CN114746093A - Method for treating vascular malformations - Google Patents

Abstract

The present invention relates to methods of inhibiting TIE2 kinase, which are beneficial in the treatment of growth of venous malformations. In particular, the present invention relates to methods of using compounds of formula I (Riparitinib) and salts thereof, alone or in combination with a second therapeutic agent

Description

Method for treating vascular malformations

The present application claims priority to U.S.S.N.62/885,519 filed on 12.8.2019, the contents of which are incorporated herein by reference in their entirety.

Background

Intimal endothelial cell kinase-2 (TIE2) is predominantly restricted to expression in endothelial cells of blood vessels. TIE2 is a receptor for angiopoietin 1(ANGPT1), angiopoietin 2(ANGPT2) and angiopoietin 4(ANGPT4), and this messaging system plays an important role in both vascular proliferation (new blood vessels sprouting from existing vessels) and angiogenesis (new blood vessel reformation).

Vascular malformations encompass a wide variety of vascular diseases. These diseases include venous malformations, lymphatic malformations, microvascular malformations, arterial malformations, and arteriovenous malformations. Malformations may be involved in any vessel type or combination. Vascular malformations grow over time and grow rapidly, and local tissue infiltration occurs. Venous malformations may be limited to local or multifocal occurrences. Venous malformations may be associated with pain, swelling, bleeding, topographical lesions, thrombosis, and other significant morbidity. Venous malformations can affect tissues such as skin, joints, muscles, bowel, and bone. Many venous malformations may be treated by surgery, laser therapy, or sclerotherapy, however not all venous malformations are suitable for these treatments. In most cases, venous malformations recur after conventional treatment.

Approximately 50% of venous malformations have been associated with gonads or with somatic mutations in TIE2 kinase. These mutations activate TIE2 kinase, leading to endothelial cell growth dysregulation and venous malformations. Therefore, new treatments for these diseases associated with the alteration of TIE2 are needed.

Disclosure of Invention

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

or a pharmaceutically acceptable salt thereof.

For example, provided herein is a method of treating a TIE2 kinase-mediated vascular abnormality or a TIE2 kinase mutant-mediated 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.

Further, provided herein is a method of 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, wherein the vascular abnormality is mediated by TIE2 kinase or by a TIE2 kinase mutant.

Further, provided herein is a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in treating a TIE2 kinase-mediated vascular abnormality or a TIE2 kinase mutant-mediated 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.

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

Drawings

Figures 1A-E show that the use of compounds of formula I inhibited phosphorylation of various TIE2 mutants (R849W, L914F, R1099 x, Y897C/R915C, and Y897F/R915L, respectively) in assays using transfected CHO cells.

Figure 2 shows 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 the inhibition of phosphorylation of the downstream messenger AKT using a compound of formula I in an assay using transfected human umbilical vein endothelial cells and various TIE2 mutants.

Figure 4 shows the inhibition of phosphorylation of downstream messenger STAT1 using a compound of formula I in an assay using transfected human umbilical vein endothelial cells and various TIE2 mutants.

Figures 5A-F show the recovery 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).

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

FIG. 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 shows a schematic of the experimental design described in example 10 for assessing inhibition of growth of mutant TIE2 human umbilical vein endothelial cells in vivo by a compound of formula I in a model of venous malformation, wherein mice were administered a control diet or a diet infused with a compound of formula I on day 0.

Figure 8B shows the effect of compound of formula I on the ocular appearance of mutant TIE2 vasculopathy on in vivo models of venous malformations at day 7.

FIG. 9 shows the effect of a compound of formula I on the size of mutant TIE2 vasculopathy on in vivo models of venous malformations at day 7.

FIG. 10 shows the effect of compounds of formula I on smooth muscle cell and outer coating cell coverage of mutant TIE2 vasculopathy on day 7 in an in vivo model of venous malformation.

FIG. 11 shows the effect of compounds of formula I on the phosphorylation of TIE2 in mutant TIE2 vasculopathy in an in vivo model of venous malformation on day 7.

FIG. 12 shows the effect of a compound of formula I on the size of mutant TIE2 vasculopathy on in vivo models of venous malformations at day 16.

FIG. 13 shows the effect of compounds of formula I on smooth muscle cell and outer coating cell coverage of mutant TIE2 vasculopathy on day 16 in an in vivo model of venous malformation.

FIG. 14 shows the effect of compounds of formula I on the phosphorylation of TIE2 in mutant TIE2 vasculopathy in an in vivo model of venous malformation on day 16.

FIG. 15 shows the effect of compounds of formula I on the vascular morphology of mutant TIE2 vasculopathy on in vivo models of venous malformation on days 7 and 16.

Figure 16A shows a schematic of the experimental design described in example 10 for assessing inhibition of growth of mutant TIE2 human umbilical vein endothelial cells in vivo by a compound of formula I in a model of venous malformation, wherein mice were administered a control diet or a diet infused with a compound of formula I on day 7.

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

FIGS. 17A-B show the effect of compounds of formula I on the size of previously established mutant TIE2 vasculopathy and on smooth muscle cell and outer coating cell coverage of mutant TIE2 vasculopathy in an in vivo model of venous malformations.

FIGS. 18A-D compare the effect of compounds of formula I from day of injection (day 0) to day 7 on Vascular Malformation (VM) lesions exhibiting wild type and L914F TIE2 mutant. Treatment with the compound of formula I diet was compared to treatment with the control diet and untreated VM lesions by macroscopic (fig. 18A) and microscopic (fig. 18B) images and quantification of vascular area (fig. 18C and 18D).

Fig. 19A-C provide a comparison of treatment of Vascular Malformation (VM) lesions exhibiting wild type and L914F TIE2 mutant from the day of injection (day 0) to day 16, or from day 7 to day 16. Microscopic view comparisons of untreated VM lesions and VM lesions expressing TIE2L914F treated with compound of formula I diets and control diets were included (fig. 19A), as well as quantification of vascular vessel area in lesions expressing TIE2 wild type or L914F (fig. 19B and 19C).

Detailed Description

The features and other details of the invention will now be more clearly described. Certain terms employed in the specification, examples, and appended claims are collected here. These definitions should be read in light of the remainder of the present disclosure and as understood by those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Definition of

The terms "individual", "patient" or "subject" are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, 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, e.g., livestock (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 an adverse, allergic, or other untoward reaction when administered to an animal or human, as appropriate. For human administration, the formulations should meet sterility, fever, and general safety and purity standards as required by the FDA Office of Biologics standards.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, 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.

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

The term "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of the compounds disclosed herein. These salts may be salts of acidic or basic groups that may be present in the compounds used in the composition. The 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. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing a pharmacologically acceptable anion, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, glucarate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts can be directed to include another molecule, such as an acetate ion, a succinate ion, or other counter ion. The 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. There may be multiple counter ions in the case where the multiple charged atoms are part of the pharmaceutically acceptable salt. 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 both acidic and basic groups; for example an amine group and a carboxylic acid group. In such cases, the compounds may exist as acid addition salts, zwitterions, or base salts. If the compound as disclosed herein is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example by treating the free base with: inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, and the like; or organic acids 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 compound as disclosed herein is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example by treating the free acid with: inorganic or organic bases such as amines (primary, secondary or tertiary), alkali metal hydroxides or alkaline earth metal hydroxides 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, secondary 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.

The term "treating" as used herein includes reversing, alleviating, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a subject in a manner that ameliorates or stabilizes the condition. As used herein, and as understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, detectably or undetectable alleviation, amelioration, or alleviation of one or more symptoms or conditions associated with a condition (e.g., cancer), diminishment of extent of disease, stabilization (i.e., not worsening) of disease state, delay or delay in disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete). "treatment" may also mean prolonging survival compared to that expected in the absence of treatment. Exemplary beneficial clinical results are described herein.

The term "administering" as used herein refers to either directly administering a composition to a subject or a pharmaceutically acceptable salt of the compound or composition, or administering to the subject a prodrug derivative or analog of the composition or a pharmaceutically acceptable salt of the compound or composition, which forms an equivalent amount of the active compound in the body of the subject.

In this specification, the term "therapeutically effective amount" means the amount of the subject compound that elicits the biological or medicinal response in a tissue, system, or animal (e.g., a mammal or a human) that is being sought by a researcher, veterinarian, medical doctor or other clinician. The compounds described herein are administered in a therapeutically effective amount to treat the disclosed conditions.

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

The present invention relates to the treatment (blocking) or prevention of venous malformation growth. Such disclosed methods may comprise administering to a patient in need of treatment or reduction of a prophylactic effect of such conditions an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, e.g., as part of a dosing regimen that modulates the inhibition of TIE 2.

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 (receptor tyrosine kinase for angiogenin ligands).

Exemplary methods include treating venous malformations in patients, for example, where such patients have expression, mutation, or alteration of TIE2 in endothelial cells that may cause or lead to growth of venous malformations. Such methods may comprise administering to the patient suffering from a venous malformation a compound of formula I, or a pharmaceutically acceptable salt thereof. For example, providing a compound such as a compound of formula I or a pharmaceutically acceptable salt thereof can inhibit processes involving growth of venous malformations. Contemplated compounds include the free base of formula I.

Effective amounts, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD50 (the lethal dose for 50% of the population) and ED50 (the therapeutically effective dose for 50% of the population). The dosage may vary depending on the dosage form employed and the route of administration employed. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED 50. In some embodiments, the compositions and methods exhibit a large therapeutic index. The amount 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, inter alia, by suitable bioanalytics. The dosage can be determined by a physician and adjusted as necessary to suit the observed therapeutic effect.

In certain embodiments, a prophylactic 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 benefits also include halting or slowing the progression of the underlying disease or condition as expected, regardless of whether an improvement is achieved.

Formula I or a pharmaceutically acceptable salt thereof (and/or additional agents) described herein may be administered to a subject in the form of a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally comprise a suitable amount of a pharmaceutically acceptable excipient to provide a form suitable for administration.

Pharmaceutically acceptable excipients may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutically acceptable excipients may 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 may 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. Saline solutions and aqueous dextrose and glycerol solutions may also be employed 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, glycol, water, ethanol and the like. Any of the reagents described herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

Provided herein in some embodiments 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 globular cellular venous malformations, mucosal and cutaneous venous malformations (also known as mucocutaneous venous malformations, VMCMs), blue bullous nevus syndrome, lesions of the gastric or gastrointestinal tract, and/or maffecci syndrome.

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

In some embodiments, the dosing regimen of the compound of formula I, or a pharmaceutically acceptable salt thereof, is a daily dosing administration or a twice daily dosing administration.

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

In some embodiments, a suitable dosing regimen for a compound of formula I, or a pharmaceutically acceptable salt thereof, includes administering twice weekly, once weekly, or every other week, such as twice weekly or once weekly, such as twice weekly.

Another aspect of the invention relates to a method of blocking venous malformation growth comprising administering a compound of formula I, or a pharmaceutically acceptable salt thereof, in a dosage sufficient to block TIE2 kinase or mutant TIE2 kinase activity, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered on an intermittent, non-daily dosing regimen. In some embodiments, the intermittent non-daily dosing regimen comprises every other day, every third day, twice weekly, and once weekly dosing.

Another aspect of the invention relates to a method of treating a patient with venous malformations prior to surgical removal of a tumor in a patient in need thereof comprising administering to the patient an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in a dosage regimen sufficient to block TIE2 kinase or mutant TIE2 kinase.

In some embodiments, the method of treating a patient with a venous malformation prior to surgical removal of the tumor in a lead therapy 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 regimen sufficient to block TIE2 kinase or mutant TIE2 kinase.

In some embodiments, a method of treating a patient with a venous malformation that was in lead therapy prior to surgical resection comprises administering a compound of formula I, or a pharmaceutically acceptable salt thereof, at a dose sufficient to block TIE2 kinase or mutant TIE2 kinase, at a dosing regimen that administers the compound of formula I, or a pharmaceutically acceptable salt thereof, daily or twice daily.

In some embodiments, the method of treating a patient with venous malformation prior to surgical resection in a lead therapy 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 regimen sufficient to block TIE2 kinase or mutant TIE2 kinase, at a dosing regimen that includes administering the compound of formula I, or a pharmaceutically acceptable salt thereof, on an intermittent, non-daily basis including on an alternate day basis, every other day basis, twice weekly, and weekly.

In some embodiments, the method of treating a patient with venous malformations prior to surgical resection in a lead therapy comprises administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, on a dosing regimen of twice weekly, or every other week.

Another aspect of the present invention is directed to a method of treating a TIE2 kinase-mediated vascular abnormality or a TIE2 kinase mutant-mediated vascular abnormality (e.g., a vascular malformation, vascular tumor (e.g., hemangioma), and/or other congenital vascular defect) 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 salt. In some embodiments, the pharmaceutically acceptable salt is a mesylate salt. In some embodiments, the pharmaceutically acceptable salt is a triflate salt. In some embodiments, the pharmaceutically acceptable salt is an ethanesulfonate. In some embodiments, the pharmaceutically acceptable salt is a benzenesulfonate salt. In some embodiments, the pharmaceutically acceptable salt is a chlorobenzenesulfonate salt. In some embodiments, the pharmaceutically acceptable salt is a camphorsulfonate salt. In some embodiments, the pharmaceutically acceptable salt is a tosylate salt. In some embodiments, the pharmaceutically acceptable salt is monotosylate. In some embodiments, the pharmaceutically acceptable salt is a salt of xylenesulfonate. In some embodiments, the pharmaceutically acceptable salt is a salt of mesitylene sulfonic acid. In some embodiments, the pharmaceutically acceptable salt is tetramethylbenzenesulfonate.

Methods of treating vascular abnormalities in a patient in need thereof are contemplated herein, 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. A vascular abnormality mediated by TIE2 kinase or a vascular abnormality mediated by a TIE2 kinase mutant may be a slow blood flow type malformation. In some embodiments, the chronic blood flow-type malformation is selected from a microvascular malformation, a lymphatic-venous malformation, or a venous malformation. In some embodiments, the slow-flow type abnormality is a venous abnormality.

Formulation, administration, dosing and treatment regimens

The invention also describes compounds of formula I (and/or additional agents) or pharmaceutically acceptable salts thereof in pharmaceutical compositions. The compositions described herein may 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, e.g., U.S. patent No. 5,698,155). Other examples of suitable Pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-.

The salts described herein may also include solubilizing agents, if desired. In addition, the agents may be delivered using suitable vehicles or delivery devices as are known in the art. The combination therapies outlined herein may be co-delivered in a single delivery vehicle or delivery device. Compositions for administration may optionally include a local anesthetic such as, for example, lignocaine (lignocaine) to reduce pain at the injection site.

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

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, especially to the ear, nose, eye, or skin. In some embodiments, administration is achieved orally or by parenteral injection. The mode of administration may be left to the discretion of the physician and will depend 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, it may be desirable to apply topically to the area in need of treatment or blockage.

In some embodiments, the salts (and/or additional agents) described herein are formulated in accordance with conventional procedures into compositions suitable for oral administration to humans. Compositions for oral delivery may be in the form of, for example, tablets, buccal tablets, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Compositions for oral administration may comprise one or more agents, for example sweetening agents, such as fructose, aspartame or saccharin; flavoring agents, such as peppermint, oil of wintergreen, or cherry; a colorant; and preservatives to provide pharmaceutically palatable preparations. In addition, when in tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action for an extended period of time. The selectively permeable membranes surrounding the osmotically active driven compound I or a pharmaceutically acceptable salt thereof (and/or additional agents) described herein are also suitable for use in compositions for oral administration. In these latter platforms, fluid from the environment surrounding the capsule is absorbed by the actuating composition, which expands to displace the agent or agent composition through the opening. These delivery platforms can provide a substantially zero order delivery profile as opposed to a spike-like profile for immediate release formulations. A time delay material such as glyceryl monostearate or glyceryl stearate may also be used. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In some embodiments, the excipient is pharmaceutical grade. Suspensions, in addition to the active compositions, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and the like, and mixtures thereof.

Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They can also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions), which can be dissolved or suspended in a sterile injectable medium just prior to use. Which may contain, for example, suspending or dispersing agents as known in the art.

The dosage and dosing schedule of a compound of formula I, or a pharmaceutically acceptable salt (and/or additional agent) thereof, as described herein, may depend on various parameters, including, but not limited to, the disease being treated, the general health of the subject, and the judgment of the administering physician. Any of the agents described herein can be administered to a subject in need thereof 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 before), concurrently with, or after (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 after) administration of another therapeutic agent. 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 to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, or 11 hours to 12 hours apart.

The dosage of a compound of formula I, or a pharmaceutically acceptable salt thereof (and/or additional agent), described herein can 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 subject to be treated. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a treatment) information about a particular subject can affect the dosage used. In addition, the exact individual dosages may be adjusted somewhat depending upon a variety of factors including the particular combination of agents administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the condition, and the anatomical location of the condition. Some variation in dosage is contemplated.

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

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

In some embodiments, the administration of the salts (and/or additional agents) described herein may independently be one to four times per day. In particular, the administration of the salt may be once a day on a dosing regimen of about 50mg to 1500mg of the salt. The daily dose suitable for the prophylactic effect sought is from 57 to 1200 mg/day. In some embodiments, the methods described herein comprise administering to the patient about 100mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, per day. In some embodiments, the methods described herein comprise administering to the patient about 200mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, per day. In some embodiments, the methods described herein comprise administering to the patient about 300mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, per day. If administered twice daily, suitable doses are from 100mg to 200mg of the salt. In some embodiments, the methods described herein comprise administering to the patient about 150, 200, or 300mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, once or twice daily. In some other embodiments, the salt may also be administered intermittently, non-daily. In some embodiments, the administration of the salt may be performed one to four times per month or one to six times per year or once every two, three, four or five years. In certain embodiments, the administration of the salt is performed weekly or biweekly. In some embodiments, when administered weekly or biweekly, in some embodiments, a suitable salt dosing regimen is in the range of 50-200mg per administration. In some embodiments, administration is weekly or biweekly and the dose is 200-400mg per administration. In some embodiments, administration is weekly or biweekly and the dose is 400-500mg per administration. In some embodiments, the administration is weekly or biweekly and the dose is 500-600mg per administration. In some embodiments, administration is weekly or biweekly and the dose is 600-700mg per administration. In some embodiments, administration is weekly or biweekly and the dose is 700-800mg per administration. In some embodiments, administration is weekly or biweekly and the dose is 800-900mg per administration. In some embodiments, administration is weekly or biweekly and the dose is 900-1000mg per administration. In some embodiments, weekly or biweekly administration and a dose of 1000-1100mg per administration. In some embodiments, administration is weekly or biweekly and the dose is 1100-1200mg per administration. The duration of administration may be one day or one month, two months, three months, six months, one year, two years, three years, and may even last for the lifetime of the subject. Chronic long-term administration will be indicated in many cases. The dose may be administered in a single dosage form or divided into multiple doses. In general, the desired dose will be administered at set intervals over a prolonged period, usually at least over weeks or months, although longer administration of months or years may be required.

In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof may 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, anti-estrogen agents, anti-androgens, steroids, anti-EGFR agents, kinase inhibitors, topoisomerase inhibitors, Histone Deacetylase (HDAC) inhibitors, DNA methylation inhibitors, anti-HER 2 agents, anti-angiogenic agents, proteasome inhibitors, thalidomide (thalidomide), lenalidomide (lenalidomide), antibody-drug conjugates (ADC), immunomodulators or cancer vaccines. In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, may be administered to a patient with a VEGF inhibitor. In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, can be administered to a patient with an inhibitor of Akt. In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, may be administered to a patient with an mTOR inhibitor. In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, may be administered to a patient 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 schedule of administration of the compound of formula I. The additional agents may be administered at their previously established therapeutic dosages and schedules, or their dosages and schedules may be modified when used in combination with the compounds of formula I to optimize efficacy, safety or tolerability.

A compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with other agents including, but not limited to, chemotherapeutic agents, targeted therapeutic agents, biological agents, or radiotherapy.

In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, may be used in combination with chemotherapeutic agents including, but not limited to, anti-tubulin agents (e.g., paclitaxel (paclitaxel), paclitaxel protein-binding particles for injectable suspensions, eribulin (eribulin), docetaxel (docetaxel), ixabepilone (ixabepilone), vincristine (vincristine), vinorelbine (vinorelbine), epothilone (epothilone), halichondrin (halichondrins), maytansinol (maytansinoids)), DNA alkylating agents (e.g., cisplatin (cissplatin), carboplatin (carboplatin), oxaliplatin (oxaubulin), cyclophosphoid (cyclopamine), ifosfamide (ifosfamide), temozolomide (temozolomide)), DNA intercalating agents (e.g., doxoloxin), doxorubicin (doxorubicin), doxorubicin (epirubicin), and epirubicin (epirubicin), doxorubicin (epirubicin), and doxorubicin (epirubicin), doxorubicin (doxorubicin), doxorubicin (e.g., a), doxorubicin (doxin), and doxorubicin (doxin) may be used in, or a pharmaceutically acceptable salt thereof), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a compound, or a pharmaceutically acceptable salt thereof, or a compound, or a pharmaceutically acceptable salt thereof, or a compound, or a pharmaceutically acceptable salt thereof, or a compound of formula I, or a compound of formula (e, or a compound of formula I, or a compound of formula, 5-fluorouracil, capecitabine, cytarabine, decitabine, 5-azacytidine, gemcitabine and methotrexate.

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with kinase inhibitors including, but not limited to, erlotinib (erlotinib), gefitinib (gefitinib), lapatinib (lapatanib), everolimus (everolimus), sirolimus (sirolimus), temsirolimus (temsirolimus), LY2835219, LEEOl, 033pd 2991, crizotinib (crizotinib), cabozantinib (cabozantinib), sunitinib (sunitinib), pazopanib (pazopanib), sorafenib (sorafenib), regorafenib (regorafenib), axitinib (axinib), dasa (dasatinib), imatinib (imatinib), nilotinib (nilotinib), vilafibratib (teminib), tematib (nilotinib), vilinenib (lasinab), teminafenib (temifitianib), temobib (apinib), temicib), erinib (apigenin), and (eribizib), and pexib (peridinib (perivaib).

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with an antiestrogen including, but not limited to, tamoxifen (tamoxifen), fulvestrant (fulvestrant), anastrozole (anastrozole), letrozole (letrozole), and exemestane (exemestane).

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with an anti-androgen agent, including, but not limited to, abiraterone acetate (abiraterone acetate), enzalutamide (enzalutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), flutamide (flutamide), cyproterone acetate (cyproterone acetate).

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

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with topoisomerase I inhibitors, including but not limited to irinotecan (irinotecan), camptothecin (camptothecin), and topotecan (topotecan).

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

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with Histone Deacetylase (HDAC) inhibitors, including, but not limited to, vorinostat (vorinostat), romidepsin (romidepsin), panobinostat (panobinostat), valproic acid (valproic acid), and belinostat (belinostat).

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with DNA methylation inhibitors, including but not limited to, dzneep and 5-aza-2' -deoxycytidine.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, may be used in combination with proteasome inhibitors, including, but not limited to, bortezomib (bortezomib) and carfilzomib (carfilzomib).

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with thalidomide (thalidomide), lenalidomide, and pomalidomide (pomalidomide).

In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, may be used in combination with a biological agent, the biological agents include, but are not limited to, trastuzumab (trastuzumab), ado-trastuzumab (ado-trastuzumab), pertuzumab (pertuzumab), cetuximab (cetuximab), panitumumab (panitumumab), ipilimumab (ipilimumab), anti-PD-1 agents including, but not limited to, labriczumab (labrolizumab) and nivolumab (nivolumab)), anti-PD-L1 agents including, but not limited to, MPDL32 3280A, anti-angiogenic agents including, but not limited to, bevacizumab (bevacizumab) and aflibercept (aflibercept)), and antibody-drug conjugates (ADC) including, bertuzumab (brentuzumab), trastuzumab (trastuzumab-dertuzumab-01), trastuzumab (trastuzumab-8201), and trastuzumab (82trastuzumab).

In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, may be used in combination with radiotherapy.

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

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with VEGF inhibitors including, but not limited to, pazopanib (pazopanib), bevacizumab (bevacizumab), cabozantinib (cabozantinib), sunitinib (sunitinib), sorafenib (sorafenib), axinib (axitinib), regorafenib (regorafenib), panatinib (ponatinib), cabozantinib (cabozantinib), vandetanib (vandetanib), ramucirumab (ramucimab), lenvatinib (bevacizumab), and ziv-aflibercept (ziv-aflibercept).

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with Akt inhibitors including, but not limited to, AZD5363, miltefosine (miltefosine), pirifosine (perifosine), VQD-002, MK-2206, GSK690693, GDC-0068, triciribine (triciribine), CCT128930, PHT-427, and magnolol (honokiol).

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with an mTOR inhibitor including, but not limited to, sirolimus (sirolimus), temsirolimus (temsirolimus), everolimus (everolimus), AP23841, AZD8055, BEZ235, BGT226, discolimus (deferolimus) (AP23573/MK-8669), EM101/LY303511, EX2044, EX3855, EX7518, GDC0980, INK-128, KU-3794, NV-128, OSI-027, PF-4691502, rapamycin analogs (rapalogs), rapamycin (rapamycin), ridaforolimus (ridaforolimus), SAR543, SAR 1126, WYE-125132, XL765, zotarolimus (ZTarimus) (ABT578), Torlin 1 (torlin 1), GSK 58, AZD 6403201449, GDSF and XL 388.

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereof may be used in combination with PI3K inhibitors, including, but not limited to, idelixib (idelalisib), copaxib (copanlisib), duvexib (duvelisib), abacixib (alpelisib), NVP-BEZ235, BKM-120, GDC-0941, GDC-0980, SF1126, PX-866, PF-04691502, XL-765, XL-147, GSK2126458, and ZSTK 474.

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

Examples of the invention

The scope of the invention is not intended to be limited to the particular embodiments disclosed in the examples, which are intended as illustrations of several aspects of the invention, and any embodiments that are functionally equivalent are 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 claims.

EXAMPLE 1 Biochemical inhibition of the R849W TIE2 mutant by Compounds of formula I

Biochemical analysis method of R849W TIE2 (seq. ID No.1)

The activity of R849W TIE2 kinase was determined by tracking the ADP generated from the kinase reaction coupled to the pyruvate kinase/lactate dehydrogenase system (e.g., Schinder et al, Science (2000)289: 1938-. In this assay, the oxidation of NADH (and thus the reduction at A340 nm) was monitored continuously by spectrophotometry. The reaction mixture (100. mu.L) contained R849W TIE2(SignalChem) (7.5nM), BSA (0.004% (w/v)), polyEY (1mg/ml), MgCl2(15mM), DTT (0.5mM), pyruvate kinase (4 units), lactate dehydrogenase (7 units), phosphoenolpyruvate (1mM) and NADH (0.28mM) and ATP (4mM) in 100mM Tris buffer (pH 7.5) containing 0.2% octyl-glucoside and 1% DMSO. The inhibition reaction is initiated by mixing serially diluted test compounds with the above reaction mixture. The absorbance at 340nm was continuously monitored on a well plate reader (BioTek) at 30 ℃ for 8 hours. The reaction rate was calculated using a time range of 3 to 4 hours. Percent inhibition was obtained by comparing the reaction rate to that of a control (i.e., no test compound). IC was calculated using a series of percent inhibition values determined at a series of inhibitor concentrations using a software routine as performed in the GraphPad Prism software package₅₀The value is obtained. The compounds of formula I disclosed herein exhibit an IC of 0.9nM₅₀The value is obtained.

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

QLKRANVQRRMAQAFQNVREEPAVQFNSGTLALNRKVKNNPDPTIYPVLDWNDIKFQDVIGEGNFGQVLKARIKKDGLWMDAAIKRMKEYASKDDHRDFAGELEVLCKLGHHPNIINLLGACEHRGYLYLAIEYAPHGNLLDFLRKSRVLETDPAFAIANSTASTLSSQQLLHFAADVARGMDYLSQKQFIHRDLAARNILVGENYVAKIADFGLSRGQEVYVKKTMGRLPVRWMAIESLNYSVYTTNSDVWSYGVLLWEIVSLGGTPYCGMTCAELYEKLPQGYRLEKPLNCDDEVYDLMRQCWREKPYERPSFAQILVSLNRMLEERKTYVNTTLYEKFTYAGIDCSAEEAA

EXAMPLE 2 Biochemical inhibition of a group of TIE2 mutants by Compounds of formula I

Biochemical analysis method of TIE2 mutant and WT TIE2

TIE2 WT or TIE2 mutant (R849W, P883A, Y897C, Y897S, Y1108F or A1124V) and polyEY substrate were added to the reaction buffer (20mM Hepes pH 7.5, 10mM MgCl2, 2mM MnCl2, 1mM EGTA, 0.02% Brij35, 0.02mg/ml BSA, 0.1mM Na3VO4, 2mM DTT, 1% DMSO). Compound 1 was added to the reaction followed 20 minutes later by the addition of a mixture of ATP and 33P ATP to a final concentration of 10 μ M. The reaction was carried out at 25 ℃ for 2 hours. The reaction was spotted onto P81 ion exchange filter paper and unbound material was washed out of the filter in 0.75% phosphoric acid. After subtracting the background values from the control reactions containing inactive enzyme, the kinase activity data are presented as a percentage of the kinase activity remaining in the test samples compared to the DMSO control reactions. IC was calculated using a series of percent inhibition values determined at a series of inhibitor concentrations using a software routine as performed in the GraphPad Prism software package₅₀The value is obtained. The compounds of formula I disclosed herein exhibit an IC of 0.97nM for WT TIE2₅₀Values, 1.3nM for R849W TIE2, 8.1nM for P883A TIE2, 1.2nM for Y897C TIE2, 1.5nM for Y897S TIE2, 8.4nM for Y1108F TIE2, and 2.7nM for a1124V TIE 2.

EXAMPLE 3 cellular inhibition of TIE2 mutant by Compounds of formula I in CHO cells

CHO-K1 cell culture

CHO-K1 cells (catalog number CCL-61) were obtained from the American type culture Collection (ATCC, Manassas, Va.). Briefly, cells were cultured in RPMI1640 medium (supplemented with 10% characterized fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL penicillin G, 100. mu.g/mL streptomycin, and 0.29mg/mL L-glutamic acid amide (Invitrogen, Carlsbad, CA)) at 37 ℃ with 5% CO₂And growth at 95% humidity. Allowing cells to expandIncrease until 70-95% of the clusters are reached, at which time they are subcultured or collected for analysis.

CHO K1 phosphorylation-TIE 2 Western blotting method of mutant TIE2 transfection

CHO K1 cells (1X 10)⁵Individual cells/well) was added to 1mL of RPMI1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% characterized fetal bovine serum and 1X non-essential amino acids in 24-well tissue culture treatment plates. The cells were then incubated at 37 ℃ with 5% CO₂And incubated overnight at 95% humidity. The medium was aspirated and 0.5mL of medium was added to each well. Transfection grade plastid DNA (cloned into pcDNA3.2) encoding TIE2 mutants R849W, L914F, R1099, Y897C/R915C or Y897F/R915L^TMTIE2 Gene Gateway, Invitrogen, Carlsbad, Calif.) of the/V5-DEST expression vector at serum-free room temperature

I medium (Invitrogen, Carlsbad, Calif.) was diluted to 5. mu.g/mL. mu.L of Lipofectamine LTX reagent (Invitrogen, Carlsbad, Calif.) was added to 0.5. mu.g of plasmid DNA. The tubes were gently mixed and incubated at room temperature for 25 minutes to allow the DNA-Lipofectamine LTX complex to form. 100 μ L of DNA-Lipofectamine LTX complex was added directly to each well containing cells and gently mixed. Twenty-four hours after transfection, the medium containing the DNA-Lipofectamine LTX complex was aspirated, the cells were washed with serum-free RPMI1640, and serum-free RPMI1640 was added. Test compounds or DMSO were added to each well (0.5% final DMSO concentration). The plates were then incubated at 37 ℃ with 5% CO₂And incubation at 95% humidity for 4 hours. After incubation, the medium was aspirated and the cells were washed with PBS. Cells were lysed with MPER lysis buffer (Pierce, Rockford, IL) containing Halt phosphatase and protease inhibitor (Pierce, Rockford, IL) and phosphatase inhibitor cocktail 2(Sigma, st. The clarified lysates were separated by SDS-PAGE on 4-12% Novex NuPage Bis-Tris gel (Invitrogen, Carlsbad, Calif.) and then transferred to Immobilon-FL PVDF. After transfer, PVDF membrane was blocked with Odyssey blocking buffer (L)i-cor, Lincoln, NE) and then probed with rabbit antibodies to phosphorylated-TIE 2 (EMD Millipore, Burlington, MA) and mouse anti-TIE 2 antibodies (BD Pharmingen, San Jose, CA). phosphorylation-TIE 2 was detected using a secondary goat anti-rabbit antibody (Li-cor, Lincoln, NE) conjugated with a near infrared dye (emission wavelength 800 nm). Total TIE2 was detected using a secondary goat anti-mouse antibody (Li-cor, Lincoln, NE) conjugated with a near infrared dye (emission wavelength of 680 nm). Fluorescence was detected using an Odyssey CL imager (Li-cor, Lincoln, NE). Image Studio software (Li-cor, Lincoln, NE) was used to quantify the bands for 160kDa phosphorylated-TIE 2 and total TIE 2. Data were analyzed using Prism Software (GraphPad Software, San Diego, Calif.) to calculate IC₅₀The value is obtained. The compounds of formula I disclosed herein exhibit the following IC₅₀The value: 3.6nM for R849W TIE2, 0.58nM for L914F TIE2, 0.41nM for R1099 TIE2, 1.2nM for Y897C/R915C TIE2, and 0.35nM for Y897F/R915L TIE2 (fig. 1A-E).

EXAMPLE 4 cellular inhibition of TIE2 mutant by Compounds of formula I in human umbilical vein endothelial cells

Human Umbilical Vein Endothelial Cell (HUVEC) culture

HUVEC for in vivo procedures was provided by Lauri Eklund, doctor Oulu, Finland, Hewlett packard. Briefly, cells were grown in endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) containing 10% fetal bovine serum (Sigma-Aldrich, Diegem, Belgium) at 37 ℃ with 5% CO₂And incubation at 95% humidity. Cells were allowed to expand until 90-95% of the population was reached, at which time they were sub-cultured or harvested for analysis.

Western blotting of HUVEC transfected with mutant TIE2

HUVEC cells (2.5X 10) stably expressing WT or TIE2 mutants R849W, L914F, R1099, Y897C/R915C, Y897F/F915L or T1105N/T1106P⁵Cell/well) was added to 2mL of endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) containing 10% fetal bovine serum (Sigma-Aldrich, diegram, Belgium) in 6-well plates coated with a patch factor solution (Tebu-Bio, Boechout, Belgium). The cells were then incubated at 37 ℃ with 5% CO₂And incubated overnight at 95% humidity. The next day test compounds or DMSO were added to each well (0.068% final DMSO concentration). The plates were then incubated at 37 ℃ with 5% CO₂And incubation at 95% humidity for 4 hours. Next, cells were stimulated with 1. mu.g/mL ANGPT1 for 15 minutes. Cells were lysed and assayed for pTIE2, TIE2, β -actin, pAKT (S473), pAKT (T308), AKT, pSTAT1 and STAT1 in Western blotch. Compounds of formula I disclosed herein exhibited complete inhibition of TIE2 phosphorylation in HUVECs with WT TIE2 and TIE2 mutants at 100nM both with and without ANGPT1 stimulation (figure 2). Compounds of formula I disclosed herein exhibited complete inhibition of AKT Ser473 and Thr308 phosphorylation downstream of TIE2 at 100nM concentration in HUVECs with WT TIE2 and TIE2 mutants, with or without stimulation by ANGPT1 (figure 3). Compounds of formula I disclosed herein exhibited complete inhibition of STAT1 phosphorylation downstream of TIE2 in HUVECs with WT TIE2 and TIE2 mutants at 100nM concentration, with or without stimulation by ANGPT1 (figure 4).

Example 5 restoration of cellular morphology of Compounds of formula I in TIE2 mutant human umbilical vein endothelial cells

Morphometry of mutant TIE2 transfected HUVEC cells

HUVEC cells stably expressing WT or TIE2 mutants R849W, L914F, R1099, Y897C/R915C, Y897F/F915L or T1105N/T1106P (5X 10)⁵Individual cells/well) was added to 6mL of endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) containing 10% fetal bovine serum (Sigma-Aldrich, Diegem, Belgium) in 10 cm well plates covered with a patch solution (Tebu-Bio, Boechout, Belgium). The cells were then incubated at 37 ℃ with 5% CO₂And incubation at 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 at 37 ℃ with 5% CO₂And an additional 48 hours incubation at 95% humidity. Cells were imaged by microscopy 24 and 48 hours after compound addition. The compound of formula I disclosed herein restored cell morphology in TIE2 mutant HUVEC cells to be comparable to HUVEC cells expressing WT TIE2 at a concentration of 100 nM. FIG. 5A shows HUVEC cells expressing WT TIE2 or L914F TIE2Cell morphology after 48 hours of untreated, DMSO control, or 100nM compound treatment. FIG. 5B shows cell morphology of HUVEC cells expressing R849W TIE2 after 48 hours of untreated, DMSO control, or 100nM compound treatment. Fig. 5C shows cell morphology of HUVEC cells expressing R1099 × TIE2 after 48 hours of untreated, DMSO control, or 100nM compound treatment. FIG. 5D shows cell morphology of HUVEC cells expressing Y897C/R915C TIE2 after 48 hours of untreated, DMSO control treatment, or treatment with 100nM compound. FIG. 5E shows cell morphology of HUVEC cells expressing Y897C/R915L TIE2 after 48 hours of untreated, DMSO control treatment, or treatment with 100nM compound. FIG. 5F shows cell morphology of HUVEC cells expressing T1105N/T1106P TIE2 after 48 hours of untreated, DMSO control treatment, or treatment with 100nM compound.

Example 6 Effect of Compounds of formula I on RNA expression in TIE2 mutant human umbilical vein endothelial cells

Gene expression assay for mutant TIE2 transfection

HUVEC cells (5X 10) stably expressing WT or TIE2 mutants R849W, L914F, R1099, Y897C/R915C, Y897F/F915L or T1105N/T1106P⁵Individual cells/well) was added to endothelial cell growth medium (Tebu-Bio, Boechout, Belgium) containing 10% fetal bovine serum (Sigma-Aldrich, diegom, Belgium) in 10 cm well plates coated with a patch solution (Tebu-Bio, Boechout, Belgium). The cells were then incubated at 37 ℃ with 5% CO₂And incubation at 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 at 37 ℃ with 5% CO₂And an additional 48 hours incubation at 95% humidity. RNA was extracted by collecting cells into TriPure isolation reagent (Sigma-Aldrich, Diegem, Belgium). Subsequently, cDNA was 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 LightCycler480 SYBRGreen premix and LightCycler480 II instrument (Roche, Switzerland). Quantitation of cDNA for ANGPT2, PDGFB, ADAMTS1, ADAMTS9, PLAT, and PLAUAnd normalized to the expression of the housekeeping gene GAPDH. The compound of formula I disclosed herein, at a concentration of 100nM, caused an increase in the expression of ANGPT2 RNA encoding TIE2 ligand, and was aberrantly down-regulated in TIE2 mutant cells. The compound of formula I disclosed herein also caused increased expression of PDGFB RNA encoding PDGFRB ligands at concentrations of 100nM and was abnormally down-regulated in TIE2 mutant cells. The compounds of formula I disclosed herein also caused a decrease in the expression of ADAMTS1 and ADAMTS9 RNA encoding extracellular metalloproteinases at 100nM, which were abnormally upregulated in cells transfected with mutant TIE 2. The compound of formula I disclosed herein also caused a decrease in the expression of PLAT and PLAU RNA encoding plasminogen activator at a concentration of 100nM, which is abnormally upregulated in cells transfected with mutant TIE 2. Fig. 6A shows ANGPT2 and PDGFB RNA expression in HUVEC cells expressing WT TIE2, L914F TIE2, R849W TIE2, and R1099 TIE 2. FIG. 6B shows the ANGPT2 and PDGFB RNA expression of HUVEC cells expressing WT TIE2, L914F TIE2, Y897C/R915C TIE2, Y897C/R915L TIE2, and T1105N/T1106P TIE 2. FIG. 6C shows ADAMTS1 and ADAMTS9 RNA expression of HUVEC cells expressing WT TIE2, L914F TIE2, R849W TIE2, and R1099 TIE 2. FIG. 6D shows ADAMTS1 and ADAMTS9 RNA expression of HUVEC cells expressing WT TIE2, L914F TIE2, Y897C/R915C TIE2, Y897C/R915L TIE2, and T1105N/T1106P TIE 2. FIG. 6E shows the PLAT and PLAU RNA expression of HUVEC cells expressing WT TIE2, L914F TIE2, R849W TIE2, and R1099 TIE 2. FIG. 6F shows the PLAT and PLAU RNA expression of HUVEC cells expressing WT TIE2, L914F TIE2, Y897C/R915C TIE2, Y897C/R915L TIE2, and T1105N/T1106P TIE 2.

Example 6 restoration of extracellular fibronectin by Compounds of formula I in TIE2 mutant human umbilical vein endothelial cells

Fibronectin assay of mutant TIE2 transfection

HUVEC cells stably expressing WT or TIE2 mutants R849W, L914F, R1099, Y897C/R915C, Y897F/F915L or T1105N/T1106P (3X 10)⁵Individual cells/well) were added to endothelial cell raw cells containing 10% fetal bovine serum (Sigma-Aldrich, diegom, Belgium) in 6-well plates coated with a patch solution (Tebu-Bio, Boechout, Belgium)In long medium (Tebu-Bio, Boechout, Belgium). The cells were then incubated at 37 ℃ with 5% CO₂And incubation at 95% humidity for 24 hours. Next, test compounds or DMSO were added to each well (0.068% final DMSO concentration). The well plates were then incubated at 37 ℃ with 5% CO₂And an additional 48 hours incubation at 95% humidity. Cells were switched to 2% FBS overnight. The intracellular fibronectin content in the transfectants was low, and therefore cell lysates were collected as a control. For cell residues, the well plate was washed with 1X PBS containing 0.05% Triton-X and 50nM NH4OH, followed by 50mM 1X PBS followed by three washes with 1X PBS; then, the resulting mixture was dissolved in a dissolution buffer (9.1mM Na) containing 6.5M urea₂HPO₄、1.7mM NaH₂PO₄1% NP-40, 0.25% sodium deoxycholate, 150mM NaCl, 0.1% SDS, 1mM EDTA). As shown in FIG. 7, a lower fibronectin content was seen in the extracellular matrix of the TIE2 mutant than in the WT extract (upper spots) in the case of DMSO treatment. The fibronectin content of the cell lysate was comparable to that of the control group. The compound of formula I disclosed herein restored fibronectin levels in the extracellular matrix of HUVEC cells expressing the TIE2 mutant at a concentration of 100nM, resulting in similar levels to cells expressing WT TIE2 (FIG. 7).

Example 7 inhibition of growth of mutant TIE2 human umbilical vein endothelial cells in vivo in a model of venous malformation by Compounds of formula I

Venous malformation mouse model evaluation

For the in vivo model, lentivirus infection was used to make new transfectants. Briefly, 2x10⁶A10 cm dish was plated with HEK293 cells for 24 hours, trypsinized, and then incubated with a mixture containing pGAG-Pol, pRSV-Rev, pMD2.VSVG, lentiviral vector pTM945, 2.5. mu.g of pTIE2-L914F, CaCl2(Merck, UK), and 2 XHBS (Thermo Fischer Scientific, Merelbeke, Belgium) for 20 minutes. 1.5ml of cells were plated in 24-well plates and incubated for 48 hours. Subsequently, 25,000 HUVECs per well (LGC Standards sarl, France) were grown for 24 hours on 24-well plates, infected with 500. mu.l of lentivirus-TIE 2-L914F, and inoculatedFollowed by an additional incubation period of 48 hours, collection, and subsequent freezing.

To assess the in vivo progression of this compound to Vascular Malformation (VM) lesions, HUVEC cells expressing TIE2-L914F (2.5X 10)⁶) Incubated, desorbed, and then resuspended in 200 μ L matrigel (Corning). The mixture was injected subcutaneously into the dorsal back 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 infused with an equivalent concentration of compound I of 10 mg/kg; once introduced, mice were allowed free diet until the point at which matrix plugs were collected on day 7 or 16 after implantation (fig. 8A). On day 7 or 16 after the day of implantation, mice were euthanized and the skin surrounding each matrix plug was cut open, then laid flat on polystyrene foam plates and fixed in 10% neutral formalin buffer (Sigma-Aldrich, Diegem, Belgium) overnight. The matrigel plug was removed from rat skin, dehydrated in a series of graded ethanol, and embedded in paraffin. The matrigel plugs embedded in paraffin were then cut into 5 μm sections for histological analysis. After deparaffinization in xylene and rehydration in a series of descending ethanol, sections were subjected to heat-induced antigen retrieval in 0.1M citrate buffer (pH 6.0) with or without 0.05% Tween-20. For immunohistochemical staining, sections were blocked with 3% H2O2(Sigma-Aldrich, diegel, Belgium), incubated with biotinylated antibody against human endothelial cell marker vitellogenin (Ulex Europaeus Agglutinin)1(UEA1) (Vector labs, Brussels, Belgium), followed by incubation with horseradish peroxidase conjugated ovalbumin secondary antibody (GE Healthcare, diegel, Belgium), followed by incubation in DAB solution (Vector labs, Brussels, Belgium). Sections were counterstained with hematoxylin and mounted in VectaMount permanent mounting medium (Vector labs, Brussels, Belgium). For immunofluorescent staining (IF), sections were incubated with primary antibodies recognizing UEA1, smooth muscle layer marker SMA (clone 1a4, Sigma-Aldrich, Diegem, Belgium), phosphorylated-TIE 2(Y772) (Bioke, Leiden, Netherlands), or total TIE2(Santa Cruz, Heidelberg, Germany). Secondary antibodies used were conjugated with Alexa488-, Alexa 649-or CY 5-fluorophore conjugates. Slides were mounted with VectaShield Hardset mounting media with DAPI (Vector labs, Brussels, Belgium). Images were obtained via a Panoramic 250Flash III digital slide scanner (3D Histech, Hungary) and visualized with Caseviewer version 2.2 software (3D Histech, Hungary). The mean vessel area was quantified by taking snapshots of at least 5 regions of each matrix plug under 20x objective and measuring the area of at least 6 UEA + vessels separately using ImageJ software.

In fig. 8B, a macroscopic view of the collected lesions shows that hyperemic venous pathways were established at day 7 post-implantation (D7) and developed more severely at day 16 in mice fed the normal or control diet. Some mice fed diets containing compounds of formula I did not develop any lesions within the matrix plug; the hyperemic vascular channels that have indeed formed appear to be more controlled and less severe than the untreated mutants. Figure 9 shows UEA1 staining of matrix plugs from mice given control diet 7 days post-implantation represented enlarged venous channels and disordered Endothelial Cell (EC) plaques in the images. However, in the plug of stroma from mice treated with the compound of formula I, the dilated vessels were less severe. There is evidence that the UEA1+ EC cluster has higher mobility than in the control. There are also several non-vascularized UEA1+ cells. In addition, the mean area of blood vessels within the matrix plug from mice treated with the compound of formula I was much lower than in mice fed the control diet.

One of the hallmarks of VM lesions is poor and inconsistent smooth muscle cell/outer coating cell coverage of the dilated venous channels. In fig. 10, IF staining of lesions in mice fed the control diet showed no or few positive SMA cells surrounding the vessels. Mice fed the compound of formula I showed some SMA positive cells but not all cells surrounding the EC layer. EC from all vessels of mice fed the control and formula I compound diets strongly showed total TIE 2; likewise, all ECs in mice fed the control diet appeared to exhibit phosphorylated TIE2(pTIE2), as visualized in fig. 11. pTIE2 appeared to perform poorly in ECs of mice fed a diet of the compound of formula I.

By day 16 post-implantation, the lesions within matrigel had been severely dilated, although some variability was seen, probably more due to technical/procedural problems (e.g. heterogeneous mixture of cells and matrigel) (fig. 12). Mice fed the control diet developed lesions comparable to untreated mice, but mice fed the formula I compound diet showed much smaller blood vessels. In mice untreated and fed the control diet, the day 16 cultures developed enlarged vascular channels and the vessels were sparsely surrounded by a smooth muscle layer and a small number of SMA + cells. In contrast, blood vessels appeared to normalize and were more uniformly surrounded by SMA + cells in the compound fed formula I (fig. 13). Fig. 14 shows that EC in untreated mice strongly expresses pTIE 2. However, pTIE2 was greatly reduced in mice fed the compound of formula I.

To assess the overall morphology in the explants, full-angle embedding of IF in stromal plugs was performed. After the skin with attached matrigel plugs was cut, it was fixed overnight in 10% neutral formalin buffer, approximately 100-200 μm matrigel plugs were sliced and washed in 1 × PBS. Sections were blocked with 1% BSA (v/v) (Gibco) in 1 XPBS solution with 0.3% Triton-X100(Sigma-Aldrich, Diegem, Belgium), followed by incubation in UEA1 for at least 72 hours, followed by incubation in CY 5-ovalbumin secondary antibody (Vector labs, Brussels, Belgium). The sections were then post-fixed in 10% NBF for 10 minutes, followed by embedding with fluorescent fixative g (fluoro mount g) (Thermo Fischer Scientific, merebeke, Belgium). A Z-axis stack image was obtained with a Zeiss cell observation rotating disc type conjugate focus microscope (Zeiss Co., Germany); the 3D projection of the Z-axis stack imagery was generated by the AriVis4D software. Figure 15 shows that the blood vessels formed within the matrix plug were abnormally elongated and disorganized on both days 7 and 16 post-implantation in mice fed the control diet. On day 7. Dilated and clustered blood vessels were seen in the cultures harvested from mice fed compounds of formula I; however, a more normal and tubular vessel is seen. A number of non-vascularized UEA + cells are also seen; note that this is the same pattern that TIE2-WT cells exhibited upon injection. On day 16, the vessels appeared to normalize to a large extent, with a uniform tubular structure. Non-vascularized UEA + cells are also present but show reduced expression. Treated and untreated VM lesions with wild type and L914F TIE2 mutant were also compared on day 16, with treatment beginning on day 0 (fig. 18A-D) or day 7 (fig. 19A-C). This data supports that feeding mice with VM with food infused with a compound of formula I results in a reduction in the development of VM, and supports that feeding a compound of formula I to mice with established VM reduces the severity of VM.

To assess the effect of the compounds of formula I on previously established VM lesions, VM mice were generated following the same approach. However, mice were instead introduced with the control or formula I compound diet at day 7 post-implantation; next, the mice were euthanized and the explants were harvested on day 16 (fig. 16A). Congested vascular channels developed throughout the experimental population, although varying in severity (fig. 16B). The mean vascular area in the explants from day 16 of mice fed the control diet was comparable to that of the control mice, as shown in fig. 17A-B. The mean vascular area from mice fed a diet of compound of formula I was moderately reduced. Lesions from mice fed a diet of compound of formula I exhibited improved smooth muscle cell layer coverage, comparable to mice fed a control diet, indicating enhanced stability of the coat cells and vascular maturation. Fig. 19A-D also depict a partial comparison of treated and untreated VM lesions with wild type and L914F TIE2 mutant at day 16, where treatment began at day 7 post-implantation. This data supports that feeding a compound of formula I to mice with established VM reduces the severity of VM.

Equivalents of the formula

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 encompassed within the scope of the following claims.

Claims (39)

1. A method of treating a TIE2 kinase-mediated vascular abnormality or a TIE2 kinase mutant-mediated 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.

2. The method of claim 1, wherein the pharmaceutically acceptable salt is a tosylate salt.

3. The method of claim 1, wherein the TIE2 kinase-mediated vascular abnormality or TIE2 kinase mutant-mediated vascular abnormality is a chronic blood flow malformation.

4. The method of claim 3, wherein the chronic blood flow deformity is selected from a microvascular deformity, a lymphatic malformation, or a venous malformation.

5. The method of claim 4, wherein the chronic-flow-type abnormality is a venous abnormality.

6. The method of claim 1, comprising administering to the patient a compound of formula I once daily, intermittently not once daily, once every other day, once every other week, twice daily, once weekly, or twice weekly.

7. The method of claim 1, comprising administering to the patient about 57mg to about 1200mg of the compound of formula I per day.

8. The method of claim 1, comprising administering to the patient about 100mg of the compound of formula I per day.

9. The method of claim 1, comprising administering to the patient about 150mg, 200mg, or 300mg of the compound of formula I once or twice daily.

10. A method of 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;

wherein the vascular abnormality is mediated by TIE2 kinase or by a TIE2 kinase mutant.

11. The method of claim 1 or 10, further comprising administering a second therapeutic agent to the patient.

12. The method of claim 11, wherein the second therapeutic agent is a VEGF inhibitor.

13. The method of claim 12, wherein the VEGF inhibitor is selected from the group consisting of pazopanib (pazopanib), bevacizumab (bevacizumab), cabozantinib (cabozantinib), sunitinib (sunitinib), sorafenib (sorafenib), axitinib (axitinib), regrafenib (regorafenib), panatinib (ponatinib), cabozantinib (cabozantinib), vandetanib (vandetanib), ramucirumab (ramucirumab), lenvatinib (lenvatinib), bevacizumab (bevacizumab), and ziv-aflibercept (ziv-aflibercept).

14. The method of claim 11, wherein the second therapeutic agent is an Akt inhibitor.

15. The method of claim 14, wherein the Akt inhibitor is selected from AZD5363, miltefosine (miltefosine), perifosine (perifosine), VQD-002, MK-2206, GSK690693, GDC-0068, triciribine (triciribine), CCT128930, PHT-427, and magnolol (honokiol).

16. The method of claim 11, wherein the second therapeutic agent is an mTOR inhibitor.

17. The method of claim 16, wherein the mTOR inhibitor is selected from sirolimus (sirolimus), temsirolimus (temsirolimus), everolimus (everolimus), AP23841, AZD8055, BEZ235, BGT226, discolimus (deferolimus) (AP23573/MK-8669), EM101/LY303511, EX2044, EX3855, EX7518, GDC0980, INK-128, KU-0063794, NV-128, OSI-027, PF-4691502, rapamycin analogs (rapalogs), rapamycin (rapamycin), ridaforolimus (ridaforolimus), SAR543, SF1126, WYE-125132, XL765, zotarolimus (zotarolimus) (ABT578), tollin 1 (tormin 1), GSK2126458, gdd 2014, c-03388, and XL 49.

18. The method of claim 11, wherein the second therapeutic agent is a PI3K inhibitor.

19. The method of claim 18, wherein the PI3K inhibitor is selected from idecoxib (idelalisib), copaxib (copanlisib), dovexib (duvelisib), capexib (alpelisib), NVP-BEZ235, BKM-120, GDC-0941, GDC-0980, SF1126, PX-866, PF-04691502, XL-765, XL-147, GSK2126458, and ZSTK 474.

20. A compound of formula I or a pharmaceutically acceptable salt thereof

For use in treating a TIE2 kinase-mediated vascular abnormality or a TIE2 kinase mutant-mediated 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.

21. The use of a compound of claim 20, wherein the pharmaceutically acceptable salt is a tosylate salt.

22. The use of a compound according to claim 20, wherein the TIE2 kinase-mediated vascular abnormality or TIE2 kinase mutant-mediated vascular abnormality is a chronic blood flow malformation.

23. The compound for use of claim 22, wherein the lentiviral malformation is selected from the group consisting of microvascular, lymphatic or venous malformations.

24. The compound for use according to claim 23, wherein the chronic blood flow malformation is a venous malformation.

25. The use of a compound of claim 20, wherein the patient is administered the compound once a day, intermittently not once a day, once every other week, twice a day, once a week, or twice a week.

26. The compound for use of claim 20, wherein about 57mg to about 1200mg of the compound of formula I is administered to the patient per day.

27. The compound for use according to claim 20, wherein about 100mg of the compound of formula I is administered to the patient per day.

28. The compound for use of claim 20, wherein about 150, 200, or 300mg of the compound of formula I is administered to the patient once or twice daily.

29. The use of a compound of claim 20, further comprising administering a second therapeutic agent to the patient.

30. The compound for use of claim 29, wherein the second therapeutic agent is a VEGF inhibitor.

31. The use of a compound of claim 30, wherein the VEGF inhibitor is selected from the group consisting of pazopanib (pazopanib), bevacizumab (bevacizumab), cabozantinib (cabozantinib), sunitinib (sunitinib), sorafenib (sorafenib), axinib (axitinib), regorafenib (regorafenib), panatinib (ponatinib), cabozantinib (cabozantinib), vandetanib (vandetanib), ramucirumab (ramucimab), lenvatinib (lenvatinib), bevacizumab (bevacizumab), and ziv-aflibercept (ziv-aflibercept).

32. The use of a compound of claim 29, wherein the second therapeutic agent is an Akt inhibitor.

33. The use of a compound as claimed in claim 32, wherein the Akt inhibitor is selected from AZD5363, miltefosine (miltefosine), piperacillin (perifosine), VQD-002, MK-2206, GSK690693, GDC-0068, triciribine (triciribine), CCT128930, PHT-427 and magnolol (honokiol).

34. The compound for use of claim 29, wherein the second therapeutic agent is an mTOR inhibitor.

35. The use of a compound as claimed in claim 34, wherein the mTOR inhibitor is selected from sirolimus (sirolimus), temsirolimus (temsirolimus), everolimus (everolimus), AP23841, AZD8055, BEZ235, BGT226, discolimus (deferolimus) (AP23573/MK-8669), EM101/LY303511, EX2044, EX3855, EX7518, GDC0980, INK-128, KU-0063794, NV-128, OSI-027, PF-4691502, rapamycin analogue (rapalogs), rapamycin (rapamycin), ridaforolimus (ridaforolimus), SAR543, SF1126, WYE-125132, XL765, zotarolimus (ABT), tollin 1 (torlin 1), GSK2126458, AZD 0349, GDC-03201449 and 2014388.

36. The compound for use according to claim 32, wherein the second therapeutic agent is a PI3K inhibitor.

37. The use of a compound as claimed in claim 36, wherein the PI3K inhibitor is selected from idenexib (idelalisib), copaxib (copanlisib), dovexib (duvelisib), apexib (apelisib), NVP-BEZ235, BKM-120, GDC-0941, GDC-0980, SF1126, PX-866, PF-04691502, XL-765, XL-147, GSK2126458 and ZSTK 474.

38. A method of treating venous malformations in a patient in need thereof, comprising administering to the patient once or twice daily from about 100mg to about 200mg of a compound of formula I:

or a pharmaceutically acceptable salt thereof.

39. A compound of formula I, or a pharmaceutically acceptable salt thereof, for use in treating venous malformations in a patient in need thereof

Comprising administering to the patient about 100mg to about 200mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, once or twice daily.

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