WO2018175301A1

WO2018175301A1 - Polymer conjugate of montesanib with reduced exposure

Info

Publication number: WO2018175301A1
Application number: PCT/US2018/023108
Authority: WO
Inventors: Silvio Traversa; Valentina Mainero; Todd James Harris; Luisa Bertarione RAVA ROSSA
Original assignee: Sienna Biopharmaceuticals, Inc.
Priority date: 2017-03-20
Filing date: 2018-03-19
Publication date: 2018-09-27

Abstract

Disclosed herein are polymer conjugates, such as SNA-103, comprising an active agent linked to a polymer, wherein the active agent comprises an inhibitor, antagonist, or inverse agonist of a vascular endothelial growth factor receptor (VEGFR), such as montesanib. The disclosed polymer conjugates reduce exposure of the active agent at non-target sites.

Description

POLYMER CONJUGATE OF MONTESANIB WITH REDUCED EXPOSURE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US provisional patent application Serial No. 62/473,982 filed March 20, 2017, and claims priority to US provisional patent application Serial No. 62/501 ,568 filed May 4, 2017, and claims priority to US provisional patent application Serial No. 62/590, 1 19 filed November 22, 2017. Each of these applications is incorporated by reference in their entirety herein.

FIELD

[0002] Disclosed herein are polymer conjugates, comprising active agents linked to polymers, and therapeutic uses thereof. More particularly, a polymer conjugate of montesanib, which exhibits reduced exposure to non-target sites, and inhibits Vascular Endothelial Growth Factor Receptors (VEGFR) mediators of various pathological conditions is described.

BACKGROUND

[0003] Inhibitors of (VEGFR) , such as montesanib, have been described for possible therapeutic use in the prevention, alleviation and treatment of kinase-associated pathologies. However, such compounds are associated with broad specificity, as well as undesirable and toxic effects. Accordingly, strategies to render these VEGFR inhibitors more specific and less toxic are needed.

SUMMARY OF EMBODIMENTS

[0004] In several embodiments, a polymer conjugate (such as SNA-103) is provided having the following structure:

[0005] Effective delivery of pharmacologically active agents may be hindered by unwanted exposure of those agents to non-desired locations (such as the systemic circulation and/or lymphatic system). For example, topical agents useful in treating various skin disorders may result in toxic side effects because of systemic exposure. One issue with delivering compositions comprising one or more active agents topically (or non-topically) is the concern that such agents need to be delivered in an amount and at a location sufficient to have a therapeutic effect. At the same time however, exposure (e.g., absorption or longevity of the composition in the systemic circulation, lymphatic system, or other non-targeted sites) may not be desirable for multiple reasons, including, but not limited to, safety reasons. There remains an unmet need for compounds with reduced exposure at non-target sites that result in a clinically therapeutic effect.

[0006] In several embodiments of the invention, the compositions described herein are both therapeutically efficacious and minimize non-target (e.g., systemic or bloodstream) exposure. In some embodiments, the active agents are PEGylated or otherwise coupled to large molecules, and surprisingly, are effective in crossing biological membranes such that the active agents are effectively delivered to the target location. Although inflammatory skin conditions are disclosed in several embodiments, other embodiments are used to treat non-dermal inflammation, as well as other several conditions (e.g., those conditions that would benefit from treatment with reduced exposure at non-target sites). For example, in some embodiments, the compositions and technology described herein are used in the gastrointestinal and pulmonary systems. Ophthalmic treatments are provided in some embodiments. In yet other embodiments, compositions for treating joints are provided. Treatment of the nose and ear are provided in other embodiments. Inflammatory and non-inflammatory conditions are contemplated herein.

[0007] Reduced exposure compounds and compositions are provided in several embodiments. "Reduced exposure" compounds are those compounds that, when delivered to a target location, are formulated to act at the target location with reduced exposure (e.g., entry and/or longevity) in non-target sites. Exposure is reduced as compared to active agents not formulated according to the embodiments described herein. As a non-limiting example, a PEGylated topical dermal active agent has reduced exposure to the bloodstream as compared to the active agent alone. Reduced exposure compounds include topical compounds that can be delivered to body surfaces and cavities such as the skin, eyes, ears, nose, mouth, vagina, rectum, etc., as well as oral (e.g., enteric coated) compounds for oral delivery that treat the gastrointestinal system (e.g., the Gl lining), inhalants that treat the lungs, injections for joints, and other modes of delivery that target one location with the goal of reducing exposure to a non-desired site. Non-desired target sites include, for example, the systemic system, the lymphatic system, non-target tissue, etc. "Reduced exposure compositions" comprise or consist essentially of one or more "reduced exposure compounds."

[0008] Reduced exposure topical compositions are provided in many embodiments. In some embodiments, a reduced exposure composition is delivered orally, e.g., for treatment of the gastrointestinal system. The active agent remains in the lining of the gastrointestinal tract and is able to achieve pharmacological specificity. Because the active agent is conjugated with PEG or another molecule as described herein, the active agent is absorbed more slowly into the non-target site (e.g., the systemic circulation and/or lymphatic system). In some cases, less or none of the active agent is absorbed into the non-target site (e.g., systemic circulation and/or lymphatic system). Further, once the composition enters the systemic circulation and/or lymphatic system, clearance (e.g., by the kidney) occurs at a much faster rate. One or more of the advantages of (i) reduced absorption into the non-target site (e.g., systemic circulation and/or lymphatic system), (ii) slower absorption into the non-target site (e.g., systemic circulation and/or lymphatic system), and (iii) faster clearance rates from the non-target site (e.g., systemic circulation and/or lymphatic system) are also achieved when using the compositions (formulated according to the methods described herein) for treating the eye (e.g., via eye drops), the lungs (e.g., via inhalants), the skin (e.g., via dermal topicals), joints (e.g., via injectables), nasal passageways, and the ear (such as the ear canal and other structures). Vaginal and rectal tissues are treated in some embodiments via, for example suppositories.

[0009] In several embodiments, there is provided in a reduced exposure composition, a polymer conjugate comprising a warhead (e.g., at least one active agent) linked to a polymer, wherein the warhead comprises an inhibitor, antagonist, or inverse agonist of, for example, a vascular endothelial growth factor receptor (VEGFR). In some embodiments, at least one inhibitor, antagonist, or inverse agonist of a vascular endothelial growth factor receptor (VEGFR) comprises or consists of a composition that includes any one of compounds 1 -59 (and derivatives thereof) disclosed herein in Table 1 coupled to a polymer. In some embodiments, the warhead of the polymer conjugate is compound 1 (montesanib). In some embodiments, the polymer conjugate is CT103, wherein the composition has the formula:

[0010] Non-dermal (non-skin) inflammation or other conditions may also be treated in some embodiments with compositions comprising these compounds. Noninflammatory conditions may also be treated with some embodiments.

[0011] As described above, several embodiments disclosed herein provide reduced or minimized exposure (e.g., entry into and/or longevity in a non-target site such as the systemic circulation and/or lymphatic system). In some embodiments, exposure at a non-target site is less than 90%, 75%, 50%, 25%, 15%, 10%, 5% or 2% (or less) of the polymer conjugate as compared to a similar active entity that has not been produced according to the embodiments described herein. In some embodiments, desirable rate of clearance from the non-target site (e.g., systemic circulation and/or lymphatic system) for the compositions described herein is increased by at least 10%, 25%, 50%, or 75% or more as compared to non-conjugated controls. As an example, a PEGylated active agent described herein not only penetrates the desired membranes to reach a desired target, but has reduced non-target exposure by at least 20-80% or more as compared to the non-PEGylated active agent. In some embodiments, blood concentrations measured post administration of the compositions described herein are less than about 0.1 ng/ml, less than 1 ng/ml, or less than 10 ng/ml after, e.g., 15 minutes, 30 minutes, 1 hour, 6 hours or 12 hours.

[0012] In some embodiments, reduced exposure at non-target sites contributes to enhanced efficacy. Efficacy may be enhanced because lower concentrations/amounts/dosing schedules are required to achieve the same or similar therapeutic efficacy at the target site (because, for example, the active ingredient stays at the desired target site for a longer time). In one embodiment, concentrations/amounts/dosing schedules are reduced by 25%-75% or more.

[0013] More rapid clearance rates of the active agent once in the non-target site(s) (such as systemic circulation and/or lymphatic system) are also beneficial because this may allow for a higher concentration or more doses to be delivered. This is especially beneficial for active agents in which a subject would benefit from a higher dose but cannot tolerate the higher dose due to toxicity at the non-target site (e.g. , systemic toxicity) . Faster clearance rates would permit the desired higher dose to be delivered according to the desired schedule. For example, a subject may be able to tolerate daily doses rather than weekly doses because of the reduced exposure.

[0014] In some embodiments, the active agents of the compositions described herein (e.g. , the compounds in Table 1 conjugated e.g. , with PEG or other polymers) are measured in non-target sites (e.g. , the systemic circulation and/or lymphatic system) at less than amounts found when the active agent is delivered without conjugation (e.g. , less than 0.5% , 1 % or 2% after 6 or 12 hours, as compared with 3- 15% (e.g. , 3-6%) when the active agent is delivered without conjugation) . In some embodiments, the active agents of the compositions described herein (e.g. , the compounds in Table 1 conjugated e.g. , with PEG or other polymers) are measured in non-target sites (e.g. , the systemic circulation and/or lymphatic system) at less than 0.5%, 1 % or 2% after 3-24 hours, as compared to an amount 2-20 times greater when the active agent is delivered without conjugation.

[0015] In some embodiments, clearance of the compositions (e.g. , the conjugated polymer compounds) occurs within minutes of exposure to the non-target site (e.g. , systemic circulation and/or lymphatic system), as opposed to hours. In other embodiments, 50% clearance of the conjugated polymer compounds occurs in less than 5 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, and 12 hours of exposure to the systemic circulation and/or lymphatic system. Clearance times of the conjugated polymer compounds are reduced by more than 25%, 50%, 75% and 90%, as compared to the non-conjugated active agents or other formulations. These reduced clearance times are beneficial to reduce toxicity and undesired side effects.

[0016] In some embodiments, an active agent may be increasingly toxic as it is metabolized in the non-target site (e.g. , systemic circulation and/or lymphatic system) because the metabolites exhibit more toxicity than the original agent. Thus, faster clearance rates, in some cases even before the toxic metabolites are created, are especially beneficial.

[0017] The term "active entity" as used herein should not be understood as limiting the participation of the polymer itself and/or the chemical linking moiety between the polymer and the warhead in defining the pharmacology of the polymer conjugate. In some embodiments, the polymer influences the selectivity and/or inhibitory activity of the polymer conjugate. In some embodiments, the chemical linking moiety between the polymer and warhead influences the selectivity and/or inhibitory activity of the polymer conjugate. In some embodiments, the polymer conjugates exhibit no change in selectivity or inhibitory activity against the therapeutic target in comparison with the unconjugated active agent. In some embodiments, the polymer conjugates exhibit a significant increase in selectivity against the therapeutic target in comparison with the unconjugated active agent. In some embodiments, the polymer conjugates exhibit a significant increase in inhibitory activity against the therapeutic target in comparison with the unconjugated active agent. In some embodiments, the polymer conjugates exhibit a significant increase in selectivity and inhibitory activity against the therapeutic target in comparison with the unconjugated active agent. In some embodiments, the increased selectivity and/or inhibitory activity of the polymer conjugate against the therapeutic target in comparison with the unconjugated active agent causes decrease in undesired biological effects. In some embodiments, the increased selectivity of the polymer conjugate is caused by an increase of the hydrodynamic volume resulting from the conjugated polymer chain. In some embodiments, the polymer chain creates a higher steric hindrance which allows discrimination among the diverse shapes and sizes of the binding sites of different proteins, thus improving selectivity with respect to the active agent alone.

[0018] In several embodiments, various inflammatory skin diseases are treated. The inflammatory skin disease comprises, in some embodiments, psoriasis, psoriasis guttata, inverse psoriasis, pustular psoriasis, psoriatic erythroderma, acute febrile neutrophilic dermatosis, eczema, xerotic eczema, dyshidrotic eczema, vesicular palmar eczema, acne vulgaris, atopic dermatitis, contact dermatitis, allergic contact dermatitis, dermatomyositis, exfoliative dermatitis, hand eczema, pompholyx, keloids, rosacea, rosacea due to sarcoidosis, rosacea due to scleroderma, rosacea due to Sweet syndrome, rosacea due to systemic lupus erythematosus, rosacea due to urticaria, rosacea due to herpetic pain, Sweet's disease, neutrophilic hydradenitis, sterile pustule, drug rash, seborrheic dermatitis, pityriasis rosea, Kikuchi's disease of the skin, pruritic urticarial papules and plaques of pregnancy, Stevens-Johnson syndrome and toxic epidermal necrolysis, tattoo reaction, Wells syndrome (eosinophilic cellulitis), reactive arthritis (Reiter syndrome), bowel-associated dermatosis-arthritis syndrome, rheumatoid neutrophilic dermatosis, neutrophilic eccrine hidradenitis, neutrophilic skin disease of dorsum of hand, balanitis circumscripta plasmacellularis, balanoposthitis, Behcet's disease, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiforme, granuloma annulare, dermatitis of hand, lichen nitidus, lichen planus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, nummular dermatitis, pyoderma gangrenosum, sarcoidosis, subcorneal pustular dermatosis, urticaria, and transient acantholytic dermatosis. [0019] In several embodiments, various skin neoplasias are treated. The skin neoplasia comprises, in some embodiments, squamous cell carcinoma, basal cell carcinoma, malignant melanoma, malignant cutaneous lymphoma, Kaposi's sarcoma, Merkel cell skin cancer, and non-melanoma skin cancer.

[0020] In several embodiments, various vascular tumors are treated. The vascular tumor comprises, in some embodiments, hemangiomas, Kaposi's sarcoma, lymphangioma, glomangioma, angiosarcoma, hemangioendothelioma, and infantile hemangiomas.

[0021] In several embodiments, various bullous diseases are treated. The bullous disease comprises, in some embodiments, bullous pemphigoid, erythema multiforme, dermatitis herpetiformis, epidermolysis bullosa acquisita, linear Immunoglobulin A disease, mucous membrane pemphigoid, pemphigoid gestationis, pemphigus foliaceus, and pemphigus vulgaris.

[0022] In several embodiments, age-related macular degeneration is treated. In several embodiments, diabetic retinopathy is treated. In several embodiments, corneal edema is treated. In several embodiments, macular edema is treated. In several embodiments, dry eye is treated.

[0023] In several embodiments, hair growth and cycling are modulated. In several embodiments, alopecia is treated.

[0024] In several embodiments, the polymer conjugates are administered in combination with UV irradiation therapy.

[0025] Also provided herein, in several embodiments, are polymer conjugates wherein the polymer is polyethylene glycol (PEG) or methoxy-polyethylene glycol (m- PEG). In several embodiments, there is provided a pharmaceutical composition comprising or consisting essentially of a polymer conjugate disclosed herein that is formulated for topical and non-topical administration. In several embodiments, methods of making and using the compositions described herein are provided.

[0026] In several embodiments, the invention comprises a reduced exposure composition comprising at least one active entity linked to at least one polymer, wherein the composition has reduced exposure at a non-target site as compared to the active entity delivered without the polymer. The non-target site comprises the systemic system, the lymphatic system and/or another non-target tissue site in some embodiments.

[0027] In some embodiments, the active entity comprises an inhibitor, an antagonist, or an inverse agonist. For example, the active entity may be an inhibitor, antagonist, or inverse agonist of a VEGFR. The active entity comprises or consists essentially of any one or more of compounds 1-59 in some embodiments. In some embodiments, the active entity comprises compound 1 . In some embodiments, the composition comprises CT103.

[0028] The active entity binds to a VEGFR in some embodiments. The active entity binds to VEGFR-1 in some embodiments. The active entity binds to VEGFR-2 in some embodiments. The active entity binds to VEGFR-3 in some embodiments. The binding may be partially or fully inhibitory or not.

[0029] In some embodiments, the polymer used in the reduced exposure compounds comprises polyethylene glycol (PEG) and/or methoxy-polyethylene glycol (m-PEG). In embodiments where the active entity has one or more carboxyl, hydroxyl, amino and/or sulfhydryl groups, the active entity is PEGylated (or conjugated/coupled to another polymer) at one or more of said carboxyl, hydroxyl, amino and/or sulfhydryl groups.

[0030] The reduced exposure compositions described herein are formulated for topical administration in several embodiments. Inhalants, injectables, eye drops, nasal sprays, oral administration etc. are provided in some embodiments. In several embodiments, methods of treating one or more of the following are provided: non- dermal inflammation, inflammatory skin disease, vascular tumors, skin neoplasia, bullous diseases, age-related macular degeneration, diabetic retinopathy, corneal edema, macular edema, dry eye, alopecia, wounds, scars, autoimmune disorders, and cancerous or pre-cancerous lesions. Methods for modulating hair growth and cycling are provided in some embodiments. Kits comprising one or more compounds and devices for administration (syringes, containers, inhalers, etc.), as well as instructions for use, are provided in certain embodiments.

[0031] Compositions may be administered via at least two routes of administration, either simultaneously or sequentially according to some embodiments. In one embodiment, the composition is administered via a first (e.g. topical dermal) route to a subject, wherein the subject further receives an additional agent via a second (e.g. , non-dermal) route to achieve synergetic effects.

[0032] In several embodiments, the inventions comprises methods for reducing exposure of a composition at least one non-target site, wherein the method comprises applying a composition comprising at least one active entity linked to at least one polymer, wherein the combination of the active entity and polymer reduces exposure at the non-target site by more than 50% as compared to the active entity without the polymer. The composition may be applied topically, injected, inhaled, or administered orally. The non-target site includes non-target tissue at which pharmacological activity is not desired and/or not achieved. Non-target sites can include the bloodstream or systemic system. Non-target sites can also include the lymphatic system. [0033] In several embodiments, a compound having the formula:

[0034] is provided. In some embodiments, n ranges from about 4 to about

1 140 (e.g., 4-10, 10-20, 20-40, 40-60, 60-80, 80-100, 125-150-150-175, 175-200, 200- 300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1 100, 1 100-1200, 1200-1300, and overlapping ranges therein). There is provided, in some embodiments, stereoisomers, enantiomers, and/or pharmaceutically acceptable salts of the compound.

[0035] In several embodiments, a reduced exposure composition for treating a target site is provided. In one embodiment, the composition comprises or consists essentially of a conjugate comprising or consists essentially of an active entity coupled (e.g., linked) to at least one polymer. Two, three or more active entities or two, three or more polymers may be used. In one embodiment, a pharmaceutically acceptable carrier formulated for delivering the conjugate to the target site is also provided. In several embodiments, the conjugate has reduced exposure at a non-target site as compared to the active entity delivered without the polymer. The non-target site includes for example the systemic system, the lymphatic system and/or other non-target tissue sites. In several embodiments, the non-target site comprises any site at which pharmacological activity is not desired and/or not achieved. In several embodiments, the conjugate has the formula:

[0036] In some embodiments, n ranges from about 4 to about 1 140 (e.g., 4- 10, 10-20, 20-40, 40-60, 60-80, 80-100, 125-150-150-175, 175-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1 100, 1 100-1200, 1200-1300, and overlapping ranges therein). There is provided, in some embodiments, stereoisomers, enantiomers, and/or pharmaceutically acceptable salts of the conjugate.

[0037] In several embodiments, a reduced exposure composition for treating a cell within a target site is provided. Methods for treating diseases, conditions, and disorders are also provided. In one embodiment, the composition comprises or consists essentially of a conjugate comprising or consists essentially of an active entity coupled (e.g., linked) to at least one polymer. Two, three or more active entities or two, three or more polymers may be used. The active entity may be for example, an inhibitor, antagonist, or inverse agonist of a cellular kinase. In several embodiments, the active entity is one or more of compounds 1 -59. In one embodiment, the composition comprises compound 1 . In one embodiment, the composition comprises SNA-103. The polymer can include, for example, polyethylene glycol (PEG) and/or methoxy- polyethylene glycol (m-PEG). In one embodiment, a pharmaceutically acceptable carrier formulated for delivering the conjugate to the target site is also provided. In several embodiments, the conjugate has reduced exposure at a non-target site as compared to the active entity delivered without the polymer. The non-target site includes for example the systemic system, the lymphatic system and/or other non-target tissue sites. In several embodiments, the non-target site comprises any site at which pharmacological activity is not desired and/or not achieved. In one embodiment, the conjugate can advantageously traverse plasma membranes of cells at the target site, thereby promoting interactions between the active entity and the cellular kinase This traversal may include the crossing of cellular lipid bilayers to, e.g., distribute the active entity among both lipophilic and hydrophilic cellular compartments. Membranes include the lipid bilayer, plasma membrane and the nuclear membrane as examples. In several embodiments, the conjugate interacts with a kinase associated with the plasma membrane, cytoplasm and/or nucleus. The conjugate may exhibit a depot effect across cellular compartments, thereby reducing the dose of the active entity required to inhibit the cellular kinase compared to the active entity without conjugation to the polymer.

[0038] In several embodiments, the cellular kinase may be a VEGFR. Examples of VEGFRs include, but are not limited to, VEGFR-1 , VEGFR-2, and VEGFR- 3. In some embodiments, the VEGFR is bound and/or inhibited by the active entity. In some embodiments, the active entity has one or more carboxyl, hydroxyl, amino and/or sulfhydryl groups. In one embodiment, at least one polymer is conjugated to the active entity at the one or more carboxyl, hydroxyl, amino and/or sulfhydryl groups.

[0039] In some embodiments, the reduced exposure composition may be formulated for topical, oral, local ocular (e.g., eye drop), inhalation, injection or suppository delivery. Topical, oral, injection, inhalation, local ocular, and suppository administration is provided in several embodiments. In several embodiments, the administration is daily. In the methods of treatment, effective amounts of the active entity are delivered to a subject (e.g., human or veterinary). In several embodiments, the composition may be administered via at least two routes of administration, either simultaneously or sequentially. In some embodiments, the composition is administered via a topical route to a subject, and the subject further receives an additional agent via a non-topical route. In some such embodiments, this co-administration achieves synergetic effects. The composition may further comprise one or more additional ingredients, such as, for example, a protective agent, an emollient, an astringent, a humectant, a sun screening agent, a sun tanning agent, a UV absorbing agent, an antibiotic agent, an anti- angiogenesis agent, a preventive or therapeutic agent for inflammatory bowel disease, a physiological cooling agent, an antifungal agent, an antiviral agent, an antiprotozoal agent, an anti-acne agent, an anesthetic agent, a steroidal anti-inflammatory agent, a non-steroidal anti-inflammatory agent, an antipruritic agent, an additional antioxidant agent, a chemotherapeutic agent, an anti-histamine agent, a vitamin or vitamin complex, a hormone, an anti-dandruff agent, an anti-wrinkle agent, an anti-skin atrophy agent, a skin whitening agent, and/or a cleansing agent.

[0040] In several embodiments, the active entity and/or conjugate may have a longer residence time within a cell or other tissue at the target site compared to the active entity without conjugation to the polymer. For example, the residence time of the active entity and/or conjugate within a cell or other tissue at the target site is, as compared to the active entity without conjugation to the polymer, (i) at least 25% (e.g., 25-50%, 50-75%, 75-100%, 100-150%, or higher and overlapping ranges therein) longer and/or (ii) at least 2-20 fold (e.g., 2-10 fold, 2-4 fold, 4-6 fold, 6-8 fold, 8-10 fold, 10-12 fold, 12- 14 fold, 14- 16 fold, 16-18 fold, 18-20 fold, 20-30 fold, 40-50 fold, 10-50 fold, 50- 100 fold, and overlapping ranges therein) longer. In one embodiment, the residence time is over 100 fold longer.

[0041] In some embodiments, a smaller dose of the conjugate may be needed to achieve a therapeutic effect comparable to the active entity without conjugation to the polymer. For example, in several embodiments, the dose of the conjugate needed to achieve a therapeutic effect comparable to the active entity without conjugation to the polymer is at least 10% (e.g. , 10- 15%, 15-20%, 20-25% , 25-30%, 30- 40%, 40-50% , 50-60%, 60-70%, 70-80%, 80-90%, 90- 100%, 100%- 125% , 125- 150%, or higher and overlapping ranges therein) lower. In one embodiment, the dose is over 200% lower. In some embodiments, fewer doses and/or smaller doses of the conjugate are required as compared to the active entity delivered without the polymer.

[0042] In several embodiments, the active entity and/or conjugate may have an increased concentration, activity and/or bioavailability within a cell or tissue at the target site compared to the active entity without conjugation to the polymer. In some such embodiments, the therapeutically effective amount of the active entity is at the target site. For example, the concentration, activity and/or bioavailability within a cell or other tissue at the target site is, as compared to the active entity without conjugation to the polymer, at least 2-20 fold (e.g. , 2-4 fold, 4-6 fold, 6-8 fold, 8-10 fold, 10-12 fold, 14- 16 fold, 18-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50- 100 fold, and overlapping ranges therein) greater than within a cell or tissue at a non-target site (e.g. , the systemic system, the lymphatic system, the circulatory system, bone marrow). I n one embodiment, the concentration, activity and/or bioavailability within a cell or tissue at the target site is over 100 fold greater.

[0043] In several embodiments, the active entity and/or conjugate may have reduced concentration, activity and/or bioavailability within a cell or tissue at a non-target site compared to the active entity without conjugation to the polymer. In several embodiments, the active entity and/or conjugate is present at a biologically inactive concentration within a cell or tissue at a non-target site. In several embodiments, reduced concentration, activity and/or bioavailability within a cell or tissue at a non-target site (e.g. , the systemic system, the lymphatic system, bone marrow, the circulatory system) advantageously reduces toxicity and/or other side effects, such as, for example, immunosuppression. For example, in some embodiments, the active entity and/or conjugate has reduced systemic absorption and/or little or no systemic toxicity when the composition is formulated for oral delivery and is administered orally (e.g. , a single administration, administration on a daily basis) . [0044] In several embodiments, the conjugate is amphiphilic and/or amphipathic. In some embodiments, the conjugate is more amphiphilic and/or amphipathic than the active entity without conjugation to the polymer. For example, in several embodiments, the conjugate, as compared to the active entity without conjugation to the polymer, is at least 25% (e.g. , 20-25%, 25-30%, 30-40%, 40-50%, 50- 60%, 60-70%, 70-80% , 80-90% , 90-100% , 100%-125%, 125- 150%, or higher and overlapping ranges therein) more amphiphilic. In one embodiment, the amphiphilicity is over 200% greater. Additionally, in some embodiments, the conjugate is more hydrophilic than the active entity without conjugation to the polymer. For example, in several embodiments, the conjugate, as compared to the active entity without conjugation to the polymer, is at least 25% (e.g. , 20-25%, 25-30%, 30-40% , 40-50%, 50- 60%, 60-70%, 70-80% , 80-90% , 90-100% , 100%-125%, 125- 150%, or higher and overlapping ranges therein) more hydrophilic. In one embodiment, the hydrophilicity is over 200% greater. In some embodiments, the greater hydrophilicity of the conjugate advantageously facilitates one or more of: non-compartmentalization within a cell or tissue at the target site; access to and activity in both the lipid bilayer and the cytosol of the cell; access to and/or activity in both the lipid bilayer and the cytoplasm of the cell; and/or access to and/or activity across the lipid bilayer. In some embodiments, the conjugate exhibits greater access to the kinase compared to the active entity without conjugation to the polymer.

[0045] In several embodiments, the method of treatment and/or use of the compositions described herein are provided for the prophylaxis or treatment of one or more of the following in a subject in need thereof: a joint, an eye, alopecia, dry eye, corneal edema, an autoimmune disorder, the gastrointestinal system, a lung, a vascular tumor, age-related macular degeneration, a cancerous or pre-cancerous lesion, a skin neoplasia, a bullous disease, a scar, a wound, diabetic retinopathy, non-dermal inflammation, an inflammatory condition, an inflammatory skin condition, and/or an inflammatory skin disease.

[0046] In several embodiments, the method of treatment and/or use of the compositions described herein are provided for modulating hair growth and/or cycling.

[0047] In several embodiments, the method of treatment and/or use of the compositions described herein are employed combination therapy with UV irradiation.

[0048] In several embodiments, the method of treatment and/or use of the compositions described herein are provided for the prophylaxis or treatment of one or more of the following conditions: age-related macular degeneration, diabetic retinopathy, corneal edema, macular edema, ocular rosacea, dry eye, hemangiomas, Kaposi's sarcoma, lymphangioma, glomangioma, angiosarcoma, hemangioendothelioma, infantile hemangiomas, squamous cell carcinoma, basal cell carcinoma, malignant melanoma, malignant cutaneous lymphoma, Kaposi's sarcoma, Merkel cell skin cancer, and non- melanoma skin cancer, bullous pemphigoid, erythema multiforme, dermatitis herpetiformis, epidermolysis bullosa acquisita, linear Immunoglobulin A disease, mucous membrane pemphigoid, pemphigoid gestationis, pemphigus foliaceus, pemphigus vulgaris, psoriasis, psoriasis guttata, inverse psoriasis, pustular psoriasis, psoriatic erythroderma, acute febrile neutrophilic dermatosis, eczema, xerotic eczema, dyshidrotic eczema, vesicular palmar eczema, acne vulgaris, atopic dermatitis, contact dermatitis, allergic contact dermatitis, dermatomyositis, exfoliative dermatitis, hand eczema, pompholyx, rosacea, rosacea due to sarcoidosis, rosacea due to scleroderma, rosacea due to Sweet syndrome, rosacea due to systemic lupus erythematosus, rosacea due to urticaria, rosacea due to herpetic pain, Sweet's disease, neutrophilic hydradenitis, sterile pustule, drug rash, seborrheic dermatitis, pityriasis rosea, Kikuchi's disease of the skin, pruritic urticarial papules and plaques of pregnancy, Stevens-Johnson syndrome and toxic epidermal necrolysis, tattoo reaction, Wells syndrome (eosinophilic cellulitis) , reactive arthritis (Reiter syndrome) , bowel-associated dermatosis-arthritis syndrome, rheumatoid neutrophilic dermatosis, neutrophilic eccrine hidradenitis, neutrophilic skin disease of dorsum of hand, balanitis circumscripta plasmacellularis, balanoposthitis, Behcet's disease, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiforme, granuloma annulare, dermatitis of hand, lichen nitidus, lichen planus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, nummular dermatitis, pyoderma gangrenosum, sarcoidosis, subcorneal pustular dermatosis, urticaria, skin fibrosis, and transient acantholytic dermatosis, alopecia, alopecia areata, androgenic alopecia, and/or dry eye.

BRIEF DESCRIPTION OF THE FIGURES

[0049] The Figures below are illustrative for some embodiments and should not be construed as overly limiting.

[0050] Figure 1 depicts the chemical structure of Motesanib.

[0051] Figure 2 depicts the synthesis scheme of CT103.

[0052] Figure 3 depicts the flash chromatogram (NP-LC) of crude purification (254/21 Onm).

[0053] Figure 4 depicts the flash chromatogram (RP-LC) of CT103 purification (254/21 Onm).

[0054] Figure 5 depicts the analytical HPLC chromatogram of Fr14 - CT103(254nm) .

[0055] Figure 6 depicts the analytical HPLC of Fr14 - CT103 (ELSD) . [0056] Figure 7 depicts the analytical HPLC chromatogram of Fr15 - CT103 (254nm).

[0057] Figure 8 depicts the analytical HPLC chromatogram of Fr15 - CT103 (ELSD).

[0058] Figure 9 depicts the MALDI-TOF spectrum of Fr15 - CT103 (green) overlaid with the spectrum of PEG (blue).

[0059] Figure 10 depicts CT103.

[0060] Figure 1 1 depicts the Motesanib.

[0061] Figure 12 depicts the CT103 synthesis.

[0062] Figure 13 depicts the MW 528 Da by-product.

[0063] Figure 14 depicts the HPLC analysis of starting Motesanib (HPLC method M l CT103 001 ).

[0064] Figure 15 depicts the HPLC analysis of reaction mixture/24 h aging time (HPLC metho MU CT103 001 ).

[0065] Figure 16 depicts the HPLC analysis of crude CT103 (HPLC method M l CT103 001 ) .

[0066] Figure 17 depicts the UV profile (@254 and 210 nm) of CT103 purification by reverse phase flash.

[0067] Figure 18 depicts the HPLC analysis of final CT103 lot n° 2015GC04/S7 (HPLC method M l CT103 001) .

[0068] Figure 19 depicts the NMR analysis of final CT103/ lot n° 2015GC04/S7.

[0069] Figure 20 depicts the ESI MS analysis of CT103 lot n° 2015GC04/S7/single charged ion.

[0070] Figure 21 depicts the ESI MS analysis of CT103 lot n° 2015GC04/S7/double charged ion.

[0071] Figure 22 depicts the certificate of analysis of final CT103 lot n° 2015GC04/S7.

[0072] Figure 23 depicts the HPLC analysis of final CT103 lot n° 2015GC04/S8 (HPLC method M l CT103 001) .

[0073] Figure 24 depicts the NMR analysis of final CT103/lot n° 2015GC04/S8.

[0074] Figure 25 depicts the ESI MS analysis of CT103 lot n° 2015GC04/S8/single charged ion.

[0075] Figure 26 depicts the ESI MS analysis of CT103 lot n° 2015GC04/S8/double charged ion. [0076] Figure 27 depicts the certificate of analysis of final CT103 lot n° 2015GC04/S8.

[0077] Figure 28 depicts the HPLC analysis of final CT103 lot n° 2015GC04/S9 (HPLX M l CT103 001 ).

[0078] Figure 29 depicts the NMR analysis of final CT103/lot n° 2015GC04/S9.

[0079] Figure 30 depicts the ESI MS analysis of CT103 lot n° 2015GC04/S9/single charged ion.

[0080] Figure 31 depicts the ESI MS analysis of CT103 lot n° 2015GC04/S9/double charged ion.

[0081] Figure 32 depicts the certificate of analysis of final CT103 lot n° 2015GC04/S9.

[0082] Figure 33 depicts Chromatograms of a CT1 03 plasma standard extract (50 μg/mL) detected using SIR (TIC, upper) and UV at 337 nm (lower).

[0083] Figure 34 depicts CT103 calibration curve (range 0.25-50 μg/ml) . Peak areas obtained from TIC MS data, y = 147323x, R2 = 0.9965

[0084] Figure 35 depicts method validation. Arrows denote peaks of LOO and

LOQ.

[0085] Figure 36 depicts individual chromatograms of SIR channels used for the analysis of compound (sample: plasma spiked with compound at 50 μg/mL). Top chromatogram: 794; middle chromatogram: 595.8, bottom chromatogram: 476.8.

[0086] Figure 37 depicts representative chromatograms showing CT103 in methanol (A) and extracted from a spiked murine plasma calibration standard (B) . For both chromatographs top to bottom traces = m/z 794, m/z 595.8, m/z 4 76.8, TIC and representative blank. The areas in between the dashed lines represents the area that CT103 eluted, the shaded peaks represent the detected CT103.

[0087] Figure 38 depicts mouse plasma concentrations of CT103. Data are presented as Mean ± CI 95% .

[0088] Figure 39 depicts tritiated thymidine incorporation in corrected counts per minute (CCPM). Data are presented as Mean ± SEM .

[0089] Figure 40 depicts Tritiated thymidine incorporation in corrected counts per minute (CCPM). Data are presented as Mean ± SEM .

[0090] Figure 41 depicts tritiated thymidine incorporation in corrected counts per minute (CCPM). Statistical analysis of the 18 hour pre-incubation time with 5x103 cells per well and 50ng/ml_ VEGF stimulation. Data are presented as Mean ± SEM . Values significantly lower than DMSO-treated cells are indicated by: * p < 0.05, ** p < 0.01 , ns not significant. [0091] Figure 42 depicts percentage inhibition by CT103 with 18 hour preincubation time ,5x103 cells per well and 50ng/ml_ VEGF stimulation. Percentage inhibition of VEGF induced proliferation by CT103. Proliferation in the absence of VEGF (0 ng/mL VEGF) was subtracted from VEGF induced proliferation (50 ng/mL only) and divided by 50 ng/mL VEGF stimulated cell proliferation in the absence of drug (DMSO).

[0092] Figure 43 depicts tritiated thymidine incorporation in corrected counts per minute (CCPM). Data are presented as Mean ± SEM .

[0093] Figure 44 depicts non-linear fit of transformed normalised maximal response for IC50 calculation. The DMSO (0 μΜ CT103) treated VEGF-induced HUVEC proliferation was excluded from the IC50 calculation as it was lower than the 3 lowest concentrations of CT103, which had reached a consistent plateau as can be seen above. The IC50 was calculated as 18.5 μΜ.

[0094] Figure 45 depicts percentage inhibition of VEGF induced proliferation by CT103. Proliferation in the absence of VEGF was subtracted from VEGF induced proliferation and divided by VEGF stimulated cell proliferation in the absence of drug (DMSO).

[0095] Figure 46 depicts bodyweights. Data are presented as Mean ± SEM percentages of the initial bodyweights. * p < 0.05, *** p < 0.001 , **** p < 0.0001 when compared to the vehicle-treated group; # # # p < 0.001 , # # # # p < 0.0001 when compared to Day 0.

[0096] Figure 47 depicts erythema scores. Data are presented as Mean ± SEM . * p < 0.05, ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 when compared to the vehicle-treated group.

[0097] Figure 48 depicts scaling scores. Data are presented as Mean ± SEM . * p < 0.05, *** p < 0.01 and **** p < 0.0001 when compared to the vehicle-treated group.

[0098] Figure 49 depicts thickness scores. Data are presented as Mean ± SEM . * p < 0.05, *** p < 0.001 , **** p < 0.0001 .

[0099] Figure 50 depicts clinical scores. Data are presented as Mean ± SEM . * p < 0.05, ** p < 0.01 , *** p < 0.001 and **** p < 0.0001 when compared to the vehicle-treated group.

[0100] Figure 51 depicts the BioMAP profile of SNA-103 in the Diversity PLUS Panel. The X-axis lists the quantitative protein-based biomarker readouts measured in each system. The Y-axis represents a log-transformed ratio of the biomarker readouts for the drug-treated sample (n = 1 ) over vehicle controls (n > 6). The grey region around the Y-axis represents the 95% significance envelope generated from historical vehicle controls. Biomarker activities are annotated when 2 or more consecutive concentrations change in the same direction relative to vehicle controls, are outside of the significance envelope, and have at least one concentration with an effect size > 20% (|log10 ratio| > 0.1). Biomarker key activities are described as modulated if these activities increase in some systems, but decrease in others. Cytotoxicity is indicated on the profile plot by a thin black arrow above the X-axis, and antiproliferative effects are indicated by a thick grey arrow. Cytotoxicity and antiproliferative arrows only require one concentration to meet the indicated threshold for profile annotation. Other BioMAP profiles disclosed herein are also depicted in a similar manner.

[0101] Figure 52 depicts a Reference Benchmark Overlay of SNA- 103 and Benchmark Calcipotriene. Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (|log10 ratio| > 0.1) in the same direction.

[0102] Figure 53 depicts an overlay of SNA- 103 (230 μΜ) and Apoptolidin (1 μΜ), which was the top similarity match from a search of the BioMAP Reference Database of > 4,000 agents for SNA-103 (230 μΜ). Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (|log10 ratio| > 0.1) in the same direction. Similarity search results are filtered and ranked. Profiles are identified as having mechanistically relevant similarity if the Pearson's correlation coefficient is > 0.7.

[0103] Figure 54 depicts Mechanism HeatMAP Analysis for SNA-103. HeatMAP analysis of the 148 biomarker readouts (rows) within the Diversity PLUS panel by SNA-103 in comparison to 19 consensus mechanism class profiles (columns). Horizontal grey lines separate the 12 Diversity PLUS systems, while the vertical grey line separates SNA-103 from the 19 consensus mechanism profiles. Biomarker activities outside of the significance envelope are red if protein levels are increased, blue if protein levels are decreased and white if levels are within the envelope or unchanged. Darker shades of color represent greater change in biomarker activity relative to vehicle control.

[0104] Figure 55 depicts clustering of test agent profiles following pairwise correlation analysis and clustering of the most similar profiles. Each colored circle represents the BioMAP profile of a compound at a specific concentration, with larger circles representing higher concentrations.

[0105] Figure 56 depicts SNA-103.

[0106] Figure 57 depicts (a) SNA-103 synthesis and (b) Motesanib acylation.

[0107] Figure 58 depicts HPLC analysis of starting Motesanib (HPLC method Ml CT103 001). [0108] Figure 59 depicts HPLC analysis of reaction mixture/24 h aging time (HPLC method M l CT103 001 ).

[0109] Figure 60 depicts HPLC analysis of crude SNA-103 (HPLC method M l CT103 001 ) .

[0110] Figure 61 depicts the UV profile (@254 and 210 nm) of SNA- 103 purification by reversed phase flash chromatography.

[0111] Figure 62 depicts HPLC analysis of final SNA- 103 lot n° 2017GC12/S3 (HPLC method M l CT103 001) .

[0112] Figure 63 depicts ¹ H NMR analysis of final SNA-103/ lot n° 2017GC 12/S3.

[0113] Figure 64 depicts ESI MS analysis of final SNA-103/ lot n° 2017GC12/S3-single charged ion.

[0114] Figure 65 depicts ESI MS analysis of final SNA-103/ lot n° 2017GC 12/S3-double charged ion.

[0115] Figure 66 depicts the certificate of analysis of final SNA- 103 lot n° 2017GC 12/S3.

[0116] Figure 67 depicts the inhibition of VEGF-induced proliferation following treatment with SNA-125 (A), SNA-352 (B) , SNA- 103 (C), and motesanib diphosphate (D) . Data are presented as mean corrected counts per minute (CCPM) ± SEM , with n=6 for (A)-(C) and with n=4 for (D).

DETAILED DESCRIPTION

Platform Technology

[0117] Several embodiments relate to the use of agents that were developed using Applicant's proprietary Low Systemic Exposure™ ("LSE™") platform technology to generate LSE molecules (also generally referred to herein as polymer conjugates or compositions). In several embodiments, the LSE platform creates polymer conjugates optimized for topical applications. In several embodiments, the polymer conjugates developed by LSE or more generally the reduced exposure technology exhibit enhanced penetration. In still further embodiments, the enhanced penetration leads to delivery of a high local concentration of the drug. In further embodiments, the polymer conjugates show a limited non-target absorption upon topical administration due to their increased molecular size and amphiphilicity and/or amphipathicity. In still further embodiments, side-effects are minimized by limiting or eliminating non-target (e.g., systemic) absorption.

[0118] In several embodiments of the reduced exposure compositions/compounds, the polymer conjugate comprises a "warhead" linked to a polymer. In some embodiments, the warhead is a pharmacologically active entity selected according to the particular target or pathway of interest. As discussed herein, there are also provided, in several embodiments, polymer conjugates for use in the treatment of conditions (including but not limited to inflammatory skin diseases). In several embodiments, the polymer is directly coupled to the warhead without a separate chemical linking moiety between the polymer and the warhead; such direct coupling may involve without limitation ester, ether, acetal, ketal, vinyl ether, carbamate, urea, amine, amide, enamine, imine, oxime, amidine, iminoester, carbonate, orthoester, phosphonate, phosphinate, sulfonate, sulfinate, sulfide, sulfate, disulfide, sulfinamide, sulfonamide, thioester, aryl, silane, siloxane, heterocycles, thiocarbonate, thiocarbamate, and phosphonamide bonds. In several embodiments, the linker is a separate chemical linking moiety between the polymer and the warhead. In several embodiments, the polymer is polyethylene glycol (PEG), wherein the terminal OH group can optionally be modified e.g. with C1 -C5 alkyl or C1 -C5 acyl groups, e.g., with C1 -, C2- or C3-alkyl groups or C1 -, C2- or C3 groups. In several embodiments, the modified PEG is a terminally alkoxy-substituted PEG. In several embodiments, the modified PEG is a methoxy-PEG (mPEG). In some embodiments, the polymer has a molecular weight ranging from about 100 to about 100,000 Da. In some embodiments, the polymer is polydisperse with respect to molecular weight (e.g., has a distribution of molecular weights) and the indicated molecular weight of the polymer represents an average molecular weight. In other embodiments, the polymer has a molecular weight ranging from about 200 to about 50,000 Da. In several embodiments, the polymer has a molecular weight ranging from about 500 to about 10,000 Da (e.g., 500-1000, 1000- 2000, 2000-3000, 3000-5000,5000-7000, 7000-10,000 Da, and overlapping ranges therein).

[0119] In several embodiments, the polymer is a short-chain PEG, and in some embodiments a terminally alkoxy-substituted PEG, such as a mPEG with a molecular weight ranging from about 200 to about 4,000 Da, from about 400 to about 3,000 Da, from about 500 to about 2,000 Da, from about 700 to about 3,000 Da, from about 900 to about 4,000 Da, or from about 1 ,000 to about 5,000 Da. In several embodiments, the short-chain PEG or mPEG has an average molecular weight of about 1 ,000-3,000 Da. (e.g., 2,000 Da). [0120] In some embodiments, the polymer is a long-chain PEG. The long- chain PEG may be a terminally alkoxy-substituted PEG, such as methoxy-substituted PEG , with a molecular weight ranging greater than about 4,000 Da. In several embodiments, the molecular weight ranges from about 4,500-10,000Da (e.g. , 4,500 to about 5,500 Da) . I n several embodiments, the long-chain PEG or mPEG has an average molecular weight of about 2,000 Da or of about 5,000 Da. In several embodiments, the polymer is of natural or semi-synthetic or synthetic origin. In several embodiments, the polymer has a linear or branched structure. In several embodiments, the polymer is selected from poly(alkylene oxides) or from (polyethylene) oxides. I n several embodiments, the polymer selected may include, without limitation, one or more of the following: polyacrylic acid, polyacrylates, polyacrylamide or N-alkyl derivatives thereof, polymethacrylic acid, polymethacrylates, polyethylacrylic acid, polyethylacrylates, polyvinylpyrrolidone, poly(vinylalcohol) , polyglycolic acid, polylactic acid, poly(lactic-co-glycolic) acid, dextran, chitosan, and hydroxyethyl starch.

[0121] In an embodiment, the polymer conjugates provided herein are administered to the skin by topical application. In one embodiment, the polymer conjugates provided herein treat inflammatory skin diseases. In one embodiment, the polymer conjugates provided herein treat skin neoplasias. In one embodiment, the polymer conjugates provided herein treat bullous diseases.

[0122] In one embodiment, active agents useful for stimulating hair follicles (for hair growth) are provided as oral applications or topical applications for the scalp. Hair removal agents and anti-acne agents are provided in other embodiments. Hair growth, hair removal and anti-acne therapies can all involve active agents that, if exposed to the non-target site (e.g. , systemic circulation and/or lymphatic system) for long periods, result in toxicity or undesired side effects. Thus, the reduced exposure compositions described herein provides benefits for these applications as well. In one embodiment, the polymer conjugates provided herein modulate hair growth and cycling. In one embodiment, the polymer conjugates provided herein treat alopecia.

[0123] In alternative embodiments, the polymer conjugates configured for reduced exposure are administered to other areas of the body besides the skin. For example, in one embodiment, administration comprises treatment of the lung and respiratory conditions via inhalation of the polymer conjugates. Eye drops are provided in some embodiments to treat eye inflammation or ophthalmic disorders and diseases. Treatment to the joints to treat inflammation or other joint conditions is also provided. In yet another embodiment, administration comprises treatment of the gastro-intestinal tract via, for example, an enteric coated capsule comprising the polymer conjugates taken orally. Reduced exposure provides benefits in these applications. Applications for the nose and ear, such as inhalants, ointments and drops are provided in several embodiments. Treatment to the nasal passage to treat allergies or allergic rhinitis is also provided. Vaginal and rectal compounds are provided in some embodiments, including as suppositories, creams, ointments, etc. In one embodiment, the polymer conjugates provided herein treat vascular tumors. In one embodiment, the polymer conjugates provided herein treat diabetic retinopathy. In one embodiment, the polymer conjugates provided herein treat macular edema. In one embodiment, the polymer conjugates provided herein treat corneal edema. In one embodiment, the polymer conjugates provided herein treat age-related macular degeneration.

[0124] In some embodiments, conjugating the warhead to a polymer (e.g., PEG) in the disclosed molecular weight ranges may slow diffusion of the molecule in the tissue, thereby potentially increasing residence time of the molecule in the target tissue, e.g. epidermis and dermis for skin, associated epithelial and sub-epithelial layers in other topical surfaces like gut, eye, mucosa, lungs etc. This "depot" effect may also lead to lower concentrations needing to be applied or for products to be applied with lower frequency, or both.

[0125] In other embodiments, conjugating the warhead to a polymer (e.g., PEG) in the disclosed molecular weight ranges may be useful in reducing the diffusion or extravasation of the molecule out of the circulatory system after it enters it via injection and or diffusion from the target tissue. Indeed, changes in the tissue distribution of polymer conjugates compared to unconjugated drug have been observed in IV injection studies. In general, the unconjugated drug tends to have a long half-life and a volume of distribution within tissues AND blood, suggesting that the unconjugated drug extravasates out of the blood vessels into the tissue prior to being cleared. Whereas, in some instances, the PEGylated drug has a volume of distribution that is largely restricted to the blood, indicating that very little extravasation occurs with the polymer conjugates prior to being renally cleared. This reduced extravasation may explain at least in part the observed shorter half-life for the polymer conjugates.

[0126] The compositions described herein may be combined with other modalities to achieve synergic effects. These other modalities include, but are not limited to, energy delivery (such as laser, radiofrequency, ultrasound, microwave, etc.), thermal therapy, light therapy, radiation, intravenous chemotherapy, and others. In some embodiments, the compositions are applied with pressure, heat, massage etc. to facilitate localization to the desired target site. In some embodiments, the compositions are administered in combination with one or more additional therapeutics that may not be reduced exposure compounds. In some embodiments, the compositions are administered in combination with UV irradiation therapy. [0127] Santi et al. state that "permanent PEGylation is generally not applicable to small-molecule drugs because the bulky carrier usually prevents their binding to targets and cell penetration." (Proceedings of the National Academy of Sciences 109.16 (2012): 621 1 -6216). Further, Nakagami et al. state that "hydrophilic polymers on the surface of particles... prevents the close interactions between particles and target cell membranes, inhibiting the cellular uptake and, subsequently, preventing endosomal escape. All of these factors combine to decrease the biological efficacy of PEGylated particles" (Gene therapy (2013): 2-4). In several embodiments, the polymer conjugate exhibits unexpected permeability across the plasma membrane. In several embodiments, the polymer conjugate exhibits unexpected permeability across the nuclear membrane. In several embodiments, the polymer conjugate exhibits unexpected permeability across both the nuclear and plasma membranes.

[0128] The reduced exposure compounds, comprising a hydrophobic drug conjugated to a short chain PEG, exhibit surprising accessibility across cellular compartments, compared to the unconjugated drug. This accessibility is thought to result for the amphipathic nature of the conjugate, allowing it to traverse and distribute evenly among both lipophilic and hydrophilic cellular compartments. Accordingly, the conjugate can cross and reside within the lipid bilayer of the cell membrane, accumulate within the cytosol, and even traverse the nuclear envelope - thereby providing access both membrane, cytosolic and nuclear molecular targets. This property of the reduced exposure compounds result in excellent depo'ing, longer residence times within target cells, and relative non-compartmentalization. Consequently, these compounds are biologically active at lower concentrations and require less frequent dosing - thereby reducing potential drug toxicity.

Polymer Conjugates Targeting Vascular Endothelial Growth Factor Receptors

[0129] In several embodiments, the warhead employed in the LSE polymer conjugate is a small molecule targeting a vascular endothelial growth factor receptor (VEGFR).

[0130] Angiogenesis, the process of sprouting new blood vessels from existing vasculature, and arteriogenesis, the remodeling of small vessels into larger conduit vessels, are both physiologically important aspects of vascular growth in adult tissues. These processes of vascular growth are required for beneficial processes such as tissue repair, wound healing, recovery from tissue ischemia and menstrual cycling. They are also required for the development of pathological conditions such as the growth of neoplasias, diabetic retinopathy, rheumatoid arthritis, psoriasis, certain forms of macular degeneration, and certain inflammatory pathologies. The inhibition of vascular growth in these contexts has also shown beneficial effects in preclinical animal models. For example, inhibition of angiogenesis by blocking vascular endothelial growth factor or its receptor has resulted in inhibition of tumor growth and in retinopathy. Also, the development of pathological pannus tissue in rheumatoid arthritis involves angiogenesis and might be blocked by inhibitors of angiogenesis. Certain diseases are known to be associated with deregulated angiogenesis, for example ocular neovascularization, such as retinopathies (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, such as a rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders, such as chronic asthma, arterial or post- transplantational atherosclerosis, endometriosis, and neoplastic diseases, for example so-called solid tumors and liquid tumors (such as leukemias).

[0131] Vascular endothelial growth factor (VEGF) is a signaling protein involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). As its name implies, VEGF activity has been mostly studied on cells of the vascular endothelium, although it does have effects on a number of other cell types (e.g., stimulation monocyte/macrophage migration, neurons, cancer cells, kidney epithelial cells, keratinocytes). In vitro, VEGF has been shown to stimulate endothelial cell mitogenesis and cell migration. VEGF is also a vasodilator and increases microvascular permeability and was originally referred to as vascular permeability factor.

[0132] The broad term "VEGF" covers a number of proteins from two families, that result from alternate splicing of mRNA from a single, 8 exon, VEGF gene. The two different familes are referred to according to their terminal exon (exon 8) splice site— the proximal splice site (denoted VEGFxxx) or distal splice site (VEGFxxxb). In addition, alternate splicing of exon 6 and 7 alters their heparin binding affinity, and amino acid number (in humans: VEGF121 , VEGF121 b, VEGF145, VEGF165, VEGF165b, VEGF189, VEGF206; the rodent orthologs of these proteins contain one fewer amino acid). These domains have functional consequences for the VEGF splice variants as the terminal (exon 8) splice site determines whether the proteins are pro-angiogenic (proximal splice site, expressed during angiogenesis) or anti-angiogenic (distal splice site, expressed in normal tissues). In addition, inclusion or exclusion of exons 6 and 7 mediate interactions with heparan sulfate proteoglycans (HSPGs) and neuropilin co- receptors on the cell surface, enhancing their ability to bind and activate VEGFRs.

[0133] All members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation, although to different sites, times and extents. The VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains, a single transmembrane spanning region and an intracellular portion containing a split tyrosine-kinase domain. VEGF-A binds to VEGFR- 1 (Flt- 1) and VEGFR-2 (KDR/Flk- 1 ). VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well defined, although it is thought to modulate VEGFR-2 signaling. Another function of VEGFR-1 may be to act as a dummy/decoy receptor, sequestering VEGF from VEGFR-2 binding (this appears to be involved during vasculogenesis in the embryo). VEGF-C and VEGF-D, but not VEGF-A, are ligands for a third receptor (VEGFR-3), which mediates lymphangiogenesis.

[0134] There is provided, in several embodiments, methods of treating a vascular tumor in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. Non-limiting examples of vascular tumors include hemangiomas, Kaposi's sarcoma, lymphangioma, glomangioma, angiosarcoma, hemangioendothelioma, and infantile hemangiomas.

[0135] While blood vessels in healthy adults are largely quiescent, adult skin retains the capacity for rapid initiation of angiogenesis during tissue repair and in numerous diseases including inflammatory skin diseases such as psoriasis, many types of dermatitis, blistering diseases, cutaneous neoplasias including squamous cell carcinomas, malignant melanomas, and Kaposi's sarcomas, and proliferative hemangiomas of childhood. Angiogenesis in the skin is also implicated in a number of other diseases that are characterized by macroscopically visible, prominent blood vessels, including rosacea and basal cell carcinoma.

[0136] There is provided, in several embodiments, methods of treating a skin neoplasia in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. Non-limiting examples of skin neoplasias include squamous cell carcinoma, basal cell carcinoma, malignant melanoma, malignant cutaneous lymphoma, Kaposi's sarcoma, Merkel cell skin cancer, and non-melanoma skin cancer.

[0137] Increased vascular permeability is one of the earliest manifestations of inflammation, resulting in extravasation of protein-rich plasma into the effected tissue. Acute vascular permeability allows the deposition of circulating plasma matrix proteins including fibrin and fibronectin (FN) which facilitate cell migration in the inflamed area. This process also provides an access point for immune cells and immunoglobulins to enter the tissue and fight foreign antigens (Nagy et al, Cold Spring Harb. Perspect. Med. 2:a006544, 2012). Conversely, chronic vascular hyperpermeability is suggested to sustain the inflammatory response and retard resolution, further promoting the development of chronic inflammation (Nagy et al, Cold Spring Harb. Perspect. Med. 2:a006544, 2012; Costa et al, Angiogenesis 10: 149- 166, 2007). This type of vascular hyperpermeability underlies the pathogenesis of a large number of chronic disorders including rheumatoid arthritis (RA) , psoriasis, ocular disease, cancer and chronic wounds (Nagy et al, Cold Spring Harb. Perspect. Med. 2:a006544, 2012; Costa et al, Angiogenesis 10: 149- 166, 2007).

[0138] VEGF is a potent vascular permeabilizing agent that is highly expressed during chronic inflammation (Nagy et al, Annu. Rev. Pathol. 2:251 -275, 2007) . Low microenvironmental levels of VEGF are desired in order to maintain stable vascular integrity and promote endothelial cell survival through autocrine mechanisms (Lee et al, Cell 130:691 -703, 2007). Whereas elevated levels of VEGF induce vascular leakages by activating VEGF receptor 2 (VEGFR2) in endothelial cells (EC) leading to the opening of intercellular and/or intracellular pathways that facilitate plasma extravasation (Koch and Claesson-Welsh, Cold Spring Harb. Perspect. Med. 2:a006502, 2012) . Moreover, VEGF may serve as a pro-inflammatory mediator as it can enhance T cell (Xia et al, Blood 102: 161 -168, 2003) , and monocyte (Murakami et al, Blood 108: 1849-1856, 2006) migration as well as promote pro-inflammatory chemokines expression by EC including MCP-1 , and IL-8, leading to further immune recruitment. Cellular sources for VEGF during inflammation can include macrophages and mast cells; however it can also be expressed by endothelial cells and acts in a paracrine and autocrine fashion. Thus, VEGF plays a major role in promoting chronic inflammation by inducing vascular permeability and contributing to immune cell recruitment.

[0139] There is provided, in several embodiments, methods of treating an inflammatory skin disease in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. Non-limiting examples of inflammatory skin diseases include psoriasis, psoriasis guttata, inverse psoriasis, pustular psoriasis, psoriatic erythroderma, acute febrile neutrophilic dermatosis, eczema, xerotic eczema, dyshidrotic eczema, vesicular palmar eczema, acne vulgaris, atopic dermatitis, contact dermatitis, allergic contact dermatitis, dermatomyositis, exfoliative dermatitis, hand eczema, pompholyx, keloids, rosacea, rosacea due to sarcoidosis, rosacea due to scleroderma, rosacea due to Sweet syndrome, rosacea due to systemic lupus erythematosus, rosacea due to urticaria, rosacea due to herpetic pain, Sweet's disease, neutrophilic hydradenitis, sterile pustule, drug rash, seborrheic dermatitis, pityriasis rosea, Kikuchi's disease of the skin, pruritic urticarial papules and plaques of pregnancy, Stevens-Johnson syndrome and toxic epidermal necrolysis, tattoo reaction, Wells syndrome (eosinophilic cellulitis), reactive arthritis (Reiter syndrome) , bowel-associated dermatosis-arthritis syndrome, rheumatoid neutrophilic dermatosis, neutrophilic eccrine hidradenitis, neutrophilic skin disease of dorsum of hand, balanitis circumscripta plasmacellularis, balanoposthitis, Behcet's disease, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiforme, granuloma annulare, dermatitis of hand, lichen nitidus, lichen planus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, nummular dermatitis, pyoderma gangrenosum, sarcoidosis, subcorneal pustular dermatosis, urticaria, and transient acantholytic dermatosis.

[0140] Bullous diseases are skin disorders characterized by blistering that often have an autoimmune etiology. VEGF has been found to be upregulated in two bullous diseases, bullous pemphigoid and erythema multiforme. Brown, Lawrence F. , et al. Journal of Investigative Dermatology 104.5 (1995): 744-749. Bullous pemphigoid is a subepidermal disorder which manifests as subepidermal blisters with a dermal infiltrate of neutrophils and eosinophils. Erythema multiforme is an inflammatory eruption characterized by symmetric erythematous, edematous, or bullous lesions of the skin or mucous membranes.

[0141] There is provided, in several embodiments, methods of treating an ophthalmic condition in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. Non-limiting examples of ophthalmic conditions include macular degeneration, age related macular degeneration (ARMD), choroidal neovascularization, retinopathy, diabetic retinopathy, acute macular neuroretinopathy, chronic macular neuroretinopathy, central serous chorioretinopathy, macular edema, cystoid macular edema, diabetic macular edema. , acute multifocal placoid pigment epitheliopathy, Behcet's disease, birdshot retinochoroidopathy, uveitis, intermediate uveitis (pars planitis) , anterior uveitis, multifocal choroiditis, multiple evanescent white dot syndrome (MEWDS), ocular sarcoidosis, posterior scleritis, serpignous choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal arterial occlusive disease, central retinal vein occlusion, disseminated intravascular coagulopathy, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal arterial microaneurysms, Coat's disease, parafoveal telangiectasis, hemi-retinal vein occlusion, papillophlebitis, central retinal artery occlusion, branch retinal artery occlusion, carotid artery disease (CAD), frosted branch angitis, sickle cell retinopathy and other hemoglobinopathies, angioid streaks, familial exudative vitreoretinopathy, and Eales disease, sympathetic ophthalmia, uveitic retinal disease, retinal detachment, trauma, laser, PDT, photocoagulation, hypoperfusion during surgery, radiation retinopathy, and bone marrow transplant retinopathy, proliferative vitreal retinopathy and epiretinal membranes, proliferative diabetic retinopathy, ocular histoplasmosis, ocular toxocariasis, presumed ocular histoplasmosis syndrome (POHS), endophthalmitis, toxoplasmosis, retinal diseases associated with HIV infection, choroidal disease associated with HIV infection, uveitic disease associated with HIV Infection, viral retinitis, acute retinal necrosis, progressive outer retinal necrosis, fungal retinal diseases, ocular syphilis, ocular tuberculosis, diffuse unilateral subacute neuroretinitis, myiasis, retinitis pigmentosa, systemic disorders with associated retinal dystrophies, congenital stationary night blindness, cone dystrophies, Stargardt's disease, fundus flavimaculatus, Bests disease, pattern dystrophy of the retinal pigmented epithelium, X-linked retinoschisis, Sorsby's fundus dystrophy, benign concentric maculopathy, Bietti's crystalline dystrophy, and pseudoxanthoma elasticum, retinal detachment, macular hole, and giant retinal tear, retinal disease associated with tumors, congenital hypertrophy of the RPE, posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, combined hamartoma of the retina and retinal pigmented epithelium, retinoblastoma, vasoproliferative tumors of the ocular fundus, retinal astrocytoma, and intraocular lymphoid tumors, punctate inner choroidopathy, acute posterior multifocal placoid pigment epitheliopathy, myopic retinal degeneration, and acute retinal pigment epithelitis.

[0142] There is provided, in several embodiments, methods of treating a bullous disease in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. Non-limiting examples of bullous diseases include bullous pemphigoid, erythema multiforme, dermatitis herpetiformis, epidermolysis bullosa acquisita, linear Immunoglobulin A disease, mucous membrane pemphigoid, pemphigoid gestationis, pemphigus foliaceus, and pemphigus vulgaris.

[0143] There is evidence to suggest that increased expression of angiogenic factors, in particular VEGF, is a central cause of proliferative diabetic retinopathy (PDR). In this condition, and others such as retinopathy of prematurity, sickle cell retinopathy, age-related macular degeneration, retina vein occlusion and Eales disease, preretinal vascularisation is a major cause of blindness. New blood vessels grow from the inner retinal vasculature into the vitreous humour. This can cause visual loss by vitreous haemorrhage and/ortractional retinal detachment due to contraction of the fibrous tissue associated with the new blood vessels. Inhibitors of the VEGF pathway intended to treat eye disease are discussed by Slevin et al. in Expert Opin. Investig. Drugs (2008) 17(9): 1301 -1314, herein incorporated by reference.

[0144] There is provided, in several embodiments, methods of treating age- related macular degeneration in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. There is provided, in several embodiments, methods of treating diabetic retinopathy in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. There is provided, in several embodiments, methods of treating corneal edema in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. There is provided, in several embodiments, methods of treating macular edema in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. There is provided, in several embodiments, methods of treating dry eye in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR.

[0145] The process of new hair growth, whether as part of the natural hair cycle or as a result of a treatment to encourage hair growth, relies on numerous cross- talking signal pathways to bring about the processes necessary for hair growth. These principal processes are: cell proliferation of the dermal papia, cell migration to form the appropriate structures, and angiogenesis to form blood supply routes to the new hair foicle. Vascular endothelial growth factor (VEGF) is a well-documented signal to activate these processes via the VEGFR2 receptor. VEGF leads to downregulation of Bad, TGF- 1 and caspase expression through the AKt/PKB and calcium ion dependent pathways, thereby bringing about the end of apoptosis and the telogen phase.

[0146] There is provided, in several embodiments, methods of modulating hair growth and cycling in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. There is provided, in several embodiments, methods of treating alopecia in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR.

[0147] There is provided, in several embodiments, a combination therapy, the combination therapy comprising administering to the subject an effective amount of a polymer conjugate in conjunction with UV irradiation therapy, wherein the warhead is a small molecule targeting a VEGFR.

[0148] A growing body of research suggests that dry eye is the result of an underlying cytokine and receptor-mediated inflammatory process. There is provided, in several embodiments, methods of treating dry eye in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. In some embodiments the composition is formulated as an eye drop. In some embodiments, one or two drops of the composition are used per application. In other embodiments, three or four drops of the composition are used per application. In additional embodiments, six drops of the composition are used per application. In some embodiments, the composition is applied for a period of 60 seconds before flushing. In other embodiments, the composition is applied for a period of 120 seconds before flushing. In additional embodiments, the composition is applied for a period of 360 seconds before flushing. In some embodiments, the composition may be administered one or more times a day. In some embodiments, the composition is administered daily. In some embodiments, the composition may be administered once a week.

[0149] In some embodiments, alopecia is treated. Non-limiting examples include androgenic alopecia and alopecia areata. Androgenic alopecia (also known as hereditary baldness, male pattern baldness, and seborrheic alopecia) is a non-scarring hair loss of telogen hairs caused by an excessive androgen effect in genetically susceptible men and women. Alopecia areata is known to be associated with autoimmune activities; hence, topically administered immunomodulatory compounds demonstrate efficacy for treating that type of hair loss.

[0150] There is provided, in several embodiments, methods of treating an alopecia in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR. In some embodiments, hair regeneration compositions are in the form of a liquid. In other embodiments, hair regeneration compositions are in the form of a lotion. In additional embodiments, hair regeneration compositions are in the form of a cream. I n some embodiments, hair regeneration compositions are in the form of a gel. In other embodiments, the hair regeneration composition is administered twice daily. In other embodiments, the hair regeneration composition is administered one daily. In additional embodiments, the hair regeneration composition is administered once weekly. In some embodiments, the hair regeneration composition is administered directly to the scalp. In some embodiments, the hair regeneration composition is administered directly non-scalp areas.

[0151] Allergic inflammatory diseases are characterized by an immune response against a sensitizing agent, such as an allergen, resulting in the release of inflammatory mediators that recruit cells involved in inflammation in a subject, potentially leading to tissue damage and sometimes death. Allergic inflammatory diseases of the eye, skin, upper and lower airways, and gastrointestinal tract, lung, including, but not limited to, atopic dermatitis, atopic keratoconjunctivitis, allergic conjunctivitis, asthma, and allergic rhinitis. There is provided, in several embodiments, methods of treating an allergic inflammatory disease in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR.

[0152] There is also provided, in several embodiments, methods of treating the following conditions in a subject, the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting a VEGFR: nail dystrophy; seborrheic keratosis; androgenic alopecia; contact dermatitis; actinic keratosis; acne; asthma; eczema (atopic derm); onychomycosis; sinusitis; allergic rhinitis; rosacea; COPD; pruritus; early AMD; urticaria; diabetic retinopathy; psoriasis; alopecia areata; dry eye; vitiligo; glaucoma; late AMD; ulcerative colitis; Crohn's disease; ocular rosacea; hair growth and cycling; skin neoplasias; squamous cell carcinoma; basal cell carcinoma; malignant melanoma; malignant cutaneous lymphomas; vascular tumors; angiosarcoma; kaposi's sarcoma; infantile hemangiomas; hemangioendothelioma; inflammatory dermatoses; dermatitis (atopic, contact); psoriasis; keloids; rosacea; bullous diseases; bullous pemphigoid; erythema multiforme; UV irradiation therapy; age-related macular degeneration; diabetic retinopathy; macular and corneal edema.

[0153] There is also provided, in several embodiments, methods of treating a respiratory disease in a subject via delivery of the polymer conjugates (wherein the warhead is a small molecule targeting a VEGFR) to the lungs and/or airways. Delivery routes may include for example intratracheal instillation or inhalation. The formulation may include liquids, nebulized or aerosolized liquids or suspensions, dry powder, nanocomposites, nanoparticles or microparticles, etc. Respiratory disorders, include treatable obstructive, restrictive or inflammatory airways diseases of whatever type, etiology, or pathogenesis. Non-limiting examples of respiratory conditions include: acute bronchitis; acute laryngotracheal bronchitis; arachidic bronchitis; catarrhal bronchitis; croupus bronchitis; dry bronchitis; infectious asthmatic bronchitis; productive bronchitis; staphylococcus or streptococcal bronchitis; vesicular bronchitis; cylindric bronchiectasis; sacculated bronchiectasis; fusiform bronchiectasis; capillary bronchiectasis; cystic bronchiectasis; dry bronchiectasis; follicular bronchiectasis; chronic obstructive pulmonary disease (COPD), chronic obstructive lung disease (COLD), chronic obstructive airways disease (COAD) or small airways obstruction of whatever type, etiology, or pathogenesis, in particular chronic bronchitis, pulmonary emphysema, bronchiectasis, cystic fibrosis, bronchiolitis obliterans, organizing pneumonia (BOOP), chronic organizing pneumonia (COP), bronchiolitis fibrosa obliterans, follicular bronchiolitis or dyspnea associated therewith; cough of whatever type, etiology, or pathogenesis in particular idiopathic cough or cough associated with gastro-esophageal reflux disease (GERD), drugs, bronchial hyper-responsivity, asthma, COPD, COLD, COAD, bronchitis, bronchiectasis, pulmonary eosinophilic syndromes, pneumoconiosis, interstitial lung disease, pulmonary fibrosis, aspiration disorders, rhinitis, laryngitis or pharyngitis; pulmonary eosinophilic syndromes of whatever type, etiology, or pathogenesis, in particular acute eosinophilic pneumonia (idiopathic or due to drugs or parasites), simple pulmonary eosinophilia, Loeffler's syndrome, tropical pulmonary eosinophilia, chronic eosinophilic pneumonia, allergic bronchopulmonary mycosis, allergic bronchopulmonary aspergillosis (ABPA), Churg-Strauss syndrome or idiopathic hypereosinophilic syndrome; asthma of whatever type, etiology, or pathogenesis, in particular asthma that is a member selected from the group consisting of atopic asthma, non-atopic asthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, intrinsic asthma caused by pathophysiologic disturbances, extrinsic asthma caused by environmental factors, essential asthma of unknown or inapparent cause, non-atopic asthma, bronchitic asthma, emphysematous asthma, exercise-induced asthma, allergen induced asthma, cold air induced asthma, occupational asthma, infective asthma caused by bacterial, fungal, protozoal, or viral infection, non-allergic asthma, incipient asthma and wheezy infant syndrome; alveolar hemorrhage of whatever type, etiology, or pathogenesis, in particular a member of the group consisting of idiopathic pulmonary hemosiderosis, alveolar hemorrhage due to drugs or other exogenous agents, alveolar hemorrhage associated with HIV or bone marrow transplant or autoimmune alveolar hemorrhage (e.g. associated with systemic lupus erythematosis, Goodpasture's syndrome, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, pauci-immune glomerulonephritis); pneumoconiosis of whatever type, etiology, or pathogenesis, in particular pneumoconiosis that is a member selected from the group consisting of aluminosis or bauxite workers' disease, anthracosis or miners' asthma, asbestosis or steam-fitters' asthma, chalicosis or flint disease, ptilosis caused by inhaling the dust from ostrich feathers, siderosis caused by the inhalation of iron particles, silicosis or grinders' disease, byssinosis or cotton-dust asthma and talc pneumoconiosis; interstitial lung diseases (ILD) or pulmonary fibrosis of whatever type, etiology, or pathogenesis, in particular idiopathic pulmonary fibrosis, crytogenic fibrosing alveolitis, fibrosing alveolitis, ILD or pulmonary fibrosis associated with connective tissue disease (systemic lupus erythematosis, mixed connective tissue disease, polymyositis, dermatomyositis, Sjorgen's syndrome, systemic sclerosis, scleroderma, rheumatoid arthritis), usual interstitial pneumonia (UIP), desquamative interstitial pneumonia (DIP), granulomatous lung disease, sarcoidosis, Wegener's granulomatosis, histiocytosis X, Langerhan's cell granulomatosis, hypersensitivity pneumonitis, extrinsic allergic alveolitis, silicosis, chronic eosinophilic pneumonia, lymphangiolyomatosis, drug-induced I LD or pulmonary fibrosis, radiation-induced ILD or pulmonary fibrosis, alveolar proteinosis, graft-versus- host-disease (GVHD), lung transplant rejection, ILD or pulmonary fibrosis due to environmental/occupational exposure, BOOP, COP, bronchiolitis fibrosa obliterans, follicular bronchiolitis, idiopathic acute interstitial pneumonitis (Hamman Rich syndrome) or alveolar hemorrhage syndromes; seasonal allergic rhinitis or perennial allergic rhinitis or sinusitis of whatever type, etiology, or pathogenesis, in particular sinusitis that is a member selected from the group consisting of purulent or nonpurulent sinusitis, acute or chronic sinusitis and ethmoid, frontal, maxillary, or sphenoid sinusitis; Acute Respiratory Distress Syndrome (ARDS), adult respiratory distress syndrome or acute lung injury of whatever type, etiology, or pathogenesis; progressive massive fibrosis (PMF); pulmonary hypertension of whatever type, etiology or pathogenesis including primary pulmonary hypertension, essential hypertension, pulmonary hypertension secondary to congestive heart failure, pulmonary hypertension secondary to COPD, pulmonary venous hypertension, pulmonary arterial hypertension and hypoxia-induced pulmonary hypertension. Respiratory disorders also include, in some embodiments, malignancies and tumors of the respiratory system, non-limiting examples of which include lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma (BAC), pulmonary adenocarcinoma (AIS), non-small-cell carcinoma, small cell carcinoma, and mesothelioma.

[0154] Compositions comprising compounds Nos 1 -59 shown in Table 1 are used, in several embodiments, as inhibitors, antagonists, and inverse agonists of a VEGFR. Several embodiments relate to polymer conjugates of compounds 1 -59, optimized for topical applications while also minimizing side-effects caused by exposure at non-target sites (e.g. , systemic absorption) . Non-topical applications are provided in other embodiments.

[0155] In several embodiments, the warhead of the polymer conjugate is a small molecule disclosed in Table 1 targeting a VEGFR. There is also provided, in several embodiments, methods of treating an inflammatory skin disease in a subject, the method comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is a small molecule disclosed in Table 1 targeting a VEGFR. In some embodiments, the warhead of the LSE polymer conjugate is compound 1 . In some embodiments, the LSE polymer conjugate is CT103.

[0156] There is also provided, in several embodiments, methods of treating a vascular tumor in a subject, the method comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is a small molecule disclosed in Table 1 targeting a VEGFR. In some embodiments, the warhead of the LSE polymer conjugate is compound 1 . In some embodiments, the LSE polymer conjugate is CT103.

[0157] There is also provided, in several embodiments, methods of treating a skin neoplasia in a subject, the method comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is a small molecule disclosed in Table 1 targeting a VEGFR. In some embodiments, the warhead of the LSE polymer conjugate is compound 1 . In some embodiments, the LSE polymer conjugate is CT103.

[0158] There is also provided, in several embodiments, methods of modulating hair and growth cycling in a subject, the method comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is a small molecule disclosed in Table 1 targeting a VEGFR. In some embodiments, the warhead of the LSE polymer conjugate is compound 1 . In some embodiments, the LSE polymer conjugate is CT103.

[0159] There is also provided, in several embodiments, methods of treating alopecia in a subject, the method comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is a small molecule disclosed in Table 1 targeting a VEGFR. In some embodiments, the warhead of the LSE polymer conjugate is compound 1 . In some embodiments, the LSE polymer conjugate is CT103.

[0160] There is also provided, in several embodiments, methods of treating a bullous disease in a subject, the method comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is a small molecule disclosed in Table 1 targeting a VEGFR. In some embodiments, the warhead of the LSE polymer conjugate is compound 1 . In some embodiments, the LSE polymer conjugate is CT103.

[0161] There is also provided, in several embodiments, methods of treating dye eye, diabetic retinopathy, macular edema, corneal edema, and/or age-related macular degeneration in a subject, the method comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is a small molecule disclosed in Table 1 targeting a VEGFR. In some embodiments, the warhead of the LSE polymer conjugate is compound 1 . In some embodiments, the LSE polymer conjugate is CT103.

[0162] There is also provided, in several embodiments, a combination therapy, the combination therapy comprising administering to the subject an effective amount of an LSE polymer conjugate in conjunction with UV irradiation therapy and wherein the warhead is a small molecule disclosed in Table 1 targeting a VEGFR. In some embodiments, the warhead of the LSE polymer conjugate is compound 1 . In some embodiments, the LSE polymer conjugate is CT103.

[0163] In some embodiments, for those compounds Nos 1 -59 having one amino group, the compound is modified (e.g., PEGylated) at that location (e.g., a PEG or modified PEG is linked to the compound by reaction with the amino group). If two or more amino groups are present, either location is PEGylated in some embodiments. In other embodiments, the amino group located the furthest away from the moieties interacting with the target is used. In some embodiments, the amino group that causes the least hindrance on activity is used (whether or not it is located the furthest way from the moieties interacting with the target). The effect of conjugation on the activity of the compound can be determined based on various methods, such as bioassays, mass spectroscopy, surface plasmon resonance, in vivo assays, clinical assays, and predictive in silico modeling programs.

[0164] In some embodiments, for those compounds Nos 1 -59 having one sulfhydryl group, the compound is modified (e.g., PEGylated) at that location. If two or more sulfhydryl groups are present, either location is PEGylated in some embodiments. In other embodiments, the sulfhydryl group located the furthest away from the moieties interacting with the target is used. In some embodiments, the sulfhydryl group that causes the least hindrance on activity is used (whether or not it is located the furthest way from the moieties interacting with the target).

[0165] In some embodiments, for those compounds Nos 1 -59 having one hydroxyl group, the compound is modified (e.g. , PEGylated) at that location. If two or more hydroxyl groups are present, either location is PEGylated in some embodiments. In other embodiments, the hydroxyl group located the furthest away from the moieties interacting with the target is used. In some embodiments, the hydroxyl group that causes the least hindrance on activity is used (whether or not it is located the furthest way from the moieties interacting with the target).

[0166] In some embodiments, for those compounds Nos 1 -59 having one carboxyl group, the compound is modified (e.g. , PEGylated) at that location. If two or more carboxyl groups are present, either location is PEGylated in some embodiments. In other embodiments, the carboxyl group located the furthest away from the moieties interacting with the target is used. In some embodiments, the carboxyl group that causes the least hindrance on activity is used (whether or not it is located the furthest way from the moieties interacting with the target).

[0167] In some embodiments, for those compounds Nos 1 -59 having two or more carboxyl, hydroxyl, amino and/or sulfhydryl groups, the compound is modified (e.g., PEGylated) at the site furthest away from the active site. In some embodiments, the site that causes the least hindrance on activity is used (whether or not it is located the furthest way from the moieties interacting with the target).

[0168] Methods for conjugating the PEG or modified PEG to the small molecule warheads in Table 1 , through reaction between functional groups (or functionalized groups) , including reaction with the above-mentioned functional groups (amino, sulfhydryl, hydroxyl, carboxyl) are used in several embodiments. Methods of conjugation can be found for example in "Bioconjugate Techniques" (3^rd Edition) 2013 by Greg T. Hermanson (http://www.sciencedirect.com/science/book/9780123822390) ; incorporated herein in its entirety by reference. Although PEGylation is used as an example, other polymers are used in some embodiments.

43

[0169] Non-limiting examples of conjugation sites according to some embodiments and chemistries for compounds in Table 1 are disclosed. For example, for structures 23, 46, and 58 of Table 1 , the existing carboxylic moiety (-COOH) could be conjugated to PEG-amine through formation of an amide bond using any one of several possible coupling agents (including, e.g., TBTU, HBTU, HOBt, DCC, and N- hydroxysuccinimide). Further, for example, for structures 7, 16, 17, 21 (secondary amino group), 29, 36, 39, and 58 of Table 1 , the existing amino group (-NH2) could be conjugated to PEG-COOH through formation of an amide bond using any one of several possible coupling agents (including, e.g., TBTU, HBTU, HOBt, DCC, and N- hydroxysuccinimide). Further, for example, for structures 6, 8, 24, 38, 43, and 45 of Table 1 , the existing hydroxyl moiety (-OH) could be conjugated to PEG-halide through formation of an ether bond in presence of a strong base (including, e.g. NaH, KH, and n- BuLi). These conjugation sites and chemistries are neither exhaustive nor limiting, and are included herein as examples only, and not intended to limit the scope of the embodiments described herein.

[0170] Identifying a conjugation site and developing a conjugation strategy and/or chemistry does not require that all the atoms and the structures of the starting compound are maintained. Once the active part of the compound has been identified or hypothesized, some atoms, groups and structures of the compound can be removed or modified while maintaining sufficient or similar target site binding and activity in several embodiments.

[0171] In alternative embodiments, the warhead employed in the LSE polymer conjugate is a small molecule targeting a VEGFR selected from one or more of the following: pyrimidine compounds; diamino-pyrimidines; vegfr-binding polypeptides; fluoro substituted omega-carboxyaryl diphenyl urea; azaindole kinase inhibitors; furo-and thienopyrimidine derivatives; tricyclic amine derivatives; heterocyclic inhibitors of kinases; pyridinyl-pyrimidinylamino-benzamide derivatives; pyrrolotriazine kinase inhibitors; fused heterocyclic derivatives; anthranylamidopyridines; anthranilamide pyridine amides; 2-amino-5-substituted pyrimidine inhibitors; isomeric fused pyrrolocarbazoles and isoindolones; benzyl-benzimidazolyl derivatives; vegfr-3 binding peptides; 1 ,3-benzoxazolyl derivatives; fused heterocyclic derivatives; peptide chains; indirubin derivatives; pyridinamide derivatives; n-oxide anthranylamide derivatives; pyridineamine derivatives; thienopyrimidine and furopyrimidine derivatives; 1 -(5-tert- butyl-2-aryl-pyrazol-3-yl)-3-[2-fluoro-4-[(3-oxo-4h-pyrido[2, 3-b]pyrazin-8- yl)oxy]phenyl]urea derivatives; pyrazolophenyl-pyridine compounds; 2-chloro-4-anilino- quinazoline compounds; n-((4-chloro-3-trifluoromethyl)phenyl)-n'-(2-fluoro-4-((2- hydroxymethylaminoformyl)-4-pyridyloxo)phenyl)urea; n-indol-1 -amide compounds; substituted aromatic urea compounds; naphthylamide compound; and 3-chloro- and 3- methoxy-n-methyl-2-pyridine carboxamide compounds.

[0172] In alternative embodiments, the LSE polymer conjugate comprises a VEGF inhibitor warhead selected from aflibercept, ziv-aflibercept, bevacizumab, sonepcizumab, VEGF sticky trap, cabozantinib, foretinib, vandetanib, nintedanib, regorafenib, cediranib, ranibizumab, lapatinib, sunitinib, sorafenib, plitidepsin, regorafenib, verteporfin, bucillamine, axitinib, pazopanib, fluocinolone acetonide, nintedanib, AL8326, 2C3 antibody, AT001 antibody, XtendVEGF antibody, HuMax-VEGF antibody, R3 antibody, AT001/r84 antibody, HyBEV, ANG3070, APX003 antibody, APX004 antibody, ponatinib, BDM-E, VGXIOO antibody, VGX200, VGX300, COSMIX, DLX903/1008 antibody, ENMD2076, I DUS815C, R84 antibody, KD019, NM3, MGCD265, MG516, MP0260, NT503, anti-DLL4/VEGF bispecific antibody, PAN90806, Palomid 529, BD0801 antibody, XV615, lucitanib, motesanib diphosphate, AAV2- SFLT01 , soluble Fltl receptor, AV-951 , Volasertib, CEP1 1981 , KH903, lenvatinib, lenvatinib mesylate, terameprocol, PF00337210, PRS050, SP01 , carboxyamidotriazole orotate, hydroxychloroquine, linifanib, ALGIOOI , AGN 150998, MP01 12, AMG386, ponatinib, PD173074, AVAIOI, BMS690514, KH902, golvatinib (E7050), dovitinib, dovitinib lactate (TKI258, CHIR258) , ORA101 , ORA102, Axitinib (Inlyta, AG013736) , PTC299, pegaptanib sodium, troponin, EG3306, vatalanib, BmablOO, GSK2136773, Anti-VEGFR Alterase, Avila, CEP7055, CLT009, ESBA903, GW654652, HMPL010, GEM220, HYB676, JNJ17029259, TAK593, Nova21012, Nova21013, CP564959, smart Anti-VEGF antibody, AG028262, AG 13958, CVX241 , SU 14813, PRS055, PG501 , PG545, PTI 101 , TG 100948, ICS283, XL647, enzastaurin hydrochloride, BC194, COT601 M06.1 , COT604M06.2, MabionVEGF, Apatinib, RAF265 (CHIR-265), Motesanib Diphosphate (AMG-706) , Lenvatinib (E7080), TSU-68 (SU6668, Orantinib), Brivanib (BMS-540215), MGCD-265, AEE788 (NVP-AEE788), ENMD-2076, OSI-930, CYC1 16, ΚΪ8751 , Telatinib, KR 633, SAR131675, Dovitinib (TKI- 258) Dilactic Acid, Apatinib, BMS-794833, Brivanib Alaninate (BMS-582664), Golvatinib (E7050), Semaxanib (SU5416), ZM 323881 HC1 , Cabozantinib malate (XL184) , ZM 306416, AL3818, AL8326, 2C3 antibody, AT001 antibody, HyBEV, ANG3070, APX003 antibody, APX004 antibody, ponatinib (AP24534), BDM-E, VGXIOO antibody (VGXIOO ORCADIAN), VGX200 (c-fos induced growth factor monoclonal antibody), VGX300, COSM IX, DLX903/1008 antibody, ENMD2076, sunitinib malate (Sutent®) , INDUS815C, R84 antibody, KD019, NM3, allogenic mesenchymal precursor cells combined with an anti- VEGF antagonist (e.g. , anti-VEGF antibody) , MGCD265, MG516, VEGF-Receptor kinase inhibitor, MP0260, NT503, anti-DLL4/VEGF bispecific antibody, PAN90806, Palomid 529, BD0801 antibody, XV615, lucitanib (AL3810, E3810), AMG706 (motesanib diphosphate), AAV2-sFLT01 , soluble Fltl receptor, cediranib (Recentin™), AV-951 , tivozanib (KRN-951 ), regorafenib (Stivarga®), volasertib (BI6727) , CEP1 1981 , KH903, lenvatinib (E7080), lenvatinib mesylate, terameprocol (EM 1421 ), ranibizumab (Lucentis®) , pazopanib hydrochloride (Votrient™), PF00337210, PRS050, SP01 (curcumin), carboxyamidotriazole orotate, hydroxychloroquine, linifanib (ABT869, RG3635) , fluocinolone acetonide (lluvien®) , ALG 1001 , AGN 150998, DARPin MP01 12, AMG386, ponatinib (AP24534) , AVA101 , nintedanib (Vargatef™), BMS690514, KH902, golvatinib (E7050), everolimus (Afinitor®), dovitinib lactate (TKI258, CH IR258), ORAIOI , ORA102, axitinib (Inlyta®, AG013736), plitidepsin (Aplidin®), PTC299, aflibercept (Zaltrap®, Eylea®), pegaptanib sodium (Macugen™, LI900015) , verteporfin (Visudyne®), bucillamine (Rimatil, Lamin, Brimani, Lamit, Boomiq) , R3 antibody, AT001 /r84 antibody, troponin (BLS0597), EG3306, vatalanib (PTK787) , BmablOO, GSK2136773, Anti-VEGFR Alterase, Avila, CEP7055, CLT009, ESBA903, HuMax-VEGF antibody, GW654652, HMPL010, GEM220, HYB676, JNJ17029259, TAK593, XtendVEGF antibody, Nova21012, Nova21013, CP564959, Smart Anti-VEGF antibody, AG028262, AG 13958, CVX241 , SU 14813, PRS055, PG501 , PG545, PTI 101 , TG 100948, ICS283, XL647, enzastaurin hydrochloride (LY317615), BC194, quinolines, COT601 M06.1 , COT604M06.2, MabionVEGF, Si-Spheres coupled to anti-VEGF or VEGF-R antibody, Apatinib (Y 968D 1 ), and AL3818.

[0173] Suitable protecting groups, in some embodiments, are for protecting functional groups during the conjugation of warhead and polymer. Various protecting groups as well as suitable means and conditions for protecting and deprotecting the substituents are used in several embodiments. The means and conditions of protecting and deprotecting employed depend on the nature of the involved functional groups. Protecting groups for hydroxy-, amino-, and/or carboxy residues are selected in several embodiments from acetonide, ethylidene methoxymethyl, 2-methoxyethoxymethyl, benzyloxymethyl, tetrahydropyranyl, methyl, ethyl, isopropyl, t-butyl, benzyl, triphenylmethyl, t-butyldimethylsilyl, triphenylsilyl, methoxycarbonyl, t-butyloxycarbonyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, acetyl, benzoyl, toluenesulfonyl, dimethoxybenzyl, nitrophenyloxycarbonyl, nitrobenzyloxycarbonyl, allyl, fluorenylmethyl, tetrahydrofuranyl, phenacyl, acetol, phenyl, trimethylsilyl, pyrrolidyl, indolyl, hydrazino and other protecting groups such as those that can be found in Greene T. W. , et al. , Protective Groups in Organic Synthesis, 4th ed. , John Wiley and Son, New York, N.Y. (2007) ; incorporated herein in its entirety be reference. The reagents and conditions of protecting and deprotecting reactions are in particular selected for their suitability at selectively attaching and removing the protecting group without adversely affecting the rest of the compound.

[0174] The polymer conjugates disclosed herein may also be prepared as pharmaceutically acceptable salts including salts of inorganic acids such as hydrochloric, hydroiodic, hydrobromic, phosphoric, metaphosphoric, nitric acid and sulfuric acids as well as salts of organic acids, such as tartaric, acetic, citric, malic, benzoic, glycolic, gluconic, succinic, aryl sulfonic, (e.g. , p-toluene sulfonic acids, benzenesulfonic), phosphoric, malonic, and the like. Suitable acids for formation of pharmaceutically acceptable salts are used in some embodiments. Further, pharmaceutically acceptable salts of compounds may be formed with a pharmaceutically acceptable cation. Pharmaceutically acceptable cations include, but are not limited to, alkali cations (Li+, Na+, K+) , earth alkali cations (Mg2+, Ca2+, Ba2+), ammonium and organic cations, such as quaternary ammonium cations. Synthesis Of Polymer Conjugates

[0175] The description above is not intended to be limiting and should be viewed as an example to guide the manufacture of the other compounds identified herein. The polymer conjugates may also be made as described in US Patent Nos. 8,673,347 and 8,926,955, both herein incorporated by reference. Several embodiments provide a method for the production of polymer conjugates of the active agents that result in a highly pure reaction product, obtained in high and consistent yields.

[0176] In one embodiment, the conjugation reaction of the process to synthesize a conjugate polymer compound is catalysed by a base in an organic solvent. The base may be a strong base. In one embodiment, the base is selected from the group of alkali metal hydrides, tertiary amines and/or alkoxide. In another embodiment, the base catalysing the polymer conjugation reaction is sodium hydride. Other bases, such as sodium methoxide, or triethylamine can also be used. In several embodiments, the molar ratio of the base catalyst to the compound is between about 1 : 1 and about 4: 1 , about 1 : 1 to about 1 .5: 1 and about 1 : 1 . The reaction may be carried out in an organic solvent, such as in anhydrous conditions (e.g., in a dry organic solvent). The water content in the solution mixture of the conjugation process may be equal or less than 200 ppm. The organic solvent may be selected from the group of dichloromethane, chloroform, Ν,Ν-dimethylformamide. In certain embodiments, the organic solvent is dichloromethane or anhydrous dichloromethane.

[0177] The conjugation reaction may be carried out under inert gas atmosphere, such as nitrogen or argon atmosphere. The reaction of the process may be carried out at a temperature of about -10° to about 60° C, about 0° to about 25° C or at room temperature after an initial step at 0° C.

[0178] Following the production of the target compound, the polymer conjugate may then be separated and purified from the reaction mixture. In one embodiment, the compound is obtained by purification of the crude mixture by flash chromatography. An automated gradient flash purification system may be used and may be equipped with a suitable column and solvent. The purification method may be selected from reverse phase and direct phase columns and the conditioning/elution solvent may be selected from dichloromethane, water, methanol, acetonitrile, ammonium formate buffer solution at different mixture ratios. In one embodiment, the compound is purified by a reverse phase flash chromatography equipped with a C18 cartridge and the purification is carried out by gradient elution with acetonitrile/water. In one embodiment, the compound is purified by a normal phase flash chromatography.

[0179] The product may then be dried e.g. over sodium sulphate and filtered off and the solvent is removed by evaporation under reduced pressure at 25° C. Purification of the target product is carried out in several embodiments. After the purification step the resultant polymer compound has a purity of at least about 95%, about 96%, about 97%, about 98%, about 98.5%, about 99% or about 99.5%. The disclosed process results in an overall mass yield of the compound from about 40% to about 98% by weight, or from about 50% to about 95% by weight based on the weight of a reactant compound.

[0180] In several embodiments, the polymer moiety which is covalently attached to the active entity is biocompatible, can be of natural or semi-synthetic or synthetic origin and can have a linear or branched structure. The polymer may be selected from poly(alkylene oxides), or from (polyethylene) oxides. However, other polymers include without limitation polyacrylic acid, polyacrylates, polyacrylamide or N- alkyl derivatives thereof, polymethacrylic acid, polymethacrylates, polyethylacrylic acid, polyethylacrylates, polyvinylpyrrolidone, poly(vinylalcohol), polyglycolic acid, polylactic acid, poly(lactic-co-glycolic) acid, dextran, chitosan, hydroxyethyl starch.

[0181] In some embodiments, the above-mentioned polymer moiety can carry an amino functional end-group or can be functionalized to carry an amino functional end-group. Hence, the polymer moiety can be an amino-activated polymer of general formula X— NH2.

[0182] The reaction of formation of the compositions identified herein may be carried out at a temperature of about 10° to about 60° C, about 15° to about 25° C. or at room temperature. The polymer moiety X may be a polyethylene glycol (PEG) moiety, wherein the terminal OH group can optionally be modified e.g. with C1 -C5 alkyl or C1 - C5 acyl groups, such as with C1 -, C2- or C3-alkyl groups or C1 -, C2- or C3 groups. The modified polyethylene glycol may be a terminally alkoxy-substituted polyethylene glycol, including a methoxy-polyethylene-glycol (mPEG).

[0183] In other aspects, the conjugated polymer compounds may be used as active agents in a topical medicament useful for the prevention, alleviation and/or treatment of dermal pathologies. It has been shown that the conjugated polymer compounds described herein are very advantageously used as topical medicament since they do not show adverse or toxic effects (e.g. irritation) when dermally administered or any phototoxic effect (e.g. photomutagenicity, phototoxicity or photosensitisation) (as shown in the studies described in the following examples).

[0184] The dermal pathologies for such treatment may be pathologies characterized by hyperproliferation of the keratinocytes, such as psoriasis, atopic dermatitis, chronic eczema, acne, pitiriasis rubra pilaris, keloids, hypertrophic scars and skin tumors, such as keratoacanthoma, squamous cell carcinoma, basal cell carcinoma. [0185] The compounds disclosed herein or pharmaceutically acceptable salts thereof can be administered as they are, or in the form of various pharmaceutical compositions according to the pharmacological activity and the purpose of administration. Yet another aspect is a pharmaceutical composition comprising an effective amount of at least one compound in Table 1 optionally together with pharmaceutically acceptable carriers, adjuvants, diluents or/and additives. Pharmaceutical carriers, adjuvants, diluents or/and additives are applied in the formulation of the pharmaceutical composition comprising a compound of embodiments identified herein.

[0186] The disclosed compounds can be employed as the sole active agent in a pharmaceutical composition. Alternatively, the compounds of Table 1 may be used in combination with one or several further active agents, e.g. other active pharmaceutical agents in the treatment of the conditions described herein.

[0187] In particular, the polymer conjugate compounds may be used in combination with at least one endogenous angiogenesis inhibitor, for example and not restricted to, angioarrestin, angiostatin (plasminogen fragment), antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59 complement fragment, endostatin (collagen XVIII fragment), fibronectin fragment, Gro-beta, heparinases, heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon inducible protein (IP-10), lnterleukin-12, kringle 5 (plasminogen fragment), metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prolactin 16 kD fragment, proliferin-related protein (PRP), retinoids, tetrahydrocortisol-S, thrombospondin-1 (TSP-1), transforming growth factor-beta or (TGF-b), vasculostatin, and vasostatin (calreticulin fragment).

[0188] In particular, the polymer conjugate compounds may be used in combination with at least one steroidal anti-inflammatory drug and/or one further agent capable of inhibiting an early mediator of the inflammatory cytokine cascade, e.g. an antagonist or inhibitor of a cytokine selected from the group consisting of TNF, IL-1 a, IL- 1 β, IL-Ra, IL-8, MIP-1 a, MIF-Ι β, MIP-2, MIF and IL-6. Particularly useful antiinflammatory drugs are selected from alclometasone dipropionate, amcinonide, beclomethasone dipropionate, betamethasone, betamethasone benzoate, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone sodium phosphate and acetate, betamethasone valerate, clobetasol butyrate, clobetasol propinate, clocortolone pivalate, Cortisol (hydrocortisone), Cortisol (hydrocortisone) acetate, Cortisol (hydrocortisone) butyrate, Cortisol (hydrocortisone) cypionate, Cortisol (hydrocortisone) sodium phosphate, Cortisol (hydrocortisone) sodium succinate, Cortisol (hydrocortisone) valerate, cortisone acetate, desonide, desoximetasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone diacetate, diflucortolone valerate, fludrocortisone acetate, fludroxycortide, flumetasone pivalate, flunisolide, fluocinolone acetonide, fluocinonide, fluocortolone, fluorometholone, flurandrenolide, fluticasone propionate, halcinonide, halobetasol propionate, medrysone, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetate, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide. Useful antagonists or inhibitors of a cytokine are selected from infliximab, etanercept or adalimumab.

[0189] The polymer conjugate compounds may be used in combination with at least one natural extract or essential oil which is anti-itching agent, for example and not restricted to, extracts of Abelmoschus esculentus, Actaea alba, Aglaia odorata, Alkanna tinctoria, Althaea officinalis, Altingia excelsa, Andropogon virginicus, Aralia nudicaulis, Aralia racemosa, Argemone mexicana, Barleria prionitis, Camelia sinensis, Caesalpinia digyna, Campsis grand/flora, Carissa congesta, Carthamus oxyacantha, Cassia tora, Chrysanthemum indicum, Cimicifuga racemosa, Cinnamomum camphora, Clematis vitalba, Cuscuta reflexa, Diospyros peregrina, Enicostema axillare, Hammamelis virginiana, Jatropha multifida, Lavandula officinalis, Lavandula latifolia, Liquidambar orientalis, Lithospermum officinale, Madhuca longifolia, Martynia annua, Medicago sativa, Michelia champaca, Mikania glomerata, Mimosa pudica, Oryza sativa, Phaseolus vulgaris, Phyllanthus urinaria, Phyllanthus virgatus, Pistacia vera, Polygonum hydropiper, Quercus ilex, Rauvolfia caffra, Ricinus communis, Rubus idaeus, Sagittaria sagittifolia, Sandoricum koetjape, Sapindus mukorossi, Schleichera oleosa, Sesbania grandi flora, Spondias dulcis, Tilia sp., Toona ciliata, Tragia involucrata, Trichosanthes quinquangulata, Vaccaria pyramidata, Ventilago madraspatana, Veratrum album or Xanthium strumarium among others

[0190] The polymer conjugate compounds may be used in combination with at least one synthetic compound or product of biotechnological origin which is an anti- itching agent, for example and not restricted to mepyramine (pyrilamine), antazoline, diphenhydramine, carbinoxamine, doxylamine, clemastine, dimenhydrinate, pheniramine, chlorphenamine (chlorpheniramine), dexchlorpheniramine, brompheniramine, triprolidine, cyclizine, chlorcyclizine, hydroxyzine, meclizine, cetirizine, levocetirizine, promethazine, thenaldine, alimemazine (trimeprazine), cyproheptadine, azatidine, ketotifen, acrivastine, astemizole, cetirizine, loratadine, desloratadine, mizolastine, terfenadine, fexofenadine, fexofenadine, azelastine, levocabastine, olopatadine, corticosteroids such as cortisone, hydrocortisone dexamethasone, prednisone; Neutrazen™ [INCI : Water, Butylene Glycol, Dextran, Palmitoyl Tripeptide-8] marketed by Atrium Innovations/Unipex Group, Meliprene [INCI : Dextran, Acetyl Heptapeptide- 1 ] marketed by Institut Europeen de Biologie Cellulaire/Unipex Group, Skinasensyl™ [INCI : Acetyl Tetrapeptide-15] marketed by Laboratoires Serobiologiques/Cognis, SymSitive® 1609 [INCI : 4-t-Butylcyclohexanol] marketed by Symrise, Symbiocell™ [INCI : Extract from Cestrum Latifolium] marketed by BASF, Gatuline®Derma-Sensitive [INCI : Octyldodecyl Myristate, Capparis Spinosa Fruit Extract] marketed by Gattefosse or MAXnolia [INCI : Magnolia Officinalis Bark Extract, Vitis Vinifera/Vitis Vinifera (Grape) Seed Extract, Tocopherol] marketed by Mibelle among others.

[0191] The polymer conjugate compounds may be used in combination with at least one physiological cooling agent, for example and not restricted to menthone glycerol acetal, menthyl lactate, menthyl ethyl oxamate, substituted menthyl-3-carboxylic acid amides (e.g. menthyl-3-carboxylic acid N-ethylamide, Na-(L- menthanecarbonyl)glycine ethyl ester, 2-isopropyl-N-2,3-trimethylbutanamide, substituted cyclohexanecarboxylic acid amides, 3-menthoxypropane-1 ,2-diol, 2- hydroxyethyl menthyl carbonate, 2- hydroxy propyl menthyl carbonate, N- acetylglycine menthyl ester, isopulegol, menthyl hydroxycarboxylic acid esters (e.g. menthyl 3- hydroxybutyrate), monomenthyl succinate, monomenthyl glutarate, 2- mercaptocyclodecanone, menthyl 2-pyrrolidin-5-onecarboxylate, 2,3-dihydroxy-p- menthane, 3,3,5-trimethylcyclohexanone glycerol ketal, 3-menthyl 3,6-di- and - trioxaalkanoates, 3-menthyl methoxyacetate and icilin.

[0192] Further agents which can be used in combination with the polymer compounds are e.g. antagonists and/or inhibitors of RAGE, antagonists and/or inhibitors of HMGB1 , antagonists and/or inhibitors of the interaction of a Toll-like receptor (TCR) with HMGB1 , the functional N-terminal lectin-like domain (D1 ) of thrombomodulin and/or a synthetic double-stranded nucleic acid or nucleic acid analogue molecule with a bent shape structure as described in the international patent application WO 2006/002971 which is herein incorporated by reference.

[0193] The compositions described herein may be administered by a physician or other professional. Patients may also be able to self-administer. In several embodiments, administration of the composition may be performed dermally, via, for example, ointments, creams, oils, liposomes or trans-dermal patches, or wherein the polymer conjugates are incorporated into liposomes.

[0194] In some embodiments, at least one excipient is provided. Excipients can include a nonaqueous or aqueous carrier, and one or more agents selected from moisturizing agents, pH adjusting agents, strontium ions (Sr2+), deodorants, fragrances, chelating agents, preservatives, emulsifiers, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, surfactants, beneficial agents, pharmaceutical agents, and other components for use in connection with the compositions described herein (such as topical compositions for treatment of the skin). In several embodiments, the composition is an anhydrous formulation to prevent skin irritation such as water- based irritant contact dermatitis or stinging sensation upon application to damaged skin. In another embodiment, the composition is formulated such that preservatives need not be employed (e.g., a preservative-free formulation) so as to avoid skin irritation associated with certain preservatives.

[0195] To facilitate application, the composition may be provided as an ointment, an oil, a lotion, a paste, a powder, a gel, a foam, or a cream. The composition may also include additional ingredients such as a protective agent, an emollient, an astringent, a humectant, a sun screening agent, a sun tanning agent, a UV absorbing agent, an antibiotic agent, an antifungal agent, an antiviral agent, an antiprotozoal agent, an anti-acne agent, an anesthetic agent, a steroidal anti-inflammatory agent, a nonsteroidal anti-inflammatory agent, an antipruritic agent, an additional antioxidant agent, a chemotherapeutic agent, an anti-histamine agent, a vitamin or vitamin complex, a hormone, an anti-dandruff agent, an anti-wrinkle agent, an anti-skin atrophy agent, a skin whitening agent, a cleansing agent, additional peptides, additional modified peptides, and combinations thereof. In a further embodiment, the composition may avoid irritants (such as animal or cellular-based materials) to avoid skin irritation.

[0196] In some embodiments, the compositions may be administered by injection or infusion, in particular by intravenous, intramuscular, transmucosal, subcutaneous or intraperitoneal injection or infusion and/or by oral, topical, dermal, nasal, inhalation, aerosol and/or rectal application, etc.

[0197] In a further embodiment, the compositions are administered reversibly immobilized on the surface of a medical device, in particular by binding, coating and/or embedding the compositions on a medical device, such as but not limited to, stents, catheters, surgical instruments, cannulae, cardiac valves, or vascular prostheses. After contacting the medical device with body fluid or body tissue, the reversibly immobilized compounds are liberated. Consequently, the coated medical devices act as drug delivery devices eluting the medicament, whereby the drug delivery kinetics can be controlled, providing an immediate release or a controlled, delayed or sustained drug delivery, for example. [0198] In some embodiments, the composition further comprises an enteric coating that resists degradation under the prevailing pH of the stomach and permits delivery to specific regions of the gastrointestinal tract.

[0199] The pharmaceutical compositions may also be used for diagnostic or for therapeutic applications. For diagnostic applications, the compound may be present in a labelled form, e.g. in a form containing an isotope, e.g. a radioactive isotope or an isotope which may be detected by nuclear magnetic resonance. In some embodiments, a therapeutic application is, in the case of a topical application, the prevention, alleviation and treatment of psoriasis and dermatitis.

[0200] The concentrations of the compounds in the pharmaceutical composition can vary. The concentration will depend upon factors such as the total dosage of the drug to be administered, the chemical characteristics (e.g. , hydrophobicity) of the compounds employed, the route of administration, the age, body weight and symptoms of a patient. The compounds typically are provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for topical administration. Typical dose ranges are from about 1 μg to about 1 g/kg of body weight per day; a dose range may be from about 0.01 mg/kg to 100 mg/kg of body weight per day, or about 0.1 to 20 mg/kg once to four times per day. In some embodiments, the dosage of the drug to be administered is likely to depend on variables such as the type and extent of the progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the selected compound and the formulation of the compound excipient, and its route of administration.

[0201] Although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it will be understood by those of skill in the art that modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather to also cover all modification and alternatives coming with the true scope and spirit of the embodiments of the invention(s) .

[0202] Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term 'including' should be read to mean 'including, without limitation,' 'including but not limited to,' or the like.

[0203] The indefinite article "a" or "an" does not exclude a plurality. The term "about" as used herein to, for example, define the values and ranges of molecular weights means that the indicated values and/or range limits can vary within ±20%, e.g. , within ±10%. The use of "about" before a number includes the number itself. For example, "about 5" provides express support for "5".

[0204] The phrases "active agent" and "active entity" are synonyms and can be used interchangeably.

[0205] The terms "CT103" and "SNA- 103" are synonyms and can be used interchangeably.

EXAMPLES

[0206] Non-limiting examples are provided below.

Example 1 : A first synthesis scheme for CT103, a PEGylated variant of the kinase inhibitor, Motesanib, (ChEMBL id 572881 , CAS 453562-69-1 ), by LSE Technology

[0207] ChEMBL id 572881 (CAS 453562-69-1 , illustrated in Figure 1 ) was identified as a promising protein kinase inhibitor. After bibliographic data mining, Motesanib resulted to inhibit several kinases whose misregulation is involved in psoriasis, including VGFR1 , VGFR2 and VGFR3 kinases at low nanomolar range.

[0208] Because of its peculiar polypharmacology profile, together with the feasibility of LSE modification and its readily commercial availability, Motesanib was considered an interesting starting point to research LSE-variants with improved therapeutic potential.

[0209] Figure 2 depicts the first synthesis scheme for CT103.

[0210] The following materials were used:

• Acetonitrile for HPLC - ACN (Merck/VWR, Cat No 1 .00030.2500)

• Ammonia solution 28-30% (Sigma Aldrich, Cat No 222, 122-8)

• Ammonia 2M solution in MeOH (Sigma Aldrich, Cat No 34, 142-8

• Bromotripyrrolidinophosphonium exafluorophosfate - PyBroP (Sigma Aldrich, Cat No 18565-16)

• Chloroform for HPLC - CH Cb ((Merck/VWR, Cat No 1 .02444.1000)

• Dichloromethane - DCM (VWR, Cat No. 23354.326)

• Dichloromethane, anhydrous (Sigma Aldrich, Cat No 27099-7)

• /V,/V-Diisopropylethylamine - DIPEA (Sigma Aldrich, Cat No 387649-100ML)

• Iodine (Sigma-Aldrich, Cat No 03551 -100G)

• Methanol - MeOH (VWR, Cat No 20864320)

• Motesanib - ChEMBL 572881 (CAS No: 453562-69- 1 ) (MedChem Express, Cat No HY- 10228, Lot No 05902) • mPEG-COOH 2015 Da (Iris Biotech GmbH, Cat No PEG 1158, Lot No 1210293)

• Silica flash column (Biotage Snap Ultra HP Sphere 25g, Cat. No FSUL-0442- 0025)

• Sodium hydrogencarbonate - NaHC0₃ (VWR Cat.no. 27780.291)

• Sodium sulfate - Na₂S0₄ (Merck/VWR, Cat No 1.06649.1000)

• RP flash column (Biotage Snap KP-C18-HS 30g, Cat. No FLS0-1 118-0030)

• RP loading samplet cartridge (Biotage KP-C18-HS 1.2g, Cat No. SAS-1 1 18- 0012)

• TLC (Fluka, Cat No 99577-1 EA)

• Trifluoroacetic acid - TFA (Sigma Aldrich, Cat No302031)

[0211] The following methods were used:

Analytical HPLC with UV detection at 210 and 254 nm and ELS detectors

(Waters 2695, 2487, 2424) (Reference Method: 10 to 100 2%min ELSD) HPLC analytical C18 column (eg. Phenomenex Jupiter C18 300A, 5μηι, 4.6x250mm, Cat No OOG-4053-EO)

Acetonitrile for HPLC, 0.1 % TFA (v/v)

H₂0 for HPLC, 0.1 % TFA (v/v)

Preparative flash chromatograph with UV detection at 210-254nm (Biotage

Isolera One), flow rate 25ml/min

Silica flash column (Biotage SNAP Ultra 25g HP Sphere)

Eluent A: Dichloromethane, 1 % NH₃ in MeOH

Eluent B: Methanol, 1 % NH₃ in H₂0

Preparative flash chromatograph with UV detection at 210-254nm (Biotage

Isolera One) flow rate 12ml/min

RP flash C18 column (Biotage SNAP KP-C18-HS 30g) Loading samplet cartridge (Biotage KP-C18-HS 1 .2g)

HPLC water with 0.1 % TFA (v/v)

HPLC acetonitrile with 0.1 % TFA v/v)

[0212] In a one neck 50 ml round bottom flask dried under nitrogen, 163.22mg of mPEG-COOH (MW 2015Da, 0.081 mmol, 1 .5eq) and 20mg of Motesanib (MW 373.45g/mol, 0.054mmol) were dissolved in 20ml of anhydrous DCM under magnetic stirring, at T=0°C. 37.76mg of PyBroP (MW 466.22g/mol, 0.081 mmol, 1 .5eq) and 37.62 ul of DIPEA (MW 129.24g/mol, d=0.742g/ml, 0.216mmol, 4eq) were added at T=0°C.

[0213] The ice bath was removed after 1 minute and the stirring continued at room temperature protected from direct light.

[0214] Reaction outcome was checked by TLC (CHCI₃/MeOH/NH₃ 90/10/1 vol/vol, Motesanib: Rf~0.54, Compound: Rf~0.45, unknown impurity Rf~0.09)

[0215] After 22 hours the reaction was quenched with 10 ml of NaHC0₃ 5% solution and extracted with dichloromethane. Organic layers were dried over anhydrous sodium sulfate. Evaporation under vacuum yielded 193.5 mg of crude (yellow wax).

[0216] The crude was dissolved in 2 ml of DCM and it was purified by flash chromatography on a silica column (Biotage SNAP Ultra 25g HP Sphere) , by direct injection (Method B) . The chromatogram (see Figure 3) didn't show any separation. Consequently the column was washed with 50% methanol and the eluate was collected and evaporated, giving 185 mg of material.

[0217] This material was redissolved in DCM and loaded onto a flash RP sample cartridge (Biotage SNAP sample 1 .2g) and evaporated under vacuum overnight.

[0218] Sample was then eluted into a flash RP column (Biotage SNAP KP- C18-HS) with method C. Sample chromatogram is reported in Figure 4.

[0219] Fractions 14 and 15 were analyzed by HPLC (Method A). They were lyophilized after acetonitrile evaporation.

[0220] Fraction 14 (Figures 5 and 6) yielded to 37.8mg of yellow wax (yield of 29.63%) , with purity >98% at 254nm and >94% at ELSD (there is almost 5% of residual PEG).

[0221] Fraction 15 (Figures 7 and 8) yielded to 73.36mg (yield of 57.41 %) of yellow wax with purity >99% at 254nm and 100% at ELSD. [0222] The mass of CT103 was estimated by MALDI/MS spectroscopy. The analysis was performed by Able Biosciences. Three PEG standards of certified molecular weight were used to calibrate the instrument (Sigma-Aldrich, MALDI Validation Set, Lot N. 03598-1057604). The sample was dissolved in water/MeOH 1/1 (0.5 mg/ml) and an aliquot analyzed in Reflectron (+) mode, using DHB as a matrix and sodium trifluoroacetate as a cationizing agent. The sample gave a strong positive ion MALDITOF mass spectrum (Figure 9) with a major series of singly-charged pseudomolecular ion cluster [M+Na]⁺ observed between m/z 1873 and 2841 , centered at approximately m/z 2356 and showing 44 Da differences. The final product was assigned the mass of 2379 Da.

[0223] The molecular identity of CT103 was confirmed by LC/MS and NMR analyses. Figure 10 depicts the structure of CT103.

Example 2: A second synthesis scheme for CT103 by LSE Technology

Abbreviations

24] The second synthesis scheme for CT103 production starting from lly available Motesanib is shown in Figure 12. Motesanib is depicted in Figure [0225] The selected synthetic scheme consisted of acylation reaction of Motesanib with MeO-PEG₂ooo-COOH, in the presence of a coupling reagent.

[0226] Three main batches of CT103, representative of the developed process, were produced. Results are summed up in Table 2. CT103 yield has been calculated assigning it 2286 Da as molecular weight (CT103 molecular weight has been calculated according to the following formula: CT103 molecular weight= Motesanib nominal mass+ MeO-PEG₂ooo-COOH molecular weight - 18.) as the molecular weight of MeO-PEG2000-COOH used for its preparation was 1931 Da. (The MeO-PEG2000- COOH molecular weight was obtained from the certificate of analysis provided by the supplier.)

Table 2: CT103 produced sam

[0227] A preliminary small scale synthesis of CT103 had been carried out in the above example, which produced the material used for the structural characterization and the in vitro activity tests.

[0228] The synthetic procedure consisted of acylation reaction of Motesanib with 1.5 equivalents MeOPEG₂₀oo-COOH, using 1.5 equivalents of PyBroP as coupling reagent and 4 eq. of DIEA as base, in DCM as solvent. Final CT103 was isolated through purification by reversed phase flash chromatography with 57% overall yield.

[0229] Starting from this procedure the development studies aimed at defining a reproducible synthetic protocol for the production of CT103 samples with a representative quality and high HPLC purity, suitable for scaling up and, preferably, with higher yield.

[0230] The results of next outlined experiments will be given as % area of product, as measured by HPLC analyses (@ 254 nm).

Step 1: Acylation reaction of Motesanib

[0231] Preliminary trials of acylation reaction of Motesanib with MeO- PEG₂ooo-COOH were carried out with TBTU, in the presence of DBU as base, and with WSC. [0232] TBTU was poorly reactive and resulted in a very low Motesanib conversion and low selectivity to CT103, while WSC performed quite well, as complete Motesanib conversion was achieved with high selectivity to CT103. The use of WSC appeared particularly attractive, being easily available on large scale and cost-effective. This reagent and its urea by-product formed during the reaction are water soluble, so easily removed by aqueous extraction. So acylation studies went on using this reagent.

[0233] The reactions were carried out in DCM as solvent at 25°C, using 1 .05 equivalent of MeO-PEG₂ooo-COOH, in the presence of an excess of WSC. A by-product with 528 Da as nominal mass (according to HPLC-MS) was formed, which could derive from the reaction of Motesanib with WSC. Figure 13 depicts a MW 528 Da by-product.

[0234] The effect of the WSC excess, which was reduced from 2.30 to 1.12 equivalents (Exp. N° 52, 53 and 54), was first evaluated. Then the order of reagent addition was assessed too. Trials from 52 to 54 were carried out by mixing in DCM Motesanib and MeO-PEG₂₀oo-COOH, and then adding WSC. In trial N° S5, MeO- PEG₂ooo-COOH was first activated with WSC, then Motesanib was added. Results are in Table 3.

Table 3: HPLC results (@254nm) of Motesanib acylation using

WSC as coupling reagent

[0235] Data show that contact mode of the reagents has a major effect on the selectivity. Crude CT103 samples obtained after aqueous work-up had a lower 528 Da by-product content as It is partially removed by water extraction. [0236] Crude CT103 purification by reversed phase flash chromatography was then studied, evaluating the effect of the gradient (linear or step) and eluent system (A: 0.1 % TFA in H₂0; B: ACN or A^' H_:iO; B: ACN). Results are shown in Table 4.

Table 4: Results of crude CT103 purification

[0237] Purified samples of CT103 with high HPLC purities and with overall yields ranging from 50 to 75%, were isolated.

[0238] According to the previous results, the use of 1 .1 equivalents of WSC, performing first the carboxylic acid activation, for the synthetic phase, and a step elution method using H₂0 as eluent A, for the purification phase, were selected for the production of representative samples of CT103.

[0239] The performance of the new starting materials (Motesanib and MeO- PEG2000-COOH) provided for the production phase was evaluated carrying out a trial using the selected experimental conditions (Exp. N° S6). CT103 was obtained with 99.9% HPLC purity and 66% yield from Motesanib.

Experimental procedure for CT103 synthesis

[0240] The selected synthetic protocol consisted, according to the previously described studies, of:

1 . acylation of Motesanib with 1 .05 equivalents of MeO-PEG2000-COOH, using 1 .1 equivalents of WSC as coupling reagent, in DCM as solvent, at 25°C;

2. purification by reversed phase flash chromatography using H₂0 and ACN as eluent phases. [0241] The reproducibility of the developed procedure was verified on gram scale, and three representative CT103 samples were synthesized (lot n° 2015GC04/S7, 2015GC04/S8, 2015GC04/S9).

[0242] The experimental procedure used for the production of CT103 sample, lot n° 2014CG 15/S7 is hereinafter described.

Synthesis of CT103 lot n° 2015GC04/S7

Acylation of Motesanib

[0243] In a 1 I four necked round bottom flask, equipped with magnetic stir bar, thermometer and condenser, protected from light by aluminum foil wrapping, 8.22 g of MeO-PEG₂₀oo-COOH were dissolved in 300 g of DCM at 25°C under nitrogen atmosphere. 0.86 g of WSC and 369 g of DCM were added. The solution was aged under stirring at 23°C for 15min. under nitrogen atmosphere, then 1 .5 g of Motesanib and 227 g of DCM were added. The reaction mixture was aged under stirring for 24 h. The reaction completion was checked by HPLC analysis (Figures 14, 15). The reaction mixture was transferred to a 1 I separating funnel, and washed with NaCI aqueous saturated solution (2 ^χ 200 ml). Phases were separated and the aqueous phase was extracted with DCM (1 ^χ 150 ml). The combined organic phases were concentrated to dryness by rotary evaporation under vacuum at 40°C. 12 g of crude CT103 as orange oil with 94.6% HPLC purity (@ 254 nm) were obtained (Figure 16).

CT103 purification

[0244] 12 g of crude CT103 were dissolved in 15 ml of H₂0 and, divided into three portions, purified by reversed-phase flash chromatography using a Biotage Isolera LS System equipped with a Biotage SNAP KP-C18-HS cartridge packed with 400 g of KP-C18-HS Silica (column volume 510 ml). The cartridge was equilibrated at 100 ml/min. with 765 ml of H₂0/ACN 80:20 v/v. Sample (9 g per purification) was loaded onto the cartridge by injection through a syringe.

[0245] The SNAP cartridge was eluted at 100 ml/min with:

1 . 255 ml of H₂0/ACN 80:20 v/v;

2. 1530 ml of H₂0/ACN 63:37 v/v;

3. 765 ml of H₂0/ACN 10:90 v/v.

[0246] The first portion of eluate (920-950 ml) was sent to the waste, then the eluted solvent was collected in fractions of 50-100 ml each.

[0247] UV profile (@ 254 and 210 nm) of one purification is depicted in Figure 17. In all, three purifications were carried out using the same conditions.

[0248] Collected individual fractions were analysed by HPLC. Fractions with HPLC purity > 99.5 % were combined (1 100 g) and concentrated under reduced pressure at 40°C to remove acetonitrile. The residual aqueous solution (622 g) was extracted with DCM (3 ^χ 150 ml). Brine (100 ml) was added during the first extraction to help phase separation. The combined organic phases were evaporated under reduced pressure at 35°C to dryness, affording 9 g of oil, which was treated with 300 ml of diethyl ether and aged for 0.5 h at 23°C, and for 1 .0 h at -18°C. The precipitated solid was filtered over sintered glass filter (G3), washed with 10 ml of diethyl ether and dried under vacuum at 30°C for 20 h and 40°C for 2 h. 6.6 g of CT103 as a white solid were obtained (lot n° 2015GC15/S7). Final CT103 sample was stored at - 22°C. CT103 characterization

CT103 lot n° 2015GC04/S7

[0249] Purity of CT103 sample, determined by HPLC analysis @ 254 nm (method Ml CT103 001), was 99.87% (Figure 18).

[0250] The product was characterized by 1 H-NMR and ESI MS analysis (Figures 19, 20, 21).

[0251] Certificate of analysis of the CT103 sample is shown in Figure 22.

CT103 lot n° 2015GC04/S8

[0252] Purity of CT103 sample, determined by HPLC analysis @ 254 nm (method Ml CT103 001), was 99.93% (Figure 23).

[0253] The product was characterized by 1 H-NMR and ESI MS analysis (Figures 24, 25, 26).

[0254] Certificate of analysis of the CT103 sample is shown in Figure 27.

CT103 lot n° 2015GC04/S9

[0255] Purity of CT103 sample, determined by HPLC analysis @ 254 nm (method Ml CT103 001), was 99.93% (Figure 28).

[0256] The product was characterized by 1 H-NMR and ESI MS analysis (figures 29, 30, 31).

[0257] Certificate of analysis of the CT103 sample is shown in figure 32.

Analytical Methods

HPLC method Ml CT103 001

[0258] The following method has been used both for reaction monitoring and for assessing chemical purity of final CT103 samples.

Example 3: Profiling study of CT103 against 270 kinases

[0259] CT103 (test concentration: 1 μΜ) was tested against 270 kinases. Materials and Methods

Preparation of test compound solution

[0260] The test compound (CT103) was dissolved in and diluted with dimethylsulfoxide (DMSO) to achieve 100-fold higher concentration. Then the solution was further 25-fold diluted with assay buffer to make the final test compound solution. Reference compounds for assay control were prepared similarly.

Assay reagents and procedures

IMAP assay

[0261] 1) The 5 μΙ_ of x4 compound solution, 5 μΙ_ of x4 Substrate/ATP/Metal solution, and 10 μΙ_ of x2 kinase solution were prepared with assay buffer (20 mM HEPES, 0.01 % Tween-20, 2 mM DTT, pH7.4) and mixed and incubated in a well of polystyrene 384 well black microplate for 1 hour at room temperature.

[0262] 2) 60 μΙ_ of IMAP binding reagent (IMAP Screening Express kit; Molecular Devices) was added to the well, and incubated for 30 minutes.

[0263] 3) The kinase reaction was evaluated by the fluorescence polarization at 485 nm for excitation and 530 nm for emission of the well.

Off-chip Mobility Shift Assay (MSA)

[0264] 1) The 5 μΙ_ of x4 compound solution, 5 μΙ_ of x4 Substrate/ATP/Metal solution, and 10 μΙ_ of x2 kinase solution were prepared with assay buffer (20 mM HEPES, 0.01 % Triton X- 100, 2 mM DTT, pH7.5) and mixed and incubated in a well of polypropylene 384 well microplate for 1 or 5 hour(s)* at room temperature. (*; depend on kinase)

[0265] 2) 60 μΙ_ of Termination Buffer (QuickScout Screening Assist MSA; Carna Biosciences) was added to the well.

[0266] 3) The reaction mixture was applied to LabChip3000 system (Caliper Life Science), and the product and substrate peptide peaks were separated and quantitated.

[0267] 4) The kinase reaction was evaluated by the product ratio calculated from peak heights of product(P) and substrate(S) peptides (P/(P+S)).

Reaction Conditions

[0268] The reaction conditions are depicted below:

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Data Analysis

[0269] The readout value of reaction control (complete reaction mixture) was set as a 0% inhibition, and the readout value of background (Enzyme(-)) was set as a 100% inhibition, then the percent inhibition of each test solution was calculated.

Results

[0270] The results are shown in Table 5 below.

Table 5: Target kinase inhibition by CT103

MLK1 Cascade -20.7

Example 4: IC50 Determination study of CT103 against 2 kinases

[0271] CT103 inhibition of FLT1 and KDR was examined at test concentrations of 50μΜ, 15μΜ, 5μΜ, 1 .5μΜ, 0.5μΜ, 0.15μΜ, 0.05μΜ, 0.015μΜ , 0.005μΜ, and 0.0015μΜ.

Materials and Methods

Preparation of test compound solution

[0272] The test compound was dissolved in and diluted with dimethylsulfoxide (DMSO) to achieve 100-fold higher concentration. Then the solution was further 25-fold diluted with assay buffer to make the final test compound solution. Reference compounds for assay control were prepared similarly.

Assay reagents and procedures

STK-ELISA

[0273] 1 ) The 10 μΙ_ of x4 compound solution, 10 μΙ_ of x4 Substrate/ATP/Metal solution, and 20 μΙ_ of x2 kinase solution were prepared with assay buffer (15 mM Tris-HCI, 0.01 % Tween-20, 2 mM DTT, pH7.5) and mixed and incubated in a well of polypropylene 96 well microplate for 1 hour at room temperature.

[0274] 2) 120 μΙ_ of 40 mM EDTA solution (pH 7.5) was added to the well, and then 120 μΙ_ of the mixture was transferred to the well of ELISA plate (see below table) .

[0275] 3) After 30 minutes incubation, the well was washed 4 times, and blocked with blocking buffer containing 0.1 % BSA.

[0276] 4) 100 μΙ_ of the first antibody (see below table) solution was added to the well and incubated for 30 minutes.

[0277] 5) After 4 times washing of the well, 100 μΙ_ of the second antibody (sell below table) solution was added to the well, and incubated for 30 minutes.

[0278] 6) After washing the well, 100 μΙ_ of TMB solution (MOSS Inc.) was added and incubated for 5 minutes. To stop the HRP reaction, 100 μΙ_ of 0.1 M sulfuric acid was added.

[0279] 7) The kinase reaction was evaluated by the absorbance at 450 nm of the well.

Off-chip Mobility Shift Assay (MSA)

[0280] 1) The 5 μΙ_ of x4 compound solution, 5 μΙ_ of x4 Substrate/ATP/Metal solution, and 10 μΙ_ of x2 kinase solution were prepared with assay buffer (20 mM HEPES, 0.01 % Triton X- 100, 2 mM DTT, pH7.5) and mixed and incubated in a well of polypropylene 384 well microplate for 1 hour at room temperature.

[0281] 2) 60 μΙ_ of Termination Buffer (QuickScout Screening Assist MSA; Carna Biosciences) was added to the well.

[0282] 3) The reaction mixture was applied to LabChip3000 system (Caliper Life Science), and the product and substrate peptide peaks were separated and quantitated.

[0283] 4) The kinase reaction was evaluated by the product ratio calculated from peak heights of product(P) and substrate(S) peptides (P/(P+S)).

Reaction Conditions - The reaction conditions are depicted below:

Data Analysis

[0284] The readout value of reaction control (complete reaction mixture) was set as a 0% inhibition, and the readout value of background (Enzyme(-)) was set as a 100% inhibition, then the percent inhibition of each test solution was calculated. The IC50 value was calculated from concentration vs. %lnhibition curves by fitting to a four parameter logistic curve.

Results

[0285] The results are shown in Table 6 below. Table 6: IC50 Determination

Example 5: Pharmacokinetic study of a single dose of CT103 in BALB/c mice

[0286] This study is aimed at validating a pharmacokinetic (PK) analysis method (Part A) and evaluating the PK of CT103 (Part B).

Methodology

Compounds and Reagents

• Distilled water, 15230, Life Technologies

• Propylene Glycol, P4347, Sigma

• Transcutol P (Gattefosse, lot 450931007)

• CT103 initially was a solid compound diluted in vehicle (sodium chloride 0.9% , saline for intra-venous administrations and 25% Transcutol/75% Propylene Glycol for epicutaneous administrations).

Experimental outline

Part A: Method optimization

Samples

[0287] Terminal blood samples from adult male BALB/c mice were collected into K2EDTA-coated tubes. Blood samples were processed to isolate plasma. Samples were pooled and stored at -80°C until further analysis.

CT103

[0288] CT103 was weighed out and reconstituted in methanol to give a stock solution at 10 mg/mL. All chemicals used for chromatography were of HPLC grade (Fisher Scientific Ltd. , Loughborough, UK) . Liquid Chromatography (LC) conditions

[0289] Samples were analyzed on a Waters Alliance 2695 High Pressure LC separations module in combination with a Waters Diode Array Detector and Waters Micromass ZMD mass spectrometer. The samples were analyzed by reversed phase liquid chromatography employing a gradient separation. Mobile phase A (MPA) consisted of 90% dH20: 10% methanol containing 0.1 % formic acid. Mobile phase B (MPB) consisted of 90% methanol: 10% dH20 containing 0.1 % formic acid. The column was a Acquity BEH C18 (1 .7 μηι, 100 x 2.10 mm). The gradient conditions started at 50% MPB and rose linearly to 100% MPB at 10 minutes post injection, returning to the initial conditions at eleven minutes. The column was allowed to re-equilibrate before the next injection. Flow rate was 0.3 mL/min and total run time per injection was twenty minutes.

Mass Spectrometry (MS) conditions

[0290] The MS was used in electrospray positive mode, with a capillary voltage of 3.0 kV and a cone voltage of 20 V. Source and desolvation temperatures and gas flows settings were standard tor the system. SIR channels were created to measure CT103 at mass-to-charge ratio (m/z) 794,595.8 and 476.8. This relates to MW = 2379.0.

Stability

[0291] Replicate plasma samples were spiked with 10 μg/mL of compound CT103. Three replicates were extracted and analyzed fresh, three were frozen at -80°C for 60 minutes before being defrosted, extracted and analyzed. Peak areas were compared to assess stability of compound CT103.

Part B: PK Analysis

[0292] Adult male BALB/c mice were randomly allocated to experimental groups and allowed to acclimatise tor one week. On Day 0, CT103 was administered either by epicutaneous application onto to the clipped skin of the back (50 mg/kg dose in 100 μΙ_) or by intra-venous administration (10 mg/kg dose) .

[0293] Epicutaneous applications were performed in non-anaesthetised but restrained animals. Animals were housed individually to prevent interferences with the application site. Blood samples were collected into K2EDTA-coated tubes according to the schedule below. Non-terminal blood samples were taken from a superficial vein and terminal blood samples were taken from a cardiac bleed. Blood samples were processed to isolate plasma. Samples were stored at -80°C until further analysis. Administration and Bleed schedule

[0294] All Groups are n=4. Vehicle for intravenous administrations was a 0.9% sodium chloride solution (saline). Vehicle for epicutaneous administrations was 25% Transcutol P/75% Propylene Glycol. Administration volume for epicutaneous application was 100 μΙ_ per mouse per day. Administration volume for intravenous injection was 5 mL/kg.

Readouts

Method validation

[0295] Method validation was performed on plasma prepared from blood collected then pooled from strain-, gender- and age-matched mice. The following parameters were tested: selectivity, linearity of standard calibration curve, accuracy and precision, lower detection/quantitation limit's (LLOQ) accuracy and precision and extraction efficacy.

PK analysis

[0296] Study samples were processed by HPLC for quantitative analysis of CT103 in plasma. Results

Method validation

[0297] Figure 33 depicts chromatograms of a CT103 plasma standard extract (50 μg/mL) detected using SIR (TIC, upper) and UV at 337 nm (lower).

[0298] Plasma calibration standards were prepared from BALB/c mouse blank plasma samples. The calibration range was 0.25 to 50 μg/mL. The calibration curve was assessed over the range 0.1 -100 μg/mL. The calibration standard at 0.1 μg/mL was detectable but too small to be accurately quantified and therefore was not included in the curve. The calibration standard at 100 μg/mL was slightly outside the linear range due to saturation of the system and was therefore not included in the curve. Curve parameters were calculated with no weighting and a zero intercept. The r² value was 0.9965, indicating good method reproducibility (Figure 34).

[0299] A 100 μΙ_ volume of compound spiked plasma was extracted in 300 μΙ_ ice- cold methanol, evaporated to dryness and reconstituted in 100 μΙ_ methanol. A 10 μΙ_ aliquot was injected into the LC-MS system.

[0300] The reproducibility of the injection, tested by repeat injection (n=6) of a sample at 10 μg/mL, was calculated at 6.88% CV. The extraction efficiency was determined to be 63.2% from six CT103 standards and six plasma extracts, and reproducibility of repeat sample extracts was 6.62% CV at a concentration of 10 μg/mL.

[0301] Figure 35 depicts method validation and Figure 36 depicts individual chromatograms of SIR channels used for the analysis of compound. The limit of detection (LOD) was assessed from compound standard injections. LOD was determined to be 0.1 μg/mL using MS detection. Compound at 0.1 μg/mL was detectable, however the peak was too small to accurately quantify and therefore this point was set as the LOD. The limit of quantitation (LOQ) was determined to be 0.25 μg/mL using MS detection. LOQ was assessed from three repeat plasma extract samples. At 0.25 μg/mL compound was detected, with the peak signal at least three times the background signal (a standard LC reference test), therefore this point was set as the LOQ. Variability at LOQ was 9.6% CV for three separate repeat plasma extracts.

[0302] Freeze-thaw stability of CT103 in murine plasma was assessed over one freeze-thaw cycle. There was no decrease in CT103 peak area observed in the triplicate samples that underwent a freeze-thaw cycle compared to the peak areas observed in the triplicate samples extracted immediately after preparation. CT103 is assumed stable for at least one freeze-thaw cycle. Pharmacokinetic analysis

[0303] Data for all samples were assessed using the summed total ion chromatogram (TIC) channel generated from the three individual channels (m/z 794, 595.8 and 476.8) by the LC-MS software. UV detection was not sensitive enough to analyse CT103 concentrations below 10 μg/mL and was therefore not used in sample analysis. Figure 37 depicts representative chromatograms showing CT103 in methanol and extracted from a spiked murine plasma calibration standard.

[0304] Figure 38 depicts mouse plasma concentrations of CT103. Data were limited for the samples taken following epicutaneous administration of CT103 at 50 mg/kg, with most concentration levels found below the limit of detection (0.1 μg/mL). A C_max of 1 .47 μg/mL was seen at 15 minutes post administration (one individual had a concentration of 2.31 μg/mL at 30 minutes post administration), and compound was detectable up to 2 hours after administration. AUC|_ast for epicutaneous administration was 2.46 μg/mL, and terminal t1/2 was 0.41 hours.

[0305] CT103 was detectable for up to 8 hours following intra-venous administration at 10 mg/kg, however the 8 hour values were at the LOD. One individual at 3 hours post administration had detectable levels of compound however this value appears unusually high and was therefore excluded from calculations. A C_max of 8.12 μg/mL was seen at 10 minutes post administration. AUC|_ast was 3.82 μg/mL terminal t1/2 was 0.33 hours.

Conclusions

Part A

[0306] A suitable compound separation method was established and the protein precipitation method of extraction gave an efficiency of 63.2% (10 μg/mL).

[0307] The reproducibility of the system was good for repeat injections (6.88%, n=6) and repeat sample extraction (6.62% for a concentration of 10μg/mL n=6). The LOD was determined to be 0.1 μg/mL and the LOQ 0.25 μg/mL with a reproducibility of 9.63% (n=3).

Part B

[0308] CT103 is stable in murine plasma following a single freeze-thaw cycle. CT103 concentrations were measured in mouse plasma samples after epicutaneous or intravenous administration. The C_max observed in the IV administration samples was much higher (over 5 fold) than the epicutaneous administration samples (8.12 μg/mL and 1 .47 μς/ηιί respectively). AUC|_ast values were much closer at 3.82 (IV) and 2.46 (epicutaneous) , due to the longer t1 /2 seen following epicutaneous administration.

Example 6: Evaluation of the ability of CT103 to inhibit VEGF-induced

HUVEC cell proliferation

[0309] This study tested the ability of CT103 to inhibit VEGF- induced HUVEC cell proliferation.

Methodology

Compounds and Reagents

[0310] Motesanib Diphosphate (AMG-706), Source: MedChemExpress, CAS #453562-69- 1 , lot HY-10228-05902.

Experimental outline

Part A: Selection of Appropriate Time Frames and assay conditions

[0311] Primary HUVEC cells (below passage 7) were seeded onto collagen- coated 96 well plates at two different concentrations 2 x 10³ or 5 x 10³ and treated with two different concentrations of VEGF: 10 ng/mL or 50 ng/mL. Some cells were not treated with VEGF to give background levels of non-VEGF driven proliferation. Cells were incubated for 72 hours at 37°C with 5% C0₂. Proliferation was measured by pulsing cells for the last 24 hours with tritiated thymidine. Plates were then harvested and assayed for tritiated thymidine incorporation. The ability of CT103 to inhibit proliferation was assessed by pre-incubation of cells with CT103 for 6 hours or 18 hours prior to stimulation with VEGF. This was tested using the following concentrations of compound: 100 μΜ , 30 μΜ and 10 μΜ. Motesanib Diphosphate (AMG-706) 10 nM was included as a positive control. Each condition was tested in sextuplicate.

[0312] The conditions tested were as follows: HUVEC VEGF CT103 CT103 VEGF Incu ation cell concentration pre- concentration time

den it incubation

ime

0 ng/ml 100μ

30μ

0

0 ng/ml ΙΟΟμΜ

30u

2x10^¾

10

0

50 ngml

30μ

ϊϋμΜ

en 0

and

18 1Ο0μ

N

3Du

0

10 ngml 100μ

30μ

ΙΟμ

0

50 ngmL 10ϋμΜ

30μίν1

ΙΟμ

0 Part B: IC₅₀ calculation for CT103 inhibition of VEGF induced HUVEC proliferation.

[0313] Primary HUVEC cells (below passage 7) were seeded onto collagen- coated 96 well plates at 5x10³ cells per well. The ability of CT103 to inhibit proliferation was assessed by pre-incubation of cells with CT103 for 18 hours prior to stimulation with VEGF (50 ng/mL) . To generate an IC₅₀ calculation, a series of 3-fold dilutions of CT103 starting at 300 μΜ was performed. Motesanib Diphosphate (AMG-706) was included as a positive control at a concentration of 10nM . Each condition was tested in sextuplicate.

Readouts

[0314] HUVEC proliferation was assayed by measuring tritiated thymidine incorporation into DNA. Cells were harvested using a harvester onto a 96 well grid filter mat and thymidine incorporation was read using a β-counter.

Results

Part A: Selection of Appropriate Time Frames and assay conditions

Pre-incubation time: 6 hours

[0315] Figure 39 depicts tritiated thymidine incorporation in corrected counts per minute (CCPM) with a pre-incubation time of 6 hours.

Pre-incubation time: 18 hours

[0316] Figure 40 depicts tritiated thymidine incorporation in corrected counts per minute (CCPM) with a pre-incubation time of 18 hours.

[0317] Proliferation data were analyzed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental groups.

Table 7: Proliferation data, Dunnett's post-test results

[0318] Analyses suggested that the best experimental conditions for Part B would be (results reported in Figure 41):

Cells seeded at 5 x 10³ cells per well

Pre-incubation with compounds for 18 hours

Stimulation with VEGF at 50 ng/mL

[0319] Figure 42 depicts percentage inhibition by CT103 with 18 hour preincubation time, 5x10³ cells per well and 50ng/ml_ VEGF stimulation.

[0320] Data generated using these experimental conditions were further analyzed by one-way ANOVA followed by Dunnett's for multiple comparisons between experimental groups.

Part B: IC₅o calculation for CT103 inhibition of VEGF induced HUVEC proliferation

[0321] Figure 43 depicts inhibition of HUVEC proliferation at a range of CT103 concentrations. Figure 44 depicts non-linear fit of transformed normalised maximal response for IC50 calculation. Figure 45 depicts percentage inhibition of VEGF induced proliferation by CT103. Conclusions

[0322] CT103 inhibited VEGF-induced HUVEC proliferation at both cell densities and pre-incubation time points. At 18 hours pre-incubation, HUVEC proliferation is substantially higher in the presence of VEGF when compared to unstimulated cultures. The 5x10³ cell density gave higher proliferation read outs than 2x10³ cells per well, thus a clear inhibition can be seen. Therefore better conditions for monitoring VEGF-induced HUVEC proliferation are a cell density of 5x10³ cells per well stimulated with 50 ng/mL VEGF and a pre-incubation time of 18 hours. These conditions were used to generate IC₅₀ values for CT103 inhibition of VEGF-induced HUVEC proliferation by performing a series of 3-fold dilutions, starting at a top concentration of 300 μΜ . A clear effect of CT103 was observed on the VEGF-induced HUVEC proliferation and an IC₅₀ value could be calculated. An IC₅₀ of 18.5μΜ for CT103 inhibition of VEGF-induced HUVEC proliferation was determined.

Example 7: Evaluation of CT103 Efficacy in a mouse model of imiquimod-induced psoriasis

[0323] This study used a mouse model of imiquimod-induced psoriasis to test the efficacy of CT103.

Methodology

Compounds and Reagents

• Imiquimod 5% w/w (Aldara)

• Betamethasone 17-valerate 0.1 % (Manx Pharma)

• Propylene Glycol (P4347)

• Transcutol P (Gattefosse, lot. 450931007)

• CT103 was provided as a solid compound to be solved in vehicle (25% Transcutol P and 75% Propylene Glycol for epicutaneous applications)

Experimental outline

[0324] Adult male BALB/c mice were randomly allocated to experimental groups and allowed to acclimatise for one week. On Day 0, animals were sensitised by topical application of 62.5 mg of imiquimod cream 5% weight/weight (Aldara™, 3M) , corresponding to ca. 3 mg of active ingredient, to their clipped back under gas (isoflurane) anaesthesia. The procedure was repeated for ten consecutive days. Treatments were given according to the schedule below with non-anaesthetised but restrained animals.

[0325] From Day 0 until the end of the experiment on Day 10, animals were monitored daily for non-specific clinical signs to include abnormal posture, coat condition and/or activity level. From Day 0 until Day 10, animals were scored daily for signs of skin inflammation to include erythema, scaling and thickening of the skin. On Day 10, animals were culled and skin biopsies performed. One biopsy per animal was stored at ambient temperature in tissue fixative until further optional analysis by histopathology. One biopsy per animal was snap frozen and stored at - 80°C until further optional analysis of tissue cytokines.

Treatment Groups and Dosages

[0326] All Groups were n= 12. Vehicle for epicutaneous administrations was 25% Transcutol P, 75% propylene glycol. Administration volume for treatment epicutaneous applications was 100 μΙ_ per mouse back.

n/a: not applicable, EC: epicutaneous application BID: twice daily, SID: once daily, epicutaneous administration on the clipped skin of the back. Clobetasol propionate was administered in the morning only. * Two hours before and four hours after the IMQ challenge.

Readouts

Bodyweights

[0327] From Day 0, animals were weighed three times a week. Data were graphed (Mean ± SEM) . Non-specific clinical observations

[0328] From Day 0 until the end of the experiment, animals were checked daily for non-specific clinical signs to include abnormal posture (hunched), abnormal coat condition (piloerection) and abnormal activity levels (reduced or increased activity).

Clinical Observations

[0329] From Day 0 until Day 10, animals were scored daily for clinical signs of skin inflammation using the following scoring system: erythema, scaling and skin thickening were each scored on a four point-scale where (0) is None, (1) is Slight, (2) is Moderate, (3) is Marked and (4) is severe. The cumulative score is a measure of the severity of the back skin inflammation. Scores for each criterion and cumulative scores were provided along with the standard error of the mean (SEM).

Histopathology

[0330] At the end of the experiment, skin biopsies were performed and one sample per animal was stored in tissue fixative. Samples were processed, sectioned and stained with Haematoxylin & Eosin in order to study tissue morphology, pathological changes and inflammation. Samples were scored for acanthosis, parakeratosis, presence of pustules and dermal inflammation using an established semi-quantitative scoring system. Acanthosis: the thickest and best orientated area of epidermis was examined and the number of epidermal layers at that level recorded. Parakeratosis: the thickest and best orientated area of epidermis was examined and the absence (score 0) or presence (score 1) of keratinisation was recorded. Presence of pustules: a number of pustules per millimeter length of skin was calculated. Additionally, dermal inflammation was scored as: (0) No inflammation, (1) light scattering of dermal inflammatory cells not involving epidermis or adnexal structures, (2) moderate infiltration of dermal inflammatory cells with only occasional infiltration of epidermis or adnexal structures and (3) marked infiltration of dermal inflammatory cells with infiltration of epidermis and adnexal structures and focal disruption of these structures.

Tissue cytokine analysis

[0331] At the end of the experiment, skin biopsies were performed and one sample per animal was stored at -80°C. To each skin biopsy, extraction buffer was added and the tissue was homogenised and centrifuged. After centrifugation, cytokine analysis was performed on the supernatant to determine the concentration of IL-17A, IL- 22 and IL-23 in all samples. Samples were analysed using multiplex xMAP bead technology, which utilises microspheres as a solid support for sandwich immunoassays and determination of numerous cytokines in the same sample. A standard curve of quantified recombinant cytokines and chemokines was used to convert detected PE fluorescence values to cytokine concentrations (pg/ml).

Results

Bodyweights

[0332] Bodyweights, expressed as percentages of the initial (Day 0) bodyweights were analysed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental groups. Results are presented in Figure 46.

[0333] Imiquimod application induced a significant decrease of the bodyweights in the vehicle-treated group on Day 3 (p < 0.0001), Day 6 (p < 0.001), Day 8 (p < 0.0001) and Day 10 (p < 0.0001) when compared to Day 0.

[0334] Clobetasol propionate induced a significant decrease of the bodyweights when compared to the vehicle-treated group on Day 3 until the end of the experiment on Day 10 (p < 0.05 on Day 3 then p < 0.0001).

[0335] Bodyweights in the CT103_5%-treated group were significantly higher than in the vehicle-treated group on Day 8 (p < 0.05).

[0336] Bodyweights in the CT103_10% group did not differ from the bodyweights measured in the vehicle-treated group.

[0337] Bodyweights in the CT103_20%-treated group were significantly higher than in the vehicle-treated group from Day 3 until the end of the experiment on Day 10 (p < 0.001 then p < 0.0001).

Non-Specific Clinical Observations

[0338] From Day 0 until the end of the experiment, animals did not show any non- specific clinical signs to include abnormal posture (hunched), abnormal coat condition (piloerection) and abnormal activity levels (reduced or increased activity).

Clinical Observations

[0339] Erythema scores, scaling scores, thickness scores and total skin scores were analyzed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental groups. Erythema Scores

[0340] As shown in Figure 47, Imiquimod induced a highly significant increase of the erythema scores from Day 2 until the end of the experiment on Day 10 in the vehicle-treated group when compared to Day 0 (p < 0.0001).

[0341] Clobetasol propionate induced a highly significant decrease of the erythema scores when compared to vehicle-treated group from Day 2 until the end of the experiment on Day 10 (p < 0.0001).

[0342] CT103 administered at 5% did not significantly reduce the imiquimod- induced erythema when compared to the vehicle-treated group.

[0343] CT103 administered at 10% did not significantly reduce the imiquimod- induced erythema when compared to the vehicle-treated group.

[0344] CT103 administered at 20% significantly reduced the imiquimod- induced erythema scores when compared to the vehicle-treated group from Day 5 until the end of the experiment on Day 10 (p < 0.01 on Day 5, p < 0.05 on Day 6 and Day 7, p < 0.001 on Day 8 and Day 9, p < 0.0001 on Day 10).

Scaling scores

[0345] As depicted in Figure 48, Imiquimod induced a significant increase in scaling scores from Day 5 until the end of the experiment on Day 10 when compared to Day 0 in the vehicle- treated group (p < 0.05 on Day 5 and Day 7, p < 0.0001 on Day 8, Day 9 and Day 10).

[0346] Clobetasol propionate significantly reduced the scaling scores when compared to the vehicle-treated group from Day 8 until the end of the experiment on Day 10 (p < 0.0001).

[0347] CT103 administered at 5% significantly reduced the imiquimod- induced scaling when compared to the vehicle-treated group on Day 8 (p < 0.05), Day 9 and Day 10 (p < 0.0001).

[0348] CT103 administered at 10% significantly reduced the imiquimod- induced scaling when compared to the vehicle-treated group on Day 8 (p < 0.001), Day 9 (p < 0.001) and Day 10 (p < 0.0001).

[0349] CT103 administered at 20% significantly reduced the imiquimod- induced scaling when compared to the vehicle-treated group on Day 7 (p < 0.05), Day 8, Day 9 and Day 10 (p < 0.0001). Thickness scores

[0350] As depicted in Figure 49, Imiquimod induced a significant increase in thickness scores from Day 2 until the end of the experiment on Day 10 when compared to Day 0 in the vehicle- treated group (p < 0.0001 ) .

[0351] Clobetasol propionate induced a significant reduction of the thickness scores from Day 2 until the end of the experiment on Day 10 (p < 0.0001 ).

[0352] CT103 administered at 5% did not reduce the imiquimod-induced skin thickening when compared to the vehicle-treated group.

[0353] CT103 administered at 10% did not reduce the imiquimod-induced skin thickening when compared to the vehicle-treated group.

[0354] CT103 administered at 20% induced a significant reduction of the imiquimod- induced skin thickening when compared to the vehicle-treated group on Day 2 (p < 0.001 ) , Day 3 (p < 0.001 ) and Day 5 (p < 0.05) .

Skin scores

[0355] Skin scores were calculated by adding the erythema scores, the scaling scores and the thickness scores.

[0356] As depicted in Figure 50, Imiquimod induced a significant increase of the total skin scores from Day 2 until the end of the experiment on Day 10 when compared to Day 0 (p < 0.0001 ).

[0357] Clobetasol propionate significantly reduced the skin scores when compared to the vehicle-treated group from Day 2 until the end of the experiment on Day 10 (p < 0.0001 ) .

[0358] CT103 administered at 5% induced a significant reduction of the skin scores when compared to the vehicle-treated group on Day 10 (p < 0.01 ).

[0359] CT103 administered at 10% induced a significant reduction of the skin scores when compared to the vehicle-treated group from Day 8 until the end of the experiment on Day 10 (p < 0.05 on Day 8 and Day 9, p < 0.0001 on Day 10).

[0360] CT103 administered at 20% induced a significant reduction of the skin scores when compared to the vehicle-treated group from Day 5 until the end of the experiment on Day 10 (p < 0.001 on Day 5, p < 0.05 on Day 6, p < 0.001 on Day 7, p < 0.0001 from Day 8 until Day 10).

Conclusions

[0361] As expected in this model of psoriasis, the following pathological changes were observed following the topical application of imiquimod: erythema from Day 2, scaling from Day 5 and skin thickening from Day 2. Skin scores then increased until Day 10. At termination, the expected pathological changes were observed: thickening of the epidermis, crusting and/or subcorneal pustule formation, pustule formation within follicular sheaths and a diffuse, mixed neutrophilic and macrophage infiltration of the dermis.

[0362] Clobetasol propionate reduced skin erythema, reduced skin scaling and reduced skin thickening. Clobetasol propionate caused bodyweight loss, a known side effect of corticosteroids administered to rodents.

[0363] CT103 administered at 5%, 10% or 20% reduced the severity of the pathological changes and did not cause bodyweight loss.

[0364] CT103 administered at 5% significantly reduced the clinical score on Day 10 and the severity of skin scaling from Day 8.

[0365] CT103 administered at 10% significantly reduced both the severity of skin scaling and the cumulative clinical scores from Day 8.

[0366] CT103 administered at 20% significantly reduced the severity of skin erythema from Day 5, skin scaling from Day 7 and skin thickening in the first days of treatment. Furthermore, it statistically reduced the cumulative clinical score from Day 5 to the end of the experiment on Day 10.

[0367] The cumulative clinical scores also show a clear dose-dependence trend.

Example 8: BioMAP Platform Analysis of SNA-103

Aim of Study

[0368] The goal of this study was to characterize SNA-103 in the BioMAP Diversity PLUS panel of 12 human primary cell-based systems. These systems are designed to model complex human tissue and disease biology of the vasculature, skin, lung and inflammatory tissues. Quantitative measurements of biomarker activities across this broad panel, along with comparative analysis of the biological activities of known bioactive agents in the BioMAP reference database are used to predict the safety, efficacy and function of these test agents.

Overview of BioMAP Technology Platform [0369] BioMAP panels consist of human primary cell-based systems designed to model different aspects of the human body in an in vitro format. The 12 systems in the Diversity PLUS panel allow test agent characterization in an unbiased way across a broad set of systems modeling various human disease states. BioMAP systems are constructed with one or more primary cell types from healthy human donors, with stimuli (such as cytokines or growth factors) added to capture relevant signaling networks that naturally occur in human tissue or pathological conditions. Vascular biology is modeled in both a Th1 (3C system) and a Th2 (4H system) inflammatory environment, as well as in a Th1 inflammatory state specific to arterial smooth muscle cells (CASM3C system). Additional systems recapitulate aspects of the systemic immune response including monocyte-driven Th1 inflammation (LPS system) or T cell stimulation (SAg system), chronic Th1 inflammation driven by macrophage activation (/Mphg system) and the T cell-dependent activation of B cells that occurs in germinal centers (BT system). The BE3C system (Th1) and the BF4T system (Th2) represent airway inflammation of the lung, while the MyoF system models myofibroblast-lung tissue remodeling. Lastly, skin biology is addressed in the KF3CT system modeling Th1 cutaneous inflammation and the HDF3CGF system modeling wound healing.

[0370] Each test agent generates a signature BioMAP profile that is created from the changes in protein biomarker readouts within individual system environments. Biomarker readouts (7 - 17 per system) are selected for therapeutic and biological relevance, are predictive for disease outcomes or specific drug effects and are validated using agents with known mechanism of action (MoA). Each readout is measured quantitatively by immune-based methods that detect protein (e.g., ELISA) or functional assays that measure proliferation and viability. BioMAP readouts are diverse and include cell surface receptors, cytokines, chemokines, matrix molecules and enzymes. In total, the Diversity PLUS panel contains 148 biomarker readouts that capture biological changes that occur within the physiological context of the particular BioMAP system.

[0371] Using custom-designed software containing data mining tools, a BioMAP profile can be compared against a proprietary reference database of > 4,000 BioMAP profiles of bioactive agents (biologies, approved drugs, chemicals and experimental agents) to classify and identify the most similar profiles. This robust data platform allows rapid evaluation and interpretation of BioMAP profiles by performing the unbiased mathematical identification of similar activities. Specific BioMAP activities have been correlated to in vivo biology, and multiparameter BioMAP profiles have been used to distinguish compounds based on MoA and target selectivity and can provide a predictive signature for in vivo toxicological outcomes (e.g., vascular toxicity, developmental toxicity, etc.) across diverse physiological systems. Materials and Methods Test Agent

[0372] SNA- 103 was profiled in the BioMAP Diversity PLUS panel at concentrations of 230 μΜ , 77 μΜ , 26 μΜ , and 8.6 μΜ . Calcipotriene was employed as the benchmark compound.

Methods for Diversity PLUS

[0373] Human primary cells in BioMAP systems are used at early passage (passage 4 or earlier) to minimize adaptation to cell culture conditions and preserve physiological signaling responses. All cells are from a pool of multiple donors (n = 2 to 6) , commercially purchased and handled according to the recommendations of the manufacturers. Human blood derived CD14+ monocytes are differentiated into macrophages in vitro before being added to the /Mphg system. Abbreviations are used as follows: Human umbilical vein endothelial cells (HUVEC), Peripheral blood mononuclear cells (PBMC), Human neonatal dermal fibroblasts (HDFn), B cell receptor (BCR) , T cell receptor (TCR) and Toll-like receptor (TLR) .

[0374] Cell types and stimuli used in each system are as follows: 3C system [HUVEC + (IL-1 P, TNFa and IFNy)], 4H system [HUVEC + (IL-4 and histamine)], LPS system [PBMC and HUVEC + LPS (TLR4 ligand)], SAg system [PBMC and HUVEC + TCR ligands], BT system [CD19+ B cells and PBMC + (a-lgM and TCR ligands)], BF4T system [bronchial epithelial cells and HDFn + (TNFa and IL-4)], BE3C system [bronchial epithelial cells + (IL- Ι β, TNFa and IFNy)], CASM3C system [coronary artery smooth muscle cells + (IL-1 β, TNFa and IFNy)], HDF3CGF system [HDFn + (IL- Ι β, TNFa, IFNy, EGF, bFGF and PDGF-BB)], KF3CT system [keratinocytes and HDFn + (IL- 1 β, TNFa and IFNy)], MyoF system [differentiated lung myofibroblasts + (TNFa and TGFp)] and /Mphg system [HUVEC and M 1 macrophages + Zymosan (TLR2 ligand)].

[0375] Systems are derived from either single cell types or co-culture systems. Adherent cell types are cultured in 96 or 384-well plates until confluence, followed by the addition of PBMC (SAg and LPS systems). The BT system consists of CD19+ B cells co-cultured with PBMC and stimulated with a BCR activator and low levels of TCR stimulation. Test agents prepared in either DMSO (small molecules; final concentration < 0.1 %) or PBS (biologies) are added at the indicated concentrations 1 -hr before stimulation, and remain in culture for 24-hrs or as otherwise indicated (48-hrs, MyoF system; 72-hrs, BT system (soluble readouts); 168-hrs, BT system (secreted IgG)). Each plate contains drug controls (e.g. , legacy control test agent colchicine at 1 .1 μΜ), negative controls (e.g. , non-stimulated conditions) and vehicle controls (e.g. , 0.1 % DMSO) appropriate for each system. Direct ELISA is used to measure biomarker levels of cell-associated and cell membrane targets. Soluble factors from supernatants are quantified using either HTRF® detection, bead-based multiplex immunoassay or capture ELISA. Overt adverse effects of test agents on cell proliferation and viability (cytotoxicity) are detected by sulforhodamine B (SRB) staining, for adherent cells, and alamarBlue® reduction for cells in suspension. For proliferation assays, individual cell types are cultured at subconfluence and measured at time points optimized for each system (48- hrs: 3C and CASM3C systems; 72-hrs: BT and HDF3CGF systems; 96-hrs: SAg system). Cytotoxicity for adherent cells is measured by SRB (24-hrs: 3C, 4H, LPS, SAg, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, and IMphg systems; 48-hrs: MyoF system) , and by alamarBlue staining for cells in suspension (24-hrs: SAg system; 42-hrs: BT system) at the time points indicated. Additional information can be found in previous descriptions.

Data Analysis

[0376] Biomarker measurements in a test agent-treated sample are divided by the average of control samples (at least 6 vehicle controls from the same plate) to generate a ratio that is then Iog 10 transformed. Significance prediction envelopes are calculated using historical vehicle control data at a 95% confidence interval.

Profile Analysis

[0377] Biomarker activities are annotated when 2 or more consecutive concentrations change in the same direction relative to vehicle controls, are outside of the significance envelope and have at least one concentration with an effect size > 20% (|log 10 ratio| > 0.1) . Biomarker key activities are described as modulated if these activities increase in some systems, but decrease in others. Cytotoxic conditions are noted when total protein levels decrease by more than 50% (log 10 ratio of SRB or alamarBlue levels < -0.3) and are indicated by a thin black arrow above the X-axis. A compound is considered to have broad cytotoxicity when cytotoxicity is detected in 3 or more systems. Concentrations of test agents with detectable broad cytotoxicity are excluded from biomarker activity annotation and downstream benchmarking, similarity search and cluster analysis. Antiproliferative effects are defined by an SRB or alamarBlue log 10 ratio value < -0.1 from cells plated at a lower density and are indicated by grey arrows above the X-axis. Cytotoxicity and antiproliferative arrows only require one concentration to meet the indicated threshold for profile annotation. Benchmark Analysis

[0378] Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% in the same direction. Differentiating biomarkers are annotated when one profile has a readout outside of the significance envelope with an effect size > 20%, and the readout for the other profile is either inside the envelope or in the opposite direction. Unless specified, the top non-cytotoxic concentration of both the test agent and benchmark agent are included in the benchmark overlay analysis.

Similarity Analysis

[0379] Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% in the same direction. Concentrations of test agents that have 3 or more detectable systems with cytotoxicity are excluded from similarity analysis. Concentrations of test agents that have 1 - 2 systems with detectable cytotoxicity will be included in the similarity search analysis, along with an overlay of the database match with the top concentration of the test agent. This will be followed by an additional overlay of the next highest concentration of the test agent containing no systems with detectable cytotoxicity and the respective database match. To determine the extent of similarity between BioMAP profiles of compounds run in the Diversity PLUS panel, we have developed a custom similarity metric (BioMAP Z-Standard) that is a combinatorial approach that has improved performance in mechanism classification of reference agents compared to other measures tested (including Pearson's and Spearman's correlation coefficients). This approach more effectively accounts for variations in the number of data points, systems, active biomarker readouts and the amplitude of biomarker readout changes that are characteristic features of BioMAP profiles. A Pearson's correlation coefficient (r) is first generated to measure the linear association between two profiles that is based on the similarity in the direction and magnitude of the relationship. Since the Pearson's correlation can be influenced by the magnitude of any biomarker activity, a per-system weighted average Tanimoto metric is used as a filter to account for underrepresentation of less robust systems. The Tanimoto metric does not consider the amplitude of biomarker activity, but addresses whether the identity and number of readouts are in common on a weighted, per system basis. A real-value Tanimoto metric is calculated

A

\A

first by normalizing each profile to the unit vector (e.g., A = \\ II ) and then applying the A - B

following formula: » "

, where A and B are the 2 profile vectors. Then, it is incorporated into a system weighted-averaged real-value Tanimoto metric in this

∑W T

calculation: ^ . The calculation uses the real-value Tanimoto score for each rth system (T,) and the weight of each rth system (W,). W, is calculated for each system in

1

the following formula: ^{1 + e— 100} * ^— 0.09^) _{where /r js the} |_argest absolute value of the ratios from the 2 profiles being compared. Based on the optimal performance of reference compounds, profiles are identified as having mechanistically relevant similarity if the Pearson's correlation coefficient (r) > 0.7. Finally, a Fisher r-to-z-transformation is used to calculate a z-score to convert a short tail distribution into a normal distribution as

1 + r

follows: z= 0.5 log₁₀ ^{1 _ r} . Then the BioMAP Z-Standard, which adjusts for the number of common readouts (CR), is generated according to the following formula: Z-Standard = z

"JCR 3 A larger BioMAP Z-Standard value corresponds to a higher confidence level, and this is the metric used to rank similarity results.

Cluster Analysis

[0380] Cluster analysis (function similarity map) uses the results of pairwise correlation analysis to project the "proximity" of agent profiles from multi-dimensional space into two dimensions. Functional clustering of the agent profiles generated during this analysis uses Pearson correlation values for pairwise comparisons of the profiles for each agent at each concentration, and then subjects the pairwise correlation data to multidimensional scaling. Profiles that are similar with a Pearson's correlation coefficient (r) > 0.7 are connected by lines. Agents that do not cluster with one another are interpreted as mechanistically distinct. This analysis is performed for projects with 3 or more agents tested. Cytotoxic concentrations are excluded from cluster analysis.

Mechanism HeatMAP Analysis

[0381] Mechanism HeatMAP analysis provides a visualization of the test compound and 19 consensus mechanisms allowing comparison of biomarker activities across all compound concentrations and consensus mechanisms. The synthetic consensus profiles used in the Mechanism HeatMAP analysis are representative BioMAP profiles of the average of multiple compounds from structurally distinct chemical classes. Profiles were calculated by averaging the values for each biomarker endpoint for all profiles selected (multiple agents at different concentrations) to build the consensus mechanism profile. [8] Biomarker activities are colored in the heatmap for consensus mechanisms and compounds when they have expression relative to vehicle controls outside of the significance envelope. Red represents increased protein expression, blue represents decreased expression and white indicates levels that were unchanged or within filtering conditions. Darker shades of color represent greater change in biomarker activity relative to vehicle control. The Mechanism HeatMAP was prepared using R and the gplots package for R.

Assay Acceptance Criteria

[0382] A BioMAP assay includes the multi-parameter data sets generated by the BioMAP platform for agents tested in the systems that make up the Diversity PLUS panel. Assays contain drug controls (e.g., legacy control test agent colchicine), negative controls (e.g., non-stimulated conditions), and vehicle controls (e.g., DMSO) appropriate for each system. BioMAP assays are plate-based, and data acceptance criteria depend on both plate performance (% CV of vehicle control wells) and system performance across historical controls for that system. The QA/QC Pearson Test is performed by first establishing the 1 % false negative Pearson cutoff from the reference dataset of historical positive controls. The process iterates through every profile of system biomarker readouts in the positive control reference dataset, calculating Pearson values between each profile and the mean of the remaining profiles in the dataset. The overall number of Pearson values used to determine the 1 % false negative cutoff is the total number of profiles present in the reference dataset. The Pearson value at the one percentile of all values calculated is the 1 % false negative Pearson cutoff. A system will pass if the Pearson value between the experimental plate's negative control or drug control profile and the mean of the historical control profiles in the reference dataset exceeds this 1 % false negative Pearson cutoff. Overall assays are accepted when each individual system passes the Pearson test and 95% of all project plates have % CV <20%.

Results

BioMAP Profile

[0383] Figure 51 depicts the BioMAP profile of SNA-103 in the Diversity PLUS Panel. SNA-103 was found to be active with 13 annotated readouts, mediating changes in key biomarker activities listed by biological and disease classifications in Table 8 below. SNA-103 impacted inflammation-related activities (decreased sTNFa, ΜΙΡ-1 α, IL-8, IL-6; increased sPGE2), immunomodulatory activities (decreased slL-10, M-CSF, HLA-DR), tissue remodeling activities (decreased MMP-1 ; increased Collagen !), and hemostasis-related activities (decreased TF). SNA-103 is antiproliferative to B cells (indicated by grey arrow in Figure 51).

Tabfe 8: Key Biomarker Activities impacted by SNA-103

Reference Benchmark Overlay

[0384] Figure 52 depicts an overlay of SNA-103 at 230 μ and the selected reference benchmark calcipotriene at 1 μΜ. Caicipotriene is a synthetic derivative of calcitriol, a form of vitamin D, used in the treatment of psoriasis. Please note: Calcipotriene is only available as a benchmark in 7 Bio AP systems.

[0385] There is 1 common activity that is annotated within the following system: LPS (sPGE2).

[0386] Differentiating biomarkers (not shown) are defined when one profile has a readout outside of the significance envelope with an effect size > 20% (|iog1 Q ratio! > 0.1), and the readout for the other profile is either inside the envelope or in the opposite direction. There are 12 differentiating activities between the two compounds: 3C (HLA-DR, TF), 4H (Eotaxin 3), LPS (M-CSF, sTNFa), HDF3CGF (Collagen III, Prolif 72, VCA -1), KF3CT (IP-10), and IMphg (E-selectin, IL-8, MIP-1 a).

Top Database Search Result for SNA-103

[0387] In a search for mathematically similar compound profiles from the BioMAP reference database, SNA-103 (230 μΜ) was most similar to apoptolidin (1 μΜ) (Pearson's correlation, r = 0.564). The Pearson's correlation coefficient between these two profiles is below our determined threshold (r < 0.7) indicating that the relevance of the similarity is unknown. Apoptolidin is a selective poiyketide produced by Nocardiopsis that induces apoptotic death in E1 A-transformed ceils by targeting the mitochondrial F0F1 -ATP synthase and inhibiting oxidative phosphorylation. Figure 53 depicts an overlay of SNA-103 (230 μΜ) and apoptolidin (1 μ ).

[0388] There are 8 common activities that are annotated within the following systems: 3C (HLA-DR), BT (Prolif, SIL-17F, slgG, sTNFa) , and BF4T (Eotaxin 3).

Top BioSeek Reference Database Matches for SNA-103

[0389] Table 9 depicts the top 3 similarity matches from a search of the Bio AP Reference Database of > 4,000 agents for each concentration of SNA- 103. The similarity between agents is determined using a combinatorial approach that accounts for the characteristics of BioMAP profiles by filtering (Tanimoto metric) and ranking (BioMAP Z-Standard) the Pearson's correlation coefficient between two profiles. Profiles are identified as having mechanistically relevant similarity if the Pearson's correlation coefficient is≥ 0.7.

Table 9: Top BioMAP Reference Database Matches for SNA-103

[0390] For profiles with a Pearson's correlation coefficient below our determined threshold (r < O. f), the relevance of the similarity is unknown. Mechanism HeatMAP Analysis of SNA-103

[0391] Figure 54 depicts Mechanism Heat AP Analysis of SNA-1 G3, with the 148 biomarker readouts within the Diversity PLUS panel compared to 19 consensus mechanism class profiles. This analysis informs on the regulatory mechanisms controlling increases or decreases in each of the biomarker readouts.

Clustering of Project Profiles

[0392] Figure 55 depicts a clustering of tested agent profiles based on pairwise correlation analysis and clustering of most similar profiles. Profiles that are similar with a Pearson's correlation coefficient (r) > 0.7 are connected by lines. Agents that do not cluster with one another are interpreted as mechanistically distinct. Cytotoxic concentrations are excluded from duster analysis. Functional clustering of the agent profiles generated during this analysis uses Pearson's correlation values for pairwise comparisons of the profiles for each agent at each concentration, and then subjects the pairwise correlation data to multidimensional scaling. SNA-103 clusters internally at the top three concentrations. Internal clustering at both high and low concentrations of SNA- 103 suggests the phenotypic signature of this compound is maintained across a wide range of concentrations, a characteristic commonly observed in marketed drugs.

Conclusions

[0393] In this study SNA-103 was characterized by profiling in the BioMAP Diversity PLUS panel of human primary ceil based assays modeling complex tissue and disease biology of organs (vasculature, immune system, skin, lung) and general tissue biology. The Diversity PLUS panel evaluates the biological impact of test agents in conditions that preserve the complex crosstalk and feedback mechanisms that are relevant to in vivo outcomes.

[0394] SNA-103 was modestly active and non-cytotoxic in the Diversity PLUS panel; an antiproliferative effect on B ceils was observed only at the top concentration tested (230 μ ). Selective antiproliferative effects on B ceils may be appropriate for B cell driven autoimmune indications, such as systemic lupus erythematosus (SLE), or for heme-oncoiogy indications such as chronic lymphocytic leukemia (CLL) or B-celi non- Hodgkin's lymphoma (B-NHL). A sharp dose response was observed between 230 μ and 77 μΜ in the BT system, suggesting that the test agent may be engaging additional targets on immune ceils at this concentration. Other activities of interest were the supra- induction of sPGE2 levels in the LPS system and Collagen-I levels in the MyoF system. These systems model monocyte and myofibroblast activation respectively, and these readouts are induced by the system activators in the respective systems. Example 9: Synthesis scheme for CT103/SNA-103

Abbreviations

introduction

[0395] This Example describes the production of a sample of SNA-103 (CT103; Figure 56). The preparation followed the synthetic procedure described in Example 2. The synthetic scheme consists of acylation reaction of Motesanib (commercially available) with l ieO-PEG₂ooo-COOH, in the presence of WSC as coupling reagent (Figure 57A). 16.8 g of SNA-103 were produced with 99.4% HPLC purity and 73% overall yield from Motesanib. SNA-103 yield has been calculated assigning it 2286 Da as molecular weight, as the peak molecular weight of MeO-PEG₂ooo-COOH used for its preparation was 1931 Da. SNA-103 molecular weight has been caicuiated according to the following formula:

[0398] SNA-103 molecular weight = Motesanib nominal mass + MeO-PEG₂ooo-COOH peak molecular weight - 18. Note that the MeO~PEG₂ooo-COOH peak molecular weight was obtained from the certificate of analysis provided by the supplier.

Experimental Procedure

[0397] The selected synthetic protocol consisted, according to the previously described studies, of (1) acylation of Motesanib with 1.06 equivalents of MeO-PEG₂ooo- COOH, using 1.1 equivalents of WSC as coupling reagent, in DCM as solvent, at 25°C; and (2) purification by reversed phase flash chromatography using H₂0 and ACN as eluent phases. The procedure was at first verified on a smaller scale (use test, lot n° 2017GC12/S1) to test starting materials, then a representative SNA-103 sample was synthesized (lot n° 2017GC12/S2). The two samples were combined to obtain 16.8 g as a single lot n° 2017GC12/S3. The experimental procedure used for the production of the representative SNA-103 sample is hereinafter described.

Motesanib acylation

[0398] Figure 57B depicts the Motesanib acylation reaction and Table 10 below shows the materials list.

Table 10: Materials List

Reagents ∞g mmot d mi Ratia

Motesanib 373,45 j 3230,0 8,65 1

MeO"P£Gic _¾~COQH 1931 j 177CB 9,17 1,06 maisf

WSC HCi 191,7 j 1840,4 9,60 1,11 molar

DCM 84,93 1,32? 1450 0,45 ml/mg Motesanib

Theoretical product

SNA-103 2286,45 119775,7 8,65 j j 1 molar

MeO-PEGj«»-COOH 1931 0,52 0,06 molar

WSC HCi 131,7 1 182,4 0,95 0,11 molar

Urea 209,7 j 1813, 7 8,65 1,00 molar

[0399] In 3 L four necked jacketed reactor, equipped with mechanical stirring, thermometer and condenser, protected from light by aluminium foil wrapping, 17.7 g of MeO-PEG2000-COOH were dissolved in 1 L of DCM at 22°C under nitrogen atmosphere. 1.84 g of WSC HCI and 200 mL of DCM were added. The solution was aged under stirring at 22°C for 30 min. under nitrogen atmosphere, then 3.23 g of Motesanib (Figure 58) and 250 mL of DCM were added. The reaction mixture was aged under stirring for 24 h. The reaction completion was checked by HPLC analysis (Figure 59) . The reaction mixture was transferred to a 2 L separating funnel, and washed with NaCI aqueous saturated solution (2 x 400 mL). Phases were separated and the organic phase was concentrated to dryness by rotary evaporation under vacuum at 40°C. 23 g of crude SNA- 103 as yellow oil with 97.4% HPLC purity (@ 254 nm) were obtained (Figure

60) .

Purification of SNA-103

[0400] 23 g of crude SNA-103 were dissolved in 42 mL of H₂0 and, divided into four portions, purified by reversed-phase flash chromatography using a Biotage Isolera LS System equipped with a Biotage SNAP KP-C18-HS cartridge packed with 400 g of KP-C18-HS Silica (column volume 510 mL). The cartridge was equilibrated at 100 mL/min. with 1530 mL of H₂0/ACN 80:20 v/v. Sample (16 g of solution per purification) was loaded onto the cartridge by injection through a syringe. The SNAP cartridge was eluted at 100 mL/min with: (1) 255 mL of H₂0/ACN 80:20 v/v; (2) 1530 mL of H₂0/ACN 63:37 v/v; (3) 1020 mL of H₂0/ACN 10:90 v/v. The first portion of eluate (about 920 mL) was sent to the waste, then the eluted solvent was collected in fractions of 50-150 mL each.

[0401] The UV profile (@ 254 and 210 nm) of one purification is depicted in Figure 61 .

[0402] Collected individual fractions were analysed by HPLC. Fractions with HPLC purity > 98 % were combined (1050 mL) and concentrated under reduced pressure at 40°C to remove acetonitrile. The residual aqueous solution (660 g) was extracted with DCM (3 x 150 mL). Brine (80 mL) was added during the first extraction to help phase separation. The combined organic phases were evaporated under reduced pressure at 40°C to dryness, affording 4.2-4.5 g of an oily residue from each purification.

[0403] The four samples obtained from each purification (17.8 g) were combined with the previously produced SNA-103 sample, 2.36 g, obtained from 0.5 g of Motesanib, treated with 200 mL of diethyl ether and aged for 0.5 h at 25°C and 2 h at - 18°C. The precipitated solid was filtered over sintered glass filter (G3), washed with 10 mL of diethyl ether and dried under vacuum at 35°C for 20 h and 40°C for 6 h. 16.8 g of SNA-103 as a white solid were obtained (lot n° 2017GC12/S3).

[0404] Purity of SNA-103 sample, determined by HPLC analysis @ 254 nm (method Ml CT103 001), was 99.4% (Figure 62). The product was also characterized by 1 H-NMR and ESI MS analyses (Figures 63, 64 and 65). The Certificate of analysis of the SNA-103 sample is shown in Figure 66. The final SNA-103 sample was stored at -22°C. Analytical method

[0405] HPLC method M l CT103 001 (described herein) has been used both for reaction monitoring and for assessing chemical purity of final SNA-103 samples.

Example 10: Efficacy Analysis of SNA-125, SNA-352 and SNA-103 in a VEGF- induced Proliferation Assay Using HRMVEC Cells

Aim of Study

[0406] The aim of this study was to compare the efficacy of SNA-125, SNA- 352 and SNA- 103 in a VEGF-induced proliferation assay using Human Retinal Microvascular Endothelial (HRMVEC) cells. SNA- 125 and SNA-352 have been observed to inhibit kinases in VEGF signaling pathway: ERK and RAF for SNA- 125, and PCKa, PCKb2 and PKg for SNA-352. It is contemplated that these compounds could have an anti-angiogenic effect in addition to their anti-inflammatory effect.

Methodology

Experimental outline

[0407] Primary HRMVEC cells were seeded onto collagen-coated 96 well plates at a concentration of 2 or 5 x 10³ and treated with VEGF at 10 or 50 ng/ml. As a control, cells without VEGF were included to give background levels of non-VEGF driven proliferation. Cells were incubated for 72 hours at 37°C with 5% C02. Proliferation was measured by pulsing cells for the last 24 hours with tritiated thymidine. Plates were then harvested and assayed for tritiated thymidine incorporation. The ability of the lead compounds to inhibit proliferation was assessed by pre-incubation of cells with the lead compounds for 18 hours prior to stimulation with VEGF. Each lead compound was tested at eight concentrations. Motesanib Diphosphate (AMG-706) was included as a positive control. Four concentrations were tested. Each condition was tested in sextuplicate. To readout cell proliferation, cells were pulsed with ³H-Thymidine and harvested 24 hours later. Radiation was then quantified . IC₅₀ values were calculated (where possible) for each compound.

Treatment Groups and Dosages

[0408] The doses of the test SNA compounds used were 300, 100, 33.3, 1 1 .1 , 3.7, 1 .2, 0.41 , and 0.14 μΜ . The doses of motesanib diphosphate used were 30, 10, 3.33, and 1 .1 1 nM. The pre-incubation time was 18 hours. Table 1 1 depicts the treatment groups and dosages employed in this study.

Table 11 : Treatment Groups and Dosages

Results

[0409] Figure 67 depicts the inhibition of VEGF-induced proliferation following treatment with SNA-125, SNA-352, SNA-103, and motesanib diphosphate. The IC₅₀ values calculated based on this analysis are shown in Table 12.

Table 12: IC50 Values for SNA-103, SNA-125, SNA-352

Conclusions

[0410] SNA-125 showed inhibition of VEGF-induced proliferation. Lower IC₅₀ values were seen when treating the lower cell density (values between 7 and 9 μΜ). SNA-352 also showed consistent inhibition of VEGF-induced proliferation at both concentrations and cell densities tested, with IC₅₀ values between 4 and 5 μΜ. SNA-103 appeared to have an effect on proliferation at the top concentration, with IC₅₀ values between 100 and 200 μΜ. Motesanib diphosphate did not appear to inhibit proliferation with this cell line.

Example 11 : Prophetic Study - Laser-induced Choroidal Neovascularization in

Green Monkeys

Model

[0411] The laser-induced choroidal neovascularization (CNV) experimental model is employed in neovascular age-related macular degeneration (AMD) research. In particular, it is used in the development of therapeutics for the treatment of retinal diseases, such as wet-AMD.

Study 1: Ocular Pharmacokinetics and Tolerance of Intravitreal SNA-103 in African Green Monkeys

[0412] Monkeys will receive IVT injections of test article in both eyes (OU) in accordance with the treatment assignment (Table 13) and study schedule (Table 14). For IVT dosing, topical local anesthesia will be administered (0.5% proparacaine) and eyes will be disinfected with 5% Betadine and rinsed with sterile normal saline. IVT injections will be administered using a 31 -gauge 0.5-inch needle placed 2 mm posterior to the limbus in the inferior temporal quadrant, targeting the central vitreous. Injections will be followed by topical administration of 0.3% ciprofloxacin ophthalmic solution or equivalent antibiotic.

Table 13: Treatment Assignment rou ft Eye Treatment ioute Volume Fre uenc

1 3 OU SNA-103 iVT 50 pi Single dose Table 14: Testing Schedule

Study Day

Event

Dosing 1 « I I

8αάγ svesgn

S!Si SOiW

Serum

Aqueous humor jj; ;j¾

ViSreous humor

Study 2; Evaluation of the Efficacy of SNA-103 in a Laser-induced CNV Model in African Green Monkeys

[0413] On Day 0, CNV will be induced between temporal vascular arcades by laser photocoagulation targeting Bruch's membrane. Six laser spots will be symmetrically placed within the perimacular region in each eye by an ophthalmologist employing an Iridex Oculight TX 532 nm laser with a pulse duration of 100 ms, spot size of 50 μηι, power of 750 mW. Laser spots will be applied using a contact laser lens. Any spots demonstrating severe retinal/subretinal hemorrhage immediately post-laser and not resolving by the time of follow-up examinations will be excluded from analyses. If hemorrhage occurs encompassing all target lesion areas within the central retina, the monkey will be substituted for with another screened monkey, up to four monkeys across all treatment groups, taking measures to assure balanced assignment to treatment. On day eight post-laser treatment, monkeys will undergo color fundus imaging and OCT to document the appearance and size of the developed CNV complex at each laser lesion, and allow randomization to treatment assignment to achieve a balanced incidence of lesion severity in each treatment arm. On study day 10 monkeys will receive IVT injections of SNA-103, SNA-103 vehicle or bevacizumab (Avastin) in either eye in accordance with Table 15. The testing schedule is depicted in Table 16, where 'OCT' indicates optical coherent tomography and ΊΟΡ' indicates intraocular pressure. Table 15: Treatment Assignment ft E e ¾3l Dose Inien mi

Table 16: Testing Schedule

Study Day

# Eyes

iiliiii

Dosing

CNV

Socty weigftis

¾!t tamp

caof fwnaws ptiotogiopiiy

fluorescein ongiogropiw

OCT

for

Aqy eous fwanof

VBreous i¾ym

£ye cotia Bon

Example 12: Process Description for ISO-Production campaign SNA17-03 / N17- 10893 to afford SNA-103 as batches SOL22363-3 (bulk) / SOL22362-2 (lab scale) Part 1: Stage 1 activation of PEGamine

Technical Flow Chart

iG^g "amine activated PEG^o-amin

Process Description

First run activation: SNA17-03-020

[0414] 1 eq. SOL21924 (600 g, PEGamine, SunBio) was dissolved in 5 vol (3 L) dichloromethane at Tout = 20°C to give a clear yellow solution. To the reaction mixture was added 1 .15 eq. triethylamine at Ti = 17°C and 1 eq. of dihydrofuran-2,5-dione as solid (Ti raised to 21 °C, slow gas evaluation observed via bubbler). The reaction mixture was stirred overnight at Ti = 20°C under slight nitrogen stream. A sample was withdrawn for TLC (silica gel plate, DCM / MeOH 10: 1 , ninhydrine reagent for primary amine detection) and full conversion was observed including traces of polar species.

[0415] 5 eq. of Amberlyst 15 were added to the mixture kept stirring for 75 min. Again, TLC was performed revealing very slight amount of polar species. Further 1 .5 eq. of Amberlyst 15 were added followed by two further cycles of Amberlyst 15 addition revealing that no further polar species were present according to TLC.

[0416] The reaction mixture SNA17-03-020OP was stored at room temperature as is without further stirring and combined with the second run for later work-up.

Second run activation: SNA17-03-021

[0417] 1 eq. SOL21924 (600 g, PEGamine, SunBio) was dissolved in 5 vol (3 L) dichloromethane at Tout = 21 °C to give a clear yellow solution. To the reaction mixture was added 1 .15 eq. triethylamine at Ti = 18°C and 1 eq. of dihydrofuran-2,5-dione as solid (Ti raised to 21 °C, slow gas evaluation observed via bubbler). The reaction mixture was stirred overnight at Ti = 20°C under slight nitrogen stream. A sample was withdrawn for TLC (silica gel plate, DCM / MeOH 10: 1 , ninhydrine reagent for primary amine detection) and full conversion was observed including traces of polar species. [0418] 5 eq. of Amberlyst 15 were added to the mixture kept stirring for 75 min. Again, TLC was performed revealing very slight amount of polar species. Further 1 .5 eq. of Amberlyst 15 were added followed by two further cycles of Amberlyst 15 addition revealing that no further polar species were present according to TLC.

[0419] The reaction mixture SNA17-03-020OP from the previous run was combined with SNA17-03-021 OP run at room temperature for final work-up.

Work-up

[0420] The combined reaction mixture (020OP and 021 OP) was filtered via glass nutsch and washed three times with 3x 1 L dichloromethane. The filtrate was re- transferred into a 20 L flask and 4 L DCM were evaporated at Tout = 30°C and 2 L heptane, isomers were added. Again, further 2 L DCM were evaporated and 2 L heptane, isomers were added followed by seeding with 1 g of activated PEGamine- COOH. Precipitation was observed and the resulting thin suspension was cooled down to Tout = 20°C kept stirring for 2 h at ambient to give activated PEGamine-COOH.

[0421] The product was dried on the rotary evaporator at Tout = 25°C (< 1 0 mbar) affording 1005 g (80% , uncorr. yield) SNA17-03-021 S1.1 in one portion. The product (activated PEGamine-COOH) met the specifications according to the Maldi-MS reference spectrum (Lims N° 17-10364). Polydispersity index was determined with 1 .010. DSC melting point gave 47.3°C and water content by KF of 0.4% w/w.

[0422] The material was stored in two PE-bags at <-15°C protected from light prior to later use in stage 2.

Part 2: Stage 2a to 2c coupling, column purification and precipitation (SNA17-03- 027)

Technical Flow Chart

Process Description

Stage 2a

[0423] 1 .1 eq. activated PEGamine-COOH (825 g) and 1 .25 eq. EDCxHCI (83 g) were suspended in 80 vol dichloromethane (10.4 L) at Tout = 20°C to give a clear colorless solution containing a white solid. Stirring for 35 min. at Ti = 18°C gave a clear solution.

[0424] 1 eq. Motesanib (130 g) were suspended in 30 vol dichloromethane (3.9 L) and added over 5 min. at Ti = 18°C to afford a clear yellow-orange solution. The reaction mixture was stirred for 2 h at Ti = 1 8°C and an IPC as 11 was withdrawn indicating 35% a/a Motesanib (RT = 8.57) , 5% a/a impurity 1 (RT = 9.33) along with 56% product SNA-103 (RT = 12.48) and 1 .3% a/a as late-running impurity 2 (RT = 12.94). Stirring further for 1 .5 h did not change HPLC profile.

[0425] Therefore, a second dosage of 0.1 eq. activated PEGamine-COOH (75 g) along with 0.15 eq. EDCxHCI (10.0 g) was performed indicating 24% a/a Motesanib (RT = 8.57) , 7% a/a impurity 1 (RT = 9.33) along with 65% product SNA- 103 (RT = 12.48) and 1 .6% a/a as late-running impurity 2 (RT = 12.94). The mixture was stirred overnight at Ti = 18°C furnishing 12% a/a Motesanib (RT = 8.57), 13% a/a impurity 1 (RT = 9.33) along with 72% product SNA-103 (RT = 12.48) and 1 .8% a/a as late-running impurity 2 (RT = 12.94) .

[0426] Again, 0.054 eq. activated PEGamine-COOH (40.5 g) along with 0.06 eq. EDCxHCI (4.3 g) were charged indicating after another stirring period of 2 h 5% a/a Motesanib (RT = 8.57) , 14% a/a impurity 1 (RT = 9.33) along with 77% product SNA-103 (RT = 12.48) and 1 .8% a/a as late-running impurity 2 (RT = 12.94). The mixture was stirred again overnight at Ti = 18°C furnishing 1 .5% a/a Motesanib (RT = 8.57), 16% a/a impurity 1 (RT = 9.33) along with 80% product SNA-103 (RT = 12.48) and 1 .9% a/a as late-running impurity 2 (RT = 12.94) .

[0427] A final dosage of 0.03 eq. activated PEGamine-COOH (22 g) along with 0.04 eq. EDCxHCI (2.5 g) was performed indicating 0.5% a/a Motesanib (RT = 8.57), 15% a/a impurity 1 (RT = 9.33) along with 80% product SNA-103 (RT = 12.48) and 1 .9% a/a as late-running impurity 2 (RT = 12.94). The mixture was stirred over the weekend at Ti = 20°C to furnish a final Motesanib content of <0.01 % a/a along with 15% a/a impurity 1 (RT = 9.33) along with 81 % product SNA-103 (RT = 12.48) and 2.0% a/a as late-running impurity 2 (RT = 12.94) after IPC 114.

[0428] The reaction mixture was transferred into the mobile feeding tank for further work-up quenched with deion. water (10 L) and a sat. Na2C03-solution (2 L) . A milky phase separation was observed (30 min.) and OP01 was again washed with deion . water (10 L) and a sat. NaHC03-solution (3 L) to afford OP02. OP02 was dried over 1 wt Na2S04 (962 g) , filtered and concentrated on the rotary evaporator at Tout = 25°C under reduced pressure to give S1.1 (1246 g) within a purity of 82% a/a SNA-103 along with 13% a/a earyl running impurity 1 (RT = 9.33) and 1 .8% a/a late-running impurity 2 (RT = 12.94). Isolated yield SNA17-03-027S1.1: 1246 g (82% a/a purity)

Stage 2b (column purification)

[0429] 1240 g of S1.1 were dissolved in 1 vol (1 .2 L) dichloromethane and 0.5 vol (0.6 L) 2-Me-THF for purification via Plug-filtration using 2.4 wt spherical silica gel (3000 g) . Spherical silica gel was loaded into a 20 L glass column and rinsed with 2-Me- THF (5 L). A solvent gradient of a DCM / 2-Me-THF 1 : 1 ratio mixture (15 L) was prepared to collect pre-fractions F1 to F3 (each 5 L fractions) .

[0430] A 50 L gradient solvent mixture of DCM / 2-Me-THF / MeOH containing 1 % triethylamine (ratio 47.5 / 47.5 / 5 + 1 % TEA) was prepared to collect fractions F4 to F7 (each 5 L fractions) containing product SNA-103 according to TLC and F8 to F 10 (each 10 L fractions, F8 still containing product). Fractions F4 to F8 were collected and concentrated on the rotary evaporator at Tout = 25°C under reduced pressure to give S2.1 . Isolated yield SNA17-03-027S2.1: 668 g (98.3% a/a purity for info along with 1.7% a/a late- running impurity 2)

[0431] Still having the late-running impurity 2 (RT = 12.94) in the final API SNA-103 contaminated, a 50 g sample of S2.1 was taken and purified using reversed phase chromatography in laboratory.

Stage 2b (further RP-column purification, small scale run):

[0432] 50 g of S2.1 were dissolved in 100 mL deion. water and poured on a RP column (400 g LiChroprepRP-18 40-63 microns from Merck) and an eluent mixture of deion. water / acetonitrile 98:2 ratio (500 mL) was used to collect fractions F1 to F24 (each 100 mL).

[0433] The fractions were concentrated to evaporate acetonitrile and the water layer was re-extracted with dichloromethane (500 mL), dried over Na2S04 (1 wt) and concentrated on the rotary evaporator under reduced pressure to furnish S3.1 . Isolated yield SNA 17-03-027S3.1: 43 g (>99% a/a purity for info along with 0.6% a/a as an early-running impurity).

[0434] S3.1 was later released under ISO-conditions to afford batch SOL22362-2. Stage 2c (precipitation of API in SNA 17-03-027)

[0435] 610 g (using S2.1) were dissolved in 2.5 vol (1 .53 L) inline-filtered MeOH at Tout = 20°C. The 30 L mobile reactor was charged with 25 vol (15.2 L) tert- Butylmethyl ether (TBME). TBME was cooled down to Ti = -5°C (Tout = -10°C) over 0.5 h and kept stirring for 15 min. The inline-filtered product- MeOH solution (2 L) was added over 45 min . at Ti = -5°C to afford a white suspension which was kept for another 1 h. Filtration and washing of the filter cake with pre-chilled TBME twice (2 vol) gave 600 g (>99%, uncorr. recovery) of S4.1 . Isolated yield SNA17-03-027S4.1: 600 g (98% a/a purity for info).

[0436] S4.1 was later released under ISO-conditions to afford batch SOL22362-3.

Claims

WHAT IS CLAIMED IS:

wherein n is 4-1 140;

and any pharmaceutically acceptable salt thereof.

2. A reduced exposure composition for treating a target site, comprising a conjugate comprising at least one active entity linked to at least one polymer, wherein the conjugate has reduced exposure at a non-target site as compared to the active entity delivered without the polymer, wherein the non-target site comprises the systemic system, the lymphatic system and/or another non-target tissue site, wherein the c

wherein n is 4-1 140;

and any pharmaceutically acceptable salt thereof; and

a pharmaceutically acceptable carrier formulated for delivery of the conjugate to the target site.

3. A reduced exposure composition for treating a cell within a target site, comprising a conjugate, the conjugate comprising an active entity linked to at least one polymer, and a pharmaceutically acceptable carrier formulated for delivery of the conjugate to the target site; wherein the composition has reduced exposure at a non-target site as compared to the active entity delivered without the polymer;

wherein the active entity is an inhibitor, antagonist, or inverse agonist of a cellular kinase;

wherein the active entity comprises or consists essentially of any one or more of compounds 1 -59;

wherein the at least one polymer is polyethylene glycol (PEG) or methoxy- polyethylene glycol (m-PEG); and

wherein the conjugate can traverse the cell membrane and distribute among both lipophilic and hydrophilic cellular compartments within the cell, thereby promoting interactions between the active entity and the cellular kinase.

4. The composition of claim 3, wherein the non-target site includes non- target tissue at which pharmacological activity is not desired and/or not achieved.

5. The composition according to any one of claims 3-4, wherein the active entity comprises compound 1 .

6. The composition according to any one of claims 3-5, wherein the composition comprises SNA-103.

7. The composition according to any one of claims 3-6, wherein the cellular kinase is VEGFR.

8. The composition according to any one of claims 3-7, wherein the active entity binds to VEGFR.

9. The composition according to any one of claims 3-8, wherein the active entity inhibits VEGFR.

10. The composition according to any one of claims 7-9, wherein the VEGFR is one or more of VEGFR- 1 , VEGFR-2, and VEGFR-3.

1 1 . The composition according to any one of claims 3- 10, wherein the active entity has one or more carboxyl, hydroxyl, amino and/or sulfhydryl groups.

12. The composition according to of claim 1 1 , wherein the at least one polymer is conjugated to the active entity at the one or more carboxyl, hydroxyl, amino and/or sulfhydryl groups.

13. The composition of any one of claims 3-12, wherein the conjugate has a longer residence time within cell compared to the active entity without conjugation to the polymer.

14. The composition of claim 13, wherein the residence time of the conjugate is at least 25% longer as compared to the active entity without conjugation to the polymer.

15. The composition of claim 13, wherein the residence time of the conjugate is at least 2-20 fold longer as compared to the active entity without conjugation to the polymer.

16. The composition of any one of claims 3- 15, wherein the conjugate exhibits greater access to the kinase compared to the active entity without conjugation to the polymer.

17. The composition of any one of claims 3- 16, wherein the conjugate exhibits a depo effect across cellular compartments, thereby reducing the dose of the active entity required to inhibit kinase activity compared to the active entity without conjugation to the polymer.

18. The composition of claim 17, wherein the dose of the conjugate needed to achieve a comparable therapeutic effect is 10-90% lower as compared to the active entity without conjugation to the polymer.

19. The composition of any one of claims 3- 18, wherein the activity entity has a concentration, activity and/or bioavailability at the target site that is at least 2-20 fold greater than at a non-target site, wherein the non-target site comprises the circulatory system.

20. The composition of any one of claims 3- 19, wherein the activity entity has a concentration, activity and/or bioavailability at the target site that is at least 2-20 fold greater than at a non-target site, wherein the non-target site comprises the lymphatic system.

21 . The composition of Claim 19 or 20, wherein the reduced concentration , activity and/or bioavailability reduces toxicity.

22. The composition of any one of claims 3-21 , wherein the activity entity has a concentration, activity and/or bioavailability at the target site that is at least 2-20 fold greater than at a non-target site, wherein the non-target site comprises bone marrow.

23. The composition of Claim 22, wherein the reduced concentration, activity and/or bioavailability in the bone marrow reduces immunosuppression.

24. The composition of any one of claims 3-23, wherein the conjugate is present at a biologically inactive concentration at a non-target site.

25. The composition of any one of claims 3-24, wherein the conjugate is amphiphilic

26. The composition of any one of claims 3-25, wherein the conjugate is at least 25% more amphiphilic than the active entity without conjugation to the polymer.

27. The composition of any one of claims 3-26, wherein the conjugate is at least 25% more hydrophilic than the active entity without conjugation to the polymer, thus facilitating non-compartmentalization within the cell.

28. The composition of any one of claims 3-27, wherein the conjugate is at least 25% more hydrophilic than the active entity without conjugation to the polymer, thus facilitating access to and activity in both the lipid bilayer and the cytosol of the cell.

29. The composition of any one of claims 3-28, wherein the conjugate is at least 25% more hydrophilic than the active entity without conjugation to the polymer, thus facilitating access to and/or activity in both the lipid bilayer and the cytoplasm of the cell.

30. The composition of any one of claims 3-29, wherein the conjugate is at least 25% more hydrophilic than the active entity without conjugation to the polymer, thus facilitating access to and/or activity across the lipid bilayer.

31 . The composition according to any one of claims 2-30, wherein the composition is formulated for topical administration.

32. The composition according to any one of claims 2-30, wherein the composition is formulated as an inhalant.

33. The composition according to any one of claims 2-30, wherein the composition is formulated as an injectable.

34. The composition according to any one of claims 2-30, wherein the composition is formulated as an eye drop.

35. The composition according to any one of claims 2-30, wherein the composition is formulated for oral administration.

36. The composition according to any one of claims 2-30, wherein the composition is administered via at least two routes of administration, either simultaneously or sequentially.

37. The composition according to any one of claims 2-36, wherein said composition is administered via a topical route to a subject, and wherein the subject further receives an additional agent via a non-topical route to achieve synergetic effects.

38. The composition according to any one of claims 2-37, the composition further comprising one or more additional ingredients from the group consisting of a protective agent, an emollient, an astringent, a humectant, a sun screening agent, a sun tanning agent, a UV absorbing agent, an antibiotic agent, an anti-angiogenesis agent, a preventive or therapeutic agent for inflammatory bowel disease, a physiological cooling agent, an antifungal agent, an antiviral agent, an antiprotozoal agent, an anti-acne agent, an anesthetic agent, a steroidal anti-inflammatory agent, a non-steroidal antiinflammatory agent, an antipruritic agent, an additional antioxidant agent, a chemotherapeutic agent, an anti-histamine agent, a vitamin or vitamin complex, a hormone, an anti-dandruff agent, an anti-wrinkle agent, an anti-skin atrophy agent, a skin whitening agent, and a cleansing agent.

39. A method for treating an inflammatory condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

40. A method for treating an inflammatory skin condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

41. A method for treating a vascular tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

42. A method for treating a skin neoplasia in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

43. A method for treating a bullous disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

44. A method for treating age-related macular degeneration in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

45. A method for treating diabetic retinopathy in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

46. A method for treating corneal edema in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

47. A method for treating macular edema in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

48. A method for treating dry eye in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

49. A method for modulating hair growth and cycling in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

50. A method for treating alopecia in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

51. A method for treating a wound in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

52. A method for treating a scar in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

53. A method for treating a cancerous or pre-cancerous lesion in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

54. A method for treating a lung in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

55. A method for treating the gastrointestinal system in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

56. A method for treating an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

57. A method for treating an eye in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

58. A method for treating a joint in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition according to any one of claims 2-38.

59. Use of the composition according to any one of claims 2-38 for treating one or more of the following conditions: hemangiomas, Kaposi's sarcoma, lymphangioma, glomangioma, angiosarcoma, hemangioendothelioma, and infantile hemangiomas.

60. Use of the composition according to any one of claims 2-38 for treating one or more of the following conditions: squamous cell carcinoma, basal cell carcinoma, malignant melanoma, malignant cutaneous lymphoma, Kaposi's sarcoma, Merkel cell skin cancer, and non-melanoma skin cancer.

61 . Use of the composition according to any one of claims 2-38 for treating one or more of the following conditions: bullous pemphigoid, erythema multiforme, dermatitis herpetiformis, epidermolysis bullosa acquisita, linear I mmunoglobulin A disease, mucous membrane pemphigoid, pemphigoid gestationis, pemphigus foliaceus, and pemphigus vulgaris.

62. Use of the composition according to any one of claims 2-38 for treating one or more of the following conditions: age-related macular degeneration, diabetic retinopathy, corneal edema, macular edema, ocular rosacea, and dry eye.

63. Use of the composition according to any one of claims 2-38 in a combination therapy with UV irradiation.

64. Use of the composition according to any one of claims 2-38 for modulating hair growth and cycling.

65. Use of the composition of any one of claims 2-38 for treating non-dermal inflammation in a subject in need thereof.

66. Use of the composition of any one of claims 2-38 for treating an inflammatory skin disease in a subject in need thereof.

67. Use of the composition of any one of claims 2-38 for treating a vascular tumor in a subject in need thereof.

68. Use of the composition of any one of claims 2-38 for treating a skin neoplasia in a subject in need thereof.

69. Use of the composition of any one of claims 2-38 for treating a bullous disease in a subject in need thereof.

70. Use of the composition of any one of claims 2-38 for treating age-related macular degeneration in a subject in need thereof.

71 . Use of the composition of any one of claims 2-38 for treating diabetic retinopathy in a subject in need thereof.

72. Use of the composition of any one of claims 2-38 for treating corneal edema, in a subject in need thereof.

73. Use of the composition of any one of claims 2-38 for treating dry eye in a subject in need thereof.

74. Use of the composition of any one of claims 2-38 for treating alopecia in a subject in need thereof.

75. Use of the composition of any one of claims 2-38 for treating a wound in a subject in need thereof.

76. Use of the composition of any one of claims 2-38 for treating a scar in a subject in need thereof.

77. Use of the composition of any one of claims 2-38 for treating a cancerous or pre-cancerous lesion in a subject in need thereof.

78. Use of the composition of any one of claims 2-38 for treating the lung in a subject in need thereof.

79. Use of the composition of any one of claims 2-38 for treating the gastrointestinal system in a subject in need thereof.

80. Use of the composition of any one of claims 2-38 for treating an autoimmune disorder in a subject in need thereof.

81 . Use of the composition of any one of claims 2-38 for treating the eye in a subject in need thereof.

82. Use of the composition of any one of claims 2-38 for treating a joint in a subject in need thereof.

83. Use of the composition according to any one of claims 2-38 for treating or preventing one or more of the following conditions: psoriasis, psoriasis guttata, inverse psoriasis, pustular psoriasis, psoriatic erythroderma, acute febrile neutrophilic dermatosis, eczema, xerotic eczema, dyshidrotic eczema, vesicular palmar eczema, acne vulgaris, atopic dermatitis, contact dermatitis, allergic contact dermatitis, dermatomyositis, exfoliative dermatitis, hand eczema, pompholyx, rosacea, rosacea due to sarcoidosis, rosacea due to scleroderma, rosacea due to Sweet syndrome, rosacea due to systemic lupus erythematosus, rosacea due to urticaria, rosacea due to herpetic pain, Sweet's disease, neutrophilic hydradenitis, sterile pustule, drug rash, seborrheic dermatitis, pityriasis rosea, Kikuchi's disease of the skin, pruritic urticarial papules and plaques of pregnancy, Stevens-Johnson syndrome and toxic epidermal necrolysis, tattoo reaction, Wells syndrome (eosinophilic cellulitis) , reactive arthritis (Reiter syndrome), bowel-associated dermatosis-arthritis syndrome, rheumatoid neutrophilic dermatosis, neutrophilic eccrine hidradenitis, neutrophilic skin disease of dorsum of hand, balanitis circumscripta plasmacellularis, balanoposthitis, Behcet's disease, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiforme, granuloma annulare, dermatitis of hand, lichen nitidus, lichen planus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, nummular dermatitis, pyoderma gangrenosum, sarcoidosis, subcorneal pustular dermatosis, urticaria, skin fibrosis, and transient acantholytic dermatosis, alopecia, alopecia areata, androgenic alopecia, and dry eye.