Use of PI3kα inhibitor in preparation of drug for treating diseases related to PIK3ca or / and TEK gene mutation

Abstract

The present invention provides a use of a PI3Kα inhibitor in the preparation of a drug for treating diseases related to PIK3CA or / and TEK gene mutation. Specifically, the present invention provides a use of a PI3Kα inhibitor CYH33 or a pharmaceutical composition comprising CYH33 in the preparation of a drug for preventing, treating or alleviating PIK3CA-related overgrowth syndrome (PROS) and / or PIK3CA-related vascular malformation (PRVM), and / or vascular malformation related to TEK gene mutation.

Description

Use of PI3Kα inhibitors in the preparation of drugs for treating diseases related to PIK3CA and / or TEK gene mutations Technical Field

[0001] This application claims priority to Chinese Patent Application No. 202411761669.2, filed with the China National Intellectual Property Administration on December 3, 2024, which is incorporated herein by reference in its entirety.

[0002] This invention relates to novel uses of PI3Kα inhibitors, and more particularly to the use of CYH33 in the preparation of medicaments for the prevention or treatment of disorders or diseases associated with PIK3CA and / or TEK gene mutations. Background Technology

[0003] PROS is a collective term for a series of PIK3CA-related overgrowth disease lineages. Its pathogenesis is caused by activating mutations in the oncogene PIK3CA, leading to abnormal activation of the PI3K / AKT / mTOR signaling pathway. This results in segmental and asymmetrical excessive or invasive growth of tissues in various parts of the body, with or without vascular lesions, leading to rare diseases of excessive growth in multiple sites, such as fibrofatty hyperplasia or overgrowth, fibrofatty infiltrative lipomatosis / facial infiltrative lipomatosis, macrodactyly, dysplastic megalencephaly, unilateral hyperplasia with multiple lipomatosis, megalencephaly-capillary malformation, Klippel-Trenaunay syndrome, CLOVES syndrome, and a series of related rare syndromes.

[0004] Vascular abnormalities can be broadly classified into hemangiomas and vascular malformations. Vascular malformations, based on their complexity, mainly include simple vascular malformations, mixed vascular malformations, and other types. Simple vascular malformations can be further divided into capillary malformations (CM), lymphatic malformations (LM), venous malformations (VM), arteriovenous malformations (AVM), and arteriovenous fistulas (AVF). LM includes ordinary (cystic) lymphatic malformations, generalized lymphatic malformations (GLA), and lymphatic malformations in Gorham-Stout syndrome (GSD). VM includes ordinary venous malformations, familial mucocutaneous venous malformations (VMCM), and venous malformations in blue rubber nevus (BENBNS) syndrome. Vascular malformations can be classified into central nervous system (CNS) vascular malformations and peripheral vascular malformations based on their location. For example, cavernous malformation (CCM) is a common vascular malformation of the CNS and can be divided into familial hereditary type and sporadic type; about 85% of CCM cases are sporadic.

[0005] The causes of vascular malformations are multifaceted, including mutations in the TEK gene encoding the endothelial receptor tyrosine kinase TIE2, mutations in the oncogene PIK3CA, a combination of TEK and PIK3CA mutations, or other mutations.

[0006] PIK3CA, encoding the catalytic subunit of phosphatidylinositol-3-kinase (PI3K), is an oncogene. Activating mutations in PIK3CA, besides being associated with the development of various malignant tumors, also trigger a series of other PIK3CA-related diseases (especially PIK3CA-related malformations), including PIK3CA-associated excessive growth syndrome (PROS) and PIK3CA-associated vascular malformations (PRVM). Among PIK3CA-related diseases, hotspot mutations (p.E542K, p.E545K, p.H1047R, and p.H1047L) have a high incidence; in addition, non-hotspot mutations, including p.C420R, p.E726K, and p.G914R, have also been shown to occur at relatively high frequencies. Vascular malformations caused by mutations in PIK3CA are called PIK3CA-associated vascular malformations (PRVM), and patients present with varying clinical manifestations, depending on the affected site, and are predominantly pediatric patients. It has been reported that over 80% of lymphangiogenic lymphatic malformations (LMs) carry the PIK3CA mutation, with the majority of LMs exhibiting common (cystic) lymphangiogenic malformations; over 55% of gamma-lymphatic malformations (GLAs) carry the PIK3CA mutation. Approximately 20% of macrophages (VMs) are associated with the PIK3CA mutation.

[0007] Tyrosine kinase 2 (TIE2), an endothelial-specific type I transmembrane protein receptor tyrosine kinase encoded by the TEK gene, is an immunoglobulin-like and EGF-like domain 2. It mediates angiopoietin signaling, thereby promoting endothelial cell survival, vascular remodeling, and integrity. Genetic or somatic variations of TEK, such as G833D, Q837H, Y897S / H / C, L914F, R915C, R918C / H, and K1100N, have been identified as being associated with the pathogenesis of hereditary or sporadic venous malformations. TEK mutations enable ligand-independent autophosphorylation, leading to overactivation of downstream pathways such as PI3K / AKT. Approximately 50%–70% of venous malformations are caused by TEK gene mutations. Based on the type of TEK mutation, VM can be further divided into single venous malformation (TEK L914F somatic mutation), multiple venous malformation (TEK R915C chimeric mutation combined with TEK Y897C somatic mutation), VMCM (TEK R849W germline mutation combined with TEK Y1108 somatic mutation), and BRBNS (TEK T1105NT1106P somatic double mutation); among them, TEK L914F somatic mutation is the most common mutation (accounting for about 60%).

[0008] Due to the diverse and complex nature of PROS and vascular malformations, there is currently no standard treatment. Treatment strategies are typically individualized, including surgical resection, laser resection, and sclerotherapy. Based on the severity and urgency of disease progression, treatment strategies can be broadly categorized into three types: i) Asymptomatic lesions on the body surface without enlargement can be closely monitored; ii) Symptomatic lesions on the body surface or those with enlargement, as well as all visceral lesions, require aggressive treatment. Since lesions that can be completely resected without functional impairment are extremely rare, systemic medication should be the first choice; iii) For patients with severe complications, such as Kaposi's hemangioendothelioma (KHE) patients with coagulation disorders, anticoagulants and blood products should be administered in addition to aggressive tumor treatment to improve coagulation function. Surgical resection or laser resection is commonly used in the clinical treatment of PROS and vascular malformations. However, the boundaries between PROS and vascular malformations are often unclear, frequently involving important blood vessels, nerves, and even organs, greatly increasing surgical risks and the possibility of functional impairment. Extensive excision often results in severe scarring post-surgery, and can sometimes lead to shock, death, or other serious consequences due to severe bleeding. Furthermore, the recurrence rate is high. Sclerotherapy is primarily used for patients with vascular malformations; it is ineffective for limb overgrowth and deformities, and also has a high recurrence rate.

[0009] Sirolimus, an inhibitor of mTOR, a downstream protein of the PI3K / AKT pathway, has been reported to have some efficacy in patients with proliferative malformations (PROS) and can improve vascular malformations. However, sirolimus does not directly target gene mutations such as PIK3CA or TEK, so patients are prone to drug resistance or relapse after a period of treatment, and its long-term use is limited due to side effects. Alpelisib (BYL719), a PI3Kα inhibitor, is the world's first and only approved treatment for PROS, for use in adults and children aged 2 years and older with severe clinical manifestations of PROS requiring systemic therapy. Currently, no targeted therapies are approved for the treatment of PRVM and TEK gene mutation-related vascular malformations. Overall, patients with PIK3CA and / or TEK gene mutation-related growth malformations and / or vascular malformations urgently need systemically treatable, effective, and safe therapeutic agents. Summary of the Invention

[0010] This invention relates to the use of the PI3Kα inhibitor CYH33 in the prevention, treatment, or alleviation of PIK3CA-related malformations, particularly PIK3CA-related excessive growth syndrome (PROS) and / or PIK3CA-related vascular malformations (PRVM); and / or TEK gene mutation-related vascular malformations, especially venous or lymphatic malformations. The inventors have found that the PI3Kα inhibitor CYH33 has good therapeutic effects on subjects carrying PROS and / or PRVM, subjects carrying TEK gene mutation-related vascular malformations, and subjects carrying both PIK3CA gene mutations and TEK gene mutation-related vascular malformations. The inventors have also found that CYH33 has good therapeutic effects on subjects with central nervous system vascular malformations.

[0011] On the one hand, the present invention provides the use of PI3Kα inhibitor CYH33 or pharmaceutical compositions containing CYH33 in the preparation of medicaments for the prevention, treatment or relief of PIK3CA-related malformations, especially PIK3CA-related overgrowth syndrome (PROS) and / or PIK3CA-related vascular malformations (PRVM); and / or TEK gene mutation-related vascular malformations.

[0012] In one embodiment, the present invention provides the use of CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or relief of PIK3CA-related malformations, particularly PIK3CA-related overgrowth syndrome (PROS) and / or PIK3CA-related vascular malformations (PRVM).

[0013] In one embodiment, the present invention provides the use of the PI3Kα inhibitor CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or relief of vascular malformations associated with PROS, PRVM, or TEK gene mutations.

[0014] In one embodiment, the present invention provides the use of the PI3Kα inhibitor CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or relief of PROS and / or PRVM.

[0015] In one embodiment, the present invention provides the use of the PI3Kα inhibitor CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or relief of PROS and PRVM.

[0016] In one embodiment, the present invention provides the use of the PI3Kα inhibitor CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or mitigation of TEK gene mutation-related vascular malformations.

[0017] In one embodiment, the present invention provides the use of the PI3Kα inhibitor CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or relief of PIK3CA-related malformations and TEK gene mutation-related vascular malformations.

[0018] In one embodiment, the present invention provides the use of CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or relief of vascular malformations.

[0019] In a further embodiment, the vascular malformation is selected from any of the following:

[0020] (1) PIK3CA-related vascular malformations and / or TEK gene mutation-related vascular malformations;

[0021] (2) Simple vascular malformation, mixed vascular malformation;

[0022] (3) Peripheral vascular malformations and / or central nervous system vascular malformations.

[0023] In a further embodiment, the vascular malformation includes PIK3CA-associated vascular malformation (PRVM), and / or TEK gene mutation-associated vascular malformation, and / or central nervous system vascular malformation.

[0024] In a further embodiment, the simple vascular malformation includes capillary malformation, venous malformation, and lymphatic malformation.

[0025] In a further embodiment, the mixed vascular malformation includes capillary-venous malformation, capillary-lymphatic malformation, lymphatic-venous malformation, and capillary-lymphatic-venous malformation.

[0026] In a further embodiment, the central nervous system vascular malformation includes cavernous malformation.

[0027] In a further embodiment, the venous malformation includes common venous malformation, familial mucocutaneous venous malformation, and venous malformation in blue rubber nevus syndrome.

[0028] In a further embodiment, the TEK gene mutation-related vascular malformation includes TEK gene mutation-related venous malformation, preferably including single venous malformation, multiple venous malformation, familial mucocutaneous venous malformation, and venous malformation in blue rubber nipple syndrome.

[0029] In one embodiment, the present invention provides the use of the PI3Kα inhibitor CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or relief of central nervous system vascular malformations (such as cavernous malformation).

[0030] On the other hand, the present invention provides a method for preventing, treating or alleviating PIK3CA-related malformations, particularly PIK3CA-related overgrowth syndrome (PROS) and / or PIK3CA-related vascular malformations (PRVM); and / or vascular malformations such as TEK gene mutation-related vascular malformations, central nervous system vascular malformations; the method comprising administering an effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0031] In one embodiment, the present invention provides a method for preventing, treating, or alleviating PIK3CA-related malformations, particularly PIK3CA-related overgrowth syndrome (PROS) and / or PIK3CA-related vascular malformations (PRVM), the method comprising administering an effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0032] In one embodiment, the present invention provides a method for preventing, treating, or mitigating vascular malformations such as PIK3CA-associated vascular malformations (PRVM) or TEK gene mutation-associated vascular malformations, the method comprising administering a therapeutically effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0033] In some embodiments, the vascular malformation is selected from any of the following: (1) PIK3CA-related vascular malformation, and / or TEK gene mutation-related vascular malformation; (2) simple vascular malformation, mixed vascular malformation; (3) peripheral vascular malformation, and / or central nervous system vascular malformation.

[0034] In another embodiment, the present invention provides a method for preventing, treating, or alleviating vascular malformations such as central nervous system vascular malformations, the method comprising administering a therapeutically effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0035] In one embodiment, the vascular malformation mainly includes PIK3CA-associated vascular malformation (PRVM), and / or TEK gene mutation-associated vascular malformation, and / or central nervous system vascular malformation.

[0036] In one embodiment, the vascular malformation includes PIK3CA-associated vascular malformation (PRVM) and / or TEK gene mutation-associated vascular malformation.

[0037] In one implementation, the vascular malformation includes vascular malformations of the central nervous system (such as cavernous malformation).

[0038] In one embodiment, the present invention provides a method for preventing, treating, or alleviating PROS and / or PRVM, the method comprising administering a therapeutically effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0039] In one embodiment, the present invention provides a method for preventing, treating, or alleviating PROS and PRVM, the method comprising administering a therapeutically effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0040] In one embodiment, the present invention provides a method for preventing, treating, or mitigating TEK gene mutation-related vascular malformations, the method comprising administering a therapeutically effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0041] In a preferred embodiment, the present invention provides a method for preventing, treating, or alleviating spongiform malformation, comprising administering a therapeutically effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0042] In one embodiment, the present invention provides a method for preventing, treating, or mitigating PIK3CA-related malformations and TEK gene mutation-related vascular malformations, the method comprising administering a therapeutically effective amount of CYH33 or a pharmaceutical composition containing CYH33 to a subject in need of such treatment.

[0043] In some embodiments of the present invention, the subject carries a PIK3CA gene mutation and / or a TEK gene mutation.

[0044] In some embodiments of the present invention, the subject carries PIK3CA-associated overgrowth syndrome (PROS) or PIK3CA-associated vascular malformation (PRVM).

[0045] In some embodiments of the invention, the subject carries PIK3CA-associated overgrowth syndrome (PROS) and PIK3CA-associated vascular malformation (PRVM).

[0046] In some embodiments of the present invention, the subject carries a TEK gene mutation-related vascular malformation.

[0047] In some embodiments of the present invention, the subject carries PROS disease and / or PRVM disease, and TEK gene mutation-related vascular malformation disease.

[0048] In some embodiments of the present invention, the subject carries TEK gene mutation-related vascular malformations and PIK3CA-related malformations (especially vascular malformations).

[0049] In some embodiments of the present invention, the subject carries a central nervous system vascular malformation.

[0050] In some embodiments, the pharmaceutical composition includes a therapeutically effective amount of CYH33, as well as pharmaceutically acceptable excipients or carriers.

[0051] In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, oral, rectal, inhalation, nasal, topical, ocular, or ocular administration.

[0052] In some embodiments, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhaler, nasal spray solution, suppository, solution, emulsion, ointment, eye drop, or ear drop.

[0053] In some embodiments of the present invention, the PROS include, but are not limited to, a series of rare syndromes such as macrodactyly / toe, Cloves syndrome, Clapto syndrome, fibrofatty hyperplasia or overgrowth (FAO), Klippel-Trenaunay syndrome (KTS), unilateral hyperplastic multiple lipomatosis (HHML), macrocephaly-capillary malformation (MCAP), diffuse capillary malformation with overgrowth (DCMO), fibrofatty vascular lesion (FAVA), facial invasive lipoma (FIL), dysplastic macrocephaly (DMEG), hemimegaceticism (HMEG), muscular hyperplasia (Muscular HH), seborrheic keratosis (SK), and benign lichenoid keratosis (BLK). Preferably, the PROS are selected from a series of related rare syndromes such as Cloves syndrome, Klippel-Trenaunay syndrome, FAO, HHML, and MCAP.

[0054] In some embodiments of the present invention, the vascular malformations include, but are not limited to: simple vascular malformations (such as capillary malformations (CM), lymphatic malformations (LM), venous malformations (VM), etc.) and mixed vascular malformations (such as capillary-venous malformations (CVM), capillary-lymphatic malformations (CLM), lymphatic-venous malformations (LVM), capillary-lymphatic-venous malformations (CLVM), etc.).

[0055] In some embodiments of the present invention, lymphatic malformations (LM) mainly include common (cystic) lymphatic malformations (such as giant cystic lymphatic malformations, microcystic lymphatic malformations, and mixed cystic lymphatic malformations), generalized lymphatic malformations (GLA) (such as Kaposi's lymphangiomatosis (KLA)), lymphatic malformations in Gorham-Stout syndrome (GSD), tubular lymphatic malformations, primary lymphedema, and a series of other rare syndromes.

[0056] In some embodiments of the present invention, capillary malformations (CM) mainly include simple vascular nevi / salmon spots, skin and / or mucous membrane CM, reticular capillary malformations, capillary malformations in capillary malformations-arteriovenous malformations (CM-AVM), congenital telangiectatic marbled skin (CMTC), telangiectasia, etc.

[0057] In some embodiments of the present invention, venous malformations (VMs) mainly include common venous malformations, etc.

[0058] In some embodiments of the present invention, the PRVM includes, but is not limited to: simple vascular malformations (such as capillary malformations (CM), venous malformations (VM), lymphatic malformations (LM)) and mixed vascular malformations.

[0059] In some preferred embodiments of the present invention, PRVM includes, but is not limited to: simple vascular malformations (such as capillary malformations (CM), venous malformations (VM), lymphatic malformations (LM)), mixed lymphovenous malformations (LVM), and complex lymphatic malformations (including extensive lymphatic dysplasia (GLA), Gorham-Stout syndrome (GSD), and Kaposi's lymphangiomatosis (KLA)).

[0060] In some other preferred embodiments of the present invention, PRVM mainly includes capillary malformations (such as reticular capillary malformations, capillary malformations in capillary malformation-arteriovenous malformation (CM-AVM), etc.), lymphatic malformations (such as ordinary (cystic) lymphatic malformations, generalized lymphatic malformations (GLA), etc.), venous malformations such as ordinary venous malformations (VM), and mixed vascular malformations (such as capillary-venous malformations (CVM), capillary-lymphatic malformations (CLM), lymphatic-venous malformations (LVM), capillary-lymphatic-venous malformations (CLVM), etc.).

[0061] In some embodiments of the present invention, the TEK gene mutation-related vascular malformation is a venous malformation or a lymphatic malformation, etc.; preferably, it is a venous malformation.

[0062] In some embodiments of the present invention, TEK gene mutation-related vascular malformations (especially venous malformations) include, but are not limited to: single venous malformation, multiple venous malformations, familial mucocutaneous venous malformation (VMCM), and venous malformations in blue rubber nevus syndrome (BRBNS).

[0063] In some embodiments of the present invention, the PIK3CA gene mutation and TEK gene mutation-related vascular malformations include, but are not limited to, venous malformations.

[0064] In some embodiments of the present invention, PIK3CA gene mutation-related vascular malformations include, but are not limited to, common venous malformations.

[0065] In some embodiments of the present invention, the venous malformation includes, but is not limited to, common venous malformation, familial mucocutaneous venous malformation, and venous malformation in blue rubber nipple syndrome.

[0066] In some embodiments of the present invention, the central nervous system vascular malformation includes, but is not limited to, cavernous malformation.

[0067] Terminology Explanation

[0068] In this invention, unless otherwise expressly stated, the terminology used herein has the meanings defined below. Terms not explicitly defined in this invention have their general meanings as commonly understood by those skilled in the art.

[0069] As used herein, the term "CYH33" refers to a highly selective PI3Kα inhibitor that exhibits significant inhibitory activity against PI3Kα and its mutants, particularly wild-type and mutant PI3Kα kinases. Its chemical name is methyl 5-{6-[(4-methanesulfonylpiperazin-1-yl)methyl]-4-morpholinylpyrrole[2,1-f][1,2,4]triazin-2-yl}-4-trifluoromethylpyridin-2-yl}carbamate, and its common name is risovalisib.

[0070] "Fibrolipogenic hyperplasia or overgrowth (FAO)" refers to a syndrome characterized by segmental, progressive overgrowth of subcutaneous, muscular, and visceral fibrolipid tissues with skeletal overgrowth.

[0071] "Megacephaly-Capillary Malformation (MCAP) syndrome" refers to the syndrome with the following main findings: (1) megalencephaly (MEG) or hemimegacephaly (HMEG) associated with neurological findings of hypotonia, epilepsy and mild to severe intellectual disability; and (2) cutaneous capillary malformations with focal or generalized overgrowth of the body.

[0072] "CLOVES syndrome" refers to congenital, lipid overgrowth, vascular malformations, epidermal nevi, and spinal / skeletal abnormalities and / or scoliosis. The syndrome is characterized by a complex combination of congenital overgrowth of lipid tissue (typically manifesting as a lipid mass in the trunk) and vascular and lymphatic malformations.

[0073] Klippel-Trenaunay syndrome is a rare congenital medical condition in which blood vessels and / or lymphatic vessels cannot form properly.

[0074] As used herein, the term “treatment” refers to preventative or therapeutic treatment, as well as curative or disease-modifying treatment, including treatment of patients at risk of contracting or suspected of having contracted a disease, and subjects who are ill or diagnosed with a disease or medical condition, including suppression of clinical relapse. Treatment may be administered to subjects with a medical condition or who may eventually contract a disease in order to prevent, cure, delay the onset of one or more symptoms of the disease or relapse, reduce the severity of one or more symptoms of the disease or relapse, or alleviate one or more symptoms of the disease or relapse, or to extend the subject’s survival beyond what would be expected without such treatment. “Treatment regimen” refers to a mode of treatment for a disease, such as a dosage pattern used during treatment. Treatment regimens may include induction regimens and maintenance regimens. The phrase “induction regimen” refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of a disease. The overall goal of an induction regimen is to provide the subject with a high level of the drug at the initial stage of the treatment regimen. An induction regimen may employ a (partial or complete) “loading regimen,” which may include administering a larger dose of the drug than a physician would use during a maintenance regimen, such as administering the drug more frequently as a physician would during a maintenance regimen, or both. The phrase "maintenance regimen" refers to a treatment regimen (or part of a treatment regimen) used to maintain a subject's condition during treatment, such as maintaining long-term remission (for months or years). Maintenance regimens can be continuous treatment (e.g., administering medication at regular intervals, such as weekly, monthly, or yearly) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment during relapse, or treatment to achieve specific predetermined criteria).

[0075] The term "therapeutic effective amount" refers to the amount of the compound of the present invention that can elicit a biological or medical response in an individual or subject, or improve symptoms, slow or delay the progression of disease, or prevent disease.

[0076] As used herein, the term "individual or subject" refers to an animal. Preferably, the animal is a mammal. "Individual or subject" also refers to, for example, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In a preferred embodiment, the individual or subject is a human.

[0077] As used herein, the term “inhibition” refers to the reduction or suppression of a specific patient, symptom, condition, or disease, or a significant reduction in biological activity or baseline activity of a process.

[0078] As used herein, in one embodiment, the term "treatment" for any disease or condition refers to improving the disease or condition (i.e., stopping or slowing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" refers to improving at least one bodily parameter, which may not be perceptible to the patient. In yet another embodiment, "treatment" refers to regulating the disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing bodily parameters) or both. Beneficial effects

[0079] The PI3Kα inhibitor CYH33 showed good therapeutic effects in PROS and / or PRVM subjects carrying PIK3CA gene mutations, as well as subjects with venous malformations caused by TEK gene mutations. CYH33's ability to inhibit the proliferation of PIK3CA-mutated and TEK-mutated angiocytes and its ability to inhibit the growth of mutant transplanted tissues were significantly stronger than BYL719. Furthermore, in a Phase I first-in-human clinical trial, the types of adverse events (AEs) with CYH33 were similar to those with BYL719, but the incidence of some AEs (such as abnormal liver enzymes and diarrhea) was significantly lower than that with BYL719. In addition, CYH33 exhibited good safety and pharmacokinetic characteristics at QD levels of 5, 10, and 15 mg, with outstanding efficacy; 87.5% of patients experienced a reduction in target lesion volume of at least 20%, and some even saw a reduction of 30-65%.

[0080] The present invention is further illustrated by the following figures and embodiments. However, these embodiments and figures should not be construed as limiting the scope of the invention in any way. Attached Figure Description

[0081] Figure 1 shows the effect of compound CYH33 in Example 1 on the volume of transplanted tissue in a nude mouse model with subcutaneous allogeneic transplantation of NIH / 3T3-PIK3CA H1047R cells.

[0082] Figure 2 shows the effect of compound CYH33 in Example 1 on the change in body weight in mice with a subcutaneous allogeneic transplantation model of NIH / 3T3-PIK3CA H1047R cells.

[0083] Figure 3 is a graph showing the inhibition of HMEC-1-PIK3CA-H1047R cell proliferation in vitro by compound CYH33 in Example 2;

[0084] Figure 4 shows the effect of compounds CYH33 and BYL719 in Example 3 on the volume of transplanted tissue in a nude mouse model with subcutaneous allogeneic transplantation of NIH / 3T3-FL-TIE2-L914F cells.

[0085] Figure 5 shows the effects of compounds CYH33 and BYL719 in Example 3 on the changes in body weight in mice with a subcutaneous allogeneic transplantation model of NIH / 3T3-FL-TIE2-L914F cells.

[0086] Figure 6 is a graph showing the inhibition of HMEC-1-TIE2-L914F cell proliferation in vitro by compounds CYH33 and BYL719 in Example 4.

[0087] Figure 7 shows the effect of compound CYH33 on the phosphorylation level of AKT after 2 hours of treatment of cells in Example 5 (a) and the gray value analysis of the protein band (b).

[0088] Figure 8 shows the changes in target lesion volume in adult patients with PROS / PRVM after receiving different doses of CYH33 in Example 6.

[0089] Figure 9 shows the changes in target lesion volume in adult PROS / PRVM patients after receiving different doses of CYH33 in a further trial in Example 6. Detailed Implementation

[0090] The following embodiments illustrate the present invention as described above; however, they do not limit the scope of this disclosure in any way. The beneficial effects of combinations of the present disclosure can also be determined using other test models known to those skilled in the art.

[0091] Experimental methods for in vitro cell viability studies

[0092] Cells in the logarithmic growth phase were used for plating. The cell concentration was adjusted, and 90 μL of cell suspension was added to each well of the culture plate. Cell-free culture medium was added to the blank control wells. The culture plates were incubated overnight at 37°C, 5% CO2, and 100% relative humidity. 10 μL of different concentrations of CYH33 working solution were added to the cell culture plates, with two replicates per group. 10 μL of DMSO-cell culture medium mixture was added to the solvent control (containing only cells and cell culture medium) and the blank control (containing cell culture medium but no cells). The final concentration of DMSO was 0.1%. The 96-well cell plate was returned to the incubator and cultured for 7 days. Then, add 50 μL (equivalent to half the volume of cell culture medium in each well) of CellTiter-Glo working solution from the Promega CellTiter-Glo luminescent cell viability assay kit (Promega-G7573) to each well, wrap the cell plate with aluminum foil to protect it from light, shake the culture plate on a track shaker for 2 minutes to induce cell lysis, and place the culture plate at room temperature for 10 minutes to stabilize the luminescence signal. Detect the luminescence signal (expressed as RLU (relative light units)) using a microplate reader.

[0093] The inhibition rate (IR0) of the test compound was calculated using the following formula in in vitro cell viability assays: IR0(%) = (1 - (RLU compound - RLU blank control) / (RLU solvent control - RLU blank control)) × 100%. The inhibition rates of different compound concentrations were calculated in Excel, and then the inhibition curve was fitted using log(inhibitor) vs. response - variable slope software in GraphPad Prism 8.0 to obtain relevant parameters, including minimum inhibition rate, maximum inhibition rate, and relative IC50. 50 and absolute IC 50 The minimum inhibition rate (MIC) refers to the Y-value corresponding to the bottom plateau of the curve, and the maximum inhibition rate is the Y-value corresponding to the top plateau of the curve, relative to the IC. 50 It is the concentration required to bring the curve down to the point halfway between the top and bottom plateaus of the curve, absolute IC. 50 This refers to the drug concentration at which cell viability is inhibited by half.

[0094] In vivo drug efficacy experiments: Measurement of transplanted tissue and animal body weight.

[0095] The effectiveness of treatment was assessed based on the growth of the transplanted tissue. Once the transplanted tissue was palpable, its volume and size were evaluated twice weekly. The volume and size of the transplanted tissue were determined using a digital caliper, and the animal's weight was measured twice weekly throughout the trial. The weight of the transplanted tissue was measured at the end of treatment.

[0096] Transplanted tissue volume V in mm3 This is expressed as follows: V = 0.5a × b 2 , where a and b are the long and short diameters of the transplanted tissue, respectively.

[0097] T / C and TGI are indicators reflecting the response of transplanted tissue volume to treatment. T / C (%) reflects the relative proliferation rate of transplanted tissue (volume), i.e., the percentage of treatment / control (T / C) value of the transplanted tissue, calculated using the following formula:

[0098] T / C (%) = (T RTV / C RTV )×100,(T RTV C RTV The values ​​represent the average relative transplanted tissue volume (RTV) on the day of treatment for the treatment group and the solvent control group, respectively. The relative transplanted tissue volume (RTV) was calculated based on the transplanted tissue measurements using the formula: RTV = Vt / V0, where V0 is the average transplanted tissue volume measured at the start of treatment (i.e., day 1 of treatment d0), Vt is the average transplanted tissue volume measured after a certain treatment time (t), and T... RTV With C RTV Take data from the same day.

[0099] TGI (%) reflects the growth inhibition rate of transplanted tissue. The formula for calculating TGI (%) is: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100. Where, T i T0 and T0 represent the average transplanted tissue volume at the end of drug administration and at the beginning of drug administration (d0), respectively, for a certain treatment group; V i V0 and V0 represent the mean tumor volume at the end of treatment and at the start of treatment (d0) in the solvent control group, respectively. At the end of the trial, the weight of the transplanted tissue was measured. The relative weight of the transplanted tissue reflects the proliferation rate relative to the transplanted tissue (weight), calculated using the formula: W0 T / W C IR reflects the inhibition rate relative to transplanted tissue (weight), and is calculated using the formula: IR = (W C -W T ) / W C ×100%, of which, W T W C The average transplanted tissue weights are for the treatment group and the solvent control group, respectively.

[0100] Statistical analysis

[0101] All in vivo data are expressed as the standard error of the mean (SEM). Transplanted tissue volume, transplanted tissue weight, and animal body weight were used for statistical analysis to calculate the mean transplanted tissue volume, mean transplanted tissue weight, and SEM for each group at different time points. Examples 1 and 3 were plotted using Graphpad Prism 8.0 software, and data analysis was performed using SPSS 19.0. One-way ANOVA was used to check for homogeneity of variance. If the variances were homogeneous (p>0.05), a one-way ANOVA test was used, and the significance of each group was checked using the LSD test. If the variances were unequal (p<0.05), a nonparametric NPAR test was used for pairwise comparisons, and the significance of each group was checked using the Mann-Whitney test to evaluate the differences in tumor volume and tumor weight between the drug treatment groups and the solvent control group at the end of the efficacy experiment. For all statistical assessments, the significance level is set at p<0.05 to report the significance compared to the control group; that is, p<0.05 is considered statistically significant, and p<0.01 indicates a highly significant statistical difference.

[0102] In Examples 1 and 3, the experimental animals were BALB / c nude and NSG female mice, 6-8 weeks old, weighing 18-22g, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. and Shanghai Southern Model Biotechnology Co., Ltd., respectively. The test animals underwent acclimatization rearing at the experimental site for 3-7 days prior to the experiment. CYH33 in these examples was provided by Shanghai Haihe Pharmaceutical Research and Development Co., Ltd., and BYL719 was a commercially available product.

[0103] Example 1

[0104] In vivo pharmacodynamic study of CYH33 on the subcutaneous allogeneic transplantation model of the stable cell line NIH / 3T3-PIK3CA H1047R

[0105] In this experiment, the stable transgenic cell line NIH / 3T3-PIK3CA H1047R was used to evaluate the anti-tissue growth efficacy of CYH33. NIH / 3T3 cells are a highly contact-inhibited continuous cell line established from NIH Swiss mouse embryo cultures, retaining some normal growth characteristics. They do not form clones in soft agar and cannot proliferate in immunodeficient mice. These cells are easily transfected and are commonly used in cell, molecular biology, and tissue transplantation. Based on the human PIK3CA gene sequence (NM_006218.4), the PIK3CA-H1047R (CAT>CGA) mutant gene fragment was designed and obtained. The PIK3CA-H1047R gene was transferred into wild-type cells NIH / 3T3 (ATCC, Cat#:CRL-1658) using a retroviral packaging system. After screening with puromycin antibiotic, a stable transgenic cell line NIH / 3T3-PIK3CA H1047R was obtained. The constructed stable transgenic NIH / 3T3-PIK3CA H1047R cells were then introduced subcutaneously into nude mice to form overgrown tissue, which was used to screen and evaluate the efficacy of the test compounds.

[0106] Experimental methods

[0107] The NIH / 3T3-PIK3CA H1047R cell line was cultured in 1640 medium with 10% fetal bovine serum and 1% penicillin / streptomycin antibiotics at 37°C in a 5% CO2 incubator, passaged twice a week. When the cell saturation reached 80%–90% and the desired number was achieved, cells in the logarithmic growth phase were harvested, centrifuged at 1000 rpm for 5 min to remove the supernatant, resuspended in culture medium, and counted using a cell counter. Based on the counting results, the original solution was diluted to a viable cell concentration of 1×10⁻⁶. 7 Cell suspensions with a cell viability of 98.6% and passage P5 were prepared by diluting the cell suspension with matrix gel at a 1:1 ratio. The mixtures were then placed on ice. Under aseptic conditions, 0.2 mL of the cell suspension was subcutaneously injected into the right axilla of each mouse, equivalent to 1 × 10n NIH / 3T3-PIK3CA H1047R cells per mouse. 6 Observe the occurrence of transplanted tissue. When the average volume of transplanted tissue reaches 132.5 mm², 3 Mice were randomly divided into 3 groups (n=5 per group) and treated with solvent control, CYH33 (7.5 mg / kg and 11.25 mg / kg), respectively. The mice were administered the drug twice a day for 16 consecutive days according to the study dosage regimen shown in Table 1. The efficacy was studied.

[0108] Table 1. Pharmacodynamic groupings and dosing regimens Note: a. BID: twice daily; b. PO: administered by gavage; c. The solvent for CYH33 is: 0.5% sodium carboxymethyl cellulose (CMC-Na) + 0.5% Tween 80.

[0109] Experimental results

[0110] The inhibitory effect of CYH33 on tissue growth in female nude mice bearing subcutaneous xenografted NIH / 3T3-PIK3CA H1047R cells is shown in Figures 1-2 and Tables 2-3. The grouping and administration regimens for each group are shown in Table 1. Figure 1 shows the tissue growth curves of each treatment group in the NIH / 3T3-PIK3CA H1047R allogeneic transplantation model. Figure 2 shows the effect of each treatment group on the body weight change of mice in the NIH / 3T3-PIK3CA H1047R cell subcutaneous allogeneic transplantation model. In Figures 1 and 2, the data points represent the mean transplanted tissue volume and mean body weight change rate for each group, respectively. The error bars represent the standard error (SEM) of the mean. Tables 2 and 3 show the mean transplanted tissue volume, mean transplanted tissue weight, and the indicators of transplanted tissue response to treatment (T / C, TGI, and IR) for each treatment group 16 days after administration. In principle, the evaluation criteria are: T / C (%) > 40% is invalid; T / C (%) ≤ 40% and p < 0.05 is valid.

[0111] Table 2. Effects of CYH33 on tissue growth in the NIH / 3T3-PIK3CA H1047R cell allogeneic model

[0112] (Calculated based on the transplanted tissue volume on day 16 after drug administration) Note: a.Mean±SEM; bT / C(%)=T RTV / C RTV ×100, RTV=V 16 / V0;TGI(%)=[1-(T 16 -T0) / (C 16 -C0)]×100; c. Using the nonparametric Mann-Whitney test, the differences between each dose group of CYH33 and the solvent control group were obtained.

[0113] Table 3. Transplanted tissue weights in each group (based on day 16 after drug administration) Note: a.Mean±SEM; b.IR(%)=(W C -W T ) / W C ×100%; c. Using the nonparametric Mann-Whitney test, the differences between each dose group of CYH33 and the solvent control group were obtained.

[0114] As shown in Figure 1 and Table 2, 16 days after the start of drug administration, the average transplanted tissue volume of mice in the solvent control group was 2072 ± 83 mm. 3 Compared with the blank solvent group, CYH33 significantly inhibited tissue growth at doses of 7.5 and 11.25 mg / kg, with mean tissue volumes of 740 ± 163 mm². 3 (T / C = 35%, TGI = 69%, p = 0.009) and 474 ± ​​57 mm 3 (T / C = 23%, TGI = 82%, p = 0.009). Furthermore, the effect of each treatment group on tissue weight was investigated in the experiment. After 16 consecutive days of administration, the transplanted tissues of mice in each treatment group were weighed. As shown in Table 3, the anti-tissue growth effects of each treatment group were significantly different from those of the solvent control group. The average tissue weight of the control group mice was 2.89 ± 0.36 g. After treatment with CYH33 (7.5, 11.25 mg / kg), the average tissue weights were 0.90 ± 0.18 g (IR = 69%, p = 0.009) and 0.75 ± 0.13 g (IR = 74%, p = 0.009), respectively. This indicates that all animals tolerated the treatment well.

[0115] In summary, the test compound CYH33 demonstrated a significant and effective anti-transplant tissue growth effect in the NIH / 3T3-PIK3CA H1047R subcutaneous transplantation model, and all animals tolerated it well. These data provide a rationale for the clinical application of CYH33 in the treatment of patients with PIK3CA-associated excessive growth syndrome (PROS) and / or PIK3CA-associated vascular malformations (PRVM).

[0116] Example 2

[0117] Effects of CYH33 on the in vitro proliferation of HMEC-1-PIK3CA-H1047R cells

[0118] In this experiment, the in vitro efficacy of CYH33 was evaluated using a constructed stable HMEC-1 cell line overexpressing the target gene PIK3CA-H1047R (CAT>CGA) mutation. HMEC-1 is a continuously regenerating human endothelial microvascular cell that retains many characteristics of endothelial cells and can be used as a substitute for primary human dermal endothelial cells in studies such as angiogenesis. The PIK3CA-H1047R (CAT>CGA) mutant target gene fragment was designed and obtained based on the human PIK3CA gene sequence (NM_006218.4). The PIK3CA-H1047R gene was transformed into HMEC-1 cells (Zhong Qiao Xinzhou, #ZQ0456) using a retroviral packaging system. After screening with puromycin antibiotic, a stable HMEC-1-PIK3CA-H1047R transgenic cell line was obtained.

[0119] Experimental methods

[0120] Cells were stained with trypan blue and viable cells were counted. The cell concentration was adjusted to an appropriate level and seeded into 96-well plates. Cell-free culture medium (containing 0.1% DMSO) was added to the min control wells. The plates were incubated overnight at 37°C. The maximum final concentration of the test compounds CYH33 and BYL719 was 10 μM, with nine concentrations set from 10 μM to the minimum. The final concentration of DMSO was 0.1%. The plates were incubated at 37°C in a 5% CO2 incubator. After 7 days, cell viability was assessed using the CellTiter-Glo luminescent cell viability assay kit (Promega-G7573).

[0121] Experimental results

[0122] As shown in Table 4 and Figure 3, CYH33 inhibits the proliferation of HMEC-1-PIK3CA-H1047R cells, with a relative IC50 value of 1.5%. 50 The IC50 value was 187 nM, while the relative IC50 value of the drug BYL719 targeting the same target was 187 nM. 50 The value was 1167 nM. Therefore, CYH33's ability to inhibit the proliferation of PIK3CA-mutated angiocytes was significantly stronger than that of BYL719.

[0123] Table 4. Anti-proliferation parameters of CYH33 in HMEC-1-PIK3CA H1047R cells

[0124] Example 3

[0125] In vivo pharmacodynamic study of CYH33 on the subcutaneous allogeneic transplantation model of the stable cell line NIH / 3T3-FL-TIE2-L914F

[0126] In this experiment, the stable transgenic cell line NIH / 3T3-FL-TIE2-L914F was used to evaluate the anti-tissue growth efficacy of CYH33. NIH / 3T3 cells are a highly contact-inhibited continuous cell line established from NIH Swiss mouse embryo cultures, retaining some normal growth characteristics. They do not form clones on soft agar and cannot proliferate in immunodeficient mice. These cells are easily transfected and are commonly used in cell, molecular biology, and tissue transplantation. The FL-TIE2-L914F (CTT>TTT) mutant expression vector was designed and obtained based on the FL-TIE2 sequence (NM_000459.5). The FL-TIE2-L914F gene was transformed into wild-type cells NIH / 3T3 (ATCC, Cat#:CRL-1658) using a retroviral packaging system. After screening with puromycin antibiotic, a stable transgenic cell line NIH / 3T3-FL-TIE2-L914F was obtained. The constructed stable transgenic NIH / 3T3-FL-TIE2-L914F cells were then introduced into nude mice to form overgrown tissue, which was used to screen and evaluate the efficacy of the experimental compounds.

[0127] Experimental methods

[0128] The NIH / 3T3-FL-TIE2-L914F cell line was cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin antibiotics at 37°C in a 5% CO2 incubator, and passaged twice a week. When the cell saturation reached 80%–90% and the required number was achieved, cells in the logarithmic growth phase were harvested. Under aseptic conditions, 0.2 mL of cell suspension was subcutaneously inoculated into the right axilla of each mouse, i.e., 1 × 10n NIH / 3T3-FL-TIE2-L914F cells per mouse. 6 Observe the occurrence of transplanted tissue. When the average volume of transplanted tissue reaches 102 mm², observe the development of transplanted tissue. 3 At that time, tumor-bearing mice were randomly divided into 3 groups (n=5 per group) and treated with solvent control, CYH33, and BYL719 respectively. The drugs were administered according to the study dosage regimen shown in Table 5, twice a day for 14 consecutive days, to study the efficacy.

[0129] Table 5. Pharmacodynamic groupings and dosing regimens Note: a. BID: twice daily; b. PO: administered by gavage; c. The solvent for BYL719 is: 10% N-methylpyrrolidone + 30% PEG300 + 20% Solutol + 40% ultrapure water; d. From day 0 to 10 after administration, the dose of CYH33 is 7.5 mg / kg; from day 11 to 14 after administration, the dose is 11.25 mg / kg, and the corresponding concentration is adjusted to 1.125 mg / mL; e. The solvent for CYH33 and the solvent control are: 0.5% CMC-Na + 0.5% Tween 80.

[0130] Experimental results

[0131] The inhibitory effect of CYH33 on tissue growth in female nude mice bearing NIH / 3T3-FL-TIE2-L914F cell subcutaneous xenografts is shown in Figures 4-5 and Tables 6-7. Figure 4 shows the tissue growth curves of each treatment group in the nude mouse model of NIH / 3T3-FL-TIE2-L914F cell subcutaneous xenograft tumors. Figure 5 shows the effect of each treatment group on the body weight change of mice in the cell subcutaneous allogeneic transplantation model. In Figures 4 and 5, the data points represent the mean transplanted tissue volume and mean body weight change rate of each group, respectively, and the error bars represent the standard error (SEM) of the mean. Tables 6 and 7 show the mean transplanted tissue volume, mean transplanted tissue weight, and the indicators of transplanted tissue response to treatment (T / C, TGI, and IR) in each treatment group 14 days after administration. In principle, the evaluation criteria were: T / C (%) > 40% was considered ineffective; T / C (%) ≤ 40% and p < 0.05 was considered effective.

[0132] Table 6. Efficacy evaluation of CYH33 on tissue growth in NIH / 3T3-FL-TIE2-L914F cell allograft model

[0133] (Calculated based on the transplanted tissue volume on day 14 after drug administration) Note: a.Mean±SEM; bT / C(%)=T RTV / C RTV ×100, RTV=V 14 / V0;TGI(%)=[1-(T 14 -T0) / (C 14 -C0)]×100; c. Use the non-parametric Mann-Whitney test to obtain the difference between the drug administration group and the solvent control group; d. The dose of CYH33 was 7.5 mg / kg on days 0 to 10 after administration, and 11.25 mg / kg on days 11 to 14 after administration.

[0134] Table 7. Transplanted tissue weights in each group (based on day 14 after drug administration) Note: a.Mean±SEM; b.IR(%)=(W C -W T ) / W C ×100%; c. One-way ANOVA was used to analyze the differences between the drug administration group and the solvent control group; d. The dose of CYH33 was 7.5 mg / kg on days 0-10 after administration and 11.25 mg / kg on days 11-14 after administration.

[0135] As shown in Figure 4 and Table 6, 14 days after the start of drug administration, the average transplanted tissue volume of mice in the solvent control group was 2486 ± 230 mm. 3 Compared with the blank solvent group, the CYH33 treatment group significantly inhibited tissue growth, with an average transplanted tissue volume of 982±70 mm. 3 (T / C = 40%, TGI = 63%, p = 0.009), and stronger than the BYL719 treatment group (1703 ± 201 mm). 3 The mean tissue weight was 68% (T / C = 68%, TGI = 33%, p = 0.076). Furthermore, the effect of each treatment group on tissue weight was investigated. After 14 days of continuous administration, the transplanted tissues of mice in each treatment group were weighed. Table 7 shows that the mean tissue weight of the control group mice was 3.26 ± 0.26 g. The anti-tissue growth effect of the CYH33 treatment group was significantly different from that of the solvent control group (mean tissue weight 1.16 ± 0.11 g, IR = 64%, p = 0.009), and stronger than that of the BYL719 treatment group (mean tissue weight 1.74 ± 0.21 g, IR = 47%, p = 0.009). All animals tolerated the treatment well.

[0136] In summary, the test compound CYH33 demonstrated a significant and effective inhibitory effect on transplanted tissue growth in the NIH / 3T3-FL-TIE2-L914F subcutaneous transplantation model, and this effect was stronger than that in the BYL719 treatment group. These data provide a rationale for using CYH33 to treat patients with TEK gene mutation-related venous malformations in clinical practice.

[0137] Example 4

[0138] Effects of CYH33 on the in vitro proliferation of HMEC-1-TIE2-L914F cells

[0139] In this experiment, the FL-TIE2-L914F (CTT>TTT) mutant expression vector was designed and obtained based on the FL-TIE2 sequence (NM_000459.5). The TIE2-L914F gene was transformed into HMEC-1 cells (Zhong Qiao Xinzhou, #ZQ0456) using a lentiviral packaging system. After screening with puromycin antibiotic, a stable transfected cell line, HMEC-1-TIE2-L914F, was obtained. The stable HMEC-1 cell line overexpressing the target gene TIE2-L914F mutant was used to evaluate the in vitro efficacy of CYH33.

[0140] Experimental methods

[0141] Adjust the cell concentration to an appropriate level, adding 2000 cells / 100 μL of cell suspension to each well of the cell plate. Add cell-free culture medium (containing 0.1% DMSO) to the Min control wells. Incubate the plate overnight at 37°C. The maximum final concentration of the test compounds CYH33 and BYL719 is 10 μM. Prepare 10× working solutions of the compounds using PBS, and serially dilute the test compounds from the highest concentration to the lowest concentration using PBS solution containing 1% DMSO, resulting in 9 concentrations. Remove the cell plate from the previous day and add 11 μL of 10× working solution of the compounds to each well. Add 11 μL of 1% DMSO PBS solution to the Max control well, bringing the final DMSO concentration to 0.1%. Incubate the plate at 37°C in a 5% CO2 incubator. After 7 days, cell viability is assessed using the CellTiter-Meiluncell luminescence assay kit (PWL111-3).

[0142] Experimental results

[0143] As shown in Table 8 and Figure 6, CYH33 inhibits the proliferation of HMEC-1-TIE2-L914F cells, relative to IC50. 50 The concentration was 291.8 nM, and its ability to inhibit the proliferation of TIE2-mutant angiocytes was stronger than that of the drug BYL719 with the same target (relative IC50). 50 (3179 nM).

[0144] Table 8. Anti-proliferation parameters of CYH33 in HMEC-1-TIE2-L914F cells

[0145] Example 5

[0146] Effect of CYH33 on p-AKT expression in HMEC-1-TIE2-L914F cells

[0147] Experimental methods

[0148] HMEC-1-TIE2-L914F cells were cultured in HMEC-1 complete medium (Zhongqiao Xinzhou, #ZQ-1319). An appropriate number of cells were seeded into 6-well plates and allowed to adhere overnight. The next day, the target compound was added to the seeded cell culture plates at the specified concentration, with DMSO used as a control well. Incubation was performed for 2 hours. Cell pellets were lysed using RIPA lysis buffer, which included protease and phosphatase inhibitors. After lysis, the protein concentration of each sample was determined using a BCA protein assay kit, and the protein concentration was uniformly quantified. 5× protein loading buffer was added, and the samples were boiled at 95°C for 10 min, cooled on ice, and stored at -20°C. Cell protein samples were added to the sample wells of the gel and loaded onto a protein ladder. Electrophoresis was performed at a constant voltage of 70V for 30 min, followed by a constant voltage of 86V until the bromophenol blue indicator band reached the bottom. Electrophoresis was then stopped. The proteins on the SDS gel were transferred to a PVDF membrane at a constant voltage of 100V for 105 min. After electroporation, the PVDF membrane was placed in blocking buffer (5% skim milk) and blocked at room temperature for 1 hour. The blocked PVDF membrane was washed, cut, and incubated overnight at 4°C with primary antibodies (1:1000 dilution) p-AKT (Ser473) (CST, #4060S), AKT (CST, #4691S), and GAPDH (Trans, #HC301), respectively. The overnight PVDF membrane was slowly washed three times in TBST and then incubated at room temperature for 1 hour with the corresponding secondary antibodies (1:3000 dilution) HRP-anti-mouse IgG (CST, #7076S) and HRP-anti-Rabbit IgG (CST, #7074S), respectively. After secondary antibody incubation, the PVDF membrane was slowly washed three times, and the membrane was exposed under a chemiluminescence imaging system to save the image (Figure 7a). The grayscale values ​​of the p-AKT and AKT bands in the Western Blot results were analyzed using Image J2x software. The grayscale results are presented in bar charts (Figure 7b). The horizontal axis of the bar chart represents the compound concentration, and the vertical axis represents the ratio of p-AKT to AKT (p-AKT / AKT). The formula is: p-AKT / AKT (% control) = Gray compound / Gray DMSO × 100%.

[0149] Experimental results

[0150] As shown in Figure 7, the tested compound CYH33 exhibited a strong dose-dependent inhibitory effect on p-AKT (Ser473) in HMEC-1-TIE2-L914F cells. Specifically, CYH33 significantly inhibited AKT phosphorylation at concentrations of 100 nM and 300 nM.

[0151] Example 6

[0152] Clinical trials of CYH33

[0153] A phase I / II, multicenter clinical study was conducted to clinically evaluate the safety, tolerability, pharmacokinetic characteristics, and efficacy of CYH33 in adult and adolescent (12–17 years) patients with severe clinical presentations, requiring systemic therapy, and who have a confirmed PIK3CA hotspot mutation in PROS and PRVM.

[0154] Specifically, a Phase I clinical trial was conducted in adult patients with PROS / PRVM. CYH33 was administered orally once daily (QD) for 21 days as one treatment cycle (the first treatment cycle in the Phase I study was 28 days), starting with a dose of 10 mg QD. Seventeen adult patients with PROS / PRVM received CYH33 treatment (5, 10, or 15 mg, QD). In the Phase I dose escalation phase, 4, 3, and 3 patients were enrolled at the 5 mg QD, 10 mg QD, and 15 mg QD dose levels, respectively. In the Phase I dose extension phase, 7 patients were enrolled at the 10 mg QD dose level. Patients were aged 18–46 years and weighed 45.8–108.4 kg; 5 were PROS patients and 12 were PRVM patients. PK samples were obtained from 16 adult patients with PROS / PRVM during the dose escalation and expansion phases, with 3, 10, and 3 patients receiving 5 mg, 10 mg QD, and 15 mg QD, respectively. The aim was to evaluate the safety, tolerability, pharmacokinetic characteristics, and preliminary efficacy of CYH33 administration in PROS / PRVM patients.

[0155] Regarding clinical safety, the most common drug-related adverse events in PROS / PRVM patients treated with CYH33 monotherapy at 5-15 mg QD in this study were hyperglycemia and stomatitis. Compared to the higher dose levels (20-60 mg QD) used in solid tumor patients, the incidence and severity of adverse events were significantly reduced, and all were CTCAE grade 1-2. The most common drug-related adverse events were CTCAE grade 1 hyperglycemia and CTCAE grade 1 or 2 stomatitis (see Table 9 for details), and no DLT events occurred in any patient. Furthermore, no adverse events such as diarrhea or loss of appetite have been reported to date.

[0156] Table 9. Preliminary safety data of CYH33 in adult patients with PROS / PRVM (≥10% TRAE)

[0157] Regarding clinical efficacy, all 16 subjects in Phase I completed at least one efficacy assessment. In the CYH33 5mg QD group, 2 out of 3 subjects achieved partial remission (PR) at the first imaging assessment; in the CYH33 10mg QD group, 9 out of 10 subjects achieved PR at the first imaging assessment; and in the CYH33 15mg QD group, all 3 subjects achieved PR at the first imaging assessment. See Figure 8 for details.

[0158] Besides the reduction in target lesion size, improvements in patients' clinical symptoms and quality of life are also important indicators of treatment efficacy, as shown in Table 10. The most common clinical symptoms in PROS / PRVM patients are overgrowth / malformation, pain, and fatigue. Preliminary data show that with CYH33 5-15mg QD treatment, the improvement rate for pain was 75-100%, the improvement rate for fatigue was 50.0-100%, the improvement rate for overgrowth / malformation was 90-100%, and the improvement rate for quality of life score was 25-67%.

[0159] Table 10. Improvement in clinical symptoms and quality of life in adult patients with PROS / PRVM after CYH33 treatment

[0160] As can be seen, CYH33 is a highly selective PI3Kα inhibitor. Preliminary data from a Phase I clinical trial in adult patients with PROS / PRVM showed that CYH33 exhibits good safety and pharmacokinetic characteristics at dose levels of 5-15 mg QD, and demonstrates outstanding efficacy, reducing lesions by 20% and even 30-65%. These results provide data support for the treatment of PROS / PRVM patients with CYH33.

[0161] Further clinical trials of CYH33

[0162] Based on the aforementioned clinical trials of CYH33, 6 more adult patients with PROS / PRVM received CYH33 treatment, bringing the total number of patients to 23. Furthermore, in the Phase I dose expansion phase, 6 and 7 patients were enrolled at the 5 mg QD and 10 mg QD dose levels, respectively. The patients were aged 18-46 years and weighed 45.8-108.4 kg, including 8 PROS patients and 15 PRVM patients. PK samples were obtained from all 23 adult PROS / PRVM patients in both the dose escalation and expansion phases, with 10, 10, and 3 patients receiving 5 mg, 10 mg QD, and 15 mg QD doses, respectively. This aimed to further evaluate the safety, tolerability, pharmacokinetic characteristics, and preliminary efficacy of CYH33 administration in PROS / PRVM patients.

[0163] The results of the clinical safety trials are summarized in Table 9-1.

[0164] Table 9-1. Preliminary safety data of CYH33 in adult patients with PROS / PRVM (≥10% TRAE)

[0165] Supplemental clinical efficacy trial results are detailed in Figure 9. Besides target lesion reduction, improvement in patients' clinical symptoms and quality of life are also important indicators of efficacy, as shown in Table 10-1. The most common clinical symptoms in PROS / PRVM patients are overgrowth / malformation, pain, and fatigue. Preliminary data show that with CYH33 5-15mg QD treatment, the improvement rate of pain was 100%, the improvement rate of fatigue was 80%, the improvement rate of grossly visible appearance abnormalities or malformations was 95.7%, and the improvement rate of quality of life score was 76.2%. Furthermore, one adult patient with a brain lesion was found in the enrolled group; after receiving CYH33 10mg QD treatment, the volume of the brain lesion shrank by approximately 50%; this provides data support for CYH33 treatment of CCM patients. Summary trial results regarding improvements in clinical symptoms and quality of life after treatment are detailed in Table 10-1.

[0166] Table 10-1. Improvement in clinical symptoms and quality of life in adult patients with PROS / PRVM after CYH33 treatment

[0167] In addition, a total of 8 adolescent (12-17 years old) patients were enrolled in the Phase I study, and the results showed that adolescent (12-17 years old) and adult patients (≥18 years old) had basically similar overall safety characteristics, efficacy, PK and PD characteristics at the same dose level (CYH33 5-10mg QD).

[0168] In summary, CYH33 is a highly selective PI3Kα inhibitor. Phase I clinical trial data in adult and adolescent patients with PROS / PRVM showed that CYH33 exhibits good safety and pharmacokinetic characteristics at dose levels of 5-15 mg QD, with significant efficacy. These results provide data support for the treatment of PROS / PRVM patients and CCM patients with CYH33.

Claims

Use of CYH33 or a pharmaceutical composition containing CYH33 in the preparation of a medicament for the prevention, treatment or relief of PIK3CA-related malformations, particularly PIK3CA-related overgrowth syndrome and / or PIK3CA-related vascular malformations. According to the use described in claim 1, wherein, The PIK3CA-related overgrowth syndromes include: macrodactyly, Cloves syndrome, Clappo syndrome, fibrofatty hyperplasia or overgrowth, Klippel-Trenaunay syndrome, unilateral hyperplastic lipomatosis, macrocephaly-capillary malformation, diffuse capillary malformation with overgrowth, fibrofatty vascular lesions, facial invasive lipoma, dysplastic macrocephaly, hemilateral macrocephaly, unilateral muscle overgrowth, seborrheic keratosis, and benign lichenoid keratosis. Preferably, the PIK3CA-related overgrowth syndrome is Cloves syndrome, Klippel-Trenaunay syndrome, fibrofatty hyperplasia or overgrowth, unilateral hyperplastic lipomatosis, or macrocephaly-capillary malformation. The PIK3CA-related vascular malformations include: simple vascular malformations (such as capillary malformations, venous malformations, and lymphatic malformations) and mixed vascular malformations (such as capillary-venous malformations, capillary-lymphatic malformations, lymphatic-venous malformations, and capillary-lymphatic-venous malformations). According to the use described in claim 2, wherein, The lymphatic malformations include: common lymphatic malformations (such as giant cystic lymphatic malformations, microcystic lymphatic malformations, and mixed cystic lymphatic malformations), generalized lymphatic abnormalities, lymphatic malformations in Gorham-Stout syndrome, tubular lymphatic malformations, and primary lymphedema. The venous malformations include common venous malformations; The capillary malformations include: simple vascular nevi / salmon spots, skin and / or mucous membrane capillary malformations, reticular capillary malformations, capillary malformations in arteriovenous malformations, congenital telangiectasia with marble-like skin, and telangiectasia. Use of CYH33 or pharmaceutical compositions containing CYH33 in the preparation of medicaments for the prevention, treatment or relief of vascular malformations. Use according to claim 4, wherein, The vascular malformation is selected from any of the following: (1) PIK3CA-related vascular malformations and / or TEK gene mutation-related vascular malformations; (2) Simple vascular malformation, mixed vascular malformation; (3) Peripheral vascular malformations and / or central nervous system vascular malformations. Use according to claim 4, wherein, The vascular malformations include PIK3CA-related vascular malformations, and / or TEK gene mutation-related vascular malformations, and / or central nervous system vascular malformations. The use according to claim 5, wherein: The simple vascular malformations include capillary malformations, venous malformations, and lymphatic malformations. The mixed vascular malformations include capillary-venous malformations, capillary-lymphatic malformations, lymphatic-venous malformations, and capillary-lymphatic-venous malformations. Use according to claim 5 or 6, wherein: The central nervous system vascular malformations include cavernous malformations. Use according to claim 7, wherein, The venous malformations include common venous malformations, familial mucocutaneous venous malformations, and venous malformations in blue rubber nevus syndrome. Use according to claim 5 or 6, wherein The TEK gene mutation-related vascular malformations include TEK gene mutation-related venous malformations, including single venous malformations, multiple venous malformations, familial mucocutaneous venous malformations, and venous malformations in blue rubber nevus syndrome.

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