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WO2018175302A1 - Conjugués polymères ciblant c-src à exposition réduite - Google Patents

Conjugués polymères ciblant c-src à exposition réduite Download PDF

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Publication number
WO2018175302A1
WO2018175302A1 PCT/US2018/023109 US2018023109W WO2018175302A1 WO 2018175302 A1 WO2018175302 A1 WO 2018175302A1 US 2018023109 W US2018023109 W US 2018023109W WO 2018175302 A1 WO2018175302 A1 WO 2018175302A1
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Prior art keywords
composition
subject
agent
polymer
conjugate
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PCT/US2018/023109
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English (en)
Inventor
Silvio Traversa
Valentina Mainero
Todd James Harris
Luisa Bertarione RAVA ROSSA
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Sienna Biopharmaceuticals, Inc.
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Publication of WO2018175302A1 publication Critical patent/WO2018175302A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/48—Two nitrogen atoms

Definitions

  • polymer conjugates comprising active agents linked to polymers, and therapeutic uses thereof. More particularly, a polymer conjugate which exhibits reduced exposure to non-target sites and inhibits kinase mediators of various pathological conditions is described.
  • Inhibitors of c-Src have been described for possible therapeutic use in the prevention, alleviation and treatment of kinase-associated pathologies.
  • Such compounds are associated with broad kinase specificity, as well as undesirable and toxic effects. Accordingly, strategies to render these active kinase inhibitors more specific and less toxic are needed.
  • a polymer conjugate (such as SNA-101) is provided having the following structure:
  • 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).
  • 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.
  • exposure e.g., absorption or longevity of the composition in the systemic circulation, lymphatic system, or other non-targeted sites
  • the compositions described herein are both therapeutically efficacious and minimize non-target (e.g., systemic or bloodstream) exposure
  • 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.
  • 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).
  • the compositions and technology described herein are used in the gastrointestinal and pulmonary systems. Ophthalmic treatments are provided in some 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.
  • 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.”
  • Reduced exposure topical compositions are provided in many 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).
  • compositions formulated according to the methods described herein
  • the compositions 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.
  • 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, c-Src.
  • at least one inhibitor, antagonist, or inverse agonist of c-Src comprises or consists of a composition that includes any one of compounds 1 -71 (and derivatives thereof) disclosed herein in Table 1 coupled to a polymer.
  • the warhead of the polymer conjugate is compound 1 .
  • the polymer conjugate is SNA-101 (also referred to as CT101), having the following formula:
  • 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.
  • a non-target site such as the systemic circulation and/or lymphatic system
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • concentrations/amounts/dosing schedules are reduced by 25%-75% or more.
  • More rapid clearance rates of the active agent once in the non-target site(s) 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.
  • the active agents of the compositions described herein 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).
  • non-target sites e.g., the systemic circulation and/or lymphatic system
  • the active agents of the compositions described herein 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.
  • non-target sites e.g., the systemic circulation and/or lymphatic system
  • clearance of the compositions occurs within minutes of exposure to the non-target site (e.g., systemic circulation and/or lymphatic system), as opposed to hours.
  • 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.
  • 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.
  • the non-target site e.g., systemic circulation and/or lymphatic system
  • faster clearance rates in some cases even before the toxic metabolites are created, are especially beneficial.
  • 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.
  • the polymer influences the selectivity and/or inhibitory activity of the polymer conjugate.
  • the chemical linking moiety between the polymer and warhead influences the selectivity and/or inhibitory activity of the polymer conjugate.
  • the polymer conjugates exhibit no change in selectivity or inhibitory activity against the therapeutic target in comparison with the unconjugated active agent.
  • the polymer conjugates exhibit a significant increase in selectivity against the therapeutic target in comparison with the unconjugated active agent.
  • 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.
  • 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, rosacea, rosacea due to sarcoidosis, rosacea due to scleroderma, rosacea due to Sweet syndrome, rosacea due to systemic lupus erythematosus, rosacea due to ur
  • polymer conjugates wherein the polymer is polyethylene glycol (PEG) or methoxy-polyethylene glycol (m- PEG).
  • PEG polyethylene glycol
  • m- PEG methoxy-polyethylene glycol
  • a pharmaceutical composition comprising or consisting essentially of a polymer conjugate disclosed herein that is formulated for topical and non-topical administration.
  • methods of making and using the compositions described herein are provided.
  • 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.
  • the active entity comprises an inhibitor, an antagonist, or an inverse agonist.
  • the active entity may be an inhibitor, antagonist, or inverse agonist of c-Src.
  • inflammatory conditions are treated.
  • non-inflammatory conditions are treated.
  • the active entity comprises or consists essentially of any one or more of compounds 1 -71 in some embodiments.
  • the active entity comprises compound 1 .
  • the composition comprises CT101 .
  • the active entity binds to c-Src in some embodiments.
  • the binding may be partially or fully inhibitory or not.
  • the polymer used in the reduced exposure compounds comprises polyethylene glycol (PEG) and/or methoxy-polyethylene glycol (m-PEG).
  • PEG polyethylene glycol
  • m-PEG methoxy-polyethylene glycol
  • 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.
  • 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.
  • methods of treating one or more of the following are provided: non- dermal inflammation, inflammatory skin disease, wounds, scars, autoimmune disorders, and cancerous or pre-cancerous lesions.
  • 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.
  • compositions may be administered via at least two routes of administration, either simultaneously or sequentially according to some embodiments.
  • 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.
  • 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.
  • 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).
  • stereoisomers, enantiomers, and/or pharmaceutically acceptable salts of the compound are provided, in some embodiments.
  • a reduced exposure composition for treating a target site 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.
  • a pharmaceutically acceptable carrier formulated for delivering the conjugate to the target site is also provided.
  • 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.
  • the non-target site comprises any site at which pharmacological activity is not desired and/or not achieved.
  • the conjugate has the formula:
  • 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).
  • stereoisomers, enantiomers, and/or pharmaceutically acceptable salts of the conjugate are provided, in some 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.
  • 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.
  • the active entity may be for example, an inhibitor, antagonist, or inverse agonist of a cellular kinase.
  • the active entity is one or more of compounds 1 -71 .
  • the composition comprises compound 1 .
  • the composition comprises SNA-101 .
  • the polymer can include, for example, polyethylene glycol (PEG) and/or methoxy- polyethylene glycol (m-PEG).
  • a pharmaceutically acceptable carrier formulated for delivering the conjugate to the target site is also provided.
  • 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.
  • the non-target site comprises any site at which pharmacological activity is not desired and/or not achieved.
  • 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.
  • 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.
  • the cellular kinase may be c-Src.
  • c-Src is bound and/or inhibited by the active entity.
  • the active entity has one or more carboxyl, hydroxyl, amino and/or sulfhydryl groups.
  • at least one polymer is conjugated to the active entity at the one or more carboxyl, hydroxyl, amino and/or sulfhydryl groups.
  • 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.
  • the administration is daily.
  • effective amounts of the active entity are delivered to a subject (e.g., human or veterinary).
  • the composition may be administered via at least two routes of administration, either simultaneously or sequentially.
  • the composition is administered via a topical route to a subject, and the subject further receives an additional agent via a non-topical route.
  • 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.
  • additional ingredients such as, for example, a protective agent, an emollient,
  • 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.
  • 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.
  • the residence time is over 100 fold longer.
  • a smaller dose of the conjugate may be needed to achieve a therapeutic effect comparable to the active entity without conjugation to the polymer.
  • 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.
  • the dose is over 200% lower.
  • fewer doses and/or smaller doses of the conjugate are required as compared to the active entity delivered without the polymer.
  • 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.
  • the therapeutically effective amount of the active entity is at the target site.
  • 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).
  • the concentration, activity and/or bioavailability within a cell or tissue at the target site is over 100 fold greater.
  • 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.
  • the active entity and/or conjugate is present at a biologically inactive concentration within a cell or tissue at a non-target site.
  • reduced concentration, activity and/or bioavailability within a cell or tissue at a non-target site advantageously reduces toxicity and/or other side effects, such as, for example, immunosuppression.
  • 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).
  • 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.
  • the conjugate is more hydrophilic than the active entity without conjugation to the polymer.
  • 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.
  • the hydrophilicity is over 200% greater.
  • 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.
  • the conjugate exhibits greater access to the kinase compared to the active entity without conjugation to the polymer.
  • 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, an autoimmune disorder, the gastrointestinal system, a lung, a cancerous or pre-cancerous lesion, a scar, a wound, non-dermal inflammation, an inflammatory condition, an inflammatory skin condition, and/or an inflammatory skin disease.
  • 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: 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
  • ure 1 depicts the chemical structure of benzamide ChEMBL
  • ure 2 depicts the synthesis scheme of CT101 .
  • ure 3 depicts the HPLC chromatogram of carboxylic intermediate 2
  • ure 4 depicts the report of the flash purification of carboxylic intermediate 2
  • ure 5 depicts the chromatogram of CT101 reaction mixture at 23h
  • ure 6 depicts the chromatogram of CT101 reaction mixture at 23h
  • ure 7 depicts the report of the flash purification of CT101 .
  • ure 8 depicts the preparative HPLC chromatogram of CT101
  • Figure 9 depicts the analytical HPLC of CT101 (280nm).
  • Figure 10 depicts CT101 .
  • Figure 1 1 depicts CT101 synthesis.
  • Figure 12 depicts the MW 229 Da by-product.
  • Figure 13 depicts the hypothesized MW 258 Da by-product structures.
  • Figure 14 depicts the hypothesized MW 272 Da by-product structures.
  • Figure 15 depicts the HPLC analysis of hydrolysis reaction mixture/ 66 h aging time (HPLC method Ml).
  • Figure 16 depicts the HPLC analysis of crude Carboxylic acid intermediate (HPLC method Ml CT101 001).
  • Figure 17 depicts the HPLC analysis of amidation reaction mixture/21 h aging time (HPLC method Ml CT101 001).
  • Figure 18 depicts the HPLC analysis of crude CT101 (HPLC method Ml CT101 001).
  • Figure 19 depicts the UV Profile (@270 and 210 nm) of CT101 purification by reversed phase flash chromatography.
  • Figure 20 depicts the HPLC analysis of final CT101 lot n° 2013RB20/S48 (HPLC method Ml CT101 002).
  • Figure 21 depicts the NMR analysis of final CT101/lot n° 2013RB20/S48.
  • Figure 22 depicts the ESI MS analysis of CT101 lot n° 2013RB20/S48/single charged ion.
  • Figure 23 depicts the certificate of analysis of final CT101/ lot n° 2013BR20/S48.
  • Figure 24 depicts the HPLC analysis of final CT101 lot n° 2013RB20/S49 (HPLC method Ml CT101 002).
  • Figure 25 depicts the NMR analysis of final CT101/lot n° 2013RB20/S49.
  • Figure 26 depicts the ESI MS analysis of CT101 lot n° 2013RB20/S49/single charged ion.
  • Figure 27 depicts the certificate of analysis of final CT101/lot n° 2013RB20/S49.
  • Figure 28 depicts representative chromatograms detected using UV analysis (A) and MS analysis (B).
  • Figure 29 depicts chromatograms of a CT101 plasma standard extract (100 ⁇ g/mL) detected using SIR (TIC, upper) and UV (lower).
  • Figure 30 depicts method validation.
  • Figure 31 depicts individual chromatograms used for the analysis of CT101 (sample: plasma spiked with CT101 for 100 ⁇ g/mL). Top chromatogram: 1 167.5; middle chromatogram: 778.6, bottom chromatogram: 584.2.
  • Figure 32 depicts mouse plasma concentrations of CT101 . Data are presented as Mean ⁇ CI 95%.
  • Figure 33 depicts representative chromatograms showing CT101 in extracted murine plasma following intra-venous administration. Top chromatogram: 2 hours. Middle chromatogram: 10 minutes, bottom chromatogram: blank murine plasma.
  • Figure 34 depicts representative chromatograms showing CT101 in extracted murine plasma following epicutaneous administration. Top chromatogram: 8 hours, middle chromatogram: 0 hours, bottom chromatogram: blank murine plasma. [0075] Figure 35 depicts raw luminescence values for a CT101 preincubation time of 6 hours and a stimulation time of 24 hours. Each symbol represents an individual well. Each condition was tested in sextuplicate.
  • Figure 36 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 6 hours and a stimulation time of 24 hours.
  • Figure 37 depicts raw luminescence values for a CT101 preincubation time of 18 hours and a stimulation time of 24 hours. Each condition was tested in sextuplicate except for Jurkat cells were conditions were tested in triplicate.
  • Figure 38 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 18 hours and a stimulation time of 24 hours.
  • Figure 39 depicts raw luminescence values for a CT101 preincubation time of 24 hours and a stimulation time of 24 hours. Each condition was tested in sextuplicate except for Jurkat cells where conditions were tested in triplicate.
  • Figure 40 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 24 hours and a stimulation time of 24 hours.
  • Figure 41 depicts raw luminescence values for a CT101 preincubation time of 6 hours and a stimulation time of 6 hours. Each condition was tested in sextuplicate.
  • Figure 42 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 6 hours and a stimulation time of 6 hours.
  • Figure 43 depicts raw luminescence values for a CT101 preincubation time of 18 hours and a stimulation time of 6 hours. Each condition was tested in sextuplicate except for Jurkat cells were conditions were tested in triplicate.
  • Figure 44 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 18 hours and a stimulation time of 6 hours.
  • Figure 45 depicts raw luminescence values for a CT101 preincubation time of 24 hours and a stimulation time of 6 hours. Each condition was tested in sextuplicate.
  • Figure 46 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 24 hours and a stimulation time of 6 hours.
  • Figure 47 depicts percentage cytotoxicity for a CT101 pre-incubation time of 6 hours and a stimulation time of 24 hours.
  • Figure 48 depicts percentage cytotoxicity for a CT101 pre-incubation time of 18 hours and a stimulation time of 24 hours.
  • Figure 49 depicts percentage cytotoxicity for a CT101 pre-incubation time of 24 hours and a stimulation time of 24 hours.
  • Figure 50 depicts percentage cytotoxicity for a CT101 pre-incubation time of 24 hours and a stimulation time of 6 hours.
  • Figure 51 depicts percentage cytotoxicity for a CT101 pre-incubation time of 6 hours and a stimulation time of 6 hours.
  • Figure 52 depicts bodyweights. Data are presented as Mean ⁇ SEM percentages of the initial bodyweights. # p ⁇ 0.05 and ### p ⁇ 0.001 when compared to Day 0. ° p ⁇ 0.05, °° p ⁇ 0.01 and **** p ⁇ 0.0001 when compared to the vehicle-treated group.
  • Figure 53 depicts ear swelling. Data are presented as Mean ⁇ SEM. ### p ⁇ 0.0001 in the vehicle-treated group when compared to Day 0. °° p ⁇ 0.01 , °°° p ⁇ 0.001 and when compared to the vehicle-treated group.
  • Betamethasone 0.1 % induced a significant reduction of ear swelling when compared to the vehicle-treated group on Day 8, Day 1 1 and Day 14 (p ⁇ 0.0001).
  • Figure 54 depicts macroscopic scores. Data are presented as Mean ⁇ SEM. ///////// p ⁇ 0.0001 when compared to Day 0 in the vehicle-treated group. °°° p ⁇ 0.01 and when compared to the vehicle-treated group.
  • Figure 55 depicts cytokine levels in ears (pg/ml).
  • Data are presented as Mean ⁇ SEM. **** p ⁇ 0.0001 , ** p ⁇ 0.001 , * p ⁇ 0.01 when compared to the Vehicle treated group. #### p ⁇ 0.0001 , ### p ⁇ 0.001 when compared to the right ear within the same group.
  • Figure 56 depicts bodyweights. Data are presented as Mean ⁇ SEM of the initial (Day -13) bodyweights. * p ⁇ 0.05, ** p ⁇ 0.01.
  • Figure 57 depicts ear wwelling. Data are presented as Mean ⁇ SEM of the difference between ovalbumin challenged and contralateral (saline-injected) ears. Statistical significances: # p ⁇ 0.05, ## p ⁇ 0.01 , ### p ⁇ 0.001 when compared to the baseline (0 hours) values. * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 when comparing to the Control group.
  • Figure 58 depicts ear swelling at peak disease (fifteen minutes after the ovalbumin challenge). Data are presented as Mean ⁇ SEM of the difference between ovalbumin-challenged and contralateral (saline-injected) ears. Statistical significances: * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 when compared to the vehicle-treated group.
  • Figure 59 depicts erythema scores (Challenged ears). Data are presented as Mean ⁇ SEM.
  • Figure 60 depicts erythema scores (Challenged ears). Data are presented as Mean ⁇ SEM.
  • Figure 61 depicts histopathology scores (Left ears). Data are presented as Mean ⁇ SEM. ** p ⁇ 0.01 , *** p ⁇ 0.001 when compared to the Control group.
  • Figure 62 depicts representative histopathology pictures. Left panel: Left ears. Right panel: Right ears. Top line: Control Group. Middle line: Betamethasone 0.1 %-treated group. Bottom line: Vehicle-treated group. Magnification: x100.
  • Figure 63 depicts representative histopathology pictures. Left panel: Left ears. Right panel: Right ears. Top line: CT101_5%-treated group. Middle line: CT101_10%-treated group. Bottom line: CT101_20%-treated group. Magnification: x100.
  • Figure 64 depicts the BioMAP profile of SNA-101 in the Diversity PLUS Panel at the indicated concentrations.
  • the X-axis lists the quantitative protein- based biomarker readouts measured in each system.
  • 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% (
  • 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.
  • Figure 65 depicts a Reference Benchmark Overlay of SNA-101 and Benchmark Apremilast. Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 66 depicts an overlay of SNA-101 (29 ⁇ ) and Topiramate (3.3 ⁇ ), which was the top similarity match from a search of the BioMAP Reference Database of > 4,000 agents.
  • Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • FIG. 67 depicts Mechanism HeatMAP Analysis for SNA-101 .
  • Horizontal grey lines separate the 12 Diversity PLUS systems, while the vertical grey line separates SNA-101 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.
  • Figure 68 depicts the BioMAP profile of SNA-101 in the Diversity PLUS Panel at the indicated concentrations.
  • the X-axis lists the quantitative protein- based biomarker readouts measured in each system.
  • 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% (
  • 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.
  • Figure 69 depicts a Reference Benchmark Overlay of SNA-101 and Benchmark Staurosporine. Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 70 depicts an overlay of SNA-101 (300 ⁇ ) and N037 (490 ng/ml), which was the top similarity match from a search of the BioMAP Reference Database of > 4,000 agents for SNA-101 (300 ⁇ ).
  • Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 71 depicts an overlay of SNA-101 (100 ⁇ ) and Infliximab (30000 ng/ml), which was the top similarity match from a search of the BioMAP Reference Database of > 4,000 agents for SNA-101 (100 ⁇ ).
  • Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 72 depicts Mechanism HeatMAP Analysis for SNA- 101 .
  • Horizontal grey lines separate the 12 Diversity PLUS systems, while the vertical grey line separates SNA-101 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.
  • Figure 73 depicts a schematic showing how the IMQ-induced psoriasis study was performed.
  • Figure 74 depicts the total psoriasis clinical scores over time for all groups (A), the SNA-101 group (B), the SNA-125 group (C), and the SNA-352 group (D). The mean score for each group is displayed for each day +/- SEM.
  • Figure 75 depicts the erythema scores over time for all groups (A), the SNA-101 group (B), the SNA-125 group (C), and the SNA-352 group (D). The mean score for each group is displayed for each day +/- SEM.
  • Figure 76 depicts the plaque scores over time for all groups (A), the SNA-101 group (B), the SNA-125 group (C), and the SNA-352 group (D). The mean score for each group is displayed for each day +/- SEM.
  • Figure 77 depicts the punctate redness/scabbing scores over time for all groups (A), the SNA-101 group (B), the SNA-125 group (C), and the SNA-352 group (D). The mean score for each group is displayed for each day +/- SEM.
  • Figure 78A depicts the weight of spleens upon experimental termination on day 10. Mean spleen weight for each group is displayed +/- SEM.
  • Figure 78B depicts left ear thickness as measured with a caliper on days 0, 4, 6, 8, and 10. Mean thickness for each group is displayed for each day +/- SEM.
  • Figure 78C depicts the daily weight of mice. Body weight changes are displayed for each day as a percent of their weight measured on day 0. Mean values for each group are displayed +/- SEM.
  • Figure 79 depicts the levels of IL-17F (A), TNF-a (B), IL-22 (C), and IL-17A (D) as measured in left ears biopunched on day 4. After tissue homogenization, the cytokine levels in tissue lysates were measured via multiplex and then normalized with total protein amounts. Mean values for each group are displayed +/- SEM.
  • Figure 80 depicts CT101 (SNA-101).
  • Figure 81 depicts the CT101 synthesis scheme.
  • Figure 82 depicts HPLC analysis of starting benzamide (HPLC method Ml CT101 001).
  • Figure 83 depicts HPLC analysis of hydrolysis reaction mixture/69 h aging time (HPLC method Ml CT101 001).
  • Figure 84 depicts HPLC analysis of crude Carboxylic acid intermediate (HPLC method Ml CT101 001).
  • Figure 85 depicts HPLC analysis of amidation reaction mixture/20 h aging time (HPLC method Ml CT101 001).
  • Figure 86 depicts HPLC analysis of crude CT101 (HPLC method Ml CT101 001).
  • Figure 87 depicts the UV profile (@270 and 210 nm) of CT101 purification by reversed phase flash chromatography.
  • Figure 88 depicts HPLC analysis of final CT101 lot n° 2017CG07/S4 (HPLC method Ml CT101 002).
  • Figure 89 depicts 1 H NMR analysis of final CT101/lot n° 2017CG07/S4.
  • Figure 90 depicts ESI MS analysis of final CT101 lot n° 2017CG07/S4 /single charged ion.
  • Figure 91 depicts ESI MS analysis of final CT101 lot n° 2017CG07/S4 /double charged ion.
  • Figure 92 depicts the certificate of analysis of final CT101/ lot n° 2017CG07/S4.
  • Figure 93 depicts HPLC analysis of final CT101 lot n° 2017CG07/S2A (HPLC method Ml CT101 002).
  • Figure 94 depicts 1 H NMR analysis of final CT101/lot n° 2017CG07/
  • Figure 95 depicts ESI MS analysis of final CT101 lot n° 2017CG07/ S2A /single charged ion.
  • Figure 96 depicts ESI MS analysis of final CT101 lot n° 2017CG07/ S2A /double charged ion.
  • Figure 97 depicts the certificate of analysis of final CT101/ lot n° 2017CG07/ S2A.
  • LSE Low Systemic ExposureTM
  • the LSE platform creates polymer conjugates optimized for topical applications.
  • the polymer conjugates developed by LSE or more generally the reduced exposure technology exhibit enhanced penetration.
  • the enhanced penetration leads to delivery of a high local concentration of the drug.
  • the polymer conjugates show a limited non-target absorption upon topical administration due to their increased molecular size and amphiphilicity and/or amphipathicity.
  • side-effects are minimized by limiting or eliminating non-target (e.g., systemic) absorption.
  • the polymer conjugate comprises a "warhead" linked to a polymer.
  • the warhead is a pharmacologically active entity selected according to the particular target or pathway of interest.
  • polymer conjugates for use in the treatment of conditions including but not limited to inflammatory skin diseases).
  • 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.
  • the linker is a separate chemical linking moiety between the polymer and the warhead.
  • 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.
  • the modified PEG is a terminally alkoxy-substituted PEG.
  • the modified PEG is a methoxy-PEG (mPEG).
  • the polymer has a molecular weight ranging from about 100 to about 100,000 Da.
  • 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).
  • 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.
  • the short-chain PEG or mPEG has an average molecular weight of about 1 ,000-3,000 Da. (e.g., 2,000 Da).
  • 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). In 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.
  • the polymer is selected from poly(alkylene oxides) or from (polyethylene) oxides.
  • 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.
  • 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.
  • active agents useful for stimulating hair follicles are provided as oral applications or topical applications for the scalp.
  • Hair removal agents and ant-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.
  • the non-target site e.g., systemic circulation and/or lymphatic system
  • the reduced exposure compositions described herein provides benefits for these applications as well.
  • the polymer conjugates configured for reduced exposure are administered to other areas of the body besides the skin.
  • 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.
  • 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.
  • 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.
  • conjugating the warhead to a polymer 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.
  • a polymer e.g., PEG
  • conjugating the warhead to a polymer 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.
  • 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.
  • 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.
  • energy delivery such as laser, radiofrequency, ultrasound, microwave, etc.
  • thermal therapy such as laser, radiofrequency, ultrasound, microwave, etc.
  • light therapy such as radiation, intravenous chemotherapy, and others.
  • the compositions are applied with pressure, heat, massage etc. to facilitate localization to the desired target site.
  • the compositions are administered in combination with one or more additional therapeutics that may not be reduced exposure compounds.
  • 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.
  • 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.
  • the warhead employed in the LSE polymer conjugate is a small molecule targeting c-Src.
  • methods of treating an inflammatory skin disease in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting c-Src.
  • inflammatory skin disease refers to a skin condition accompanied by inflammation that is mediated, in part, by immune cells, including in some embodiments, T-cells.
  • 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, 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
  • the c-Src kinase is the most widely studied member of the largest family of nonreceptor protein tyrosine kinases, known as the Src family kinases (SFKs).
  • Src family kinases Other SFK members include Lyn, Fyn, Lck, Hck, Fgr, BIk, Yrk, and Yes.
  • the Src kinases can be grouped into two sub-categories, those that are ubiquitously expressed (Src, Fyn, and Yes), and those which are found primarily in hematopoietic cells (Lyn, Lck, Hck, BIk, Fgr). (Benati, D.
  • SFKs are key messengers in many cellular pathways, including those involved in regulating proliferation, differentiation, survival, motility, and angiogenesis.
  • the activity of SFKs is highly regulated intramolecularly by interactions between the SH2 and SH3 domains and intermolecularly by association with cytoplasmic molecules. This latter activation may be mediated by focal adhesion kinase (FAK) or its molecular partner Crk- associated substrate (CAS), which plays a prominent role in integrin signaling, and by ligand activation of cell surface receptors, e.g. epidermal growth factor receptor (EGFR).
  • FAK focal adhesion kinase
  • CAS molecular partner Crk- associated substrate
  • Src can also be activated by dephosphorylation of tyrosine residue Y530. Maximal Src activation requires the autophosphorylation of tyrosine residue Y419 (in the human protein) present within the catalytic domain. Elevated Src activity may be caused by increased transcription or by deregulation due to overexpression of upstream growth factor receptors such as EGFR, HER2, platelet- derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor, ephrins, integrin, or FAK.
  • upstream growth factor receptors such as EGFR, HER2, platelet- derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor, ephrins, integrin, or FAK.
  • AD Atopic dermatitis
  • Atopic dermatitis is a chronically relapsing inflammatory skin disease with a dramatically increasing incidence over the last decades.
  • Clinically AD is characterized by highly pruritic often excoriated plaques and papules that show a chronic relapsing course.
  • the diagnosis of AD is mostly based on major and minor clinical findings. See Hanifin J. M., Arch Dermatol: 135, 1551 (1999).
  • Histopathology reveals spongiosis, hyper and focal parakeratosis in acute lesions, whereas marked epidermal hyperplasia with hyper and parakeratosis, acanthosis/hypergranulosis and perivascular infiltration of the dermis with lymphocytes and abundant mast cells are the hallmarks of chronic lesions.
  • Psoriasis is characterized by frequent episodes of redness, itching, and thick, dry, silvery scales on the skin. Psoriasis comprises lesions that can involve primary and secondary alterations in epidermal proliferation, inflammatory responses of the skin, and an expression of regulatory molecules such as lymphokines and inflammatory factors. Psoriatic skin is morphologically characterized by an increased turnover of epidermal cells, thickened epidermis, abnormal keratinization, inflammatory cell infiltrates into the epidermis and polymorphonuclear leukocyte and lymphocyte infiltration into the epidermis layer. Psoriasis is often associated with other inflammatory disorders, for example arthritis, including rheumatoid arthritis, inflammatory bowel disease (IBD), and Crohn's disease.
  • IBD inflammatory bowel disease
  • compositions comprising compounds Nos 1 -71 shown in Table 1 are used, in several embodiments, as inhibitors, antagonists, and inverse agonists of c-Src.
  • Several embodiments relate to polymer conjugates of compounds 1 -71 , 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.
  • the warhead of the polymer conjugate is a small molecule disclosed in Table 1 targeting c-Src.
  • methods of treating an inflammatory skin disease in a subject 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 c-Src.
  • the warhead of the LSE polymer conjugate is compound 1 .
  • the LSE polymer conjugate is CT101 .
  • dry eye is the result of an underlying cytokine and receptor-mediated inflammatory process.
  • methods of treating dry eye in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting c-Src.
  • the composition is formulated as an eye drop.
  • one or two drops of the composition are used per application.
  • three or four drops of the composition are used per application.
  • six drops of the composition are used per application.
  • the composition is applied for a period of 60 seconds before flushing.
  • 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.
  • 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
  • Alopecia areata is known to be associated with autoimmune activities; hence, topically administered immunomodulatory compounds demonstrate efficacy for treating that type of hair loss.
  • hair regeneration compositions are in the form of a liquid.
  • hair regeneration compositions are in the form of a lotion.
  • hair regeneration compositions are in the form of a cream.
  • hair regeneration compositions are in the form of a gel.
  • the hair regeneration composition is administered twice daily.
  • the hair regeneration composition is administered one daily.
  • the hair regeneration composition is administered once weekly.
  • the hair regeneration composition is administered directly to the scalp.
  • the hair regeneration composition is administered directly non-scalp areas.
  • 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.
  • methods of treating an allergic inflammatory disease in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting c-Src.
  • methods of treating the following conditions in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is a small molecule targeting c-Src: 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 melanom
  • methods of treating a respiratory disease in a subject via delivery of the polymer conjugates (wherein the warhead is a small molecule targeting c-Src) to the lungs and/or airways 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,
  • 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, et
  • 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.
  • 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.
  • 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.
  • the amino group located the furthest away from the moieties interacting with the target is used.
  • 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.
  • 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).
  • 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).
  • 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).
  • the compound is modified (e.g., PEGylated) at the site furthest away from the active site.
  • 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).
  • Non-limiting examples of conjugation sites according to some embodiments and chemistries for compounds in Table 1 are disclosed.
  • 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).
  • 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).
  • the existing hydroxyl moiety could be conjugated to PEG-halide through formation of an ether bond in the presence of a strong base (including, e.g. NaH, KH, and n-BuLi).
  • a strong base including, e.g. NaH, KH, and n-BuLi.
  • 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.
  • the warhead employed in the LSE polymer conjugate is a small molecule targeting c-Src selected from one or more of the following: substituted 2-anilinopyrimidines; imidazoles, oxazoles and thiazoles with protein kinase inhibiting activities; heterocyclic substituted pyrazoles; aryl-amino substituted pyrrolopyrimidine multi-kinase inhibiting compounds; 2-phenylamino-4- (5- pyrazolylamino)-pyrimidine derivatives; bicyclic heteroaryls; 2-amino-6-anilino-purines; triazolopyridazines; substituted amides; pyrimidine derivatives; pyridinyl- pyrimidinylamino-benzamide derivatives; pyrrolo[2,3-d]pyrimidines; amino-substituted dihydropyrimido[4,5-d]pyrimidinone derivatives; 2-heteroarylamin
  • the LSE polymer conjugate comprises an RORvt antagonist/inverse agonist warhead selected from one or more of the following: stearic acid; All-trans retinoic acid; ALTA 1550; Ursolic acid; Digoxin; T0901317; SR1001 ; SR1078; SR3335; SR1555; SR221 1 ; ML209; N-(1 -(4-(1 , 1 , 1 ,3,3,3-hexafluoro-2- hydroxypropan-2-yl)benzyl)-1 ,2,3,4-tetrahydroquinolin-6-yl)acetamide; 2,4-difluoro-N-(1 - ((4-fluorophenyl)sulfonyl)-1 ,2,3,4-tetrahydroquinolin-7-yl)benzenesulfonamide; 2-Chloro- 6-fluoro-N-(1 -((4-fluorophenyl)
  • the LSE polymer conjugate comprises a Src family tyrosine kinase inhibitor warhead selected from dasatinib and saracatinib.
  • the LSE polymer conjugate comprises an IL-23 inhibitor warhead selected from SCH-90222, STA-5326, and STA-5326.
  • the LSE polymer conjugate comprises a STAT3 inhibitor warhead selected from cucurbitacin I, niclosamide, cryptotanshinone, SD 1008, Stat3 Inhibitor III, WP1066, Nifuroxazide, Stat3 Inhibitor, Stattic, Stat3 Inhibitor, S3I-201 ; Stat3 Inhibitor VIII, 5, 15-DPP, 2-Hydroxy-4-(((4- methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid (NSC74859) and Kahweol.
  • the LSE polymer conjugate comprises a JAK inhibitor warhead selected from ruxolitinib, fedratinib, tofacitinib, baricitinib, pacritinib, decernotinib, XL019, AZD1480, INCB0391 10, LY2784544, BMS91 1543, NS018, GLPG0634, GLPG0788, or N-(cyanomethyl)-4-2-(4-morpholinoanilino)pyrimidin-
  • JAK inhibitor warhead selected from ruxolitinib, fedratinib, tofacitinib, baricitinib, pacritinib, decernotinib, XL019, AZD1480, INCB0391 10, LY2784544, BMS91 1543, NS018, GLPG0634, GLPG0788, or N-(cyanomethyl)-4-2-(4-morpholinoanilino)pyrimi
  • Spleen tyrosine kinase is a non-receptor linked protein tyrosine kinase which plays a role as a mediator of immunoreceptor signaling in a host of inflammatory cells including mast cells, B-cells, macrophages and neutrophils.
  • the LSE polymer conjugate comprises a SYK inhibitor warhead selected from Cerdulatinib (4-(cyclopropylamino)-2-((4-(4-(ethylsulfonyl)piperazin- 1 - yl)phenyl)amino)pyrimidine-5 -carboxamide), entospletinib (6-( 1 H-indazol-6-yl)-N-(4- morpholinophenyl)imidazo[l,2-a]pyrazin-8-amine), fostamatinib ([6-( ⁇ 5-Fluoro-2- [(3,4,5- trimethoxyphenyl)amino]-4-pyrimidinyl ⁇ amino)-2,2-dimethyl-3-oxo-2,3-dihydro- 4H- pyrido[3,2-b][l,4]oxazin-4-yl]methyl dihydrogen phosphate), fostamatinib dis
  • PRT-062607 (4- ((3-(2H-l,2,3-triazol-2-yl)phenyl)amino)-2-(((IR,2S)-2- aminocyclohexyl)amino)pyrimidine-5-carboxamide hydrochloride), R1 12 (3,3'-((5- fluoropyrimidine-2,4-diyl)bis(azanediyl))diphenol), R348 (3-Ethyl-4-methylpyridine), R406 (6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2- dimethyl-2H- pyrido[3,2-b] [ 1 ,4]oxazin-3(4H)-one), piceatannol (3-Hydroxyresveratol), YM 193306(see Singh et al.
  • the LSE polymer conjugate comprises a RAC1 inhibitor warhead selected from W56, NSC23760 and NSC 23766, or the inhibitors described in Yuan Gao, et al. PNAS, May 18, 2004, vol. 101 , 7618-7623.
  • the LSE polymer conjugate comprises an ENaC inhibitor warhead selected from triamterene, phenamil, amiloride and amiloride derivatives, particularly benzyl amiloride (Benzamil). Additional amiloride derivatives are described in WO2012035158; WO2009074575; WO201 1028740; WO2009150137; WO201 1079087; and WO2008135557, each of which are herein specifically incorporated by reference.
  • the LSE polymer conjugate comprises a NFkB inhibitor warhead selected from Bithionol, Bortezomib, Cantharidin, Chromomycin A3, Daunorubicinum, Digitoxin, Ectinascidin 743, Emetine, Fluorosalan, Manidipine hydrochloride, Narasin, Ouabain, Sorafenib tosylate, Sunitinib malate, Tioconazole, Tribromsalan, Triclabendazolum, Zafirlukast, and Withaferin A.
  • NFkB inhibitor warhead selected from Bithionol, Bortezomib, Cantharidin, Chromomycin A3, Daunorubicinum, Digitoxin, Ectinascidin 743, Emetine, Fluorosalan, Manidipine hydrochloride, Narasin, Ouabain, Sorafenib tosylate, Sunitinib malate
  • the LSE polymer conjugate comprises an IRAK inhibitor warhead selected from the inhibitors described in Wang, Zhulun, et al. "IRAK-4 inhibitors for inflammation.” Current topics in medicinal chemistry 9.8 (2009): 724-737, which is herein specifically incorporated by reference.
  • the LSE polymer conjugate comprises a PKC inhibitor warhead selected from sotrastaurin (also known as AEB071 and described in U.S. Pat. No. 6,645,970), 3-(1 H-lndol-3-yl)-4-[2-(piperazin-1 -yl)quinazolin-4-yl]-1 H- pyrrole-2,5-dione (described in U.S. Pat. No.
  • the LSE polymer conjugate comprises a PKCa/ ⁇ inhibitor warhead selected from 3-[2-chloro-7-[(dimethylamino)methyl]-1 - naphthalenyl]-4-[7-[2-(2-methoxyethoxy)ethoxy]-1 H-indol-3-yl]-1 H-pyrrole-2,5-dione (CAS No. 919992-85-1 described in PCT Publication No. WO07/006,533 and US Publication No.
  • the LSE polymer conjugate comprises a specific SIP receptor agonist warhead selected from SIP itself, SEW2871 , JTE-013, VPC23019, R- 3477 (Actelion), KRP-203 (Kyorin Pharmaceutical Co.), sonepcizumab (Lpath), BAF-312 (Novartis), ONO-4641 (Ono Pharmaceutical Co.), ES-285 (PharmaMar SA), 2-amino-2-[2- (4-octylphenyl)ethyl]propane-l,3-diol (FTY720; fingolimod), phospho- FTY720, and pharmaceutically acceptable salts thereof.
  • the LSE polymer conjugate comprises a PI3K inhibitor warhead selected from Compound 1 ((S)-3-(1 -((9H-purin-6-yl)amino)ethyl)- 8-chloro-2-phenylisoquinolin-1 (2H)-one), AMG-319, GSK 2126458, GSK 1059615, GDC- 0032, GDC-0980, GDC-0941 , XL147, XL499, XL765, BKM 120, GS1 101 , CAL 263, SF1 126, PX-866, BEZ235, CAL-120, BYL719, RP6503, RP6530, TGR1202, INK1 1 17, PX-886, BAY 80-6946, IC871 14, Palomid 529, ZSTK474, PWT33597, TG100-1 15, GNE- 477, CUDC-907, AEZS-136,
  • the LSE polymer conjugate comprises an AKT inhibitor warhead selected from AZD5363, miltefosine, perifosine, VQD-002, MK- 2206, GSK690693, GDC-0068, triciribine, CCT128930, PHT-427, or honokiol, or a combination thereof.
  • the AKT inhibitor is MK-2206 or perifosine.
  • a mTOR inhibitor warhead selected from AP23841 , AZD8055, BEZ235, BGT226, deferolimus (AP
  • the LSE polymer conjugate comprises a PDE4 inhibitor warhead selected from rolipram, mesembrine, drotaverine, roflumilast, ibudilast, piclamilast, luteolin, cilomilast, diazepam, arofylline, CP-80633, denbutylline, drotaverine, etazolate, filaminast, glaucine, HT-0712, ICI-63197, irsogladine, Mesembrine, Ro20-1724, RPL-554, and YM-976.
  • PDE4 inhibitor warhead selected from rolipram, mesembrine, drotaverine, roflumilast, ibudilast, piclamilast, luteolin, cilomilast, diazepam, arofylline, CP-80633, denbutylline, drotaverine, etazolate, filamin
  • Suitable protecting groups 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the base is selected from the group of alkali metal hydrides, tertiary amines and/or alkoxide.
  • the base catalysing the polymer conjugation reaction is sodium hydride.
  • Other bases, such as sodium methoxide, or triethylamine can also be used.
  • 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, ⁇ , ⁇ -dimethylformamide. In certain embodiments, the organic solvent is dichloromethane or anhydrous dichloromethane.
  • 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.
  • the polymer conjugate may then be separated and purified from the reaction mixture.
  • 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.
  • 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.
  • the compound is purified by a normal phase flash chromatography.
  • 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.
  • 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.
  • 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.
  • the above-mentioned polymer moiety can carry an amino functional end-group or can be functionalized to carry an amino functional end-group.
  • the polymer moiety can be an amino-activated polymer of general formula X— NH2.
  • 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).
  • 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).
  • adverse or toxic effects e.g. irritation
  • any phototoxic effect e.g. photomutagenicity, phototoxicity or photosensitisation
  • 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.
  • 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.
  • compositions 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.
  • the disclosed compounds can be employed as the sole active agent in a pharmaceutical composition.
  • 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.
  • 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 ⁇ , IL-Ra, IL-8, MIP-1 a, MIF- ⁇ ⁇ , MIP-2, MIF and IL-6.
  • a cytokine selected from the group consisting of TNF, IL-1 a, IL- 1 ⁇ , IL-Ra, IL-8, MIP-1 a, MIF- ⁇ ⁇ , 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, de
  • 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, Jatroph
  • 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, fexofenad
  • 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.
  • 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 (TLR) 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.
  • TLR Toll-like receptor
  • 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.
  • 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).
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • a medical device such as but not limited to, stents, catheters, surgical instruments, cannulae, cardiac valves, or vascular prostheses.
  • 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.
  • 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.
  • compositions may also be used for diagnostic or for therapeutic 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.
  • a therapeutic application is, in the case of a topical application, the prevention, alleviation and treatment of psoriasis and dermatitis.
  • 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 ⁇ g 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.
  • 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.
  • CT101 and "SNA-101” are synonyms and can be used interchangeably.
  • Example 1 A first synthesis scheme for CT101 , a PEGylated variants of the kinase inhibitor ChEMBL id 249094 (CAS944795-06-6), by LSE Technology
  • ChEMBL 249097 (CAS No. 944795-06-6, illustrated in Figure 1) was identified as a member of the extensively studied protein kinase inhibitors class of 2,4- dianilino pyrimidines. After bibliographic data mining, ChEMBL 249097 resulted to inhibit several kinases whose misregulation is involved in atopic dermatitis, including CSK, Lyn, SYK and TXK kinases at low nanomolar range. [0222] Because of its peculiar polypharmacology profile, together with the feasibility of LSE modification and its readily commercial availability, ChEMBL 249097 was considered an interesting starting point to research LSE-variants with improved therapeutic potential.
  • Figure 2 depicts the first synthesis scheme for CT101 .
  • ChEMBL id 249094 (CAS944795-06-6) (Proactive Molecular Res, Cat No P06-
  • Trifluoroacetic acid (Sigma Aldrich, Cat No302031)
  • HPLC analytical C18 column eg. Phenomenex Jupiter C18 300A, 5 ⁇ , 4.6x250mm, Cat No 00G-4053-EO
  • HPLC semipreparative C18 column e.g. Phenomenex Jupiter C18 300A, 5 ⁇ , 15x250mm, Cat No 00G-4053-AK
  • Step 1 In a 25ml one neck round bottom flask with glass stopper, 50.81 mg of ChEMBL id 249097 (CAS944795-06-6, MW: 335.36 g/mol; 0.1515mmol) were dissolved in 3 ml of THF; then 5ml of water and 561 ⁇ _ of 32% NaOH solution (MW 40, d 1 .35g/ml, 40 eq) were added dropwise under vigorous stirring. The mixture was shielded from direct light with an aluminum foil, heated to 70°C on oil bath and let under stirring for 48-60h.
  • Reaction outcome was checked by HPLC (Method A) by injection of 5 ⁇ _ of a sample resulting from 10 ⁇ _ of reaction mixture diluted with 52.6 ⁇ _ of H 2 0/THF 5/3 (vol/vol). Typical conversion range is 75-80% ( Figure 3).
  • Reaction mixture was neutralized with diluted hydrochloric acid, saturated with sodium chloride and extracted with 5x100ml_ of methylene chloride and 2x100ml_ of ethyl acetate. Collected organic layers were dried over anhydrous sodium sulfate and evaporated, yielding to 46mg of crude. The material was then purified by flash chromatography on a silica column (Method B, Figure 4).
  • Step 2 In a one neck round bottom flask dried under nitrogen, 13.4mg of carboxylic intermediate 2 (MW 336.36, 0.04mmol) were suspended in 10ml of anhydrous dichloromethane under magnetic stirring. Subsequently, 8.23mg of dicyclohexyl carbodiimide (MW 206.33, 1 eq) and 4.59mg of N-hydroxy succinimide (MW 1 15.1 , 1 eq) were added and the mixture left under stirring. After 15 min, 80.39mg of mPEG-amine (MW 2015, 1 eq) were added and the reaction vessel shielded from direct light.
  • carboxylic intermediate 2 MW 336.36, 0.04mmol
  • reaction outcome was evaluated by HPLC (Method A, Figures 5 and 6) by injection of 15 ⁇ _ of a sample prepared by evaporating 32 ⁇ _ of reaction mixture under nitrogen flow and re-dissolving the residue in 38 ⁇ _ of THF and 62 ⁇ _ of H 2 0.
  • the reaction mixture was directly loaded into a flash silica column previously equilibrated with dichloromethane, and then eluted with a gradient of methanol (Method C) as reported in Figure 7.
  • the product was characterized by analytical RP-HPLC (Method A) by injection of 15 ⁇ of a 1 mg/ml solution in water / methanol 1/1.
  • Figure 9 reports the resulting chromatographic profile at 280 nm.
  • CT101 The molecular identity of CT101 was confirmed by LC/MS and NMR analyses.
  • Figure 10 depicts the structure of CT101.
  • Example 2 A second synthesis scheme for CT101 by LSE Technology
  • FIG. 1 The second synthesis scheme for CT101 production starting from commercially available benzamide is shown in Figure 1 1.
  • the selected synthetic scheme consists of hydrolysis to the carboxylic acid intermediate (Step 1) and amidation with mPEG 2 ooo-NH 2 (Step 2).
  • Figure 1 1 depicts the second scheme for CT101 synthesis.
  • CT101 Two main batches of CT101 , representative of the developed process, were produced: the first batch generated 1 .50 g of CT101 with 41 % overall yield from the starting benzamide and 99.5% HPLC purity, the second batch generated two CT101 samples (1 .63 g and 0.36 g respectively) with 47% overall yield from the starting benzamide, and 99,7% HPLC purity.
  • CT101 yield has been calculated assigning it 2333 Da as the average molecular weight, as the average molecular weight of MeO-PEG 200 o- NH 2 used for its preparation was 2015 Da.
  • the used synthetic procedure consisted of hydrolysis of the benzamide with a NaOH aqueous solution as base and THF as solvent.
  • the carboxylic acid intermediate isolated by flash chromatography, was amidated with mPEG 2 ooo-NH 2 in the presence of N-hydroxysuccinimide and ⁇ , ⁇ '-dicyclohexylcarbodiimide, producing the CT101 which was purified by normal phase flash chromatography first and then by preparative HPLC.
  • the overall yield from the starting benzamide was nearly 26%.
  • milder hydrolytic conditions (lower temperatures/ Exp. N°18) are used.
  • HPLC results shown in Table 9 refer to the isolated crude CT101 . It should be pointed out that the HPLC analysis doesn't reveal possible residual amounts of mPEG 20 oo-NH 2 in the crude CT101 , which is checked through NMR analysis.
  • the reaction mixture was cooled to 25°C, diluted with 130 ml of H20, and then 131 g of solvent were removed under reduced pressure at 45°C.
  • the residual aqueous solution was extracted with 160 ml of AcOEt.
  • the aqueous phase was diluted with 170 ml of AcOEt and neutralized under stirring with 15% HCI to pH 5,3.
  • the organic phase was dried over sodium sulphate and the solvent was evaporated under reduced pressure at 40°C.
  • the residual solid was further dried by suspending it in 50 ml of DCM and evaporating the solvent under reduced pressure at 40°C. This drying operation was repeated twice.
  • the dried solid residue was dissolved in 290 ml of DCM.
  • the solution was placed in a 500 ml three necked round bottom flask, equipped with magnetic stir bar, thermometer and condenser and protected from light by aluminum foil wrapping. 0.38 g of TEA were added at 25°C.
  • the solution was aged under stirring at 25°C for 10 min., then 0.73 g of TBTU were added.
  • the reaction mixture was aged at 25°C for 21 h under nitrogen atmosphere.
  • Sample loading was performed by dissolving the crude CT101 sample (1 .7-1 .8 g per purification) in 10 ml of H20 and injecting it onto the cartridge through a syringe.
  • the SNAP cartridge was eluted at 50 ml/min with:
  • the precipitated solid was filtered over sintered glass filter (G4), washed with 10 ml of diethyl ether and dried under vacuum at 28°C for 2 h to afford 1 .63 g of CT101 as an off white solid (lot n° 2013RB20/S48).
  • the precipitated solid was filtered over sintered glass filter (G4), washed with 10 ml of diethyl ether and dried under vacuum at 28°C for 2 h to afford 0.36 g of CT101 as an off white solid (lot n° 2013RB20/S49).
  • CT101 and CAS944795-06-6 were tested against 271 target kinases.
  • 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.
  • DMSO dimethylsulfoxide
  • Reference compounds for assay control were prepared similarly. Assay reagents and procedures IMAP Assay
  • MSA Off-chip Mobility Shift Assay
  • Termination Buffer Quality of Service
  • reaction control complete reaction mixture
  • background Enzyme(-)
  • CT101 inhibition of BTK, FYN, and PKCs were examined at the following test concentrations: 50, 15, 5, 1 .5, 0.5, 0.15, 0.05, 0.015, 0.005, 0.0015 ⁇ .
  • CT101 inhibition of SRC was examined at the following test concentrations: 30, 10, 3, 1 , 0.3, 0.1 , 0.03, 0.01 , 0.003, 0.001 ⁇ .
  • 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.
  • DMSO dimethylsulfoxide
  • Reference compounds for assay control were prepared similarly. Assay reagents and procedures
  • MSA Off-chip Mobility Shift Assay
  • Termination Buffer Quality of Service
  • reaction control complete reaction mixture
  • background Enzyme(-)
  • IC 50 value was calculated from concentration vs. %lnhibition curves by fitting to a four parameter logistic curve.
  • This study aims at selecting an appropriate vehicle formulation for topical application of CT101 onto the skin (Part A), validating a pharmacokinetic (PK) analysis method (Part B) and evaluating the PK of CT101 (Part C).
  • Part A pharmacokinetic analysis method
  • Part B pharmacokinetic analysis method
  • Part C evaluating the PK of CT101
  • CT101 was initially a solid compound that was diluted in vehicle (sodium chloride 0.9%, saline for intra-venous administrations and that determined in Part A for epicutaneous applications)
  • Part A Vehicle Assessment
  • Vehicle 2 was 10% Propylene Glycol, 40% DMSO and 50% distilled water
  • Vehicle 3 was 25% Transcutol P, 10% Propylene Glycol and 65% distilled water
  • Terminal blood samples from adult male CD-1 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.
  • CT101 was weighed out and reconstituted in methanol to give a stock solution at 1 mg/mL. All chemicals used for chromatography were of HPLC grade (Fisher Scientific Ltd., Loughborough, UK).
  • the MS was used in electrospray positive mode, with a capillary voltage of 3.4 kV and a cone voltage of 20 V. Source and desolvation temperatures and gas flows settings were standard for the system. SIR channels were created to measure CT101 at mass-to-charge ratio (m/z) 584.2, 778.6 and 1 167.5. Sample preparation and analysis
  • Non-biological standards were prepared by spiking CT101 into 10% methanol: 90% water.
  • Biological standards were prepared by spiking CT101 into blank murine plasma.
  • the calibration curves were prepared with ten non-blank standards each at a volume of 50 ⁇ _ (individual calibrant concentrations were 0.1 , 0.2, 0.5, 0.8, 1 , 5, 10, 20, 50 and 100 ⁇ g/mL).
  • Plasma samples and standards were extracted by adding three volumes of ice-cold methanol to precipitate the plasma proteins. After methanol addition, the sample was briefly vortexed to mix, then centrifuged at 10,000 g for five minutes (4°C). The supernatant was transferred to a labelled LC vial for analysis. Ten microliters of supernatant were injected into the LC-MS system. Calibration standards were analysed first (low to high concentration).
  • Plasma samples were analysed by LC-MS.
  • Vehicle for intravenous administrations was a 0.9% sodium chloride solution
  • Part A Three vehicle formulations were assessed by epicutaneous application onto mice ears for up to three days. Animals were monitored for signs of inflammation.
  • Part B 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. The stability of the compound in plasma at -80°C was tested by determining the recovery following a freeze/thaw and a one week storage at -80°C.
  • Part C Study samples were then processed by HPLC for quantitative analysis of CT101 in plasma. Results
  • the volume used was sufficient to apply the solution to the entire surface of the ear.
  • the administration volume could be reduced to ten to fifteen microliters per ear.
  • Vehicle 1 (25% Transcutol P and 75% Propylene Glycol) was absorbed and left an oily residue at the surface of the ear. The residue was still observed for up to one hour after the epicutaneous application.
  • Vehicle 2 (10% Propylene Glycol, 40% DMSO and 50% distilled water) was spread with difficulty and was not absorbed and remained at the surface of the ear. The solution was easily shaken off and/or groomed away by the animals when returned to their cages.
  • Vehicle 3 (25% Transcutol P, 10% Propylene Glycol and 65% distilled water) was absorbed and left an oily residue at the surface of the ear. The residue was still observed for up to one hour after the epicutaneous application.
  • Plasma calibration standards were prepared from CD-1 mouse plasma blank plasma samples and the calibration standards were initially run with UV detection. However, this method was found to have poor separation, with a much lower limit of detection than the MS detection. As a consequence, the UV detection method was abandoned. Analysis of the UV data from the calibration curve showed that the LOQ by UV was 10 ⁇ / ⁇ ..
  • Figure 28 (A) shows UV (280 nm) traces for, from bottom to top: blank calibration plasma, 10 ⁇ g/mL calibration plasma, 100 ⁇ / ⁇ calibration plasma, sample 21 (1/2 hour epicutaneous administration, animal 3.4), and sample 58 (1/2 hour intravenous administration, animal 8.3).
  • Figure 28 (B) shows the MS TIC traces for the same injections. The shaded areas in all traces indicate the CT101 peak. There was co-eluting interference seen in the plasma samples at 280 nm (indicated by the arrow), making baseline to baseline integration difficult. The width of the peak detected at 280 nm is approximately one minute wide (horizontal lines); a reflection on the polymeric nature of the PEGylated compound. In comparison, due to the selective nature of the MS method, there are no co-eluting interferences detected, and the peak monitored at the chosen masses was much narrower.
  • Plasma calibration standards were prepared from CD-1 mouse plasma blank plasma samples. The calibration range was 0.1 to 100 ⁇ g/mL.
  • a 50 ⁇ _ volume of calibrant was extracted in 150 ⁇ _ ice-cold methanol.
  • a 10 ⁇ _ aliquot of supernatant was injected into the LC-MS system.
  • the limit of detection (LOD) was 10 ⁇ g/mL with the UV method and 0.2 ⁇ g/mL with the MS method.
  • the limit of quantitation (LOQ) was greater than 10 ⁇ g/mL with the UV method and 0.5 ⁇ g/mL with the MS method.
  • CT101 at 0.1 ⁇ g/mL was not detectable.
  • CT101 at 0.2 ⁇ g/mL was detectable, but the peak was too small to accurately quantify, so this point was set as the LOD.
  • At 0.5 ⁇ g/mL CT101 was detected, with the peak signal at least three times the background signal (a standard LC reference test), so this point was set as the LOQ.
  • Figure 29 depicts chromatograms of a CT101 plasma standard extract (100 ⁇ g/mL) detected using SIR (TIC, upper) and UV (lower).
  • Figure 30 depicts method validation.
  • Figure 31 depicts individual chromatograms used for the analysis of CT101 .
  • CT101 was applied topically once at 50 mg/kg or administered once by intravenous injection at 5 mg/kg.
  • Plasma samples were analysed by LC-MS method.
  • Plasma levels of CT101 in animals treated by topical application were below the detection limit of the assay (0.2 ⁇ g/mL) for all but one sample.
  • FIG. 33 depicts representative chromatograms showing CT101 in extracted murine plasma following intra-venous administration.
  • Figure 34 depicts representative chromatograms showing CT101 in extracted murine plasma following epicutaneous administration.
  • Vehicle 2 (10% propylene glycol, 40% DMSO and 50% distilled water) is not appropriate for epicutaneous applications.
  • Vehicle 1 (25% Transcutol P and 75% Propylene Glycol) was used for Part B and Part C of this study.
  • a suitable method for the analysis of CT101 in murine plasma by LC- MS was developed. The method is reproducible and has a good limit of detection (0.2 ⁇ g/mL) when compared to the UV method. This method was used to analyze samples from mice administered with CT101 by epicutaneous application and intra-venous injection.
  • CT101 concentrations were measured in mouse plasma samples after epicutaneous or intravenous administration.
  • the administration dose for epicutaneous administration was ten times higher than when using the intravenous administration route.
  • Plasma levels after epicutaneous application of CT101 were below the detection limit of the assay except for one animal (animal 4) on two time points (15 and 30 minutes).
  • Example 6 Evaluation of the ability of CT101 to inhibit conventional NF-KB signaling
  • NF- KB reporter construct a secreted luciferase reporter gene, driven by an IFN- ⁇ minimal promoter fused to five copies of the NF- ⁇ consensus transcriptional response element and three copies of the c-Rel binding site.
  • Use of a stably transfected cell line eliminates the variation associated with transient transfection of the luciferase reporter into cell lines.
  • Cell lines were pre-treated with compound CT101 , before stimulation with a ligand suitable for the cell type; THP-1 cells were stimulated with the TLR1/2 ligand Pam3CSK4 (100 ng/mL), Jurkat cells were stimulated with Concanavalin A (ConA, 50 ⁇ g/mL) and HEK293 cells with TNF-a (10 ng/mL). Cells were then incubated for a further six hours or twenty-four hours before assessment of luciferase activity in the cultures as a measurement of NF- ⁇ transcriptional activity.
  • THP-1 cells were stimulated with the TLR1/2 ligand Pam3CSK4 (100 ng/mL)
  • Jurkat cells were stimulated with Concanavalin A (ConA, 50 ⁇ g/mL)
  • HEK293 cells TNF-a (10 ng/mL). Cells were then incubated for a further six hours or twenty-four hours before assessment of luciferase activity in the cultures as a measurement of
  • CT101 was tested at three concentrations on the three different cell types (100 ⁇ , 30 ⁇ , and 10 ⁇ ). Each condition was tested in sextuplicate. Concentrations may be lowered (if cell toxicity is observed) or increased up to mM ranges (if effect is not observed).
  • Table 14 shows the experimental conditions tested for each of the reporter cell lines (HEK, THP-1 , Jurkat).
  • LDH lactose dehydrogenase
  • Luciferase activity in reporter cell lines was assayed using a Promega luminometer.
  • LDH assays were assayed using a colorimetric system and absorbance read on an omega plate reader.
  • HEK, Jurkat and THP-1 reporter cell lines were pre-incubated with CT101 (100 ⁇ , 30 ⁇ , 10 ⁇ ) or DMSO vehicle (0.1 % v/v) control for 6 hours, before stimulation for a further 24 hours with 10 ng/mL TNF-a (HEK cells), 100 ng/mL Pam3CSK4 (THP-1 cells) or 50 ⁇ g/mL ConA (HEK293 cells). Control cells were not stimulated (non-stimulated) to provide background levels of luciferase activity present in cell lines. As a positive control some cells were incubated with 200 nM of staurosporine.
  • the Jurkat cell line did not grow well in the conditions recommended by the supplier therefore in some experiments where cell number was limiting; this cell line was tested in triplicate or quadruplicate for each condition.
  • the Jurkat cell line was not tested for the 6h pre-incubation, 6h stimulation or 24h pre- incubation, 6h stimulation experimental conditions.
  • Figure 35 depicts raw luminescence values for a CT101 preincubation time of 6 hours and a stimulation time of 24 hours. Each symbol represents an individual well. Each condition was tested in sextuplicate.
  • Figure 36 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 6 hours and a stimulation time of 24 hours.
  • Figure 37 depicts raw luminescence values for a CT101 preincubation time of 18 hours and a stimulation time of 24 hours. Each condition was tested in sextuplicate except for Jurkat cells were conditions were tested in triplicate.
  • Figure 38 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 18 hours and a stimulation time of 24 hours.
  • Figure 39 depicts raw luminescence values for a CT101 preincubation time of 24 hours and a stimulation time of 24 hours. Each condition was tested in sextuplicate except for Jurkat cells where conditions were tested in triplicate.
  • Figure 40 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 24 hours and a stimulation time of 24 hours.
  • Figure 41 depicts raw luminescence values for a CT101 preincubation time of 6 hours and a stimulation time of 6 hours. Each condition was tested in sextuplicate.
  • Figure 42 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 6 hours and a stimulation time of 6 hours.
  • Figure 43 depicts raw luminescence values for a CT101 preincubation time of 18 hours and a stimulation time of 6 hours. Each condition was tested in sextuplicate except for Jurkat cells were conditions were tested in triplicate.
  • Figure 44 depicts fold induction of Luciferase above non-stimulated cells for a CT101 preincubation time of 18 hours and a stimulation time of 6 hours.
  • Figure 45 depicts raw luminescence values for a CT101 preincubation time of 24 hours and a stimulation time of 6 hours. Each condition was tested in sextuplicate.
  • Figure 46 depicts fold induction of Luciferase above non-stimulated cells for a CT101 pre-incubation time of 24 hours and a stimulation time of 6 hours.
  • Figure 47 depicts percentage cytotoxicity for a CT101 pre-incubation time of 6 hours and a stimulation time of 24 hours.
  • Figure 48 depicts percentage cytotoxicity for a CT101 pre-incubation time of 18 hours and a stimulation time of 24 hours.
  • Figure 49 depicts percentage cytotoxicity for a CT101 pre-incubation time of 24 hours and a stimulation time of 24 hours.
  • Figure 50 depicts percentage cytotoxicity for a CT101 pre-incubation time of 24 hours and a stimulation time of 6 hours.
  • Figure 51 depicts percentage cytotoxicity for a CT101 pre-incubation time of 6 hours and a stimulation time of 6 hours.
  • the LDH assay shows that the reduction of luciferase activity in HEK cells is not caused by a reduction of the number of cells.
  • the LDH assay suggests that, in Jurkat and THP-1 cells, the reduction of the luciferase activity may in part be associated with a reduction of the number of cells.
  • CT101 proved to inhibit ligand induced NF- ⁇ activity in a statistically significant manner at the top tested dose (100 ⁇ ) after 6 and 18 hours of pre-incubation in HEK cells, that proved to be the best performing of the three tested cell lines.
  • control protein kinase inhibitor drug Staurosporine did inhibit NF- KB activity, under each condition tested for the HEK reporter cell line, the percentage inhibition of NF- ⁇ induction was dependent on culture conditions and the cell line.
  • This study used a mouse model of 4-Ethoxymethylene-2-phenyl-2- oxazolin-5-one (Oxazolone)-induced contact dermatitis to test the efficacy of CT101.
  • CT101 was provided as a solid compound that was solved in vehicle (25% Transcutol P and 75% Propylene Glycol for epicutaneous applications)
  • mice Female BALB/c mice were randomly allocated to experimental groups and allowed to acclimatise for one week. On Day 0, animals were sensitised to oxazolone by epicutaneous application of 50 ⁇ _ of a 1.5% solution in acetone: olive oil (4: 1) to the clipped abdomen. On Days 7, 10 and 13, animals were challenged by epicutaneous application of 10 ⁇ _ per surface of a 1 .0% oxazolone solution, or vehicle, to both surfaces of both ears. Oxazolone and treatments were given according to the administration schedule below. Ear swelling was measured 24 hours after each oxazolone challenge using a digital calliper. At termination ears were collected, cut in two halves and stored for optional histopathology and optional tissue cytokine analysis.
  • Vehicle for epicutaneous administrations is 25% Transcutol P, 75% propylene glycol.
  • Administration volume for treatment epicutaneous applications was 20 ⁇ _ per ear per administration (two administrations per day). Only one ear per animal was treated with the active ingredients (left ear), while the contra-lateral ear was treated with the vehicle each time (right ear).
  • mice were treated as follows:
  • ear thickness was measured using digital callipers. Swelling was calculated as the difference in thickness between the pre-challenge thickness (Day -1) and the daily values taken on Day 8, Day 1 1 and Day 14.
  • Bodyweight data were analyzed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental days in the vehicle- treated group then for multiple comparisons between experimental groups. As shown in Figure 52, bodyweights in the vehicle-treated group increased prior to the oxazolone challenge and Day 6 bodyweights were significantly higher than bodyweights recorded on Day 0 (p ⁇ 0.001). Oxazolone challenges induced a decrease in bodyweights. Day 1 1 bodyweights were significantly lower than bodyweights recorded on Day 0 (p ⁇ 0.05).
  • Betamethasone 0.1 % induced a significant decrease of the bodyweights when compared to the vehicle-treated group from Day 4 until the end of the experiment on Day 14 (p ⁇ 0.0001).
  • CT101 administered at 10% did not prevent the oxazolone challenges- induced bodyweight loss.
  • the bodyweights in the CT101_ 10%-treated group was significantly lower than in the vehicle-treated group on Day 6 (p ⁇ 0.001), on Day 8 (p ⁇ 0.05), Day 1 1 (p ⁇ 0.05) and Day 14 (p ⁇ 0.01).
  • CT101 administered at 20% did not prevent the oxazolone challenges- induced bodyweight loss but did not induce any further bodyweight loss when compared to the vehicle-treated group.
  • Ear swelling data were analysed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental days in the vehicle- treated group then for multiple comparisons between experimental groups.
  • Oxazolone challenges induced a significant increase of ear swelling in the vehicle-treated group on Day 8, Day 1 1 and Day 14 when compared to Day 0 (p ⁇ 0.0001 ).
  • CT101 administered at 10% induced a significant reduction of ear swelling when compared to the vehicle-treated group on Day 1 1 (p ⁇ 0.01) and Day 14 (p ⁇ 0.0001).
  • CT101 administered at 20% induced a significant reduction of ear swelling when compared to the vehicle-treated group on Day 14 (p ⁇ 0.001).
  • Macroscopic score data were analysed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental days in the vehicle-treated group then for multiple comparisons between experimental groups.
  • Oxazolone induced a significant increase of the macroscopic scores in the vehicle-treated group from Day 7 until the end of the experiment on Day 14 when compared to Day 0 (p ⁇ 0.0001).
  • Betamethasone 0.1 % induced a significant reduction of the macroscopic scores when compared to the vehicle-treated group from Day 7 until the end of the experiment on Day 14 (p ⁇ 0.0001).
  • CT101 administered at 10% induced a significant decrease of the macroscopic scores when compared to the vehicle-treated group on Day 13 (p ⁇ 0.001) and Day 14 (p ⁇ 0.0001).
  • CT101 administered at 20% induced a significant reduction of the macroscopic scores when compared to the vehicle-treated group on Day 14 (p ⁇ 0.0001).
  • Betamethasone induced significant decrease in IL- 1 ⁇ and IFN- ⁇ .
  • levels IL-4 in the left ear were slightly increased when compared to the right ear.
  • levels of IL-4 were higher in the right when compared to the left ear within CT101 20% group. This suggests that an active treatment at the site of inflammation was therefore able to reduce some of the cytokine levels in this model.
  • Betamethasone 0.1 % reduced ear swelling and skin erythema. Betamethasone 0.1 % caused bodyweight loss, a known side effect of steroids administered to rodents.
  • CT101 administered at 10% and 20% partially prevented the development of pathological changes to the skin (swelling and erythema), inducing a statistically significant reduction of ear swelling and of the macroscopic clinical scores
  • Cytokines in the present model mostly were present at detectable levels in ear homogenates, with the exclusion of TNF-a which was present at levels below limit of detection.
  • Betamethasone induced decrease in pro-inflammatory cytokines namely IL-1 ⁇ and IFN- ⁇
  • This study used a mouse model of ovalbumin-induced atopic dermatitis to test the efficacy of CT101 .
  • CT101 was provided as a solid compound and was diluted in vehicle
  • One group of animals (Group 1 , Control) received an intradermal injection of saline in each ear. Treatments were administered according to the schedule below. On Day 0, at 1 ⁇ 4 hour, 1 ⁇ 2 hour and one hour after the ovalbumin challenge and on Day 1 , twenty-five and twenty-nine hours after the ovalbumin challenge, ears were observed for clinical signs of skin inflammation to include erythema, scaling and skin thickening. Ear thickness was measured using digital callipers. Twenty-eight hours after ovalbumin challenge, animals were culled and ears dissected out. One half of each ear was stored at room temperature in tissue fixative until further optional histopathology analysis.
  • Vehicle for epicutaneous administrations is 25% Transcutol P and 75% Propylene Glycol.
  • Administration volume for epicutaneous applications was 20 ⁇ _ per ear.
  • Treatment groups were as follows:
  • H&E haematoxylin and eosin
  • Epidermal and dermal layer thickening may be quantified by taking five representative micrometre measurements for each layer using the linear measurement tool in the Leica Application Suite programme. Skin layer measurements are not relevant to this model (but to our models of skin scleroderma) and were not performed.
  • cellular infiltrate may be characterised and or quantified by counting inflammatory cells on three to five H&E-stained sections. Cellular characterisation and quantification were performed.
  • Sections were scored for epidermal hyperplasia (0: absent, 1 : present), dermal oedema (0: absent, 1 : mild, 2: moderate and 3: severe) and dermal inflammation (0: less than 5 inflammatory cells, 1 : 5-10 cells, 2: 10-25 cells and 3: more than 25 cells). A total score was calculated by adding the individual parameter scores for a maximum possible score of 7. Sections were evaluated in blinded fashion.
  • Bodyweight data were analyzed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental groups.
  • Betamethasone 17-valerate administered twice daily by topical application onto the ears from Day -5 induced a significant reduction of the bodyweight measured on Day -3 (p ⁇ 0.05) and Day 0 (p ⁇ 0.01) when compared to the vehicle-treated group ( Figure 56).
  • CT101 administered at 5%, 10% or 20% twice daily by topical application onto the ears from Day -5 did not induce any bodyweight reduction.
  • Ovalbumin induced a significant increase of ear thickness in the vehicle-treated group fifteen minutes (p ⁇ 0.01), twenty-five hours (p ⁇ 0.05) and twenty- nine hours (p ⁇ 0.01) after the challenge when compared to baseline (0 hours) values ( Figure 57).
  • Ovalbumin induced a significant increase of ear thickness in the vehicle-treated group fifteen minutes (p ⁇ 0.01), thirty minutes (p ⁇ 0.05), twenty-five hours (p ⁇ 0.01) and twenty-nine hours (p ⁇ 0.001) after the challenge when compared to the Control group.
  • Table 17 summarizes the ear swelling data.
  • Ear swelling data recorded at peak disease were further analysed by one-way ANOVA followed by Dunnett's post-test for multiple comparisons to the vehicle-treated group.
  • Ovalbumin challenge induced a significant increase of the ear swelling measured in the vehicle-treated group when compared to the Control group (p ⁇ 0.01).
  • Betamethasone 0.1 % induced a significant reduction of the ovalbumin-induced ear swelling when compared to the vehicle-treated group (p ⁇ 0.001).
  • CT101 administered at 5%, did not reduce the ovalbumin-induced ear swelling (Figure 58).
  • CT101 administered at 10% and 20%, reduced the ovalbumin- induced ear swelling. The reduction was found to be statistically significant when CT101 was administered at 10% (p ⁇ 0.01). Skin inflammation
  • Erythema was scored on a point-scale where (0) is absent, (1) is mild, (2) is moderate and (3) is severe. Erythema score data were analysed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental times or between experimental groups.
  • ovalbumin challenge (intra-dermal administration on Day 0) induced a significant increase in erythema scores observed in the vehicle-treated group at all experimental times when compared to the scores observed prior to the challenge.
  • Betamethasone 17-valerate induced a reduction of the erythema scores when compared to the vehicle-treated group. The reduction was statistically significant thirty minutes after the challenge (p ⁇ 0.01), twenty-five hours after the challenge (p ⁇ 0.0001) and twenty-nine hours after the challenge (p ⁇ 0.0001).
  • CT101 administered at 5% reduced the erythema scores from thirty minutes after the challenge when compared to the vehicle-treated group but the reduction was not statistically significant.
  • CT101 administered at 10% reduced the erythema scores from one hour after the challenge when compared to the vehicle-treated group but the reduction was not statistically significant. Fifteen minutes after the challenge, the erythema scores in the CT101_10%-treated group was significantly higher than in the vehicle-treated group (p ⁇ 0.05).
  • CT101 administered at 20% reduced the erythema scores from thirty minutes after the challenge when compared to the vehicle-treated group and the reduction was statistically significant twenty-nine hours after the challenge (p ⁇ 0.05).
  • Figure 59 and Figure 60 depict erythema scores (challenged ears) at different times post-challenge.
  • ovalbumin challenge induced a significant increase of the epidermal hyperplasia scores (p ⁇ 0.01), the dermal oedema scores (p ⁇ 0.01), the dermal inflammation scores (p ⁇ 0.01) and the total histopathology scores (p ⁇ 0.001) in the vehicle-treated group when compared to the control group ( Figure 61).
  • Betamethasone 0.1 % did not significantly reduce the histopathology scores.
  • CT101 applied at 5%, 10% or 20% induced a non-significant reduction of the dermal oedema but did not significantly reduce the epidermal hyperplasia, the dermal inflammation or the total histopathology scores.
  • FIG. 62 Representative pictures in Figure 62 show, from left to right and from top to bottom: Mouse #1 .5, normal left ear (score 0) and normal right ear (score 0); Mouse #2.5 left ear with epidermal hyperplasia and inflammatory infiltration next to the auricular cartilage (score 6) and normal right ear (score 0); Mouse #3.8 left ear with epidermal hyperplasia, dermal oedema and marked eosinophilic infiltration of the dermis next to the auricular cartilage (score 6) and normal right ear (score 0).
  • FIG. 63 Representative pictures in Figure 63 show, from left to right and from top to bottom: Mouse #4.9 left ear with epidermal hyperplasia and eosinophilic infiltration of the dermis close to the auricular cartilage (score 4) and normal right ear (score 0); Mouse #5.1 left ear with epidermal hyperplasia and mild eosinophilic infiltration of the dermis (score 3) and normal right ear with focal neutrophilic inflammation (score 1); Mouse #6.1 left ear with eosinophilic infiltration (score 1) and normal right ear (score 0).
  • Ovalbumin challenge induced erythema and ear swelling as expected in this mouse model of atopic dermatitis. Maximum response was seen fifteen minutes after the ovalbumin challenge. Ovalbumin also induced histopathological change: epidermal hyperplasia, dermal oedema and dermal inflammation, as expected in this model.
  • CT101 administered at 5%, 10% or 20% did not reduce the erythema scores at peak disease, but the 20% dose elicited a statistically significant erythema reduction at 29 h after the ovalbumin challenge.
  • CT101 administered at 10% or 20% reduced the ear swelling at peak disease. The reduction was statistically significant when CT101 was administered at 10%.
  • CT101 administered at 5%, 10% or 20% reduced the dermal oedema but did not significantly reduce the total histopathology scores.
  • CT101 administered at 20%, reduced the incidence of histopathological change.
  • the goal of this study was to characterize SNA-101 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.
  • 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).
  • LPS system monocyte-driven Th1 inflammation
  • SAg system T cell stimulation
  • /Mphg system chronic Th1 inflammation driven by macrophage activation
  • BT system germinal centers
  • the BE3C system (Th1) and the BF4T system (Th2) represent airway inflammation of the lung, while the MyoF system models myofibroblast-lung tissue remodeling.
  • skin biology is addressed in the KF3CT system modeling Th1 cutaneous inflammation and the HDF3CGF system modeling wound healing.
  • 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.
  • 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.
  • bioactive agents biologicals, approved drugs, chemicals and experimental agents
  • 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.
  • SNA-101 was profiled in the BioMAP Diversity PLUS panel at concentrations of 29 ⁇ , 9.7 ⁇ , 3.2 ⁇ , and 1 .1 ⁇ . Apremilast was employed as the benchmark compound.
  • 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).
  • HUVEC Human umbilical vein endothelial cells
  • PBMC Peripheral blood mononuclear cells
  • HDFn Human neonatal dermal fibroblasts
  • BCR B cell receptor
  • TCR T cell receptor
  • TLR Toll-like receptor
  • 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- ⁇ ⁇ , TNFa and IFNy)], CASM3C system [coronary artery smooth muscle cells + (IL-1 ⁇ , TNFa and IFNy)], HDF3CGF system [HDFn + (IL- ⁇ ⁇ , TNFa, IFNy, EGF, bFGF and PDGF-BB)],
  • 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 ⁇ ), 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.
  • SRB sulforhodamine B
  • 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 Iog10 transformed.
  • Significance prediction envelopes are calculated using historical vehicle control data at a 95% confidence interval.
  • 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% (
  • 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.
  • BioMAP Z-Standard 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.
  • Tanimoto metric 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 first by normalizing each profile to the unit vector (e.g., and then applying the
  • a and B are the 2 profile vectors. Then, it is incorporated into a system weighted-averaged real-value Tanimoto metric in this 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 the following formula: where /r js the
  • BioMAP Z-Standard which adjusts for the number of common readouts (CR)
  • Z-Standard z A
  • ar g er BioMAP Z-Standard value corresponds to a higher confidence level, and this is the metric used to rank similarity results.
  • Cluster analysis 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
  • 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.
  • 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.
  • 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.
  • drug controls e.g., legacy control test agent colchicine
  • negative controls e.g., non-stimulated conditions
  • vehicle controls e.g., DMSO
  • 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
  • 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
  • FIG 64 depicts the BioMAP profile of SNA-101 in the Diversity PLUS Panel.
  • SNA-101 was found to be modestly active with 3 annotated readouts, mediating changes in key biomarker activities listed by biological and disease classifications in Table 19 below.
  • SNA-101 impacted inflammation-related activities (decreased SAA; increased sPGE2) and tissue remodeling activities (increased Collagen I).
  • Figure 65 depicts an overlay of SNA-101 at 29 ⁇ and the selected reference benchmark apremilast at 10 ⁇ .
  • Apremilast is a PDE4 inhibitor approved for the treatment of plaque psoriasis and psoriatic arthritis. The two agents did not have any common activities that meet the defined criteria for annotation.
  • Differentiating biomarkers are defined when one profile has a readout outside of the significance envelope with an effect size > 20% (
  • Table 20 depicts the top 3 similarity matches from a search of the BioMAP Reference Database of > 4,000 agents for each concentration of SNA-101.
  • 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. For profiles with a Pearson's correlation coefficient below our determined threshold (r ⁇ 0.7), the relevance of the similarity is unknown.
  • Figure 67 depicts Mechanism HeatMAP Analysis of SNA-101 , 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.
  • SNA-101 was characterized by profiling in the BioMAP Diversity PLUS panel of human primary cell 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.
  • SNA-101 was largely inactive in the Diversity PLUS panel, with no cytotoxic or antiproliferative effects observed at the concentration range tested. A modest increase in sPGE2 and Collagen I expression was observed, as was a modest decrease in serum amyloid A expression. Overall, the profile of SNA-101 was not similar to the benchmark Apremilast.
  • SNA-101 was profiled in the BioMAP Diversity PLUS panel at concentrations of 300 ⁇ , 100 ⁇ , 33 ⁇ , and 1 1 ⁇ . Staurosporine was employed as the benchmark compound.
  • FIG. 68 depicts the BioMAP profile of SNA-101 in the Diversity PLUS Panel.
  • SNA-101 was found to be modestly active with 4 annotated readouts, mediating changes in key biomarker activities listed by biological and disease classifications in Table 21 below.
  • SNA-101 impacted inflammation-related activities (decreased sTNFa, IL-8, IL-1 a) and a tissue remodeling activity (decreased MMP-1).
  • SNA-101 is antiproliferative to endothelial cells at the top concentration only (grey arrow of Figure 68). SNA-101 had no cytotoxic effects in the concentration range tested.
  • FIG. 69 depicts an overlay of SNA-101 at 300 ⁇ and the selected reference benchmark staurosporine at 10 nM.
  • Staurosporine is a broad-spectrum protein kinase inhibitor that acts on protein kinase C (PKC), protein kinase A (PKA), p60v-src tyrosine protein kinase, and CaM kinase II (CAM KM).
  • PDC protein kinase C
  • PKA protein kinase A
  • CAM KM CaM kinase II
  • Staurosporine is an analog of K252a.
  • Differentiating biomarkers are defined when one profile has a readout outside of the significance envelope with an effect size > 20% (
  • 3C HLA-DR, TF, uPAR
  • 4H Eotaxin 3, MCP-1 , P-selectin, VEGFR2, uPAR
  • LPS LPS
  • SAg CD40, CD69, E-selectin, IL-8, MCP-1 , Prolif
  • BT SIL-17A, SIL-17F, slL-2, slgG
  • BF4T IL-1 a, MCP-1 , PAI-1 , VCAM-1
  • BE3C IL-8, MMP-1 , MMP-9, PAI-1 , tPA, uPA, uPAR
  • CASM3C M-CSF, MCP-1 , Prolif, SAA, TF, uPAR
  • HDF3CGF MCP-1 , MIG, MMP-1 , Prolif 72
  • BT sTNFa
  • IMphg E-selectin, IL-1 a, IL-8
  • Figure 71 depicts an overlay of SNA-101 (100 ⁇ ) and infliximab (30000 ng/ml).
  • Table 22 depicts the top 3 similarity matches from a search of the BioMAP Reference Database of > 4,000 agents for each concentration of SNA-101.
  • 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. For profiles with a Pearson's correlation coefficient below our determined threshold (r ⁇ 0.7), the relevance of the similarity is unknown.
  • Figure 72 depicts Mechanism HeatMAP Analysis of SNA-101 , 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.
  • SNA-101 was characterized by profiling in the BioMAP Diversity PLUS panel of human primary cell 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.
  • SNA-101 was modestly active in the Diversity PLUS panel with only 4 annotated readouts that impact inflammation and tissue-remodeling activities.
  • SNA-101 had no cytotoxic effects at the concentration range tested. Antiproliferative effects to endothelial cells were observed at the top tested concentration of 300 ⁇ , but not at the lower concentrations.
  • SNA-101 was also previously profiled in the Diversity PLUS panel at a lower concentration range (1 .1 ⁇ - 29 ⁇ , refer to Example 9) where it was minimally active.
  • SNA-101 and the selected reference benchmark staurosporine a broad-spectrum kinase inhibitor, had only 4 common activities, but 65 differentiating activities.
  • staurosporine at 10 nM was more active than SNA-101 at 300 ⁇ .
  • the top database search match for SNA-101 was N037, a metabolite of the anesthetic ketamine.
  • the only common activity between the two profiles was the antiproliferative effect on endothelial cells, an activity only observed for the top concentration of SNA-101 .
  • An overlay with the second concentration of SNA-101 and its top match is also provided, although the Pearson's correlation coefficient did not meet our threshold for significance.
  • Example 11 Mouse Model of IMQ-induced Psoriasis
  • Table 23 depicts the test groups and Figure 73 depicts the timing of the experiments performed in this study.
  • Psoriasis reactions were scored based on the parameters shown in Table 24 and recorded according to a 0-12 scale. The clinical score is determined by summing the score of each section.
  • the total psoriasis score was determined by summing the plaque score, the erythema score and the punctate redness/scabbing score. As seen in Figure 74, the difference between SNA-125 at 5% and the vehicle is statistically significant on day 8 and 10, while for SNA-125 at 10% is significant from day 8 to day 10. Further, a statistically significant difference between SNA-352 at 5% and the vehicle on day 10 was found, as well as for SNA-125 at 10% from day 8 to day 10. Further, the difference between SNA-101 at 20% and the vehicle is statistically significant from day 8 to day 10.
  • This example is directed to a scaled-up synthetic procedure for SNA- 101 ( Figure 80).
  • the selected synthetic scheme consists of Benzamide hydrolysis to the carboxylic acid intermediate (Step 1) and amidation with mPEG2000-NH2 (Step 2).
  • the aqueous phase was diluted with 500 ml of AcOEt and neutralized under stirring with 15% HCI to pH 5.
  • the organic phase was concentrated by rotavapor distillation under reduced pressure at 40°C.
  • the residual solid was further dried by suspending it in 200 ml of DCM and evaporating the solvent under reduced pressure at 40°C. This drying operation was repeated twice.
  • the dried solid residue was dissolved in 960 ml of DCM.
  • the solution was placed in a 3 L four necked jacketed reactor, equipped with mechanical stirring, thermometer and condenser and protected from light by aluminum foil wrapping.
  • 3.5 ml of TEA were added at 25°C.
  • the solution was aged under stirring at 25°C for 10 minutes, then 4.8 g of TBTU were added.
  • the reaction mixture was aged at 20°C for 20 h under nitrogen atmosphere.
  • Sample loading was performed by dissolving the crude CT101 sample (about 6.4 g per purification) in 20 ml of H 2 0 and injecting it onto the cartridge through a syringe.
  • the SNAP cartridge was eluted at 100 ml/min with:

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Abstract

L'invention concerne des conjugués polymères, tels que SNA-101, comprenant un agent actif lié à un polymère, l'agent actif comprenant un inhibiteur, un antagoniste ou un agoniste inverse de c-Src. Les conjugués polymères de l'invention réduisent l'exposition de l'agent actif à des sites non cibles.
PCT/US2018/023109 2017-03-20 2018-03-19 Conjugués polymères ciblant c-src à exposition réduite WO2018175302A1 (fr)

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