TW202313056A - PHARMACEUTICAL COMBINATIONS COMPRISING A PI3K <i>α</i> INHIBITOR - Google Patents

Abstract

The present disclosure relates to a pharmaceutical combination comprising a PI3K[alpha] inhibitor or a pharmaceutically acceptable salt thereof, and one or two agents selected from the group consisting of a PARP1/2 inhibitor, an estrogen receptor modulator and a CDK4/6 inhibitor, or a pharmaceutically acceptable salt thereof, and optionally an aromatase inhibitor or a pharmaceutically acceptable salt thereof. The present disclosure also relates to methods of using the combinations to treat or prevent cancer and uses thereof.

Description

Drug Combinations Containing PI3K alpha Inhibitors

Cross References to Related Applications

This application claims priority to patent application CN 202111061695.0 filed on September 10, 2021, the disclosure of which is incorporated herein by reference.

This disclosure pertains to the field of medicine. Specifically, the present disclosure relates to a pharmaceutical combination comprising a PI3K alpha inhibitor or a pharmaceutically acceptable salt thereof, and a PARP1/2 inhibitor, an estrogen receptor modulator, a cyclin-dependent kinase 4/6 One or two of (CDK4/6) inhibitors or a pharmaceutically acceptable salt thereof, and an aromatase inhibitor or a pharmaceutically acceptable salt thereof if necessary. The present disclosure also relates to methods and uses of using the combination to treat or prevent cancer.

PI3K alpha inhibitor

The PI3K/AKT/mTOR signaling pathway is one of many mechanisms that regulate the cell cycle and apoptosis. Dysregulation of individual components in the pathway can lead to tumorigenesis. Receptor tyrosine kinase (RTK) can regulate the activation of PI3K/AKT signaling pathway when activated by growth factors. These growth factors include insulin-like growth factor (Insulin-like Growth Factors, IGF), epidermal growth factor (Epidermal Growth Factor, EGF) and hepatocyte growth factor (Hepatocyte growth factor, HGF), which induce RTK tyrosine residues Activation of RTKs by autophosphorylation. The binding of PI3K to the phosphorylated tyrosine residues on RTK leads to the activation of the catalytic subunit of PI3K. Human cells contain three genes ( PIK3CA , PIK3CB and PIK3CD ) that encode the catalytic subunits PI3K α (p110 α ), PI3K β (p110 β ) and PI3K δ of type IA PI3K, respectively. Both p110α and p110β have specific roles in insulin signaling; however, glucose homeostasis is primarily mediated by p110α . The catalytic subunit p110 α of type IA PI3K activates PI3K by binding to p85 α , thereby further phosphorylating phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate ( PIP3). As an important second messenger and mediator, PIP3 recruits AKT from the cytoplasm to the cell membrane by interacting with the PH domain of AKT. The membrane translocation of AKT and the Thr308 and Ser473 sites of AKT are controlled by 3-phosphoinositide-dependent protein kinase 1 (phosphatidylinositol-dependent kinase 1, PDK1) and 3-phosphoinositide-dependent protein kinase 2 (phosphatidylinositol-dependent kinase 2 , PDK2) phosphorylation is a prerequisite for AKT activation. When AKT is fully activated, it can regulate biological processes such as cell proliferation and apoptosis.

Dysregulation of the PI3K signaling pathway is associated with almost all human cancers. PIK3CA (encoding the PI3K catalytic subunit α ) is one of the frequently mutated oncogenes in human tumors. Studies have found that about 2-5% of human solid tumors have mutations in the oncogene PIK3CA , and the proportions of mutations are about 32%, 27%, and 25% in colon cancer, glioblastoma, gastric cancer, breast cancer, and lung cancer, respectively. %, 8% and 4%, among other gastrointestinal tumors, the mutation rate of esophageal squamous cell carcinoma was 11%, and that of esophageal adenocarcinoma was 6%.

PARP1/2 inhibitors

Poly(ADP-ribose) polymerase (PARP) inhibitors are a class of hot anticancer drugs in continuous development, and are a kind of nuclear enzyme closely related to DNA repair, especially DNA single-strand damage repair. Enzyme that can prevent DNA single-strand breaks from repairing DNA single-strand breaks (SSBs) by capturing PARP bound to DNA single-strand breaks (SSBs), and then triggering a large number of DNA double-strand breaks (double-strand breaks, DSBs), DSBs cannot be accurately repaired in homologous recombination repair-deficient (HRD) tumors, which can lead to DNA damage accumulation and tumor cell death.

PARP inhibitors include 18 subtypes, and the members have certain homology. PRAP1 and PARP2 are the main two types of enzymes in the PARP family, of which PARP1 plays more than 90% of the functions, and PARP2 has similar functions to PARP1, but the substrate selectivity of the two is different. Among them, PARP-1 has the highest content in eukaryotic cells. When tumor cell DNA is damaged by chemotherapeutic drugs or ionizing radiation, PARP-1 is quickly activated, and uses NAD+ as a substrate to transfer polyadenosine diphosphate-ribose (ADP) to specific proteins, and PARP-1 catalyzes specific Protein polyadenosine diphosphate ribosylation (PARylation), which initiates the DNA repair process. Studies have shown that PARP-1 is involved in DNA repair pathways such as base excision repair (BER), homologous recombination (HR) and non-homologous end joining (NHEJ).

PARP inhibitor monotherapy has been clinically used in the treatment of homologous recombination-deficient (HRD) cancer patients, such as BRCA germline mutation or HRD-positive ovarian cancer, breast cancer, pancreatic cancer and prostate cancer. In cancer patients with intact homologous recombination repair (HRR), single-agent PARP inhibitors are suboptimal. In addition, drug resistance is evident in the majority of patients with advanced BRCA1/2-mutated cancers, and these acquired resistances affect the efficacy of PARP inhibitors. Most of the currently known PARP-1 inhibitors also have inhibitory effects on PARP-2 with comparable activity. For example, three PARP-1/2 inhibitors, Olaparib, Rucaparib, and Niraparib, were launched as antineoplastic drugs in 2014, 2016, and 2017. .

Estrogen receptor modulators and aromatase inhibitors

Estrogen receptor alpha (ER α ) and estrogen receptor beta (ER β ) are steroid hormone receptors and members of a large family of nuclear receptors. Both receptors are involved in the regulation and development of the female reproductive system, and also play roles in the central nervous system, cardiovascular system and bone metabolism. Breast cancer is the most common malignant tumor in women. According to reports, about 70% of breast cancer is related to estrogen and estrogen receptor in the human body, which is positive for estrogen receptor α (ER α ); other cancers such as ovarian cancer and endometrial carcinomas are also thought to depend on ERα signaling for proliferation.

Several types of therapy have been developed to counteract ERα function. Of these, endocrine therapy is the first-line treatment for patients with estrogen receptor-positive breast cancer. Existing drugs include: Aromatase inhibitors (AIs) Selective estrogens Receptor down-regulators (SERDs), selective estrogen receptor modulators (SERMs), etc.; work by reducing the amount of hormones in the body or by blocking the effects of hormones on cells. Specifically, selective estrogen receptor modulators (Selective estrogen receptor modulators, SERMs) are a class of drugs that can prevent estrogens such as estradiol from mediating their biological effects in vivo, mainly by interacting with endogenous estradiol Alcohol competitively binds to estrogen receptors to block the estrogen signaling pathway mediated by estradiol to play a role. SERMs such as tamoxifen have achieved good efficacy in the treatment of hormone-dependent (ER ⁺ ) breast cancer patients, but long-term use often leads to drug resistance.

Selective estrogen receptor down-regulators (Selective estrogen receptor down-regulators, SERDs) are a class of drugs that can induce rapid down-regulation of estrogen receptors by inhibiting the function of the two transcriptional activation domains AF1 and AF2 of estrogen receptors. The drug has two functions of antagonizing ERα and degrading ERα at the same time, and has a certain curative effect on breast cancer patients who have developed resistance to antihormonal drugs. For example, fulvestrant can inhibit the growth of drug-resistant estrogen receptor positive (ER ⁺ ) breast cancer cells, but its bioavailability and receptor affinity are relatively poor.

Aromatase inhibitor (Aromatase inhibitor, AI) can specifically inactivate aromatase, block the aromatization reaction, inhibit the production of estrogen, and reduce the level of estrogen in the blood to achieve the purpose of treating breast cancer. Such as letrozole for the treatment of estrogen receptor positive breast cancer.

CDK inhibitors

Tumor development is closely linked to genetic alterations and deregulation of CDKs and their regulators, which function to phosphorylate and thereby activate or inactivate certain proteins including, for example, retinoblastoma protein, lamin, histone H1, and mitotic Spindle components. CDK-mediated catalytic steps include the phosphotransfer reaction from ATP to the substrate of the macromolecular enzyme. Regulation of CDK/cyclin complex activity at the molecular level requires a series of stimulatory and inhibitory phosphorylation or dephosphorylation events. CDK phosphorylation is achieved by a group of CDK-activating kinases (CAKs) and/or kinases such as weel, Myt1 and Mik1. Dephosphorylation is achieved by phosphatases such as cdc25(a & c), pp2a or KAP.

CDK4/6 inhibitors restore cell cycle control and block tumor cell proliferation by selectively inhibiting cyclin-dependent kinases 4 and 6 (CDK4/6). CDK4/6 and cyclin D (cyclin D) can phosphorylate the retinoblastoma gene (Rb) and then release the transcription factor E2F to promote the transcription of cell cycle-related genes and make the cells enter the S phase. CDK4/6 inhibitors effectively block the progression of tumor cells from G1 phase to S phase.

Breast cancer (BC) is the most common malignancy in women. More than 70% of patients were hormone receptor positive (HR+) and human epidermal growth factor receptor 2 negative (HER2-). In recent decades, endocrine therapy (ET) has been the cornerstone of the treatment of these patients. In recent years, CDK4/6 overactivity has been very frequent in estrogen receptor-positive (ER+) breast cancers. CDK4/6 inhibitors approved for marketing in combination with ET significantly increased the progression-free survival of patients with HR+ advanced breast cancer in first-line treatment. These patients inevitably develop resistance to ET and/or CDK4/6 inhibitor therapy as standard of care.

Compound CYH33

CYH33 is a novel, highly efficient and highly selective phosphatidylinositol-3 kinase α (PI3K α ) inhibitor, which has the strongest inhibitory effect on PI3K α and its mutants, and can significantly inhibit wild-type and mutant types (including: E542K, H1047R or E545K) PI3K α kinase activity, leading to cell arrest in G ₀ /G ₁ phase, thereby inhibiting cell proliferation. Compound CYH33 showed significant antitumor activity both in vitro and in vivo, especially against tumors with high-frequency activation of PI3Kα signaling pathway. Enzyme tests show that CYH33 exhibits effective inhibitory activity on PI3K kinase family, and has the strongest inhibitory activity on PI3K α and its mutants (IC ₅₀ : 5-20nM). Cytological tests showed that CYH33 inhibited PI3Kα-mediated AKT phosphorylation in Rh30-Myr-p110α cells at 41nM level. In contrast, 10- to 30-fold higher concentrations were required to inhibit PI3K β /γ/ δ -mediated AKT phosphorylation. Preclinical studies have shown that the compound CYH33 can effectively inhibit the growth of breast cancer, esophageal cancer and ovarian cancer at the cellular level and in mouse xenograft tumor models.

There are currently a variety of PI3K inhibitors on the market worldwide, such as Gilead’s PI3Kδ inhibitor Idelalisib, Bayer’s pan-PI3K inhibitor Copanlisib, Verastem’s PI3Kδ inhibitor Duvelisib, and Novartis’ Alpelisib (BYL719). Among them, BYL719 was approved by the FDA on May 24, 2019, and is currently the first selective PI3K α inhibitor on the market.

Currently there are many treatment options for cancer patients. However, in view of the complexity of the mechanism of tumorigenesis, the unpredictability of the interaction between different drugs and other factors, it is found that it is feasible and can bring better effects than single drugs (relieving symptoms, delaying progression or inhibiting symptoms, reducing single drug dosage, improving the incidence and/or severity of adverse events (AEs) during treatment, improving quality of life, and/or acting in a synergistic manner, etc.) A big challenge. Furthermore, in many cases the cancer acquires resistance to existing treatments and eventually becomes difficult to treat. There remains therefore a need for effective and safe combinations of therapeutic agents, especially for the treatment of cancers that are resistant and/or refractory to current therapies.

The present disclosure relates to a pharmaceutical combination comprising: (a) a first active agent which is a PI3K alpha inhibitor or a pharmaceutically acceptable salt thereof, and (b) a second active agent which is selected from PARP1/2 inhibitory one or both of estrogen receptor modulators, CDK inhibitors, or pharmaceutically acceptable salts thereof; optionally (c) a third active agent, which is an aromatase inhibitor or its Pharmaceutically acceptable salts.

These combinations are useful for separate, simultaneous or sequential administration to an individual in need thereof for the treatment or prevention of cancer.

The drug combinations in the embodiments of the present disclosure are collectively referred to as "combinations of the present disclosure", wherein a preferred two-drug combination such as compound CYH33 or compound I-27 is selected from PARP1/2 inhibitors, estrogen receptor modulators, A dual combination of one of the CDK4/6 inhibitors; and a three-drug combination such as compound CYH33 or compound I-27 with two selected from PARP1/2 inhibitors, estrogen receptor modulators, and CDK4/6 inhibitors triple combination, and three-drug combination such as compound CYH33 or compound I-27 and a triple combination selected from PARP1/2 inhibitors, estrogen receptor modulators, CDK4/6 inhibitors and aromatase inhibitors. The inventors have found that the combinations of the present disclosure exhibit synergy.

Combinations of the present disclosure pertaining to the treatment or prevention of cancer.

The present disclosure pertains to combinations of the present disclosure for the preparation of a pharmaceutical composition or medicament (eg, pharmaceutical formulation) for the treatment or prevention of cancer in an individual in need thereof.

The present disclosure relates to the use of the combination of the present disclosure in the preparation of a pharmaceutical composition or medicament for treating or preventing cancer.

The present disclosure pertains to a method of treating or preventing an individual suffering from cancer comprising administering to the individual in need thereof an effective amount of a combination of the present disclosure.

The present disclosure further provides a commercial package, such as a kit of parts, comprising a combination of the present disclosure as therapeutic agents and instructions for their simultaneous, separate or sequential administration, for the treatment or prevention of cancer.

Administration of the combinations of the present disclosure not only results in beneficial effects (e.g., synergistic therapeutic effects with respect to relief of symptoms, delay of progression, or suppression of symptoms), but also unexpected beneficial effects, e.g. Fewer side effects, longer-lasting response, improved quality of life, reduced mortality, and/or reduced morbidity compared to monotherapy with one of the agents. Combinations of the present disclosure have beneficial therapeutic properties, such as synergistic interactions, strong in vitro or in vivo antiproliferative activity, and/or strong in vitro or in vivo antitumor responses, making them particularly suitable for the treatment of cancer.

Figure 1 shows the effect of the combination of compound CYH33 and olaparib on tumor volume in SK-OV-3 xenograft tumor model with PIK3CA mutation (PO QD 21 days).

Figure 2 shows the effect of the combination of compound CYH33 and olaparib on tumor weight in SK-OV-3 xenograft tumor model with PIK3CA mutation (PO QD 21 days).

Figure 3a and Figure 3b show the treatment of ER+, HER2-, PIK3CA-mutant T47D subcutaneous xenograft tumor female BALB/c nude mice model compound CYH33, fulvestrant, palbociclib as a single drug or in combination Growth curves of tumor volume changes in each group (PO QD35 days).

Figure 4a, Figure 4b and Figure 4c show the administration of ER+, HER2-, PIK3CA mutation T47D subcutaneous xenograft tumor female BALB/c nude mouse model compound CYH33, palbociclib, fulvestrant single drug, dual drug combination Or after three-drug combination treatment, the growth curve of tumor volume change in each group (PO QD28 days).

1. A drug combination comprising:

(a) a first active agent which is a PI3K alpha inhibitor or a pharmaceutically acceptable salt thereof, and

(b) the second active agent, which is any one or any two selected from PARP1/2 inhibitors, estrogen receptor modulators, and CDK inhibitors, or a pharmaceutically acceptable salt thereof;

Optionally, (c) a third active agent which is an aromatase inhibitor or a pharmaceutically acceptable salt thereof is also included.

2. The pharmaceutical combination according to embodiment 1, comprising:

(a) a first active agent which is a compound of formula (If) or formula (Ig), or a pharmaceutically acceptable salt thereof,

或

Y為NH或O，(Ig)。

or

Y is NH or O, (Ig).

in,

R ₁ is -NR ₅ R ₆ ;

； _R2 is

;

R ₅ and R ₆ are each independently a C ₁ -C ₄ alkyl group, or an unsubstituted or substituted piperazine ring formed together with their connected nitrogen atom, the substituent being -S(O) ₂ R ₁₂ ;

； R ₇ , R ₈ , R ₉ and R ₁₀ are each independently H or C ₁ -C ₃ alkyl, or R ₂ is

;

R ₁₁ is C ₁ -C ₄ alkyl, benzyl unsubstituted or substituted by one or more substituents, phenyl unsubstituted or substituted by one or more substituents, unsubstituted or substituted by one or more Isoxazolyl substituted with radical, or pyridyl unsubstituted or substituted by one or more substituents selected from halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy group, -CF ₃ , -C(O)OR ₁₂ , -C(O)NR ₁₂ R ₁₅ ,

R ₁₂ and R ₁₅ are each independently a C ₁ -C ₃ alkyl group;

and

(b) the second active agent, which is selected from any one or any two of PARP1/2 inhibitors, estrogen receptor modulators, and CDK inhibitors, or a pharmaceutically acceptable salt thereof;

Optionally, (c) a third active agent which is an aromatase inhibitor or a pharmaceutically acceptable salt thereof is also included.

3. The pharmaceutical combination according to embodiment 2, wherein

R ₁ is dimethylamino or 1-methylsulfonylpiperazinyl;

、

或

； _R2 is

,

or

;

R ₁₁ is methyl, ethyl, propyl, cyclopropyl, tert-butyl, isobutyl, 4-fluorobenzyl, unsubstituted or phenyl substituted by one or more substituents, unsubstituted or substituted by Isoxazole substituted with one or more substituents, or unsubstituted or substituted pyridine rings with one or more substituents selected from fluorine, chlorine, trifluoromethyl, methyl, methoxy, ethyl Oxycarbonyl, dimethylaminocarbonyl, 4-methylpiperazine-1-carbonyl, piperidine-1-carbonyl and 4-dimethylaminopiperidine-1-carbonyl.

4. The pharmaceutical combination according to any one of the preceding embodiments, wherein the first active agent is the compound CYH33 having the formula or a pharmaceutically acceptable salt thereof

or a compound I-27 of the following formula or a pharmaceutically acceptable salt thereof

Or a prodrug of compound 1-27.

5. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27, or a pharmaceutically acceptable salt thereof, and;

(b) The second active agent, which is selected from any one or any two of PARP1/2 inhibitors, estrogen receptor modulators, and CDK inhibitors, or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical combination according to any one of the preceding embodiments, wherein the second active agent is selected from any one of PARP1/2 inhibitors, estrogen receptor modulators, CDK inhibitors, or a pharmaceutically acceptable salt thereof .

7. The pharmaceutical combination according to any one of the preceding embodiments, wherein the second active agent is selected from any two of PARP1/2 inhibitors, estrogen receptor modulators, CDK inhibitors, or a pharmaceutically acceptable Salt.

8. The pharmaceutical combination according to any one of the preceding embodiments, wherein the CDK inhibitor is a CDK4/6 inhibitor.

9. The pharmaceutical combination according to any one of the preceding embodiments, wherein the aromatase inhibitor may be a non-steroidal aromatase inhibitor or a steroidal aromatase inhibitor.

10. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) The second active agent, which is selected from any one of PARP1/2 inhibitors, estrogen receptor modulators, CDK4/6 inhibitors or pharmaceutically acceptable salts thereof.

11. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) A second active agent which is a PARP1/2 inhibitor or a pharmaceutically acceptable salt thereof.

12. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) The second active agent is selected from CDK4/6 inhibitors or pharmaceutically acceptable salts thereof.

13. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) A second active agent which is an estrogen receptor modulator or a pharmaceutically acceptable salt thereof.

14. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or a pharmaceutically acceptable salt thereof, and

(b) The second active agent, which is selected from any two of PARP1/2 inhibitors, estrogen receptor modulators, and CDK4/6 inhibitors, or a pharmaceutically acceptable salt thereof.

15. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof,

(b) A second active agent which is a CDK4/6 inhibitor or a pharmaceutically acceptable salt thereof, and an estrogen receptor modulator or a pharmaceutically acceptable salt thereof.

16. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof,

(b) a second active agent selected from any one of PARP1/2 inhibitors, estrogen receptor modulators, CDK4/6 inhibitors, or a pharmaceutically acceptable salt thereof, and

(c) A third active agent which is an aromatase inhibitor or a pharmaceutically acceptable salt thereof.

17. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof,

(b) a second active agent which is a CDK4/6 inhibitor or a pharmaceutically acceptable salt thereof, and

(c) A third active agent which is an aromatase inhibitor or a pharmaceutically acceptable salt thereof.

18. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof,

(b) a second active agent selected from any two of PARP1/2 inhibitors, estrogen receptor modulators, and CDK4/6 inhibitors, or a pharmaceutically acceptable salt thereof, and

(c) A third active agent which is an aromatase inhibitor or a pharmaceutically acceptable salt thereof.

19. The pharmaceutical combination according to any one of the preceding embodiments, wherein the PARP1/2 inhibitor is Olaparib, Rucaparib, Niraparib, Talazopar Talazoparib, Fluzoparib or Pamiparib, preferably olaparib; the selective estrogen receptor modulator can be a selective estrogen receptor modulator, such as azoxime Arzoxifene, Toremifene, Raloxifene or Tamoxifen, preferably Tamoxifen; it can also be a selective estrogen receptor down-regulator, such as fluoride Fulvestrant or Giredestrant (GDC-9545), etc., preferably Fulvestrant; CDK4/6 inhibitors are Palbociclib, Ribociclib, or Abemaciclib, preferably Palbociclib.

20. The pharmaceutical combination according to any one of the preceding embodiments, wherein the aromatase inhibitor is Letrozole, Anastrozole, Exemestane or Vorozole, Preferably letrozole.

21. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) Any one of olaparib, fulvestrant and palbociclib.

22. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof,

(b) any of olaparib, fulvestrant and palbociclib, and

(c) Letrozole.

23. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) Any two of olaparib, fulvestrant and palbociclib.

24. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof,

(b) any two of olaparib, fulvestrant and palbociclib, and

(c) Letrozole.

25. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) Orapali.

26. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) Fulvestrant.

27. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) Palbociclib.

28. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) olaparib and fulvestrant.

29. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) Olaparib and palbociclib.

30. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) fulvestrant and palbociclib.

31. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) Orapali, and

(c) Letrozole.

32. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) fulvestrant, and

(c) Letrozole.

33. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) palbociclib, and

(c) Letrozole.

34. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) olaparib and fulvestrant, and

(c) Letrozole.

35. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) fulvestrant and palbociclib, and

(c) Letrozole.

36. The pharmaceutical combination according to any one of the preceding embodiments, comprising:

(a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and

(b) olaparib and palbociclib, and

(c) Letrozole.

37. The pharmaceutical combination according to any one of the preceding embodiments, wherein the first active agent is compound CYH33 or a pharmaceutically acceptable salt thereof.

38. The pharmaceutical combination according to any one of the preceding embodiments, wherein the first active agent is compound CYH33, which may be in free form (i.e. not a salt), or in salt form, such as hydrochloride or mesylate, preferably mesylate.

39. The pharmaceutical combination according to any one of the preceding embodiments, wherein each of the active agents can be formulated as different compositions or formulations, or two, three or all of the active agents can be formulated in the same composition or formulation .

40. The pharmaceutical combination according to any one of the preceding embodiments, wherein each of the active agents is in unit dosage form.

41. The pharmaceutical combination according to any one of the preceding embodiments, wherein the compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10-40 mg, 20-40 mg, 20-30 mg or 30-40 mg, such as 10 mg, In the form of a dosage unit of 20 mg, 30 mg or 40 mg, preferably an oral dosage form.

42. The pharmaceutical combination according to any one of the preceding embodiments, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10-40 mg, 20-40 mg, 20-30 mg or 30-40 mg, For example 10 mg, 20 mg, 30 mg or 40 mg.

43. The pharmaceutical combination according to any one of the preceding embodiments, wherein the initial daily dose of CYH33 or a pharmaceutically acceptable salt thereof is 40 mg or 30 mg.

44. The pharmaceutical combination according to any one of the preceding embodiments, wherein CYH33 or a pharmaceutically acceptable salt thereof is administered continuously in each cycle, preferably once a day.

45. The pharmaceutical combination according to any one of the preceding embodiments, wherein olaparib is in the form of a dosage unit of 200-600 mg, eg 200 mg, 250 mg, 300 mg, 400 mg, 500 mg or 600 mg, preferably an oral dosage form.

46. The pharmaceutical combination according to any one of the preceding embodiments, wherein the daily dose of olaparib (ie total daily dose) is 200-600 mg, such as 200 mg, 250 mg, 300 mg, 400 mg, 500 mg or 600 mg, preferably 400mg, 500mg or 600mg, more preferably administered in the form of 400mg, 500mg or 600mg tablets.

47. The pharmaceutical combination according to any one of the preceding embodiments, wherein olaparib is administered continuously in each cycle, preferably once or twice a day.

48. The pharmaceutical combination according to any one of the preceding embodiments, wherein fulvestrant is in the form of dosage units of 250mg-500mg, eg 250mg or 500mg, preferably in parenteral dosage form, eg injection.

49. The pharmaceutical combination according to any one of the preceding embodiments, wherein the single administration dose of fulvestrant is 250 mg-500 mg, eg 250 mg or 500 mg, preferably 500 mg.

50. The pharmaceutical combination according to any one of the preceding embodiments, wherein fulvestrant is administered to an individual in need thereof in one or more doses, the next dose being administered 2, 3 or 4 weeks after the previous dose; preferably Preferably, it is administered on Days 1 and 15 of the first cycle, and on Day 1 of each subsequent cycle.

51. The pharmaceutical combination according to any one of the preceding embodiments, wherein palbociclib is in the form of a dosage unit of 75-125 mg, eg 75 mg, 100 mg, 125 mg, preferably an oral dosage form.

52. The drug combination according to any one of the preceding embodiments, wherein palbociclib is continuously administered for three weeks in each cycle, followed by a one-week drug rest, preferably once a day in each cycle for three consecutive weeks, followed by a one-week drug rest .

53. The pharmaceutical combination according to any one of the preceding embodiments, wherein letrozole is in the form of a dosage unit of 2.5 mg, preferably an oral dosage form.

54. The pharmaceutical combination according to any one of the preceding embodiments, wherein the daily dose of letrozole is 2.5 mg.

55. The pharmaceutical combination according to any one of the preceding embodiments, wherein letrozole is administered continuously in each cycle, preferably once daily.

56. The pharmaceutical combination according to any one of the preceding embodiments, wherein each cycle is 28 days (4 weeks).

57. The pharmaceutical combination according to any one of the preceding embodiments, wherein the pharmaceutical combination is administered for at least one cycle, eg 2-12 or more cycles.

58. The pharmaceutical combination according to any one of the preceding embodiments, wherein the pharmaceutical combination is administered in each cycle, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg, 40mg or 50mg, once a day; and the daily dose of olaparib is 200mg, 250mg or 300mg, administered twice a day.

59. The pharmaceutical combination according to any one of the preceding embodiments, wherein the pharmaceutical combination is administered every 4 weeks, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg or 40 mg, once a day; and the single administration dose of fulvestrant is 500 mg, administered on the 1st and 15th days of the first cycle, and administered on the 1st day of each subsequent cycle.

60. The pharmaceutical combination according to any one of the preceding embodiments, wherein the pharmaceutical combination is administered every 4 weeks, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg or 40 mg once daily; and wherein a single dose of fulvestrant is 500 mg administered on Days 1 and 15 of the first cycle and on Day 1 of each subsequent cycle; and The daily dose of palbociclib was 125 mg, administered once a day for three consecutive weeks in each cycle, and then stopped for one week.

61. The pharmaceutical combination according to any one of the preceding embodiments, wherein the pharmaceutical combination is administered in each cycle, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg or 40mg, once a day; and the daily dose of letrozole is 2.5mg, once a day.

62. The pharmaceutical combination according to any one of the preceding embodiments, wherein the pharmaceutical combination is administered every 4 weeks, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30mg or 40mg, once a day; and the daily dose of letrozole is 2.5mg, once a day; and the daily dose of palbociclib is 75mg, 100mg or 125mg, once a day in each cycle for three consecutive weeks, Then stop for a week.

63. The pharmaceutical combination according to any one of the preceding embodiments, wherein the individual active agents are administered separately, simultaneously or sequentially.

64. The pharmaceutical combination according to any one of the preceding embodiments, wherein the first active agent such as compound CYH33 or compound 1-27 and/or the second active agent and/or the third active agent are administered simultaneously.

65. The pharmaceutical combination according to any one of the preceding embodiments, wherein the first active agent such as compound CYH33 or compound 1-27 and/or the second active agent and/or the third active agent are administered at different times.

66. The pharmaceutical combination according to any one of the preceding embodiments, wherein the first active agent, such as compound CYH33 or compound 1-27, is administered to the individual prior to administration of the second and/or third active agent.

67. The pharmaceutical combination according to any one of the preceding embodiments, wherein the second active agent and/or the third active agent is administered to the individual prior to administration of the first active agent, such as compound CYH33 or compound 1-27.

68. The pharmaceutical combination according to any one of embodiments 1 to 67, which is used for the treatment or prevention of a disease associated with abnormal activation of PI3K, preferably cancer, more preferably a solid tumor, such as an advanced solid tumor.

69. The pharmaceutical combination according to any one of the preceding embodiments 1 to 67, for use in the treatment or prophylaxis or prophylaxis of a disease, preferably a solid tumor, such as an advanced solid tumor, in an individual in need thereof.

70. The pharmaceutical combination according to embodiment 68 or 69 for the use, wherein the disease is selected from ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, bile duct cancer or prostate cancer.

71. The pharmaceutical combination according to any one of embodiments 68 to 70 for the use, wherein the disease carries DNA damage repair gene mutation, PIK3CA gene mutation, resistance to PARP inhibitors and/or resistance to platinum chemotherapy agents medicine.

72. The pharmaceutical combination according to any one of embodiments 68 to 71 for the use, wherein the disease carries a mutation in a key gene of the DDR pathway and is resistant to a PARP inhibitor.

73. The pharmaceutical combination according to any one of embodiments 68 to 72 for this use, wherein the disease is advanced breast cancer with HR+, HER2- and/or PIK3CA gene mutations, especially HR+, HER2- and PIK3CA gene mutations advanced breast cancer.

74. A method for treating or preventing a disease associated with abnormal activation of PI3K, the method comprising administering an effective amount of the drug combination as defined in any one of embodiments 1 to 67 to an individual in need, wherein the disease is preferably cancer, More preferably a solid tumor, such as an advanced solid tumor.

75. A method for treating or preventing a disease, the method comprising administering to an individual in need thereof an effective amount of a pharmaceutical combination as defined in any one of embodiments 1-67, wherein the disease is cancer, preferably a solid tumor, such as Advanced solid tumors.

76. The method according to embodiment 74 or 75, wherein the disease is selected from ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, bile duct cancer or prostate cancer.

77. The method according to any one of embodiments 74 to 76, wherein the disease carries DNA damage repair gene mutations, PIK3CA gene mutations, resistance to PARP inhibitors and/or resistance to platinum-based chemotherapeutic agents.

78. The method according to any one of embodiments 74 to 77, wherein the disease carries a mutation in a key gene of the DDR pathway and is resistant to a PARP inhibitor.

79. The method according to any one of embodiments 74 to 78, wherein the disease is HR+, HER2- and/or PIK3CA mutated breast cancer, especially advanced HR+, HER2- and PIK3CA mutated breast cancer.

80. Use of the drug combination according to any one of embodiments 1-67 in the preparation of a drug for the treatment of a disease, the disease is selected from cancer, preferably solid tumors, such as advanced solid tumors; more preferably, the disease is selected from ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, bile duct cancer, or prostate cancer.

81. Use of the drug combination according to any one of embodiments 1 to 67 in the preparation of a drug for treating a disease related to abnormal activation of PI3K, the disease is preferably cancer, preferably a solid tumor, such as an advanced solid tumor; more preferably , the disease is selected from ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, bile duct cancer or prostate cancer.

82. The use according to embodiment 80 or 81, wherein the disease carries DNA damage repair gene mutation, PIK3CA gene mutation, resistance to PARP inhibitors and/or resistance to platinum-based chemotherapeutic agents.

83. The use according to any one of embodiments 80 to 82, wherein the disease carries a DDR gene mutation and is resistant to a PARP inhibitor.

84. The use according to any one of embodiments 80 to 83, wherein the disease is HR+, HER2- and/or PIK3CA gene mutation advanced breast cancer, especially HR+, HER2- and PIK3CA gene mutation advanced breast cancer.

85. A combined preparation comprising the pharmaceutical combination as defined in any one of the preceding embodiments 1 to 73.

86. A pharmaceutical composition comprising the pharmaceutical combination as defined in any one of the preceding embodiments 1 to 73, and one or more pharmaceutically acceptable excipients.

87. A kit of parts comprising a pharmaceutical combination as defined in any one of the preceding embodiments 1 to 73 and instructions for simultaneous, separate or sequential administration of the active agents, preferably wherein the active agents are in pharmaceutical dosage unit form.

The various embodiments/technical solutions described above and below in this disclosure and the features in each embodiment/technical solution should be understood as being able to be combined with each other arbitrarily, and each of these combined solutions is included within the scope of the present disclosure , as if these mutually combined solutions are specifically and individually listed herein, unless the context clearly shows otherwise.

definition

The following words, phrases and symbols used in this application have the meanings as described below, unless otherwise indicated by the context in which they are used. Technical and scientific terms used herein that are not specifically defined have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, "comprising" or "comprising" are intended to indicate the presence of the mentioned features, components or steps, but they do not exclude the presence or addition of one or more other features, components, steps. As used herein, "comprises" or "comprises" encompasses consisting of the stated features, ingredients or steps, unless otherwise specified.

When "and/or" used herein is used to connect two or more optional items, it should be understood as meaning any one of the optional items or a combination of any two or more of the optional items.

As used herein, "PI3K alpha inhibitor" refers to a compound that targets, reduces or inhibits phosphatidylinositol-3-kinase alpha isoforms, including wild-type and mutant forms. Phosphatidylinositol-3-kinase (PI3K) activity has been shown to increase in response to stimulation by several hormones and growth factors, including insulin, platelet-derived growth factor, insulin-like growth factor, epidermal growth factor, Colony-stimulating factor and hepatocyte growth factor.

The "first active agent" or "PI3K α inhibitor" used herein includes pyrrole[2,1-f][1,2,4, represented by general formula I described in WO2013177983, CN103450204A and other patent applications/patents of the same family. ] triazine compounds and pharmaceutically acceptable salts thereof, especially formula (If) and formula (Ig) compounds and pharmaceutically acceptable salts thereof, preferably the compound I-33 disclosed therein (also known as CYH33 ) and compound I-27 and pharmaceutically acceptable salts thereof. The entire content of this patent or patent application, including definitions of terms, is incorporated herein by reference.

The "first active agent" or "PI3K α inhibitor" as used herein is preferably the compound CYH33 of the following formula, whose chemical name is {5-{6-[(4-methylsulfonylpiperazin-1-yl)methyl Base]-4-morpholinylpyrrole[2,1-f][1,2,4]triazin-2-yl}-4-trifluoromethylpyridin-2-yl}carbamate methyl ester,

Or have the compound I-27 of following formula structure, its chemical name is: 2-(4-trifluoromethyl-6-aminopyridin-3-yl)-6-[(4-methylsulfonyl) piperazine -1-yl]methyl-4-morpholinylpyrrole[2,1-f][1,2,4]triazine,

or a prodrug of compound 1-27.

Compound CYH33 used herein or a pharmaceutically acceptable salt thereof, or compound 1-27 or other prodrugs of compound 1-27 can be synthesized by those skilled in the art, for example, Its synthesis and identification can refer to WO2013177983. It can be administered in free form or as a pharmaceutically acceptable salt.

The specific compounds involved in "PARP1/2 inhibitors", "estrogen receptor modulators", "CDK inhibitors", "CDK4/6 inhibitors" and "aromatase inhibitors" used herein are known Known compounds can be synthesized according to the prior art by those skilled in the art. It can be administered in free form or as a pharmaceutically acceptable salt.

The preferred PARP1/2 inhibitor is Olaparib (Olaparib), whose trade name is Lipuzo or LYNPARZA, chemical name: 4-(3-{[4-(cyclopropylcarbonyl)piperazin-1-yl ]carbonyl}4-fluorophenyl)methyl]phthalazin-1(2H)-one, the structural formula is as follows:

As used herein, "estrogen receptor modulators" include selective estrogen receptor modulators (SERMs) and selective estrogen receptor down-regulators (SERDs).

The preferred selective estrogen receptor modulator is tamoxifen, whose chemical name is (Z)-2-[4-(1,2-diphenyl-1-butene)phenoxy]-N,N -Dimethylethylamine; the selective estrogen receptor down-regulator is preferably Fulvestrant, the chemical name is: 7α- [9-(4,4,5,5,5-pentafluoropentane Sulfoyl)nonyl]estra-1,3,5(10)-triene-3,17 β -diol, structural formula:

氟維司群 他莫昔芬。

Fulvestrant Tamoxifen.

The preferred CDK4/6 inhibitor is Palbociclib, the chemical name is: 6-acetyl-8-cyclopentyl-5-methyl-2-[[5-(1-piperazinyl)- 2-pyridyl]amino]pyrido[2,3-d]pyrimidin-7(8H)-one, the structural formula is as follows:

The preferred aromatase inhibitor is Letrozole (Letrozole), chemical name: 4,4'-[(1H-1,2,4-triazol-1-yl)-methylene]-bis-benzyl Nitrile, the structural formula is as follows:

"Cancer" as used herein refers to a cellular disorder characterized by uncontrolled or dysregulated cell proliferation, reduced cell differentiation, inappropriate ability to invade surrounding tissues, and/or the ability to establish new growth foci at other sites. "Cancer" includes, but is not limited to: solid tumors and hematological malignancies, preferably solid tumors, more preferably advanced solid tumors, including cancers of the skin, tissues, organs, bones, cartilage, etc. Examples of "cancer" include, but are not limited to, human rhabdomyosarcoma, human glioma, lung cancer (including non-small cell lung cancer (NSCLC)), kidney cancer (eg, renal cell carcinoma (RCC), renal pelvis cancer), primary peritoneal cancer , breast cancer, gynecological cancer (such as ovarian cancer, ovarian clear cell carcinoma (OCCC), uterine cancer, uterine sarcoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer or vulvar cancer), endocrine system cancer (such as cancer of the thyroid, parathyroid, or adrenal glands), prostate cancer, testicular cancer, penile cancer, liver cancer, bowel cancer, small bowel cancer, colorectal cancer, rectal cancer, Bowel cancer, colorectal cancer (CRC), anal region cancer, esophageal cancer, gastric cancer, pancreatic cancer, bile duct cancer, head and neck cancer (such as head and neck squamous cell carcinoma (HNSCC)), bone cancer, skin cancer, melanoma, central nervous system ( CNS) cancer, soft tissue sarcoma, urethral cancer, childhood solid tumors, bladder cancer, ureteral cancer, chronic or acute leukemia, chronic myelomonocytic leukemia (CMML), lymphocytic lymphoma; especially human rhabdomyosarcoma, non-small Cell lung cancer, human glioma, prostate cancer, ovarian cancer, breast cancer, colon cancer or liver cancer; preferably ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, bile duct cancer or prostate cancer.

As used herein, "cancer" includes both primary cancers, metastatic cancers, recurrent cancers, and refractory cancers, as well as cancers in patients with a poor prognosis. For example, cancers that are resistant or refractory to currently available therapies, such as cancers that are resistant or refractory to treatment with "PARP1/2 inhibitors", "estrogen receptor modulators", "CDK inhibitors".

"Cancer" as used herein also includes:

1) Cancers (such as advanced solid tumors) carrying DDR (DNA damage repair) gene mutations (especially DDR key gene mutations), such as ovarian cancer, breast cancer, prostate cancer, and cholangiocarcinoma;

2) Cancers (such as advanced solid tumors) carrying PIK3CA gene mutations (especially PIK3CA hotspot mutations), such as ovarian cancer, breast cancer, and endometrial cancer;

3) Cancers (such as advanced solid tumors) resistant to PARP inhibitors (acquired resistance), such as high-grade serous ovarian cancer, fallopian tube or primary peritoneal cancer;

4) Cancers that carry DDR gene mutations and are resistant to PARP inhibitors (such as advanced solid tumors), such as breast cancer and prostate cancer;

5) Cancers resistant to platinum-based chemotherapy agents (such as advanced solid tumors) or refractory recurrent cancers, such as high-grade serous ovarian cancer, fallopian tube cancer or primary peritoneal cancer;

6) Cancers with HR+, HER2- and/or PIK3CA gene mutations (such as advanced solid tumors), such as advanced breast cancer.

Cancers described herein include PIK3CA gene mutations, DDR gene mutations, PARP inhibitor-resistant and/or platinum-based chemotherapy-resistant cancers (including advanced solid tumors), such as ovarian cancer, breast cancer, cholangiocarcinoma, endometrial cancer carcinoma, fallopian tube or primary peritoneal carcinoma, or prostate cancer, etc.

Preferably, the cancer described herein is ovarian cancer, breast cancer, endometrial cancer, cholangiocarcinoma, fallopian tube or primary peritoneal cancer, or prostate cancer, especially if it carries DDR gene mutation or PIK3CA gene mutation.

Preferably, the cancer described herein is breast cancer, especially advanced breast cancer with mutations in HR+, HER2- and PIK3CA genes.

For example, the combination of compound CYH33 and olaparib is used in ovarian cancer, breast cancer, endometrial cancer, fallopian tube, primary peritoneal cancer, prostate cancer, bile duct cancer, etc. carrying DDR gene mutation or PIK3CA gene mutation. For example, the combination of compound CYH33 and fulvestrant/palbociclib/fulvestrant+palbociclib/letrozole+palbociclib is used for the treatment of advanced breast cancer, especially HR+, HER2- and PIK3CA gene mutations patients with advanced breast cancer.

Diseases associated with abnormal activation of PI3K (phosphatidylinositol 3-kinase) include the cancers described in this disclosure. The nature of diseases associated with aberrant PI3K activation is multifactorial. In some cases, drugs with different mechanisms of action may be combined. However, simply considering any combination of therapeutic agents with different modes of action does not necessarily lead to a combination with beneficial effects.

Diseases related to abnormal activation of PI3K include cancers related to abnormal activation of PI3K, such as cancers mediated by the α subunit of PI3K. Diseases associated with aberrant activation of PI3K may include those showing overexpression or amplification of PI3K α isoforms and/or somatic mutations in PIK3CA , or germline or somatic mutations in PTEN or mutations and predisposition to p85α for upregulation of the p85-p110 complex. bit. In a preferred embodiment, the cancer is a tumor and/or cancerous proliferation mediated by a PI3K alpha isoform. In another preferred embodiment, the cancer is an advanced solid tumor caused by a mutation in the PIK3CA gene.

The "DDR gene" used herein includes : ATM, BRCA1, BRCA2, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, RAD54L and the like.

"Treatment" as used herein includes administering a combination of the present disclosure to an individual in need thereof to achieve, including but not limited to, alleviation, cure, alleviation of symptoms, reduction of symptoms, prolongation of survival, and progression of a disease or disorder or symptoms thereof (e.g., cancer) Delay; in the case of cancer, this treatment includes inhibiting the growth of solid tumors, reducing tumor size, preventing metastatic spread of tumors and the growth or development of micrometastases, etc. "Progression delay" means that the combination is administered to a patient in a pre-morbid or early stage of the cancer to be treated, a pre-form of the corresponding cancer has been diagnosed and/or in a patient diagnosed in a situation where the corresponding cancer is likely to develop.

As used herein, "prevention" includes the suppression or delay of the occurrence or frequency of a disease or disorder, or symptoms thereof, such as cancer, and generally refers to the occurrence of signs or symptoms before they occur, especially in individuals at risk. previous drug administration. "Prevention" also includes preventing the occurrence or recurrence of cancer.

As used herein, an "effective amount" refers to an amount (e.g., a therapeutically effective amount, especially a combined therapeutically effective amount) of the active agents of the present disclosure for (i) treating a particular disease, (ii) attenuating, ameliorating or eliminating a particular disease or (iii) preventing or delaying the onset of one or more symptoms of a particular disease described herein. In the case of cancer, a therapeutically effective amount of the active agent reduces the number of cancer cells; reduces tumor size; inhibits (i.e., to some extent slows and preferably stops) cancer cell infiltration of surrounding organs; inhibits (i.e., to some extent slow down and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate to some extent one or more symptoms associated with cancer.

"Individual" or "patient" as used herein refers to both mammals and non-mammals. Mammal refers to any member of the class Mammalia, which includes, but is not limited to: humans; non-human primates, cattle, horses, sheep, pigs, rabbits, dogs, and cats, etc. "Individual" is not limited to a specific age or gender. Preferably, the subject or patient is a human.

As used herein, "pharmaceutical combination" includes non-fixed combination products and fixed combination products, including but not limited to kits, pharmaceutical compositions. "Non-fixed combination" means each active agent (for example, (a) compound CYH33 or compound 1-27 or a pharmaceutically acceptable salt thereof, (b) selected from PARP1/2 inhibitors, estrogen receptor modulators, One or both of the CDK4/6 inhibitors, or a pharmaceutically acceptable salt thereof, and optionally (c) an aromatase inhibitor or a pharmaceutically acceptable salt thereof) as separate entities (e.g., separate A unit dosage form) is administered to an individual simultaneously, without specific time limitation, or at the same or different time intervals, sequentially, wherein such administration provides in vivo an effective amount of the respective active agent. In some embodiments, the active agents used in the pharmaceutical combination are administered at levels not exceeding their respective levels when used alone. "Fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity (eg, the same unit dosage form). The active agents may each be in separate formulations, which may be the same or different. A kit of parts may contain the active agents for combined administration. Dosages and/or time intervals of each active agent are preferably selected to produce a more favorable effect.

"Dosage unit" as used herein refers to physically discrete units suitable as unitary dosages for humans and other mammals, each unit containing a predetermined quantity of active substance calculated to produce the desired therapeutic effect, together with a suitable pharmaceutically acceptable dose. acceptable excipients.

"Daily dose" as used herein refers to the total daily dose, which may be administered once or in divided doses, eg 2 or 3 or more times.

As used herein, "administration" refers to the physical introduction of each of the active agents of the combinations of the present disclosure into an individual using any of a variety of methods and delivery systems known to those of ordinary skill in the art. Routes of administration for each active agent in the combinations of the present disclosure include oral, intravenous (eg, infusion, drip or injection), intramuscular, subcutaneous, intraperitoneal, spinal, topical or other parenteral routes of administration. As used herein, the phrase "parenteral administration" refers to modes of administration other than gastrointestinal and topical administration, usually by intravenous, and includes, but is not limited to, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intrathecal , intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, and in vivo electroporation. Accordingly, each active agent in the combination of the present disclosure can be formulated into capsules, tablets, injections (including infusion or injection), syrups, sprays, lozenges, liposomes or suppositories, etc.

As used herein, "combined administration" is intended to encompass the administration of selected active agents to a single patient, and is intended to include therapeutic regimens in which the active agents are not necessarily administered by the same route of administration or at the same time.

"Continuous administration" as used herein refers to daily administration. In the case of continuous administration, the drug can be administered once or more times a day, for example, the drug is administered at a frequency of once a day, twice a day, or three times a day, preferably once a day.

As used herein, "period" refers to a specific period of time expressed in days or weeks that repeats on a regular schedule. For example, each treatment cycle (or prophylaxis cycle) of the combination of the present disclosure is 14 to 28 days, preferably each cycle is two weeks (ie, 14 days), three weeks (ie, 21 days) or four weeks (ie, , 28 days). Each active agent of the disclosed combination can be administered on the same day or different days of the cycle, that is to say, the active agents of the disclosed pharmaceutical combination are administered separately, simultaneously or sequentially (or called sequentially) within the cycle.

As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that are suitable within the scope of sound medical judgment for contact with mammalian (especially human) tissues without undue toxicity, irritation, allergic response and Complications of other problems and with a reasonable benefit/risk ratio.

As used herein, "pharmaceutically acceptable salt" includes, but is not limited to, an acid addition salt or a base addition salt of a compound. For example: Compound CYH33 can form various salts with various inorganic and organic acids. Acids useful in the preparation of pharmaceutically acceptable acid addition salts of compounds of the present disclosure are those that form non-toxic acid addition salts (i.e., salts containing a pharmaceutically acceptable anion, such as, hydrochloride, sulfuric acid, or phosphate , propionate, fumarate, malonate, methanesulfonate, acetate, benzoate, bromide, chloride, citrate, fumarate, hydrobromide, iodate, Lactate, maleate, mandelate, nitrate, oxalate, salicylate, succinate and tartrate, benzenesulfonic acid, 4-toluenesulfonic acid, 2-naphthalenesulfonic acid, 1,5 - those acids of naphthalene disulfonic acid, 2- or 3-toluenesulfonic acid, methylsulfuric acid, ethylsulfuric acid).

As used herein, "synergy" refers to a therapeutic combination that is more effective than the additive effects of two or more individual active agents. For example, a determination of a synergistic interaction between compound CYH33, or a pharmaceutically acceptable salt thereof, and one or more other active agents can be based on results obtained from assays described herein. For example, the Combination Index (CI) value of the combination therapy can be calculated by the Compusyn software (1.0) in an in vitro tumor cell activity test. When CI<0.9, the combination therapy has a synergistic effect. In vivo anti-tumor efficacy experiments can also be used to perform two-way ANOVA and Tukey's multiple comparisons on the experimental results with Graphpad Prism 6.0 software, in order to obtain the statistical difference index p between groups, when p <0.05 It was considered statistically significant, i.e. when p < 0.05 in the combination group compared to the single drug group, the combination therapy had a synergistic effect. In addition, when the tumor inhibition rate (TGI) of the combination group is greater than the sum of the tumor inhibition rates of each group, the combination therapy has a synergistic effect. Combination therapies may provide "synergy" and be demonstrated to be "synergistic", ie, the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds alone. A synergistic effect may be obtained when the active agents are: (1) co-formulated and administered or delivered simultaneously in a combined unit dosage form of two or more active ingredients; (2) delivered separately or sequentially (or sequentially) or as separate The formulations are delivered simultaneously; or (3) administered by some other regimen.

All numerical ranges herein should be understood to disclose every value and subset of values within that range, whether or not specifically disclosed otherwise. For example, reference to any numerical range should be deemed to refer to each value within the numerical range, eg, each integer within the numerical range. The disclosure contemplates all values falling within these ranges, and all smaller ranges, as well as upper and lower limits of numerical ranges.

Dosage Forms, Administration and Dosing Regimen

The pharmaceutical compositions of the present disclosure may include one or more pharmaceutically acceptable excipients and can be produced in a conventional manner by mixing one or more of the combination partners with the pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, and disintegrants. Examples of pharmaceutically acceptable diluents include, but are not limited to, lactose, dextrose, mannitol, and/or glycerin, and/or lubricants and/or polyethylene glycol. Examples of pharmaceutically acceptable binders include, but are not limited to, magnesium aluminum silicate, starch (such as corn starch, wheat starch, or rice starch), gelatin, methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, And if necessary, pharmaceutically acceptable disintegrating agents include but are not limited to starch, agar, alginic acid or its salts (such as sodium alginate) and/or effervescent mixtures, or adsorbents, dyes, flavoring agents and sweeteners. Compounds of the present disclosure can also be in the form of parenterally administrable compositions or infusion solutions. The pharmaceutical composition may be sterile and/or may comprise excipients such as preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for adjusting the osmotic pressure and/or buffers.

Combinations of the present disclosure may be in the form of pharmaceutical compositions or pharmaceutical formulations. The active agents comprised in the combinations of the present disclosure may be in the form of a pharmaceutical dosage unit, eg a single pharmaceutical dosage unit. The combinations of the present disclosure may be in any dosage form (eg, unit dosage form) known to those of ordinary skill in the art, such as Sugar-coated tablets, tablets, capsules, granules, powders, suppositories, solutions, suspensions, injections (injection solutions or injection suspensions). They are prepared in a manner known per se, for example by conventional mixing, granulating, sugar-coating, dissolving or lyophilization processes. It is to be understood that the dosage unit of each agent contained in an individual dose of each dosage form need not itself constitute an effective amount, since the necessary effective amount can be achieved by administering a plurality of dosage units. A unit dosage form containing a combination of the present disclosure may contain an active agent in an amount normally administered when the active agent is administered alone.

The combinations of the present disclosure may take the form of a kit of parts, in the sense that each active agent may be administered independently or using different fixed combinations with different amounts of the active agents, ie administered simultaneously or at different time points. Subsequently, the active agents included in the kit of parts can be administered simultaneously or chronologically crossed, ie at different time points with respect to any active ingredient of the kit of parts and the time intervals between administrations can be equal or unequal.

Therapeutically effective amounts of each of the active agents in the disclosed combinations (combined therapeutically effective amounts, such as daily doses or intermittent doses of the amounts herein) may be administered simultaneously or sequentially in any order, either separately or as fixed doses. Administer in combination. The active agents of the combinations of the present disclosure may be administered separately at different times during the course of treatment or administered simultaneously in separate or single combinations.

Combinations of the present disclosure can be administered to individuals who have been treated with one or more prior therapies but have subsequently relapsed or metastasized.

Each active agent in the combinations of the present disclosure may be administered to an individual in need thereof in one or more doses.

Each cycle of administration of the combinations of the present disclosure is 28 days (4 weeks).

Combinations of the present disclosure may be administered for at least one cycle, eg, 2-12 or more treatment cycles.

The first active agent in the combination of the present disclosure, such as compound CYH33 or compound 1-27 or a pharmaceutically acceptable salt thereof, can be administered once a day, twice a day or more, once every two days or Once every three days, continuous administration in each cycle, or a regimen of continuous administration for three weeks followed by a one-week rest period. The daily dose of compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof (based on free compound) is 10-50 mg, such as 10-40 mg, 20-40 mg, 20-30 mg, 30-40 mg, such as 10 mg, 20 mg , 30mg, 40mg. Preferably, the initial dose of CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof is 40 mg or 30 mg, for example, the initial dose is 40 mg QD or 30 mg QD. Preferably, compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof is administered continuously in each cycle, with a daily dose of 10 mg, 20 mg, 30 mg or 40 mg, preferably once a day. Preferably, compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof is administered orally.

The PARP1/2 inhibitors in the combinations disclosed herein, such as olaparib, can be administered once a day or twice a day, once every two days, or once every three days, in each cycle, or for three consecutive days. A regimen of one week followed by one week off. The daily dose of olaparib or a pharmaceutically acceptable salt thereof is 200-600 mg, such as 200 mg, 250 mg, 300 mg, 400 mg, 500 mg or 600 mg. Preferably, olaparib is administered continuously in each cycle, with a single dose of 200-300 mg, such as 200 mg, 250 mg or 300 mg, preferably administered twice a day. Preferably, olaparib or a pharmaceutically acceptable salt thereof is administered orally.

The estrogen receptor modulators in the combinations of the present disclosure, such as fulvestrant, can be administered to an individual in need in one or more doses, wherein in the case of multiple doses, the previous dose is followed by, 2, 3 or 4 weeks to administer the next dose. Preferably, it is administered on days 1 and 15 of the first cycle, and on day 1 of each subsequent cycle. Each dose of fulvestrant can be 250mg-500mg, such as 250mg or 500mg, preferably 500mg. Preferably, fulvestrant is administered by intramuscular injection.

The CDK4/6 inhibitors in the combinations of the present disclosure, such as palbociclib or a pharmaceutically acceptable salt thereof, can be administered once a day, once every two days or once every three days, continuously in each cycle, or A regimen of three weeks of continuous administration followed by one week of rest, or two weeks of continuous administration followed by two weeks of rest. The daily dose of palbociclib or a pharmaceutically acceptable salt thereof is 75-125 mg, such as 75 mg, 100 mg, 125 mg. Preferably, palbociclib is administered continuously for three weeks in each cycle and then stopped for one week, with a daily dose of 125 mg, preferably once a day. Preferably, palbociclib or a pharmaceutically acceptable salt thereof is administered orally.

The aromatase inhibitors in the combinations of the present disclosure, such as letrozole or a pharmaceutically acceptable salt thereof, can be administered once daily, once every two days, or once every three days, continuously in each cycle, or continuously A regimen of three weeks on followed by one week off was used. The daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg. Preferably, letrozole is administered continuously in each cycle, with a daily dose of 2.5 mg, preferably once a day. Preferably, letrozole or a pharmaceutically acceptable salt thereof is administered orally.

For example, the first active agent compound CYH33 is administered at a daily dose of about 10-40 mg, such as 20-30 mg, 20-40 mg, 30-40 mg, in combination with the second active agent, or with the second active agent and the third active agent. The active agents are administered in combination.

For example, compound CYH33 is administered in combination with a daily dose of 10-40 mg of olaparib at a daily dose of 200-600 mg. Preferably, compound CYH33 is administered in combination with a daily dose of 20 mg and a daily dose of olaparib 400 mg, 500 mg or 600 mg.

For example, compound CYH33 is administered at a daily dose of 20-40 mg in combination with fulvestrant at a dose of about 500 mg. Compound CYH33 was administered at a daily dose of 20 mg, 30 mg or 40 mg in combination with fulvestrant at a dose of 250 mg or 500 mg.

For example, compound CYH33 is administered at a daily dose of 10-40 mg in combination with a daily dose of palbociclib of 75-125 mg. Compound CYH33 was administered at a daily dose of 10 mg, 20 mg, 30 mg or 40 mg in combination with a daily dose of palbociclib 75-mg, 100 mg or 125 mg.

For example, compound CYH33 is administered at a daily dose of 10-40 mg in combination with a daily dose of fulvestrant 500 mg, and a daily dose of palbociclib 75-125 mg. Compound CYH33 was administered in combination with fulvestrant 500 mg injection and palbociclib 75-125 mg daily dose at a daily dose of 10 mg, 20 mg, 30 mg or 40 mg.

For example, compound CYH33 is administered at a daily dose of 10-40 mg in combination with a daily dose of palbociclib of 75 mg, 100 mg or 125 mg, and a daily dose of letrozole of 2.5 mg. Compound CYH33 was administered at a daily dose of 10 mg, 20 mg, 30 mg or 40 mg in combination with a daily dose of palbociclib 75 mg, 100 mg or 125 mg, and letrozole 2.5 mg.

Effects of combinations of the present disclosure

In humans, the complexity and expense of conducting clinical studies on patients makes this type of test impractical as a primary model of synergy. However, synergy can be measured by observing synergy in one species to predict effects in other species and existing animal models as described herein; Application of potentokinetic methods to predict effective dose ratio ranges and required absolute doses and plasma concentrations in other species. The established correlation between tumor models and the effects observed in humans suggests that synergy in animals can be demonstrated by xenograft models.

The established test model can show that the combination of the present disclosure can obtain the above-mentioned beneficial effects. Those skilled in the art are well able to select relevant test models to demonstrate these beneficial effects. The pharmacological activity of the disclosed combinations was demonstrated in preclinical tumor cells and animal tumor models mainly described below or in clinical studies.

Specifically, CYH33 exhibited a time- and dose-dependent inhibition of the activity of the PI3K pathway in vivo, and was able to dose-dependently inhibit several human esophageal cancer cell line mouse xenograft models (CDX) and patient-derived xenografts. Tumor growth in model (PDX). The antitumor activity of CYH33 in combination with olaparib was evaluated in a mouse subcutaneous xenograft model of the PIK3CA- mutated human ovarian cancer cell line SK-OV-3. Compared with CYH33 and olaparib alone, the combination of CYH33 and olaparib showed stronger antitumor activity.

In addition, the dual or triple drug combination of CYH33 with fulvestrant and palbociclib has a strong inhibitory effect on cell proliferation of ER-positive, HER2-negative, PIK3CA mutant breast cancer cell line T47D cells in vitro cell viability experiments . Calculated by Compusyn software, the two-drug combination of CYH33 and fulvestrant, and CYH33 and palbociclib have a synergistic anti-cell proliferation effect; and the three-drug combination of CYH33+fulvestrant+palbociclib has a synergistic anti-cell proliferation effect effect. In vivo dual- or triple-drug combinations of CYH33 with fulvestrant and palbociclib strongly inhibited tumor growth in a subcutaneous xenograft model of the breast cancer cell line T47D mice. Thus, it is proved that the combined use of CYH33+fulvestrant, CYH33+palbociclib, and CYH33+fulvestrant+palbociclib all have synergistic antitumor activity.

A suitable clinical study may be, for example, an open-label, dose-escalation study in patients with cancer. Such studies specifically demonstrate the synergistic effect of the active ingredients of the combinations of the present disclosure. Beneficial effects on cancer can be determined directly from the results of such studies known to those of ordinary skill in the art. Such studies may be particularly suitable for comparing the effects of monotherapy with the active ingredients and combinations of the present disclosure. In one embodiment, the dose of the first active agent, such as compound CYH33, is escalated until the maximum tolerated dose is reached, and the second active agent is administered at a fixed dose. Alternatively, compound CYH33 can be administered at a fixed dose and the dose of the second active agent can be tapered from the maximum tolerated dose. Each patient may receive daily or intermittent doses of the compound. The efficacy of treatment can be determined in such studies, for example, after 12, 18 or 24 weeks by evaluating symptom scores every 6 weeks.

The following examples illustrate the invention described above without however limiting the scope of the disclosure in any way. The beneficial effects of the disclosed combinations can also be determined by other test models known to those of ordinary skill in the art.

Experimental method for experimental study of cell viability in vitro

Take the cells in the logarithmic growth phase for plating, adjust the cell concentration, add 90 μL of cell suspension to each well of the culture plate, and add the culture solution without cells to the blank control well; put the culture plate at 37°C, 5% CO ₂ Cultivate overnight in an incubator with 100% relative humidity; take 10 μL of different concentrations of CYH33 working solution and add it to the above-mentioned cell culture plate to adjust the final concentration of other active agents. Each group has two replicate wells. Add 10 μL of DMSO-cell culture medium mixture to the blank control (containing cell culture medium but not cells), the final concentration of DMSO is 0.25%, and put the 96-well cell plate back into the incubator for 72 hours . Then add 50 μL (equal to half the volume of the cell culture medium in each well) CellTiter-Glo working solution of Promega CellTiter-Glo Luminescent Cell Viability Detection Kit (Promega-G7573) into each well, and wrap the cell plate with aluminum foil to avoid light; The culture plate was shaken on an orbital shaker for 2 minutes to induce cell lysis, the culture plate was placed at room temperature for 10 minutes to stabilize the luminescence signal, and the luminescence signal (expressed as RLU (relative light units)) was detected on a 2104 EnVision plate reader.

In vitro tumor cell activity experiments use the following formula to calculate the inhibition rate (Inhibition rate, IR ₀ ) of the test compound: IR ₀ (%)=(1-(RLU compound-RLU blank control)/(RLU vehicle control-RLU blank control) )×100%. Calculate the inhibition rate of different concentrations of compounds in Excel, then use the GraphPad Prism (6.02.328) software to use log (inhibitor) vs. response (response)--variable slope (Variable slope) to fit the inhibition curve, and get Relevant parameters, including minimum inhibition rate, maximum inhibition rate and relative IC ₅₀ , were displayed.

The minimum inhibition rate is the Y value corresponding to the plateau at the bottom of the curve, the maximum inhibition rate is the Y value corresponding to the plateau at the top of the curve, the relative _IC50 is the concentration required to bring the curve down to a point halfway between the top and bottom plateaus of the curve, and the absolute IC ₅₀ refers to the drug concentration at which cell viability is inhibited by half.

In vivo efficacy experiments Tumor measurements and animal body weights:

According to the tumor growth status to reflect the effect of treatment. Once tumors were palpable, tumor volume dimensions were assessed twice weekly. Tumor volume dimensions were determined by digital caliper measurements, and tumor volume and animal body weight were measured twice weekly throughout the experiment. The tumor volume V is expressed in mm ³ and calculated according to the following formula: V=0.5 a×b ² , where a and b are the long and short diameters of the tumor, respectively. Body weight change BWC(%) response compared to the percentage of body weight change at the beginning of treatment, the calculation formula is: BWC(%)=(BW _n -BW ₀ )/(BW ₀ )×100%, BW _n and BW ₀ are expressed as Body weight at present and at the beginning of treatment.

T/C and TGI are indicators that reflect tumor (tumor volume) response to treatment. Among them, T/C (%) reflects the relative tumor proliferation rate, that is, the percentage of tumor treatment/control (T/C) value, which is calculated by the following formula:

T/C(%)=(T _RTV /C _RTV )×100, (T _RTV and C _RTV represent the average relative tumor volume (RTV) of the treatment group and the vehicle control group on the treatment day, respectively). The relative tumor volume (RTV) was calculated according to the results of tumor measurement, and the calculation formula was: RTV=Vt/V ₀ , where V ₀ was measured at the beginning of group administration (that is, d ₀ on the first day of treatment) The average tumor volume, Vt is the average tumor volume at each measurement, T _RTV and C _RTV take the data of the same day.

TGI (%) reflects tumor growth inhibition rate. The calculation formula of TGI(%) is: TGI(%)=[1-(T _i -T ₀ )/(V _i -V ₀ )]×100. Among them, T _i , T ₀ , V _i , and V ₀ are the average tumor volume of a certain treatment group at the end of administration, the average tumor volume of this treatment group at the beginning of administration (d ₀ ), and the average tumor volume of the vehicle control group at the end of treatment. The average tumor volume at the beginning of treatment (d ₀ ) in the vehicle control group.

The tumor weight was measured at the end of the experiment, and the relative tumor weight was calculated as: T _W /C _W . T _W and C _W are the average tumor weights of the treatment group and the vehicle control group, respectively.

IR reflects the relative tumor (tumor weight) inhibition rate, and the calculation formula is: IR=(Cw-Tw)/Cw×100%, where Tw and Cw are the average tumor weights of the treatment group and the vehicle control group, respectively.

Statistical Analysis

Compusyn software (1.0) was used for in vitro cell activity experiments to calculate the combination index (CI) value of combination therapy (synergism: CI<0.9; additive effect: 0.9<CI<1.1; antagonism: CI>1.1).

All data in vivo are presented as standard error of the mean (SEM). Tumor volume, tumor weight, and body weight were used for statistical analysis, and the average tumor volume, average tumor weight, and SEM of each group at different time points were calculated. Example 1 Two-tailed t-test was used to evaluate the difference in tumor volume and tumor weight between each drug treatment group and vehicle control group at the end of the drug efficacy experiment. In Examples 3 and 5, Graphpad Prism 6.0 software was used to conduct One-way ANOVA combined with Dunnett's method for multiple comparisons to evaluate the difference in tumor volume between each drug treatment group and the vehicle control group at the end of the drug efficacy experiment. In Examples 1, 3 and 5, Graphpad Prism 6.0 software was also used to conduct two-way ANOVA combined with Tukey's method for multiple comparisons to evaluate the differences in tumor volumes between drug treatment groups during the drug efficacy test. For all statistical evaluations, the significance level was set at p < 0.05 to report significance compared to the control group, ie p < 0.05 was considered statistically significant and p < 0.01 indicated a highly significant statistical difference.

The cell lines SK-OV-3 and T47D breast cancer cell lines used in the experiment were all from the American Type Culture Collection (ATCC), and the product numbers were HTB-77 and HTB-133, respectively.

Example 1

Pharmacodynamic study of CYH33 combined with olaparib in human ovarian cancer SK-OV-3 model on nude mice

In this experiment, the mouse subcutaneous xenograft tumor model of PIK3CA-mutated human ovarian cancer cell line SK-OV-3 was used to evaluate the anti-tumor efficacy of the combination of CYH33 and olaparib. Tumor inhibitory effect of Pali/Olaparib) alone and its combination in BALB/c female nude mice bearing subcutaneous xenografts of SK-OV-3 human ovarian cancer cells. The female mice used were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The tumor tissues in the vigorous growth stage were cut into about 2 mm ³ and inoculated subcutaneously in the right armpit of nude mice under aseptic conditions to observe the tumor occurrence. The subcutaneous transplanted tumors of nude mice were measured with a vernier caliper to measure the diameter of the transplanted tumors. ^When the tumor volume reached 150mm after implantation of the tumor cells in the armpit of the tumor-bearing mice, the tumor-bearing mice were randomly divided into 8 groups (6 mice in each group). Give vehicle control, olaparib single drug (100mg/kg), CYH33 single drug (5, 10, 20 mg/kg), or olaparib (100 mg/kg) and CYH33 (5, 10, 20 mg/kg) The combination therapy of the two drugs was studied for its curative effect. Animals were divided into 8 groups according to the study dosage scheme shown in Table 1, and administered. Oral administration once a day for 21 consecutive days.

Table 1. Study Dosing Regimen

The antitumor efficacy of CYH33 and olaparib alone or in combination in BALB/c female nude mice bearing subcutaneous xenografts of SK-OV-3 human ovarian cancer cells are shown in Figure 1, Figure 2 and Table 2, The study dosage scheme of each group 1-8 in the above chart is shown in Table 1 for groups 1-8. Among them, Figure 1 shows After CYH33 and olaparib were applied alone or in combination to BALB/c female nude mice bearing subcutaneous xenografts of SK-OV-3 human ovarian cancer cells, the tumor growth curves of each treatment group were shown in Figure 2. Tumor body weight at the end of the experiment (after 21 days of administration); data points and columnar data in Figure 1 and Figure 2 represent the average tumor volume and average tumor weight of each group, and the error bars represent the standard error of the mean (SEM). Table 2 shows the average tumor volume of each treatment group on the starting day of administration, as well as the average tumor volume and tumor average weight after 21 days of administration, and the indicators of tumor response to treatment T/C, TGI and IR (%) .

Table 2. Evaluation of antitumor effect of CYH33 combined with olaparib on SK-OV-3 xenograft tumor model

It can be known from Figure 1 and Table 2 that the average tumor volume of the experimental mice in the control group was 2175.14±423.77mm ³ . After 21 days of continuous administration, CYH33 single dose (5, 10, 20mg/kg) dependently inhibited the tumor growth of tumor-bearing mice, T/C were 49.06% (TGI: 48.91%), 29.04% ( TGI: 68.86%), 34.95% (TGI: 75.60%). Olaparib monotherapy (100mg/kg) can also inhibit tumor growth, but the T/C value is higher than 40%, which is 55.44% (TGI: 43.37%). After different doses of CYH33 (5, 10, 20 mg/kg) combined with olaparib (100 mg/kg), the tumor growth was significantly delayed, and the T/C values were all less than 40%, showing that it had achieved preclinical anti-tumor activity Effective standards (such as technical guidelines for non-clinical evaluation of anticancer drugs), were 30.84% (TGI: 70.32%, p <0.01), 20.79% (TGI: 80.36%, p <0.01) and 12.96% (TGI: 87.72%, p <0.01). It can be seen that, compared with the control group, CYH33 combined with olaparib has a statistically significant difference in tumor growth inhibition ( p <0.01). In addition, the combination of the tested CYH33 and olaparib has a stronger tumor inhibitory effect than either of the corresponding single drugs.

In addition, multiple comparative analysis was performed on CYH33 and olaparib alone and in combination groups, using Two-way ANOVA analysis combined with Tukey's multiple comparisons to obtain the differences between the drug treatment groups, and it was found that different doses of CYH33 (5, 10, 20mg/kg) and olaparib (100mg/kg) in combination, the combination group CYH33 (5mg/kg) + olaparib (100mg/kg) ( p <0.01), CYH33 (10mg/kg ) + Olaparib (100mg/kg) ( p <0.0001), CYH33 (20mg/kg) + Olaparib (100mg/kg) ( p <0.0001) and Olaparib (100mg/kg) single drug There are very significant differences compared to each other.

In addition, the effect of the combination of CYH33 and olaparib on tumor weight was also investigated in the experiment. After 21 days of continuous administration, the tumors of mice in each treatment group were weighed. Figure 2 shows the tumor weight of each treatment group after CYH33 and olaparib were applied alone or in combination to BALB/c female nude mice bearing subcutaneous xenograft tumors of SK-OV-3 human ovarian cancer cells. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to blank vehicle control. It can be known from Table 2 and Figure 2 that the average tumor weight of the experimental mice in the control group was 1.923±0.351g. After treatment with CYH33 monotherapy (20mg/kg, 10mg/kg and 5mg/kg), the average tumor weights were 0.440g (IR: 77.12%, p <0.01), 0.575g (IR: 70.10%, p < 0.01) and 0.957g (IR: 50.23%), the average tumor weight of olaparib (100mg/kg) monotherapy was 1.151g (IR: 40.15%). The mean tumor weights of CYH33 (20mg/kg, 10mg/kg, and 5mg/kg) and olaparib (100mg/kg) combined treatment groups were 0.194g, 0.351g, and 0.504g, respectively, and the IR values were 89.91% ( p <0.01), 81.75% ( p <0.01), and 73.79% ( p <0.01). The study also showed that the combination of CYH33 and olaparib has a stronger tumor inhibitory effect than the corresponding dose of single drug.

In summary, in the SK-OV-3 human ovarian cancer mouse subcutaneous xenograft tumor model, CYH33 single drug, olaparib single drug, and CYH33 combined with olaparib can inhibit tumor growth. From the perspective of tumor volume and tumor weight, the combination of CYH33 and olaparib has a stronger tumor inhibitory effect than either of the corresponding single drugs. At the same time, it was found that the T/C values of CYH33 (5mg/kg) and olaparib (100mg/kg) alone were greater than 40%, which could not reach the standard of effective anti-tumor activity in preclinical. Reach the effective standard of anti-tumor activity. Multiple statistical analysis showed that after different doses of CYH33 (5, 10, 20 mg/kg) combined with olaparib (100 mg/kg), there was a significant difference between the combination group and olaparib alone.

These results show that the combination of CYH33 and olaparib can significantly inhibit tumor growth, suggesting that the combination of the two drugs also has similar effects in clinical practice. The combination of CYH33 and olaparib showed a stronger anti-tumor effect than the corresponding single drug, suggesting that the combination of the two drugs can delay the progression of cancer or provide clinical benefits for the treatment of cancer patients, especially for patients with Patients with ovarian cancer, breast cancer, prostate cancer, bile duct cancer, endometrial cancer, fallopian tube or primary peritoneal cancer, etc.

Example 2

The antiproliferative effect of CYH33 combined with fulvestrant or palbociclib in T47D breast cancer cell line was studied by cell viability assay in vitro

1. Experimental method

The T47D breast cancer tumor cell line (ER+, HER2-, PIK3CA mutation) was cultured in an incubator at 37°C and 5% CO ₂ . Passaging regularly, cells in the logarithmic growth phase were taken for plating, and CYH33+fulvestrant, CYH33+palbociclib, and each single drug were subjected to cell plating and compound treatment respectively. Among them, CYH33: start at 10 μM, 3-fold dilution, 9 concentration gradients, fulvestrant: start at 1 μM, 3-fold dilution, 9 concentration gradients, palbociclib: start at 10 μM, 3-fold dilution, 9 concentrations gradient; the final concentrations of CYH33 and palbociclib were 10, 3.3333, 1.1111, 0.3704, 0.1235, 0.0412, 0.0137, 0.0046, 0.0015 μM, and the final concentrations of fulvestrant were 1, 0.333, 0.111, 0.037, 0.01235 , 0.00412, 0.00137, 0.00046, 0.00015 μM. After the cells were cultured for 72 hours, the luminescent signal was detected according to the Promega CellTiter-Glo Luminescent Cell Viability Detection Kit (Promega-G7573). The results are shown in Table 3 and Table 4.

2. Experimental results

The test results of CYH33 combined with palbociclib are shown in Table 3. CYH33 alone has a strong inhibitory effect on cell proliferation of T47D cells, with a relative IC ₅₀ of 0.075 μM and a maximum inhibition rate of 74.65%. The highest inhibition rate within the range was 40.13%, and the relative IC ₅₀ was 8.061μM. When the two drugs were used in combination, the highest inhibitory rate of palbociclib within the concentration setting range was 78.54%, and the relative IC ₅₀ was 0.015 μM. The CI values under each Fa were analyzed by Compusyn software, as shown in Table 4. The CI values under each Fa were all less than 0.9, indicating a synergistic effect; and the best CI value was 0.19488.

Similarly, as shown in Table 3, the results of combined use of CYH33 and fulvestrant showed that CYH33 alone had a strong inhibitory effect on T47D cell proliferation, with a relative IC ₅₀ of 0.075 μM and the highest inhibition rate of 74.65%; fulvestrant The highest inhibitory rate of Sitran within the concentration setting range was 35.15%, and the relative IC ₅₀ was 0.001μM. When the two drugs were used in combination, the highest inhibition rate was 75.64% within the concentration setting range, and the relative IC ₅₀ was 0.001 μM. The CI values under each Fa were analyzed by Compusyn software, as shown in Table 4. When Fa<0.9, the CI values were all less than 0.9, indicating a synergistic effect; and the best CI value was 0.11425.

Table 3. Anti-cell proliferation parameters of CYH33 combined with fulvestrant or palbociclib in T47D breast cancer cell lines

Table 4. Fa-CI values of CYH33 combined with palbociclib, CYH33 combined with fulvestrant in T47D cells

Example 3

Pharmacodynamic study of CYH33 combined with fulvestrant or palbociclib in nude mouse model of breast cancer

In this study, the combination of CYH33 with fulvestrant and palbociclib respectively was evaluated in the treatment of estrogen receptor-positive, PIK3CA -mutated human breast cancer T47D cell BALB/c (ER+, HER2-, PIK3CA mutated) nude mice subcutaneously. In vivo drug efficacy in a xenograft tumor model. The female mice used were purchased from Shanghai Xipuer-Bikay Experimental Animal Co., Ltd. Two days before cell inoculation, a 0.18 mg 17 β -estradiol tablet was implanted subcutaneously in the left upper limb of the mouse. When the average volume of the tumor reached about 193 mm ^{3 ,} it was randomly divided into 12 groups (6 rats in each group) for administration. CYH33 and palbociclib were administered orally once a day for 35 consecutive days, and fulvestrant was administered subcutaneously once a week for a total of 6 times.

The antitumor efficacy of CYH33, fulvestrant or palbociclib alone or in combination in T47D xenografted female BALB/c nude mice is shown in Figure 3, Table 5 and Table 6. Among them, Figure 3 shows the tumor volume growth curves of each treatment group after CYH33, fulvestrant or palbociclib were applied alone or in combination to T47D xenografted female BALB/c nude mice. Among them, the data points represent the average tumor volume in each group (see Table 5 for each group). Error bars represent standard error of the mean (SEM). Table 5 shows the average tumor volume of each treatment group on the starting day of administration, the average tumor volume after 35 days of administration, and the indicators T/C and TGI of tumor response to treatment.

As shown in Figure 3a and Table 5, after 35 days of continuous administration, CYH33 can inhibit tumor growth in a dose-dependent manner at doses of 2.5 mg/kg, 5 mg/kg, and 10 mg/kg. The tumor growth rates (TGI) were 30.15%, 36.70%, and 77.67%, respectively, and the relative tumor proliferation rates (T/C) were 75.51%, 70.10%, and 36.71%, respectively. Single drug CYH33 at 2.5mg/kg and 5mg/kg did not reach the effective standard of preclinical anti-tumor activity. The positive drug fulvestrant (2mg/mouse) alone showed a certain anti-tumor effect (TGI=52.00%, T/C=57.62%), which did not reach the standard of effective anti-tumor activity in preclinical. Compared with CYH33 single drug and fulvestrant single drug, CYH33 (2.5mg/kg, 5mg/kg, 10mg/kg) and fulvestrant (2mg/mouse) combined anti-tumor effect is stronger than the single drug, all strongly inhibit tumor growth, TGI were 74.28%, 89.63% and 84.82%, T/C value was less than 40%, respectively, 39.45%, 39.45%, 26.94% and 30.87%. And the combination group of CYH33 (5mg/kg) and fulvestrant (2mg/mous) has a synergistic anti-tumor effect compared with single drug (for example, TGI of the combination group (89.63%)>CYH33 (5mg/kg) TGI (36.7%) + Fulvestrant (2mg/mouse) TGI (52%).

Table 5. Evaluation of antitumor efficacy of CYH33 combined with fulvestrant on T47D xenograft tumor model (based on tumor volume)

In addition, multiple comparisons were made for the tumor volume data of each group of CYH33 and fulvestrant alone and in combination, and the difference between each drug treatment group was obtained by using Two-way ANOVA analysis combined with Tukey's method for multiple comparisons. Results showed that CYH33 (2.5mg/kg) combined with fulvestrant (2mg/mouse) ( p <0.05), and CYH33 (5mg/kg) combined with fulvestrant (2mg/mouse) ( p <0.001 ) anti-tumor effect, compared with the corresponding CYH33 single drug group, all have significant statistical significance.

As shown in Figure 3b and Table 6, the positive drug palbociclib (45mg/kg) alone showed a strong tumor inhibitory effect (TGI=75.85%, T/C=38.19%). Combination of CYH33 (2.5mg/kg, 5mg/kg, 10mg/kg) and palbociclib (45mg/kg) respectively, all potently inhibited tumor growth, TGI were 93.07%, 99.52% and 112.39%, respectively, T/C They were 24.15%, 18.89% and 7.96%, respectively, all less than 40%, reaching the standard of effective anti-tumor activity in preclinical.

Table 6. Evaluation of antitumor efficacy of CYH33 combined with palbociclib on T47D xenograft tumor model (based on tumor volume)

In addition, multiple comparisons were made for the tumor volume data of each group of CYH33 and palbociclib alone and in combination, and the difference between each drug treatment group was obtained by using Two-way ANOVA analysis combined with Tukey's method for multiple comparisons. CYH33 (2.5 mg/kg) in combination with palbociclib (45 mg/kg) ( p <0.0001), and CYH33 (5 mg/kg) in combination with palbociclib (45 mg/kg) ( p <0.0001) Compared with the corresponding CYH33 single-drug group, the anti-tumor effect of the group was statistically significant.

In summary, in the estrogen receptor-positive, PIK3CA- mutated human breast cancer T47D cell BALB/c nude mouse subcutaneous xenograft tumor model, the combination of CYH33 and fulvestrant, CYH33 and palbociclib showed stronger effects than Corresponding to the antitumor effect of any single drug. The T/C values of the combination group were all less than 40%, reaching the standard of effective preclinical antitumor activity. And it can be seen from the TGI value that the combination of CYH33 (5mg/kg) and fulvestrant (2mg/mouse) has a synergistic anti-tumor effect. In addition, multiple statistical analysis showed that the anti-tumor effects of the dual groups of CYH33 (2.5mg/kg, 5mg/kg) and fulvestrant (2mg/mouse) and palbociclib (45mg/kg) respectively, compared with the corresponding Compared with the CYH33 single drug group, there were significant statistical significance.

Example 4

The antiproliferative effect of CHY33 combined with fulvestrant and palbociclib in T47D breast cancer cell line was studied by cell viability assay in vitro.

The T47D breast cancer tumor cell line (ER+, HER2-, PIK3CA mutation) was cultured in an incubator at 37°C and 5% CO ₂ . Passage regularly, and take cells in logarithmic growth phase for plating. CYH33, fulvestrant and palbociclib were subjected to cell plating and compound treatment. Among them, CYH33: 2 β M starting, 2-fold dilution, 9 concentration gradients; fulvestrant: 0.05 β M starting, 2-fold dilution, 9 concentration gradients; palbociclib: 10 β M starting, 2 1-fold dilution, 9 concentration gradients. After the cells were cultured for 72 hours, the luminescent signal was detected according to the Promega CellTiter-Glo Luminescent Cell Viability Detection Kit (Promega-G7573). The anti-cell proliferation effect of CYH33 and palbociclib, CYH33 and fulvestrant, and CYH33 combined with palbociclib and fulvestrant in breast cancer cell line T47D were studied. The results are shown in Table 7 and Table 8, wherein plate numbers 5 and 6 in Table 7 correspond to plates 5 and 6 in Table 8.

Table 7. Anti-cell proliferation parameters of CHY33 combined with fulvestrant and palbociclib in T47D breast cancer cell lines

Table 8. Fa-CI values of CYH33 combined with fulvestrant and/or palbociclib in T47D cells

The anti-cell proliferation effect of CYH33 combined with fulvestrant + palbociclib showed that CYH33 alone had a strong inhibitory effect on cell proliferation on T47D cells, with a relative IC ₅₀ of 0.066 μM and a maximum inhibition rate of 77.16%. The highest inhibitory rate of Setran + Palbociclib in the concentration setting range was 57.91%, and the relative IC ₅₀ was 0.025μM. The minimum relative IC ₅₀ was 0.002μM, and the highest inhibitory rate was 100% when the three drugs were used in combination. Analyzed by Compusyn software The CI values under each Fa are shown in Table 8. When Fa<0.8, the CI values are all less than 0.9, indicating a synergistic effect.

The anti-cell proliferation effect of palbociclib combined with CYH33 + fulvestrant showed that palbociclib alone had a strong inhibitory effect on cell proliferation of T47D cells, with a relative IC ₅₀ of 0.916 μM and a maximum inhibition rate of 59.74%. CYH33+ The highest inhibition rate of fulvestrant within the concentration setting range was 75.57%, and the relative IC ₅₀ was 0.010μM. When the three drugs were used in combination, the relative IC ₅₀ was 0.001μM, and the highest inhibition rate was 88.42%. The CI values are shown in Table 8. When Fa<0.7, the CI values are all less than 0.9, indicating a synergistic effect.

Example 5

Pharmacodynamic study of CYH33 combined with fulvestrant and palbociclib in T47D nude mouse breast cancer model

In the breast cancer T47D xenograft tumor model, CYH33, fulvestrant, and palbociclib were evaluated as a single agent, two-drug combination or three-drug combination respectively in the treatment of estrogen receptor-positive, PIK3CA- mutated human breast cancer T47D cells BALB /c (ER+, HER2-, PIK3CA mutation) in vivo antitumor efficacy in subcutaneous xenograft tumor model in nude mice. The mice used were purchased from Shanghai Xipuer-Bikay Experimental Animal Co., Ltd. Two days before cell inoculation, a 0.18 mg 17 β -estradiol tablet was implanted subcutaneously in the left upper limb of the mouse. When the average tumor volume reached about 197mm ^{3 ,} they were randomly divided into 14 groups (6 rats in each group) for administration. CYH33 and palbociclib were administered orally once a day for 28 consecutive days. Fulvestrant was administered subcutaneously once a week for a total of 5 administrations.

The anti-tumor efficacy of CYH33 combined with fulvestrant and palbociclib in nude mouse breast cancer model is shown in Figure 4, Table 9-Table 11. Figure 4 shows the tumor volume growth curves for each treatment group. Data points in Figure 4 represent mean tumor volumes within each group and error bars represent standard error of the mean (SEM). Table 9 shows the average tumor volume of each treatment group on the starting day of administration, as well as the average tumor volume and the indicators of tumor response to treatment T/C and TGI after 28 days of administration. Tables 10, 11, and 12 respectively show the results of multiple comparative analysis among the groups of CYH33, fulvestrant, and palbociclib alone, in combination with two drugs, and in combination with three drugs.

As shown in Figure 4 and Table 9, on PG-D28 (day 28 after treatment), the average tumor volume of the experimental mice in the control group reached 1337±170 mm ³ . Single drug CYH33 can inhibit tumor growth in a dose-dependent manner at doses of 5 mg/kg and 10 mg/kg, with tumor growth inhibition rates (TGI) of 46.41% and 62.02% and relative tumor proliferation rates (T/C) of 60.26% respectively and 47.16%; the T/C of single-agent fulvestrant (2mg/mouse) was 59.29%, neither of which reached the effective standard of preclinical anti-tumor activity. After CYH33 (5 mg/kg) combined with fulvestrant (2 mg), the T/C was 36.1%, reaching the standard of effective preclinical anti-tumor activity, and CYH33 (5 mg/kg, 10 mg/kg) combined with fulvestrant The tumor inhibitory effect of the combined use of the group (2mg) (TGI: 74.91%, 69.82%; T/C: 36.1%, 40.42%) was stronger than that of the CYH33 single-drug corresponding dose group or the fulvestrant single-drug therapy group ( TGI is 47.82%, T/C is 59.29%).

The TGI of palbociclib (20mg/kg) was 25.8%, and the T/C was 77.85%, which did not reach the effective standard of preclinical anti-tumor activity; Beciclib (20mg/kg) combined, TGI were 68.58%, 66.32%, 85.55%, T/C were 41.51%, 43.3%, 27.06%, respectively, the tumor inhibitory effect was stronger than the corresponding dose group of CYH33 monotherapy or Piper Beciclib monotherapy group.

CYH33 (2.5mg/kg, 5mg/kg, 10mg/kg) combined with fulvestrant (2mg) and palbociclib (20mg/kg) respectively, the TGI were 91.9%, 96.61%, 102.81%, respectively, T/C were 21.63%, 17.63%, and 12.34%, respectively, and the anti-tumor effect of the three-drug combination was stronger than that of the single-drug therapy group, CYH33+fulvestrant, CYH33+palbociclib, or fulvestrant+palbociclib Sealy (TGI is 60.37%, T/C is 48.55%) dual drug group. And the results show that the CYH33 (10mg/kg) + fulvestrant (2mg/mouse) and palbociclib (20mg/kg) combination group is more effective than the corresponding double CYH33 + fulvestrant, and palbociclib alone Drugs have a synergistic antitumor effect.

Table 9. Evaluation of antitumor efficacy of CYH33 combined with fulvestrant and palbociclib on T47D xenograft tumor model (based on tumor volume)

In addition, multiple comparative analysis was performed on CYH33, fulvestrant, palbociclib single drug and their three-drug combination group, and the experimental results are shown in Table 10. The three-drug combination of CYH33 (2.5mg/kg, 5mg/kg, 10mg/kg) and fulvestrant (2mg) + palbociclib (20mg/kg) respectively and the corresponding CYH33, fulvestrant, palbociclib Compared with drugs, there is a very significant statistical significance.

Table 10. Multiple comparisons of tumor volume data (CYH33, fulvestrant, palbociclib and three-drug combination)

In addition, a multiple comparison analysis was performed for each treatment group of CYH33 monotherapy and the current standard treatment fulvestrant + palbociclib combination. The experimental results are shown in Table 11. The three-drug combination of CYH33 (2.5mg/kg, 5mg/kg, 10mg/kg) and fulvestrant (2mg) + palbociclib (20mg/kg) respectively and the corresponding standard treatment of fulvestrant + palbociclib Compared with the two joints, there is a very significant statistical significance. In addition, the three-drug combination of CYH33 (5mg/kg, 10mg/kg) with fulvestrant (2mg) + palbociclib (20mg/kg) and the corresponding CYH33 single drug group or fulvestrant + palbociclib Compared with the two groups, there is a very significant statistical significance. It shows that the combination of CYH33 (5mg/kg or 10mg/kg) and standard treatment fulvestrant (2mg) + palbociclib (20mg/kg) has a synergistic anti-tumor effect.

Table 11. Multiple comparisons of tumor volume data (CYH33 and fulvestrant + palbociclib)

Similarly, multiple comparative analyzes were performed on the treatment groups of fulvestrant monotherapy and dual CYH33 + palbociclib respectively. The experimental results showed that fulvestrant (2 mg) was compared with CYH33 (2.5 The three-drug combination of 5 mg/kg, 10 mg/kg) and palbociclib (20 mg/kg) has a very significant statistical significance compared with the corresponding fulvestrant single drug. In addition, the three-drug combination of fulvestrant (2 mg) and CYH33 (5 mg/kg) + palbociclib (20 mg/kg) was compared with the corresponding fulvestrant single drug group, and compared with the corresponding double CYH33 + palbociclib Compared with Besili, there are extremely significant statistical significance. This indicates that fulvestrant monotherapy and dual CYH33 (5 mg/kg) + palbociclib (20 mg/kg) have synergistic anti-tumor effects.

A multiple comparison analysis was performed for each treatment group of single-agent palbociclib and the combination of dual CYH33+fulvestrant. The experimental results showed that the three-drug combination of CYH33 (2.5mg/kg, 5mg/kg, 10mg/kg) with fulvestrant (2mg) and palbociclib (20mg/kg) respectively compared with the corresponding palbociclib single drug, All There is a very significant statistical significance. At the same time, it was found that palbociclib (20mg/kg), CYH33 (10mg/kg), fulvestrant (2mg) triple combination and single drug palbociclib (20mg/kg) and double CYH33 (10mg/kg) + fulvestrant Compared with the group, there were extremely significant statistical differences. It shows that the combination of single drug palbociclib (20mg/kg) and dual CYH33 (10mg/kg) + fulvestrant has a synergistic anti-tumor effect.

In summary, in the estrogen receptor positive, PIK3CA -mutated human breast cancer T47D cell BALB/c nude mouse subcutaneous xenograft tumor model, CYH33 combined with fulvestrant and palbociclib showed a stronger effect than Corresponding to the antitumor effect of any single drug. The T/C of the three-drug combination group was less than 40%, reaching the standard of effective anti-tumor activity in preclinical. According to the TGI value, it can be seen that the combination of CYH33 (10mg/kg) + fulvestrant and palbociclib has a synergistic anti-tumor effect.

Moreover, the statistical analysis showed that the anti-tumor effect of the three-drug combination in the three dose groups tested was significantly different from that of any single drug, compared with the standard treatment fulvestrant + palbociclib. , also has extremely significant statistical significance, even the three-drug combination with CYH33 at 5mg/kg is still extremely statistically significant compared with the corresponding combination of CYH33+palbociclib ( p <0.01), and CYH33 at 10mg/kg Compared with the corresponding combination of CYH33+fulvestrant, the three-drug combination is still very statistically significant ( p <0.001), showing that the three-drug combination has a synergistic anti-tumor effect.

Example 6

Clinical trial of combination of CYH33 and olaparib

A clinical study was conducted to clinically evaluate the safety and tolerability of the combination of CYH33 and olaparib in patients with advanced solid tumors. In this study, the dose and administration regimen of CYH33 and olaparib can be selected based on the following existing information: human safety, efficacy and PK information of CYH33, preclinical safety, efficacy and PK information of olaparib, in In the dose escalation and dose expansion section, determine the MTD (maximum tolerated dose) or RP2D (phase II recommended dose) of the combined drug. In the dose-expansion portion of this study, the aim was to In evaluating the clinical efficacy of the drug-drug interaction potential of the combination and the drug-drug interaction potential of the combination in patients with advanced solid tumors when CYH33 was administered in combination with olaparib to determine the parameters described in Table 12 Goals and associated endpoints.

Table 12. Objectives and relevant endpoints of the clinical trials of the combination of CYH33 and olaparib

Dosing regimen:

The experimental drugs are CYH33 and olaparib, and the treatment plan is combination therapy.

CYH33:

-A: The starting dose is 10 mg orally administered once a day (QD), 28 days as a cycle.

-B: The initial dose is 20 mg QD orally, and 28 days is a cycle.

-C: The initial dose is 30 mg QD orally, and 28 days is a cycle.

-D: The initial dose is 40mg QD orally administered, 28 days as a cycle.

Orapali:

-A: The starting dose is 300 mg, administered orally twice a day (BID), 28 days as a cycle.

-B: The starting dose is 250 mg, administered orally twice a day (BID), 28 days as a cycle.

-C: The initial dose is 200 mg, administered orally twice a day (BID), and 28 days is a cycle.

Indications (enrolled tumor types):

Advanced solid tumors (including but not limited to ovarian cancer, fallopian tube cancer, primary peritoneal cancer, prostate cancer, endometrial cancer, etc.).

Example 7

Safety, tolerability, pharmacokinetic profile and preliminary efficacy of CYH33 combined with endocrine therapy with or without palbociclib PIK3CA mutation, HR+, HER2- locally advanced, recurrent or metastatic breast cancer Open phase Ib clinical trial research

This study aimed to evaluate CYH33+fulvestrant, CYH33+fulvestrant+palbociclib, and CYH33+letrozole in patients with PIK3CA- mutant, HR+, HER2-locally advanced, recurrent or metastatic breast cancer who failed prior line therapy. +Safety, tolerability, pharmacokinetic profile and preliminary efficacy of palbociclib.

The purpose and endpoint of the study are shown in Table 13 below:

Table 13.

Dosing regimen:

The experimental drug is CYH33 combined with fulvestrant or letrozole, with or without palbociclib, and the treatment plan is combination therapy.

CYH33:

-A: The initial dose is 30 mg orally administered once a day (QD), 28 days as a cycle.

-B: The initial dose is 40mg QD orally administered, 28 days as a cycle.

-C: The starting dose is 20 mg QD orally, 28 days as a cycle.

-D: The initial dose is 10 mg QD orally, and 28 days is a cycle.

Fulvestrant:

Buttock injection: In the first cycle, inject 500 mg on the first day and on the 15th day, and inject 500 mg on the first day of the subsequent cycle. 28 days is a cycle.

Letrozole:

Oral administration of 2.5mg QD, 28 days as a cycle.

Palbociclib:

-A: 125mg QD orally administered, continuous medication for 3 weeks and 1 week off, 28 days as a cycle.

-B: Oral administration of 100mg QD, continuous medication for 3 weeks and rest for 1 week, 28 days as a cycle.

-C: Oral administration of 75mg QD, taking medicine continuously for 3 weeks and stopping for 1 week, 28 days as a cycle.

Indications (enrolled tumor types):

PIK3CA-mutated, HR+, HER2- advanced breast cancer.

Claims (26)

A drug combination comprising: (a) a first active agent which is a compound of formula If or a compound of formula Ig or a pharmaceutically acceptable salt thereof,

in, R ₁ is -NR ₅ R ₆ ; _R2 is

; R ₅ and R ₆ are each independently a C ₁ -C ₄ alkyl group, or an unsubstituted or substituted piperazine ring formed together with their connected nitrogen atom, the substituent being -S(O) ₂ R ₁₂ ; R ₇ , R ₈ , R ₉ and R ₁₀ are each independently H or C ₁ -C ₃ alkyl, or R ₂ is

; R ₁₁ is C ₁ -C ₄ alkyl, benzyl unsubstituted or substituted by one or more substituents, phenyl unsubstituted or substituted by one or more substituents, unsubstituted or substituted by one or more substituents Isoxazolyl substituted with radical, or pyridyl unsubstituted or substituted by one or more substituents selected from halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy group, -CF ₃ , -C(O)OR ₁₂ , -C(O)NR ₁₂ R ₁₅ ,

R ₁₂ and R ₁₅ are each independently C ₁ -C ₃ alkyl; and (b) the second active agent, which is selected from any one or any two of PARP1/2 inhibitors, estrogen receptor modulators, and CDK4/6 inhibitors, or a pharmaceutically acceptable salt thereof; Optionally, (c) a third active agent, which is an aromatase inhibitor or a pharmaceutically acceptable salt thereof, is also included. The pharmaceutical combination as claimed in item 1, wherein the first active agent is a compound CYH33 having the following formula or a pharmaceutically acceptable salt thereof

or a compound I-27 of the following formula or a pharmaceutically acceptable salt thereof

The drug combination according to claim 1 or 2, wherein the PARP1/2 inhibitor is olaparib, rucaparib, niraparib, talazoparib, fluzoparib or pamiparib , preferably olaparib; estrogen receptor modulator is fulvestrant, zoxifene, toremifene, raloxifene, or tamoxifen, preferably fulvestrant; CDK4/6 inhibitor The agent is selected from palbociclib, ribociclib or abeciclib, preferably palbociclib. The drug combination according to any one of claims 1 to 3, wherein the aromatase inhibitor is letrozole, anastrozole, exemestane or vorozole, preferably letrozole. The drug combination according to any one of claims 1 to 4, comprising: (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) Any one or two of olaparib, fulvestrant and palbociclib. The drug combination according to any one of claims 1 to 5, comprising: (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, (b) any one or any two of olaparib, fulvestrant and palbociclib, and (c) Letrozole. The drug combination according to any one of claims 1 to 6, comprising: (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) Orapali; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) Fulvestrant; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) palbociclib; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) olaparib and fulvestrant; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) olaparib and palbociclib; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) Orapali, and (c) letrozole; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) fulvestrant and palbociclib; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) fulvestrant, and (c) letrozole; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) palbociclib, and (c) letrozole; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) olaparib and fulvestrant, and (c) letrozole; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) fulvestrant and palbociclib, and (c) letrozole; or (a) a first active agent selected from compound CYH33 or compound I-27 or a pharmaceutically acceptable salt thereof, and (b) olaparib and palbociclib, and (c) Letrozole. The pharmaceutical combination according to any one of claims 1 to 7, wherein the first active agent is compound CYH33 or a pharmaceutically acceptable salt thereof, preferably the hydrochloride or methanesulfonate of compound CYH33. The pharmaceutical combination as described in any one of claims 1 to 8, wherein the compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10-40 mg, 20-40 mg, 20-30 mg or 30-40 mg, such as In the form of dosage units of 10 mg, 20 mg, 30 mg or 40 mg, preferably an oral dosage form. The pharmaceutical combination as described in any one of claims 1 to 9, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10-40 mg, 20-40 mg, 20-30 mg or 30- 40 mg, eg 10 mg, 20 mg, 30 mg or 40 mg. The drug combination according to any one of claims 1 to 10, wherein CYH33 or a pharmaceutically acceptable salt thereof is administered continuously in each cycle, preferably once a day. The drug combination as claimed in any one of claims 1 to 11, wherein each cycle is 4 weeks; and the drug combination is administered for at least one cycle, such as 2-12 or more cycles. The pharmaceutical combination according to any one of claims 1 to 12, wherein The drug combination is administered every 4 weeks, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg or 40 mg, once a day; and wherein the dose of olaparib The daily dose is 400mg-600mg, administered twice a day; or The drug combination is administered every 4 weeks, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg or 40 mg, once a day; and wherein fulvestrant A single dose of 500 mg administered on Days 1 and 15 of the first cycle and on Day 1 of each subsequent cycle; or The drug combination is administered every 4 weeks, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg or 40 mg, once a day; and wherein fulvestrant The single administration dose is 500 mg, administered on the 1st and 15th days of the first cycle, and administered on the 1st day of each subsequent cycle; and wherein the daily dose of palbociclib is 125 mg, 100 mg or 75 mg, in Each cycle was administered once a day for three consecutive weeks, followed by a one-week withdrawal; or The drug combination is administered every 4 weeks, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg or 40 mg, once a day; and wherein the daily dose of letrozole The dose is 2.5mg, once a day; or The drug combination is administered every 4 weeks, wherein the daily dose of compound CYH33 or a pharmaceutically acceptable salt thereof is 10-50 mg, such as 10 mg, 20 mg, 30 mg or 40 mg, once a day; and wherein the daily dose of letrozole The dose is 2.5 mg, once a day; and the daily dose of palbociclib is 125 mg, 100 mg or 75 mg, administered once a day for three consecutive weeks in each cycle, and then stopped for one week. The pharmaceutical combination according to any one of claims 1 to 13, wherein the respective active agents can be administered separately, simultaneously or sequentially. The pharmaceutical combination according to any one of claims 1 to 14, which is used for treating or preventing a disease in an individual in need thereof, the disease is cancer, preferably a solid tumor, such as an advanced solid tumor; more preferably, the The disease is selected from ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, bile duct cancer or prostate cancer. The drug combination as claimed in item 15, wherein the disease carries DNA damage repair gene mutation, PIK3CA gene mutation, resistance to PARP inhibitors and/or resistance to platinum chemotherapy agents. The drug combination according to claim 15 or 16, wherein the disease is HR+, HER2- and/or PIK3CA gene mutation advanced breast cancer, especially HR+, HER2- and PIK3CA gene mutation advanced breast cancer. A method for treating or preventing a disease, the method comprising administering an effective amount of the drug combination as described in any one of claims 1 to 14 to an individual in need, wherein the disease is cancer, more Preferably a solid tumor, such as an advanced solid tumor; more preferably, the disease is selected from ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, primary peritoneal cancer, bile duct cancer or prostate cancer. The method according to claim 18, wherein the disease carries DNA damage repair gene mutation, PIK3CA gene mutation, resistance to PARP inhibitors and/or resistance to platinum chemotherapy agents. The method according to claim 18 or 19, wherein the disease is HR+, HER2- and/or PIK3CA gene mutation advanced breast cancer, especially HR+, HER2- and PIK3CA gene mutation advanced breast cancer. A use of the drug combination as described in any one of claims 1 to 14 in the preparation of drugs for the treatment of diseases, the disease is selected from cancer, preferably solid tumors, such as advanced solid tumors; more preferably, the disease is selected from From ovarian, breast, endometrial, fallopian tube, primary peritoneal, bile duct, or prostate cancer. The use as described in claim 21, wherein the disease carries DNA damage repair gene mutation, PIK3CA gene mutation, resistance to PARP inhibitors and/or resistance to platinum chemotherapy agents. The use as described in claim 21 or 22, wherein the disease is HR+, HER2- and/or PIK3CA gene mutation advanced breast cancer, especially HR+, HER2- and PIK3CA gene mutation advanced breast cancer. A combined preparation comprising the drug combination as described in any one of claims 1 to 17. A pharmaceutical composition, which comprises the pharmaceutical combination as described in any one of claims 1 to 17, and one or more pharmaceutically acceptable excipients. A kit of parts comprising the pharmaceutical combination as claimed in any one of claims 1 to 17 and instructions for simultaneous, separate or sequential administration of the active agents, preferably wherein the active agents are in the form of pharmaceutical dosage units.

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