BYL719 (Alpelisib) for use in the treatment of PIK3CA-Related Overgrowth Spectrum (PROS - CLOVES syndrome)
Patent Information
- Application Number
- AU2024278598
- Authority / Receiving Office
- AU · AU
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2026-08-27
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Abstract
Description
This application is a divisional of Australian Application No. 2022235569, filed 5 21 September 2022, which is a divisional of Australian Application No. 2017219834, filed 17 February 2017, is related to PCT / EP2017 / 053587, and claims priority from European Provisional Application No. 16305193.1, filed 19 February 2016, all of which are incorporated herein by reference in their entirety. 10 FIELD OF THE INVENTION: The invention relates to methods for the treatment of PIK3CA-related overgrowth spectrum (PROS), such as congenital lipomatous overgrowth, vascular malformations and epidermal nevi (CLOVES). 15 BACKGROUND OF THE INVENTION: The term “PROS” for PIK3CA-Related Overgrowth Spectrum” was agreed upon to encompass both the known and emerging clinical entities associated with somatic PIK3CA mutations including, macrodactyly, FAO, HHML, CLOVES, and related megalencephaly conditions (Keppler-Noreuil et al 2014). A workshop was convened at the National Institutes 20 of Health (NIH) to discuss and develop a consensus document regarding diagnosis and treatment of patients with PIK3CA-associated somatic overgrowth disorders. CLOVES, stands for Congenital, Lipomatous, Overgrowth, Vascular Malformations, Epidermal Nevi and Spinal / Skeletal Anomalies and / or Scoliosis. This syndrome is considered as a rare disorder and characterised by progressive, complex, and mixed truncal vascular 25 malformations, dysregulated adipose tissue, varying degrees of scoliosis, and enlarged bony structures without progressive bony overgrowth (Sapp et al 2007; Alomari et al 2009). This syndrome is different from cancer. In fact, in CLOVES syndrome, tumours are benign, the tissues are just overgrowth and the subject is deformed. CLOVES syndrome is rare and evident at birth. It affects males and females equally regardless of their race or ethnicity. 30 Many of the patients with this syndrome are misdiagnosed. CLOVES syndrome is caused by a somatic mosaic mutation in PIK3CA gene. PIK3CA encodes the 110-kD catalytic alpha subunit of PI3K, which in response to tyrosine kinase receptor ligand binding is activated and converts phosphatidylinositol (3,4)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3). Activating mutations 5 in PIK3CA have been described in several types of cancers but never in CLOVES syndrome. In 2012, Kurek et al has identified the activating mutations in PIK3CA by sequencing DNA or RNA. But, today, there is no cure for CLOVES Syndrome. Limaye et al 2015 discloses that somatic mutations in PIK3CA are involved in cancers, overgrowth syndromes, and lymphatic malformation (LM). More particularly, the 10 [Text continues on page 2.] 2024278598 19 Dec 2024 2024278598 05 Aug 2026 authors showed that cultured cell lines derived from healthy donors (not affected by PROS), called human umbilical venous endothelial cells (HUVEC) then retrovirally transfected with mutant PIK3CA have an activation of the AKT / mTORC pathway. The drug BYL719 abolished, as expected, PIK3CA-variant induced AKT phosphorylation in this artificial 5 model, indicating that these proteins participate in the same signalling pathway. But, this in vitro model does not recapitulate any symptom or disease phenotype related to PROS patients, more particularly does not show any evidence that this drug could be used to treat patients suffering from PROS. Thus, there is a need to understand the use of BYL719 in patients with PROS, more particularly with CLOVES syndrome or Klippel-Trenaunay 0 syndrome. It is an object of the present invention to overcome or ameliorate one or more of the disadvantages of the prior art, or at least to provide a useful alternative. SUMMARY OF THE INVENTION: The present invention relates to a method of treating PROS in a subject in need 15 thereof comprising a step of administrating the subject with a therapeutically effective amount of BYL719. In particular, the present invention is defined by the claims. In a first aspect, the present invention relates to a method of treating PIK3CA-Related Overgrowth Spectrum (PROS) in a subject in need thereof comprising a step of administering to the subject a therapeutically effective amount of BYL719, wherein BYL719 reduces the 20 size of overgrown tissue or prevents the development of one or more tissue abnormalities. In a second aspect, the present invention relates to a method of treating PIK3CA-Related Overgrowth Spectrum (PROS) in a subject in need thereof comprising the steps of: identifying the subject as suffering from a PROS disorder; 25 administering to the subject a therapeutically effective amount of BYL719; and reducing the size of overgrown tissue or tumor or preventing the development of one or more tissue or organ abnormalities. In a third aspect, the present invention relates to use of a therapeutically effective 30 amount of BYL719 in the manufacture of a medicament for treating PIK3CA-Related Overgrowth Spectrum (PROS) in a subject in need thereof, wherein BYL719 reduces the size of overgrown tissues or prevents the development of one or more tissue abnormalities. 2024278598 05 Aug 2026 In a fourth aspect, the present invention relates to use of a therapeutically effective amount of BYL719 in the manufacture of a medicament for treating PIK3CA-Related Overgrowth Spectrum (PROS) in a subject in need thereof, wherein the treating comprises identifying the subject as suffering from a PROS disorder, wherein the treating reduces the 5 size of overgrown tissue or tumor or prevents the development of one or more tissue or organ abnormalities. DETAILED DESCRIPTION OF THE INVENTION: BYL719 synthesized by Novartis is in clinical trial at phase II / III for advanced solid 0 tumours. Inventors developed the first genetic mouse model of PROS that recapitulates the human disease and demonstrated the efficacy of BYL719, a pharmacological inhibitor of PIK3CA, in preventing and improving all organs dysfunction in the PROS mouse. Based on these results they treated two patients, one adult and one child, with severe CLOVES syndrome using BYL719. The drug had a robust efficiency on disease in the two patients 15 inducing quick recovery of all affected organs. Previously intractable vascular tumors shrunk, congestive heart failure fully recovered, hemi hypertrophy reduced, and scoliosis was attenuated. The drug was not associated with any significant side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic in PROS patients. 20 Accordingly, the present invention relates to a method of treating PROS in a subject in need thereof comprising a step of administrating the subject with a therapeutically effective amount of BYL719. More particularly, the invention relates to a method of treating CLOVES syndrome in a subject in need thereof comprising the step of administrating the 25 subject with a therapeutically effective amount of BYL719. [TEXT CONTINUES ON PAGE 3] 2024278598 19 Dec 2024 As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical 5 condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of 10 treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment 15 regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a 20 therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a 25 particular predetermined criteria [e.g., pain, disease manifestation, etc.]). As used herein the term “PROS” refers to PIK3CA-Related Overgrowth Spectrum. It is a group of disorders such as fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, 30 skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML) and Klippel-Trenaunay syndrome. As used herein, the term “fibroadipose overgrowth (FAO)” refers to a syndrome, which is characterized by the major findings of segmental progressive overgrowth of 2024278598 19 Dec 2024 subcutaneous, muscular, and visceral fibroadipose tissue with skeletal overgrowth (Lindhurst etal 2012). As used herein, the term “megalencephaly-capillary malformation (MCAP) syndrome” refers to a syndrome which is characterized by the major findings of (1) 5 megalencephaly (MEG) or hemimegalencephaly (HMEG) associated with neurologic findings of hypotonia, seizures, and mild to severe intellectual disability; and (2) cutaneous capillary malformations with focal or generalized somatic overgrowth (Mirzaa et al 2013). In a particular embodiment, the PROS disorder is CLOVES. As used herein, the term “CLOVES” refers to Congenital, Lipomatous, Overgrowth, Vascular Malformations, 10 Epidermal Nevi and Spinal / Skeletal Anomalies and / or Scoliosis. This syndrome is characterised by lipomatous tissues showing complex congenital overgrowth (typically appearing as a truncal lipomatous mass) and a combination of vascular and lymphatic malformations. As used herein, the term “Hemihyperplasia Multiple Lipomatosis (HHML)” refers to a 15 condition characterized by asymmetric nonprogressive overgrowth, multiple lipomas, and superficial vascular malformations (BG et al 2013). In a particular embodiment, the PROS disorder is Klippel-Trenaunay syndrome. As used herein, the term “Klippel-Trenaunay syndrome” refers to a rare congenital medical condition in which blood vessels and / or lymph vessels fail to form properly. 20 Thus, the method according to the present invention can be supplied to a subject, who has been diagnosed as presenting one of the disorders in PROS. As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a 25 canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with PROS disorders. In a particular embodiment, the subject is a human afflicted or susceptible to be afflicted with CLOVES syndrome. In a particular embodiment, the subject is a human afflicted or susceptible to be afflicted with Klippel-Trenaunay syndrome. 2024278598 19 Dec 2024 As used herein, the term “BYL719” is an ATP-competitive oral PI3K inhibitor selective for the pl 10a isoform that is activated by a mutant PIK3CA gene (Furet P., et al. 2013; Fritsch C., et al 2014). This molecule is also called Alpelisib and has the following formula in the art: A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological 10 conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound 15 employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound 20 at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the 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 the symptomatic adjustment of the dosage to the subject to 25 be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective 2024278598 19 Dec 2024 amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day. The PIK3CA inhibitor as described above may be combined with pharmaceutically 5 acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, 10 semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and 15 human beings. Suitable unit administration forms comprise oral-route forms 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, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain 20 vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable 25 solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and 30 must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these 2024278598 19 Dec 2024 preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, 5 hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, 10 polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for 15 example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in 20 the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable 25 solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage 30 forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal 2024278598 19 Dec 2024 administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage 5 will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. The invention will be further illustrated by the following figures and examples. 10 However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1: Characterization of the mouse model of PROS and efficacy of the 15 BYL719. A) Kaplan-Meier survival curves of PIK3CAWT and PIK3CACAGG~CreER mice after tamoxifen administration. B) Kaplan-Meier survival curves of PIK3CA(AGGAie,R mice treated or not with BYL719 after tamoxifen administration. After 40 days of treatment the BYL719 is withdrawn. C) Kaplan-Meier survival curves of PIK3CA( AGGAie,R mice treated or not with BYL719 10 days after tamoxifen administration. Scale bars 10pm. 20 Figure 2: Efficiency of the BYL719 in an adult patient with severe CLOVES syndrome. A) Clinical parameters (weight, chest girth and waist circumferences) of the patient 1 before and after BYL719 initiation. B) Brain Natriuretic Peptide (BNP) and serum creatinine levels before and after BYL719 initiation. Figure 3: Efficiency of the BYL719 in a child patient with severe CLOVES 25 syndrome. Clinical parameters (thigh and waist circumferences) of the patient 2 before and after BYL719 initiation. EXAMPLE: Material & MethodsAnimals 30 For this study, we interbedded homozygous R26StopFLPl 10* (Stock# 012343) and heterozygous CAGGCre-ER™ (Stock# 004682) on C57BL / 6 background obtained from The Jackson Laboratories. We obtained R26Stop,'P 110*" x CAGGCre-ER™+ (referred here as PIK3CACAGG-CreER) and R26StopFLPl 10*+ / + x CAGGCre-ER™' (referred here as PIK3CAWT). 2024278598 19 Dec 2024 Animals were fed ad libitum and housed at constant ambient temperature in a 12-hour light cycle. Animal procedures were approved by the Departmental Director of “Services Veterinaires de la Prefecture de Police de Paris” and by the ethical committee of the Paris Descartes University. A single dose of tamoxifen (40mg x kg1) was administered through 5 oral gavage at the age of 21 days. For survival studies, mice were followed daily after tamoxifen gavage {PIK3CAWT n=16 and PIK3CACAGG~CreER n=16). For therapeutic studies, mice were treated with the PI3KCA inhibitor BYL719 (Chern Express; 50 mg x kg1 in 0.5% carboxymethylcellulose (Sigma), daily p.o.) or vehicle (0.5% carboxymethylcellulose (Sigma), daily p.o.). Treatment was started in the meantime of tamoxifen gavage for the 10 preventive study (PIK3CA(AGGAie,R n=18) or ten days after for the therapeutic study (PIK3CACAGG~CreER n=6). A total of 6 mice were sacrificed on day 51 after tamoxifen gavage in the vehicle group of the PIK3CA( AGGA i e, R, 6 mice on day 51 after tamoxifen gavage in the preventive BYL719 group and 6 mice on day 70 after tamoxifen gavage in the therapeutic study of the PIK3CACAGG~CreER for tissues examination. 15 Cell cultures Breast cancer cell lines T-47D were obtained from Sigma Aldrich. Cells were cultured in media with DMEM + 2mM Glutamine + 10% Fetal Bovine Serum (FBS). For BYL719 experiments (Chern Express), cells were treated with increasing concentration of BYL719 (0, 0.5, 1 and 5 pmol / L) for 2, 4 and 6 hours before western blot was performed. Each 20 experiment was performed in duplicate and repeated at least three times. Morphological analysis Mousse tissues were fixed in 4% paraformaldehyde, paraffin embedded. 4- / / m sections of liver were stained with periodic acid Schiff (PAS), 4- / 7 m sections of liver of spleen were stained with hematoxylin and eosin (H&E) and 4- / / m sections of kidneys were stained 25 with Masson’s trichrome. Immunohistochemistry and immunofluorescence 4-pm sections of paraffin-embedded kidneys were incubated with anti-P-AKT (Ser473) antibody (Cell Signaling Technology, ref# 4060), anti-P-S6RP antibody (Cell Signaling Technology, ref# 5364) and anti-CD34 antibody (eBioscience, ref# 14-0341). 30 Immunofluorescence studies were analyzed using the confocal microscope Zeiss LSM 700. Western blot Western blots were performed as previously described21. Briefly, protein extracts from liver, muscles, heart, kidneys and T-47D cells were resolved by SDS-PAGE before being transferred onto the appropriate membrane and incubated with anti-P-AKT (Ser473) antibody 2024278598 19 Dec 2024 (Cell Signaling Technology, ref# 4060), anti-P-AKT (Thr308) antibody (Cell Signaling Technology, ref# 13038), anti-P-S6RP antibody (Cell Signaling Technology, ref# 5364), anti-GAPDH (Merck Millipore, ref#374) and anti-0 actin antibody (Sigma-Aldrich, ref#A2228), followed by the appropriate peroxidase-conjugated secondary antibody. Chemiluminescence 5 was acquired using a Fusion FX7 camera (Vilbert Lourmat) and densitometry was performed using Bio ID software (Certain Tech). Patients This study was performed in the Renal Division of Necker Hospital, starting in September 2015. The patient with the CLOVES syndrome had clinical examination, tumor 10 measurements, naevi measurement before treatement instauration. BYL719 treatment was initiated at the dose of 250mg orally per day. The study was conducted on two patients, an adult and a child, followed at Necker hospital. This protocol was approved by the ANSM (authorization n°553984-986 and n°584018); informed written consent was obtained from each patient or their legal representative. BYL719 was compassionately offered by Novartis. 15 The first patient received 250 mg / day and the second patient 50 mg / day. BYL719 was orally delivered every morning before breakfast. Glycaemia was monitored after any meal during two months and then progressively sparse. Data analysis and statistics Data were expressed as means ± SEM. Survival curves were analyzed with a Mantel-20 Cox (log-rank) test. Differences between the experimental groups were evaluated using ANOVA, followed when significant (P < 0.05) by the Tukey-Kramer test. When only two groups were compared, Mann-Whitney tests were used. The statistical analysis was performed using Graph Prism Software. Renal function 25 The serum creatinine level was measured weekly during the first year and every 3 months thereafter using a Synchron Cx4 autoanalyzer (Beckman Coulter, Villepinte, France). The glomerular filtration rate was estimated using the MD RD formula (eGFR). Biopsy samples and morphological analysis Kidney biopsy specimens were fixed in alcohol-formalin-acetic acid solution and 30 embedded in paraffin. Four-micrometer sections were stained with the Periodic Acid Schiff (PAS) stain, Masson’s trichrome and hematoxylin and eosin (H&E). Electron microscopy analyses were performed. Immunohistochemistry and immunofluorescence 2024278598 19 Dec 2024 Four sections of paraffin-embedded kidneys were incubated with anti-nephrin antibody (Progen), anti-WTl antibody (Dako), anti-podocin antibody (Sigma) and anti-synaptopodin antibody (Novus Biologicals). Upon the total glomerular area. For each biopsies, all glomerular section were quantified. The primary antibodies were revealed with 5 the appropriate Alexa 488- or 555-conjugated secondary antibodies (Molecular Probes). Immunofluorescence staining was visualized using the Zeiss LSM 700 confocal microscope. The podocyte-stained area was automatically quantified using a Nikon digital camera Dx / m / 1200 and Image J software and expressed as the percentage of the podocyte-stained area. 10 In situ hybridization Alcohol-formalin-acetic acid solution-fixed, paraffin-embedded tissues were assayed for PISKCa RNA expression using a previously described digoxygenin-anti-digoxygenin technique. Nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate toluidinium (NBT-BCIP) was used to visualize infected cells in the tissues. The specificity of the hybridization 15 signal was systematically checked by hybridizing sense probes with parallel sections and antisense probes with uninfected renal tissues. ISH-stained tissues were visualized and photographed with an Olympus Proxis microscope and a Zeiss Axio Cam ICcl. Cell culture Human fibroblasts were grown in DMEM supplemented with 15% FBS, penicillin (50 20 IU / ml) / streptomycin (50 pg / ml), and nonessential amino acids (Invitrogen). Western blot Western blot were performed as previously described, using a rabbit antibody to human phospho-p70 S6 kinase (Thr389) (Cell Signaling Technologies) at 1:1000 followed by a horseradish peroxidase-conjugated antirabbit secondary antibody at 1:10,000 (Dako). The 25 phosphorylation status of the p70 S6 kinase at the Thr389 site is specific for mTOR phosphorylation. A mouse monoclonal anti-b-actin antibody (Sigma-Aldrich, Lyon, France) was used as control. Protein phosphorylation levels were normalized to the matching densitometric values of b-actin. DNA sequencing 30 DNA was extracted using standard techniques from peripheral blood mononuclear cells (PBMCs) taken from the patient and skin biopsy in the tumor area. Mutation screening was performed by direct sequencing of all the PISKCa exons and flanking intronic regions. Volumetric determination 2024278598 19 Dec 2024 Magnetic resonance imaging (MRI) was used to determine the volume of each tumor. MRI was performed at day 0 before BYL719 initiation and then monthly. In brief, the volume was calculated by summing the products of the area measurements and slice thickness. Data analysis and statistics 5 The data were expressed as the means ± SD. Differences among the experimental groups were evaluated using ANOVA, followed when significant by the Tukey-Kramer test. When only two groups were compared, the Mann-Whitney test was used. Probability values <0.05 were considered statistically significant. Analyses were performed with GraphPad Prism 5 (GraphPad software, La Jolla, CA). 10 Results Inventors have observed a significant reduction of the size of the overgrown tissues in the subject, which was administered with BYL719. After a month of treatment, the patient weight decreased from 83,5 kg to 73,5 kg. This weigh loss was related to a dramatic reduction 15 of the oedema and an improvement of the global cardiac function rate (cardiac output was 22 1 / min in December 2015 before treatment and measured at 81 / min on February the 8th). The plasmatic brain natriuretic peptide levels decreased from 2500pg / ml to 240pg / ml at day 30 confirming the improvement of the heart failure. The subcutaneous tumour sizes showed a global reduction of 10 per cent after 30 days of treatment as assed by the CT scan and 20 Magnetic Resonance Imaging (MRI). The Kamofsky performance status scale of the patient improved from 40 to 60% in 30 days. Haemoglobin level increased from 8g / dl to 11.8 g / dl. Some skin areas (left ear) showed accelerated age phenotype before treatment that were improved after a month of BYL719 administration. Furthermore, several large naevi showed decolouration after the beginning of the treatment. 25 A mouse model of PROS We began this study by developing a mouse model of PROS. To this end, we took advantage of the transgenic mouse strain R26StopFLPl 10*. These mice allow for inducible expression of activated PI3KCA heterodimer in a tissue-specific manner. R26StopFLPl 10* mice were crossed with CAGG-CreER mice to generate PIK3CACAGG~CreER animals that 30 ubiquitously overexpress PIK3CA upon tamoxifen administration. Three weeks old mice were treated with a single dose of 40mg.kg-1 tamoxifen to induce Cre recombination16. We observed that compared to the control PIK3CAWT mice, the PIK3CACAGG~CreER mice started to die on day 3 post Cre induction with an average of 6 days post Cre recombination (Fig.lA). Death occurring suddenly in most cases, with necropsy revealing intraabdominal and hepatic 2024278598 19 Dec 2024 hemorrhages (data not shown). Some mice displayed difficulty walking with hypertrophic psoas muscle assessed by the magnetic resonance imaging (MRI) (data not shown). Additionally, whole body MRI showed the rapid occurrence of scoliosis, vessels abnormalities, kidney cysts, and muscles hypertrophy (Fig.IC). Histological examination 5 revealed multiple organs abnormalities including severe liver steatosis with vessel disorganization (data not shown), loss of integrity of the spleen microarchitecture (data not shown), spontaneous hemorrhage, and fibrosis of the kidney with aberrant vessels. To further characterize the vessels abnormalities, we performed CD34 immunostaining that confirmed the presence of severe vessel dilation. As expected, western blot and immunofluorescence 10 studies showed AKT / mTORC pathway activation in all examined organs. We therefore concluded that the PIK3CACAGG~CreER mice recapitulate human PROS phenotype. BYL719 prevents and is an effective therapy for PROS in mice We decided to test the impact of BYL719 in PIK3CACAGG~CreER mice. To this aim, we first performed a preventive study where the BYL719 was orally administrated starting just 15 after Cre induction (data not shown). We observed that daily administration of BYL719 dramatically improved animal survival (Fig.IC). In fact, while all PIK3CACAGG~CreER mice in the placebo group died within 21 days, PIK3CACAGG~CreER mice treated with BYL719 were alive 40 days later with overtly normal appearance. Importantly, treatment interruption 40 days after Cre recombination lead to the death of all the PIK3CACAGG~CreER animals. We 20 sacrificed several PIK3CA(agga'c,r mice receiving BYL719 on day 40 after Cre induction. Histological examination showed that mice treated with BYL719 had preserved tissues (data not shown) and normal vessels (data not shown). Western blot and immunofluorescence confirmed the efficiency of BYL719 at inhibiting PI3KCA activation (data not shown). Next, we performed a therapeutic study by giving either the placebo or the BYL719 to 25 PIK3CACAGG-CreER mice ten days after Cre induction, when mice begin to die (Fig. IC). On day 10 after Cre induction, MRI confirmed the presence of tissue abnormalities as described previously (data not shown). While the placebo group mice died within the next following days, BYL719 dramatically improved the survival of PIK3CACAGG~CreER mice (Fig.lJ). MRI performed 12 days after the beginning of the treatment (22 days after Cre induction) 30 demonstrated very rapid improvement of scoliosis, muscle hypertrophy, and vessel abnormalities (data not shown). Histological analysis revealed that BYL719 treated mice displayed no, or only minor, tissue changes (data not shown). As in the preventive study, western blot and immunofluorescence confirmed PIK3CA inhibition (data not shown). We concluded that the BYL719 was able to dramatically improve PROS mice, 2024278598 19 Dec 2024 suggesting it may be an effective therapeutic option for PROS patients for whom no medication currently exists. Effect ofBYL719 in human cells with PIK3CA mutation Next, we examined the efficacy of BYL719 at inhibiting the PIK3CA pathway in 5 human cells. To this aim, we used T-47D human cells derived from breast cancer. These cells harbor heterozygous mutation of the PIK3CA c.3140A>G (H1047R). This gain of function mutation is also the most frequent mutation observed in PROS patients6. We first confirmed in these cells, the spontaneous activation of the AKT / mTORC pathway as assessed by the phosphorylation status of AKT on the residue Thr308 and Ser473 10 but also the phosphorylation of the S6RP protein (data not shown). Then, cells were exposed to increasing concentrations of BYL719. We observed that, after 2 hours of BYL exposure at a concentration of I nmol / L, the AKT / mTORC pathway was fully inhibited (data not shown). The effect was similar when the cells were exposed to 4 and 6 hours to BYL719. These data suggested that BYL719 is able to inhibit the PI3KCA activation in human 15 cells with PIK3CA c.3140A>G (H1047R) mutation. Dramatic impact of BYL719 in patients with PROS Based on these results, we decided to administer BYL719 as a compassionate use for two patients, one adult and one child, who suffered from extremely severe clinical manifestation of CLOVES syndrome and were experiencing therapeutic failure and life 20 threatening complications. Patient 1 was a 29 years old man with mosaicism and biopsy proven gain of function mutation PIK3CA c.3140A>G (H1047R). He had overgrowth syndrome characterized by left leg hypertrophy, scoliosis, multiple naevi and extremely severe vascular abnormalities (data not shown). He underwent multiple aggressive debulking surgeries to remove abdominal and dorsal vascular tumors, as well as multiple angiographies 25 with embolization to limit tumor growth. He became paraplegic at the age of 20 years old due to spinal cord compression and required bladder stenting to drain his urine. He developed progressively worsening severe systolic heart failure with a measured cardiac output of 18 Emin and a Brain Natriuretic Peptide (BNP) level constantly over 2500 pg / mL (N<100 pg / mL), resistant to all usual drugs. It is unclear whether the heart failure was due to the 30 presence of vascular shunts, PIK3CA mutation in the cardiac cells, or both. During 5 years, the patient received rapamycin to limit tumor growth progression but without any efficiency. Lastly, the patient developed kidney dysfunction with severe proteinuria. Kidney biopsy revealed glomerular lesions with extensive fibrosis. Kidney lesions may be the result of heart dysfunction, the use of rapamycin11, and / or PIK3CA mutation in kidney epithelial cells. CT 2024278598 19 Dec 2024 scan (data not shown) and MRI (data not shown) showed severe vascular abnormalities, while PET-scan was negative. Due to the severity of the case with poor prognosis, physicians, surgeons and radiologists decided to stop any interventional treatment and to provide him only supportive and palliative care. His vital prognosis was engaged with survival estimated 5 at few months. Following our experimental findings and after ethical consideration we decided to purpose the BYL719 to our patient. We obtained the approbation from Novartis and the French regulatory agency (Agence nationale de securite du medicament et des produits de sante, ANSM) to deliver the BYL719 as a compassionate use. We stopped rapamycin and one 10 month later we started the BYL719. The drug was orally delivered every day. We choose 250 mg per day as a starting dosage, the lowest dose used in clinical trial and based on our in vitro data. Within the first few days we observed a dramatic improvement of the general status of the patient. The patient started to feel better and more comfortable. Week after week we could objective a dramatic effect of the drug on tumors sizes, venous dilation and skin aspect (data 15 not shown). He lost 23 kg in 12 months due to edema clearance but also due to a major reduction of all vascular tumor abnormalities (data not shown). The circumference of the thorax and the abdomen reduced dramatically, A-20% and A-35% in 12 months respectively (Fig.2A). CT-scan and MRI confirmed global vascular tumors shrinkage with a 63% volume decreased at 12 months, as well as subcutaneous infiltration disappearance (data not shown). 20 The effect of the drug on the heart function was remarkable, with a complete correction of the BNP level in the course of 4 weeks (Fig.2B). The cardiac output dropped to 3 1 / min, and the size of the heart reduced of 25% as determined by CT scan (data not shown). Left ventricular mass indexed to body surface area decreased from 250 g / m2 to 148 g / m2. Renal function was also rapidly improved with an estimated glomerular filtration rate increasing from 33 to 52 25 ml / min / 1.75m2 (Fig.3D). The volume of the hypertrophic left leg also reduced (data not shown). We observed a dramatic improvement of the skin hypertrophy with a change in the nevus coloration and a reduction of the size of the ear (data not shown). Lastly, 6 months after BYL719 initiation the patient began to have partial gain bladder function with improvement of saddle anesthesia. MRI revealed a 60% reduction in size of the venous malformation that 30 was compressing the spinal cord. Twelve month later, the patient had no side effect except hyperglycemia that was well controlled by regimen. The patient is still receiving the BYL719. Patient 2 was a 9 years old girl with a CLOVES syndrome diagnosed two years prior to this study and biopsy verified gain of function mosaicism mutation in PIK3CA c.3140A>G (H1047R). She had scoliosis, left leg hypertrophy, vascular abnormalities and hypertrophic 2024278598 19 Dec 2024 muscle of the back (data not shown). Importantly, she had a voluminous cystic lymphangioma involving the left kidney and the gastrointestinal tract. PET-scan showed metabolic fixation of the thymus, muscle from the back and the left leg (data not shown). The intraabdominal tumor was growing rapidly, with surgical or interventional radiology deemed impossible. After 5 ethical consideration, we obtained the approbation from Novartis and the French regulatory agency (ANSM) to provide BYL719. Since the drug has never been tested in a child, we decided to start at the lowest available dosage, 50 mg / day. As with the first patient, we observed a dramatic clinical improvement rapidly after the beginning treatment (data not shown). The patient reported improved comfort and we observed a 15% reduction in the 10 volume of the left leg, the left foot (1 shoe size reduction), and 8% of her abdominal circumference (data not shown) in 120 days. Hypertrophic muscles of the back shrunk rapidly and, unexpectedly, her scoliosis was reversed in 120 days without any other intervention (Fig.3). More importantly, MRI showed a 60% reduction of the volume of the intraabdominal tumor in only 4 months. PET-scan performed 4 months after treatment initiation confirmed 15 the reduction of the disease in almost all tissues. During the follow up, we found the normal growth of this patient was not impacted by the BYL719, and she reported no side effects. The patient is still receiving treatment. Discussion Here, we report the first mouse model of PROS that recapitulates the human 20 phenotype, a rescue of PROS in the mouse model by treatment with the PI3CA inhibitor BYL719, and, using a translational approach, have demonstrated the efficacy of BYL719 in PROS patients. This study provides the first direct evidence supporting PIK3CA inhibition as a robust and effective therapeutic strategy in the previously untreatable PROS. BYL719 treatment promoted the recovery of two patients, an adult and a child, with 25 positive clinical impact observable rapidly after initiation of treatment. As in the mouse model, the drug improved all organ dysfunctions related to PROS. Importantly, the drug maintained efficacy in improving malformations during the entire treatment period. The drug was well tolerated, without any significant side effects, in contrast to mTOR inhibitors. As anticipated, given that PI3KCA is downstream of the insulin receptor, hyperglycemia 30 occurred in the first patient, indicating that caution should be exercised given the potential for long-term side effects, particularly in children where growth, fertility, and puberty could be affected. However, during the treatment period the growth of the child was not affected, and any detrimental effects must be weighed against the benefits in this devastating and previously intractable disease. Several questions remain, including when the best period is to 2024278598 19 Dec 2024 start the treatment and when or if it therapy can be halted. Given the mouse data it seems highly possible that withdraw of the drug would be associated with the restart of the diseases, as is also seen in patients with Tuberous Sclerosis17. PIK3CA is one of the most commonly mutated oncogenes in human cancers, and 5 similar mutations are found in PROS patients6. As in cancer, although PIK3CA mutations can be detected across the entire coding sequence of the gene, most of the mutations are found in 3 major hotspot clusters in the helical (E542K, E545K) and kinase domains (H1047R). Each of these mutations leads to gain-of-function activation of PIK3CA pathway. Importantly, in cancer models, BYL719 is able to fully block the activity of the pathway regardless of the 10 type of mutation14. Additionally, our mouse model of PROS based on an increased number of copy of the PIK3CA gene, is rescued by the BYL719. Taking together these data suggest that the drug is certainly efficient in all form of PROS and not only acting on the H1047R mutation. This discovery may have broad clinical impact. Importantly, if PROS are rare, they are 15 not exceptional. The number of PROS patients is assumed to be largely underestimated due to phenotype heterogeneity leading to misdiagnosis. Mosaicism often requires the use highly sensitive technologies to detect PIK3CA somatic alteration6. With an incidence of 1 / 100,000 the recent proposal to include patients with Klippel-Trenaunay syndrome into the PROS highlights the great number of patients that may benefit from BYL7199. Importantly, PROS is 20 an evolving entity since more and more diseases, such as isolated venous malformations can also be linked to PIK3CA mutations8,18,19. Our mouse model offers a unique opportunity to better understand the physiopathology of the PROS. This model recapitulates PROS with vasculature abnormalities, muscle hypertrophy, kidney dysfunction, liver steatosis, spleen disorganization 25 and scoliosis. Severity was related to the high percentage of Cre recombination events induced by tamoxifen treatment. The mice perished due to severe bleeding, particularly from the mesenteric and liver vessels. Bleeding and leakage are among the severe complication observed in patients with CLOVES syndrome6. We also found that overexpression of the PIK3CA gene in the liver lead to steatosis as in the genetic overactivation of the 30 AKT / mTORC20. Remarkably, the mice quickly developed scoliosis after Cre recombination. This lesion is certainly due to the development of hypertrophic paraspinal-truncal soft tissue masses, since the BYL719 was able to quickly reverse the abnormalities. The improvement of scoliosis was also visible in the two patients with reduction in muscle masses and hypermetabolic signal. BYL719 was able to either prevent the development of any tissue 2024278598 19 Dec 2024 abnormalities or to rescue all defects while disease was achieved. In conclusion, the results of this work indicate that PIK3CA inhibition may be a safe and effective means to improve health and quality of life in PROS patients. Obviously, these results will have to be confirmed in larger cohort of patients but, they represent the most 5 promising available therapy for patient with PROS. Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. 10 The term “comprise” and variants of the term such as “comprises” or “comprising” are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required. 15 Any reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.
Claims
1. A method of treating PIK3CA-Related Overgrowth Spectrum (PROS) in a subject in needthereof comprising a step of administering to the subject a therapeutically effective amount of BYL719, wherein BYL719 reduces the size of overgrown tissue or prevents the development of one or more tissue abnormalities.
2. A method of treating PIK3CA-Related Overgrowth Spectrum (PROS) in a subject in needthereof comprising the steps of:identifying the subject as suffering from a PROS disorder;administering to the subject a therapeutically effective amount of BYL719; and reducing the size of overgrown tissue or tumor or preventing the development of one or more tissue or organ abnormalities.
3. Use of a therapeutically effective amount of BYL719 in the manufacture of a medicamentfor treating PIK3CA-Related Overgrowth Spectrum (PROS) in a subject in need thereof, wherein BYL719 reduces the size of overgrown tissues or prevents the development of one or more tissue abnormalities.
4. Use of a therapeutically effective amount of BYL719 in the manufacture of a medicamentfor treating PIK3CA-Related Overgrowth Spectrum (PROS) in a subject in need thereof, wherein the treating comprises identifying the subject as suffering from a PROS disorder, wherein the treating reduces the size of overgrown tissue or tumor or prevents the development of one or more tissue or organ abnormalities.
5. The method according to claim 1 or 2, or the use according to claim 3 or 4, wherein thePROS disorder is Congenital, Lipomatous, Overgrowth, Vascular Malformations, Epidermal Nevi and Spinal / Skeletal Anomalies and / or Scoliosis (CLOVES) syndrome.
6. The method according to claim 1 or 2, or the use according to claim 3 or 4, wherein thePROS disorder is Klippel-Trenaunay syndrome.2024278598 05 Aug 20267. The method according to claim 1 or 2, or the use according to claim 3 or 4, wherein thePROS disorder is selected from the group consisting of fibroadipose overgrowth, megalencephaly-capillary malformation syndrome, hemihyperplasia multiple lipomatosis, Klippel-Trenaunay syndrome, and congenital lipomatous asymmetric overgrowth, lymphatic, capillary, venous and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies and / or scoliosis (CLOVES) syndrome.
8. The method according to claim 1 or 2, or the use according to claim 3 or 4, wherein thetherapeutically effective amount of BYL719 treats a symptom selected from the group consisting of edema, venous dilation, vascular tumors, elevated brain natriuretic peptide blood level, proteinuria, kidney dysfunction, overgrown tissue, dysregulated adipose tissue, scoliosis, enlarged bony structures without progressive bony overgrowth, benign tumors, megalencephaly-capillary malformation, lipomatous asymmetric overgrowth of the trunk, epidermal nevi, lymphatic malformation, muscle hypertrophy, liver steatosis, and spleen disorganization.
9. The method according to any one of claims 1, 2 or 5-8, or the use according to any one ofclaims claim 3-8, wherein the therapeutically effective amount comprises 50 mg of BYL719.
10. The method according to any one of claims 1, 2 or 5-8, or the use according to any one of claims claim 3-8, wherein the therapeutically effective amount comprises 250 mg of BYL719.
11. The method according to any one of claims 1, 2 or 5-10, or the use according to any one of claims claim 3-10, wherein the PROS disorder is caused by a somatic mosaic mutation in PIK3CA selected from the group consisting of H1047R, E542K and E545K.
12. The method according to any one of claims 1, 2 or 5-11, or the use according to any one of claims claim 3-11, wherein BYL719 is administered orally.