TWI891650B - Methods for treating vascular malformations - Google Patents

Abstract

The present disclosure relates to methods for inhibiting TIE2 kinase useful in the treatment of growth of venous malformations. Specifically, the disclosure relates to methods of using a compound of Formula I and salts thereof

Description

Methods for treating vascular malformations

Intimal endothelial cell kinase-2 (TIE2) is primarily expressed in vascular endothelial cells. TIE2 is a receptor for angiopoietin 1 (ANGPT1), angiopoietin 2 (ANGPT2), and angiopoietin 4 (ANGPT4), and this signaling system plays a key role in both vascular proliferation (sprouting of new blood vessels from existing ones) and angiogenesis (the de novo formation of new blood vessels).

Vascular malformations encompass a wide range of disorders of the vascular system. These disorders include venous, lymphatic, microvascular, arterial, and arteriovenous malformations. Any type or combination of blood vessels may be involved. Vascular malformations grow over time, can grow rapidly, and can cause local tissue invasion. Venous malformations can be localized or multifocal. Venous malformations can be associated with pain, swelling, bleeding, cosmetic changes, thrombosis, and other significant morbidities. Venous malformations can affect tissues such as the skin, joints, muscles, intestines, and bones. Many venous malformations can be treated with surgery, laser therapy, or sclerotherapy, but not all are suitable for these treatments. In most cases, the venous malformation will recur after conventional treatment.

Approximately 50% of venous malformations are associated with gonadal involvement or somatic mutations in the TIE2 kinase. These mutations activate TIE2 kinase, leading to dysregulated endothelial cell growth and venous malformations. Therefore, new treatments for these diseases associated with TIE2 alterations are needed.

Described herein are compounds that are inhibitors of TIE2 kinase and their use in treating or preventing venous malformations. The present invention relates to methods of treating venous malformations using compounds of Formula I described below as potent inhibitors of TIE2: or its pharmaceutically acceptable salts.

For example, provided herein is a method for treating TIE2 kinase-mediated vascular abnormalities or TIE2 kinase mutant-mediated vascular abnormalities in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

Furthermore, provided herein is a method for treating vascular abnormalities in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein the vascular abnormality is mediated by TIE2 kinase or a TIE2 kinase mutant.

Furthermore, the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof for use in treating TIE2 kinase-mediated vascular abnormalities or TIE2 kinase mutant-mediated vascular abnormalities in a patient in need thereof, comprising administering a therapeutically effective amount of the compound of Formula I or a pharmaceutically acceptable salt thereof to the patient.

Also provided herein is a method for treating venous malformation in a patient in need thereof, comprising administering to the patient about 100 mg to about 200 mg of a compound of Formula I, or a pharmaceutically acceptable salt thereof, once or twice daily.

Also provided herein is a compound of Formula I, or a pharmaceutically acceptable salt thereof, for treating venous malformation in a patient in need thereof, comprising administering to the patient about 100 mg to about 200 mg of the compound of Formula I, or a pharmaceutically acceptable salt thereof, once or twice daily.

Figures 1A-E show the inhibition of phosphorylation of various TIE2 mutants (R849W, L914F, R1099*, Y897C/R915C, and Y897F/R915L, respectively) using compounds of Formula I in an assay using transfected CHO cells.

Figure 2 shows the inhibition of phosphorylation of various TIE2 mutants using compounds of Formula I in an assay using transfected human umbilical vein endothelial cells.

Figure 3 shows that compounds of Formula I inhibit phosphorylation of the downstream signaling protein AKT in an assay using transfected human umbilical vein endothelial cells and various TIE2 mutants.

Figure 4 shows that compounds of Formula I inhibit phosphorylation of the downstream signaling protein STAT1 in an assay using transfected human umbilical vein endothelial cells and various TIE2 mutants.

Figures 5A-F show restoration of cell morphology using compounds of Formula I in assays using transfected human umbilical vein endothelial cells and various TIE2 mutants (WT or L914F, R849W, R1099*, Y897C/R915C, Y897C/R915L, and T1105N/T1106P, respectively).

Figures 6A-F show the effects of compounds of Formula I on ANGPT2, PDGFB, ADAMTS1, ADAMTS9, PLAT, and PLAU in assays using transfected human umbilical vein endothelial cells and various TIE2 mutants.

Figure 7 shows the restoration of extracellular fibronectin using compounds of Formula I in an assay using transfected human umbilical vein endothelial cells and various TIE2 mutants.

Figure 8A is a schematic diagram of the experimental design described in Example 7, which was used to evaluate the inhibition of the growth of mutant TIE2 human umbilical vein endothelial cells in vivo by the compound of Formula I in a venous malformation model, wherein on day 0, mice were given a control diet or a diet infused with the compound of Formula I.

FIG8B shows the effect of the compound of Formula I on the ocular appearance of mutant TIE2 vascular lesions in an in vivo model of venous malformation at day 7.

Figure 9 shows the effect of the compound of Formula I on the size of mutant TIE2 vascular lesions at day 7 in an in vivo model of venous malformation.

FIG10 shows the effect of the compound of Formula I on smooth muscle cell and envelope cell coverage of mutant TIE2 vascular lesions at day 7 in an in vivo model of venous malformation.

Figure 11 shows the effect of the compound of Formula I on TIE2 phosphorylation in mutant TIE2 vascular lesions at day 7 in an in vivo model of venous malformation.

Figure 12 shows the effect of the compound of Formula I on the size of mutant TIE2 vascular lesions at day 16 in an in vivo model of venous malformation.

FIG13 shows the effect of the compound of Formula I on smooth muscle cell and envelope cell coverage of mutant TIE2 vascular lesions at day 16 in an in vivo model of venous malformation.

Figure 14 shows the effect of the compound of Formula I on TIE2 phosphorylation in mutant TIE2 vascular lesions at day 16 in an in vivo model of venous malformation.

Figure 15 shows the effect of the compound of formula I on vascular morphology of mutant TIE2 vascular lesions at days 7 and 16 in an in vivo model of venous malformation.

Figure 16A is a schematic diagram of the experimental design described in Example 7, which was used to evaluate the inhibition of the growth of mutant TIE2 human umbilical vein endothelial cells in vivo by the compound of Formula I in a venous malformation model, wherein mice were given a control diet or a diet infused with the compound of Formula I on day 7.

Figure 16B shows the effect of the compound of Formula I on the ocular appearance of previously established mutant TIE2 vasculopathy in an in vivo model of venous malformation.

Figures 17A-B show the effects of the compound of Formula I on the size of previously established mutant TIE2 vascular lesions and on the smooth muscle cell and envelope cell coverage of mutant TIE2 vascular lesions in an in vivo model of venous malformation.

Figures 18A-D compare the effects of the compound of Formula I on vascular malformation (VM) lesions expressing wild-type and L914F TIE2 mutations from the day of injection (Day 0) to Day 7. VM lesions treated with the compound of Formula I diet were compared to those treated with a control diet and untreated VM lesions using macroscopic ( Figure 18A ) and microscopic (Figure 18B) images and quantification of vascular vessel area ( Figures 18C and 18D ).

Figures 19A-C provide treatment comparisons of vascular malformation (VM) lesions expressing wild-type and L914F TIE2 mutants from the day of injection (day 0) to day 16, or from day 7 to day 16. Included are microscopic comparisons of untreated VM lesions and VM lesions expressing TIE2 L914F treated with a Formula I compound diet versus a control diet ( Figure 19A ), as well as quantification of vascular vessel area in lesions expressing TIE2 wild-type or L914F ( Figures 19B and 19C ).

Cross-reference to related applications

This application claims priority to U.S.S.N. 62/885,519, filed on August 12, 2019, the contents of which are incorporated herein by reference in their entirety.

The features and other details of the present invention will now be described more specifically. Certain terms used in this specification, examples, and the accompanying patent claims are collected here. These definitions should be read in the context of the remainder of this invention and as understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

Definition

The terms "individual,""patient," or "subject" are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, and most preferably humans. The compounds described herein can be administered not only to mammals such as humans, but also to other mammals, such as animals in need of veterinary treatment, such as domestic animals ( e.g., dogs, cats, and the like), farm animals ( e.g., cows, sheep, pigs, horses, and the like), and laboratory animals ( e.g., rats, mice, guinea pigs, and the like).

"Pharmaceutically or pharmacologically acceptable" includes molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans, as appropriate. For human administration, formulations should meet sterility, pyrogenicity, and general safety and purity standards, as required by the FDA Office of Biologics standards.

As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds that provide supplemental, additional, or enhanced therapeutic functions.

As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

As used herein, the term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of the compounds disclosed herein. These salts can be salts of acidic or basic groups that may be present in the compounds used in the compositions. Compounds included in the compositions of the present invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that can be used to prepare the pharmaceutically acceptable acid addition salts of the alkaline compounds are acids that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to apple acid salts, oxalate salts, chlorides, bromides, iodides, nitrates, sulfates, hydrosulfates, phosphates, acid phosphates, isonicotinates, acetates, lactates, salicylates, citrates, tartares, oleates, tannates, pantothenates, alcohols, and the like. Hydrogen sulfate, ascorbate, succinate, cis-malate, gentianate, fumarate, gluconate, glucuronide, glucarate, formate, benzoate, glutamine, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and bis(hydroxynaphthoate) (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), alkaline metal salts (e.g., sodium and potassium), alkaline earth metal salts (e.g., magnesium), and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counter ion. A counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of the pharmaceutically acceptable salt, there may be multiple counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counter ions. The compounds of the present invention may contain acidic and basic groups; for example, an amine group and a carboxylic acid group. In such cases, the compound may exist as an acid addition salt, a zwitterionic ion, or a base salt. If the compounds disclosed herein are bases, the desired pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, and the like; or an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosyl acids (such as glucuronic acid or galacturonic acid), alpha-hydroxy acids (such as citric acid or tartaric acid), amino acids (such as aspartic acid or glutamic acid), aromatic acids (such as benzoic acid or cinnamic acid), sulfonic acids (such as p-toluenesulfonic acid or ethanesulfonic acid), or the like. If the compounds disclosed herein are acids, the desired pharmaceutically acceptable salts can be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base, such as an amine (primary, di-, or tertiary), an alkali metal hydroxide, or an alkali earth metal hydroxide, or the like. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia, primary, di-, and tertiary amines, and cyclic amines such as piperidine, morpholine, and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

As used herein, the term "treating" or "treatment" includes reversing, alleviating, or arresting the symptoms, clinical signs, and potential pathological changes of a condition in a manner that improves or stabilizes the condition of a subject. As used herein, and as understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, detectable or undetectable alleviation, improvement, or alleviation of one or more symptoms or conditions associated with a condition (e.g., cancer), reduction in disease severity, stabilization of the disease state (i.e., non-worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Exemplary beneficial clinical outcomes are described herein.

As used in the present invention, the term "administer," "administering," or "administration" refers to directly administering a composition or a pharmaceutically acceptable salt of the compound or composition to a subject, or administering a prodrug derivative or analog of the composition or a pharmaceutically acceptable salt of the compound or composition to the subject, which can form an equivalent amount of the active compound in the subject's body.

As used herein, the term "therapeutically effective amount" means the amount of a compound of the present invention that elicits the biological or medical response that a researcher, veterinarian, physician, or other clinician is seeking in a tissue, system, or animal (e.g., mammal or human). The compounds described herein are administered in therapeutically effective amounts to treat the conditions disclosed herein.

The compound of formula I is also referred to herein as rebastinib.

The present invention relates in part to treating (blocking) or preventing the growth of venous malformations. Such disclosed methods may comprise administering an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof to a patient in need of treatment or prevention of these conditions, for example, as part of a regimen for modulating TIE2 inhibition.

In some embodiments, the compound of Formula I is a sulfonate salt according to Formula II. Formula II, for example, is a potent inhibitor of TIE2 (angiopoietin ligand receptor tyrosine kinase).

Exemplary methods include treating venous malformations in patients, for example, where such patients have an expression, mutation, or alteration of TIE2 in endothelial cells that may cause or contribute to the growth of the venous malformation. Such methods may include administering a compound of Formula I or a pharmaceutically acceptable salt thereof to the patient suffering from the venous malformation. For example, providing a compound such as a compound of Formula I or a pharmaceutically acceptable salt thereof may inhibit a process that includes the growth of the venous malformation. Contemplated compounds include free bases of Formula I.

Effective dose, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, to determine the LD50 (50% population lethal dose) and the ED50 (50% population therapeutically effective dose). The dose can vary depending on the dosage form and route of administration used. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the LD50/ED50 ratio. In some embodiments, the compositions and methods exhibit a large therapeutic index. The level of the composition in plasma can be measured, for example, by high performance liquid chromatography, and the effect of any particular dose can be monitored, in particular, by suitable bioassays. The dosage can be adjusted at the physician's discretion and as needed to suit the observed therapeutic effect.

In certain embodiments, a preventive effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the desired underlying disease or condition, regardless of whether improvement is achieved.

The compound of Formula I or a pharmaceutically acceptable salt thereof (and/or additional reagents) described herein can be administered to a subject in the form of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such a composition may optionally contain an appropriate amount of a pharmaceutically acceptable excipient to provide a form suitable for administration.

Pharmaceutically acceptable excipients can be liquids such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical excipients can be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, adjuvants, stabilizers, thickeners, lubricants, and colorants can be used. In some embodiments, the pharmaceutically acceptable excipient is sterile when administered to a subject. When any of the agents described herein are administered intravenously, water is a suitable excipient. Aqueous saline solutions, dextrose solutions, and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, ethylene glycol, water, ethanol, and the like. If desired, any of the agents described herein may also contain a small amount of a wetting agent or emulsifier, or a pH buffering agent.

In some embodiments, provided herein are methods for treating venous malformations or other congenital venous malformations, comprising administering to a patient in need thereof an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof. Contemplated venous malformations include spheroid cell venous malformations, mucocutaneous venous malformations (also known as VMCMs), blue rubber bubble nevus syndrome, gastric or gastrointestinal lesions, and/or Maffucci syndrome.

For example, contemplated herein is a method of blocking the growth of venous malformations comprising administering to a patient in need thereof an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof, for example, in a dosing regimen sufficient to block TIE2 kinase in the tumor microenvironment.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof is administered daily or twice daily.

In other embodiments, the dosing regimen of the compound of Formula I or a pharmaceutically acceptable salt thereof is intermittent. The intermittent non-daily dosing regimen may include, but is not limited to, every other day dosing, every other day dosing, twice weekly dosing, or once weekly dosing. In some embodiments, the method comprises administering to the patient the compound of Formula I once daily, intermittent non-daily dosing, every other day dosing, every other day dosing, every other week dosing, twice daily dosing, once weekly dosing, or twice weekly dosing.

In some embodiments, suitable dosing regimens for the compound of Formula I or a pharmaceutically acceptable salt thereof include administration twice a week, once a week, or every other week, for example, twice a week or once a week, for example, twice a week.

Another aspect of the present invention relates to a method for inhibiting the growth of venous malformations, comprising administering a compound of Formula I or a pharmaceutically acceptable salt thereof in an amount sufficient to inhibit the activity of TIE2 kinase or mutant TIE2 kinase, wherein the compound of Formula I or a pharmaceutically acceptable salt thereof is administered on an intermittent, non-daily, dosing schedule. In some embodiments, the intermittent, non-daily dosing schedule includes every other day dosing, every other day dosing, twice weekly dosing, and once weekly dosing.

Another aspect of the present invention relates to a method for treating a patient with venous malformation who is undergoing lead therapy prior to surgical tumor resection, comprising administering to a patient in need thereof an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the dosing regimen of the compound of Formula I or a pharmaceutically acceptable salt thereof is sufficient to block TIE2 kinase or mutant TIE2 kinase.

In some embodiments, the method for treating a patient with venous malformation who is undergoing lead therapy prior to surgical resection of a tumor comprises administering to a patient in need thereof an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof at a dosage regimen sufficient to block TIE2 kinase or mutant TIE2 kinase.

In some embodiments, a method of treating a patient with a venous malformation who is undergoing lead therapy prior to surgical resection comprises administering a compound of Formula I or a pharmaceutically acceptable salt thereof daily or twice daily in an amount sufficient to block TIE2 kinase or mutant TIE2 kinase.

In some embodiments, the method of treating a patient with a venous malformation who is undergoing lead therapy prior to surgical resection comprises administering to a patient in need thereof an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof, in an intermittent, non-daily manner, at a dosing regimen sufficient to block TIE2 kinase or mutant TIE2 kinase, including dosing every other day, every other day, twice a week, and once a week.

In some embodiments, the method of treating a patient with a venous malformation who is undergoing lead therapy prior to surgical resection comprises administering to a patient in need thereof an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof at a dosing schedule of twice weekly, once weekly, or every other week.

Another aspect of the present invention relates to a method for treating TIE2 kinase-mediated vascular abnormalities or TIE2 kinase mutant-mediated vascular abnormalities (e.g., vascular malformations, vascular tumors (e.g., hemangiomas), and/or other congenital vascular defects) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutically acceptable salt is a sulfonate. In some embodiments, the pharmaceutically acceptable salt is a methanesulfonate. In some embodiments, the pharmaceutically acceptable salt is a triflate. In some embodiments, the pharmaceutically acceptable salt is an ethanesulfonate. In some embodiments, the pharmaceutically acceptable salt is a benzenesulfonate. In some embodiments, the pharmaceutically acceptable salt is a chlorobenzenesulfonate. In some embodiments, the pharmaceutically acceptable salt is a camphorsulfonate. In some embodiments, the pharmaceutically acceptable salt is a toluenesulfonate. In some embodiments, the pharmaceutically acceptable salt is a monotoluenesulfonate. In some embodiments, the pharmaceutically acceptable salt is ditoluenesulfonate. In some embodiments, the pharmaceutically acceptable salt is tritoluenesulfonate. In some embodiments, the pharmaceutically acceptable salt is tetratoluenesulfonate.

Contemplated herein are methods for treating a vascular abnormality in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the vascular abnormality is a TIE2 kinase-mediated vascular abnormality or a TIE2 kinase mutant-mediated vascular abnormality. The TIE2 kinase-mediated vascular abnormality or the TIE2 kinase mutant-mediated vascular abnormality can be a slow-flow malformation. In some embodiments, the slow-flow malformation is selected from a microvascular malformation, a lymphatic malformation, a lymphovenous malformation, or a venous malformation. In some embodiments, the slow-flow malformation is a venous malformation.

Formulations, administration, dosing, and treatment regimens

The present invention also describes a compound of Formula I (and/or additional agents) or a pharmaceutically acceptable salt thereof in a pharmaceutical composition. The compositions described herein can be in the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules, liquid-containing capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other suitable form. In some embodiments, the composition is in the form of a capsule (see, for example, U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro, ed., 19th ed., 1995), which is incorporated herein by reference.

The salts described herein may also include a solubilizing agent, if necessary. Furthermore, the reagents may be delivered using a suitable vehicle or delivery device as known in the art. The combination therapies outlined herein may be delivered together in a single delivery vehicle or delivery device. The composition for administration may optionally include a local anesthetic (such as, for example, lignocaine) to reduce pain at the injection site.

In some embodiments, the compound of Formula I described herein or its pharmaceutically acceptable salt (and/or additional reagent) is formulated into a composition suitable for the mode of administration according to conventional processing procedures.

In certain embodiments, routes of administration include, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation, or topical, particularly to the ear, nose, eye, or skin. In some embodiments, administration is achieved orally or by parenteral injection. The mode of administration is left to the discretion of the physician and depends in part on the site of the medical condition. In most cases, administration will result in the release of any of the agents described herein into the bloodstream.

In some embodiments, local administration to the area in need of treatment or blockade is desirable.

In some embodiments, the salts described herein (and/or additional agents) are formulated according to conventional processes into compositions suitable for oral administration to humans. Orally delivered compositions can be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions can contain one or more agents, such as sweeteners, such as fructose, aspartame, or saccharin; flavorings, such as peppermint, wintergreen oil, or cherry oil; coloring agents; and preservatives, to provide pharmaceutically palatable preparations. In addition, when in tablet or pill form, the composition can be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action with an extended time period. The selective permeable membranes described herein that enclose an osmotically active driving compound of formula I or a pharmaceutically acceptable salt thereof (and/or additional reagents) are also suitable for compositions for oral administration. In these latter platforms, fluid from the environment surrounding the capsule is absorbed by the driving composition, which swells to displace the agent or agent composition through the opening. These delivery platforms can provide essentially zero-order delivery curves as opposed to the spike-like curves of immediate-release formulations. Time-delay materials such as glyceryl monostearate or glyceryl stearate may also be used. Oral compositions may include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In some embodiments, the excipients are pharmaceutical grade. In addition to the active ingredient, the suspension may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They can also be prepared as sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable medium immediately prior to use. They may contain, for example, suspending or dispersing agents known in the art.

The dosage and schedule of the compound of Formula I or a pharmaceutically acceptable salt thereof (and/or additional agents) described herein may depend on various parameters, including, but not limited to, the disease being treated, the individual's general health, and the judgment of the administering physician. Any agent described herein may be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to) administration of another therapeutic agent. , concurrently with, or thereafter (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks thereafter) the drug to a subject in need thereof. In various embodiments, any of the agents described herein are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 to 2 hours apart, 2 to 3 hours apart, 3 to 4 hours apart, 4 to 5 hours apart, 5 to 6 hours apart, 6 to 7 hours apart, 7 to 8 hours apart, 8 to 9 hours apart, 9 to 10 hours apart, 10 to 11 hours apart, or 11 to 12 hours apart.

The dosage of a compound of Formula I described herein, or a pharmaceutically acceptable salt thereof (and/or additional reagents), may depend on several factors, including the severity of the condition, whether the condition is being treated or prevented, and the age, weight, and health of the individual being treated. Furthermore, information about a particular individual's pharmacogenomics (the effect of genotype on the pharmacokinetics, pharmacodynamics, or efficacy profile of a treatment) may influence the dosage used. Furthermore, the exact individual dosage may vary slightly depending on a variety of factors, including the specific combination of reagents administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the specific disease being treated, the severity of the condition, and the anatomical location of the condition. Some variation in dosage is to be expected.

In some embodiments, when administered orally to mammals, the dosage of a compound of Formula I described herein, or a pharmaceutically acceptable salt thereof (and/or additional agent) can be 0.001 mg/kg/day to 150 mg/kg/day, 0.001 mg/kg/day to 100 mg/kg/day, 0.01 mg/kg/day to 50 mg/kg/day, or 0.1 mg/kg/day to 10 mg/kg/day. In some embodiments, when administered orally to humans, the dosage of any agent described herein is typically 0.001 mg to 1500 mg per day, 1 mg to 600 mg per day, or 5 mg to 30 mg per day. In some embodiments, the dosage of the salt (or agent) ranges from 50 mg to 1200 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 900 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 600 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 300 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 150 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 140 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 130 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 120 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 110 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 100 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 50 mg to 90 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 57 mg to 1200 mg per day. In some embodiments, the dosage of the salt (or test agent) is within the range of 57 mg to 150 mg per day. In some embodiments, the dosage of the salt (or test agent) is in the range of 57 mg to 140 mg per day. In some embodiments, the dosage of the salt (or test agent) is in the range of 57 mg to 130 mg per day. In some embodiments, the dosage of the salt (or test agent) is in the range of 57 mg to 120 mg per day. In some embodiments, the dosage of the salt (or test agent) is in the range of 57 mg to 110 mg per day. In some embodiments, the dosage of the salt (or test agent) is in the range of 57 mg to 100 mg per day. In some embodiments, the dosage of the salt (or test agent) is in the range of 57 mg to 90 mg per day. In other embodiments, the dosage of the salt (or trial) or salt is in the range of 60 mg to 200 mg per day. In other embodiments, the dosage of the salt (or trial) or salt is in the range of 60 mg to 150 mg per day. In other embodiments, the dosage of the salt (or trial) or salt is in the range of 70 mg to 150 mg per day. In other embodiments, the dosage of the salt (or trial) or salt is in the range of 80 mg to 150 mg per day. In other embodiments, the dosage of the salt (or trial) or salt is in the range of 90 mg to 150 mg per day. In other embodiments, the dosage of the salt (or trial) or salt is in the range of 100 mg to 150 mg per day. In other embodiments, the salt (or agent) or the dosage of the salt is in the range of 100 mg to 200 mg per day.

In some embodiments, for the administration of a compound of Formula I described herein, or a pharmaceutically acceptable salt thereof (and/or additional agent), by parenteral injection, the dosage is 0.1 mg to 250 mg per day. In some embodiments, the dosage is 1 mg to 200 mg per day. In some embodiments, the dosage is 1 mg to 150 mg per day. In some embodiments, the dosage is 10 mg to 150 mg per day. In some embodiments, the dosage is 20 mg to 200 mg per day. In some embodiments, the dosage is 30 mg to 200 mg per day. In some embodiments, the dosage is 40 mg to 200 mg per day. In some embodiments, the dosage is 50 mg to 200 mg per day. In some embodiments, the dosage is 50 mg to 150 mg per day. In some embodiments, the dosage is 57 mg to 150 mg per day. In some embodiments, the dosage is 57 mg to 100 mg per day. In some embodiments, the dosage is 60 mg to 150 mg per day. In some embodiments, the dosage is 70 mg to 150 mg per day. In some embodiments, the dosage is 60 mg to 140 mg per day. In some embodiments, the dosage is 60 mg to 130 mg per day. In some embodiments, the dosage is 60 mg to 120 mg per day. In some embodiments, the dosage is 60 mg to 110 mg per day. In some embodiments, the dosage is 60 mg to 100 mg per day. In some embodiments, the dosage is 60 mg to 90 mg per day. In some embodiments, the dosage is 70 mg to 130 mg per day. In some embodiments, the dosage is 70 mg to 120 mg per day. In some embodiments, the dosage is 70 mg to 110 mg per day. In some embodiments, the dosage is 70 mg to 100 mg per day. In some embodiments, the dosage is 1 mg to 20 mg per day, or 3 mg to 5 mg per day. Injections can be given up to four times per day. In some embodiments, when administered orally or parenterally, the dosage of any of the agents described herein is typically 0.1 mg to 1500 mg per day, or 0.5 mg to 10 mg per day, or 0.5 mg to 5 mg per day. A dosage of up to 3000 mg per day can be administered.

In some embodiments, the salts described herein (and/or additional agents) can be administered independently one to four times daily. Specifically, the salts can be administered once daily at a dosage of about 50 mg to 1500 mg of the salt. A daily dose of 57-1200 mg/day is suitable for the desired preventive effect. In some embodiments, the methods described herein comprise administering to the patient about 100 mg of a compound of Formula I or a pharmaceutically acceptable salt thereof daily. In some embodiments, the methods described herein comprise administering to the patient about 200 mg of a compound of Formula I or a pharmaceutically acceptable salt thereof daily. In some embodiments, the methods described herein comprise administering to the patient about 300 mg of a compound of Formula I or a pharmaceutically acceptable salt thereof daily. If administered twice daily, a suitable dosage is 100 mg to 200 mg of the salt. In some embodiments, the methods described herein comprise administering to the patient about 150, 200, or 300 mg of a compound of Formula I or a pharmaceutically acceptable salt thereof once or twice daily. In some other embodiments, the salt may be administered intermittently, not daily. In some embodiments, the salt may be administered one to four times per month, one to six times per year, or once every two, three, four, or five years. In certain embodiments, the salt is administered weekly or every two weeks. In some embodiments, when administered weekly or every two weeks, in some embodiments, a suitable salt dosing regimen is in the range of 50-200 mg per administration. In some embodiments, the dosage is 200-400 mg per administration weekly or every two weeks. In some embodiments, the dosage is 400-500 mg per administration weekly or every two weeks. In some embodiments, the dosage is 500-600 mg per administration weekly or every two weeks. In some embodiments, the dosage is 600-700 mg per administration weekly or every two weeks. In some embodiments, the dosage is 700-800 mg per administration weekly or every two weeks. In some embodiments, the dosage is 800-900 mg per administration weekly or every two weeks. In some embodiments, the dosage is 900-1000 mg per administration weekly or every two weeks. In some embodiments, the dosage is 1000-1100 mg per administration weekly or every two weeks. In some embodiments, the dosage is 1100-1200 mg per administration weekly or every two weeks. Administration may last from one day to one month, two months, three months, six months, one year, two years, three years, and possibly even for the lifetime of the individual. Chronic, long-term administration will be indicated in many cases. The dosage may be administered as a single dose or divided into multiple doses. Generally, the required dose should be administered at set intervals over a long period of time, usually at least over several weeks or months, but may require administration for longer periods of time, such as months or years.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be administered as a single agent or in combination with other therapeutic agents. Such other therapeutic agents include radiation therapy, anti-tubulin agents, DNA alkylating agents, DNA synthesis inhibitors, DNA intercalators, antiestrogens, anti-androgens, steroids, anti-EGFR agents, kinase inhibitors, topoisomerase inhibitors, histone deacetylase (HDAC) inhibitors, DNA methylation inhibitors, anti-HER2 agents, anti-angiogenic agents, proteasome inhibitors, thalidomide, lenalidomide, antibody-drug conjugates (ADCs), immunomodulators, or cancer vaccines. In some embodiments, a compound of Formula I or a pharmaceutically acceptable salt thereof can be administered to a patient together with a VEGF inhibitor. In some embodiments, a compound of Formula I or a pharmaceutically acceptable salt thereof can be administered to a patient together with an Akt inhibitor. In some embodiments, a compound of Formula I or a pharmaceutically acceptable salt thereof can be administered to a patient together with an mTOR inhibitor. In some embodiments, a compound of Formula I or a pharmaceutically acceptable salt thereof can be administered to a patient together with a PI3K inhibitor.

When a compound of Formula I or a pharmaceutically acceptable salt thereof is used in combination with other agents, the other agents may be administered independently of the dosing schedule of the compound of Formula I. The other agents may be administered at their previously established therapeutic doses and dosing schedules, or their doses and dosing schedules may be modified when used in combination with the compound of Formula I to optimize efficacy, safety, or tolerability.

The compound of formula I or a pharmaceutically acceptable salt thereof can be used in combination with other agents including (but not limited to) chemotherapy agents, targeted therapy agents, biological agents or radiotherapy.

In some embodiments, a compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a chemotherapeutic agent, including but not limited to anti-tubulin agents (e.g., paclitaxel, paclitaxel protein-bound particles for injectable suspension, eribulin, docetaxel, ixabepilone, vincristine, vinorelbine, epothilones, halichondrins, maytansinoids), DNA alkylating agents (e.g., cisplatin, carboplatin), in), oxaliplatin, cyclophosphamide, ifosfamide, temozolomide), DNA intercalators (e.g., doxorubicin, pegylated liposomal doxorubicin, daunorubicin, idarubicin, and epirubicin), 5-fluorouracil, capecitabine, cytarabine, decitabine, 5-azacytidine, gemcitabine, and methotrexate.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a kinase inhibitor, including but not limited to erlotinib, gefitinib, lapatanib, everolimus, sirolimus, temsirolimus, LY2835219, LEEO11, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1, PD-L1 0332991, crizotinib, cabozantinib, sunitinib (sunitinib, pazopanib, sorafenib, regorafenib, axitinib, dasatinib, imatinib, nilotinib, vemurafenib, dabrafenib, trametinib, idelalisib, duvelisib, alpelisib, copanlisib, and quizartinib.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with an anti-estrogen, including but not limited to tamoxifen, fulvestrant, anastrozole, letrozole, and exemestane.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with an antiandrogen, including but not limited to abiraterone acetate, enzalutamide, nilutamide, bicalutamide, flutamide, and cyproterone acetate.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a steroid agent, including but not limited to prednisone and dexamethasone.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a topoisomerase I inhibitor, including but not limited to irinotecan, camptothecin, and topotecan.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a topoisomerase II inhibitor, including but not limited to etoposide, etoposide phosphate, and mitoxantrone.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a histone deacetylase (HDAC) inhibitor, including but not limited to vorinostat, romidepsin, panobinostat, valproic acid, and belinostat.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a DNA methylation inhibitor, including but not limited to DZNep and 5-aza-2'-deoxycytidine.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a proteasome inhibitor, including but not limited to bortezomib and carfilzomib.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with thalidomide, lenalidomide, and pomalidomide.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a biologic agent, including but not limited to trastuzumab, ado-trastuzumab, pertuzumab, cetuximab, panitumumab, ipilimumab, anti-P D-1 agents (including but not limited to labrolizumab and nivolumab), anti-PD-L1 agents (including but not limited to MPDL3280A), anti-angiogenic agents (including but not limited to bevacizumab and aflibercept), and antibody-drug conjugates (ADCs) (including brentuximab vedotin, trastuzumab deruxtecan (DS-8201), and trastuzumab emtansine).

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with radiation therapy.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a therapeutic vaccine, including but not limited to sipuleucel-T.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a VEGF inhibitor, including but not limited to pazopanib, bevacizumab, cabozantinib, sunitinib, sorafenib, axitinib, regorafenib, ponatinib, cabozantinib, vandetanib, ramucirumab, lenvatinib, bevacizumab, and ziv-aflibercept.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with an Akt inhibitor, including but not limited to AZD5363, miltefosine, perifosine, VQD-002, MK-2206, GSK690693, GDC-0068, triciribine, CCT128930, PHT-427, and honokiol.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with an mTOR inhibitor, including but not limited to sirolimus, temsirolimus, everolimus, AP23841, AZD8055, BEZ235, BGT226, deferolimus (AP23573/MK-8669), EM101/LY303511, EX2 044, EX3855, EX7518, GDC0980, INK-128, KU-0063794, NV-128, OSI-027, PF-4691502, rapalogs, rapamycin, ridaforolimus, SAR543, SF1126, WYE-125132, XL765, zotarolimus (ABT578), torin 1, GSK2126458, AZD2014, GDC-0349, and XL388.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof can be used in combination with a PI3K inhibitor, including but not limited to idelalisib, copanlisib, duvelisib, alpelisib, NVP-BEZ235, BKM-120, GDC-0941, GDC-0980, SF1126, PX-866, PF-04691502, XL-765, XL-147, GSK2126458, and ZSTK474.

In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof may be used in combination with one or more other agents described herein.

Example

The scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are intended as illustrations of several aspects of the invention, and any functionally equivalent embodiments are intended to fall within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended patent applications.

Example 1. Biochemical inhibition of the R849W TIE2 mutant by the compound of formula I R849W TIE2 biochemical analysis (Seq. ID No. 1)

The activity of R849W TIE2 kinase is determined by tracking the ADP generated from the kinase reaction coupled to the pyruvate kinase/lactate dehydrogenase system (e.g., Schindler et al., Science (2000) 289: 1938-1942). In this assay, the oxidation of NADH (thus, a decrease in A at _{340 nm} ) is continuously monitored spectrophotometrically. The reaction mixture (100 μL) contained R849W TIE2 (SignalChem) (7.5 nM), BSA (0.004% (w/v)), polyEY (1 mg/ml), _MgCl₂ (15 mM), DTT (0.5 mM), pyruvate kinase (4 units), lactate dehydrogenase (7 units), phosphoenolpyruvate (1 mM), NADH (0.28 mM), and ATP (4 mM) in 100 mM Tris buffer (pH 7.5) containing 0.2% octyl-glucoside and 1% DMSO. The inhibition reaction was initiated by mixing serially diluted test compounds with the reaction mixture. Absorption at 340 nm was monitored continuously for 8 hours at 30°C on a plate reader (BioTek). Reaction rates were calculated using a 3- to 4-hour time frame. Percent inhibition was determined by comparing the reaction rate to that of a control (i.e., no test compound). _IC50 values were calculated using a software routine such as that implemented in the GraphPad Prism software suite using a series of percent inhibition values determined at a range of inhibitor concentrations. The compound of Formula I disclosed herein exhibited an _IC50 value of 0.9 nM.

R849W TIE2 protein sequence used for screening (Seq. ID No. 1)

Example 2. Biochemical inhibition of a group of TIE2 mutants by the compound of formula I Biochemical analysis of a panel of TIE2 mutants and WT TIE2

TIE2 WT or TIE2 mutants (R849W, P883A, Y897C, Y897S, Y1108F, or A1124V) and polyEY substrate were added to reaction buffer (20 mM Hepes pH 7.5, 10 mM MgCl ₂ , 2 mM MnCl ₂ , 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na ₃ VO ₄ , 2 mM DTT, 1% DMSO). Compound 1 was added to the reaction, followed by a mixture of ATP and ³³ P ATP 20 minutes later to a final concentration of 10 μM. The reaction was incubated at 25°C for 2 hours. Reactions were spotted onto P81 ion exchange filter paper, and unbound phosphate was washed off the filter in 0.75% phosphoric acid. After subtracting background values from control reactions containing inactive enzyme, kinase activity data were presented as the percentage of kinase activity remaining in the test samples compared to DMSO control reactions. _IC50 values were calculated using a software routine implemented in the GraphPad Prism software suite using a range of percent inhibition values determined at a range of inhibitor concentrations. The compounds of Formula I disclosed herein exhibited _IC50 values of 0.97 nM against WT TIE2, 1.3 nM against R849W TIE2, 8.1 nM against P883A TIE2, 1.2 nM against Y897C TIE2, 1.5 nM against Y897S TIE2, 8.4 nM against Y1108F TIE2, and 2.7 nM against A1124V TIE2.

Example 3. Cytostatic Effect of Compound I on TIE2 Mutants in CHO Cells CHO-K1 cell culture

CHO-K1 cells (Catalog No. CCL-61) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Briefly, cells were grown in RPMI 1640 medium supplemented with 10% characterized fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL penicillin G, 100 μg/mL streptomycin, and 0.29 mg/mL L-glutamine (Invitrogen, Carlsbad, CA) at 37°C, 5% _CO₂ , and 95% humidity. Cells were allowed to expand until they reached 70–95% confluence, at which point they were subcultured or harvested for analysis.

Western blotting of TIE2-phosphorylated CHO K1 in TIE2 mutant transfected cells

CHO K1 cells (1 x 10 ⁵ cells/well) were added to 1 mL of RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% characterized fetal bovine serum and 1X nonessential amino acids in a 24-well tissue culture plate. The cells were then incubated overnight at 37°C, 5% CO ₂ , and 95% humidity. The medium was aspirated and 0.5 mL of medium was added to each well. Transfection-grade plasmid DNA encoding the TIE2 mutants R849W, L914F, R1099*, Y897C/R915C, or Y897F/R915L ( TIE2 Gene Gateway, Invitrogen, Carlsbad, CA) transformed into the pcDNA3.2 ^™ /V5-DEST expression vector was diluted to 5 μg/mL in serum-free Opti-MEM® I medium (Invitrogen, Carlsbad, CA) at room temperature. For every 0.5 μg of plasmid DNA, 2 μL of Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA) was added. The tube was gently mixed and incubated at room temperature for 25 minutes to allow the DNA-Lipofectamine LTX complex to form. Add 100 μL of DNA-Lipofectamine LTX complex directly to each well containing cells and mix gently. Twenty-four hours after transfection, aspirate the medium containing the DNA-Lipofectamine LTX complex, wash the cells with serum-free RPMI 1640, and replace serum-free RPMI 1640. Add test compounds or DMSO to each well (0.5% final DMSO concentration). The plate is then incubated for 4 hours at 37°C, 5% _CO₂ , and 95% humidity. Following incubation, aspirate the medium and wash the cells with PBS. Cells were lysed with MPER lysis buffer (Pierce, Rockford, IL) containing Halt phosphatase and protease inhibitors (Pierce, Rockford, IL) and phosphatase inhibitor cocktail 2 (Sigma, St. Louis, MO) for 10 minutes at 4°C with shaking. The clarified lysate was separated by SDS-PAGE on 4-12% Novex NuPage Bis-Tris gel (Invitrogen, Carlsbad, CA) and then transferred to Immobilon-FL PVDF. After transfer, the PVDF membrane was blocked with Odyssey blocking buffer (Licor, Lincoln, NE) and then probed with rabbit antibodies against phospho-TIE2 (EMD Millipore, Burlington, MA) and mouse anti-TIE2 antibodies (BD Pharmingen, San Jose, CA). Phospho-TIE2 was detected using a secondary goat anti-rabbit antibody (Licor, Lincoln, NE) conjugated to a near-infrared dye (emission wavelength of 800 nm). Total TIE2 was detected using a secondary goat anti-mouse antibody (Licor, Lincoln, NE) conjugated to a near-infrared dye (emission wavelength of 680 nm). Fluorescence was detected using an Odyssey CL imager (Licor, Lincoln, NE). Image Studio software (Licor, Lincoln, NE) was used to quantify the 160 kDa phospho-TIE2 and total TIE2 bands. Data were analyzed using Prism software (GraphPad Software, San Diego, CA) to calculate _IC50 values. The compounds of Formula I disclosed herein exhibited the following _IC50 values: 3.6 nM for R849W TIE2, 0.58 nM for L914F TIE2, 0.41 nM for R1099* TIE2, 1.2 nM for Y897C/R915C TIE2, and 0.35 nM for Y897F/R915L TIE2 ( Figures 1A-E ).

Example 4. Inhibitory effect of the compound of formula I on TIE2 mutants in human venous endothelial cells Human umbilical vein endothelial cell (HUVEC) culture

HUVECs used for in vivo experiments were kindly provided by Dr. Lauri Eklund (Oulu, Finland). Briefly, cells were cultured in endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Diegem, Belgium) at 37°C, 5% _CO₂ , and 95% humidity. Cells were allowed to expand until they reached 90–95% confluence, at which point they were subcultured or harvested for analysis.

Western blotting of HUVEC transfected with mutant TIE2

HUVEC cells (2.5 x 10 ⁵ cells/well) stably expressing WT or TIE2 mutants R849W, L914F, R1099*, Y897C/R915C, Y897F/F915L, or T1105N/T1106P were added to 2 mL of endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) containing 10% fetal bovine serum (Sigma-Aldrich, Diegem, Belgium) in a 6-well plate coated with attachment factor solution (Tebu-Bio, Boechout, Belgium). The cells were then incubated overnight at 37°C, 5% CO ₂ , and 95% humidity. The next day, test compounds or DMSO were added to each well (0.068% final DMSO concentration). The plates were then incubated for 4 hours at 37°C, 5% _CO₂ , and 95% humidity. Cells were then stimulated with 1 μg/mL ANGPT1 for 15 minutes. The cells were lysed and Western blots were performed to measure pTIE2, TIE2, β-actin, pAKT(S473), pAKT(T308), AKT, pSTAT1, and STAT1. Compounds of Formula I disclosed herein exhibited complete inhibition of TIE2 phosphorylation at a concentration of 100 nM in HUVECs harboring WT TIE2 and TIE2 mutants, with or without ANGPT1 stimulation ( Figure 2 ). At a concentration of 100 nM, the compound of Formula I disclosed herein completely inhibited TIE2 downstream AKT phosphorylation at Ser473 and Thr308 in HUVECs harboring WT TIE2 and TIE2 mutants, with or without ANGPT1 stimulation ( Figure 3 ). At a concentration of 100 nM, the compound of Formula I disclosed herein completely inhibited TIE2 downstream STAT1 phosphorylation in HUVECs harboring WT TIE2 and TIE2 mutants, with or without ANGPT1 stimulation ( Figure 4 ).

Example 5. Restoration of cell morphology by the compound of formula I in TIE2 mutant human umbilical vein endothelial cells Determination of cell morphology of HUVEC cells transfected with mutant TIE2

HUVEC cells (5 x 10 ⁵ cells/well) stably expressing WT or TIE2 mutants R849W, L914F, R1099*, Y897C/R915C, Y897F/F915L, or T1105N/T1106P were added to 10 cm culture dishes coated with attachment factor solution (Tebu-Bio, Boechout, Belgium) in 6 mL of endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) containing 10% fetal bovine serum (Sigma-Aldrich, Diegem, Belgium). The cells were then incubated at 37°C, 5% CO ₂ , and 95% humidity for 2 days. Next, test compounds or DMSO were added to each well (0.068% final DMSO concentration). The plates were then incubated for an additional 48 hours at 37°C, 5% _CO2 , and 95% humidity. Cells were imaged by microscopy 24 and 48 hours after compound addition. Compounds of Formula I disclosed herein at a concentration of 100 nM restored the cell morphology in TIE2 mutant HUVEC cells to that of HUVEC cells expressing WT TIE2. Figure 5A shows the cell morphology of HUVEC cells expressing WT TIE2 or L914F TIE2 after being untreated, treated with DMSO control, or treated with 100 nM compound for 48 hours. Figure 5B shows the cell morphology of HUVEC cells expressing R849W TIE2 after being left untreated, treated with DMSO, or treated with 100 nM compound for 48 hours. Figure 5C shows the cell morphology of HUVEC cells expressing R1099* TIE2 after being left untreated, treated with DMSO, or treated with 100 nM compound for 48 hours. Figure 5D shows the cell morphology of HUVEC cells expressing Y897C/R915C TIE2 after being left untreated, treated with DMSO, or treated with 100 nM compound for 48 hours. Figure 5E shows the cell morphology of HUVEC cells expressing Y897C/R915L TIE2 after being left untreated, treated with DMSO as a control, or treated with 100 nM compound for 48 hours. Figure 5F shows the cell morphology of HUVEC cells expressing T1105N/T1106P TIE2 after being left untreated, treated with DMSO as a control, or treated with 100 nM compound for 48 hours.

Example 6. Effect of Formula I Compound on RNA Expression in TIE2 Mutant Human Umbilical Vein Endothelial Cells Gene expression assay for mutant TIE2 transfection

HUVEC cells (5 x 10 ⁵ cells/well) stably expressing WT or TIE2 mutants R849W, L914F, R1099*, Y897C/R915C, Y897F/F915L, or T1105N/T1106P were added to 10 cm culture dishes coated with attachment factor solution (Tebu-Bio, Boechout, Belgium) in endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) containing 10% fetal bovine serum (Sigma-Aldrich, Diegem, Belgium). The cells were then incubated at 37°C, 5% CO ₂ , and 95% humidity for 2 days. Next, test compounds or DMSO were added to each well (0.068% final DMSO concentration). The plates were then incubated for an additional 48 hours at 37°C, 5% _CO₂ , and 95% humidity. RNA was extracted by harvesting the cells into TriPure Isolation Reagent (Sigma-Aldrich, Diegem, Belgium). cDNA was then synthesized from the extracted RNA using the RevertAid H Minus First-Strand cDNA Synthesis Kit (Thermo Fischer Scientific, Merelbeke, Belgium). Quantitative PCR was performed using a LightCycler 480 SYBR Green Master Mix and a LightCycler 480 II instrument (Roche, Switzerland). cDNA for ANGPT2, PDGFB, ADAMTS1, ADAMTS9, PLAT, and PLAU was quantified and normalized to the expression of the housekeeping gene GAPDH. Compounds of Formula I disclosed herein at a concentration of 100 nM increased the expression of ANGPT2 RNA, encoding the TIE2 ligand, and abnormally downregulated it in TIE2 mutant cells. Compounds of Formula I disclosed herein at a concentration of 100 nM also increased the expression of PDGFB RNA, encoding the PDGFRB ligand, and abnormally downregulated it in TIE2 mutant cells. Compounds of Formula I disclosed herein at a concentration of 100 nM also decreased the expression of ADAMTS1 and ADAMTS9 RNA, encoding the exometalloproteinases, which were abnormally upregulated in cells transfected with mutant TIE2. Compounds of Formula I disclosed herein also reduced the expression of PLAT and PLAU RNA, encoding fibronectin activator, at a concentration of 100 nM, and their expression was abnormally upregulated in cells transfected with mutant TIE2. Figure 6A shows the expression of ANGPT2 and PDGFB RNA in HUVEC cells expressing WT TIE2, L914F TIE2, R849W TIE2, and R1099* TIE2. Figure 6B shows the expression of ANGPT2 and PDGFB RNA in HUVEC cells expressing WT TIE2, L914F TIE2, Y897C/R915C TIE2, Y897C/R915L TIE2, and T1105N/T1106P TIE2. Figure 6C shows the expression of ADAMSTS1 and ADAMSTS9 RNA in HUVEC cells expressing WT TIE2, L914F TIE2, R849W TIE2, and R1099* TIE2. Figure 6D shows the expression of ADAMSTS1 and ADAMSTS9 RNA in HUVEC cells expressing WT TIE2, L914F TIE2, Y897C/R915C TIE2, Y897C/R915L TIE2, and T1105N/T1106P TIE2. Figure 6E shows the expression of PLAT and PLAU RNA in HUVEC cells expressing WT TIE2, L914F TIE2, R849W TIE2, and R1099* TIE2. Figure 6F shows the expression of PLAT and PLAU RNA in HUVEC cells expressing WT TIE2, L914F TIE2, Y897C/R915C TIE2, Y897C/R915L TIE2, and T1105N/T1106P TIE2.

Example 6. Restoration of extracellular fibronectin by the compound of formula I in TIE2 mutant human umbilical vein endothelial cells Fibronectin assay for mutant TIE2 transfection

HUVEC cells (3 x 10 ⁵ cells/well) stably expressing WT or TIE2 mutants R849W, L914F, R1099*, Y897C/R915C, Y897F/F915L, or T1105N/T1106P were added to 6-well plates coated with attachment factor solution (Tebu-Bio, Boechout, Belgium) in endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) containing 10% fetal bovine serum (Sigma-Aldrich, Diegem, Belgium). The cells were then incubated for 24 hours at 37°C, 5% CO ₂ , and 95% humidity. Next, test compounds or DMSO were added to each well (0.068% final DMSO concentration). The plate was then incubated for an additional 48 hours at 37°C, 5% _CO₂ , and 95% humidity. The cells were then switched to 2% FBS overnight. Because intracellular fibronectin levels were low in transfectants, cell lysates were collected as controls. For cell residues, the plates were washed with 1X PBS containing 0.05% Triton-X and 50 nM _NH₄OH , followed by 50 mM 1X PBS, and then three times with 1X PBS. Extracellular matrix proteins were then extracted using a lysis buffer containing 6.5 M urea ₍ 9.1 mM _Na₂HPO₄ , 1.7 mM _NaH₂PO₄ , 1% NP- ₄₀ , 0.25% sodium deoxycholate, 150 mM NaCl, 0.1% SDS, 1 mM EDTA). As shown in Figure 7, lower levels of fibronectin were observed in the extracellular matrix of TIE2 mutants treated with DMSO compared to WT extracts (upper spot). The levels of fibronectin in cell lysates were comparable to those in controls. Compounds of Formula I disclosed herein at a concentration of 100 nM restored fibronectin levels in the extracellular matrix of HUVEC cells expressing TIE2 mutants, resulting in levels similar to those in cells expressing WT TIE2 ( FIG. 7 ).

Example 7. Inhibition of the growth of human umbilical vein endothelial cells with mutant TIE2 in vivo by the compound of formula I in a venous malformation model Evaluation of venous malformation mouse models

For the in vivo model, lentiviral infection was used to generate new transfectants. Briefly, 2 x 10 ⁶ HEK293 cells were plated onto a 10-cm culture dish for 24 hours, trypsinized, and then incubated for 20 minutes with a mixture containing pGAG-Pol, pRSV-Rev, pMD2.VSVG, the lentiviral vector pTM945, 2.5 μg of pTIE2-L914F, CaCl ₂ (Merck, UK), and 2x HBS (Thermo Fischer Scientific, Merelbeke, Belgium). 1.5 ml of cells were plated into a 24-well plate and incubated for 48 hours. Subsequently, 25,000 HUVECs (LGC Standards sarl, France) were grown per well in a 24-well plate for 24 hours, infected with 500 μl of lentivirus-TIE2-L914F, incubated for another 48 hours, harvested, and frozen.

To evaluate the compound's effect on the development of vascular malformation (VM) lesions in vivo, HUVEC cells (2.5 x 10 ⁶ ) expressing TIE2-L914F were cultured, detached, and then resuspended in 200 μL of Matrigel (Corning). This mixture was injected subcutaneously into the dorsal flank of 5-7-week-old male athymic nude mice (Charles River). On the day of injection (day 0), mice were given either a control diet or a diet containing an equivalent concentration of Compound I at 10 mg/kg. Once introduced, mice were allowed free access to food until Matrigel plugs were harvested 7 or 16 days after implantation ( Figure 8A ). On day 7 or 16 after implantation, mice were euthanized and the skin surrounding each matrix plug was dissected, followed by placement on polystyrene foam plates and fixation overnight in 10% neutral formalin buffer (Sigma-Aldrich, Diegem, Belgium). The matrix plugs were removed from the skin, dehydrated in a series of graded ethanol, and embedded in paraffin. The paraffin-embedded matrix plugs were then cut into 5 μm sections for histological analysis. After dewaxing in xylene and rehydration in a series of descending ethanol, the sections were subjected to heat-induced antigen retrieval in 0.1 M citrate buffer (pH 6.0) with or without 0.05% Tween-20. For immunohistochemical staining, sections were blocked with ₃ % _H₂O₂ (Sigma-Aldrich, Diegem, Belgium) and incubated with a biotinylated antibody against the human endothelial cell marker Ulex Europaeus Agglutinin 1 (UEA1) (Vector labs, Brussels, Belgium), followed by a horseradish peroxidase-conjugated avidin secondary antibody (GE Healthcare, Diegem, Belgium), and then incubated in DAB solution (Vector labs, Brussels, Belgium). Sections were counterstained with hematoxylin and mounted with VectaMount permanent mounting medium (Vector labs, Brussels, Belgium). For immunofluorescence (IF) staining, sections were incubated with primary antibodies recognizing UEA1, the smooth muscle marker SMA (clone 1A4, Sigma-Aldrich, Diegem, Belgium), phospho-TIE2 (Y772) (Bioke, Leiden, Netherlands), or total TIE2 (Santa Cruz, Heidelberg, Germany). Secondary antibodies used were conjugated to Alexa488-, Alexa649-, or CY5-fluorophores. Slides were mounted with VectaShield Hardset mounting medium and DAPI (Vector labs, Brussels, Belgium). Images were acquired using a Panoramic 250 Flash III digital slide scanner (3D Histech, Hungary) and visualized using Caseviewer version 2.2 software (3D Histech, Hungary). The mean vascular area was quantified by taking snapshots of at least five regions of each matrix plug under a 20x objective lens and measuring the area of at least six UEA+ vessels using ImageJ software.

In Figure 8B , macroscopic images of lesions collected show that congested venous channels were established on day 7 (D7) post-implantation in mice fed a normal or control diet and became more severe on day 16. Some mice fed a diet containing the compound of Formula I did not develop any lesions in the matrix gel plugs; the congested vascular channels that did develop appeared more controlled and less severe than in untreated mutants. Figure 9 shows representative images of UEA1 staining of matrix gel plugs from mice fed a control diet 7 days after implantation, showing enlarged venous channels and disorganized endothelial cell (EC) patches. However, expanded vascularization was less pronounced in matrix plugs from mice treated with the compound of Formula I. There was evidence that UEA1+ EC clusters were more mobile than those in controls. Some non-vascularized UEA1+ cells were also present. Furthermore, the average blood vessel area in matrix plugs from mice treated with the compound of Formula I was significantly lower than that from mice fed a control diet.

One hallmark of VM pathology is poor and inconsistent smooth muscle cell/coat cell coverage of dilated venous channels. In Figure 10 , IF staining of lesions in mice fed a control diet revealed few or no SMA-positive cells surrounding the vessels. Mice fed the compound of Formula I showed some SMA-positive cells, but not all cells surrounding the EC layer. ECs from all vessels in both control and compound of Formula I-fed mice strongly expressed total TIE2; similarly, all ECs in mice fed a control diet appeared to express phosphorylated TIE2 (pTIE2), as shown in Figure 11. pTIE2 expression appeared weaker in ECs from mice fed the compound of Formula I diet.

By day 16 post-implantation, lesions within the Matrigel had expanded significantly, although some variability was observed, likely due to technical/handling issues (e.g., a heterogeneous mixture of cells and Matrigel) ( Figure 12 ). Mice fed a control diet developed lesions comparable to untreated mice, but mice fed the compound of Formula I diet displayed much smaller blood vessels. In untreated and control-fed mice, day 16 cultures developed enlarged vascular channels that were sparsely surrounded by a smooth muscle layer and a small number of SMA+ cells. In contrast, in mice fed the compound of Formula I diet, blood vessels appeared normalized and more consistently surrounded by SMA+ cells ( Figure 13 ). Figure 14 shows that ECs in untreated mice strongly expressed pTIE2. However, pTIE2 was significantly reduced in mice fed the compound of Formula I.

To assess the overall morphology of the culture, whole-body embedding IF of blood vessels within the matrix plugs was performed. After dissection of the skin with the matrix plug attached, it was fixed flat overnight in 10% neutral formalin buffer. The matrix plugs were sliced into thin sections approximately 100-200 μm in diameter and washed in 1X PBS. The sections were blocked with 1% BSA (v/v) (Gibco) and 0.3% Triton-X100 (Sigma-Aldrich, Diegem, Belgium) in 1X PBS, then incubated in UEA1 for at least 72 hours and then incubated with a CY5-avidin secondary antibody (Vector labs, Brussels, Belgium). Sections were then postfixed in 10% NBF for 10 minutes and then mounted with Fluormount G (Thermo Fischer Scientific, Merelbeke, Belgium). Z-stack images were acquired using a Zeiss CellView spinning disk microscope (Zeiss, Germany); 3D projections of the Z-stack images were generated using AriVis4D software. Figure 15 shows that blood vessels formed within the matrix gel plugs in mice fed a control diet were abnormally elongated and disorganized at both days 7 and 16 after implantation. On day 7, dilated and clumped blood vessels were observed in cultures obtained from mice fed the compound of Formula I; however, more normal, tubular-shaped blood vessels were observed. Numerous non-vascularized UEA+ cells were also seen; notably, this is the same pattern exhibited by TIE2-WT cells upon injection. At day 16, the vasculature appeared to be largely normalized, with uniform tubular structures. Non-vascularized UEA+ cells were also present but their expression was reduced. A comparison of treated and untreated VM lesions at day 16 with wild-type and L914F TIE2 mutants was also performed, where treatment was initiated on day 0 ( Figures 18A-D ) or day 7 ( Figures 19A-C ). This data supports that feeding mice with VM with a diet infused with the compound of Formula I results in a reduction in the progression of VM and that feeding the compound of Formula I to mice with established VM reduces the severity of VM.

To evaluate the effect of the compound of Formula I on previously established VM pathologies, VM mice were generated using the same method. However, mice were fed a control or compound of Formula I diet on day 7 after implantation; the mice were then euthanized and the explants were harvested on day 16 ( FIG. 16A ). Congested vascular channels developed in all experimental groups, although the severity varied greatly ( FIG. 16B ). The mean vascular area of the vessels in the explants from mice fed a control diet on day 16 was comparable to that of the control mice, as shown in FIG. 17A-B . The mean vascular area of the vessels from mice fed a compound of Formula I diet was moderately reduced. Lesions from mice fed a Formula I compound diet showed improved smooth muscle cell coverage, comparable to that of mice fed a control diet, indicating enhanced envelope cell homeostasis and vascular maturation. Figures 19A-D also show partial comparisons of treated and untreated VM lesions with wild-type and L914F TIE2 mutants at day 16, where treatment began 7 days after implantation. This data supports that administering a Formula I compound to mice with established VM reduces the severity of VM.

Equivalent

Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be within the scope of the appended claims.

Claims (19)

Use of a compound of formula I or a pharmaceutically acceptable salt thereof, It is used to prepare a drug for treating TIE2 kinase-mediated vascular abnormalities or TIE2 kinase mutant-mediated vascular abnormalities in patients in need. For the use of claim 1, the pharmaceutically acceptable salt is tosylate. The use of claim 1, wherein the TIE2 kinase-mediated vascular abnormality or TIE2 kinase mutant-mediated vascular abnormality is a slow flow malformation. The use of claim 3, wherein the slow blood flow malformation is selected from microvascular malformation, lymphatic malformation or venous malformation. The use as claimed in claim 4, wherein the slow blood flow malformation is a venous malformation. The use of claim 1, wherein the treatment comprises administering the compound of formula I to the patient once a day, intermittently not daily, once every other day, once every two days, once every other week, twice a day, once a week, or twice a week. The use of claim 1, wherein the treatment comprises administering to the patient from about 57 mg to about 1200 mg of the compound of formula I per day. The use of claim 1, wherein the treatment comprises administering to the patient approximately 100 mg of the compound of formula I per day. The use of claim 1, wherein the treatment comprises administering to the patient about 150, 200 or 300 mg of the compound of formula I once or twice daily. For the use of claim 1, the drug further comprises a second therapeutic agent, or is used in combination with a second therapeutic agent. The use of claim 10, wherein the second therapeutic agent is a VEGF inhibitor. The use of claim 11, wherein the VEGF inhibitor is selected from pazopanib, bevacizumab, cabozantinib, sunitinib, sorafenib, axitinib, regorafenib, ponatinib, cabozantinib, vandetanib, ramucirumab, lenvatinib, bevacizumab and ziv-aflibercept. The use of claim 10, wherein the second therapeutic agent is an Akt inhibitor. The use of claim 13, wherein the Akt inhibitor is selected from AZD5363, miltefosine, perifosine, VQD-002, MK-2206, GSK690693, GDC-0068, triciribine, CCT128930, PHT-427, and honokiol. The use of claim 10, wherein the second therapeutic agent is an mTOR inhibitor. The use of claim 15, wherein the mTOR inhibitor is selected from sirolimus, temsirolimus, everolimus, AP23841, AZD8055, BEZ235, BGT226, deferolimus (AP23573/MK-8669), EM101/LY303511, EX2044, EX3855, EX751 8. GDC0980, INK-128, KU-0063794, NV-128, OSI-027, PF-4691502, rapalogs, rapamycin, ridaforolimus, SAR543, SF1126, WYE-125132, XL765, zotarolimus (ABT578), torin 1, GSK2126458, AZD2014, GDC-0349, and XL388. The use of claim 10, wherein the second therapeutic agent is a PI3K inhibitor. The use of claim 17, wherein the PI3K inhibitor is selected from idelalisib, copanlisib, duvelisib, alpelisib, NVP-BEZ235, BKM-120, GDC-0941, GDC-0980, SF1126, PX-866, PF-04691502, XL-765, XL-147, GSK2126458, and ZSTK474. Use of a compound of formula I or a pharmaceutically acceptable salt thereof, It is used for the preparation of a medicament for treating venous malformations in a patient in need thereof, wherein the patient is administered about 100 mg to about 200 mg of a compound of formula I or a pharmaceutically acceptable salt thereof once or twice a day.