BYL719 (alpelisib) for use in the treatment of PIK3CA-associated overgrowth syndrome (PROS CLOVaS syndrome)

Abstract

Methods for treating PROS in a subject in need thereof are provided. The present invention provides a method for treating PIK3CA-associated overgrowth syndrome (PROS) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of BYL719.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a method for treating PIK3CA-associated hypergrowth syndrome (PROS), such as congenital lipomatous hypergrowth-vascular malformations-epidermal nevi-spinal / skeletal malformations (CLOVES).

[0002] Background of the Invention The term "PROS" for PIK3CA-associated hypergrowth disorder is defined to encompass both known and new clinical entities associated with somatic PIK3CA mutations, including macrodactyly, FAO, HHML, CLOVES, and related megaencephalopathy (Keppler-Noreuil et al 2014). A workshop was convened at the National Institutes of Health (NIH) to discuss and develop a consensus document on the diagnosis and management of patients with PIK3CA-associated somatic hypergrowth disorder.

[0003] CLOVES represents congenital lipomatous hypergrowth-vascular malformation-epidermal nevus-spinal / skeletal malformation and / or scoliosis. This syndrome is considered a rare disorder and is characterized by progressive, complex, and mixed trunk vascular malformations, dysregulated adipose tissue, varying degrees of scoliosis, and progressive skeletal hypertrophy without skeletal hypergrowth (Sapp et al 2007; Alomari et al 2009). This syndrome is distinct from cancer. In fact, in CLOVES syndrome, the tumors are benign, the tissues are simply overgrown, and the objects are deformed. CLOVES syndrome is rare and is evident at birth. CLOVES syndrome affects both males and females equally, regardless of race or ethnicity. Many patients with this syndrome are misdiagnosed.

[0004] CLOVES syndrome is caused by somatic mosaic mutations in the PIK3CA gene. PIK3CA encodes the 110-kD catalytic alpha subunit of PI3K, which is activated in response to tyrosine kinase receptor ligand binding and converts phosphatidylinositol (3,4)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3). While activating mutations in PIK3CA have explained several types of cancer, CLOVES syndrome remains unexplained. In 2012, Kurek et al. identified activating mutations in PIK3CA by sequencing DNA or RNA. However, currently, there is no cure for CLOVES syndrome.

[0005] Limaye et al. (2015) disclosed that somatic mutations in PIK3CA are associated with cancer, hypergrowth syndrome, and lymphoid malformations (LM). In particular, the authors demonstrated that a cultured cell line obtained from healthy donors (without PROS), called human umbilical vein endothelial cells (HUVECs), retrovirally transfected with mutant PIK3CA, exhibited activation of the AKT / mTORC pathway. As predicted, the drug BYL719 inactivated PIK3CA mutant-induced AKT phosphorylation in this artificial model. This suggests that these proteins are involved in the same signaling pathway. However, this in vitro model did not reproduce any phenotype of any symptoms or disease associated with PROS patients, and in particular, it did not provide any evidence that this drug could be used to treat patients with PROS. Therefore, there is a need to understand the use of BYL719 in patients with PROS, particularly CLOVES syndrome or Klippel-Trenauni syndrome.

[0006] Summary of the Invention The present invention relates to a method of treating PROS in a subject that requires treatment of PROS, the method comprising administering to the subject a therapeutically effective amount of BYL719. In particular, the present invention is defined by the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] [Figure 1A] Characterization of a mouse model of PROS and efficacy of BYL719. [Figure 1B] Characterization of a mouse model of PROS and efficacy of BYL719. [Figure 1C] Characterization of a mouse model of PROS and efficacy of BYL719. [Figure 2A] Efficacy of BYL719 in adult patients with severe CLOVES syndrome. [Figure 2B] Efficacy of BYL719 in adult patients with severe CLOVES syndrome. [Figure 3] Efficacy of BYL719 in pediatric patients with severe CLOVES syndrome.

[0008] DETAILED DESCRIPTION OF THE INVENTION BYL719, synthesized by Novartis, is in a Phase II / III clinical trial for advanced solid tumors. The inventors have developed the first genetic mouse model of PROS that recapitulates the human disease and have demonstrated the efficacy of BYL719, a pharmacological inhibitor of PIK3CA, in preventing and ameliorating all organ dysfunctions in this PROS mouse. Based on these results, the inventors treated two patients (one adult and one pediatric) with severe CLOVES syndrome using BYL719. The drug induced rapid recovery of all affected organs and had robust efficacy against the disease in both patients; previously intractable hemangioma shrinkage, congestive heart failure fully recovered, hemihyperplasia decreased, and scoliosis abated. The drug was not associated with any significant side effects. Taken together, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic agent in PROS patients.

[0009] Accordingly, the present invention relates to a method for treating PROS in a subject requiring treatment for PROS, comprising the step of administering a therapeutically effective amount of BYL719 to the subject. In particular, the present invention relates to a method for treating CLOVES syndrome in a subject requiring treatment for CLOVES syndrome, comprising the step of administering a therapeutically effective amount of BYL719 to the subject.

[0010] As used herein, the terms “to treat” or “treatment” refer to both preventive or protective treatments and curative or disease-modifying treatments, and include treatments for subjects at risk of developing a disease or suspected of having a disease, and subjects diagnosed with a disease or health condition, including the suppression of clinical regression. Treatments may be applied to subjects with a medical disability or at risk of eventually developing a disability, to prevent, cure, delay the onset of one or more symptoms of the disability, reduce the severity of the symptoms, or alleviate the symptoms or reverse the disability, or to extend the subject's survival beyond what would be predicted in the absence of such treatment. “Treatment plan” means a pattern of treatment for a disease, e.g., a pattern of administration used during treatment. A treatment plan may include an induction plan and a maintenance plan. The expression “induction plan” or “induction period” refers to a treatment plan (or part of a treatment plan) used for the initial treatment of a disease. The general purpose of an induction plan is to provide a subject with a high level of medication during the initial stages of a treatment plan. An induction plan may utilize a “loading plan” (in part or in whole), which may include administering a higher dose of the drug than the clinician would use during the maintenance plan, administering the drug more frequently than the clinician would use during the maintenance plan, or both. The terms “maintenance plan” or “maintenance period” refer to a treatment plan (or part of a treatment plan) used to maintain a subject during treatment for a disease, for example, to maintain the subject in a reduced state for an extended period (several months or several years). A maintenance plan may utilize continuous treatment (e.g., administering the drug at regular intervals, e.g., once a week, once a month, once a year, etc.) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, retrograde treatment, or treatment upon achievement of specific predetermined evaluation criteria [e.g., pain, disease symptoms, etc.]).

[0011] As used herein, the term "PROS" refers to PIK3CA-associated hypergrowth syndromes. PROS is a group of disorders including fibroadipose hypergrowth (FAO), macrocephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric hypergrowth of the torso, lymphoid, capillary and venous tissues, and mixed vascular malformation-epidermal nevus-skeletal and spinal malformation (CLOVES) syndrome, as well as hemiplegia-multiple lipomatous neoplasms (HHML) and Klippel-Trenauni syndrome.

[0012] As used herein, the term “fibroadipose hypergrowth (FAO)” refers to a syndrome characterized by the primary finding of progressive somite hypergrowth of subcutaneous, muscular, and visceral fibroadipose tissue, accompanied by skeletal hypergrowth (Lindhurst et al 2012).

[0013] As used herein, the term “macrocephaly-capillary malformation (MCAP) syndrome” refers to a syndrome characterized by (1) macrocephaly (MEG) or hemimacrocephaly (HMEG) associated with neurological findings of hypotension, stroke, and moderate to severe intellectual disability, and (2) major findings of cutaneous capillary malformation with localized or generalized somatic hypergrowth (Mirzaa et al 2013).

[0014] In certain embodiments, the PROS disorder is CLOVES. As used herein, the term "CLOVES" refers to congenital lipomatous overgrowth-vascular malformation-epidermal nevus-spine / skeletal malformation and / or scoliosis. This syndrome is characterized by a combination of lipomatous tissue and vascular and lymphoid malformations presenting with complex congenital overgrowth (typically manifesting as trunk lipomatous masses).

[0015] As used herein, the term “hemiplegia-hyperplastic multiple lipoma (HHML)” refers to a condition characterized by asymmetrical, non-progressive hypergrowth, multiple lipomas, and surface vascular malformations (BG et al 2013).

[0016] In certain embodiments, PROS disorder is Klippel-Trenauni syndrome. As used herein, the term "Klippel-Trenauni syndrome" refers to a rare congenital medical condition in which the blood vessels and / or lymphatic vessels are not properly formed.

[0017] Therefore, the method of the present invention can be provided to subjects diagnosed with exhibiting one of the impairments in PROS.

[0018] As used herein, the term “subject” refers to any mammal, such as rodents, cats, dogs, and primates. In particular, in the present invention, the subject is a human being with or suspected of having PROS disorder. In certain embodiments, the subject is a human being with or suspected of having CLOVES syndrome. In certain embodiments, the subject is a human being with or suspected of having Klippel-Trenauni syndrome.

[0019] As used herein, the term "BYL719" refers to an ATP-competitive oral PI3K inhibitor selective for the p110α isoform activated by the mutant PIK3CA gene (Furet P., et al. 2013; Fritsch C., et al. 2014). This molecule is also known as alpelisib and, in the art, is given by the following formula: [ka] It has.

[0020] The “therapeutically effective dose” refers to the minimum amount of activating agent necessary to impart a therapeutic benefit to the subject. For example, the “therapeutically effective dose” for a subject is the amount that induces, alleviates, or otherwise produces improvement in pathological symptoms associated with the disorder, disease progression, or physiological symptoms, or resistance to death from the disorder. It will be understood that the total daily dose of the compound of the present invention will be determined by the attending physician within the scope of appropriate medical judgment. The specific therapeutically effective dose level for any particular subject will be determined by a variety of factors, including the disorder being treated and its severity; the activity of the specific compound being used; the specific composition being used; the subject’s age, weight, overall health, sex, and diet; the duration, route of administration, and elimination rate of the specific compound being used; the duration of treatment; drugs used in combination with or concurrently with the specific compound being used; and other similar well-known factors in the medical field. For example, it is well within the reach of those skilled in the art to start with a dose of the compound at a level lower than the dose required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product can vary over a wide range of 0.01 to 1,000 mg per adult per day. Typically, compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient for symptomatic dose adjustment for the subject being treated. Pharmaceuticals typically contain about 0.01 mg to about 500 mg of the active ingredient, preferably 1 mg to about 100 mg of the active ingredient. Effective doses of the drug are usually provided at dose levels of 0.0002 mg / kg to about 20 mg / kg body weight per day, particularly about 0.001 mg / kg to 7 mg / kg body weight per day.

[0021] The PIK3CA inhibitors described above can be combined with pharmaceutically acceptable excipients and optionally with sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmacologically" or "pharmaceutically acceptable" means molecular entities and compositions that, when appropriately administered to mammals, particularly humans, do not produce adverse, allergic, or other undesirable reactions. A pharmaceutically acceptable carrier or excipient refers to any kind of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or compounding aid. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration can be administered to animals and humans, in unit dose forms, or as mixtures with conventional pharmaceutically acceptable carriers, either alone or in combination with other active principles. Appropriate unit dosage forms include oral routes such as tablets, gel capsules, powders, granules, and oral suspensions or solutions; sublingual and buccal administration forms; aerosols; implants; subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, subarachnoid, and intranasal administration forms; and rectal administration forms. Typically, pharmaceutical compositions contain a pharmaceutically acceptable medium for injectable formulations. These can be specific isotonic and sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts), or, on a case-by-case basis, dry, particularly lyophilized, compositions that can be formed into injection solutions by the addition of sterile water or saline. Pharmaceutical forms suitable for injection include sterile water-soluble solutions or suspensions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injection solutions or suspensions. In all cases, the form must be sterile and fluid enough to allow for easy injection. The pharmaceutical form must be stable under manufacturing and storage conditions and must be resistant to contamination by microorganisms, such as bacteria and fungi. A liquid formulation containing the compound of the present invention as a free base or a pharmacokinetically acceptable salt can be prepared in water appropriately mixed with a surfactant, such as hydroxypropyl cellulose.Dispersants can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. Polypeptides (or nucleic acids encoding them) can be incorporated into the composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins). These acid addition salts are formed from inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can be obtained from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, etc., and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc. The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, polyethylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by coating, for example, by using lecithin, by maintaining the required particle size in the case of dispersants, and by using surfactants. Prevention of microbial activity can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be preferable to include isotonic agents, such as sugars or sodium chloride. Extended absorption of the injectable composition can be provided by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition. Sterile injectable solutions are prepared by encapsulating the active polypeptide together with some of the other components assumed above in the required amount in a suitable solvent, and, if necessary, subsequently by sterile filtration. Generally, dispersants are prepared by encapsulating various sterile active ingredients in a sterile medium containing a basic dispersion medium and other components required from those assumed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which involve generating the powders of the active ingredient and any further desired ingredients from a pre-sterilized filtered solution.After formulation, the solution will be administered in a form suitable for the formulation and in a therapeutically effective amount. The formulation can be readily administered in various forms of administration, such as the certain injectable solutions described above. However, drug-releasing capsules and the like can also be used. For parenteral administration of aqueous solutions, for example, the solution may need to be appropriately buffered as needed, and the liquid diluent may first be isotonicized with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art, considering this disclosure. For example, a certain dose can be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of subcutaneous infusion fluid or injected into the target site of administration. Some variations of the dose will inevitably arise depending on the symptoms of the subject being treated. In any case, the person administering the drug will determine the appropriate dose for each individual subject.

[0022] The present invention will be further illustrated by the following drawings and embodiments. However, these embodiments and drawings should not be construed in any way as limiting the scope of the present invention.

[0023] drawing Figure 1: Characterization of the mouse model of PROS and efficacy of BYL719. A) PIK3CA after tamoxifen administration WT and PIK3CA CAGG-CreER Kaplan-Meier survival curves of mice. B) PIK3CA treated with or not treated with BYL719 after tamoxifen administration. CAGG-CreER Kaplan-Meier survival curves of mice. BYL719 was discontinued 40 days after treatment. C) PIK3CA treated with or not treated with BYL719 10 days after tamoxifen administration. CAGG-CreER Kaplan-Meier survival curves for mice. Scale bar: 10 μm.

[0024] Figure 2: Efficacy of BYL719 in an adult patient with severe CLOVES syndrome. A) Clinical parameters (body weight, chest circumference, and abdominal circumference) of patient 1 before and after the start of BYL719. B) Brain natriuretic peptide (BNP) and serum creatinine levels before and after the start of BYL719.

[0025] Figure 3: Efficacy of BYL719 in a pediatric patient with severe CLOVES syndrome. Clinical parameters (mid-thigh circumference and abdominal circumference) of patient 2 before and after the start of BYL719.

[0026] Example Materials and Methods Animals For this study, homozygous R26Stop FL P110 * (Stock# 012343) and heterozygous CAGGCre-ER TM (Stock# 004682) were inserted on the C57BL / 6 background obtained from Jackson Laboratories. R26Stop FL P110 *+ / - ×CAGGCre-ER TM+ (herein referred to as PIK3CA CAGG-CreER ) and R26Stop FL P110 *+ / + ×CAGGCre-ER TM- (herein referred to as PIK3CA WT ) were obtained. The animals were fed freely and housed at a constant ambient temperature on a 12-hour light cycle. The handling of the animals was approved by the ethics committee of the Departmental Director of 「Services Veterinaires de la Prefecture de Police de Paris」 and Paris Descartes University. A single dose of tamoxifen (40 mg×kg -1 ) was administered by oral gavage at 21 days of age. For the survival study, the mice were followed daily after tamoxifen gavage (PIK3CA WT n = 16 and PIK3CA CAGG-CreER(n=16). For therapeutic research, mice were given the PI3KCA inhibitor BYL719 (Chem Express; 50 mg x kg in 0.5% carboxymethylcellulose (Sigma)). -1 The subjects were treated daily with PO or a medium (0.5% carboxymethylcellulose (Sigma), daily PO). The treatment was used in a preventive study (PIK3CA CAGG-CreER (n=18) Studies on tamoxifen forced nutrition during or after treatment (PIK3CA CAGG-CreER The study was started 10 days after n=6. PIK3CA CAGG-CreER In the standard group, a total of 6 mice were euthanized on day 51 after tamoxifen forced feeding, and in the prophylactic BYL719 group, 6 mice were euthanized on day 51 after tamoxifen forced feeding, and PIK3CA was used for histological examination. CAGG-CreER In a therapeutic study, six mice were euthanized 70 days after forced feeding with tamoxifen.

[0027] cell culture The breast cancer cell lineage T-47D was obtained from Sigma Aldrich. Cells were cultured in a medium containing DMEM + 2 mM glutamine + 10% fetal bovine serum (FBS). For the BYL719 experiment (Chem Express), cells were treated with increasing concentrations of BYL719 (0, 0.5, 1, and 5 μmol / L) for 2, 4, and 6 hours prior to Western blotting. Each experiment was duplicated and repeated at least three times.

[0028] Morphological analysis Mouse tissues were fixed in 4% paraformaldehyde and embedded in paraffin. 4 μm sections of liver were stained with the periodate Schiff reaction (PAS). 4 μm sections of spleen or liver were stained with hematoxylin and eosin (H&E), and 4 μm sections of kidney were stained with Masson's trichrome.

[0029] Immunohistochemistry and immunofluorescence 4 μm sections of paraffin-embedded kidney were treated with anti-P-AKT (Ser 473The samples were incubated with the following antibodies: (Cell Signaling Technology, ref# 4060), anti-P-S6RP antibody (Cell Signaling Technology, ref# 5364), and anti-CD34 antibody (eBioscience, ref# 14-0341). Immunofluorescence studies were analyzed using a Zeiss LSM 700 confocal microscope.

[0030] Western blot Western blots were previously described. 21 The procedure was carried out as described above. In short, protein extracts from liver, muscle, heart, kidney, and T-47D cells were separated by SDS-PAGE, then transferred onto a suitable membrane, and anti-P-AKT(Ser 473 ) Antibody (Cell Signaling Technology, ref# 4060) Anti-P-AKT (Thr 308 The samples were incubated with the following antibodies: (Cell Signaling Technology, ref# 13038), anti-P-S6RP antibody (Cell Signaling Technology, ref# 5364), anti-GAPDH (Merck Millipore, ref# 374), and anti-β-actin antibody (Sigma-Aldrich, ref# A2228), followed by incubation with an appropriate peroxidase conjugate secondary antibody. Chemiluminescence was acquired using a Fusion FX7 camera (Vilbert Lourmat), and density measurements were performed using Bio1D software (Certain Tech).

[0031] patient This study was conducted at the Renal Division of Necker Hospital, starting in September 2015. Patients with CLOVES syndrome underwent clinical examinations, tumor measurements, and nevus measurements prior to the initiation of treatment. BYL719 treatment was initiated at an oral dose of 250 mg per day. This study was conducted on two patients (one adult and one child) and followed up at Necker Hospital. This protocol was approved by ANSM (Approval Nos. 553984-986 and 584018), and written informed consent was obtained from each patient or their legal representative. BYL719 was provided exceptionally by Novartis. Patient 1 received 250 mg / day, and patient 2 received 50 mg / day. BYL719 was delivered orally every morning before breakfast. Glycoglycemia was monitored after any meal for two months, and then the monitoring interval was gradually increased.

[0032] Data analysis and statistics Data were expressed as mean ± SEM. Survival curves were analyzed using the Mantell-Cox (Long Rank) test. Differences between experimental groups were assessed using ANOVA, followed by the Tukey-Kramer test to determine significance (P<0.05). When comparing only two groups, the Mann-Whitney test was used. Statistical analysis was performed using Graph Prism software.

[0033] renal function Serum creatinine levels were measured weekly during the first year and every three months thereafter using a Synchron Cx4 autoanalyzer (Beckman Coulter, Villepinte, France). Glomerular filtration rate was estimated using the MDRD formula (eGFR).

[0034] Biopsy sample and morphological analysis Renal biopsy specimens were fixed in an alcohol-formalin-acetic acid solution and embedded in paraffin. 4-micrometer sections were stained with periodate Schiff reaction (PAS) dye, Masson's trichrome, and hematoxylin and eosin (H&E). Electron microscopy analysis was performed. Immunohistochemistry and immunofluorescence Four sections of paraffin-embedded kidneys were incubated with anti-nephrin antibody (Progen), anti-WT1 antibody (Dako), anti-podosin antibody (Sigma), and anti-synaptopodil antibody (Novus Biologicals). All glomerular sections were quantified for each biopsy according to total glomerular area. Primary antibodies were identified using appropriate Alexa 488 or 555 conjugate secondary antibodies (Molecular Probes). Immunofluorescence staining was visualized using a Zeiss LSM 700 confocal microscope. Podocyte staining area was automatically quantified using a Nikon digital camera Dx / m / 1200 and ImageJ software, and expressed as % podocyte staining area.

[0035] in situ hybridization Alcohol-formalin-acetic acid fixed and paraffin-embedded tissues were assayed for PI3KCα RNA expression using the previously described digoxigenin anti-digoxigenin technique. Affected cells in the tissues were visualized using nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphothordinium (NBT-BCIP). The specificity of the hybridization signal was systematically confirmed by hybridizing the sense probe with parallel sections and the antisense probe with unaffected kidney tissue. ISH-stained tissues were visualized and imaged using an Olympus Proxis microscope and a Zeiss Axio Cam ICc1.

[0036] cell culture Human fibroblasts were grown in DMEM supplemented with 15% FBS, penicillin (50 IU / ml) / streptomycin (50 μg / ml), and non-essential amino acids (Invitrogen).

[0037] Western blot Western blotting was performed as previously described, using a rabbit antibody against human phospho-p70 S6 kinase (Thr389) (Cell Signaling Technologies) at a ratio of 1:1000, followed by a horseradish peroxidase conjugate anti-rabbit secondary antibody (Dako) at a ratio of 1:10,000. The phosphorylation status of p70 S6 kinase at Thr389 is specific to mTOR phosphorylation. A mouse monoclonal anti-β-actin antibody (Sigma-Aldrich, Lyon, France) was used as a control. Protein phosphorylation levels were normalized to matched β-actin density measurements.

[0038] DNA sequencing DNA was extracted from peripheral blood mononuclear cells (PBMCs) and skin biopsies from tumor areas, collected from patients using standard techniques. Mutation screening was performed by direct sequencing of all PI3KCα exons and adjacent intron regions.

[0039] volume measurement The volume of each tumor was measured using magnetic resonance imaging (MRI). MRI was performed on day 0 of BYL719 initiation and monthly thereafter. Briefly, the volume was calculated by summing the products of the area measurement and the slice thickness.

[0040] Data analysis and statistics Data were expressed as mean ± SD. Differences between experimental groups were assessed using ANOVA, followed by the Tukey-Kramer test to determine statistical significance. When comparing only two groups, the Mann-Whitney test was used. A probability value < 0.05 was considered statistically significant. The analysis was performed using GraphPad Prism 5 (GraphPad software, La Jolla, CA).

[0041] result A significant reduction in the size of overgrown tissue was observed in subjects treated with BYL719. After one month of treatment, the patient's weight decreased from 83.5 kg to 73.5 kg. This weight loss was associated with a dramatic reduction in edema and an improvement in overall cardiac function velocity (cardiac output was measured at 22 l / min in December 2015 before treatment, and 8 l / min on February 8). Plasma brain natriuretic peptide levels decreased from 2500 pg / min to 240 pg / min on day 30, confirming an improvement in heart failure. Subcutaneous tumor size showed an overall reduction of 10% at 30 days post-treatment, as assessed by CT scan and magnetic resonance imaging (MRI). The patient's Karnovsky Performance Status Scale improved from 40% to 60% at day 30. Hemoglobin levels increased from 8 g / dl to 11.8 g / dl.

[0042] Some skin areas (left ear) that showed accelerated aging phenotypes before treatment improved after 1 month of BYL719 administration. In addition, several large nevi showed depigmentation after the start of treatment.

[0043] PROS mouse models This research was initiated by developing a mouse model of PROS. To achieve this objective, the transgenic mouse strain R26StopFLP110 was used. * This was used. In these mice, the activated PI3KCA heterodimer can be attracted to and expressed in a tissue-specific manner. R26StopFLP110 *Mice were crossed with CAGG-CreER mice, and PIK3CA was ubiquitously overexpressed upon administration of tamoxifen. CAGG-CreER Animals were induced. 3-week-old mice were given 40 mg / kg. -1 Cre recombination was induced by treatment with a single dose of tamoxifen. 16 . Control PIK3CA WT Compared to a mouse, PIK3CA CAGG-CreER Mice were observed to begin dying 3 days after Cre induction (average 6 days after Cre recombination) (Figure 1A). Autopsy revealed that the majority of the sudden deaths were due to intraperitoneal and hepatic hemorrhage (data not shown). Some mice exhibiting difficulty walking due to hypertrophic psoas muscles were evaluated by magnetic resonance imaging (MEI) (data not shown). In addition, whole-body MRI showed the rapid development of scoliosis, vascular abnormalities, renal cysts, and muscle hypertrophy (Figure 1C). Histological examination revealed multiple organ abnormalities, including severe fatty liver with vascular destruction (data not shown), loss of splenic microstructural integrity (data not shown), spontaneous hemorrhage, and renal fibrosis including aberrant vessels. CD34 immunostaining was performed to further characterize the vascular abnormalities, confirming the presence of severe vasodilation. As predicted, Western blot and immunofluorescence studies showed AKT / mTORC pathway activation in all examined organs. Therefore, PIK3CA CAGG-CreER It was concluded that the mice reproduced the human PROS phenotype.

[0044] BYL719 prevents PROS in mice and is an effective treatment for PROS. PIK3CA CAGG-CreER We decided to investigate the effects of BYL719 in mice. To this end, we first conducted a prophylactic study in which BYL719 was administered orally, starting immediately after Cre induction (data not shown). Daily administration of BYL719 dramatically improved the survival of the animals (Figure 1C). In fact, all PIK3CAs in the placebo group were observed to be different. CAGG-CreER Mice died within 21 days, but PIK3CA was treated with BYL719. CAGG-CreERThe mice were alive after 40 days with a seemingly normal appearance. Importantly, discontinuing treatment 40 days after Cre recombination resulted in all PIK3CA CAGG-CreER Several animals died. These were PIK3CA animals that had been treated with BYL719. CAGG-CreER Mice were euthanized 40 days after Cre induction. Histological examination revealed that mice treated with BYL719 had preserved tissue (data not shown) and normal blood vessels (data not shown). Western blotting and immunofluorescence confirmed the efficacy of BYL719 in inhibiting PI3KCA activation (data not shown).

[0045] Next, either placebo or BYL719 is administered via PIK3CA. CAGG-CreER A therapeutic study was conducted by administering BYL719 to mice 10 days after Cre induction (when the mice began to die) (Figure 1C). On day 10 after Cre induction, the presence of the previously described tissue abnormalities was confirmed by MRI (data not shown). The placebo group mice died the following day, but BYL719 showed PIK3CA CAGG-CreER Mouse survival was dramatically improved (Figure 1J). MRI performed 12 days after the start of treatment (22 days after Cre induction) demonstrated very rapid improvement in scoliosis, muscle hypertrophy, and vascular abnormalities (data not shown). Histological analysis showed that BYL719-treated mice exhibited no or only slight tissue changes (data not shown). Similar to preventive studies, PIK3CA inhibition was confirmed by Western blotting and immunofluorescence (data not shown).

[0046] It was concluded that BYL719 dramatically improved PROS mice. This suggests that BYL719 could be a viable treatment option for PROS patients for whom there are currently no available therapies.

[0047] Efficacy of BYL719 in human cells with PIK3CA mutations Next, we tested the efficacy of BYL719 in inhibiting the PIK3CA pathway in human cells. For this purpose, we used T-47D human cells obtained from breast cancer. These cells have a heterozygous mutation in PIK3CA c.3140A>G(H1047R). Acquisition of this functional mutation is also the most frequently observed mutation in PROS patients. 6 .

[0048] First, in these cells, the residue Thr 308 and Ser 473 Spontaneous activation of the AKT / mTORC pathway was confirmed not only by the phosphorylation state of AKT but also by the phosphorylation of the S6RP protein (data not shown). Next, cells were exposed to increasing concentrations of BYL719. Complete inhibition of the AKT / mTORC pathway was observed 2 hours after exposure to BYL at a concentration of 1 μmol / L (data not shown). This effect was similar to that observed when cells were exposed to BYL719 for 4 and 6 hours. These data demonstrate that BYL719, along with the PIK3CA c.3140A>G(H1047R) mutation, can inhibit PIK3CA activation in human cells.

[0049] Dramatic effects of BYL719 in patients with PROS Based on these results, we decided to administer BYL719 as a rescue drug for two patients (one adult and one child) with extremely severe clinical manifestations of CLOVES syndrome who had experienced treatment failure and life-threatening complications. Patient 1 was a 29-year-old male who, based on mosaicism and biopsy, demonstrated the acquisition of the functional mutation PIK3CA c.3140A>G(H1047R). Patient 1 had hypergrowth syndrome characterized by left leg hypertrophy, scoliosis, multiple nevi and extremely severe vascular abnormalities (data not shown). Patient 1 had undergone multiple invasive debulking surgeries to remove abdominal and back vascular tumors and multiple angiography with embolization to limit tumor growth. Patient 1 became paraplegic at age 20 due to spinal cord compression and required bladder stent placement for urinary drainage. Patient 1 progressed to severe, progressively worsening systolic heart failure, resistant to all conventional medications, with a measured cardiac output of 18 l / min and consistently brain natriuretic peptide (BNP) levels exceeding 2500 pg / mL (N < 100 pg / mL). It is unclear whether the heart failure was due to the presence of a vascular shunt, a PIK3CA mutation in cardiomyocytes, or both. For five years, the patient received rapamycin to limit tumor growth and progression, but without effect. Finally, the patient progressed to renal dysfunction with severe proteinuria. Renal biopsy revealed glomerular lesions with extensive fibrosis. The renal lesions were associated with cardiac dysfunction and the use of rapamycin. 11 This may be a result of a PIK3CA mutation in renal epidermal cells. Severe vascular abnormalities were observed on CT scan (data not shown) and MRI (data not shown), but the PET scan was negative. Due to the severity of the case with a poor prognosis, the physician, surgeon, and radiographer decided to discontinue any interventional procedures and provide the patient with supportive care and palliative care only. The patient's survival was estimated to be several months.

[0050] Following laboratory findings and ethical considerations, it was decided to propose BYL719 to this patient. Permission was obtained from Novartis and the French regulatory authorities (Agence nationale de securite du medicament et des produits de sante, ANSM) to administer BYL719 as rescue use. Rapamycin was discontinued, and BYL719 was initiated one month later. This drug was delivered orally daily. The starting dose was selected as 250 mg per day, based on the lowest dose used in clinical trials and in vitro data. A dramatic improvement in the patient's overall condition was observed within the first few days. The patient began to feel better and more comfortable. Dramatic effects of the drug on tumor size, venous dilation, and skin characteristics could be targeted weekly (data not shown). The patient lost 23 kg over 12 months, not only due to the reduction of edema but also due to a significant reduction in all vascular tumor abnormalities (data not shown). Chest circumference and abdominal circumference decreased dramatically by Δ-20% and Δ-35%, respectively, over 12 months (Figure 2A). CT scans and MRI confirmed overall angiotumor contraction and disappearance of subcutaneous infiltration, with a 63% volume reduction over 12 months (data not shown). The drug's effect on cardiac function was significant. BNP levels were completely corrected within 4 weeks (Figure 2B). Cardiac output decreased to 3 l / min, and heart size, as measured by CT scan, decreased by 25% (data not shown). Left ventricular volume, correlated with body surface area, was 250 g / m². 2 From 148g / m 2 It decreased to [a certain level]. Renal function also improved rapidly. The estimated glomerular filtration rate was 33 to 52 ml / min / 1.75m². 2The volume increased (Figure 3D). The volume of the enlarged left leg also decreased (data not shown). A dramatic improvement in skin hypertrophy was observed, accompanied by changes in nerve coloration and a decrease in ear size (data not shown). Finally, six months after starting BYL719, the patient began to partially regain bladder function with improved saddle anesthesia. MRI demonstrated a 60% reduction in the size of the venous malformation that had been compressing the spinal cord. Twelve months later, the patient had no side effects except hyperglycemia, which was well controlled as planned. The patient is still receiving BYL719.

[0051] Patient 2 was a 9-year-old girl diagnosed with CLOVES syndrome two years prior to this study. Biopsy confirmed the acquisition of a functional mosaic mutation in PIK3CA c.3140A>G(H1047R). The patient had scoliosis, left leg hypertrophy, vascular abnormalities, and back muscle hypertrophy (data not shown). Importantly, Patient 2 had a large cystic lymphangioma associated with the left kidney and gastrointestinal tract. PET scans showed metabolic fixation in the thymus, back muscles, and left leg (data not shown). The intraperitoneal tumor was rapidly growing, and surgery or intervening radiology was deemed impossible. After ethical considerations, authorization to provide BYL719 was obtained from Novartis and the French supervisory authority (ANSM). As this drug had not been tested in children, it was decided to start with the minimum available dose of 50 mg / day. Similar to Patient 1, rapid and dramatic clinical improvement was observed after the initiation of treatment (data not shown). At day 120, this patient reported improved comfort, a 15% reduction in left leg volume, a decrease of one shoe size in the left foot, and an 8% reduction in waist circumference (data not shown). Back muscle hypertrophy rapidly contracted, and unexpectedly, Patient 2's scoliosis regressed at day 120 without any other intervention (Figure 3). More importantly, MRI showed a 60% reduction in intra-abdominal tumor volume in just four months. A PET scan performed four months after the start of treatment confirmed disease reduction in almost all tissues. During follow-up, it was found that this patient's normal growth was not affected by BYL719. No side effects were reported from Patient 2. This patient is still receiving treatment.

[0052] Discussion This report describes the first mouse model of PROS that replicates the human phenotype, the rescue of PROS in this mouse model by treatment with the PI3CA inhibitor BYL719, and the use of a conversion approach that demonstrated the efficacy of BYL719 in PROS patients. This study provides the first direct evidence supporting PI3CA inhibition as a robust and effective therapeutic strategy for previously untreatable PROS.

[0053] Treatment with BYL719 accelerated recovery in two patients (one adult and one child), with rapidly observable positive clinical effects after the initiation of treatment. Similar to the mouse model, this drug improved all organ dysfunction associated with PROS. Importantly, the drug maintained its malformation-improving friendliness throughout the treatment period. In contrast to mTOR inhibitors, the drug was well tolerable with no significant side effects. As predicted, assuming PI3KCA is downstream of the insulin receptor, the hyperglycemia observed in patient 1 warrants attention to the predetermined possibility of long-term side effects, particularly those affecting growing children, conception, and adolescence. However, the child's growth was not affected during the treatment period, and any adverse effects should be weighed against the benefits in this previously severely refractory disease. Several questions remain, including the best time to initiate treatment and when treatment should be discontinued. Similar to the mouse data, as also seen in patients with tuberous sclerosis, 17 Therefore, it is highly likely that discontinuing medication will be associated with a relapse of the disease.

[0054] PIK3CA is one of the most commonly mutated oncogenes in human cancer, and similar mutations have been found in PROS patients. 6 Similar to cancer, PIK3CA mutations can be detected throughout the entire coding sequence of this gene, with the majority of mutations found in three main hotspot clusters in the helical domain (E542K, E545K) and kinase domain (H1047R). Each of these mutations results in gain-of-function activation of the PIK3CA pathway. Importantly, in cancer models, BYL719 can completely block the activity of this pathway, regardless of the type of mutation. 14In addition, our mouse model of ROS based on copy number augmentation of the PIK3CA gene is rescued by BYL719. In summary, these data suggest that this drug is indeed effective against all forms of PROS and does not act only on the H1047R mutation.

[0055] This finding may have broad clinical implications. Importantly, even though PROS is rare, this is no exception. The number of PROS patients is likely significantly underestimated due to phenotypic heterogeneity that leads to misdiagnosis. Mosaicism often requires the use of highly sensitive techniques to detect PIK3CA somatic alterations. 6 Assuming an incidence of 1 / 100,000, recent proposals to include patients with Klippel-Trenauni syndrome in PROS highlight a significant number of patients who could benefit from BYL719. Importantly, PROS should be considered because an increasing number of other diseases, such as isolated venous malformations, may also be associated with PIK3CA mutations. 8、18、19 It is a developing entity.

[0056] Our mouse model provides the only opportunity to better understand the physiology and pathology of PROS. This model exhibits vascular abnormalities, muscle hypertrophy, renal dysfunction, fatty liver, spleen PROS with destructive and scoliosis was reproduced. Severity was associated with a high rate of Cre recombination events induced by tamoxifen treatment. Mice died, particularly due to severe bleeding from mesenteric and hepatic blood vessels. Among the serious complications, bleeding and leakage were observed in patients with CLOVES syndrome. 6 Furthermore, it was found that overexpression of the PIK3CA gene in the liver leads to fatty liver, similar to the case of genetic overactivation of AKT / mTORC. 20The mice rapidly progressed to scoliosis after Cre recombination. This lesion was certainly due to the progression of hypertrophic paravertebral trunk parenchyma. BYL719 was able to rapidly regress this abnormality. Improvement in scoliosis was also observed in two patients, accompanied by a reduction in myoma accumulation and hypermetabolic signals. BYL719 was able to either prevent the progression of any tissue abnormality or rescue all defects while the disease was progressing.

[0057] In summary, the results of this study suggest that PIK3CA inhibition may be a safe and effective means of improving the health and quality of life for patients with PROS. While these results clearly need to be confirmed in a larger cohort of patients, they represent the most promising and available treatment for patients with PROS.

[0058] Throughout this application, various references illustrate the state of the art in which the present invention pertains. The disclosures of these references are incorporated herein by reference.

Claims

1. A pharmaceutical composition comprising BYL719 for use in a method of treating PIK3CA-associated overgrowth syndrome (PROS) disorder in subjects requiring treatment for PROS disorder, wherein BYL719 is administered daily in a dose in the range of 0.001 mg / kg to 7 mg / kg.

2. The pharmaceutical composition according to claim 1, wherein the PROS disorder is congenital lipomatous hypergrowth - vascular malformation - epidermal nevus - spinal / skeletal malformation and / or scoliosis (CLOVES) syndrome.

3. The pharmaceutical composition according to claim 1, wherein the PROS disorder is Klippel-Trenauni syndrome.

4. The pharmaceutical composition according to claim 1, wherein the PROS disorder is selected from the group consisting of fibroadipose hypergrowth, macrocephaly-capillary malformation syndrome, hemiplegia-hyperplastic multiple lipomas, and Klippel-Trenauni syndrome.

5. The pharmaceutical composition according to claim 1, wherein the PROS disorder is selected from the group consisting of fibroadipose hypergrowth, macrocephaly-capillary malformation syndrome, hemiplegia-multiple lipomatous neoplasms, Klippel-Trenauny syndrome, and congenital lipomatous asymmetric hypergrowth, lymphoid, capillary, venous and mixed vascular malformations-epidermal nevi-skeletal and spinal malformations, and / or scoliosis (CLOVES) syndrome.

6. A pharmaceutical composition according to any one of claims 1 to 5, wherein the method treats one or more symptoms of PIK3CA-associated overgrowth syndrome (PROS) disorder.

7. The pharmaceutical composition according to claim 6, wherein the method treats a symptom selected from the group consisting of edema, venous dilation, hemangiomas, elevated blood concentration of brain natriuretic peptide, proteinuria, renal dysfunction, overgrowth, dysregulated adipose tissue, scoliosis, skeletal hypertrophy without progressive skeletal hypergrowth, benign tumors, macrocephaly-capillary malformations, lipomatous asymmetric overgrowth of the torso, epidermal nevi, lymphoid malformations, muscle hypertrophy, fatty liver, and splenic destruction.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the method comprises orally administering a therapeutically effective amount of BYL719 sufficient to reduce the size of overgrown tissue or prevent the progression of any tissue abnormality.

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