of irinotecan, and dissolve them together in a 1000ml aqueous solution containing 100g of mannitol, mix well, and canned in a 2g vial , a total of 500 bottles, before capping, placed in a freeze-drying box, vacuum-dried for 24 hours, capped, and packaged.

or

Take 5g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, 100g of irinotecan, dissolve together in 1000ml of aqueous solution containing 100g of mannitol, mix well, and canned in a 2g vial , a total of 500 bottles, before capping, placed in a freeze-drying box, vacuum-dried for 24 hours, capped, and packaged.

or

Take 10g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, 5g of irinotecan, and dissolve them together in a 1000ml aqueous solution containing 100g of mannitol, mix well, and canned in a 2g vial , a total of 500 bottles, before capping, placed in a freeze-drying box, vacuum-dried for 24 hours, capped, and packaged.

or

Take 20g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, 5g of irinotecan, and dissolve them together in a 1000ml aqueous solution containing 100g of mannitol, mix well, and canned in a 2g vial , a total of 500 bottles, before capping, placed in a freeze-drying box, vacuum-dried for 24 hours, capped, and packaged.

or

Take 50g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and 5g of irinotecan, and dissolve them together in a 1000ml aqueous solution containing 100g of mannitol, mix well, and canned in a 2g vial , a total of 500 bottles, before capping, placed in a freeze-drying box, vacuum-dried for 24 hours, capped, and packaged.

or

Take 100g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, 5g of irinotecan, and dissolve them together in a 1000ml aqueous solution containing 100g of mannitol, mix well, and canned in a 2g vial , a total of 500 bottles, before capping, placed in a freeze-drying box, vacuum-dried for 24 hours, capped, and packaged.

Example 3

Take 2g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and 1g of irinotecan, respectively, dissolve them in 1000ml of aqueous solution containing 100g of mannitol, and fill them into 2g vials, respectively. A total of 500 bottles were placed in a freeze-drying box before capping, vacuum-dried for 24 hours, capped, and 1 bottle of each was combined and packaged.

Example 4

Take TSL-1502 10g, and optionally any one of the following drugs temozolomide, cisplatin, carboplatin, irinotecan 5g, dissolve in 1000ml aqueous solution containing 100g mannitol respectively, canned in 2g vials, respectively. A total of 500 bottles were placed in a freeze-drying box before capping, vacuum-dried for 24 hours, capped, and 1 bottle of each was combined and packaged.

Example 5

Take 5g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and 2.5g of irinotecan, respectively, dissolve them in 1000ml aqueous solution containing 100g of mannitol, and fill them into 2g vials. A total of 500 bottles were placed in a freeze-drying box before capping, vacuum-dried for 24 hours, capped, and 1 bottle of each was combined and packaged.

Example 6

Take 5g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and 5g of irinotecan, respectively, dissolve them in 1000ml aqueous solution containing 100g of mannitol, and fill them into 2g vials, respectively. A total of 500 bottles were placed in a freeze-drying box before capping, vacuum-dried for 24 hours, capped, and 1 bottle of each was combined and packaged.

Example 7

Take 50g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and 5g of irinotecan, and dissolve them in 1000ml of aqueous solution containing 100g of mannitol, respectively, and fill them into 2g vials. A total of 500 bottles were placed in a freeze-drying box before capping, vacuum-dried for 24 hours, capped, and 1 bottle of each was combined and packaged.

Example 8

Take TSL-15025g, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and irinotecan 50g, dissolve them in 1000ml aqueous solution containing 100g mannitol, respectively, and canning them in 2g vials. 500 bottles, before capping, put them in a freeze-drying box, vacuum dry for 24 hours, seal caps, take 1 bottle each for combined packaging, and that's it.

Example 9

Take 5 g of TSL-1502, and optionally any one of the following drugs, temozolomide, cisplatin, carboplatin, and 10 g of irinotecan, respectively, dissolve them in 1000 ml of aqueous solution containing 100 g of mannitol, and fill them into 2 g vials, respectively. A total of 500 bottles were placed in a freeze-drying box before capping, vacuum-dried for 24 hours, capped, and 1 bottle of each was combined and packaged.

The present invention is further illustrated by experimental data below.

Experimental example 1

Efficacy of TSL-1502 alone or in combination with carboplatin on subcutaneous xenografts of human breast cancer MX-1 nude mice

3 test drugs

Drug name and batch number: TSL-1502 is a white powder with a purity of 99.56% and a water content of 16.65%, batch number 120301; AZD2281 is a white powder with a purity of 99.15%, batch number 20131105; carboplatin is a white powder with a purity of 100.0%, batch number C20141208. Provided by: TSL-1502 and AZD2281 ( Olapali )

All were provided by the Pharmacology and Toxicology Research Center of the Research Institute of Tasly Pharmaceutical Group Co., Ltd.; AZD2281 was purchased from Shanghai Demo Company; carboplatin was provided by this laboratory, from Kunming Institute of Precious Metals.

Preparation method: TSL-1502 is prepared with distilled water and diluted to the corresponding concentration; AZD2281 is prepared and diluted with 3% DMA + 20% PEG 400 + 20% propylene glycol + 57% normal saline, and used as a "solvent"; Carboplatin is prepared with 5% glucose The solution is prepared extemporaneously.

4 Laboratory animals

BALB/c nude mice, 6-7 weeks, ♀, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. Production license number: SCXK (Shanghai) 2012-0002; animal certificate number 2015000526837. Breeding environment: SPF grade.

5 Model and basis for dose selection

Referring to the in vivo efficacy studies of the FDA-approved PARP inhibitors Olaparib (LYNPARZA), Rucaparib (RUBRACA) and Niraparib (ZEJULA) [2-4], and the experimental results in Chapter 5, human breast cancer MX-1 was selected Nude mice subcutaneously transplanted tumor model and administration dose of TSL-1502 (25, 50 mg/kg, PO, BID×14).

6 Experimental steps

Nude mice were subcutaneously inoculated with human breast cancer MX-1 tumor tissue. After the tumor grew to 100-200 mm ³ , the animals were randomly divided into groups (D0). The dosage and dosing schedule are shown in Table 1.

7 results

As shown in Table 1 and Figures 1-3, TSL-1502 (25, 50 mg/kg, PO, BID×14) dose-dependently inhibited the growth of subcutaneously transplanted tumors in nude mice of human breast cancer MX-1, and the tumor inhibition rate 71% and 91% respectively (D21); AZD2281 (25, 50mg/kg, PO, BID×14) was also effective against MX-1, and the tumor inhibition rates were 42% and 72% (D21), of which 50mg/kg 2/6 of the tumors in the group regressed partially; TSL-1502 had a stronger effect on MX-1 than AZD2281, and there was a statistically significant difference between the 25 mg/kg dose group (P<0.05), but the 50 mg/kg dose group compared There was no statistically significant difference (P=0.057).

The tumor inhibition rate of carboplatin (60mg/kg, IP, Q4D×3) against MX-1 was 149%, with 2/6 partial tumor regression and 3/6 complete tumor regression (D21) until the end of the experiment (D37) , there are still 2/6 tumors partially regressed and 1/6 tumors completely regressed; TSL-1502 25mg/kg combined with carboplatin 60mg/kg, the tumor inhibition rate was significantly improved, from 149% with carboplatin alone to 200%, All tumors (6/6) regressed completely. When AZD2281 25mg/kg and carboplatin 60mg/kg were used in combination, all tumors regressed completely (D21); by the end of the experiment (D37), no tumor recurrence was found in the combination group; TSL-1502 or AZD2281, the curative effect of the combination group was significantly stronger than that of the carboplatin single group (P<0.05), indicating that the two drugs have obvious synergistic effects on carboplatin. Tumor-bearing mice can tolerate the above drugs well. Among them, the maximum body weight of TSL-1502 combined with carboplatin group decreased by 3.8% (D6), and the maximum body weight of AZD2281 combined with carboplatin group decreased by 9.3% (D9). Body weight can be gradually recovered; other drugs did not cause significant weight loss in mice. In comparison, the efficacy of TSL-1502 on MX-1 was significantly stronger than that of the reference drug AZD2281; both drugs could significantly enhance the efficacy of carboplatin in the treatment of MX-1.

Table 1. Efficacy of TSL-1502 and AZD2281 alone or in combination with carboplatin on subcutaneous xenografts of human breast cancer MX-1 nude mice.

D0: time of first administration; P value refers to comparison with solvent; *P<0.05, compared with AZD2281 25 mg/kg; Student's t-test was used.

Table 1-2 King's formula to calculate the Q value of the combined effect of TSL-1502 and carboplatin (Q≥1.15 is synergistic)

Figure 1. Efficacy of TSL-1502 and AZD2281 alone or in combination with carboplatin on subcutaneous xenografts of human breast cancer MX-1 nude mice

Figure 2. Effects of TSL-1502, AZD2281 alone or in combination with carboplatin on body weight of tumor-bearing nude mice. Mean ± SD; solvent, n=12, treatment group, n=6.

Figure 3. The efficacy of TSL-1502 and AZD2281 alone or in combination with carboplatin on the subcutaneous xenograft of human breast cancer MX-1 nude mice (tumor photos).

Experimental example 2

The synergistic effect of TSL-1502 on irinotecan hydrochloride in the treatment of human colorectal cancer SW620 nude mice subcutaneously transplanted

3 test drugs

Drug name and batch number: TSL-1502 is white powder, purity 99.56%, water content 16.65%, batch number 120301; AZD2281 is white powder, purity 99.15%, batch number 20131105;

(Irinotecan hydrochloride injection), a pale yellow clear liquid, 2ml: 40mg/bottle, batch number JM85B, production date: 2015/02, validity period: 2018/01.

Provided by: TSL-1502 and AZD2281 were provided by the Pharmacology and Toxicology Research Center of the Research Institute of Tasly Pharmaceutical Group Co., Ltd.; AZD2281 was purchased from Shanghai Demo Company;

Provided by this laboratory, produced by Pfizer.

Preparation method: TSL-1502 is prepared with distilled water and diluted to the corresponding concentration; AZD2281 is prepared and diluted with 3%DMA+20%PEG 400+20%propylene glycol+57%physiological saline;

Dilute with normal saline.

4 Laboratory animals

BALB/cA-nude nude mice, 5-6 weeks, ♀, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Production license number: SCXK (Shanghai) 2013-0018; animal certificate number 2013001817948. Breeding environment: SPF grade.

5 Model and basis for dose selection

Refer to the in vivo pharmacodynamic doses of FDA-approved PARP inhibitors Olaparib (olaparib), Rucaparib (rucaparib) and Niraparib (niraparib) [2-4], and the experimental results in Chapter 5 , selected human colorectal cancer SW620 nude mice subcutaneously transplanted tumor model and TSL-1502 (50mg/kg, PO, BID×17) and

(10mg/kg, IP, D0,4) dosing regimen for combined use.

6 Experimental steps

Nude mice were subcutaneously inoculated with human colorectal cancer SW620 cells, and when the tumors grew to 100-200 mm ³ , the animals were randomly divided into groups (D ₀ ). The dosage and dosing schedule are shown in Table 2.

7 results

As shown in Table 2 and Figures 4-5, TSL-1502 (50mg/kg, PO, BID×17) had no obvious inhibitory effect on the growth of human colorectal cancer SW620 nude mice subcutaneously transplanted tumor, and the tumor inhibition rate was - 5%; AZD2281 (30mg/kg, PO, QD×17) also had no significant effect on SW620, and the tumor inhibition rate was 6%;

(10mg/kg, IP, D0,4) had a tumor inhibition rate of 47% on SW620; TSL-1502 (5, 15, 50mg/kg, PO, BID×5) had

Significantly enhanced

On the efficacy of SW620, the tumor inhibition rates were

47% at 79%, 82% and 89% (P<0.05 or P<0.01, compared

Comparison), while the toxicity did not increase significantly; AZD2281 (30mg/kg, PO, QD×5) also significantly increased the efficacy

For the efficacy of SW620, the tumor inhibition rate increased to 89%, and 1/6 of the tumors partially regressed. The tumor-bearing mice were able to tolerate the above drugs well, and there was no obvious symptoms such as weight loss. The results showed that according to the current dosing regimen, TSL-1502 and AZD2281 alone had no obvious effect on the subcutaneous xenograft of SW620 nude mice, but significantly increased the effect.

The therapeutic effect of SW620 was treated without a significant increase in toxicity.

Table 2. TSL-1502, AZD2281 pair

Synergy in the treatment of subcutaneous xenografts of human colorectal cancer SW620 nude mice.

D0: time of first administration; P value refers to the comparison with solvent, #P<0.05;**P<0.01,*P<0.05 compared with solvent

10mg/kg group comparison; all use Student's t test. Number of mice at the start of the experiment: n=12 in the solvent group, n=6 in the treatment group.

Table 2-2 Kim's formula to calculate the Q value of the combined effect of TSL-1502 and Captopro (Q≥1.15 is synergistic)

Figure 4. TSL-1502, AZD2281 pair

Synergistic effect on the treatment of human colorectal cancer SW620 subcutaneously transplanted tumor in nude mice

Figure 5. TSL-1502, AZD2281 alone or with

The effect of combined use on the body weight of tumor-bearing nude mice. Mean ± SD; solvent group, n=12, treatment group, n=6.

Figure 6. TSL-1502, AZD2281 pair

The synergistic effect of treating human colorectal cancer SW620 nude mice subcutaneously transplanted tumor (tumor photo).

Experimental example 3

A report on the pharmacodynamics of TSL-1502 combined with cisplatin in human breast cancer MX-1 xenograft model

2.1 Cell line:

The human breast cancer cell line MX-1 was purchased from the Cell Bank of the Chinese Academy of Sciences;

2.2 Reagents

Fetal bovine serum (Gibco, Lot NO: 623311), RPM1640 medium (Gibco, Lot NO: 869317), L15 medium (Gibco, Lot NO: 810875), trypsin (Sigma Lot NO: 27250018), penicillin G sodium salt (Shandong Lukang Medicine Lot NO: B100122), Streptomycin Sulfate (Shandong Lukang Medicine Lot NO: 080307), 0.25% EDTA (Trypsin (trypsin) Lot NO: 25200056), Hanks (NO 14170112), NaHCO ₃ ( Chengdu Kelong Lot NO. 20101013), HCL (Nanjing Chemical Reagent Co., Ltd.), Matrigel (Matrix) (BD Lot NO: 20110068), Trypan Blue, DMSO, PEG400, Tw-80, 0.9% Sodium Chloride Solution, PBS Powder bag (or NaCl, KCl, NaH2PO4, KH2PO4) and so on.

2.3 Instruments:

5% CO2 cell incubator (ThermoForma, USA), ultra-clean workbench (Suzhou purification), alcohol cotton ball, alcohol lamp, dropper, culture flask, centrifuge tube, cryopreservation tube, glass bottle (for medium), Cell counting plate, coverslip, cell culture dish, cell culture dish, pressure sterilizer, inverted microscope, centrifuge, liquid nitrogen, autoclave, electronic balance, pH meter, vernier caliper, magnetic stirrer, 100mL volumetric flask, 5mL volumetric flask, 1000mL volumetric flask, 1mL syringe, gavage needle, centrifuge tube

2.4 Animals:

Balb/c female nude mice (SPF grade), female, 4-6 weeks old, weighing 18-22 g, 60 mice were purchased from Shanghai Sipple-Bikai Laboratory Animal Co., Ltd. The animal production license number is Scxk (Shanghai) 2008-0016, and the qualification certificate number is 2010001601905.

2.5 Test drug:

Test drug: TSL-1502, synthesized in the laboratory of Suzhou Henderson Pharmaceutical Technology Co., Ltd. (batch number: 100301), dissolved in physiological saline; DDP (cisplatin): purchased from Shandong Boyuan Pharmaceutical Technology Co., Ltd., dissolved in physiological saline;

Veliparib CAS: 912444-00-9, purchased from Shanghai Haoyuan Chemical Technology Co., Ltd., batch number: HM-0698-20101112, dissolved in normal saline

3 Methods Methodology

3.1 Cell Culture:

After the MX-1 cell line was purchased, it was frozen at -80°C for future use, and the freezing medium was fetal bovine serum: DMSO=9:1. After cell recovery, cells were routinely cultured in RPMI-1640 complete medium (containing 10% fetal bovine serum, 1% (W/V) penicillin and 1% (W/V) streptomycin, and fetal bovine serum required imported ), placed in a 5% CO ₂ incubator at 37°C, relative saturated humidity, observed the growth status daily, adhered to the wall for 2-3 days, passaged for one generation, passage expansion, collected exponentially proliferating cells with 0.25% trypsin + 0.53 Digest with mMol/L of EDTA, collect in incomplete culture medium without calf serum and shake gently to prepare cell suspension.

3.2 Construction of tumor-bearing mouse model:

Balb/c female nude mice (SPF grade), it is a hairless, athymic congenital T cell immunodeficiency animal, lacking immune rejection, the tumor grows well after transplantation, and can maintain the original shape and cell dynamics of tumor cells It is an ideal and commonly used animal in tumor research. It is 4-6 weeks old, weighs 18-22g, and has 60 animals. The skin of the inoculation site is disinfected with 75% alcohol. The inoculation site of ectopic tumor is In the groin of the left lower extremity, the orthotopic tumor inoculation site was the fat pad of the left second nipple of nude mice (as shown in the figure below), the cell inoculation cell count was 0.5×10 ⁷ /ml, and the cell inoculation dose was 0.2ml respectively (1×10 ⁶ cells), the cell line and 50% matrix were mixed and inoculated (adding a little more matrix helps the tumor to grow in a regular shape), observe the tumor growth every day, and record the tumor size (length, width) volume (length x width ² / 2), calculate the standard deviation, about 14-18 days after inoculation, the tumor tissue should grow to be about 100mm ³ .

3.3 Solution preparation

Blank solution: add 0.9 g of NaCl to a 100 mL volumetric flask, dissolve with distilled water, shake to prepare a 0.9% NaCl solution, and store at room temperature for later use.

Cisplatin solution: 6 mg of cisplatin (DDP) sample was accurately weighed and placed in a 100 ml volumetric flask, and 0.9% NaCl solution was used to make a DDP solution with a concentration of 0.6 mg/ml.

Veliparib solution: Accurately weigh 25 mg of veraparib sample into a 100-ml volumetric flask, and use 0.9% NaCl solution to make a veraparib solution with a concentration of 2.5 mg/ml.

TSL-1502 solution: Accurately weigh 25, 12.5, and 6.25 mg of compound TSL-1502 samples respectively, place them in a 100-ml volumetric flask, and use 0.9% NaCl solution to dissolve into 2.5, 1.25, and 0.625 mg/ml respectively. solution of TSL-1502.

3.4 Grouping and dosing schedule of tumor-bearing mice:

Grouping and dosing schedule of tumor-bearing mice: nude mice with tumors growing to about 100 mm ³ (about 14 days), excluding nude mice with larger or smaller tumors, and selecting nude mice with better tumor size consistency , start random grouping into the following 6 groups, each group should ensure at least 6 animals.

The blank control group (0.1ml/10g, po (oral), Bid (twice a day)), after grouping, were given oral gavage of normal saline for 9 consecutive days, and intraperitoneal injection of normal saline was administered on the 3rd and 6th days respectively. , 40 days of continuous observation;

Cisplatin single-administration group (6 mg/kg, ip (intravenous injection), qd (once a day)), after grouping, oral administration of normal saline for 9 consecutive days, of which the 3rd and 6th days were given respectively. Intraperitoneal injection of cisplatin solution, continuous observation for 40 days;

Veraparib administration group (25 mg/kg, po (oral), Bid (twice a day)) + cisplatin (6 mg/kg, ip (intravenous injection), qd (once a day)), after grouping, Continuous administration of veraparib solution by oral gavage for 9 days, in which the intraperitoneal injection of cisplatin solution was administered on the 3rd and 6th days respectively, and the observation was continued for 40 days;

The TSL-1502 administration group (6.25mg/kg, po, Bid) + cisplatin (6mg/kg, ip, qd) was administered orally orally with TSL-1502 solution for 9 consecutive days, of which the 3rd and 6th days were given Intraperitoneal injection of cisplatin solution, continuous observation for 40 days;

The TSL-1502 administration group (12.5 mg/kg, po, Bid) + cisplatin (6 mg/kg, ip, qd) was continuously administered orally orally with TSL-1502 solution for 9 days, of which the 3rd and 6th days were given Intraperitoneal injection of cisplatin solution, continuous observation for 40 days;

The TSL-1502 administration group (25mg/kg, po, Bid) + cisplatin (6mg/kg, ip, qd) was continuously administered with oral gavage of TSL-1502 solution for 9 days, of which the 3rd and 6th days were administered respectively Intraperitoneal injection of cisplatin solution, continuous observation for 40 days;

3.5 Detection indicators and methods:

3.5.1 Tumor volume and tumor growth curve:

The tumor volume of nude mice was detected every other day. Measurement method of tumor volume: measure the length and width of the tumor tissue with a vernier caliper (assuming that the tumor tissue is oval), and calculate the volume using the formula of length × width 2/2 to calculate tumor inhibition. Rate and relative tumor volume (RTV), Vt: tumor volume obtained by measuring the tumor every day, V0: initial tumor volume (before administration);

3.5.2 Relative tumor volume (RTV), the calculation formula is:

RTV=V _t /V _initial

Where V _initial is the tumor volume measured at the time of group administration, and V _t is the tumor volume at each measurement.

3.5.3 Relative tumor proliferation rate T/C (%), the calculation formula is:

T/C(%)=(T _RTV /C _RTV )×100%

3.5.4 Tumor volume inhibition rate (GI)

GI=[1−(TV _t −TV _initial )/(CV _t −CT _initial )]×100%

where TV _t represents the tumor volume at each measurement in the treatment group;

TV _initially represents the tumor volume measured by the treatment group at the time of group administration;

CV _t represents the tumor volume of the control group at each measurement;

CT _initially represents the tumor volume measured in the control group at the time of group administration;

3.5.5 Body weight change curve of tumor-bearing mice:

Measure the body weight of the mice while measuring the volume of the tumor-bearing mice, draw the weight change curve of the tumor-bearing mice, observe the change trend of the body weight of the tumor-bearing mice, and evaluate the change of the compound on the body weight of the tumor-bearing mice;

3.6 Statistical analysis:

The measurement data such as tumor volume, tumor weight and tumor-bearing mice were calculated using

Expressed; using Excel software or SPSS 12.0 software package for statistical analysis, statistical method using t test.

3.7 Analysis of experimental results

3.7.1 Analyze the tumor volume of each administration group after administration, and draw the time-volume change pharmacodynamic curve;

3.7.2 Analyze the relative tumor proliferation rate of each administration group after administration, and draw the time-tumor relative proliferation rate change curve;

3.7.3 Analysis of tumor size inhibition in each administration group after administration, and calculation of tumor inhibition rate;

3.7.4 Analyze the weight of tumor-bearing mice in each administration group after administration, and draw a weight-time curve;

4Results

4.1 The effect of TSL-1502 combined with DDP on the tumor inhibition rate of human breast cancer MX-1 xenografted tumor-bearing mice:

Compared with the blank control group, on the 26th day of inoculation, the tumor inhibition rate of cisplatin (6mg/kg) alone reached 100.33%, and on the 50th day of inoculation, TSL-1502 (6.25, 12.5, 25mg/kg) combined with cisplatin The tumor inhibition rates relative to cisplatin were 32%, 82%, and 86%, respectively. The same dose of veraparib (25 mg/kg) combined with cisplatin had a tumor inhibition rate of 61%, which was significantly lower than that of TSL-1502 (P< 0.01), see Table 3 and Figure 7 for details.

Table 3. Effect of TSL-1502 or veraparib in combination with carboplatin on tumor inhibition rate (GI)

Compared with blank control group, *p<0.05, **p<0.01; compared with DDP group control: #P<0.05, ##P<0.01.

4.2 The effect of TSL-1502 combined with DDP on the relative proliferation rate (T/C, %) of human breast cancer MX-1 xenografted tumor-bearing mice: compared with the blank control group, on the d26th day of inoculation, cisplatin ( 6mg/kg) relative tumor proliferation rate reached T/C (%) 1.34%, and on the 50th day of inoculation, TSL-1502 (6.25, 12.5, 25mg/kg) combined with cisplatin and the relative T/C% value of cisplatin They were 67.24%, 19.60% and 15.50%, respectively. The T/C% value of veraparib (25 mg/kg) combined with cisplatin at the same dose was 39.10% relative to cisplatin. For the human cancer xenograft tumor model, the relative tumor proliferation rate T/C (%) is recommended as the test evaluation index. In principle, the evaluation criteria are: T/C(%)>40% is invalid; T/C(%)≤40%, and P<0.05 is effective after statistical processing. From the results, compared with cisplatin alone, TSL-1502 combined with cisplatin has a very significant inhibitory effect on breast cancer tumors.

Table 4. Changes in relative tumor proliferation rate (T/C%) of tumor-bearing mice during administration

Table 5. Summary of tumor inhibition rate (GI, %) and relative tumor proliferation rate (T/C, %) of tumor-bearing mice during administration

Compared with the Control group, *p<0.05, **p<0.01; compared with the DDP group: #P<0.05, ##P<0.01;

4.4 The effect of TSL-1502 combined with DDP on the body weight of tumor-bearing mice:

Compared with the blank control, TSL-1502 combined with DDP had a certain effect on the weight gain of tumor-bearing mice at the initial stage of administration, but after continuous administration for more than 40 days, there was no significant body weight of the animals in the combined administration group of each dose compared with the DDP alone administration group. difference (P>0.05). See Table 6 and Figure 9. It shows that the toxicity of TSL-1502 combined with DDP does not increase significantly compared with DDP alone.

Table 6. Changes in mean body weight (BW) of tumor-bearing mice during administration

group	d14	d16	d18	d20	d22	d24	d26	d28	d30	d32
blank	19.28	20.06	20.13	21.10	22.28	22.69	23.05
Control group, DDP (6mg/kg)	19.99	20.79	20.28	19.84	18.24	19.20	19.04	19.60	20.24	20.93
Veraparib (25mg/kg) + DDP (6mg/kg)	20.23	20.60	20.29	19.78	19.73	19.75	19.51	19.19	19.86	19.96
TSL-1502(6.25mg/kg)+DDP(6mg/kg)	19.74	20.26	19.90	19.19	18.98	19.04	19.29	19.55	19.75	19.98
TSL-1502(12.5mg/kg)+DDP(6mg/kg)	19.53	19.03	17.79	16.44	16.00	16.03	16.10	16.28	16.61	16.55
TSL-1502(25mg/kg)+DDP(6mg/kg)	20.81	20.44	18.75	17.11	16.58	16.38	16.20	16.08	16.33	16.70
group	d34	d36	d38	d40	d42	d44	d46	d48	d50
blank
Control group, DDP (6mg/kg)	21.31	21.94	22.51	22.84	23.08	23.54	24.01	25.22	26.08
Verapat (25mg/kg)+DDP (6mg/kg)	19.94	19.89	20.19	20.49	20.61	20.19	20.16	20.69	20.97
TSL-1502(6.25mg/kg)+DDP(6mg/kg)	20.29	20.70	21.19	21.64	22.78	23.41	24.33	24.79	25.74
TSL-1502(12.5mg/kg)+DDP(6mg/kg)	16.46	16.46	16.99	17.26	18.16	19.51	21.29	22.83	24.28
TSL-1502(25mg/kg)+DDP(6mg/kg)	16.48	16.50	17.03	17.19	18.09	18.89	20.89	22.53	23.80

.references:

[1]. Cherrie K. Donawho, Yan Luo, et al. ABT-888, an Orally Active Poly (ADP-Ribose) Polymerase Inhibitor that Potentiates DNA-Damaging Agents in Preclinical Tumor Models. Clin Cancer Res 2007; 13(9) May 1, 2007.

Experimental example 4

A report on the pharmacodynamics of TSL-1502 combined with TMZ (temozolomide) in a B16F10 melanoma xenograft model

2.1 Cell line:

The melanoma cell line B16F10 was mainly purchased from the Chinese Academy of Sciences Cell Bank;

2.2 Reagents

Fetal bovine serum (Gibco, Lot NO: 623311), RPM1640 medium (Gibco, Lot NO: 869317), L15 medium (Gibco, Lot NO: 810875), trypsin (Sigma Lot NO: 27250018), penicillin G sodium salt (Shandong Lukang Medicine Lot NO: B100122), Streptomycin sulfate (Shandong Lukang Medicine Lot NO: 080307), 0.25% EDTA (trypsin Lot NO: 25200056), Hanks (NO 14170112), NaHCO3 (Chengdu Kelong Lot NO.20101013), HCL (Nanjing Chemical Reagent Co., Ltd.), Matrigel (Matrix) (BD Lot NO: 20110068), Trypan Blue, DMSO, PEG400, Tween-80, 0.9% Sodium Chloride Solution, PBS Powder Pack ( or NaCl, KCl, NaH ₂ PO ₄ , KH ₂ PO ₄ ) and the like.

2.3 Instruments:

5% CO2 cell incubator (ThermoForma, USA), ultra-clean workbench (Suzhou purification), alcohol cotton ball, alcohol lamp, dropper, culture flask, centrifuge tube, cryopreservation tube, glass bottle (for medium), Cell counting plate, coverslip, cell culture dish, cell culture dish, pressure sterilizer, inverted microscope, centrifuge, liquid nitrogen, autoclave, electronic balance, pH meter, vernier caliper, magnetic stirrer, 100mL volumetric flask, 5mL volumetric flask, 1000mL volumetric flask, 1mL syringe, gavage needle, centrifuge tube

2.4 Animals:

C57BL/6 mice (SPF grade), half male and half male, 6-8 weeks old, weighing 22-25 g, 70 mice were purchased from Shanghai Sipple-Bikai Laboratory Animal Co., Ltd. The experimental animal production license number is Scxk (Shanghai) 2008-0016, and the qualification certificate number is 2008001608319.

2.5 Test drug:

TSL-1502, synthesized in the laboratory of Suzhou Henderson Pharmaceutical Technology Co., Ltd., batch number: 100301, dissolved in normal saline;

TMZ: purchased from Jiangsu Hengrui Pharmaceutical Technology Co., Ltd., batch number: 20100805, dissolved in normal saline;

Veraparri was purchased from Shanghai Haoyuan Chemical Technology Co., Ltd., batch number: HM-0698-20101112, and dissolved in physiological saline.

3 Methods Methodology

3.1 Cell Culture:

The B16F10 cell line was frozen at -80°C after purchase, and the freezing medium was fetal bovine serum: DMSO=9:1. After cell recovery, cells were routinely cultured in RPMI-1640 complete medium (containing 10% calf serum, 1% (W/V) penicillin and 1% (W/V) streptomycin) in 5% CO2 Culture at 37°C in an incubator with relative saturated humidity, observe the growth status every day, adhere to the wall for 2-3 days, and pass the passage for expansion. The exponentially proliferating cells are collected and digested with 0.25% trypsin + 0.53 mMol/L EDTA, and collected in Gently shake it in the incomplete culture medium without calf serum to make a cell suspension, count the cells and detect the cell viability by trypan blue staining (should be more than 95%).

3.2 Construction of tumor-bearing mouse model:

SPF (specific pathogen-free, specific pathogen-free) closed-group inbred C57BL/6 female mice, 6-8 weeks old, about 22 grams each, 15 mice were randomly divided into 3 groups, 5 mice in each group, using 75% The abdominal skin was disinfected with alcohol, and the above-mentioned B16F10 melanoma cell solution was respectively inoculated into the left Flank of the mice. The concentration of inoculated cells in each group was ⁵ × ^{10 6} /mL. Inoculate ^2.5x105 cells), 0.012mL (inoculate 6x104 cells), inoculate each group of cell lines and 50% matrix (add a little more matrix to help the tumor grow with regular shape), observe the tumor growth every day, and record the tumor size (length, width) volume (length x width ^2/2 ), calculate the standard deviation, the tumor volume should be around 100-200 ^mm3 within 8-10 days of inoculation.

3.3 Solution preparation

Blank solution: add 0.9 g of NaCl to a 100 mL volumetric flask, dissolve and shake with distilled water to prepare a 0.9% NaCl solution, and store at room temperature for later use.

TMZ solution: 50 mg of temozolomide (TMZ) sample was accurately weighed and placed in a 100-mL volumetric flask, and 0.9% NaCl solution was used to make a TMZ solution with a concentration of 5.0 mg/ml.

Veraparib solution: Accurately weigh 25 mg of veraparib sample into a 100-ml volumetric flask, and use 0.9% NaCl solution to make a veraparib solution with a concentration of 2.5 mg/ml.

TSL-1502 solution: Accurately weigh 30, 15, and 10 mg of Henderson compound TSL-1502 samples respectively, place them in a 100-ml volumetric flask, and use 0.9% NaCl solution to dissolve into a concentration of 3.0, 1.5, and 1.0 mg, respectively. /ml of TSL-1502 solution.

3.4 Grouping and dosing schedule of tumor-bearing mice:

Tumor-bearing mice grouping and dosing schedule: SPF (specific pathogen-free) grade C57BL/6 female mice, 6-8 weeks old, 70 mice, each about 22 grams, disinfect the abdominal skin with 75% alcohol , respectively, take the above B16F10 melanoma cell solution and inoculate it in mouse Flank. The above optimal conditions were selected for the inoculation plan. The tumor shape was round or oval, and the tumor in B6 mice grew to about 100mm3 (the time was the 10th day after inoculation). Nude mice with large or small tumors were excluded, and the tumor size was consistent. The better nude mice were randomly divided into 6 groups, with 8 mice in each group.

Blank control group (0.1ml/10g, po, Bid): After grouping, oral administration of normal saline was administered continuously for 9 days, and on the 2nd, 3rd, 4th, 5th, and 6th days, the oral administration of normal saline was added once, and the group was continuously 23 days of observation;

Temozolomide (TMZ) single-administration group (50 mg/kg, po, qd): After grouping, oral administration of normal saline was administered continuously for 9 days, and temozolomide solution was orally administered orally on days 2, 3, 4, 5, and 6 respectively. , 23 consecutive days of observation;

Veraparib administration group (25mg/kg, po, Bid) + temozolomide (50mg/kg, po, qd): After grouping, oral administration of veraparib solution was administered continuously for 9 days. On the 6th day, temozolomide solution was orally administered orally for 5 consecutive days and observed for 23 consecutive days;

TSL-1502 administration group (10mg/kg, po, Bid) + temozolomide (50mg/kg, po, qd): After grouping, oral administration of TSL-1502 solution was administered orally for 9 days. , 5 and 6 days, respectively, orally gavaged temozolomide solution for 23 consecutive days;

TSL-1502 administration group (15mg/kg, po, Bid) + temozolomide (50mg/kg, po, qd): After grouping, oral administration of TSL-1502 solution was administered orally for 9 days. , 5 and 6 days, respectively, orally gavaged temozolomide solution for 23 consecutive days;

TSL-1502 administration group (30mg/kg, po, Bid) + temozolomide (50mg/kg, po, qd): After grouping, oral gavage of TSL-1502 solution was administered continuously for 9 days. , 5 and 6 days, respectively, orally gavaged temozolomide solution for 23 consecutive days;

3.5 Detection indicators and methods:

3.5.1 Tumor volume and tumor growth curve:

The tumor volume of nude mice was detected every other day. Measurement method of tumor volume: measure the length and width of tumor tissue with a vernier caliper (assuming that the tumor tissue is oval), and calculate the volume using the formula of length × width ^2/2 , and calculate tumor inhibition. Rate and relative tumor volume (RTV), Vt: tumor volume obtained by measuring the tumor every day, V _{initial l} : initial tumor volume (before administration);

3.5.2 Relative tumor volume (RTV), the calculation formula is:

RTV=V _t /V _initial

Where V _initial is the tumor volume measured at the time of group administration, and V _t is the tumor volume at each measurement.

3.5.3 Relative tumor proliferation rate T/C (%), the calculation formula is:

T/C(%)=(T _RTV /C _RTV )×100%

3.5.4 Tumor volume inhibition rate (GI)

GI=[1−(TV _t −TV _initial )/(CV _t −CT _initial )]×100%

where TV _t represents the tumor volume at each measurement in the treatment group;

TV _initially represents the tumor volume measured by the treatment group at the time of group administration;

CV _t represents the tumor volume of the control group at each measurement;

CT _initially represents the tumor volume measured in the control group at the time of group administration;

3.5.4 Body weight change curve of tumor-bearing mice:

Measure the body weight of the mice while measuring the volume of the tumor-bearing mice, and draw the weight change curve of the tumor-bearing mice, observe the change trend of the body weight of the tumor-bearing mice, and evaluate the change of the compound on the body weight of the tumor-bearing mice;

3.6 Statistical analysis:

The measurement data such as tumor volume, tumor weight and tumor-bearing mice were calculated using

Expressed; using Excel software or SPSS 12.0 software package for statistical analysis, statistical method using t test.

3.7 Analysis of experimental results

3.7.1 Analyze the tumor volume of each administration group after administration, and draw the time-volume change pharmacodynamic curve;

3.7.2 Analyze the relative tumor proliferation rate of each administration group after administration, and draw the time-tumor relative proliferation rate change curve;

3.7.3 Analysis of tumor size inhibition in each administration group after administration, and calculation of tumor inhibition rate;

3.7.3 Analyze the weight of tumor-bearing mice in each administration group after administration, and draw a weight-time curve;

4Results

4.1 The effect of TSL-1502 combined with TMZ on the tumor volume of B16F10 model:

The results showed that TSL-1502 had a good inhibitory effect on the tumor growth of melanoma B16F10 tumor-bearing mice. On the 33rd day after inoculation, the tumor inhibition rate of TMZ (50 mg/kg) alone was 37.4%, with a significant inhibitory effect (P<0.05), while the tumor inhibition rate of TSL-1502 (10 mg/kg) combined with TMZ group was 76.1% , significantly higher than the TMZ single administration group (P<0.01). With the increase of the dose of TSL-1502, the tumor inhibition effect of combined TMZ was better, the inhibition rate of (15mg/kg)+TMZ group was 81.2%, and the inhibition rate of TSL-1502 (30mg/kg)+TMZ group was 87.0%. See Table 7 and Figure 10.

Table 7. Mean tumor volume (mm ³ ) and tumor inhibition rate (GI, %) at 33 days

group	Mean tumor volume (mm3)	Tumor inhibition rate (GI, %)
Blank control group	12660.89	0.00
TMZ(50mg/kg)	8025.33*	37.36
Veliparib(25mg/kg)+TMZ	5991.14#	53.61
TSL-1502(10mg/kg)+TMZ	3188.62##	76.13
TSL-1502(15mg/kg)+TMZ	2570.66##	81.17
TSL-1502(30mg/kg)+TMZ	1833.32###	87.02

Compared with Control, *p<0.05, **p<0.01; compared with TMZ control: #P<0.05, ##P<0.01;

4.2 The effect of TSL-1502 combined with TMZ on the relative tumor proliferation rate (T/C%) of B16F10 melanoma model:

The results in Tables 8, 9 and Figure 11, Figure 12 show that on day 33 after inoculation, the relative tumor proliferation rate T/C% of TSL-1502 (10, 15, 30 mg/kg) combined with TMZ was increased by 61.5% of TMZ alone, respectively Increase to 26.5%, 20.7%, 15.4% (T/C%≤40%, P<0.05 is effective). Compared with TMZ alone, TSL-1502 10mg/kg and 15mg/kg significantly increased the antitumor effect of TMZ (P<0.01), and TSL-1502 30mg/kg had a better synergistic effect (P<0.001).

Table 8. Changes in relative tumor proliferation rate (T/C%) of tumor-bearing mice during administration

Table 9. Summary of Relative Tumor Proliferation Rates (T/C, %) and Tumor Inhibition Rates (GI, %) at 33 Days

group	RTV	T/C%	%GI
blank	5400.71	0.00	0.00
TMZ(50mg/kg)	3323.94*	61.55	37.36
Veraparib (25mg/kg) + TMZ	2649.89#	49.07	53.61
TSL-1502(10mg/kg)+TMZ	1430.26##	26.48	76.13
TSL-1502(15mg/kg)+TMZ	1116.71##	20.68	81.17
TSL-1502(30mg/kg)+TMZ	830.31###	15.37	87.02

Compared with Control, *p<0.05, **p<0.01; compared with TMZ control group: #P<0.05, ##P<0.01, ###P<0.001. references:

[1]. Cherrie K. Donawho, Yan Luo, et al. ABT-888, an Orally Active Poly (ADP-Ribose) Polymerase Inhibitor that Potentiates DNA-Damaging Agents in Preclinical Tumor Models. Clin Cancer Res 2007; 13(9) May 1, 2007.

[2].JOANN P.PALMA, LUIS E.RODRIGUEZ, et al. The PARP Inhibitor, ABT-888 Potentiates Temozolomide: Correlation with Drug Levels nd Reduction in PARP Activity In Vivo.ANTICANCER RESEARCH 28:2625-2636 (2008).

.

Claims (10)

1. A compound drug combination containing TSL-1502 and another antitumor drug, the compound drug combination comprising a pharmaceutical preparation composition containing two active ingredients of TSL-1502 and another antitumor drug, or a discrete drug preparation composition containing TSL-1502 and another antitumor drug. A pharmaceutical preparation of 1502 and another antineoplastic drug, a combination of the two preparations packaged together.
2. The compound medicine combination according to claim 1, wherein the dosage of each medicine adopts a medicine effective amount.
3. The compound medicine combination according to claim 1, characterized in that, the compound medicine combination is in any ingestible pharmaceutical form, preferably the medicine form is an injection, and the unit dosage form contains 0.1-2000 mg of active pharmaceutical ingredients.
4. The compound drug combination according to claim 1, characterized in that it comprises TSL-1502 and another antitumor drug, and the weight ratio of the two is 1:1000 to 1000:1.
5. The compound drug combination according to claim 1, wherein the another antitumor drug is selected from temozolomide, cisplatin, carboplatin, and irinotecan.
6. The compound drug combination according to claim 5, wherein the compound drug combination comprises TSL-1502 and temozolomide according to an effective dose (5-60): (10-100) ratio; preferably (10-40): (40-60); most preferably (10-30): 50; or the compound drug combination comprises TSL-1502 and cisplatin in an effective amount (5-50): (2-10) ratio; preferably (5- 40): (2-8); most preferably (6.25-25): 6;

   The compound drug combination comprises TSL-1502 and carboplatin in an effective dose (10-90):(5-100) ratio; preferably (20-70):(50-70), most preferably 25-50:60;

   Or the compound drug combination comprises TSL-1502, irinotecan hydrochloride (Kipto) according to the effective dose (5-100): (5-30) 10 ratio, preferably (5-60): (5-20) , most preferably (5-50): 10.
7. The compound drug combination according to any one of claims 1-6, characterized in that, the using method comprises: preparing a compound containing a required amount of TSL-1502 and another antitumor drug together as two active pharmaceutical ingredients. Injections are used together. The required amount of TSL-1502 and another antitumor drug can also be made into injections and used separately.
8. The preparation method of the compound drug combination according to claim 1, comprising preparing a required amount of TSL-1502 and another anti-tumor drug together or separately as active pharmaceutical ingredients, according to the conventional technology of preparation into edible pharmaceutical composition.
9. The preparation method according to claim 8, wherein the dosage form of the compound drug combination is an injection, wherein no adjuvant or one or more pharmaceutical adjuvants may be added, and the adjuvant is selected from the group consisting of: glucose, lactose, mannose alcohol, sodium chloride, hydroxypropyl-B-cyclodextrin, and then made into an injection.
10. The application of the compound pharmaceutical composition according to any one of claims 1 to 6 in the preparation of an anti-tumor drug, preferably the tumor is selected from breast cancer, melanoma, and colorectal cancer.

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