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WO2025106953A1 - Administration prolongée d'inhibiteurs de jak contenant du nitrile à partir d'un hydrogel par l'intermédiaire d'adduits de thioimidate réversibles - Google Patents

Administration prolongée d'inhibiteurs de jak contenant du nitrile à partir d'un hydrogel par l'intermédiaire d'adduits de thioimidate réversibles Download PDF

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WO2025106953A1
WO2025106953A1 PCT/US2024/056342 US2024056342W WO2025106953A1 WO 2025106953 A1 WO2025106953 A1 WO 2025106953A1 US 2024056342 W US2024056342 W US 2024056342W WO 2025106953 A1 WO2025106953 A1 WO 2025106953A1
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thiolated
alopecia
hydrogel
jak inhibitor
inflammatory
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Leo Wang
George Cotsarelis
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia

Definitions

  • the invention relates to sustained delivery JAK inhibitor hydrogels comprising JAK inhibitors having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer, such as hyaluronic acid, to form a thioimidate adduct for the treatment of an inflammatory disorder such as inflammatory skin disorders and autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis or reactive arthritis, as well as autoimmune and inflammatory hair loss, such as alopecia areata, and/or non-inflammatory disorder hair loss, such as androgenetic alopecia.
  • an inflammatory disorder such as inflammatory skin disorders and autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis or reactive arthritis
  • autoimmune and inflammatory hair loss such as alopecia areata
  • non-inflammatory disorder hair loss such as androgenetic alopecia.
  • the invention also relates to methods for treating inflammatory disorders, including skin disorders such as atopic dermatitis, alopecia areata, central centrifugal cicatricial alopecia, lichen planopilaris; hidradenitis suppurativa, lichen planus, mucosal lichen planus, psoriasis, pemphigus or vitiligo, and other autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis or reactive arthritis by administering to a subject in need thereof a JAK inhibitor having a nitrile group reversibly conjugated to a thiol group of a thiolated hyaluronic acid to form a thioimidate adduct.
  • skin disorders such as atopic dermatitis, alopecia areata, central centrifugal cicatricial alopecia, lichen planopilaris; hidradenitis suppurativa,
  • the invention further relates to methods for treating such inflammatory disorders and autoimmune diseases by administering to a subject in need thereof a composition comprising an injectable nitrile-containing JAK inhibitor, for example, baricitinib or any other nitrile-containing JAK inhibitors, as described infra in the absence of a thiolated polymer that forms a hydrogel, wherein the JAK inhibitor crystallizes or precipitates out of the composition and sustains its own release.
  • an injectable nitrile-containing JAK inhibitor for example, baricitinib or any other nitrile-containing JAK inhibitors
  • JAK Janus kinase
  • STAT pathway The Janus kinase and signal transducer and activator of transcription JAK (Janus kinase)/STAT pathway has emerged as a disease target for many inflammatory disorders in the skin.
  • JAK-STAT signaling e.g., ruxolitinib, abrocitinib, upadacitinib), vitiligo (ruxolitinib), alopecia areata (e.g., ritlecitinib, baricitinib), psoriasis/psoriatic arthritis (e.g., deucravacitinib, tofacitinib), while many others are currently under investigation for other dermatologic diseases.
  • atopic dermatitis e.g., ruxolitinib, abrocitinib, upadacitinib), vitiligo (ruxolitinib), alope
  • biomaterial-based drug delivery systems can be utilized to retain payloads at a particular target tissue of interest, maximizing bioavailability while minimizing systemic absorption and off- target toxicities.
  • hydrogels are desirable as they are often biocompatible due to their high-water content and they can be engineered to be injectable for minimally invasive delivery.
  • hydrogel systems have been attempted for drug delivery, their use for local and sustained drug release in dermatology remains difficult.
  • small molecules such as JAK inhibitors are challenging to deliver from hydrogels due to their small size and rapid diffusion.
  • Previous research delivering JAK inhibitors from hydrogels showed release over days but were unable to achieve longer-term release profiles.
  • a notable and similar dermatologic example showed metalloprotease-triggered delivery of tofacitinib for atopic dermatitis in vitro from a polyethylene glycol hydrogel but with release of -40% at 48 hours.
  • the need for repeated administrations either as a topical or injectable limits compliance and is a major barrier to use and clinical translation.
  • sustained delivery hydrogels for treatment of a disorder or a disease comprising a small molecule drug having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer, such as hyaluronic acid, to form a thioimidate adduct.
  • injectable pharmaceuticals compositions for treatment of a disorder or a disease comprising the foregoing sustained delivery hydrogels and a pharmaceutically acceptable carrier.
  • methods for treating a disorder or a disease in a subject in need thereof comprising administering the foregoing sustained delivery hydrogels to the subject.
  • sustained delivery JAK inhibitor hydrogels for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss
  • the JAK inhibitor hydrogel comprising a JAK inhibitor having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct.
  • sustained delivery baricitinib hydrogels comprising baricitinib having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss.
  • the invention provides injectable pharmaceutical compositions for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss, the injectable pharmaceutical composition comprising a nitrile-containing JAK inhibitor, e.g., baricitinib, and a pharmaceutically acceptable carrier.
  • the invention provides sustained delivery pharmaceutical compositions for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss, the sustained delivery pharmaceutical composition comprising a nitrile-containing JAK inhibitor, e.g., baricitinib, and a pharmaceutically acceptable carrier.
  • the sustained delivery pharmaceutical composition is an injectable pharmaceutical composition.
  • the invention provides injectable pharmaceutical compositions for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss, the injectable pharmaceutical composition comprising a low concentration of thiolated hydrogel, a nitrile-containing JAK inhibitor and a pharmaceutically acceptable carrier.
  • the invention provides methods for treating an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss in a subject in need thereof, the method comprising administering a sustained delivery nitrile-containing JAK inhibitor hydrogel to a body part of the subject affected by the inflammatory disorder, the autoimmune disease, the autoimmune and inflammatory hair loss, and/or the non-inflammatory disorder hair loss, wherein the JAK inhibitor hydrogel comprises a JAK inhibitor having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer by a thioimidatc adduct.
  • the invention provides methods for treating an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a noninflammatory disorder hair loss in a subject in need thereof, the method comprising administering a sustained delivery baricitinib hydrogel to a body part of the subject affected by the inflammatory disorder, autoimmune disease, the autoimmune and inflammatory hair loss, and/or the noninflammatory disorder hair loss, wherein the sustained delivery baricitinib hydrogel comprises baricitinib reversibly conjugated at a nitrile group to a thiol group of a thiolated polymer by a thioimidate adduct.
  • the invention provides methods for treating an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss in a subject in need thereof, the method comprising administering an injectable pharmaceutical composition to a body part of the subject affected by the inflammatory disorder, the autoimmune disease, the autoimmune and inflammatory hair loss, and/or the non-inflammatory disorder hair loss, wherein the injectable pharmaceutical composition comprises a nitrile- containing JAK inhibitor, e.g., baricitinib, and a pharmaceutically acceptable carrier.
  • the injectable pharmaceutical composition is administered with hollow microneedles that allow delivery of the injectable pharmaceutical composition comprising the therapeutic agent (the JAK inhibitor) in a upon application of pressure.
  • the invention provides methods for treating an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss in a subject in need thereof, the method comprising administering s sustained delivery pharmaceutical composition to a body part of the subject affected by the inflammatory disorder, the autoimmune disease, the autoimmune and inflammatory hair loss, and/or the noninflammatory disorder hair loss, wherein the sustained delivery pharmaceutical composition comprises a nitrile-containing JAK inhibitor, e.g., baricitinib, and a pharmaceutically acceptable carrier.
  • the sustained delivery pharmaceutical composition is an injectable pharmaceutical composition.
  • the invention provides methods for producing a sustained delivery JAK inhibitor hydrogel for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss in a subject in need thereof, wherein the JAK inhibitor hydrogel comprises a nitrilc-containing JAK inhibitor and the nitrile group is reversibly conjugated to a thiol group of a thiolated polymer to form thioimidate adduct, the method comprising: (a) mixing a thiolated polymer with the nitrile- containing JAK inhibitor having a nitrile group to a desired weight percent concentration (w/v) of from about 0.5 to 10 wt.% for 24 hours to form a crosslinked JAK inhibitor hydrogel, wherein the thiolated polymer and the JAK inhibitor are each dissolved in a respective solvent; (b) centrifuging the crosslinked JAK inhibitor hydrogel; and (c) washing the centrifuged
  • Figures 1A-1B show baricitinib and cysteamine thioimidate formation.
  • the reaction forms through a modified Pinner reaction in which a free, nucleophilic thiol attacks the electrophilic nitrile on baricitinib.
  • IB shows liquid chromatography-mass spectrometry demonstrates product formation between baricitinib (373 Da), cysteamine (77 Da), andbaricitinib-cysteamine (449 Da), resolving with three distinct retention time peaks (0.3 s, 1.5 s, 1.4 s, respectively).
  • Figures 2A-2D show polymer design, thioimidate bonding, and hydrogel disulfide crosslinking.
  • Fig. 2A shows hyaluronic acid (100 kDa) is amidated at its carboxyl position with cysteamine to yield a thiolated product where x is the fraction of disaccharides with thiol modification and 1-x is the fraction of unmodified disaccharides.
  • Fig. 2A shows hyaluronic acid (100 kDa) is amidated at its carboxyl position with cysteamine to yield a thiolated product where x is the fraction of disaccharides with thiol modification and 1-x is the fraction of unmodified disaccharides.
  • unmodified thiols oxidize and form disulfide crosslinks between polymer chains under physiologic conditions to yield a dynamically crosslinked hydrogel.
  • the degree of unbound thiols which form disulfides is represented by x-y, and unmodified disaccharides are represented as 1-x. Figs.
  • 2C-2E show the structures of three iterations of disaccharide modifications that form during thioimidate bonding between baricitinib and hyaluronic acid, namely the thioimidate, free thiol, and disulfide.
  • Figures 3A-3B show quantification of hyaluronic acid thiolation and consumption of thiols by baricitinib.
  • Figures 4A-4B show that 13 C and NMR validate thioimidate formation between baricitinib and thiolated hyaluronic acid.
  • Figures 5A-5C show hydrogel formation and shear oscillatory rheometry.
  • Fig. 5A shows hydrogels are formed between thiolated hyaluronic acid and baricitinib, where thioimidate adducts and disulfide crosslinks form simultaneously. Upon material deposition, thioimidate bonds reverse to release baricitinib into the surrounding environment.
  • Fig. 5B shows shear oscillatory rheometry over 24 hours demonstrating crosslinking of polymers into hydrogels (-100 Pa) with and without baricitinib (2 mg/mL) which occur over the course of 24 hours. Frequency sweeps demonstrate classic viscoelastic behavior of materials.
  • Figures 6A-6C show baricitinib release and ultraviolet spectrometry.
  • Fig. 6A shows quantification of baricitinib aromatic absorption in ultraviolet A and B range (280-400 nm) demonstrating highest absorption in the ultraviolet B range. Subsequent plotting of absorbance against dose demonstrates linear absorption at 300 nm.
  • Fig. 6C shows release curves for hydrogels assembled at 0.2 mg/mL or 2 mg/mL baricitinib loaded (2 wt% hydrogel, 30% thiol modification).
  • Figures 7A-7E show validation of baricitinib activity on JAK/STAT signaling using a HEK293 luciferase reporter.
  • Fig. 7A shows HEK293 cell line with the firefly luciferase gene under the control of Interferon Stimulated Response Element (ISRE) where type I interferon-induced JAK/STAT signaling pathway in the target cells can be monitored by measuring luciferase activity.
  • ISRE Interferon Stimulated Response Element
  • Fig. 7C shows a schematic of experimental design demonstrating hydrogel incubation in saline with collection and replacement of releasate performed at regular intervals and then added directly to cells.
  • Fig. 7D shows incubation of cells with 5 pL releasates demonstrates sustained JAK/STAT inhibition but only from the 2 mg/mL formulation. *p ⁇ 0.05 between 0.2 mg/mL and 2 mg/mL.
  • Figures 8A-8D show in vivo injections of baricitinib hydrogel in an imiquimod model of psoriasiform dermatitis.
  • Fig. 8B shows images of skin changes at five days and seven days. Fig.
  • Fig. 8D shows quantification of epidermal thickness at seven days by quantifying epidermal ROI divided by length. *p ⁇ 0.05 by one-way ANOVA.
  • Figure 9 shows LCMS between sulfur-containing small molecules and baricitinib.
  • L- cysteine (122 Da), a thiol forms a product with baricitinib (372 Da) at 494 Da, indicating formation of a thioimidate.
  • Levamisole (205 Da), a thiazole does not form a product with baricitinib.
  • Figure 10 shows 1 H NMR with shifted aromatic peaks on baricitinib. Individual aromatic protons are highlighted. Shifted protons (yellow, green, red, orange) are highlighted relative to thioimidate (purple). One proton (blue) is unshifted.
  • Figure 11 shows hydrogel formation and shear oscillatory rheometry frequency sweeps. Frequency sweeps (0.5% strain) between hydrogels and baricitinib hydrogels demonstrate frequency dependent properties to suggest viscoelastic behavior.
  • Figure 12 shows ROI selection in FIJI accurately highlights the epidermis for quantification of total thickness.
  • Figure 13 shows representative H&E images of skin sections demonstrating changes in epidermal thickness in response to baricitinib hydrogel treatments. Sections represent tissue examined directly over injection sites.
  • Fig. 14A shows absolute weight at five days.
  • Fig. 14B shows weight loss as percentage of untreated controls that did not receive imiquimod.
  • Figure 15 shows a representative section of hydrogel in subcutaneous tissue in imiquimod mouse model. Hydrogels in the dermis arc typically lost in processing but can be observed in few sections in the subcutaneous space, where they are stable one week after injection.
  • Figure 16 shows using 1 H NMR that an aromatic nitrile containing molecule, CHIR 99021 , and thiolated hyaluronic acid react in a reversible manner to form a thioimidate.
  • the 1 H NMR data shows the formation of a new set of peaks in the aromatic region downfield from those in CHIR99021 alone.
  • Thiolated hyaluronic acid alone shows no peaks in this region.
  • Nitriles react with thiols to form thioimidate adducts, which possess reversible and dynamic S-C covalent bonds that form through a Pinner-like mechanism between a thiol nucleophile and an electrophilic carbon of a nitrile group.
  • the thioimidate can be further stabilized through other mechanisms.
  • a hydrogel comprising a small molecule drug having an electrophilic aliphatic nitrile to reversibly conjugate a nucleophilic thiol of a thiolated polymer as a thioimidate adduct was sought to achieve a pharmacologic goal of a slow drug release over a period of about 12 weeks to treat various dermatologic indications.
  • Previously formulated hydrogels comprising small molecules demonstrated a rapid release of the small molecules from the hydrogels during four to six weeks.
  • a 12- week release period would enable a patient to make quarterly visits for routine intradermal or subcutaneous injections of the small molecule-containing thiolated hydrogels under already well-established and well-accepted protocols that are used, for example, for hydrogel dermal filler treatments, and avoid systemic exposure to the small molecule.
  • An aliphatic nitrile that is present on numerous clinically approved JAK inhibitors was identified as a potential reactive electrophile that can be leveraged to reversibly and covalently bind the drug, i.e., the JAK inhibitor, within a thiolated hydrogel for controlled and tunable release for local delivery, e.g., in the skin or a joint.
  • thiols on a thiolated polymer such as hyaluronic acid
  • a thiolated polymer such as hyaluronic acid
  • a nitrile-containing JAK inhibitor such as baricitinib
  • baricitinib which is a representative JAK1/2 inhibitor
  • disulfide crosslinks to form a hydrogel.
  • Hyaluronic acid which is a naturally occurring glycosaminoglycan that is biocompatible, widely investigated for drug delivery applications, and already used in dermal injectables clinically, was selected.
  • any thiol-containing polymer may be used according to the present invention.
  • the invention provides sustained delivery JAK inhibitor hydrogels for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss, the JAK inhibitor hydrogel comprising a JAK inhibitor having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct.
  • the thiolated polymer is a thiolated hyaluronic acid, a thiolated chitosan, a thiolated cyclodextrin, a thiolated poly(ethylene glycol), a thiolated polyvinyl alcohol, a thiolated polyacrylamide, a thiolated polyethylene oxide, a thiolated polymethacrylate, a thiolated dextran, a thiolated alginate, a thiolated cellulose, a thiolated gelatin, a thiolated collagen, a thiolated silk, a thiolated keratin or a thiolated elastin.
  • the thiolated polymer is a thiolated hyaluronic acid.
  • the thiolated hyaluronic acid has an average molecular weight of from 1 KD to 1,000,000 KD when the hyaluronic acid is polydispersed in solution.
  • the sustained delivery JAK inhibitor hydrogel the thiolated hyaluronic acid has an average molecular weight of from 10,000 KD to 1,000,000 KD when the hyaluronic acid is polydispersed in solution.
  • the thiolated hyaluronic acid has an average molecular weight of 100,000 KD when the hyaluronic acid is polydispersed in solution.
  • the nitrile-containing JAK inhibitor is baricitinib, delgocitinib, ruxolitinib, deuruxolitinib, tofacitinib, momelotinib, gusacitinib, itacitinib, izencitinib, lorpucitinib, povorcitinib or a nitrile-containing metabolite thereof.
  • the nitrile-containing JAK inhibitor is a compound having chemical structure (A):
  • the nitrile-containing JAK inhibitor is a compound having structure (I) or (II):
  • the sustained delivery JAK inhibitor hydrogel is formulated for local administration.
  • the local administration comprises transdermal administration, intradermal injection, intramuscular injection, subcutaneous injection or intra-articular injection.
  • the local administration comprises transdermal administration.
  • the transdermal administration comprises transdermal delivery via microneedles coated with the JAK inhibitor, a solid polymer matrix having the JAK inhibitor incorporated therein, dissolving polymer microneedles having the JAK inhibitor incorporated therein, hydrogel-forming microneedles comprising the JAK inhibitor in the hydrogel, or a transdermal patch.
  • the inflammatory disorder is an inflammatory skin disorder.
  • the inflammatory skin disorder is atopic dermatitis, cicatricial alopecia, central centrifugal cicatricial alopecia, hidradcnitis suppurativa, lichen planopilaris, frontal fibrosing alopecia, alopecia areata, lichen planus, mucosal lichen planus, psoriasis, pemphigus or vitiligo.
  • the autoimmune disease is alopecia areata, persistent patchy alopecia areata, alopecia totalis, alopecia universalis, ophiasis alopecia, alopecia barbae, rheumatoid arthritis, psoriatic arthritis or reactive arthritis.
  • the autoimmune and inflammatory hair loss is alopecia areata.
  • the hair loss is a non-inflammatory disorder hair loss, wherein the non-inHammatory disorder hair loss is male or female androgenetic alopecia (male pattern baldness or female pattern baldness) or telogen effluvium.
  • the invention provides sustained delivery baricitinib hydrogels comprising baricitinib having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss.
  • the sustained delivery baricitinib hydrogel is formulated for local administration, wherein the local administration comprises intradermal injection, intramuscular injection, subcutaneous injection or intra-articular injection. In an embodiment, the local administration comprises transdermal administration. In various embodiments, the inflammatory disorder is an inflammatory skin disorder.
  • the autoimmune disease is alopecia areata, persistent patchy alopecia areata, alopecia totalis, alopecia universalis, ophiasis alopecia, alopecia barbae, rheumatoid arthritis, psoriatic arthritis or reactive arthritis.
  • the autoimmune and inflammatory hair loss is alopecia areata.
  • the hair loss is a non-inflammatory disorder hair loss, wherein the noninflammatory disorder hair loss is male or female androgenetic alopecia (male pattern baldness or female pattern baldness) or telogen effluvium.
  • the sustained delivery baricitinib hydrogel comprises chemical substructures (a) together with (b) and (c):
  • the invention provides injectable pharmaceutical compositions for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss, the injectable pharmaceutical composition comprising a nitrile-containing JAK inhibitor and a pharmaceutically acceptable carrier.
  • the transdermal administration comprises transdermal delivery via microneedles coated with the JAK inhibitor, a solid polymer matrix having the JAK inhibitor incorporated therein, dissolving polymer microneedles having the JAK inhibitor incorporated therein, hydrogel-forming microneedles comprising the JAK inhibitor in the hydrogel, or a transdermal patch.
  • the local administration comprises intradermal injection, intramuscular injection, subcutaneous injection, or intra-articular injection.
  • the local administration comprises transdermal administration.
  • the inflammatory disorder is an inflammatory skin disorder.
  • the inflammatory skin disorder is atopic dermatitis, cicatricial alopecia, central centrifugal cicatricial alopecia, hidradenitis suppurativa, lichen planopilaris, frontal fibrosing alopecia, alopecia areata, lichen planus, mucosal lichen planus, psoriasis, pemphigus or vitiligo.
  • the autoimmune disease is alopecia areata, persistent patchy alopecia areata, alopecia totalis, alopecia universalis, ophiasis alopecia, alopecia barbae, rheumatoid arthritis, psoriatic arthritis or reactive arthritis.
  • the autoimmune and inflammatory hair loss is alopecia areata.
  • the hair loss is a noninflammatory disorder hair loss, wherein the non-inflammatory disorder hair loss is male or female androgenetic alopecia (male pattern baldness or female pattern baldness) or telogen effluvium.
  • the invention provides methods for treating an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss in a subject in need thereof, the method comprising administering a sustained delivery nitrile-containing JAK inhibitor hydrogel to a body part of the subject affected by the inflammatory disorder, the autoimmune disease, the autoimmune and inflammatory hair loss, and/or the non-inflammatory disorder hair loss, wherein the JAK inhibitor hydrogel comprises a JAK inhibitor having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct.
  • the thiolated polymer is a thiolated hyaluronic acid, a thiolated chitosan, a thiolated cyclodextrin, a thiolated poly(ethylene glycol), a thiolated polyvinyl alcohol, a thiolated polyacrylamide, a thiolated polyethylene oxide, a thiolated polymethacrylate, a thiolated dextran, a thiolated alginate, a thiolated cellulose, a thiolated gelatin, a thiolated collagen, a thiolated silk, a thiolated keratin or a thiolated elastin.
  • the thiolated polymer is a thiolated hyaluronic acid.
  • the thiolated hyaluronic acid has an average molecular weight of from 1 KD to 1,000,000 KD when the hyaluronic acid is polydispersed in solution.
  • the thiolated hyaluronic acid has an average molecular weight of from 10,000 KD to 1,000,000 KD when the hyaluronic acid is polydispersed in solution.
  • the thiolated hyaluronic acid has an average molecular weight of 100,000 KD when the hyaluronic acid is polydispersed in solution.
  • the nitrile- containing JAK inhibitor hydrogel is formulated for local administration.
  • the local administration comprises intradermal injection, intramuscular injection, subcutaneous injection or intra-articular injection.
  • the local administration comprises transdermal administration.
  • the transdermal administration comprises transdermal delivery via microneedles coated with the JAK inhibitor, a solid polymer matrix having the JAK inhibitor incorporated therein, dissolving polymer microneedles having the JAK inhibitor incorporated therein, hydrogel -forming microneedles comprising the JAK inhibitor in the hydrogel, or a transdcrmal patch.
  • the local administration comprises transdermal injection, intradermal injection intramuscular injection, or intra-articular injection.
  • the inflammatory disorder is an inflammatory skin disorder.
  • the inflammatory skin disorder is atopic dermatitis, cicatricial alopecia, central centrifugal cicatricial alopecia, hidradenitis suppurativa, lichen planopilaris, frontal fibrosing alopecia, alopecia areata, lichen planus, mucosal lichen planus, psoriasis, pemphigus or vitiligo.
  • the autoimmune disease is alopecia areata, persistent patchy alopecia areata, alopecia totalis, alopecia universalis, ophiasis alopecia, alopecia barbae, rheumatoid arthritis, psoriatic arthritis or reactive arthritis.
  • the autoimmune and inflammatory hair loss is alopecia areata.
  • the hair loss is a non-inflammatory disorder hair loss, wherein the non-inflammatory disorder hair loss is male or female androgenetic alopecia (male pattern baldness or female pattern baldness) or telogen effluvium.
  • the nitrile-containing JAK inhibitor is baricitinib, delgocitinib, ruxolitinib, deuruxolitinib, tofacitinib, momelotinib, gusacitinib, itacitinib, izencitinib, lorpucitinib, povorcitinib or a nitrile-containing metabolite thereof.
  • the nitrile-containing JAK inhibitor is a compound having chemical structure (A): [0046]
  • the nitrile-containing JAK inhibitor is a compound having structure (I) or (II):
  • the invention provides methods for treating an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a noninflammatory disorder hair loss in a subject in need thereof, the method comprising administering a sustained delivery baricitinib hydrogel to a body part of the subject affected by the inflammatory disorder, autoimmune disease, the autoimmune and inflammatory hair loss, and/or the noninflammatory disorder hair loss, wherein the sustained delivery baricitinib hydrogel comprises baricitinib reversibly conjugated at a nitrile group to a thiol group of a thiolated polymer to form a thioimidate adduct.
  • the thiolated polymer is a thiolated hyaluronic acid, a thiolated chitosan, a thiolated cyclodextrin, a thiolated poly(ethylene glycol), a thiolated polyvinyl alcohol, a thiolated polyacrylamide, a thiolated polyethylene oxide, a thiolated polymcthacrylatc, a thiolated dextran, a thiolated alginate, a thiolated cellulose, a thiolated gelatin, a thiolated collagen, a thiolated silk, a thiolated keratin or a thiolated clastin.
  • the thiolated polymer is a thiolated hyaluronic acid.
  • the thiolated hyaluronic acid has an average molecular weight of from 1 KD to 1 ,000,000 KD when the hyaluronic acid is polydispersed in solution.
  • the thiolated hyaluronic acid has an average molecular weight of from 10,000 KD to 1,000,000 KD when the hyaluronic acid is polydispersed in solution.
  • the thiolated hyaluronic acid has an average molecular weight of 100,000 KD when the hyaluronic acid is polydispersed in solution.
  • the sustained delivery baricitinib hydrogel is formulated for local administration, wherein the local administration comprises intradermal injection, intramuscular injection, subcutaneous injection or intra- articular injection. In an embodiment, the local administration comprises transdermal administration. In an embodiment, the inflammatory disorder is an inflammatory skin disorder.
  • the inflammatory skin disorder is atopic dermatitis, cicatricial alopecia, central centrifugal cicatricial alopecia, hidradenitis suppurativa, lichen planopilaris, frontal fibrosing alopecia, alopecia areata, lichen planus, mucosal lichen planus, psoriasis, pemphigus or vitiligo.
  • the autoimmune disease is alopecia areata, persistent patchy alopecia areata, alopecia totalis, alopecia universalis, ophiasis alopecia, alopecia barbae, rheumatoid arthritis, psoriatic arthritis or reactive arthritis.
  • the autoimmune and inflammatory hair loss is alopecia areata.
  • the hair loss is a non-inflammatory disorder hair loss, wherein the non-inflammatory disorder hair loss is male or female androgenetic alopecia (male pattern baldness or female pattern baldness), or telogen effluvium.
  • the sustained delivery baricitinib hydrogel comprises chemical substructures (a), (b) and (c):
  • the invention provides methods for treating an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss in a subject in need thereof, the method comprising administering an injectable pharmaceutical composition to a body part of the subject affected by the inflammatory disorder, the autoimmune disease, the autoimmune and inflammatory hair loss, and/or the non-inflammatory disorder hair loss, wherein the injectable pharmaceutical composition comprises a nitrile- containing JAK inhibitor and a pharmaceutically acceptable carrier.
  • the nitrile-containing JAK inhibitor JAK inhibitor is baricitinib, delgocitinib, ruxolitinib, deuruxolitinib, tofacitinib, momelotinib, gusacitinib, itacitinib, izencitinib, lorpucitinib, povorcitinib or a nitrile-containing metabolite thereof.
  • the nitrile- containing JAK inhibitor JAK inhibitor is a compound having chemical structure (A):
  • the nitrile-containing JAK inhibitor is a compound having structure (I) or (II):
  • the injectable pharmaceutical composition is formulated for local administration.
  • the local administration comprises intradermal injection, intramuscular injection, subcutaneous injection or intra-articular injection.
  • the local administration comprises transdermal administration.
  • the injectable pharmaceutical composition is administered with hollow microneedles that allow delivery of a therapeutic agent (the JAK inhibitor) upon application of pressure.
  • the transdermal administration comprises transdermal delivery via microneedles coated with the JAK inhibitor, a solid polymer matrix having the JAK inhibitor incorporated therein, dissolving polymer microneedles having the JAK inhibitor incorporated therein, hydrogel-forming microneedles comprising the JAK inhibitor in the hydrogel, or a transdermal patch.
  • the local administration comprises intradermal injection, intramuscular injection, or intra-articular injection.
  • the inflammatory disorder is an inflammatory skin disorder.
  • the local administration comprises transdermal injection.
  • the inflammatory skin disorder is atopic dermatitis, cicatricial alopecia, central centrifugal cicatricial alopecia, hidradenitis suppurativa, lichen planopilaris, frontal fibrosing alopecia, alopecia areata, lichen planus, mucosal lichen planus, psoriasis, pemphigus or vitiligo.
  • the autoimmune disease is alopecia areata, persistent patchy alopecia areata, alopecia totalis, alopecia universalis, ophiasis alopecia, alopecia barbae, rheumatoid arthritis, psoriatic arthritis or reactive arthritis.
  • the autoimmune and inflammatory hair loss is alopecia areata.
  • the hair loss is a non-inflammatory disorder hair loss, wherein the non-inflammatory disorder hair loss is male or female androgenetic alopecia (male pattern baldness or female pattern baldness) or telogen effluvium.
  • the invention provides methods for producing a sustained delivery JAK inhibitor hydrogel for treatment of an inflammatory disorder, an autoimmune disease, an autoimmune and inflammatory hair loss, and/or a non-inflammatory disorder hair loss in a subject in need thereof, wherein the JAK inhibitor hydrogel comprises a nitrile-containing JAK inhibitor and the nitrile group is reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct, the method comprising: (a) mixing a thiolated polymer with the nitrile- containing JAK inhibitor having a nitrile group to a desired weight percent concentration (w/v) of from 0.5 to 10 wt.% for 24 hours to form a crosslinked JAK inhibitor hydrogel, wherein the thiolated polymer and the JAK inhibitor are each dissolved in a respective solvent; (b) centrifuging the crosslinked JAK inhibitor hydrogel; and (c) washing the centrifuged cross
  • the thiolated polymer is a thiolated hyaluronic acid, a thiolated chitosan, a thiolated cyclodextrin, a thiolated poly(ethylene glycol), a thiolated polyvinyl alcohol, a thiolated polyacrylamide, a thiolated polyethylene oxide, a thiolated polymethacrylate, a thiolated dextran, a thiolated alginate, a thiolated cellulose, a thiolated gelatin, a thiolated collagen, a thiolated silk, a thiolated keratin or a thiolated elastin.
  • the thiolated polymer is a thiolated hyaluronic acid.
  • the thiolated hyaluronic acid has an average molecular weight of from 1 KD to 1,000,000 KD when polydispersed in solution.
  • the thiolated hyaluronic acid has an average molecular weight of from 10,000 KD to 1 ,000,000 KD when polydispersed in solution.
  • the thiolatcd hyaluronic acid has an average molecular weight of 100,000 KD when polydispersed in solution.
  • the nitrile-containing JAK inhibitor is baricitinib, delgocitinib, ruxolitinib, deuruxolitinib, tofacitinib, momelotinib, gusacitinib, itacitinib, izencitinib, lorpucitinib, povorcitinib or a nitrile-containing metabolite thereof.
  • the nitrile-containing JAK inhibitor is a compound having chemical structure (A):
  • the method further comprises formulating the sustained delivery JAK inhibitor hydrogel for local administration.
  • the local administration comprises intradermal injection, intramuscular injection, subcutaneous injection or intra-articular injection.
  • the local administration comprises transdermal administration.
  • the transdermal administration comprises transdermal delivery via microneedles coated with the JAK inhibitor, a solid polymer matrix having the JAK inhibitor incorporated therein, dissolving polymer microneedles having the JAK inhibitor incorporated therein, hydrogel-forming microneedles comprising the JAK inhibitor in the hydrogel, or a transdermal patch.
  • the local administration comprises intradermal injection, intramuscular injection, or intra-articular injection.
  • the local administration comprises transdermal administration.
  • the inflammatory disorder is an inflammatory skin disorder.
  • the inflammatory skin disorder is atopic dermatitis, cicatricial alopecia, central centrifugal cicatricial alopecia, hidradenitis suppurativa, lichen planopilaris, frontal fibrosing alopecia, alopecia areata, lichen planus, mucosal lichen planus, psoriasis, pemphigus or vitiligo.
  • the autoimmune disease is alopecia areata, persistent patchy alopecia areata, alopecia totalis, alopecia universalis, ophiasis alopecia, alopecia barbae, rheumatoid arthritis, psoriatic arthritis or reactive arthritis.
  • the autoimmune and inflammatory hair loss is alopecia areata.
  • the hair loss is a non-inflammatory disorder hair loss, wherein the non-inflammatory disorder hair loss is male or female androgenetic alopecia (male pattern baldness or female pattern baldness) or telogen effluvium.
  • the JAK inhibitors according to embodiments of the present invention may be any JAK inhibitors having a nitrile group that is capable of being reversibly conjugated to a thiol group of any thiolated polymer to form a thioimidate adduct, thereby forming an administrable JAK inhibitor hydrogel or the nitrile-containing JAK inhibitor may be administered as an injectable pharmaceutical composition in the absence of conjugation to a thiolated polymer.
  • Nitrile- containing JAK inhibitors that are capable of being reversibly conjugated to a thiol group of a thiolated hyaluronic acid to form a thioimidate adduct that are amenable for sustained delivery in hydrogel according to embodiments of the invention include, but are not limited to, baricitinib, delgocitinib, ruxolitinib, deuruxolitinib, tofacitinib, momelotinib, gusacitinib, itacitinib, izencitinib, lorpucitinib, povorcitinib and nitrile-containing metabolites or derivatives thereof.
  • Baricitinib has the following chemical structure:
  • a nitrile-containing metabolite compound of baricitinib has the following structures, M3, M12, M10 or M22, as shown in Shawky A.M., et al. Pharmaceutics, 2022, 14(5): 1001, which is incorporated herein in its entirety:
  • nitrile- and nitrile oxide-containing metabolite compounds of baricitinib reversibly may be conjugated to a thiol group of a thiolated polymer, such as hyaluronic acid or other thiolated polymers, as described herein, to form a thioimidatc adduct or thioimidatc oxide adduct for the treatment of the diseases and disorders described herein.
  • Delgocitinib has the following chemical structure:
  • a nitrile-containing metabolite compound of delgocitinib has the following structures, Ml, M2 and M3, as shown in Shawky A.M., et al. Pharmaceutics, 2022, 14(5): 1001, which is incorporated herein in its entirety: [0060] These nitrile-containing metabolite compounds of delgocitinib may be reversibly conjugated to a thiol group of a thiolatcd polymer, such as hyaluronic acid or other thiolatcd polymers, as described herein, to form a thioimidate adduct for the treatment of the diseases and disorders described herein.
  • a thiolatcd polymer such as hyaluronic acid or other thiolatcd polymers
  • Ruxolitinib has the following chemical structure:
  • a nitrile-containing metabolite compound of ruxolitinib has the following structures, M49, M 18/M31 , M43/M45, M7/M8/M16/M27, M44/M37/M38 and M28/M51, M44, M9/M11, and M37/M38, as shown in Shawky A.M., et al. Pharmaceutics, 2022, 14(5): 1001, which is incorporated herein in its entirety:
  • nitrile-containing metabolite compounds of ruxolitinib may be reversibly conjugated to a thiol group of a thiolated polymer, such as hyaluronic acid or other thiolated polymers, as described herein, to form a thioimidate adduct for the treatment of the diseases and disorders described herein.
  • a thiolated polymer such as hyaluronic acid or other thiolated polymers
  • the nitrile-containing JAK inhibitor is deuruxolitinib (CAS 1513883- 39-0) having the following chemical structure:
  • Tofacitinib has the following chemical structure:
  • nitrile-containing metabolite compounds of tofacitinib may be reversibly conjugated to a thiol group of a thiolatcd polymer, such as hyaluronic acid or other thiolatcd polymers, as described herein, to form a thioimidate adduct for the treatment of the diseases and disorders described herein.
  • a thiolatcd polymer such as hyaluronic acid or other thiolatcd polymers
  • Momelotinib has the following chemical structure:
  • Itacitinib has the following chemical structure:
  • Gusacitinib has the following chemical structure: as described by Shawky A.M., et al. Pharmaceutics, 2022, 14(5): 1001 , which is incorporated herein in its entirety. Shawky et al. also describe the following nitrile-containing JAK inhibitors, Compounds 1 (referred to as “compound A” herein) and 3 (referred to as “compound II” herein) , respectively:
  • the nitrile-containing JAK inhibitor is izencitinib, lorpucitinib or povorcitinib, whose structure is described by Zhang et al., European Journal of Medicinal Chemistry 261 (2023) 115848, which is incorporated herein in its entirety.
  • Izencitinib has the following chemical structure:
  • Lorpucitinib has the following chemical structure:
  • Povorcitinib has the following chemical structure:
  • the nitrile-containing JAK inhibitor is a compound having chemical structure (I) or (II):
  • hydrogel is well-known in the art and means a crosslinked polymer network in water. Hydrogels may be used to encapsulate and release a therapeutic agent for local and sustained delivery.
  • a “sustained delivery JAK inhibitor hydrogel” is a crosslinked polymer network comprising a JAK inhibitor having a nitrile group that is reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct.
  • Thiol-containing polymers that are capable of being reversibly conjugated to a nitrile group of a nitrile-containing JAK inhibitor to form a thioimidate adduct that are amenable for sustained delivery in hydrogel according to embodiments of the invention include, but are not limited to a thiolated hyaluronic acid, a thiolated chitosan, a thiolated cyclodextrin, a thiolated poly(ethylene glycol), a thiolated polyvinyl alcohol, a thiolated polyacrylamide, a thiolated polyethylene oxide, a thiolated polymethacrylate, a thiolated dextran, a thiolated alginate, a thiolated cellulose, a thiolated gelatin, a thiolated collagen, a thiolated silk, a thiolated keratin and a thiolated elastin, which crosslink via their
  • thiol group- containing polymers also form intra- and inter-chain disulfide binds which contribute to their gelling properties.
  • the ability to cross-link via disulfide binds within their own structure results in formation of stable three-dimensional hydrophilic networks.
  • thiolating agent is an organic compound that contains a thiol functional group and is used to conjugate the thiol group to a polymer to form a thiolated polymer.
  • Thiolating agents include but are not limited to cystine, cysteamine, and 3,3'-dithiodipropionic acid. Any of the aforementioned thiolating agents are capable of conjugating a thiol group to a polymer, e.g., to a hyaluronic acid, to thiolate the polymer.
  • the thiolated hydrogel is a viscous solution. Accordingly, in some embodiments, such low concentration-hydrogels may be delivered locally as an injectable composition comprising a JAK inhibitor for non-sustained release from the hydrogel.
  • a low concentration thiolated polymer hydrogel comprises about 0.5 wt% of a thiolated polymer.
  • high concentrations of the thiolated polymer form a composition that is neither a hydrogel nor a solid, rendering it unusable for local delivery.
  • a high concentration thiolated polymer hydrogel comprises about 20 wt% of a thiolated polymer.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviations, per practice in the art.
  • a measurable value such as an amount, e.g., in mg, a temporal duration, a concentration, and the like, may encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1 %, and still more preferably ⁇ 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.
  • composition As used herein, the terms “component,” “composition,” “composition of compounds,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” “therapeutic,” “therapy,” “treatment,” or “medicament” are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action.
  • solvent is well-known in the art and means any substance that is capable of dissolving one or several substances, thus creating a solution.
  • a solvent typically is a liquid, but alternatively may be a solid or a gas.
  • treatment refers to the administering of a therapeutically effective amount of the compound of the invention, i.e., a nitrile-containing small molecule such as a JAK inhibitor, to ameliorate undesired symptoms associated with a disease or disorder, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disorders described herein, slow down the deterioration of symptoms, to slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease form occurring or a combination of two or more of the above.
  • the terms “treatment” or “therapy” include preventative (e.g., prophylactic), curative or palliative treatment.
  • the disorder is an inflammatory disorder.
  • the inflammatory disorder is an inflammatory skin disorder.
  • the term “inflammatory skin disorder” means a disorder or disease of the skin involving different immune cells, in which the immune cells, e.g., white blood cells (lymphocytes, monocytes, neutrophils, eosinophils and basophils), release inflammatory mediators, which in turn cause small blood vessels in tissues to dilate, resulting in the affected area of the skin to turn red, swell, feel hot, and be painful, i.c., to become inflamed.
  • cytokines include but are not limited to, cytokines, arachidonic acid metabolites, e.g., prostaglandins and leukotrienes, nitric oxide, and oxygen free radicals, which are produced by epithelial cells, endothelial cells, and infiltrating inflammatory cells.
  • JAK inhibitors have been shown to disrupt T cell-induced macrophage activation and to reduce downstream proinflammatory cytokine and chemokine responses, which suggests that suppressing the T cell-macrophage interaction contributes to the therapeutic effect of JAK inhibitors, as described by Nyirenda MH, Nijjar JS, Frleta-Gilchrist M, et al. JAK inhibitors disrupt T cell-induced proinflammatory macrophage activation.
  • the inflammatory skin disorder is atopic dermatitis, alopecia areata, central centrifugal cicatricial alopecia; hidradenitis suppurativa, lichen planus, lichen planopilaris, frontal fibrosing alopecia, mucosal lichen planus, psoriasis, pemphigus and/or vitiligo.
  • the inflammatory disorder is rheumatoid arthritis, psoriatic arthritis or reactive arthritis.
  • the term “treating” includes alleviating or reducing at least one adverse or negative effect or symptom of a condition, disease or disorder.
  • autoimmune disease is well-known in the ait and means a disease in which the body's immune system attacks healthy cells and tissues. Over 100 autoimmune diseases have been identified that affect over 24 million people. Rheumatoid arthritis (RA) is an example of an autoimmune disease; in RA, the immune system produces antibodies that attach to the lining of joints and the immune system attacks this lining causing chronic inflammation and painful swelling that lead to bone erosion and joint deformity.
  • RA Rheumatoid arthritis
  • a “therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given indication and administration regimen.
  • sustained delivery is well-known in the ail and means a delivery of a drug in a human body (or in a non-human mammal, e.g., dog) at a predetermined and constant rate (zeroorder drug release) to maintain a continuous level of the drug, usually over an extended period of time with the least possible side-effects after administration of a single dose.
  • Zero-order drug release is a way to improve the therapeutic effect and avoid the side effects of the drug.
  • the drug is released from the carrier.
  • the hydrogel in which the nitrilc-group of the JAK inhibitor is reversibly conjugated to a thiol group of a thiolatcd polymer to form a thioimidate adduct provides a “sustained delivery of the JAK inhibitor” at a constant rate after one administration, such as by intradermal or subcutaneous injection, such as over a 12- week period; thus, the drug concentration-time profile is flat.
  • subject refers to an animal, for example a human, to whom treatment, including prophylactic treatment, with the pharmaceutical compositions according to the present invention, i.e., the herein described multifunctional branched therapeutic agents, respectively, is provided.
  • subject refers to human and non-human animals.
  • non-human animals and “non-human mammals” are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses and non-mammals such as reptiles, amphibians, chickens, and turkeys.
  • mammals such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses and non-mammals such as reptiles, amphibians, chickens, and turkeys.
  • a “pharmaceutically acceptable carrier” is well known to those skilled in the art.
  • the carrier may be a solid carrier for solid formulations, a liquid carrier or diluent for liquid formulations, or mixtures thereof.
  • the pharmaceutical compositions of the invention may further include one or more ingredient selected from diluents, buffers, flavoring agents, binders, disintegr nts, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.
  • the formulations may be of immediate release, sustained release, delayed-onset release or any other release profile known to one skilled in the art.
  • the phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and quaternary ammonium salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • Suitable bases for use in the preparation of pharmaceutically acceptable salts including, but not limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide; and organic bases, such as primary, secondary, tertiary, and quaternary, aliphatic and aromatic amines, including L-arginine, benethamine, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, N-methyl-glucamine, hydrabamine, IH-imidazole, L-lysine, morpholine, 4-(2- hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, l-
  • the invention further includes derivatives of the compounds of the invention, i.e., derivatives of the nitrile-containing JAK inhibitors which also contain a nitrile group.
  • derivatives includes but is not limited to ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like.
  • the invention further includes metabolites of the compounds of the invention, i.e., metabolites of the nitrile-containing JAK inhibitors which also contain a nitrile group.
  • metabolites of the compounds of the invention i.e., metabolites of the nitrile-containing JAK inhibitors which also contain a nitrile group.
  • the term “metabolite” means any substance produced from another substance by metabolism or a metabolic process.
  • the invention further includes pharmaceutical products of the compounds of the invention.
  • pharmaceutical product means a composition suitable for pharmaceutical use (pharmaceutical composition), as defined herein.
  • the invention further includes prodrugs of the compounds of the invention.
  • prodrug means a substance which can be converted in vivo into a biologically active agent by such reactions as hydrolysis, esterification, de-esterification, activation, salt formation and the like.
  • This invention further includes crystals of the compounds of the invention. Further, this invention provides polymorphs of the compound of the invention.
  • crystal means a substance in a crystalline state.
  • polymorph refers to a particular crystalline state of a substance, having particular physical properties such as X-ray diffraction, IR spectra, melting point, and the like.
  • the invention further includes sustained delivery hydrogels comprising a small molecule drug having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer, such as hyaluronic acid, to form a thioimidate adduct.
  • the small molecule drug may be selected from any of those drugs which treat any of diverse disease states, including respiratory, cardiovascular, metabolic, infectious, and rare diseases, as well as drugs that are useful in the therapeutic areas of oncology, immunology, inflammation, endocrinology, dermatology, neurology, ophthalmology, hematology, gastroenterology, and aesthetics.
  • the nitrile group of the small molecule drug may adjoin an aliphatic carbon, a non-aromatic unsaturated carbon, an aromatic carbon, a nitrogen atom, or a sulfur atom within the small molecule drug.
  • Examples of small molecule drugs having a nitrile group include, but are not limited to, the compounds listed in Table 1 thru Table 7 below.
  • the small molecule drug having a nitrile group is a dipeptidyl peptidase-4 (DPP-4) inhibitor (also known as gliptins).
  • the small molecule drug having a nitrile group is the DPP-4 inhibitor alogliptin.
  • the small molecule drug having a nitrile group is a Wnt signaling pathway agonist.
  • the small molecule drug having a nitrile group is the Wnt signaling pathway agonist CHIR 99021.
  • sustained delivery hydrogels comprising a small molecule drug having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct.
  • a small molecule drug having a nitrile group reversibly conjugated to a thiol group of a thiolated polymer to form a thioimidate adduct.
  • the small molecule drugs having a nitrile group listed in Table 1 thru Table 7 below may, but is not limited to, be used to treat a disorder or disease indicated therein.
  • the small molecule drug having a nitrile group is BMS- 180428 or BMS- 180448, which have the structures depicted below.
  • the small molecule drug having a nitrile group is apalutamide (ERLEADA®), which has the structure depicted below.
  • Apalutamide, an androgen receptor (AR) antagonist is a nonsteroidal antiandrogen used for the treatment of prostate cancer.
  • the small molecule drug having a nitrile group is cnobosarm, which has the structure depicted below.
  • the small molecule drug having a nitrile group is glasdegib (DAURISMOTM), which has the structure depicted below.
  • Glasdegib a hedgehog pathway inhibitor, is used for the treatment of acute myeloid leukemia (DAURISMOTM).
  • the small molecule drug having a nitrile group is pradofloxacin, which has the structure depicted below.
  • the small molecule drug having a nitrile group is ravuconazolc, which has the structure depicted below.
  • the small molecule drug having a nitrile group is fosravuconazole, which has the structure depicted below.
  • the small molecule drug having a nitrile group is lanoconazole, which has the structure depicted below.
  • the small molecule drug having a nitrile group is doravirinc (PIFELTRO®), which has the structure depicted below.
  • Doravirine a non-nucleoside reverse transcriptase inhibitor, is used in the treatment of HIV/AIDS.
  • the small molecule drug having a nitrile group is perampanel (FYCOMPATM), which has the structure depicted below and is used as an anti-epileptic medication.
  • the small molecule drug having a nitrile group is selpercatinib (RETEVMO®), which has the structure depicted below; it is a kinase inhibitor and used in the treatment of cancer.
  • the small molecule drug having a nitrile group is cyanocobalamin (Vitamin B12), which has the structure depicted below.
  • the small molecule drug having a nitrile group is zuranolone (ZURZUVAETM), which has the structure depicted below. Zuranolone, a neuroactive steroid gamma-aminobutyric acid (GABA) A receptor positive modulator, is used for the treatment of postpartum depression (PPD).
  • the small molecule drug having a nitrile group is nirmatrclvir, which has the structure depicted below.
  • Nirmatrelvir, a 3C-like protease inhibitor is part of a nirmatrelvir/ritonavir combination (PAXLOVIDTM) used to treat COVID-19.
  • the small molecule drug having a nitrile group is ibuzatrelvir, which has the structure depicted below.
  • Ibuzatrelvir is an antiviral being developed for the treatment of COVID-19.
  • the small molecule drug having a nitrile group is adagrasib (KRAZATI®), which has the structure depicted below.
  • Adagrasib an irreversible inhibitor of KRAS G12C
  • the small molecule drug having a nitrile group is olutasidenib (REZLIDHIA®), which has the structure depicted below.
  • Olutasidenib an isocitrate dehydrogenase- 1 (IDH1) inhibitor, is used for the treatment of acute myeloid leukemia (AML).
  • sustained delivery hydrogels comprising a small molecule drug having a nitrile group
  • the physician will determine the actual dosage and duration of treatment, which will be most suitable for an individual with that disorder or disease and can vary with the age, weight, genetics and/or response of the particular individual, as well as be informed by the use of that small molecule drug having a nitrile group alone, i.e, in the absence of a hydrogel.
  • a therapeutic agent of the invention which is a nitrilc-containing JAK inhibitors as described herein which are either (i) nitrile group reversibly conjugated to a thiol group of a thiolated hydrogel, such as hyaluronic acid, to form a thioimidate adduct or (ii) in the absence of a hydrogel, and a pharmaceutical composition comprising the same can be administered to a subject by any method known to a person skilled in the art.
  • tissue e.g., needle or catheter
  • a topical administration may be desired for application to dermal, ocular, or mucosal surfaces.
  • Another method of administration is via aspiration or aerosol formulation.
  • hydrogels and compositions provided herein may be use for systemic delivery of the active agent contained in those hydrogels and compositions.
  • the compound or the pharmaceutical composition may be administered topically to body surfaces and are thus formulated in a form suitable for topical administration.
  • suitable topical formulations include gels, ointments, creams, lotions, drops and the like.
  • the compositions are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.
  • Transdermal formulations may be prepared by incorporating the active agent in a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose, with the resulting formulation then being packed in a transdermal device adapted to be secured in dermal contact with the skin of a wearer.
  • a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose
  • Transdermal administration may comprise application to the area to be treated, e.g., the skin affected by one of the disorders described herein, by transdermal injection or a transdermal delivery system comprising a microneedle coated with the therapeutic agent (i.e., the JAK inhibitor), a solid polymer matrix having the therapeutic agent (the JAK inhibitor) incorporated therein, dissolving polymer microneedles that dissolve after insertion into the skin and release the therapeutic agent; a transdermal patch comprising a reservoir storing the agent and a semi- permeable membrane, a transdermal gel comprising the agent dissolved therein, a transdermal spray comprising the agent dissolved therein, or a metered dose transdermal spray comprising the agent dissolved therein.
  • the therapeutic agent i.e., the JAK inhibitor
  • the JAK inhibitor solid polymer matrix having the therapeutic agent (the JAK inhibitor) incorporated therein, dissolving polymer microneedles that dissolve after insertion into the skin and release the therapeutic agent
  • the microneedles for transdermal delivery of a JAK inhibitor are hydrogel-forming microneedles, as described by Turner, J.G, et al., Macromol. Biosci. 2021, 27, 2000307.
  • Such hydrogel-forming microneedles are made of a swellable polymer such as a crosslinked hydrogel, which swells upon water uptake when inserted into the skin and comprise the JAK inhibitor in the hydrogel.
  • the hydrogel-forming microneedles comprise a therapeutic-agent-loaded (i.e., JAK inhibitor loaded) reservoir attached to the tops of the microneedles.
  • Such hydrogel-forming microneedles are minimally invasive, have a higher drug loading capacity and a tunable therapeutic-agent release rate, and are biocompatible.
  • administration is effected by intradermal (into the dermis) or subcutaneous injection of a nitrile-containing JAK inhibitor in the absence of a thiolated polymer, i.e., without formation of a hydrogel, wherein the JAK inhibitor crystallizes or precipitates out of the injected composition and sustains its own release in the dermis.
  • administration is effected by transdermal injection.
  • any of the usual pharmaceutical media may be employed.
  • suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like.
  • suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like.
  • the carrier will usually comprise sterile water, though other ingredients may be included, such as ingredients that aid solubility or for preservation.
  • Injectable solutions may also be prepared in which case appropriate stabilizing agents may be employed.
  • appropriate stabilizing agents may be employed.
  • Methods of treatment using formulations suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the active ingredient.
  • a suspension in an aqueous liquor or a non-aqueous liquid may be employed, such as a syrup, an elixir, an emulsion, or a draught.
  • a tablet may be made by compression or molding, or wet granulation, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, with the active compound being in a free-flowing form such as a powder or granules which optionally is mixed with, for example, a binder, disintegrat'd, lubricant, inert diluent, surface active agent, or discharging agent. Molded tablets comprised of a mixture of the powdered active compound with a suitable carrier may be made by molding in a suitable machine.
  • a syrup may be made by adding the active compound to a concentrated aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredient(s).
  • Such accessory ingredient(s) may include flavorings, suitable preservative, agents to retard crystallization of the sugar, and agents to increase the solubility of any other ingredient, such as a polyhydroxy alcohol, for example glycerol or sorbitol.
  • Formulations suitable for parenteral administration may comprise a sterile aqueous preparation of the active compound, which, in some embodiments, is isotonic with the blood of the recipient (e.g., physiological saline solution).
  • Such formulations may include suspending agents and thickening agents and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose form.
  • Parenteral administration may comprise any suitable form of systemic delivery.
  • Administration may for example be intravenous, intra-arterial, intrathecal, intramuscular, subcutaneous, intramuscular, intra-abdominal (e.g., intraperitoneal), etc., and may be canned out by infusion pumps (external or implantable) or any other suitable means appropriate to the desired administration modality.
  • a dosage unit of the compounds used in the present invention may comprise a single compound or mixtures thereof with additional therapeutic agents.
  • a “dose” or “dosage unit” or “unit dosage” of a compound of the invention as measured in milligrams refers to the milligrams of the compound of the invention present in a composition, regardless of the form of the composition.
  • a dosage unit can be prepared for oral dosage forms, such as tablets, capsules, pills, powders, liquid suspensions, and granules.
  • a compound of the invention is administered at a dosage of 1- 3000 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 1-1000 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 1-500 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 10- 500 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25-500 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 50-500 mg per day.
  • a compound of the invention as described herein is administered at a dosage of 5- 250 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 10-250 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 20-250 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25- 250 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25-200 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25-150 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25- 125 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25-100 mg per day.
  • a compound of the invention is administered at a dose of 1- 10 mg per day, 3-26 mg per day, 3-60 mg per day, 3-16 mg per day, 3-30 mg per day, 10-26 mg per day, 10- 100 mg per day, 15-60 mg per day, 15-100 mg per day, 25-100 mg per day, 50-100 mg per day, 50-200 mg per day, 100-200 mg per day, 100-250 mg per day, 125-300 mg per day, 20-50 mg per day, 5-50 mg per day, 200-500 mg per day, 125-500 mg per day, 500-1000 mg per day, 200-1000 mg per day, 1000-2000 mg per day, 1000-3000 mg per day, 125-3000 mg per day, 2000- 3000 mg per day, 300-1500 mg per day or 100-1000 mg per day.
  • the methods may comprise administering a compound at various dosages.
  • the compound may be administered per day at a dosage of 3 mg, 10 mg, 30 mg, 40 mg, 50 mg, 80 mg, 100 mg, 120 mg, 125 mg, 200 mg, 250 mg, 300 mg, 450 mg, 500 mg, 600 mg, 900 mg, 1000 mg, 1500 mg, 2000 mg, 2500 mg or 3000 mg.
  • the compound may be administered at a dosage of 0.1 mg/kg/day.
  • the compound may be administered at a dosage between 0.2 to 30 mg/kg/day, or 0.2 mg/kg/day, 0.3 mg/kg/day, 1 mg/kg/day, 3 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 20 mg/kg/day, 30 mg/kg/day, 50 mg/kg/day or 100 mg/kg/day.
  • the compound of the invention is prepared for once daily administration. In another embodiment, the compound of the invention is prepared for more than once daily administration, for example, twice daily, three times daily, four times daily, etc. In some embodiments, the compound or the pharmaceutical composition of the invention is administered in the form of a capsule, a tablet, or a liquid suspension. In other embodiments, the compound or the pharmaceutical composition of the invention is administered in an oral dosage unit form.
  • the methods of treatment of the present invention can additionally include administering to the subject one or more additional therapeutic agents for a combination therapy.
  • additional therapeutic agents may be administered, by a route and in an amount commonly used therefore, simultaneously or sequentially with a compound or composition of the present invention.
  • the term “combination therapy” means the administration of two or more therapeutic agents to treat a cancer described in the present invention. Such administration encompasses coadministration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the disorders described herein. [00154] The following examples are presented in order to more fully illustrate particular embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
  • Thiolated hyaluronic acid was purchased from a supplier (HA works LLC, Bedminster, NJ), synthesized as previously described through an amidation reaction at the carboxyl of hyaluronic acid with 30% or 50% degree of modification of disaccharides. Polymers were stored under vacuum in a desiccator at room temperature. All other chemical reagents were purchased from Sigma- Aldrich (St. Louis, MO) and stored according to manufacturer’s instructions unless otherwise indicated. All experiments were performed in triplicate unless otherwise indicated.
  • LCMS Liquid chromatography-mass spectrometry
  • 3 H spectrum of baricitinib was obtained in 64K data points over 10 kHz spectral width using a 30° flip-angle pulse.
  • 13 C spectra were obtained in 64K data points over 29.761 kHz spectral width using the pulse program, zgpgsezr, a z-restored spin-echo 13 C pulse sequence with power-gated X H decoupling.
  • a 2s relaxation delay was used between acquisitions.
  • the free induction decays were processed using exponential window function (line-broadening 0.3 Hz for 1 H and 1 Hz for 13 C) before Fourier transformation.
  • Thiol quantification assays The Free Thiol Assay Kit (abl 12158, Abeam, Waltham, MA) was used for all thiol quantification measurements. Solutions of thiolated hyaluronic acid in distilled water were mixed with a solution of baricitinib in 1:1 dimethyl sulfoxide (DMSO) to distilled water to the indicated final concentrations in 100 pL total volume including 50 pL of assay reaction mixture. Solutions prior to addition of reaction mixture were gently vortexed and products were allowed to form for 20 minutes. After mixing, 50 pL of product was transferred to a black walled 96-well plate and products were incubated with 50pL of the assay reaction mixture according to manufacturer’s protocols. Analyses were performed using the Biotek Synergy Hl microplate reader (ex/em: 490/520 nm).
  • Hydrogel formation and in vitro release Hydrogels were formed by evenly mixing stock solutions of thiolated hyaluronic acid in phosphate-buffered saline (PBS) with stock solutions of baricitinib in DMSO to the final desired weight percent concentration (w/v) and allowed to mix and crosslink for 24 hours. After 24 hours, hydrogels were briefly centrifuged and DMSO was removed by briefly washing hydrogels in PBS (3 x 5 minutes). For release studies, 100 pL of hydrogel was incubated in 200 pL of PBS at room temperature and releasates were collected and replaced with fresh PBS at days 2, 4, 8, 12, 16, 20, 28, and 42. At the final timepoint, hydrogels were manually disrupted.
  • PBS phosphate-buffered saline
  • Shear oscillatory rheometry Hydrogels were formed as described and deposited on the bottom plate of an HR 20 (TA Instruments, New Castle, DE) rheometer immediately after mixing or after 24 hours of gelation. The rheometer was fitted with a 20 mm diameter stainless steel parallel plate geometry and placed at a 350 pm gap. Oscillatory rheological time sweeps (1% strain, 10 Hz) were performed to obtain the average storage (G’) and loss (G”) moduli, and oscillatory frequency sweeps (1% strain, 0.1 Hz to 100 Hz) were obtained to characterize dynamic viscoelastic properties. RESULTS AND DISCUSSION
  • the thioimidate may further be stabilized to form a thiazoline ring; however, the difference in molecular weight is too small between the products to differentiate thioimidate from thiazoline.
  • a tetrahedral bisadduct that was previously reported to occur with low frequency was not observed; this would appear as a product with molecular weight that combines two cysteamines or cysteines and one baricitinib.
  • a hydrogel was designed from thiolated hyaluronic acid that directly binds to baricitinib, which also forms disulfide bonds with itself for gel formation.
  • thiolated hyaluronic acid formed from the amidation reaction between the carboxyl of hyaluronic acid to cysteamine with a degree of thiol modification of either 30% or 50%, and a molecular weight of approximately 100 kDa was used (Figure 2A).
  • the thiols function to form thioimidates with baricitinib as well as disulfide crosslinks between polymer chains to form hydrogels ( Figure 2B).
  • 13 C NMR was used to confirm that the formed products between baricitinib and thiolated hyaluronic acid were thioimidates.
  • 13 C NMR revealed distinct peaks for both baricitinib and thiolated hyaluronic acid that are consistent with predicted and expected spectra.
  • the 13 C NMR spectra of the combined product demonstrated a distinct peak at 170 ppm that was not present in the spectra of the reactants ( Figure 4A). This is consistent with prior reports for thioimidate carbons as well as the predicted peak from the NMR predict tool at nmrdb.org (Universidad del Valle). Thioimidate product formation using ’H NMR.
  • baricitinib release its absorption properties were determined in the ultraviolet (UV) range, where baricitinib absorbs due to its aromatic ring. It was determined that baricitinib absorbs most in the ultraviolet B range (280-320 nm), with absorbances increasing linearly with concentration (Figure 6A), consistent with prior methods for measuring baricitinib in solution.
  • baricitinib hydrogels were assembled with varying baricitinib loading (0.2 mg/mL, 2 mg/mL, corresponding to 40-fold or 4-fold thiol excess), hydrogel concentration (%w/v), and thiol modification (30% or 50%).
  • Baricitinib hydrogels were incubated in PBS at room temperature with releasates collected and replaced regularly over six weeks. At six weeks, hydrogels had nearly fully eroded and were manually disrupted in PBS. Baricitinib in releasates was measured by absorbance at 300 nm on a standard curve. Cumulative release demonstrates similar release profiles with 0.2 mg/mL of baricitinib included ( Figure 6B). In these formulations, approximately 40% was released by one week, 50% by two weeks, and 60% by three weeks. By four weeks, differences in release were observed; 90% of baricitinib was released in 2 wt% 30% mod hydrogels, while -70% was released in 5 wt% 50% mod hydrogels.
  • the latter formulation has four-times the concentration of thiols in solution. This sustains baricitinib release through thioimidate formation and by increasing disulfide crosslinking, thereby decreasing network mesh size. At 2 mg/mL baricitinib, there is a larger concentration gradient due to the higher amount of baricitinib loaded. Consistent with this, release was much faster with up to -70% released by one week, 80% by two weeks, and 90% by four weeks. Significant differences in release were observed before two weeks, where there was faster release observed in formulations assembled at 2 wt% compared to formulations assembled at 5 wt%, consistent with the role of thiols in forming thioimidates and disulfides to slow release.
  • baricitinib also began to precipitate over time within the hydrogel, which may also partially contribute to its sustained release.
  • formulations of baricitinib were selected at 2 wt% and 30% thiol modification for biologic activity - release profiles of relevant formulations are plotted ( Figure 6C). Higher concentrations of hydrogel could not be injected through a 27G syringe while higher thiol modifications did not change release profiles.
  • HEK 293 culture Cells (BPS Bioscience, San Diego, CA) were thawed in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. In subsequent studies, media was supplemented with 400 pg/ml of geneticin. Prior to use, cells were split into 96 well black-walled, clear bottom plates (30,0000/well) in media without geneticin 24 hours prior.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin
  • Luciferase Assays For the interferon (IFNa) titration, varying concentrations from 10' 1 to 10 4 U/mL of IFNa at 100 pL total volume were incubated for 6 hours. For the baricitinib condition, concentrations of 10’ 3 to 10 3 g/mL baricitinib were added to wells and incubated for 1 hour. IFNa was added to each well at a final concentration of 100 U/mL at 100 pL total per well and incubated for 6 hours. For the releasate assay, releasate volumes of 5 pL or 20 pL were added to cells in triplicate from each timepoint and incubated for 1 hour.
  • IFNa interferon
  • IFNa volume was added to a total of 100 pL and 100 U/mL and then incubated for 6 hours. After the 6-hour incubation of IFNa, luciferase activity was quantified for each of the three tests using the ONE-StepTM Luciferase Assay System (BPS Bioscience, San Diego, CA) with 100 pL master mix was added for a total volume of 200 pL. The 96 well plate was placed on an orbital shaker for 15 minutes covered from light before taking fluorescence measurements using the Biotek Synergy Hl microplate reader.
  • HaCAT culture and proliferation assay HaCaT cells were cultured in calcium-free DMEM with 10% FBS, and 1% penicillin/streptomycin as previously described. Cells were split into 24-well plates 24 hours at 100,000/well. After 24 hours, hydrogels were formed and 50 pL was transferred to a polycarbonate membrane transwell insert with 6.5 mm diameter, 0.1 pm pore size (Coming, Coming, NY) and coincubated with cells for the indicated times. The CellTiter 96® AQueous One Solution Cell Proliferation MTS Assay (Promega, Madison, WI) was utilized according to manufacturer’s protocols after incubation with one hour. Absorbance was measured at 490 nm using the Biotek Synergy Hl microplate reader.
  • Quantification of epidermal thickness was performed on stitched lOx slides of 6-10 mm skin sections by selecting the entire region-of-interest (ROI) of the epidermis using the Wand Tool on FIJI on Legacy Mode. The smooth feature was utilized to ensure the ROI appropriate captured the entire region between the granular layer and basal layer. The ROI area was measured and subsequently divided by the length of epidermis. Average epidermal thickness was obtained for two sections per mouse.
  • ROI region-of-interest
  • Baricitinib hydrogels prepared as described above were tested in vivo, using a mouse model for alopecia areata, as described in McElwee et al. (1998) J Invest Dermatol 111:797-803. Briefly, in this model, alopecia develops in -10-20% of aging mice after six months which are subsequently be transferred to younger C3H/HeJ mice through skin grafts, reliably producing alopecia areata in up to 95-100% of mice six to ten weeks after grafting. Baricitinib (Jabbari et al. (2015) EBioMedicine 2:351-355) and other JAK inhibitors (Xing et al.
  • NMR is used to show the reaction between an aromatic nitrile containing molecule, CHIR 99021 (a Wnt signaling pathway agonist), and thiolated hyaluronic acid.
  • CHIR 99021 a Wnt signaling pathway agonist
  • the nitrile group is expected to react with the thiol group in a reversible manner to form a thioimidate.
  • a 20 mM solution of CHIR 99021 dissolved in ethanol was combined with thiolated hyaluronic acid in D2O. The solution was left to sit overnight and filtered for 1 H NMR analysis.
  • the aromatic nitrile in CHIR 99021 reacts in a reversible manner with the thiolated hyaluronic acid to form a thioimidate.
  • the 'H NMR data shows the formation of a new set of peaks in the aromatic region downfield from those in CHIR99021 alone.
  • Thiolated hyaluronic acid alone shows no peaks in this region.

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Abstract

L'invention concerne des hydrogels d'inhibiteurs de JAK à administration prolongée pour le traitement d'un trouble inflammatoire, d'une maladie auto-immune, d'une perte de cheveux auto-immune et inflammatoire, et/ou d'une perte de cheveux due à un trouble non inflammatoire, l'hydrogel d'inhibiteurs de JAK comprenant un inhibiteur de JAK ayant un groupe nitrile conjugué de manière réversible à un groupe thiol d'un polymère thiolé pour former un adduit de thioimidate. L'invention concerne également des compositions pharmaceutiques injectables pour le traitement d'un trouble inflammatoire, d'une maladie auto-immune, d'une perte de cheveux auto-immune et inflammatoire, et/ou d'une perte de cheveux due à un trouble non inflammatoire, les compositions pharmaceutiques injectables comprenant un inhibiteur de JAK contenant du nitrile et un support pharmaceutiquement acceptable.
PCT/US2024/056342 2023-11-17 2024-11-18 Administration prolongée d'inhibiteurs de jak contenant du nitrile à partir d'un hydrogel par l'intermédiaire d'adduits de thioimidate réversibles Pending WO2025106953A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180243481A1 (en) * 2015-08-17 2018-08-30 The Johns Hopkins University Fiber-hydrogel composite surgical meshes for tissue repair
US20210338834A1 (en) * 2018-09-26 2021-11-04 Ascendis Pharma A/S Degradable hyaluronic acid hydrogels
US20220257600A1 (en) * 2018-06-20 2022-08-18 Progenity, Inc. Treatment of a disease of the gastrointestinal tract with a jak or other kinase inhibitor

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US20180243481A1 (en) * 2015-08-17 2018-08-30 The Johns Hopkins University Fiber-hydrogel composite surgical meshes for tissue repair
US20220257600A1 (en) * 2018-06-20 2022-08-18 Progenity, Inc. Treatment of a disease of the gastrointestinal tract with a jak or other kinase inhibitor
US20210338834A1 (en) * 2018-09-26 2021-11-04 Ascendis Pharma A/S Degradable hyaluronic acid hydrogels

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Title
NODDELAND HEIDI KYUNG; LIND MARIANNE; JENSEN LOUISE BASTHOLM; PETERSSON KARSTEN; SKAK-NIELSEN TINE; LARSEN FLEMMING HOFMANN; MALMS: "Design and characterization of matrix metalloproteinase-responsive hydrogels for the treatment of inflammatory skin diseases", ACTA BIOMATERIALIA, ELSEVIER, AMSTERDAM, NL, vol. 157, 13 December 2022 (2022-12-13), AMSTERDAM, NL, pages 149 - 161, XP087255404, ISSN: 1742-7061, DOI: 10.1016/j.actbio.2022.12.015 *
ZHENG XUAN-QI; HUANG JIN-FENG; LIN JIA-LIANG; ZHU YA-XIN; WANG MIN-QI; GUO MEI-LIANG; ZAN XING-JIE; WU AI-MIN: "Controlled release of baricitinib from a thermos-responsive hydrogel system inhibits inflammation by suppressing JAK2/STAT3 pathway in acute spinal cord injury", COLLOIDS AND SURFACES B: BIOINTERFACES, ELSEVIER AMSTERDAM, NL, vol. 199, 29 December 2020 (2020-12-29), NL , XP086512058, ISSN: 0927-7765, DOI: 10.1016/j.colsurfb.2020.111532 *

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