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WO2018208369A1 - Utilisations de conjugués polymères de composés indolocarbazole à exposition réduite - Google Patents

Utilisations de conjugués polymères de composés indolocarbazole à exposition réduite Download PDF

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Publication number
WO2018208369A1
WO2018208369A1 PCT/US2018/023102 US2018023102W WO2018208369A1 WO 2018208369 A1 WO2018208369 A1 WO 2018208369A1 US 2018023102 W US2018023102 W US 2018023102W WO 2018208369 A1 WO2018208369 A1 WO 2018208369A1
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sna
subject
agent
polymer
composition
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PCT/US2018/023102
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English (en)
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Silvio Traversa
Valentina Mainero
Raffaella Bagnod
Todd James Harris
Luisa Bertarione RAVA ROSSA
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Sienna Biopharmaceuticals, Inc.
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Priority to EP18716429.8A priority Critical patent/EP3621657A1/fr
Publication of WO2018208369A1 publication Critical patent/WO2018208369A1/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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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

Definitions

  • SNA-120 SNA-125 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 indolocarbazole compound.
  • the polymer conjugate comprises an indolocarbazole compound of formula (I) or of formula (II):
  • R 1 and R 2 are the same or a different residue and are each independently selected from the group consisting of:
  • R 7 is selected from the group consisting of hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyi, - (CH 2 ) a C0 2 R 10 (wherein a is 1 or 2, and wherein R 10 is selected from the group consisting of hydrogen and substituted or unsubstituted lower alkyl) and -(CH 2 ) a C0 2 NR 5 R 6 ,
  • R 8 is selected from hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl
  • (f) -CH CH(CH 2 ) m R 16 , wherein m is 0 to 4, and R 16 is hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COOR 15 , -OR 15 (wherein R 15 is as defined above) - CONR 5 R 6 or -NR 5 R 6 (wherein R 5 and R 6 are as defined above);
  • R 3 is hydrogen, halogen, acyl, carbamoyl, substituted or unsubstituted lower alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted lower alkynyl or amino;
  • W 1 and W 2 are independently hydrogen, hydroxy or W 1 and W 2 together represent oxygen;
  • X is a polymer moiety, either linear or branched
  • A represents -L 1 -X' and B represents -L 2 - Y', wherein at least one of X and Y' is a polymer moiety, either linear or branched, which is bound by L 1 and/or L 2 to the tetrahydrofuran ring of the compound of formula (II);
  • L 1 and/or L 2 are a covalent chemical bond or a linker group
  • R 19 or R 20 are each independently selected from hydrogen, lower alkyl, lower alkenyl, lower alkynyl or R 19 or R 20 are independently the residue of an a-amino acid in which the hydroxy group of the carboxyl group is excluded, or R 19 or R 20 are combined with a nitrogen atom to form a heterocyclic group; and
  • the polymer moiety X, X or/and Y' covalently attached to the indolocarbazole compound of formulae (I) and (II) has to be biocompatible, can be of natural or semi-synthetic or synthetic origin and can have a linear or branched structure.
  • the polymer moiety X, X or/and Y' is selected from poly(alkylene oxides), in particular 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, polyaminoacids, hydroxyethyl starch.
  • the polymer moiety X, X' or/and Y' is a polyethylene glycol (PEG) moiety, wherein the terminal OH group can optionally be modified e.g. with C ⁇ Cs alkyl or C ⁇ Cs acyl groups.
  • the terminal OH group is optionally modified with Ci-, C 2 - or C 3 -alkyl groups or Ci-, C 2 - or C 3 groups.
  • the modified polyethylene glycol is a terminally alkoxy-substituted polyethylene glycol.
  • the polymer moiety is methoxy-polyethylene- glycol (mPEG).
  • lower alkyl when used alone or in combination with other groups, means a straight chained or branched lower alkyl group containing from 1 -6 carbon atoms, preferably from 1 -5, more preferably from 1 -4 and especially preferably 1 - 3 or 1 -2 carbon atoms.
  • These groups include, in some embodiments, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, amyl, isoamyl, neopentyl, 1 - ethylpropyl, hexyl, and the like.
  • the lower alkyl moiety of the "lower alkoxy”, the "lower alkoxycarbonyl", the “lower akylaminocarbonyl', “lower hydroxyalkyl' and of the "tri-lower alkylsilyl' groups has the same meaning as "lower alkyl" defined above.
  • the "lower alkenyl” groups are defined as C 2 -C 6 alkenyl groups which may be straight chained or branched and may be in the Z or E form. Such groups include vinyl, propenyl, 1 -butenyl, isobutenyl, 2-butenyl, 1 -pentenyl, (Z)-2- pentenyl, (E)-2- pentenyl, (Z)-4-methyl-2-pentenyl, (E)-4-methyl-2-pentenyl, pentadienyl, e.g., 1 , 3 or 2,4-pentadienyl, and the like.
  • the C 2 -C 6 - alkenyl groups are C 2 - C 5 -, C 2 -C 4 -alkenyl groups. In other embodiments, the C 2 -C 6 - alkenyl groups are C 2 -C 3 - alkenyl groups.
  • lower alkynyl groups refers to C 2 -C 6 -alkynyl groups which may be straight chained or branched and include ethynyl, propynyl, 1 -butynyl, 2- butynyl, 1 -pentynyl, 2-pentynyl, 3-methyl-1 -pentynyl, 3-pentynyl, 1 -hexynyl, 2-hexynyl, 3-hexynyl and the like.
  • C 2 -C 6 -alkynyl groups are C 2 -C 5 -, C 2 -C 4 - alkynyl groups.
  • C 2 -C 6 -alkynyl groups are C 2 -C 3 -alkynyl groups.
  • aryl group refers to C 6 -C 14 -aryl groups which contain from 6 up to 14 ring carbon atoms. These groups may be mono-, bi- or tricyclic and are fused rings. In some embodiments, the aryl groups include phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl and the like. The aryl moiety of the "arylcarbonyl” and the “arylaminocarbonyl” groups has the same meaning as defined above.
  • heteroaryl groups may contain 1 to 3 heteroatoms independently selected from nitrogen, sulfur or oxygen and refers C 3 -C 13 -heteroaryl groups. These groups may be mono-, bi- or tricyclic. In some embodiments, the C 3 - C 13 heteroaryl groups include heteroaromatics and saturated and partially saturated heterocyclic groups. These heterocyclics may be monocyclic, bicyclic, tricyclic. In some embodiments, the 5 or 6-membered heterocyclic groups are thienyl, furyl, pyrrolyl, pyridyl, pyranyl, morpholinyl, pyrazinyl, methyl pyrrolyl, and pyridazinyl.
  • the C 3 -Ci 3 - heteroaryl may be a bicyclic heterocyclic group.
  • the bicyclic heterocyclic groups are benzofuryl, benzothienyl, indolyl, imidazolyl, and pyrimidinyl.
  • the C 3 -C 13 -heteroaryls are furyl and pyridyl.
  • lower alkoxy includes alkoxy groups containing from 1 to 6 carbon atoms, in some embodiments from 1 to 5, in other embodiments from 1 -4 and in yet other embodiments 1 to 3 or 1 to 2 carbon atoms and may be straight chained or branched. These groups include methoxy, ethoxy, propoxy, butoxy, isopropoxy, tert- butoxy, pentoxy, hexoxy and the like.
  • acyl includes lower alkanoyl containing 1 to 6 carbon atoms, in some embodiments from 1 to 5, from 1 to 4, from 1 to 3 or from 1 to 2 carbon atoms and may be straight chained or branched. These groups include, in some embodiments, formyl, acetyl, propionyl, butyryl, isobutyryl, tertiary butyryl, pentanoyl and hexanoyl.
  • the acyl moiety of the "acyloxy" group has the same meaning as defined above.
  • halogen includes fluoro, chloro, bromo, iodio, and the like.
  • aralkyl' group refers C 7 -Ci 5 -aralkyl wherein the alkyl group is substituted by an aryl.
  • the alkyl group and aryl may be selected from the C C 6 alkyl groups and the C 6 -C 14 -aryl groups as defined above, wherein the total number of carbon atoms is between 7 and 15.
  • the C 7 -C 15 -aralkyl groups are benzyl, phenylethyl, phenylpropyl, phenylisopropyl, phenylbutyl, diphenylmethyl, 1 , 1 - diphenylethyl, 1 ,2-diphenylethyl.
  • the aralkyl moiety of the "aralkyloxy" groups has the same meaning as defined above.
  • the substituted lower alkyl, alkenyl and alkynyl groups have 1 to 3 independently selected substituents, such as lower alkyl, hydroxy, lower alkoxy, carboxyl, lower alkoxycarbonyl, nitro, halogen, amino, mono- or di- lower alkylamino, dioxolane, dioxane, dithiolane, and dithione.
  • the lower alkyl substituent moiety of the substituted lower alkyl, alkenyl and alkynyl groups, and the lower alkyl moiety of the lower alkoxy, the lower alkoxycarbonyl, and the mono- or di-lower alkylamino substituents of the substituted lower alkyl, alkenyl and alkynyl groups have the same meaning as "lower alkyl" defined above.
  • the substituted aryl, the substituted heteroaryl and the substituted aralkyl groups each has 1 to 3 independently selected substituents, such as lower alkyl, hydroxy, lower alkoxy, carboxy, lower alkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino, and halogen.
  • substituents such as lower alkyl, hydroxy, lower alkoxy, carboxy, lower alkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino, and halogen.
  • the lower alkyl moiety of the lower alkyl, the lower alkoxy, the lower alkoxycarbonyl, and the mono- or di- lower alkylamino groups among the substituents has the same meaning as 'lower alkyl' defined above.
  • the heterocyclic group formed by R 5 and R 6 combined with a nitrogen atom includes pyrrolidinyl, piperidinyl, piperidino, morpholinyl, morpholino, thiomorpholino, N-methylpiperazinyl, indolyl, and isoindolyl.
  • each of R 1 and R 2 is hydrogen.
  • the residue R 14 is selected from phenyl, pyridyl, imidazolyl, thiazolyl, tetrazolyl, -COOR 15 , -OR 15 (wherein R 15 is in some embodiments selected from hydrogen, methyl, ethyl, phenyl or acyl), -SR 7 (wherein R 7 is in some embodiments selected from substituted or unsubstituted lower alkyl, 2-thiazoline and pyridyl) and -NR 5 R 6 (wherein R 5 and R 6 are in some embodiments selected from hydrogen, methyl, ethyl, phenyl, carbamoyl and lower alkylaminocarbonyl).
  • the residue R 16 is selected from hydrogen, methyl, ethyl, phenyl, imidazole, thiazole, tetrazole, -COOR 15 , -OR 15 and -NR 5 R 6 (wherein the residues R 15 , R 5 and R 6 have the meanings as described above).
  • the residue R 7 is selected from the group consisting of substituted or unsubstituted lower alkyl, substituted or unsubstituted phenyl, pyridyl, pyrimidinyl, thiazole and tetrazole.
  • k is 2, 3 or 4
  • j is 1 or 2
  • m and n are independently 0 or 1.
  • R 3 is hydrogen or acetyl. Furthermore, in some embodiments, each W 1 and W 2 is hydrogen.
  • X' when Y' is a polymer moiety and X' is not a polymer moiety, X' is selected from carboxy, hydroxymethyl or a lower alkoxycarbonyl. In some embodiments X' is selected from methoxycarbonyl.
  • Y' is selected from hydroxy or acetyloxy.
  • the warhead of the polymer conjugate is a derivative of K252a, which has the formula:
  • the polymer conjugate is SNA-125, wherein the composition has the formula:
  • the polymer conjugate is SNA-120, wherein the composition has the 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.
  • active entity 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. In some embodiments, 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, keloids, rosacea, rosacea due to sarcoidosis, rosacea due to scleroderma, rosacea due to Sweet syndrome, rosacea due to systemic lupus erythematosus, rosacea
  • various skin neoplasias are treated.
  • the skin neoplasia comprises, in some embodiments, squamous cell carcinoma, basal cell carcinoma, malignant melanoma, malignant cutaneous lymphoma, Kaposi's sarcoma, Merkel cell skin cancer, and non-melanoma skin cancer.
  • various vascular tumors are treated.
  • the vascular tumor comprises, in some embodiments, hemangiomas, Kaposi's sarcoma, lymphangioma, glomangioma, angiosarcoma, hemangioendothelioma, and infantile hemangiomas.
  • bullous diseases are treated.
  • the bullous disease comprises, in some embodiments, bullous pemphigoid, erythema multiforme, dermatitis herpetiformis, epidermolysis bullosa acquisita, linear Immunoglobulin A disease, mucous membrane pemphigoid, pemphigoid gestationis, pemphigus foliaceus, and pemphigus vulgaris.
  • age-related macular degeneration is treated.
  • diabetic retinopathy is treated.
  • corneal edema is treated.
  • macular edema is treated.
  • dry eye is treated.
  • hair growth and cycling are modulated.
  • alopecia is treated.
  • the polymer conjugates are administered in combination with UV irradiation therapy.
  • 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 indolocarbazole compound. In some embodiments, the active entity comprises a derivative of K252a. In some embodiments, the composition comprises SNA-125.
  • the active entity binds to a tropomyosin-receptor-kinase A (TrkA) in some embodiments.
  • the active entity binds to a Janus Kinase (JAK) family member in some embodiments.
  • the active entity binds to one or more of Janus Kinase 1 (JAK1), Janus Kinase 2 (JAK2), Janus Kinase 3 (JAK3), and/or Tyrosine kinase 2 (TYK2) in some embodiments.
  • the active entity binds to mitogen-activated protein kinase kinase (MAP2K) in some embodiments.
  • the active entity binds to mitogen-activated protein kinase kinase 3 (MAP2K3) 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, vascular tumors, skin neoplasia, bullous diseases, age-related macular degeneration, diabetic retinopathy, corneal edema, macular edema, dry eye, alopecia, wounds, scars, autoimmune disorders, and cancerous or pre-cancerous lesions.
  • Methods for modulating hair growth and cycling are provided in some embodiments.
  • Kits comprising one or more compounds and devices for administration (syringes, containers, inhalers, etc.), as well as instructions for use, are provided in certain embodiments.
  • 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.
  • 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.
  • an active entity coupled (e.g., linked) to at least one polymer.
  • the polymer can include, for example, polyethylene glycol (PEG) and/or methoxy-polyethylene glycol (m-PEG).
  • PEG polyethylene glycol
  • m-PEG methoxy-polyethylene glycol
  • the active entity may be for example, an inhibitor, antagonist, or inverse agonist of a kinase that mediates the one or more ophthalmic, gastrointestinal, and dermatological conditions.
  • the active entity may be an indolocarbazole compound or a derivative thereof.
  • the active entity comprises SNA-120.
  • the active entity comprises SNA-125.
  • 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 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 kinase compared to the active entity without conjugation to the polymer.
  • the kinase may be one or more of tropomyosin receptor kinase A (TrkA) , mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAP2K), mitogen-activated protein kinase kinase 3 (MAP2K3) , and a Janus Kinase (JAK) family member.
  • the JAK protein family includes JAK1 , JAK2, JAK3, and Tyrosine kinase 2 (TYK2) .
  • the composition prevents the activation of NF-Kappa B signaling.
  • the composition binds and/or inhibits the one or more kinases.
  • the active entity has one or more carboxyl, hydroxyl, amino and/or sulfhydryl groups.
  • at least one polymer is conjugated (e.g. , PEGylated) 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.
  • 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.
  • 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.
  • 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 composition displays minimal toxicity when administered topically (e.g., topical administration on a daily basis).
  • the reduced exposure composition reduces inflammation upon administration.
  • the active entity and/or conjugate inhibits the proliferation of keratinocytes.
  • 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 (e.g., a JAK family protein and/or a STAT family protein) compared to the active entity without conjugation to the polymer.
  • compositions described herein are provided for modulating hair growth and cycling in a subject in need thereof.
  • the method of treatment and/or use of the compositions described herein are provided for the prophylaxis or treatment of one or more of the following in a subject in need thereof: a joint, an eye, alopecia, dry eye, corneal edema, macular edema, an autoimmune disorder, the gastrointestinal system, a lung, a vascular tumor, age-related macular degeneration, a cancerous or pre-cancerous lesion, a skin neoplasia, a bullous disease, a scar, a wound, diabetic retinopathy, non- dermal inflammation, an inflammatory condition, an inflammatory skin condition, and/or an inflammatory skin disease.
  • compositions described herein are employed in combination with UV irradiation.
  • 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: squamous cell carcinoma, basal cell carcinoma, malignant melanoma, malignant cutaneous lymphoma, Kaposi's sarcoma, Merkel cell skin cancer, non-melanoma skin cancer, bullous pemphigoid, erythema multiforme, dermatitis herpetiformis, epidermolysis bullosa acquisita, linear Immunoglobulin A disease, mucous membrane pemphigoid, pemphigoid gestationis, pemphigus foliaceus, pemphigus vulgaris, hemangiomas, Kaposi's sarcoma, lymphangioma, glomangioma, angiosarcoma, hemangioendothelioma, infantile hemangiomas, psoriasis, psori
  • a method for synthesizing SNA-125 comprises the hydrolysis of K252a to K252b followed by the coupling of K252b to methoxy polyethylene glycol amine (mPEG amine).
  • the hydrolysis K252a to K252b may performed in the presence of at least one of lithium hydroxide and tetrahydrofuran.
  • the coupling of K252b to mPEG amine is performed in the presence of at least one of TBTU, 4-methylmorpholine, and dichloromethane, thereby resulting in the formation of an amide bond between the K252b and the mPEG.
  • Figure 1 depicts luciferase activity of THP1 cells stimulated with a concentration range of HKLM or LPS, calculated relative to unstimulated cells.
  • Figure 2 depicts plasma levels after intravenous dosing.
  • Figure 3 depicts body weight versus day of study - males.
  • Figure 4 depicts body weight versus day of study - males.
  • Figure 5 depicts plasma levels after intravenous dosing - Day 1 .
  • Figure 6 depicts plasma levels after intravenous dosing - Week 2.
  • Figure 7 depicts body weight versus day of study - Males.
  • Figure 8 depicts body weight versus day of study - Males.
  • Figure 9 depicts body weight versus day of study (Main phase) -
  • Figure 10 depicts body weight versus day of study (Main phase) -
  • Figure 1 1 depicts normalized body weight trends.
  • Figure 12 depicts scar formation; SEI of saline-injected scars (Group 1 , Left Ears). Values are expressed as mean ⁇ StdDev.
  • Figure 13 depicts scar formation; SEI of vehicle-treated scars (Group 1 , Right Ears). Values are expressed as mean ⁇ StdDev.
  • Figure 14 depicts scar formation; SEI of CT340-injected scars (Group 2). Values are expressed as mean ⁇ StdDev.
  • Figure 15 depicts Scar Formation; SEI of CT340 topical-dosed scars (Group 3). Values are expressed as mean ⁇ StdDev.
  • Figure 16 depicts Scar Formation; SEI of TACA-treated scars (Group 4). Values are expressed as mean ⁇ StdDev. [0117] Figure 17 depicts scar formation; summary of SEI. Values are expressed as mean ⁇ StdDev.
  • Figure 18 depicts scar formation following intra-lesion injections with CT340 or TACA.
  • Figure 19 depicts scar formation following topical dosing with CT340
  • Figure 20 depicts scar formation SEI of CT340-treated scars, Values are expressed as mean ⁇ StdDev.
  • Figure 21 depicts scar inflammation scores following intra-lesion injections with CT340 or TACA. Values are expressed as mean ⁇ StdDev.
  • Figure 22 depicts scar inflammation scores following topical dosing with CT340, Values are expressed as mean ⁇ StdDev.
  • Figure 23 depicts the inhibition of proliferation by K252a, CT327 and CT340. Column bar graphs of proliferation assay results expressed as Absorbance at 570 nm with reference at 650 nm.
  • Figure 24 depicts the inhibition of proliferation by K252a, CT327 and CT340. Column bar graphs of proliferation assay results expressed as Absorbance at 570 nm with reference at 650 nm.
  • Figure 25 depicts the inhibition of proliferation by K252a, CT327 and CT340. Column bar graphs of proliferation assay results expressed as Absorbance at 570 nm with reference at 650 nm.
  • Figure 26 depicts a JAK2 vs Staurosporine concentration-%inhibition curve used to derive the slope (1 .853), R 2 (1 .00), and IC 50 (4.30E-10).
  • Figure 27 depicts a JAK2 vs CT340 concentration-%inhibition curve used to derive the slope (1 .147), R 2 (1 .00), and IC 50 (1 .35E-07).
  • Figure 28 depicts a JAK3 vs Staurosporine concentration-%inhibition curve used to derive the slope (1 .597), R 2 (1 .00), and IC 50 (2.78E-10).
  • Figure 29 depicts a JAK3 vs CT340 concentration-%inhibition curve used to derive the slope (1 .164), R 2 (1 .00), and IC 50 (3.87E-08).
  • Figure 30 depicts a PDGFRb vs Staurosporine concentration- %inhibition curve used to derive the slope (2.900), R 2 (1 .00), and IC 50 (3.87E-10).
  • Figure 31 depicts a PDGFRb vs CT340 concentration-%inhibition curve used to derive the slope (1 .165), R 2 (1 .00), and IC 50 (1 .12E-07).
  • Figure 32 depicts a TRKA vs Staurosporine concentration-%inhibition curve used to derive the slope (2.106), R 2 (1 .00), and IC 50 (5.02E-10).
  • Figure 33 depicts a TRKA vs CT340 concentration-%inhibition curve used to derive the slope (1 .159), R 2 (1 .00), and IC 50 (2.55E-08).
  • Figure 34 depicts a MAP2K1 vs Staurosporine concentration- %inhibition curve used to derive the slope (1 .287), R 2 (1 .00), and IC 50 (1 .39E-09).
  • Figure 35 depicts a MAP2K1 vs CT340 concentration-%inhibition curve used to derive the slope (1 .434), R 2 (1 .00), and IC 50 (1 .96E-08).
  • Figure 36 depicts a MAP2K3 vs Staurosporine concentration- %inhibition curve used to derive the slope (1 .402), R 2 (1 .00), and IC 50 (1 .13E-09).
  • Figure 37 depicts a MAP2K3 vs CT340 concentration-%inhibition curve used to derive the slope (1 .41 1 ), R 2 (1 .00), and IC 50 (1 .26E-08).
  • Figure 38 depicts a TAK1 -TAB1 vs Staurosporine concentration- %inhibition curve used to derive the slope (1 .369), R 2 (.98), and IC 50 (4.14E-08).
  • Figure 39 depicts a TAK1 -TAB1 vs CT340 concentration-%inhibition curve used to derive the slope (1 .480), R 2 (.98), and IC 50 (2.19E-07).
  • Figure 43 depicts anti-NGF antibody effect on Capsaicin responses. Inhibition of capsaicin responses in DRG neurons by anti-NGF was between 37.4 ⁇ 7.7% (100 ng/ml), and 63.3 ⁇ 9.7% (10 ⁇ g/ml). Results are given as mean (percent inhibition) ⁇ s.e.m.
  • Figure 44 depicts the effect of compound incubation on neurite length.
  • Treatment with CT327 and CT340 at 1 nM, 10 nM, 100 nM, 1 ⁇ and 10 ⁇ concentrations did not affect neurite length of neurons compared with control.
  • Treatment with GW441756 resulted in vesiculation and reduced neurite length at the higher concentrations of 1 ⁇ and 10 ⁇ .
  • Anti-NGF antibody treatment at 1 and 10 ⁇ g/ml concentrations also did not affect neurite length. Neurite lengths were normalized to controls and are given as mean percent of control ⁇ s.e.m.
  • Figure 45 depicts the effect of CT327 incubation on neurite length. 24 hour Incubation with CT327 did not have any effect on neurite length. Neurite lengths are expressed as mean percent of control ⁇ s.e.m.
  • Figure 46 depicts the effect of CT340 incubation on neurite length. 24 hour Incubation with CT340 did not significantly affect neurite length compared to control. Neurite lengths are expressed as mean percent of control ⁇ s.e.m.
  • Figure 47 depicts the effect of GW441756 incubation on neurite length.
  • Neurons treated with 0.33% ethanol (solvent for GW441756) had similar neurite length compared with NGF-treated controls. Neurite lengths are expressed as mean percent of control ⁇ s.e.m.
  • Figure 48 depicts the effect of anti-NGF antibody incubation on neurite length. Neurons treated with anti-NGF at 1 or 10 ⁇ g/ml did not show a significant change in neurite length compared to control. Neurite lengths are expressed as mean percent of control ⁇ s.e.m.
  • FIG. 49 depicts TrkA/Gap43 immunostaining in DRG neurons: A) Merged image showing co-localization of TrkA and Gap43 immunostaining in DRG neuron; B) Gap43 was strongly localized in cell bodies and neurites; C) TrkA immunostaining was observed to be densely localized in the cell bodies, while neurites were very faint.
  • Figure 50 depicts representative PGP9.5-immunoreactive intraepithelial nerve fibres (IEFN) (arrowed) in untreated control skin (top panel) and treated skin region (bottom panel) mini-pig skin using an antibody dilution of 1 :40,000, magnification x40.
  • IEFN intraepithelial nerve fibres
  • Figure 51 depicts a scatter plot showing the PGP9.5 intra epithelial fibre counts in untreated and treated mini-pig skin from the various groups. The median value is indicated.
  • C control; L, low dose; M, medium dose; H high dose; R, recovery; ut untreated area; t treated area.
  • Figure 52 depicts the BioMAP profile of SNA-120 in the Diversity PLUS Panel.
  • 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 53 depicts a Reference Benchmark Overlay of SNA- 120 and Benchmark SR221 1 .
  • Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 54 depicts the changes in secretion of (a) IL-17F (b) IgG, (c) IL- 17A, and (d) TNFa in the BioMAP BT system mediated by SNA-120 (3.1 ⁇ ), SNA-125 (3.9 ⁇ ), Tofacitinib (3.3 ⁇ ), Apremilast (3.3 ⁇ ), SR221 1 (3.3 ⁇ ), and Cyclosporin A (3.3 ⁇ ).
  • Figure 55 depicts an overlay of SNA-120 (28 ⁇ ) and GSK690693 (10 ⁇ ), which was the top similarity match from a search of the BioMAP Reference Database of > 4,000 agents for SNA-120 (28 ⁇ ).
  • Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 56 depicts Mechanism HeatMAP Analysis for SNA-120. HeatMAP analysis of the 148 biomarker readouts (rows) within the Diversity PLUS panel by SNA-120 in comparison to 19 consensus mechanism class profiles (columns). Horizontal grey lines separate the 12 Diversity PLUS systems, while the vertical grey line separates SNA-120 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 57 depicts clustering of test agent profiles following pairwise correlation analysis and clustering of the most similar profiles.
  • Each colored circle represents the BioMAP profile of a compound at a specific concentration, with larger circles representing higher concentrations.
  • Figure 58 depicts the BioMAP profile of SNA-125 in the Diversity PLUS Panel.
  • 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 59 depicts a Reference Benchmark Overlay of SNA- 125 and Benchmark Tofacitinib. Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 60 depicts an overlay of SNA-125 (3.9 ⁇ ) and SB203580 (10 ⁇ ), which was the top similarity match from a search of the BioMAP Reference Database of > 4,000 agents for SNA-125 (3.9 ⁇ ).
  • Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 61 depicts Mechanism HeatMAP Analysis for SNA-125. HeatMAP analysis of the 148 biomarker readouts (rows) within the Diversity PLUS panel by SNA-125 in comparison to 19 consensus mechanism class profiles (columns). Horizontal grey lines separate the 12 Diversity PLUS systems, while the vertical grey line separates SNA-125 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 62 depicts the BioMAP profile of SNA-125 in the Diversity PLUS Panel.
  • 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 63 depicts a Reference Benchmark Overlay of SNA-125 and Benchmark K252a. Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 64 depicts an overlay of SNA-125 (30 ⁇ ) and IKK 16 (370 nM), which was the top similarity match from a search of the BioMAP Reference Database of > 4,000 agents for SNA-125 (30 ⁇ ).
  • Common biomarker readouts are annotated when the readout for both profiles is outside of the significance envelope with an effect size > 20% (
  • Figure 65 depicts Mechanism HeatMAP Analysis for SNA-125. HeatMAP analysis of the 148 biomarker readouts (rows) within the Diversity PLUS panel by SNA-125 in comparison to 19 consensus mechanism class profiles (columns). Horizontal grey lines separate the 12 Diversity PLUS systems, while the vertical grey line separates SNA-125 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 66 depicts a BioMAP profile overlay of SNA-125 (10 ⁇ ), Methotrexate 10 ⁇ ), and Tofacitinib (10 ⁇ ).
  • Figure 67 depicts the design of the IMQ-induced psoriasis mouse study. Animal shaving, IMQ cream application, treatment, left ear biopsy punch, body weight measurements, ear thickness measurements, psoriasis clinical scoring, and termination were performed at the indicated tiem points.
  • Figure 68 depicts the change in animal body weight throughout the IMQ-induced psoriasis mouse study.
  • Figure 69 depicts the changes in the total psoriasis score throughout the IMQ-induced psoriasis mouse study.
  • the difference between SNA-125 at 5% and the vehicle is statistically significant from day 7.
  • the differences between SNA- 125 at 0.5% and 1 % and the vehicle are statistically significant on day 10.
  • Figure 70 depicts the changes in the Erythema score throughout the IMQ-induced psoriasis mouse study. SNA-125 at 5% is statistically significant from the vehicle from day 7. SNA-125 at 0.5% and 1 % are statistically significant on day 10 only.
  • Figure 71 depicts the changes in the plaque score throughout the IMQ-induced psoriasis mouse study. SNA-125 at 5% is statistically significant from the vehicle on day 10.
  • Figure 72 depicts the changes in the punctate redness/scabbing score throughout the IMQ-induced psoriasis mouse study.
  • Figure 73 depicts the changes in spleen thickness throughout the IMQ-induced psoriasis mouse study
  • Figure 74 depicts the changes in ear thickness throughout the IMQ- induced psoriasis mouse study.
  • Figure 75 depicts the levels of cytokines (a) IL-22, (b) IL-17A, (c) IL17F, and (d) TNFa, in ear samples at Day 4.
  • Figure 76 depicts CT327 calibration curves in rat plasma used to determine pharmacokinetic plasma levels (dotted lines represent upper and lower confidence limits).
  • Figure 77 depicts mean plasma concentration-time after dose profile of CT327 (SNA-120) after a single intravenous administration at 18 mg/kg. Open symbols represent the average measured values ( ⁇ 95% CI, vertical bar), while filled-in symbols represent interpolated values.
  • Figure 78 depicts the (A) colon dissection diagram and (B) fields and scoring order employed in the oxazolone-induced colitis mouse study.
  • Figure 79 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the body weight of animals challenged with oxazolone. Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Figure 79A depicts percent body weight change from Day -1 to Day 4 of the study.
  • Figure 79B depicts the area under the curve (AUC) of the percent weight change depicted in Figure 79A.
  • Figure 80 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the body weight of animals challenged with oxazolone according to last observation carried forward analysis. Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Figure 80A depicts percent body weight change from Day -1 to Day 4 of the study.
  • Figure 80B depicts the area under the curve (AUC) of the percent weight change depicted in Figure 80A.
  • Figure 81 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the Day 2 endoscopy score of animals challenged with oxazolone by (A) bar chart and (B) dot plot. Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Figure 82 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the Day 2 stool consistency score of animals challenged with oxazolone by (A) bar chart and (B) dot plot. Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Figure 83 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the Day 4 endoscopy score of animals challenged with oxazolone by (A) bar chart and (B) dot plot. Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Figure 84 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the Day 4 stool consistency score of animals challenged with oxazolone by (A) bar chart and (B) dot plot. Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Figure 85 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the disease activity index (DAI) score of animals at (A) Day 2 and (B) Day 4 following challenge with oxazolone.
  • DAI disease activity index
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Figure 86 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the colon weight/length ratio of animals challenged with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Figure 87 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the colon inflammation histopathology scores of animals challenged with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO). Group means with standard error of the mean (SEM) bars are depicted.
  • Figure 88 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the colon edema histopathology scores of animals challenged with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO). Group means with standard error of the mean (SEM) bars are depicted.
  • Figure 89 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the colon mucosal necrosis/loss histopathology scores of animals challenged with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO). Group means with standard error of the mean (SEM) bars are depicted.
  • Figure 90 depicts the effect of SNA- 125, SNA-352, tofacitinib, and prednisolone on the summed colon histopathology scores of animals challenged with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO). Group means with standard error of the mean (SEM) bars are depicted.
  • Figure 91 depicts representative control animal H&E-stained colon histopathology micrographs at 40x and 100x magnifications.
  • Figure 92 depicts representative H&E-stained colon histopathology micrographs at 40x and 100x magnifications for animals administered BID
  • A Vehicle PO
  • B 15 mg/kg Tofacitinib PO
  • C 1 mg/kg Prednisolone PO
  • D 400 mg/kg SNA-125 PO
  • E 400 mg/kg SNA-352 PO.
  • Moderate inflammation unfilled black arrows
  • edema filled red arrows
  • multifocal ulceration (brackets) are indicated.
  • Figure 93 depicts representative H&E-stained colon histopathology micrographs at 40x and 100x magnifications for animals administered BID
  • A Vehicle IC
  • B 1 mg/kg Tofacitinib IC
  • C 400 mg/kg SNA-125 IC
  • D 400 mg/kg SNA-352 IC.
  • Figure 94 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the proein levels of IFNv in colon tissue homogenate supernatants of animals following challenge with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO). Group means with standard error of the mean (SEM) bars are depicted.
  • Figure 95 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the protein levels of TNFa in colon tissue homogenate supernatants of animals following challenge with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Group means with standard error of the mean (SEM) bars are depicted, with outliers removed (A), or present (B).
  • Figure 96 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the protein levels of IL-6 in colon tissue homogenate supernatants of animals following challenge with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Group means with standard error of the mean (SEM) bars are depicted, with outliers removed (A), or present (B).
  • Figure 97 depicts the effect of SNA-125, SNA-352, tofacitinib, and prednisolone on the protein levels of IL-10 in colon tissue homogenate supernatants of animals following challenge with oxazolone.
  • Compounds or vehicle controls were dosed BID as indicated intracecally (IC) or orally (PO).
  • Group means with standard error of the mean (SEM) bars are depicted, with outliers removed (A), or present (B).
  • Figure 98 depicts a schematic showing how the IMQ-induced psoriasis study was performed.
  • Figure 99 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 100 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 101 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 102 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 103A depicts the weight of spleens upon experimental termination on day 10. Mean spleen weight for each group is displayed +/- SEM .
  • Figure 103B 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 103C 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 104 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 105 depicts a schematic of the IL-23-induced psoriasis mouse model study.
  • Figure 106 depicts the effect SNA-120 and SNA-325 in an IL-23- induced psoriasis mouse model.
  • Figure 106A depicts the total psoriasis clinical scores for each group over time. The mean score for each group is displayed for each day +/- SEM.
  • Figure 106B depicts the right ear thickness of each group at the indicated time points. Mean thickness for each group is displayed for each day +/- SEM .
  • Figure 106C the depicts body weight of each group over the course of the study. 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 108 depicts representative Day 2 endoscopy images of naive control, vehicle control (PO), vehicle control (IC), and tofacitinib (15 mg/kg PO) animals. Animals underwent video endoscopy on Day 2 and colitis severity was scored on a scale of 0-4. Images were captured from each animal during the procedure and representative images from each treatment group are presented.
  • Figure 109 depicts representative Day 2 endoscopy images of tofacitinib (15 mg/kg IC), prednisolone (1 mg/kg PO), SNA-125 (400 mg/kg PO), and SNA-352 (400mg/kg PO) animals. Animals underwent video endoscopy on Day 2 and colitis severity was scored on a scale of 0-4. Images were captured from each animal during the procedure and representative images from each treatment group are presented.
  • 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.
  • the polymer has a molecular weight ranging from about 200 to about 50,000 Da.
  • 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) .
  • the long-chain PEG or mPEG has an average molecular weight of about 2,000 Da or of about 5,000 Da.
  • 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.
  • the polymer conjugates provided herein treat inflammatory skin diseases.
  • the polymer conjugates provided herein treat skin neoplasias.
  • the polymer conjugates provided herein treat bullous diseases.
  • active agents useful for stimulating hair follicles are provided as oral applications or topical applications for the scalp.
  • Hair removal agents and anti-acne agents are provided in other embodiments.
  • Hair growth, hair removal and anti-acne therapies can all involve active agents that, if exposed to the non-target site (e.g. , systemic circulation and/or lymphatic system) for long periods, result in toxicity or undesired side effects.
  • the reduced exposure compositions described herein provides benefits for these applications as well.
  • the polymer conjugates provided herein modulate hair growth and cycling.
  • the polymer conjugates provided herein treat alopecia.
  • 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.
  • the polymer conjugates provided herein treat vascular tumors.
  • the polymer conjugates provided herein treat diabetic retinopathy.
  • the polymer conjugates provided herein treat macular edema.
  • the polymer conjugates provided herein treat corneal edema.
  • the polymer conjugates provided herein treat age-related macular degeneration.
  • 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.
  • the compositions described herein may be combined with other modalities to achieve synergic effects. These other modalities include, but are not limited to, energy delivery (such as laser, radiofrequency, ultrasound, microwave, etc.) , thermal therapy, light therapy, radiation, intravenous chemotherapy, and others.
  • 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 compositions are administered in combination with UV irradiation therapy.
  • the polymer conjugate exhibits unexpected permeability across the nuclear membrane.
  • the polymer conjugate exhibits unexpected permeability across both the nuclear and plasma membranes. Accordingly, in Example 9, two polymer conjugates, SNA- 125 and SNA-120, were surprisingly shown to penetrate the keratinocyte cellular membrane and interact with the target kinases intracellularly within the cytoplasm, thereby leading to inhibition of proliferation of keratinocytes in a non-toxic manner.
  • 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 an indolocarbazole compound.
  • Angiogenesis the process of sprouting new blood vessels from existing vasculature, and arteriogenesis, the remodeling of small vessels into larger conduit vessels, are both physiologically important aspects of vascular growth in adult tissues. These processes of vascular growth are required for beneficial processes such as tissue repair, wound healing, recovery from tissue ischemia and menstrual cycling. They are also required for the development of pathological conditions such as the growth of neoplasias, diabetic retinopathy, rheumatoid arthritis, psoriasis, certain forms of macular degeneration, and certain inflammatory pathologies. The inhibition of vascular growth in these contexts has also shown beneficial effects in preclinical animal models.
  • angiogenesis For example, inhibition of angiogenesis by blocking vascular endothelial growth factor or its receptor has resulted in inhibition of tumor growth and in retinopathy. Also, the development of pathological pannus tissue in rheumatoid arthritis involves angiogenesis and might be blocked by inhibitors of angiogenesis.
  • Certain diseases are known to be associated with deregulated angiogenesis, for example ocular neovascularization, such as retinopathies (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, such as a rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders, such as chronic asthma, arterial or post- transplantational atherosclerosis, endometriosis, and neoplastic diseases, for example so-called solid tumors and liquid tumors (such as leukemias).
  • retinopathies including diabetic retinopathy
  • age-related macular degeneration psoriasis
  • hemangioblastoma hemangioma
  • arteriosclerosis arteriosclerosis
  • inflammatory disease such as a rheumatoid or r
  • vascular tumors include hemangiomas, Kaposi's sarcoma, lymphangioma, glomangioma, angiosarcoma, hemangioendothelioma, and infantile hemangiomas.
  • Angiogenesis in the skin is also implicated in a number of other diseases that are characterized by macroscopically visible, prominent blood vessels, including rosacea and basal cell carcinoma.
  • methods of treating a skin neoplasia in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • skin neoplasias include squamous cell carcinoma, basal cell carcinoma, malignant melanoma, malignant cutaneous lymphoma, Kaposi's sarcoma, Merkel cell skin cancer, and non-melanoma skin cancer.
  • Increased vascular permeability is one of the earliest manifestations of inflammation, resulting in extravasation of protein-rich plasma into the effected tissue.
  • Acute vascular permeability allows the deposition of circulating plasma matrix proteins including fibrin and fibronectin (FN) which facilitate cell migration in the inflamed area. This process also provides an access point for immune cells and immunoglobulins to enter the tissue and fight foreign antigens (Nagy et al, Cold Spring Harb. Perspect. Med. 2:a006544, 2012).
  • chronic vascular hyperpermeability is suggested to sustain the inflammatory response and retard resolution, further promoting the development of chronic inflammation (Nagy et al, Cold Spring Harb. Perspect. Med.
  • inflammatory skin diseases include psoriasis, psoriasis guttata, inverse psoriasis, pustular psoriasis, psoriatic erythroderma, acute febrile neutrophilic dermatosis, eczema, xerotic eczema, dyshidrotic eczema, vesicular palmar eczema, acne vulgaris, atopic dermatitis, contact dermatitis, allergic contact dermatitis, dermatomyositis, exfoliative dermatitis, hand eczema, pompholyx, keloids, rosacea, rosacea due to sarcoidosis, rosace
  • Bullous diseases are skin disorders characterized by blistering that often have an autoimmune etiology.
  • Two bullous diseases are bullous pemphigoid and erythema multiforme.
  • Bullous pemphigoid is a subepidermal disorder which manifests as subepidermal blisters with a dermal infiltrate of neutrophils and eosinophils.
  • Erythema multiforme is an inflammatory eruption characterized by symmetric erythematous, edematous, or bullous lesions of the skin or mucous membranes.
  • methods of treating a bullous disease in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • bullous diseases include bullous pemphigoid, erythema multiforme, dermatitis herpetiformis, epidermolysis bullosa acquisita, linear Immunoglobulin A disease, mucous membrane pemphigoid, pemphigoid gestationis, pemphigus foliaceus, and pemphigus vulgaris.
  • PDR proliferative diabetic retinopathy
  • methods of treating age- related macular degeneration in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • methods of treating diabetic retinopathy in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • methods of treating corneal edema in a subject the method comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • methods of treating macular edema in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • methods of treating dry eye in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • the process of new hair growth relies on numerous cross- talking signal pathways to bring about the processes necessary for hair growth. These principal processes are: cell proliferation of the dermal papia, cell migration to form the appropriate structures, and angiogenesis to form blood supply routes to the new hair foicle.
  • methods of modulating hair growth and cycling in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • methods of treating alopecia in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • a combination therapy comprising administering to the subject an effective amount of a polymer conjugate in conjunction with UV irradiation therapy, wherein the warhead is an indolocarbazole compound.
  • methods of treating dry eye in a subject comprising administering to the subject an effective amount of a polymer conjugate, wherein the warhead is an indolocarbazole compound.
  • 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. In other embodiments, the composition is applied for a period of 120 seconds before flushing.
  • the composition is applied for a period of 360 seconds before flushing.
  • the composition may be administered one or more times a day.
  • the composition is administered daily.
  • 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 an indolocarbazole compound.
  • 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 an indolocarbazole compound: nail dystrophy; seborrheic keratosis; androgenic alopecia; contact dermatitis; actinic keratosis; acne; asthma; eczema (atopic dermatitis); onychomycosis; sinusitis; allergic rhinitis; rosacea; COPD; pruritus; early AMD; urticaria; diabetic retinopathy; psoriasis; alopecia areata; dry eye; vitiligo; glaucoma; late AMD; ulcerative colitis; Crohn's disease; ocular rosacea; hair growth and cycling; skin neoplasias; squamous cell carcinoma; basal cell carcinoma; malignant melanoma
  • methods of treating a respiratory disease in a subject via delivery of the polymer conjugates (wherein the warhead is an indolocarbazole compound) 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.
  • compositions comprising an indolocarbazole compound are used, in several embodiments, as inhibitors, antagonists, and inverse agonists of TkrA, Jak3, and/or MAP2K3.
  • Several embodiments relate to polymer conjugates of an indolocarbazole compound, 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 an indolocarbazole compound or derivative thereof.
  • 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 an indolocarbazole compound.
  • the warhead of the LSE polymer conjugate is a derivative of K252a.
  • the LSE polymer conjugate is SNA-125.
  • methods of treating a vascular tumor in a subject comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is an indolocarbazole compound.
  • the warhead of the LSE polymer conjugate is a derivative of K252a.
  • the LSE polymer conjugate is SNA-125.
  • methods of treating a skin neoplasia in a subject comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is an indolocarbazole compound.
  • the warhead of the LSE polymer conjugate is a derivative of K252a.
  • the LSE polymer conjugate is SNA-125.
  • methods of modulating hair and growth cycling in a subject comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is an indolocarbazole compound.
  • the warhead of the LSE polymer conjugate is a derivative of K252a.
  • the LSE polymer conjugate is SNA-125.
  • methods of treating alopecia in a subject comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead an indolocarbazole compound.
  • the warhead of the LSE polymer conjugate is a derivative of K252a.
  • the LSE polymer conjugate is SNA-125.
  • methods of treating a bullous disease in a subject comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is an indolocarbazole compound.
  • the warhead of the LSE polymer conjugate is a derivative of K252a.
  • the LSE polymer conjugate is SNA-125.
  • methods of treating dye eye, diabetic retinopathy, macular edema, corneal edema, and/or age-related macular degeneration in a subject comprising administering to the subject an effective amount of an LSE polymer conjugate wherein the warhead is an indolocarbazole compound.
  • the warhead of the LSE polymer conjugate is a derivative of K252a.
  • the LSE polymer conjugate is SNA-125.
  • a combination therapy comprising administering to the subject an effective amount of an LSE polymer conjugate in conjunction with UV irradiation therapy and wherein the warhead is an indolocarbazole compound.
  • the warhead of the LSE polymer conjugate is a derivative of K252a.
  • the LSE polymer conjugate is SNA-125.
  • 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. In some embodiments, the amino group that causes the least hindrance on activity is used (whether or not it is located the furthest way from the moieties interacting with the target).
  • the effect of conjugation on the activity of the compound can be determined based on various methods, such as bioassays, mass spectroscopy, surface plasmon resonance, in vivo assays, clinical assays, and predictive in silico modeling programs.
  • 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.
  • the sulfhydryl group located the furthest away from the moieties interacting with the target is used.
  • 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).
  • An existing carboxylic moiety can 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).
  • An existing amino group (-NH2) can 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).
  • An existing hydroxyl moiety (-OH) can be conjugated to PEG-halide through formation of an ether bond in presence of a strong base (including, e.g. NaH, KH, and n-BuLi).
  • 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.
  • 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 polymer conjugate 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 polymer conjugates 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 endogenous angiogenesis inhibitor, for example and not restricted to, angioarrestin, angiostatin (plasminogen fragment), antiangiogenic antithrombin I I I , cartilage-derived inhibitor (CDI), CD59 complement fragment, endostatin (collagen XVI I I fragment) , fibronectin fragment, Gro-beta, heparinases, heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon inducible protein (IP- 10), lnterleukin-12, kringle 5 (plasminogen fragment) , metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prolactin 16 kD fragment, prolifer
  • 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, M IP- 1 a, M IF- ⁇ ⁇ , MIP-2, M IF and IL-6.
  • a cytokine selected from the group consisting of TNF, IL-1 a, IL- 1 ⁇ , IL-Ra, IL-8, M IP- 1 a, M IF- ⁇ ⁇ , MIP-2, M IF 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, fex
  • 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.
  • 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.
  • the indefinite article “a” or “an” does not exclude a plurality.
  • the term “about” as used herein to, for example, define the values and ranges of molecular weights means that the indicated values and/or range limits can vary within ⁇ 20% , e.g. , within ⁇ 10% .
  • the use of "about” before a number includes the number itself. For example, “about 5" provides express support for "5".
  • CT340 and “SNA-125" are synonyms and can be used interchangeably.
  • CT327 and “SNA-120” are synonyms and can be used interchangeably.
  • the product from Step 2 is purified by preparative column chromatography using silica gel which has been pretreated with ethyl acetate containing 5% triethylamine.
  • the Step 2 product was dissolved in dichloromethane/ethyl acetate for application to the column. Elution with ethyl acetate enabled residual K252a and UV- inactive species to be eluted (as monitored by TLC) .
  • the column was then conditioned with dichloromethane and eluted with dichloromethane/MeOH (98.5: 1 .5 - 95.5:4.5). Fractions were collected and analyzed by HPLC and TLC, and pooled according to their purity. Evaporation of the combined desired product fractions led to CT340 of purity >95% by HPLC area.
  • the resulting material is of high chemical purity, a precipitation step is performed to transform it from a semisolid concentrate into a readily handled free- flowing solid.
  • K-252a is a potent inhibitor of multiple kinases.
  • the inhibitory activity of K-252a against selected common tyrosine kinases and serine/threonine kinases was evaluated.
  • a similar experiment has been conducted for evaluating the inhibitory activity of CT340.
  • K-252a (Acros lot A020265401 ) was dissolved in DMSO to make a 1 mM stock solution which was then diluted with DMSO to obtain a 20 ⁇ solution, further diluted with the assay buffer to achieve a concentration of 0.8 ⁇ .
  • K-252a was tested at a concentration of 200 nM .
  • the preparation of CT340 and the reference compounds was conducted following a similar procedure. CT340 also was tested at a concentration of 200 nM . Staurosporine, 5-iodotubericidin, NK inhibitor I I and SB202190 have been used as reference compounds.
  • the inhibitory activities of CT340 (200nm) against 89 tyrosine kinases are shown in Table 1 below.
  • the inhibitory activities of CT340 (200nM) against 187 serine/threonine kinases are shown in Table 2.
  • the readout value of reaction control (with ATP) was set as a 0% inhibition and the readout value of background (without ATP) was set as a 100% inhibition.
  • the results clearly show a dramatic improvement in the selectivity of kinase inhibition of CT340 versus K-252a. The less other kinases are inhibited, the less toxic the molecule is likely to be.
  • CT340 was tested in an in vitro cell-based assay to monitor the antiinflammatory activity in a complex biological system. Inhibition of NF- ⁇ , a key regulator of the inflammatory response, was assayed using human monocytes (THP1) carrying a luciferase reporter gene driven by a tandem repeat of the NF- ⁇ consensus transcriptional response element. In addition, staurosporine, a broad spectrum kinase inhibitor, was used as comparator.
  • THP1 human monocytes
  • staurosporine a broad spectrum kinase inhibitor
  • cell lines were pre-cultured with the inhibitors, i.e. CT340 or staurosporine, or with a volume equivalent of DMSO. After 2 h, the pre-determined sub-optimal dose of stimuli were added, and incubation was continued for another 6 h. Luciferase activity was determined as described in the data sheet of the cell lines.
  • the inhibitors i.e. CT340 or staurosporine
  • THP1 cells were grown and stimulated for 24 h, as described on the data sheet of the cell line. Cells were stimulated with HKLM (0.625 x 10 ⁇ 7 cells/mL - 10 x 10 ⁇ 7 cells/mL) or LPS (0.01 - 100 ng/mL). Fold induction relative to unstimulated cells is depicted in Figure 1 . Luciferase activity is dose dependently increased with both stimuli, with a maximum fold increase of 200-fold for both stimuli.
  • HKLM and LPS were chosen for further experiments, being 2.5 ⁇ 10 ⁇ 7 cells/mL, and 1 ng/mL, respectively. Stimulation was furthermore confirmed when cells were stimulated for 6 h instead of 24 h.
  • results obtained after 8 or 24 h are highly similar. Staurosporine results in substantial reduction of luciferase activity, in both concentrations used. In addition, preincubation of cells with 100 ⁇ CT340 inhibits the luciferase activity, both in unstimulated, as well as in LPS or HKLM-stimulated cells.
  • Inhibitors were tested at 3 concentrations, being 100 ⁇ , 1 ⁇ and 10 nM. As controls, cells were treated with 1 % DMSO (volume equivalent to 100 ⁇ inhibitor), 0.01 % DMSO (volume equivalent to 1 ⁇ inhibitor), or with medium alone (control for cells treated with 10 nM inhibitor). Cells were pre- treated with inhibitor for 2 h, and subsequently stimulated with LPS or HKLM for 6 h.
  • Luciferase activity was effectively induced by both stimuli used, with an increase of 6.5- and 3.4-fold in LPS and HKLM stimulated cells, respectively. Relative luciferase units are depicted in Table 7.
  • NF- ⁇ can efficiently and dose dependently be activated by LPS and heat killed Listeria monocytogenes (HKLM).
  • CT340 The toxicity and pharmacokinetic of CT340 were investigated when given by single intravenous and dermal administration to the Sprague Dawley rat.
  • the toxicity features were investigated in 8 main groups (Groups 1 -4 for intravenous dosing and Groups 5-8 for dermal dosing), each group comprising 2 male and 2 female rats. Animals were dosed at 10, 30 and 100 mg/kg for both administration routes (a dose volume of 5 ml/kg in physiological saline, for intravenous administration and Propylene glycol/DMSO/Water 10/40/50 for dermal administration). Animals of control groups (Groups 1 and 5) received the vehicle alone.
  • test item was administered by intravenous bolus injection into the tail vein with an approximate speed of 3 ml/minute.
  • the dose was administered to each animal at a dose volume of 5 ml/kg. Control animals received the vehicle alone at the same dose volume.
  • the test item was applied uniformly over an area of approximately 10% of the total body surface area (approximately 5x7 cm) .
  • the dose was administered to each animal at a dose volume of 5 ml/kg.
  • a patch of surgical gauze covered by a strip of synthetic film was placed over the treated site and the whole assembly held in place by encircling the trunk of the animal with a length of adhesive bandage, this forming a semi-occlusive barrier.
  • the amount administered to individual animals was determined by their body weight, measured prior to dosing.
  • AUC o-tiast
  • the maximum tolerated dose is then considered to be greater than 100 mg/kg for both dermal and intravenous administrations.
  • CT340 plasma levels were investigated up to 24 hours after dosing.
  • CT340 when given by single intravenous or dermal administration to rats at the dose levels of 10, 30 and 100 mg/kg, was investigated over a period of 7 days after dosing.
  • the maximum tolerated dose is then considered to be greater than 100 mg/kg for both dermal and intravenous administrations.
  • CT340 The toxicity of CT340 was investigated, when given by daily intravenous administration to the Sprague Dawley rat for a 2-week period followed by a 2-week recovery period.
  • test item was dissolved in physiological saline (NaCI 0.9%) and treatment groups were dosed at 10, 30 and 100 mg/kg/day at the dose volume of 5 ml/kg and a speed of 3 ml/minute.
  • the control group received the vehicle alone.
  • test item was administered by intravenous bolus injection into the tail vein with an approximate speed of 3 ml/minute.
  • the dose was administered to each animal at a dose volume of 5 ml/kg.
  • Control animals received the vehicle alone at the same dose volume.
  • the dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
  • NOAEL No Observed Adverse Effect Level
  • AUC calculation with trapezoidal rule was considered to be a good estimation.
  • CT340 The toxicity of CT340 was investigated, when given at the dose levels of 10, 30 and 100 mg/kg/day by daily intravenous administration to the Sprague Dawley rat for a 2-week period followed by a 2-week recovery period.
  • NOAEL No Observed Adverse Effect Level
  • test item was applied evenly over the prepared skin of animals of the relevant group (Groups 2, 3 and 4) at a dose volume of 0.5 mL/kg.
  • the required amount of formulation was spread evenly over the skin of the prepared site by gentle massage over an area of approximately 25x20 cm.
  • Control animals (Group 1) received the vehicle alone at the same dose volume, in the same manner.
  • the treated skin site was covered using a patch of surgical gauze and the whole assembly held in position by encircling the trunk of the animal with a cotton jacket. This semi-occlusive dressing was removed approximately 6 hours later and the treated site was cleaned by washing with a piece of surgical gauze soaked with warm water, removing any residual test item.
  • a C max of 8.74 ng/mL was measured in the single male of Group 3, while a Cmax of 9.85 ng/mL was measured in the single female of the same group with a Tmax of 2 and 1 hour in the male and female animals, respectively.
  • Mean Cmax was 7.735 ng/mL in the males of Group 4, with a Tmax of 2 hours.
  • AUCo-in f calculable for 1 male dosed at 100 mg/kg, was 538.918 ng h/mL.
  • test item CT340 is locally well tolerated after a dermal application at concentrations of 20, 60 and 200 mg/mL over the intact skin of the minipig.
  • No signs of potential treatment-related adverse effects of the test item were observed at any of the dose levels investigated (10, 30 and 100 mg/kg/day).
  • the maximum tolerated dose in this study may be considered to be greater than 100 mg/kg for a single dermal administration in minipigs.
  • the high dose level of 100 mg/kg at a concentration of 200 mg/mL may be considered as the high dose level to be selected for a subsequent repeated dose study.
  • Plasma samples were obtained from male and female minipigs before dosing (pre-dose) and 0.5, 1 , 2, 4, 6, 8 and 24 hours after the start of dosing on Day 1 of the study, following a single dermal administration of CT340 at the dose levels of 10, 30 and 100 mg/kg/day.
  • a C max of 8.74 ng/mL was measured in the single male of Group 3, while a C max of 9.85 ng/mL was measured in the single female of the same group.
  • T max were of 2 and 1 hour in the male and female animals, respectively.
  • Mean C max was 7.735 ng/mL in the males of Group 4, with a T max of 2 hours.
  • AUC 0-inf calculable for 1 male dosed at 100 mg/kg, was 538.918 ng h/mL.
  • test item CT340 was locally and systemically well tolerated following a single dermal application to the minipig at all the dosages tested.
  • the maximum tolerated dose in this study may be considered to be greater than 100 mg/kg for a single dermal administration in minipigs.
  • the high dose level of 100 mg/kg at a concentration of 200 mg/mL may be considered as the high dose level to be selected for a subsequent repeated dose study.
  • test item formulations were applied evenly over the prepared skin of animals of the relevant group. A 5.5 g aliquot of each test item formulation was administered and spread evenly over the skin of the prepared site by gentle massage, over an area of approximately 20 x 25 cm in animals of the relevant group. Control animals (Group 1) received the vehicle alone (control item) at the same dose volume, in the same manner.
  • the treated skin site was covered using a patch of surgical gauze and the whole assembly held in position by encircling the trunk of the animal with a cotton jacket. This semi- occlusive dressing was removed approximately 6 hours later and the treated site was cleaned by washing with a piece surgical gauze soaked with warm water, removing any residual substance.
  • CT340 was considered reasonably tolerated at the application site and not systemically toxic in the minipig following a repeated dermal (epicutaneous) administration over a period of at least 14 consecutive days, when administered at concentrations of 20, 60 and 200 mg/mL (corresponding to dose levels of approximately 1 10, 330 and 1 100 mg/animal/day).
  • CT340 was considered reasonably tolerated at the application site and not systemically toxic in the minipig following a repeated dermal (epicutaneous) administration over a period of at least 14 consecutive days, when administered at concentrations of 20, 60 and 200 mg/mL (corresponding to dose levels of 1 10, 330 and 1 100 mg of active ingredient/animal/day, i.e. 0.22, 0.66 and 2.2 mg/cm2/day and approximately 7, 21 and 69 mg of active ingredient/kg/day) .
  • Example 8 Effects of CT340 in a Model of Hypertrophic Scar Formation in NZW
  • the objective of this study was to assess the effect of CT340 on hypertrophic scar formation in NZW rabbits.
  • Four wounds were surgically created in each ear, scars were allowed to develop, and the effects of test article on hypertrophic scars were determined by photomicroscopy and histopathology.
  • Surgical wounds were induced bilaterally as four circular lesions per ear down to the bare cartilage on the ventral side of each ear. Treatment started on Day 26, after confirming the complete formation of hypertrophic scars. Animals were dosed via intra-lesional injections into each scar or by topical application with the test article, CT340. Dosing was repeated once weekly for a total of three doses. Test article efficacy in scar reduction was assessed using the Scar Elevation Index (SEI) and by histopathology.
  • SEI Scar Elevation Index
  • mice were assigned to study groups of six rabbits each. Animals of Groups 1 (right ear only) and 3 were dosed via topical application. Animals of Groups 1 (left ear only), 2, and 4 were dosed via intra-lesional injection into each scar (10 pL/scar). Animals of Groups 1 - 4 (respectively) were dosed with test and control article into both ears with vehicle; CT340 at 200 mg/mL; CT340 at 5%; or TACA
  • the test article was provided to the laboratory in a powder form and stored frozen at -80 °C pending use.
  • the first control article was the vehicle used to formulate the topical formulation of CT340.
  • the control article consisted of 25% Transcutol (diethylene glycol monoethyl ether) and 75% 1 ,2-propanediol.
  • the vehicle was formulated from components Transcutol P (Gattefosse Catalog 3260JV1 ) and 1 ,2- propanediol (Fluka Catalog 82281 ) .
  • the second control article was triamcinolone acetonide (TACA). This control article is a synthetic glucocorticoid corticosteroid with anti-inflammatory action.
  • the third control article was sterile saline, intended to be identical to the vehicle used to formulate the injectable formulation of CT340.
  • a liquid adhesive e.g. , Mastisol; Ferndale Laboratories, Inc. , Ferndale, M l
  • a polyurethane dressing Teegaderm; 3M Health Care, St. Paul, MN.
  • the polyurethane dressing remained attached at all times to ensure a moist wound environment.
  • the dressing was changed (at least) once weekly through Day 26.
  • mice of Group 1 served as negative controls and were dosed with vehicle as follows: the left ears were dosed with Control Article 3 (saline) via intra-lesional injection, and the right ears were dosed topically with Control Article 1 (25% Transcutol:75% 1 ,2-propanediol). Dosing of the left ears was repeated once weekly for a total of 3 doses; dosing of the right ears was repeated once daily for a total of 17 doses. Animals of Groups 2 and 4 were dosed via intra-lesional injection of each scar (10 ⁇ L/scar); dosing was repeated once weekly for a total of 3 doses. Animals of Group 3 were dosed topically; dosing was repeated once daily for a total of 17 doses. Injections were administered as 10 ⁇ ./wound of test or control article using a Hamilton syringe with 27-gauge needles; topical application was performed by applying 10 ⁇ _ using a micropipette.
  • the NBF-fixed tissues were processed for histopathologic evaluation. Tissues were dehydrated, embedded in paraffin, sectioned at 3- to 5- ⁇ thicknesses, and stained with H&E and with Masson's trichrome. Slides were evaluated via light microscopy by a board-certified veterinary pathologist. Ear lesions (8/animal) were assessed using the scar elevation index (SEI) described by Morris et al. (1997) . In brief, the SEI measures the ratio of total scar connective tissue area to the area of underlying dermis.
  • SEI scar elevation index
  • the scar area was measured in 1 or 2 sections per scar. Measurements were made digitally.
  • a computerized visual imaging system was used to photograph the areas of interest using an Olympus (Tokyo, Japan) camera, with the image digitized by using MicroSuite Basic Edition Software (Tokyo, Japan).
  • MSE Maximum Scar Elevation
  • P-h the external perimeter of the newly formed hypertrophied dermis
  • P-d the perimeter of the underlying dermis
  • Body Weight [0475] Group mean normalized (to pre-dose) body weight values are plotted in Figure 1 1 . All rabbits gained weight during the course of the study; adverse effects were not apparent following the surgery or treatments.
  • Inflammation was composed of a variable inflammatory response consisting of an admixture of mononuclear cells, lymphoid cells, macrophages, and neutrophils in the wound site. Fibrosis and granulation tissues also were present. All wounds were morphologically similar, but somewhat differentiated based upon severity of the lesion, size of the scar, and presence of inflammation. There were clear differences in the severity of the inflammation between groups. Group 1 left (saline) and right (vehicle 2) ears did not differ with regard to inflammation, and there was marked inflammation at the lesion sites in both groups. In Group 4 (treated with TACA), there was very little inflammation, consistent with the expected activity of TACA in this model.
  • CT340 While intra- lesion injection of CT340 had little effect on inflammation, there was marked reduction in inflammation in scars treated daily by topical application of CT340, similar to that seen with TACA. This observation suggests that topical application of 5% CT340 has marked anti-inflammatory effects under the conditions of this study.
  • Example 9 CT340 in vitro activity testing in comparison to K252a and CT327:
  • the purpose of this study was to evaluate the in vitro activity of CT340 in comparison with K252a and CT327.
  • the in vitro activity was evaluated as ability to inhibit proliferation on neonatal Human Epidermal Keratinocytes.
  • T-75 culture flasks are rinsed one time with 9 ml of Trypsin/EDTA solution according to the manufacturer instructions. 3 ml of Trypsin/EDTA is then added for the second time to detach cells. After approximately 10 minutes at 37°C, cell detachment is complete and Trypsin is blocked with 9 ml of Trypsin Neutralizer solution. Cells are transferred to a sterile 15 ml conical tube, filled with other 9 ml of Trypsin neutralizer and centrifuged at 180 x g tor 7 minutes.
  • the supernatant is removed and the cell pellet is resuspended in 12 ml of supplemented medium.
  • Vital cell count with Trypan Blue is performed and cells are diluted to seed one or more 96-well plates with 1 ,5 x 103 cells/well in 100 ⁇ _. Cells are incubated tor 18h in a 37°C, 5% C0 2 , humidified cell culture incubator. The day after, 10 ⁇ of test item solution at the appropriate concentration are added to each well (10 ⁇ L/well of Complete Growth medium are added to control wells).
  • the plate(s) are then incubated for 24h, 48h, 72h and 96h in a humidified incubator at 37°C with 5% C0 2 and analyzed with MTT at each time-point (Time 0 is analyzed just after cell treatment with test item solution) .
  • a K252a 10 mM stock solution is prepared in DMSO and stored at - 20°C. Solutions tor cell treatment are freshly prepared just before use by serial dilution of stock solution in complete growth medium.
  • CT340 and CT327 10 mM stock solutions are prepared in DMSO and stored at - 20°C. Solutions tor cell treatment are freshly prepared just before use by serial dilution of stock solution in complete growth medium. Only tor the third experiment a 10 mM starting solution is prepared in complete growth medium just before cell treatment. This solution is then further diluted in the same medium to obtain the desired concentrations to be tested. Complete growth medium is used as positive control.
  • K252a, CT327 or CT340 concentration data having a post test p value ⁇ 0.05 if compared to control wells data are considered as significantly inhibiting the proliferation of human epidermal keratinocytes.
  • IC 50 value was calculated fitting data on a sigmoidal dose- response curve with variable slope.
  • GraphPad Prism Software was used for data representation, statistical analysis and curve fitting.
  • K252a shows a good concentration-response curve after contact time 48 h, 72 h and 9 6h and is only partially effective after contact time 24h.
  • CT327 and CT340 partially inhibit proliferation at time 24 h and 96 h but without a concentration- dependent response.
  • CT327 and CT340 were tested at different concentrations than K252a in order to evaluate if higher concentrations of the molecules are able to inhibit the proliferation of HEKn cell line.
  • K252a was tested at the same concentrations of the previous experiment while CT327 and CT340 were tested at the following concentrations: 100 ⁇ , 50 ⁇ , 25 ⁇ , 5 ⁇ , 1 ⁇ , 500 nM , 200 nM , 100 nM , 50 nM and 25 nM .
  • MTT assays were performed only at time 72 h. The results of the experiment performed on neonatal HEK cell line to evaluate the ability of the compounds to inhibit proliferation are reported in Figure 24.
  • increased concentrations of CT327 and CT340 cause inhibited proliferation at time 72 h with a partial concentration-dependent response.
  • the 1 % DMSO concentration present in the wells with the higher concentrations of CT327 and CT340 did not allow for statistically significant relevance and the IC 50 value could not be calculated.
  • a third experiment was performed in order to identify an IC 50 value tor CT340 and CT327 in the absence of DMSO in the final wells. For this reason, 10 mM starting solutions of CT340 and CT327 were prepared by dissolving them directly in complete growth medium just before cell treatment. These solutions were then further diluted in the same medium to obtain the desired concentrations to be tested.
  • K252a was tested at the same concentrations of the previous experiments while CT327 was tested at the following concentrations: 500 ⁇ , 100 ⁇ , 50 ⁇ , 25 ⁇ , 5 ⁇ , 1 ⁇ , 500 ⁇ , and 50 ⁇ .
  • CT340 was tested at the following concentrations: 500 ⁇ , 100 ⁇ , 50 ⁇ , 25 ⁇ , 5 ⁇ , 1 ⁇ , 500 ⁇ , 200 ⁇ and 100 ⁇ .
  • K252a showed a good concentration-dependent response for all three time points analyzed. Furthermore, increased concentrations of CT327 and CT340 inhibit proliferation at time points 72 h, 96 h and 120 h with a concentration-dependent response.
  • IC 50 values for CT327 and CT340 are the following:
  • CT327 and CT340 maintain the ability to inhibit proliferation of HEKn cells, even though employed at higher concentrations if compared to the unconjugated molecule. Furthermore, CT340 appears to be slightly more active than CT327.
  • CT340 inhibition of JAK2, JAK3, PDGFRb, TRKA, MAP2K1 , MAP2K3, TAK1 -TAB1 was evaluated as well as a control.
  • the CT340 test concentrations used were the following: 10, 3, 1 , 0.3, 0.1 , 0.03, 0.01 , 0.003, 0.001 , and 0.0003 ⁇ .
  • test compound was dissolved in and diluted with dimethylsulfoxide (DMSO) to achieve 100-fold higher concentration. Then the solution was further 25-fold diluted with assay buffer to make the final test compound solution. Reference compounds for assay control were prepared similarly.
  • DMSO dimethylsulfoxide
  • MSA Off-chip Mobility Shift Assay
  • reaction control complete reaction mixture
  • background Enzyme(-)
  • IC50 value was calculated from concentration vs. %inhibition curves by fitting to a four parameter logistic curve.
  • Example 11 Examination of CT327 and CT340 in pain and nerve regeneration
  • This study evaluated the effect on nociception and neurite outgrowth of the highly selective TrkA inhibitors CT327 and CT340, using: 1) a functional calcium imaging assay to measure inhibition of capsaicin responses; and 2) neurite outgrowth assay in cultured sensory (DRG) neurons.
  • the effects of CT327 and CT340 were compared with a commercially available TrkA inhibitor (GW441756) and an anti-NGF antibody
  • Nerve Growth Factor is a homodimer, which following binding results in dimerization and autophosphorylation of its high affinity receptor TrkA. Phosphorylation of TrkA increases the catalytic activity of the kinase domain and creates binding sites for SH2 domain containing cytoplasmic proteins. These proteins initiate the activation of several signal transduction pathways such as PLCy, ras, PB kinase/AKT, and Raf/MEK/ERK.
  • NGF released from inflammatory cells and tissues during inflammation and injury results in hyperalgesia that can last from several hours to days via TrkA activation on sensory nerve terminals. This activates a multitude of downstream signalling pathways involving MAP kinases (ERK), PBK, and PLC, which are involved in pathological conditions.
  • ERK MAP kinases
  • PBK PBK
  • PLC MAP kinases
  • NGF binding to TrkA also potentiates the heat and capsaicin receptor TRPV1 through phosphorylation of TRPV1 channels by serine/threonine kinases, PKC, PKA, and calcium/calmodulin-dependent kinase II and tyrosine kinase c- Src.
  • TRPV1 is a plasma membrane bound ion channel expressed by nociceptors involved in thermosensation, which is activated by noxious heat (>43°C), capsaicin, low pH, the inflammatory mediators arachidonic acid and bradykinin leading to the perception of pain and thermal hypersensitivity.
  • the sensitivity of the capsaicin receptor (TRPV1 ) is modulated by NGF in rodent and human DRG neurons. TRPV1 expression and function has been found to be up-regulated by NGF in vitro and in clinical conditions of hypersensitivity. Responses of cultured DRG neurons to capsaicin thus provide an in vitro model for NGF-trkA pathway signalling, as used in this study.
  • NGF influences the survival of developing primary sensory neurons mainly through its high affinity receptor TrkA
  • its role in mature DRG neurons is restricted to regulating their sensitivity and neuropeptide expression, but not survival, or neurite length.
  • systemic blockade of NGF in clinical conditions has been associated with positive sensory symptoms and accelerated arthritis, and generalized loss of protective sensation remains a concern with long-term treatment; these may be avoided by regional topical delivery for cutaneous hypersensitivity disorders.
  • CT327 and CT340 designed to block NGF- TrkA signalling in an in vitro model of NGF induced hypersensitivity in adult rat DRG neurons, and demonstrate the inhibition of capsaicin responses without affecting neurite length.
  • Bilateral DRG from 1 1 adult rats were microdissected from cervical, thoracic, lumbar and sacral levels and enzyme digested in 0.2% collagenase/0.5% dispase in Ham's F12 medium for 3 hours (previously described in Anand et al, 2010) .
  • Enzyme digested tissue was mechanically dissociated in BSF2 medium (Ham's F12 containing 2% heat inactivated fetal calf serum, 60 ng/ml progesterone, 0.16 ⁇ g/ml sodium selenite, 0.1 mg/ml transferrin, 16 ⁇ g/ml putrescine, 10 ⁇ g/ml insulin, 3 mg/ml BSA, 100 ⁇ g/ml each penicillin/streptomycin) , containing 1 mg/ml soy-bean trypsin inhibitor.
  • BSF2 medium Ham's F12 containing 2% heat inactivated fetal calf serum, 60 ng/ml progesterone, 0.16 ⁇ g/ml sodium selenite, 0.1 mg/ml transferrin, 16 ⁇ g/ml putrescine, 10 ⁇ g/ml insulin, 3 mg/ml BSA, 100 ⁇ g/ml each penicillin/streptomycin
  • the resulting neuronal suspension was plated on collagen (from rat tail, 50 ⁇ g/ml) , and laminin (20 ⁇ g/ml) coated glass bottomed petri dishes (MatTek, USA) at 1000 cells per dish in 200 ⁇ ls BSF2 medium; after 30 minutes 2 mis warm BSF2 medium containing human NGF (Sigma U.K. 100 ng/ml) was added to the cultures, which were incubated at 37 °C in a humid environment.
  • Creabilis TrkA inhibitors CT327, CT340 and commercially available TrkA inhibitor GW441756 were dissolved in DMSO to make stock solutions of 4.5 mM (CT327) , and 4.7 mM (CT 340) , 30 mM (GW441756) aliquoted and stored at -20 °C, until use.
  • Intermediate dilutions of CT327 and CT340 were prepared in sterile distilled water, and of GW441756 in ethanol, at 500x final concentration.
  • Anti-NGF antibody (1 mg/ml, anti human/mouse) was obtained commercially (L148M , Exalpha Biologicals), aliquoted and stored at -20 °C until use.
  • Neurons with a stable baseline ratio of 340/380 were stimulated with 200 nM capsaicin (20-30 seconds to identify capsaicin sensitive neurons which indicated calcium influx by increased 340/380 ratio; this was followed by washout and a rest period of 30 minutes.
  • Test compounds were applied from stock solutions (500x final concentration- 4 ⁇ /2 ⁇ ) , followed 10 minutes later by 1 ⁇ capsaicin, and responses were measured as the difference from baseline to peak ratio, and expressed as a percentage response (ratio of second (1 ⁇ capsaicin)/first (200 nM capsaicin) response), and compared with NGF treated controls (no test compound applied between capsaicin stimuli). Percent inhibition was calculated for each group and compared with controls. Student's t test was used to compare between groups; P ⁇ 0.05 was considered statistically significant.
  • TrkA/Gap43 immunofluorescence confirmed that TrkA positive neurons were present in the treated groups ( Figure 49) .
  • the commercial TrkA inhibitor GW441756 used for comparison, also did not have any effect on neurite length at 1 , 10 and 100 nM concentrations, but reduced neurite length at the higher concentrations of 1 and 10 ⁇ with vesiculation, indicating possible structural effects at these concentrations.
  • Anti-NGF antibody treatment at 1 and 10 ⁇ g/ml concentrations did not significantly affect neurite length.
  • CT327 and CT340 are potent inhibitors of NGF/TrkA- dependent capsaicin responses in cultured sensory neurons. Topical treatment with TrkA inhibitors may provide pain relief with the efficacy but without the safety liability of systemic NGF blockade.
  • Tissue sections (15 ⁇ thick) were collected onto poly-L-lysine (Sigma, Poole, UK) coated glass slides. Endogenous peroxidase was blocked by incubation in industrial methylated spirits (IMS) containing 0.3% w/v hydrogen peroxide for thirty minutes. After rehydration with PBS buffer, sections were incubated overnight with primary antibody at final dilutions listed above. Sites of primary antibody attachment were revealed using nickel-enhanced, avidin-biotin peroxidase (ABC - Vector Laboratories, Peterborough, UK) as described. Sections were counter-stained for nuclei in 0.1 % w/v aqueous neutral red, dehydrated and mounted in xylene-based mountant (DPX; BDH/Merck, Poole, UK), prior to photomicrography.
  • IMS industrial methylated spirits
  • PGP9.5 intra-epidermal fibers were counted at optimal titre of 1 :40,000 and results expressed as fibers/mm.
  • the nerve fibers present in first 2 mm of each section fibers were counted.
  • Optimal dilution for PGP9.5 staining in the pig tissue was 1 :40,000. Intra-epidermal nerve fibers were seen, and immunoreactive fibers were also present in arrector pili and deeper dermal nerve fascicles (Figure 50).
  • the goal of this study was to characterize SNA-120 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- 120 was profiled in the BioMAP Diversity PLUS panel at concentrations of 28000 nM, 9200 nM, 3100 nM, and 1000 nM.
  • SR221 1 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
  • 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 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%.
  • FIG. 52 depicts the BioMAP profile of SNA-120 in the Diversity PLUS Panel.
  • SNA-120 was found to be active with 21 annotated readouts, mediating changes in key biomarker activities listed by biological and disease classifications in Table 16 beiow.
  • SNA-120 impacted inflammation-related activities (decreased E-seieetin, sTNFa, MIP-1a, IL-8, IL-1a), immunomodulatory activities (decreased slgG, slL-10; increased CD69), tissue remodeling activities (decreased TIMP-2, tPA, MMP-1, PAI-1, Collagen ill, uPAR, MMP-9), and hemostasis-reiated activities (decreased TF), SNA-120 is antiproliferative to endothelial cells, fibroblasts, and T cells (as indicated by grey arrows in Figure 52). There are no cytotoxic impacts detected at the concentration range tested.
  • Figure 53 depicts an overlay of SNA-120 at 28 ⁇ and the selected reference benchmark SR2211 at 10 ⁇ .
  • SR2211 is an antagonist of retinoic acid receptor related nuclear receptor gamma.
  • 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 MCP-1, uPAR
  • LPS CD69, sPGE2, sTNFa
  • SAg CD69
  • BF4T Eotaxin 3
  • BE3C MMP-1, MMP-9, PAI-1, tPA
  • CASM3C HLA-DR, IL-8, LDLR, TM, uPAR
  • HDF3CGF Collagen I, Collagen lil, EGFR, M-CSF, MIG, PAI-1, VCAM-1
  • MyoF IL-8, MMP-1, VCAM- 1
  • IMphg CD69, MCP-1, MIP-1a, VCAM-1).
  • Figure 54 depicts changes in the secretion of IL-17F, IgG, IL-17A, and TNFa in the BioMAP BT system mediated by SNA-120 (3.1 ⁇ ), Tofacitinib (3.3 ⁇ ), Apremilast (3.3 ⁇ ), SR221 1 (3.3 ⁇ ), and Cyclosporin A (3.3 ⁇ ). Tofacitinib was found to be more active than SNA-120 in decreasing IL-17F secretion, displaying an activity similar to SR221 1 ( Figure 54A). Surprisingly, SNA-120 was found to be as active as tofacitinib in decreasing IL-17A secretion ( Figure 54C). SR221 1 decreased IL-17A secretion as expected, while Apremilast increased IL-17A secretion.
  • LPS sPGE2, sTNFa
  • SAg Prolif
  • BT SIL-17A, SIL-17F, slL-2, slL-6, slgG, sTNFa
  • BE3C MMP-1 , MMP-9, PAI-1
  • HDF3CGF PAI-1
  • IMphg CD69, slL-10
  • Table 17 depicts the top 3 similarity matches from a search of the BioMAP Reference Database of > 4,000 agents for each concentration of SNA-120.
  • 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.
  • Figure 56 depicts Mechanism HeatMAP Analysis of SNA-120, 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.
  • Figure 57 depicts a clustering of tested agent profiles based on pairwise correlation analysis and clustering of most similar profiles. Profiles that are similar with a Pearson's correlation coefficient (r) > 0.7 are connected by lines. Agents that do not cluster with one another are interpreted as mechanistically distinct. Cytotoxic concentrations are excluded from duster analysis. Functional clustering of the agent profiles generated during this analysis uses Pearson's correlation values for pairwise comparisons of the profiles for each agent at each concentration, and then subjects the pairwise correlation data to multidimensional scaling. SNA-120 clusters internally at two concentrations. Internal clustering suggests the phenotypic signature of this compound is maintained across a range of concentrations, a characteristic commonly observed in marketed drugs.
  • r Pearson's correlation coefficient
  • SNA-120 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-120 was broadly active and non-cytotoxic in the Diversity PLUS panel. Antiproliferative effects to multiple cell types were observed, a feature that is appropriate for compounds developed for oncology, but not autoimmune indications. Inhibition of several inflammation-related readouts were observed with a sharp dose response noted between the two highest and two lowest concentrations. This suggests that the compound may have additional targets at the higher concentrations (> 9.2 ⁇ ). Robust activity was also observed in systems containing epithelial cells, indicating target expression on this cell type and potentially utility for pulmonary indications. At the top tested concentration, SNA-120 shared 12 common activities with the requested benchmark compound SR221 1 , a RORy inhibitor, predominantly in the BT system.
  • SNA- 120 shared 15 common activities, also predominantly in the BT system, with the pan- AKT inhibitor GSK690693.
  • Other top database matches with a Pearson's correlation coefficient r > 0.7 include BSK_4925, a Lck inhibitor, and filgotinib, a JAK inhibitor. It is noted that several activities of this test agent, particularly in the BE3C, BF4T and HDF3CGF systems, are consistent with the EGFR inhibitor mechanism class (see Figure 56).
  • Test Agent SNA-125 was profiled In the BioMAP Diversity PLUS panel at concentrations of 3900 nM, 1300 nM, 430 nM, and 140 nM, Tofacitinib was employed as the benchmark compound.
  • FIG. 58 depicts the BioMAP profile of SNA-125 in the Diversity PLUS Panel.
  • SNA-125 was found to be modestly active with 4 annotated readouts, mediating changes in key biomarker activities listed by biological and disease classifications in Table 18 below.
  • SNA-125 mediated changes in key biomarker activities are listed by biological and disease classifications.
  • SNA-125 impacted inflammation-related activities (decreased sTNFa) and tissue remodeling activities (modulated MMP-1). There are no cytotoxic or antiproliferative impacts detected at the concentration range tested.
  • Figure 59 depicts an overlay of SNA-125 at 3.9 ⁇ and the selected reference benchmark tofacitinib at 3.3 ⁇ .
  • Tofacitinib is a JAK1/3 kinase inhibitor approved in 2012 for the treatment of rheumatoid arthritis.
  • Differentiating biomarkers are defined when one profile has a readout outside of the significance envelope with an effect size > 20% (jiogI G ratioj > 0.1), and the readout for the other profile is either inside the envelope or in the opposite direction.
  • 3C HLA-DR, MIG, uPAR
  • 4H Eotaxin 3, P-seiectin, VCAM-1 , VEGFR2
  • LPS Liscosity Polypeptide
  • SAg CD38, CD40, CD69, E-selectin, IL-8, MCP-1 , MIG, Prolif
  • BT Prolif, slL-2, slL-6
  • BF4T MMP-3, MMP-9
  • BE3C l-TAC, IP-10, MIG, uPA
  • CASM3C HLA- DR, LDLR, M-CSF, MCP-1 , MIG, VCAM-1
  • HDF3CGF IP-10, M-CSF, VCAM-1
  • KF3CT KF3CT
  • Figure 54 depicts changes in the secretion of IL-17F, IgG, IL-17A, and TNFa in the BioMAP BT system mediated by SNA-125 (3.9 ⁇ ), Tofacitinib (3.3 ⁇ ), Apremilast (3.3 ⁇ ), SR221 1 (3.3 ⁇ ), and Cyclosporin A (3.3 ⁇ ). Tofacitinib was found to be more active than SNA-125 in decreasing IL-17F secretion, displaying an activity similar to SR221 1 ( Figure 54A). SNA-125 and Tofacitinib were both very active in decreasing secreted IgG, with SNA-125 as active as Cyclosporin A ( Figure 54B).
  • SNA-125 was found to be as active as tofacitinib in decreasing IL-17A secretion ( Figure 54C).
  • SR221 1 decreased IL-17A secretion as expected, while Apremilast increased IL-17A secretion.
  • SNA-125 was found to have activity with regards to reducing TNFa secretion ( Figure 54D).
  • LPS sTNFa
  • BT SIL-17A
  • SIL-17F SIL-17F
  • slgG slgG
  • BE3C MMP-1
  • IMphg sIL- 10
  • Table 19 depicts the top 3 similarity matches from a search of the BioMAP Reference Database of > 4,000 agents for each concentration of SNA-125.
  • 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 61 depicts Mechanism HeatMAP Analysis of SNA-125, 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-125 was characterized by profiling in the BioMAP Diversity PLUS panel of human primary ceil based assays modeling complex tissue and disease biology of organs (vasculature, immune system, skin, lung) and general tissue biology.
  • the Diversity PLUS panel evaluates the biological impact of test agents in conditions that preserve the complex crosstalk and feedback mechanisms that are relevant to in vivo outcomes.
  • SNA-125 was selectively active in the Diversity PLUS panel, with specific inhibition of sTNFa activity in the LPS and BT systems. No cytotoxic or antiproliferative effects were observed. Inhibition of TNFa activity or production is consistent with drugs currently approved for the treatment of psoriasis, such as anti-TNF antibodies (e.g. adalimumab and infliximab) and JAK inhibitors (e.g. tofacitinib).
  • anti-TNF antibodies e.g. adalimumab and infliximab
  • JAK inhibitors e.g. tofacit
  • SNA-125 was profiled in the BioMAP Diversity PLUS panel at concentrations of 30 ⁇ , 10 ⁇ , 3.3 ⁇ , and 1.1 ⁇ . K252a was employed as the benchmark compound.
  • Figure 62 depicts the BioMAP profile of SNA-125 in the Diversity PLUS Panel.
  • SNA-125 was found to be active with 52 annotated readouts, mediating changes in key biomarker activities listed by biological and disease classifications in Table 20 below.
  • SNA-125 mediated changes in key biomarker activities are listed by biological and disease classifications.
  • SNA-125 impacted inflammation-related activities (decreased Eotaxin 3, E-selectin, MCP-1 , VCAM-1 , sTNFa, MIP-1 a, IL-8, IL ⁇ 1 a, P- selectin, sPGE2, IL-8), immunomodulatory activities (decreased CD40, slgG, slL-10, M- CSF, SIL-17A, slL-6, SIL-17F, slL-2), tissue remodeling activities (decreased Collagen I, TiMP-2, Decorin, MMP-1 , uPA, PAi-1 , Collagen III, uPAR, MMP-9), and hemostasis- related activities (decreased TF; increased TM).
  • SNA-125 is antiproliferative to B cells, endothelial cells, fibroblasts, and T cells (as indicated grey arrows of Figure 62). SNA- 125 had no cytotoxic effects in the concentration range tested.
  • Figure 63 depicts an overlay of SNA-125 at 30 ⁇ and the selected reference benchmark K252a at 30 nM.
  • K252a is a non-selective protein kinase inhibitor that inhibits PKC, Ca2+/calmodulin-stimulated phosphodiesterases, lVILCK, and receptor tyrosine kinases.
  • K252a is an analog of staurosporine.
  • Differentiating biomarkers are defined when one profile has a readout outside of the significance envelope with an effect size > 20% (
  • TM 3C
  • LPS CD89, sPGE2
  • SAg IL-8
  • BT slL-2
  • BF4T VCAM-1
  • BE3C tPA
  • CASM3C IL-6
  • HDF3CGF Collagen I, Collagen III, EGFR, l-TAC, IL-8, MCP-1
  • KF3CT IL-8
  • MyoF Decorin, MMP-1, VCAM-1
  • IMphg IL-1Q, IL-8, MCP-1).
  • 3C IL-8, TF
  • LPS E-selectin, IL-1a, IL-8, MCP-1, TF, TM, VCAM-1, sPGE2, sTNFa
  • SAg IL-8, Prolif
  • BT Prolif, SIL-17A, si L-2 , slL-6, slgG, sTNFa
  • CASM3C IL-6
  • IMphg E- selectin, IL-1 a, IL-8, MIP-1a
  • Table 21 depicts the top 3 similarity matches from a search of the BioMAP Reference Database of > 4,000 agents for each concentration of SNA-125.
  • 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.
  • Figure 65 depicts Mechanism HeatMAP Analysis of SNA-125, 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.
  • Figure 66 depicts the BioMAP profile overlay of SNA-125 (10 ⁇ ) with Methotrexate 1 Q ⁇ ) and Tofacitinib (10 ⁇ ). Differences in the LPS system and /Mphg system were observed. A similar impact on T-ceil proliferation can be seen in the Figure 66 overlay of the three compounds. It is noted that Tofacitinib activities in 3C consistent with systemic side effects, not present with SNA-125
  • SNA-125 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-125 was broadly active across the Diversity PLUS panel with 52 annotated readouts affecting biomarkers involved with inflammation, immunomodulation, tissue remodeling, and hemostasis. SNA-125 was antiproliferative to B cells, endothelial cells, fibroblasts, and T cells and had no effects on cytotoxicity at the concentration range tested. SNA-125 was previously profiled in the Diversity PLUS panel at a lower concentration range (140 nM - 3900 nM, refer to project #SNE001-01-b), where only inhibition of TNFa and modulation of MMP-1 were observed.
  • SNA-125 and its reference benchmark K252a a non-selective protein kinase inhibitor, had 37 common activities and 21 differentiating activities; in general, SNE-SNA-125 was more active at the top tested concentration (30 ⁇ ) than the top tested concentration of K252a (30 nM).
  • the top database search match for SNA-125 was IKK 16, an IKK2 inhibitor, and that match was above our threshold for significance (r > 0.7). There were 24 common activities between SNA-125 and IKK2, most of which were in the LPS system modeling Th1 type inflammation and monocyte activation and the BT system modeling T cell dependent B cell activation. Inhibitors of IKK2 and p38 MAPK were among the top matches for this compound, indicating that SNA125 may have phenotypic impacts on the NFKB and/or MAPK signaling pathways.
  • Imiquimod is a TLR7/8 ligand and a potent immune activator that is used topically for genital and perianal warts, as well as for superficial basal cell carcinomas and actinic keratosis.
  • An acute model of psoriasis has been developed in mice by topical application of a 5% IMQ cream (AldaraTM), based on the clinical observation that IMQ exacerbates psoriasis. Similar to human psoriasis, IL-23/IL-17 axis plays a pivotal role in IMQ-induced psoriasis in mice.
  • Figure 69 depicts the changes in the total psoriasis score throughout the study, which was calculated by summing the plaque score, the erythema score and the punctate redness/scabbing score.
  • the difference between SNA-125 at 5% and the vehicle is statistically significant from day 7.
  • the differences between SNA-125 at 0.5% and 1 % and the vehicle are statistically significant on day 10 only.
  • Figure 70 depicts the changes in the erythema score throughout the IMQ-induced psoriasis mouse study. SNA-125 at 5% is statistically significant from the vehicle from day 7. SNA-125 at 0.5% and 1 % are statistically significant on day 10 only.
  • Figure 71 depicts the changes in the plaque score throughout the IMQ-induced psoriasis mouse study. SNA-125 at 5% is statistically significant from the vehicle on day 10.
  • Figure 72 depicts the changes in the punctate redness/scabbing score throughout the IMQ-induced psoriasis mouse study. As expected, IMQ only animals exhibited increased punctate redness and scabbing, which was diminished by SNA-125 application.
  • Figures 73 and 74 depict the changes in spleen thickness and ear thickness throughout the IMQ-induced psoriasis mouse study, respectively.
  • Topical application of the IMQ cream causes the enlargement of spleen and lymph nodes, and increased ear thickness.
  • the commonly used antipsoriatic agent clobetasol almost completely attenuated these IMQ-induced changes.
  • Neither treatments with vehicle or test compound significantly modulated spleen weight.
  • Figure 75 depicts the levels of cytokines IL-22, IL-17A, IL17F, and TNFa in ear samples taken at Day 4. Note that while the cytokines were measured on day 4, different cytokines can have different pharmacodynamics behavior (i.e. peak at different days post IMQ application). Daily application of IMQ induced an increase (significant in IL-22, IL-17F and TNFa) in inflammation/psoriasis-associated cytokines in the ears of diseased animals on day 4. Daily treatment with clobetasol decreased the cytokine production to baseline levels.
  • TNFa (vehicle, mean 9.331 ⁇ 2.267; 5% SNA-125, mean 4.860 + 0.973) and IL-22 (vehicle, mean 9.650 ⁇ 2.339; 5% SNA-125, mean 5.243 ⁇ 1.759) was observed.
  • Example 17 Plasma Pharmacokinetic Profile of CT327 (SNA-120) Following a
  • CT327 SNA-120
  • This Example reports the analyses of CT327 plasma levels in the blood samples collected from 5 minutes to 8 hours after drug administration.
  • CT327 A single administration of CT327 was given to rats and blood samples were collected at scheduled times in order to verify the kinetic profile.
  • One group of 9 female rats received the test item by the intravenous injection, at 18 mg/kg dose level.
  • the vehicle was physiological saline solution (NaCI 0.9%).
  • the required amount of CT327 was dissolved in the vehicle at the concentration of 3.6 mg/ ml_.
  • Concentration of the test item was calculated in terms of active ingredient according to the assay results (97.7%), as reported in the certificate of analysis.
  • the formulation was prepared on the day of dosing. Since the formulation had to be administered within 30 minutes after preparation and on the basis of the bleeding scheme, three formulations of the test item were prepared at the scheduled times.
  • the study consisted of one group comprising 9 female rats.
  • the test item was administered by intravenous bolus injection into the tail vein over a period of approximately 1 mL/minute at a dose volume of 5 mL/kg body weight.
  • the dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal. Each animal received a single dose.
  • Blood samples were collected from the animals at the following sampling times: 0 (predose), 5, 10, 20 and 30 minutes, 1 , 2, 4 and 8 hours after the single dose administration. Blood samples were collected from each animal at 3 alternating time points. The theoretical and actual times of collection were recorded. At each sampling time, blood samples of approximately 0.8 mL each were collected under light isofluorane anaesthesia from the retroorbital sinus of 3 animals and transferred into light-protected tubes containing EDTA anticoagulant, immediately centrifuged at 4°C and 3000g for 10 minutes and the plasma frozen at -80°C. Two different aliquots were prepared: the first contained 250 ⁇ of plasma and the second (100 ⁇ ). In addition, about 10 mL of plasma were obtained from 5 untreated female rats of the same batch of the study for analytical (calibration curve) purposes.
  • Rat plasma (pool obtained from 5 untreated female rats of the same batch "stock order 661 " used for the experiment, i.e. Hsd: Sprague Dawley SD rats, 45-53 days old and with body weight of approximately 150-174 g)
  • Solid Phase Extraction (SPE) cartridges Oasis® HLB 1 cc/10mg, particle size 30 ⁇
  • CT327 plasma levels were performed by HPLC using a Beckman System Gold® chromatograph 126 solvent module, 168 UV-Vis Detector, 508 autosampler, equipped with a XTerra® RP 18 column 3.0 x 50 mm.
  • Plasma samples were purified before HPLC analysis, in order to eliminate any possible source of interference, by SPE technique using Waters Oasis® HLB (Hydrophilic-Lipophilic Balance reverse-phase sorbent) cartridges.
  • the purification of the biological samples consisted in the following consecutive steps: (i) conditioning of the cartridge with 1 mL of methanol; (ii) equilibration with 1 mL of MilliQ water; (iii) loading of the sample properly diluted in water (i.e.
  • the purification was performed using an extraction manifold (supplied by Waters S.p.A. , Vimodrone, Italy) able to accommodate up to 20 cartridges and connected to a dual-headed vacuum pump (supplied by VWR International Sri, Milano, Italy).
  • CT327 standard samples in plasma were prepared with the above described procedure by purifying blank plasma samples (rat plasma recovered from untreated animals) spiked with a solution of test item in MilliQ H 2 0 up to a volume of 0.1 ml, as depicted below in Tables 23 and 24.
  • the peak corresponding to CT327 shows a retention time of about 8.5 minutes.
  • the quantitation of CT327 in the chromatograms was performed by integration of the corresponding peak with the following parameters: peak threshold 200 and peak width 0.2. Blank plasma samples were also analysed and included in the sample analysis run in order to control the specificity of the analytical procedure.
  • the calibration curve (peak area versus test item concentration) is shown in Figure 76 for plasma samples spiked with CT327 and was obtained by plotting the detected response versus the nominal sample concentration.
  • the values of correlation coefficient (r 2 ) suggest a good linearity over the range investigated, with a LOD (Limit of Detection) of 35.5 nM and a LOQ (Limit of Quantification) of 69.8 nM for the calibration curve at low concentration and with LOD 0.9 ⁇ /LOQ 1 .7 ⁇ for the calibration curve at high concentration.
  • CT327 reached the maximum peak concentration at the first sampling time point (5 minutes) and quantifiable levels were observed until 4 hours post-administration in all the animals.
  • CL (clearance, in L/h), expressed as the volume of blood cleared of drug per unit time
  • MRT mean residence time, in h
  • V ss (apparent volume of distribution at steady state, in L);
  • t 1 ⁇ 2 (terminal half-life, in h), determined by one-phase exponential decay fitting (GraphPad Prism software analysis).
  • the correlation coefficient (r 2 ) for the goodness of the fit of the regression line through the data points was high enough (0.991) for the value to be considered reliable; the 95% confidence interval for the t 1 ⁇ 2 estimate was 0.13-0.23 h;
  • AUC 0-inf area under the curve from time 0 to infinity, in ⁇ -h
  • AUC 0-inf was considered reliable since the extrapolation from the last data point to infinity represented only the 0.1 % of the total
  • CT327 The pharmacokinetics of CT327 was investigated after intravenous administration to rats in order to assess the systemic exposure, distribution and elimination of the drug candidate. All the analyzed samples (3 animals/timepoint) showed detectable and consistent CT327 plasma levels up to 8 hours after administration.
  • CT327 plasma levels confirmed its surprising and unique chemico-physical properties conferred by its mini- PEG moiety, resulting in high systemic exposure upon IV bolus (thanks to high water solubility), very rapid elimination from the blood stream (low molecular weight, probable fast kidney filtration with urinary elimination) and large confinement to the circulatory system (the compound undergoes no or very limited distribution from blood into tissues).
  • These overall features are in line with the optimal safety profile shown by CT327 from a toxicological point of view: even if absorbed upon topical administration, one can exclude drug distribution and accumulation thanks to an extremely rapid clearance.
  • the goal of this study was to characterize the efficacy of SNA-125 and SNA-352 delivered by oral and intracecal routes for the treatment of colitis with the use of an oxazolone-challenged mouse model of colitis.
  • oxazolone- challenged mice were also treated with Tofacitinib and Prednisolone by oral and intracecal routes.
  • Each mouse underwent video endoscopy on Days 2 & 4 using a small animal endoscope (Karl Storz Endoskope, Germany), under isoflurane anesthesia. During each endoscopic procedure still images as well as video were recorded to evaluate the extent of colitis and the response to treatment. Additionally, an image was captured from each animal at the most severe region of disease identified during endoscopy. Colitis severity was scored using a 0-4 scale as defined in Table 29. Additionally, stool consistency was scored during endoscopy using the parameters defined in Table 30.
  • Peripheral blood and colon tissue were collected at sacrifice on day 4 as follows. Blood was collected via cardiac puncture into KiEDTA-coated tubes and centrifuged at 4000x g for 10 minutes. Plasma was collected, flash frozen, and stored at -80°C. The colon was excised, rinsed, measured, weighed, and then trimmed to 6.0 cm in length and divided into 2 pieces; the most distal 5.0 cm section was swiss rolled and placed in formalin for subsequent histological evaluation (see Figure 78A). The proximal 1.0 cm portion was weighed, snap frozen, and stored at -80°C.
  • Each colon sample was rolled into a swiss roll. Tissues were embedded in paraffin and sectioned at approximately 5 microns. One slide for each colon was stained with hematoxylin and eosin and examined by a board-certified veterinary pathologist. The pathologist was blinded to the treatment that each group received at the time of assessment. Each slide containing one rolled colon was split into four approximately equal quarters. Each quarter was evaluated and scored for inflammation, edema, and mucosal necrosis, according to the scoring criteria listed below in Tables 31 to 33. As depicted in Figure 78B, with the slide label to the left, quarters were evaluated starting at the top left and moving clockwise.
  • Colon tissue homogenate supernatants were analyzed for protein levels of a panel of mouse inflammatory mediators: IFN- ⁇ , IL-10, IL-6, & TNF-a using a multiplex system (MAGPIX, EMD Millipore).
  • Figure 79 depicts the effect of oral and intracecal administration of SNA-125, SNA-352, tofacitinib, and prednisolone on the body weight of animals challenged with oxazolone.
  • Figure 80 depicts this data according to last observation carried forward analysis. A reduction of the body weight of animals treated with oxazolone was observed. A trend towards a decrease of the body weight loss was observed in the oral SNA-125 administration group.
  • Figures 81 and 83 depict the effect of oral and intracecal administration of SNA-125, SNA-352, tofacitinib, and prednisolone on the Day 2 and 4 endoscopy scores, respectively, of animals challenged with oxazolone.
  • oral and intracecal Tofacitinib administration yielded a 10-15% improvement in endoscopy scores.
  • orally administered SNA-125 demonstrated a 22% reduction of the Day 2 endoscopy score.
  • oral and intracecal Tofacitinib adminstration yielded a 10-15% improvement in endoscopy scores.
  • orally administered SNA-125 yielded a 15% reduction of the endoscopy score.
  • Figures 108 to 1 13 depict representative Day 2 and Day 4 endoscopy images.
  • Figures 82 and 84 depict the effect of oral and intracecal administration of SNA-125, SNA-352, tofacitinib, and prednisolone on the Day 2 and 4 stool consistency scores, respectively, of animals challenged with oxazolone. Oral SNA- 125 showed a significant reduction (65%) in the stool consistency score on Day 2.
  • Figure 85 depicts the effect of oral and intracecal administration of SNA-125, SNA-352, tofacitinib, and prednisolone on the disease activity index (DAI) score of animals at Days 2 and 4 following challenge with oxazolone.
  • DAI disease activity index
  • Figure 86 depicts the effect of oral and intracecal administration of SNA-125, SNA-352, tofacitinib, and prednisolone on the colon weight/length ratio of animals challenged with oxazolone.
  • Oxazolone produced mild to moderate colitis characterized by multifocal inflammation, edema, and necrosis.
  • Figures 87-89 depict the histopathology scoring results for inflammation, edema, and mucosal necrosis/loss, respectively, while Figure 90 depicts the summation of these scores. Note that control animals not given oxazolone were essentially normal and were not included in the statistical analysis.
  • both SNA-125 and SNA-352 tended to reduce mucosal necrosis compared to vehicle and this improvement was more noticeable than for either prednisolone or tofacitinib.
  • both SNA-125 and SNA-352 tended to reduce the sum score compared to vehicle, and this improvement was more noticeable than for either prednisolone or tofacitinib.
  • this treatment effect was statistically significant while for edema and the sum score, this effect approached significance
  • Sections of colon were often thickened by inflammation and edema that variably extended into the lamina intestinal, submucosa and muscular wall.
  • the inflammation was pyogranulomatous - composed of a mixture of neutrophils, macrophages, lymphocytes, and plasma cells.
  • Mucosal necrosis was also variably present and characterized by partial or complete loss of the surface epithelium with erosion to underlying lamina intestinal or submucosa. There was multifocal peritonitis suggesting that there was multifocal full thickness erosion. Due to the multifocal distribution of these changes, the inflammation, edema, and mucosal necrosis were variable along the swiss rolled section. Representative photomicrographs are shown in Figures 91 -93.
  • control animals in this study had basically normal colons without significant inflammation, mucosal necrosis, or edema.
  • the intracecal vehicle group had relatively mild colitis compared to other groups. While there were some areas in some animals with inflammation, edema, and mild necrosis (inset), most of the colons had mild inflammation with mild edema and minimal to no necrosis (Figure 93). Animals treated with intracecal tofacitinib had diffuse inflammation with pockets of more severe inflammation and mucosal necrosis. Animals treated intracecal with SNA-125 were divided between mild colitis and severe colitis. Animals treated intracecally with SNA-352 had reduced colitis compared to tofacitinib treated animals, but since the vehicle group had such mild colitis there was no noticeable change compared to vehicle.
  • Figures 94-97 depicts the effect of oral and intracecal administration of SNA-125, SNA-352, tofacitinib, and prednisolone on the levels of IFNY, IL-10, IL-6 and TNFa in colon homogenates.
  • Oral and intracecal SNA-352 administration decreased IFNY.
  • Intracecal SNA-125 significantly increased TNFa as compared to the intracecal vehicle control (with the intracecal control outlier removed).
  • Oral tofacitinib significantly increased IL-10 levels, and a strong trend was also observed for the intracecal Tofacitinib group.
  • Intracecal treatment was less effective. Treatment with intracecal SNA-352 may have mildly reduced colitis compared to tofacitinib treatment and SNA- 125; however any change was mild and not statistically significant.
  • Table 34 depicts the test groups and Figure 98 depicts the timing of the experiments performed in this study.
  • Psoriasis reactions were scored based on the parameters shown in Table 35 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 99, 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.
  • Cytokine analysis [0707] Left ears were biopunched on day 4 and after tissue homogenization, the levels of cytokines IL-17F, TNF-a, IL-22, and IL-17A in the tissue lysates were measured via multiplex and then normalized with total protein amounts. Mean values for each group are displayed in Figure 104.
  • the objective of this study was to determine the efficacy of SNA- 120 and SNA-325 as a therapeutic in the mouse model of IL-23-induced psoriasis.
  • the IL- 23/Th17 pathway has been shown to play a major role in psoriasis, and injection of IL-23 into mice produces clinical features associated with psoriasis such as hyperproliferation of keratinocytes and thickened epidermis with infiltration of mononuclear cells.
  • an IL-23 mouse model simulates human AD (i.e. 37% homology with human AD transcriptome).
  • tofacitinib administration has found to reduce ear swelling and inflammatory infiltrates in mouse skin in a dose-dependent manner.
  • Table 36 depicts the test groups and Figure 105 depicts a schematic of the timing of the experiments performed.
  • Figure 106A depicts the total psoriasis clinical scores for each group over time.
  • Figure 106B depicts right ear thickness for each group (measured with a caliper 3 times weekly) while Figure 106C depicts changes in body weight throughout the study.
  • SNA- 120, SNA- 125, SNA-352 will be tested in the AEW model. AEW or 0.9% saline was administered topically BID. Additionally, SNA-120, SNA-125, SNA-352, or vehicle will be administered topically BID. Scratching measurements will be performed 14 hour after the last AEW treatment each day for one hour. Non-specific clinical signs and treatment site assessments will be performed from Day 1 to Day 6. All animals will be weighted on Day 1 and Day 6. On day 6 animals will be terminated and skin biopsies will be performed. Additionally, histology (H&E) and skin biopsies for NGF analysis were performed at study termination.
  • H&E histology
  • Parakeratosis is a mode of keratinization characterized by the retention of nuclei in the stratum corneum. In the skin, this process leads to the abnormal replacement of annular squames with nucleated cells. Parakeratosis is associated with the thinning or loss of the granular layer and is usually seen in diseases of increased cell turnover, whether inflammatory or neoplastic. Parakeratosis also is seen in the plaques of psoriasis and in dandruff. It is predicted that in young mice treated with saline, a normal epidermis with keratinocytes organized as a single line will be observed.
  • mice treated with saline normal epidermis with keratinocytes frequently organized as a single line and a few signs of parakeratosis will be observed.
  • histology assessments will show normal epidermis with some increase in stratification.
  • histology assessments will demonstrate normal epidermis with increase in stratification and signs of parakeratosis.
  • SNA- 120, SNA- 125, and SNA-352 administration will reverse AEW-induced stratification and parakeratosis in a dose-dependent manner.

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Abstract

L'invention concerne des utilisations thérapeutiques de conjugués polymères, comportant les SNA-120 et SNA-125, comprenant un agent actif lié à un polymère, l'agent actif comprenant un composé indolocarbazole ou un dérivé de celui-ci. Les conjugués polymères de l'invention réduisent l'exposition de l'agent actif au niveau de sites non ciblés.
PCT/US2018/023102 2017-05-10 2018-03-19 Utilisations de conjugués polymères de composés indolocarbazole à exposition réduite WO2018208369A1 (fr)

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