WO2019023426A1 - Compositions et méthodes pour le traitement d'une réponse immunitaire anormale par l'inhibition de gsk - Google Patents

Compositions et méthodes pour le traitement d'une réponse immunitaire anormale par l'inhibition de gsk Download PDF

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Publication number
WO2019023426A1
WO2019023426A1 PCT/US2018/043835 US2018043835W WO2019023426A1 WO 2019023426 A1 WO2019023426 A1 WO 2019023426A1 US 2018043835 W US2018043835 W US 2018043835W WO 2019023426 A1 WO2019023426 A1 WO 2019023426A1
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gsk3
cells
gimap5
inhibitor
sph
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PCT/US2018/043835
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English (en)
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Kasper Hoebe
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Children's Hospital Medical Center
MENGISTU, Mehari Endale
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    • 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
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/14—Alkali metal chlorides; Alkaline earth metal chlorides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants

Definitions

  • An autoimmune disease is a condition arising from an abnormal immune response, mostly but not limited to a T cell response or antibody response against normal cells and/or tissues of the body.
  • autoimmune include, but are not limited to, celiac disease, diabetes mellitus type 1, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, auto-immune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) and systemic lupus erythematosus.
  • NSAIDs NSAIDs
  • immunosuppressants NSAIDs
  • treatment may improve symptoms the disease typically is not cured, and there is a need for improved treatments for those with immune diseases.
  • T cell immunosuppressive function and immune homeostasis that may prevent or correct immune-associated sequelae observed in patients having immune disorders characterized by faulty regulatory T cell function.
  • autoimmune disease including, but not limited to, islet autoimmunity in type I diabetes, systemic lupus erythematosus, auto-immune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) or asthma.
  • ITP idiopathic thrombocytopenic purpura
  • glycogen synthase kinase-3 for example, GSK or GSKa
  • the T-cell mediated disease may be one associated with a loss of Treg
  • immunosuppressive function for example, an immune disease or an autoimmune disease such as, for example, an immunodeficiency, primary immunodeficiency disease (PID), type I diabetes, systemic lupus erythematosus (SLE), asthma, colitis, psoriasis, bronchiectasis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) or combinations thereof.
  • PID primary immunodeficiency disease
  • SLE systemic lupus erythematosus
  • colitis psoriasis
  • bronchiectasis autoimmune hemolytic anemia
  • ITP idiopathic thrombocytopenic purpura
  • the disclosed compositions and/or methods may be used to ameliorate, prevent, or reduce the severity and/or progression of any aforementioned disease.
  • FIG 1A-1D Loss of Gimap5 impairs CD4+ T cell survival and iTreg cell induction.
  • A the number of splenic CD4+ T cells and
  • B the percentage of naive (CD441o;CD62hi) and memory-like (CD44hi;CD621o) CD4+ T cells was determined using flow cytometry.
  • FIG 2A-2H Impaired CD4+ T cell proliferation is associated with increased GSK3 activity.
  • A Immunoblot analysis of c-Myc expression in total lysates of CD4+ T cells from WT and Gimap5 ⁇ 11 mice stimulated with aCD3/aCD28 or during resting conditions.
  • C C
  • FIG 3A-3I Loss of Gimap5 results in impaired inactivation of GSK3 .
  • A Association of GSK3 with vesicles in WT and Gimap5 S P H/S P H CD4+ T cells as exemplified by GSK3 intensity, vesicle number, and vesicle size of GSK3 spots 6 and 24h after aCD3/aCD28 activation.
  • B Bright detail similarity analysis (ImageStream) of activated CD4+T cells from WT and Gimap5 s P Ns P h mice show colocalization between Gimap5 and GSK3 .
  • C Representative Z-stacks of WT and Gimap5 sph/sph CD4+ T cells stimulated for 24h with aCD3/aCD28.
  • D Immunoblot analysis of WT and Gimap5 CD4+ T cells stimulated with aCD3/aCD28 depicting total and phosphorylated protein levels of GSK3 (P- Ser9 and P- Ser389), p38, and p53.
  • F Nuclear localization and (H) expression of total GSK3 .
  • FIG 4A-4D Loss of Gimap5 causes increased DNA damage in activated CD4+ T cells.
  • A ⁇ 2 ⁇ expression in live WT or Gimap5 sph/sph CD4+ T cells following aCD3/aCD28 stimulation.
  • C,D Effect of lithium on ⁇ 2 ⁇ expression in WT or Gimap5 s P h/s P h CD4+ T cells, after 3 days stimulation with aCD3/aCD28.
  • FIG 5A-5G GSK3 inhibition improves lymphocyte survival and prevents immunopathology.
  • A,B LiCl treatment of Gimap5 s P h/s P h mice in vivo starting at 3 weeks of age and analyzed at 7-8 weeks of age, rescues CD4+ T cell (A) and B cell (B) survival.
  • C Reduced frequency of CD4+ T cells undergoing lymphopenia-induced proliferation
  • FIG 6A-6D GSK3 -deletion in CD4+ T cells improves CD4+ T cell survival and prevents colitis.
  • A Tamoxifen treatment of Gimap5 s P h/s P h ; Gsk3 fl/fl; Cd4cre-ert2 mice starting at 3 weeks of age selectively rescues splenic CD4+ T cell survival while maintaining overall CD4+ T cell quiescence (B).
  • C,D Reduced colitis in Gimap5 sph/sph ; Gs ⁇ *TM;
  • Cd4cre-ert2 mice treated with tamoxifen as determined by disease scores (C) based on histology (D).
  • Data represent mean values + SEM from at least 6 mice per group at 8 weeks of age; histology is a representative depiction of disease severity. Statistical significance is determined by ANOVA followed by Sidak's multiple comparisons test.
  • FIG 7A-7D A human loss-of-function mutation in GIMAP5 results in a similar T cell deficiency.
  • A Whole exome sequencing uncovered a homozygous variant (SNP: rs72650695), causing an L204P amino acid change in GIMAP5, resulting in complete loss of GIMAP5 protein expression in T cells.
  • B Impaired expansion of CD3+ T cells from GIMAP5-/- patient compared to heterozygous control cells following stimulation with PHA (4 days) and IL-2 (days 4-10). The proliferation capacity is restored in the presence of LiCl. Experiment is representative of three independent experiments from samples obtained several months apart.
  • FIG 8A-8J Human GIMAP5-/- T cells show impaired GSK3 sequestration and increased DNA damage.
  • A-D Colocalization and spot analysis of GIMAP5+, GSK3 + and CD 107+ vesicles in primary CD4+ T cells isolated from healthy controls at a resting state (day 0) or after aCD3/aCD28-stimulation for 1-2 days (representative images from day 1 shown) using ImageStream analysis.
  • E-H ImageStream analysis of GSK3 -specific vesicle association in control or GIMAP5-/- patient CD4+ T cells restimulated with aCD3/aCD28 after primary expansion.
  • Data depicts (E) GSK3 + spot number, (F) GSK3 intensity therein, and (G) spot area in live CD4+ T cells at 0, 6, or 24 hrs of aCD3/aCD28-restimulation. Data represent mean values + SD.
  • H Representative images of GSK3 vesicular association in control and GIMAP5-/- CD4+ T cells taken using a 60x objective.
  • I,J Analysis of DNA damage response ( ⁇ 2 ⁇ ) in control of GIMAP5 sph/sph CD4+ T cells after (I) 1-3 days or (J) 2 days restimulation with aCD3/aCD28. Data represents mean values of a single experiment performed in duplicate and repeated twice. ImageStream data represent average values of >500 CD4+ T cells per experiment.
  • BF Bright field [0015]
  • FIG 9 Gimap5 is a critical regulator of GSK3 during T cell activation.
  • Gimap5 controls regulation of GSK3 in T cells through vesicular sequestration and affects both the 1) (early) transcriptional program required for T cell growth, and 2) the late stage nuclear accumulation of P- Ser389 GSK3 required for the DNA damage response during cycling. Gimap5-deficient CD4+ T cells, fail to inhibit GSK3 leading to a failed transcriptional program and increased DNA damage during T cells proliferation.
  • FIG 10A-B Normal thymocyte survival and thymic emigration.
  • FIG 11A-11G Reduced T cell survival is independent of Bim and Bax/Bak.
  • A-C Mean frequency +/- SD of CD4+ T cells (A), CD 8+ T cells (B), and NK cells (C) in the spleen of 5-233k-old Bim deficient Gimap5 s P h/s P h mice (n>3 mice/group).
  • D Analysis of CD4+ T cells undergoing lymphopenia-induced proliferation (CD621oCD44hi) in the spleen of 5 -week-old Gimap 5 s P h/s P h mice deficient for Bim.
  • FIG 12A-D Normal proximal CD4+ T cell signaling in 3-week-old Gimap5 sph/sph mice
  • B Phosphorylation of ERK (Thr202/Tyr204), JNK
  • Immunoblot data depicts representative immunoblots of at least 3 independent experiments performed on pooled CD4+ T cells.
  • D Proliferation of WT CD4+ T cells after 3 days of aCD3/aCD28 stimulation + calcineurin inhibitor cyclosporin A (200 ng/mL). Plots are representative of 4 independent samples.
  • F Representative flow plots detailing calcium flux upon stimulation with aCD3/aCD28 or Ionomycin.
  • FIG 14A-14E Vesicular localization of Gimap5 and GSK3p.
  • FIG 15A-E GSK3 vesicular localization is Wnt independent.
  • Each ImageStream data point represents average values of >500 CD4+ T cells.
  • FIG 16A-16F TCR-induced P-Ser9 GSK3 is unaffected.
  • A Immunoblot analysis of P-Ser9 GSK3 in WT and Gimap5 sph/sph CD4+ T cells during 24 hours activation with aCD3/aCD28.
  • FIG 17A-17B Lithium chloride prevents colitis.
  • A,B Gross morphology of the colon (A) and liver (B) of Wildtype and Gimap5 S P H/S P H mice treated with either LiCl or vehicle from 3 weeks of age to 8 weeks of age. Images representative of 6 mice per group at 7-8 weeks of age. Scale bar shown is in cm.
  • C Frequency of regulatory T cells
  • FIG 18A-18D Genetic targeting of GSK3 in CD4+ T cells improves T cell survival.
  • A Loss of GSK3 protein in splenic CD4+ T cells from Gsk3 fl/flCd4-cre/ert2 mice treated with tamoxifen from 3 to 8 weeks of age. Representative flow plot is shown.
  • B,C Absolute number of (B) B cells and (C) CD4+ T cells in the spleen of WT and Gimap5 sph/sph mice either GSK3 -sufficient or insufficient CD4+ T cells.
  • D Representative H&E stained liver sections of tamoxifen- treated Wildtype, Gimap5 ⁇ 5 ?
  • FIG. Structure of C66H11ON15O20P. DETAILED DESCRIPTION
  • 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, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5- fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about” meaning within an acceptable error range for the particular value should be assumed.
  • the term "effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.
  • an inhibitor of glycogen synthase kinase 3, for example glycogen synthase 3 ⁇ (GSK3 ) or glycogen synthase 3a (GSKa) for use in the therapy of a T-cell mediated disease in an individual in need thereof is disclosed.
  • the T-cell mediated disease may be one associated with a loss of Treg immunosuppressive function.
  • the T-cell mediated disease may be an immune disease or an autoimmune disease.
  • the T cell mediated disease may be selected from an immunodeficiency, primary immunodeficiency disease (PID), type I diabetes, systemic lupus erythematosus (SLE), asthma, colitis, psoriasis, bronchiectasis, auto-immune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) or combinations thereof.
  • the treatment step may ameliorate, prevent, or reduce the severity and/or progression of any aforementioned disease.
  • the individual may have a loss of function (LOF) mutation in one Gimap5 allele, or in both Gimap5 alleles.
  • LEF loss of function
  • a version of Gimap5 is described in, for example, GIMAP5 Acc.#: HGNC: 18005.
  • the LOF mutation may be a heterozygous mutation or a homozygous mutation.
  • SNP rs72650695
  • the LOF mutation may be one in which a mutation causes one or more of a loss of GIMAP5 protein expression or function, impairment of RNA stability, impairment of transcription factor (TF) binding sites, or a combination thereof.
  • the TF may be one that is a direct target of GSK3 ⁇ regulation.
  • the inhibitor of GSK3 or GSK3a may be administered to an individual at a time period selected from within a day, within a week, within two weeks, within three weeks, within a month, within two months, within three months, within four months, within five months, within six months, or within a year of diagnosis.
  • the administration step may occur prior to said disease state being characterized as late-stage.
  • the inhibitor of GSK3 or GSK3a described above may be selected from an inhibitor listed in Table 1.
  • the inhibitor is LiCl.
  • the inhibitor may be LiCl and may be administered in a dose of about 100 to about 150 mg/kg/day to the individual.
  • Prescription Tablets currently available include 300 mg and are generally prescribed three times daily (900mg/adult/day).
  • Each 5 mL of Lithium Oral Solution contains 8 mEq of Lithium ion (Li+) which is equivalent to the amount of Lithium in 300 mg of Lithium carbonate.
  • the GSK3 inhibitor may be selected from a metal cation, for example, Lithium Chloride, an ATP-competitive GSK inhibitor, a marine organism-derived compound (6-BIO, Dibromocantharelline, Hymenialdesine, Indirubins, Meridianins) an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an aminopyrimidine (CT98014, CT98023, CT99021, TWS119), an aminopyrim
  • arylindolemaleimide SB-216763, SB-41528
  • a thiazole AR-A014418, AZD-1080
  • a paullone Alsterpaullone, Cazpaullone, Kenpaullone
  • an aloisine a non-ATP competitive Marine organism-derived compound (Manzamine A, Palinurine, Tricantine)
  • TTZD-8 thiadiazolidindione
  • NP00111 NP031115
  • Tideglusib Tideglusib
  • HMK- 32 halomethylketone
  • L803-mts non-ATP competitive peptide
  • the active agent may form salts, which are also within the scope of the preferred embodiments.
  • Reference to a compound of the active agent herein is understood to include reference to salts thereof, unless otherwise indicated.
  • an active agent contains both a basic moiety, such as, but not limited to an amine or a pyridine or imidazole ring, and an acidic moiety, such as, but not limited to a i C t aons
  • salts carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term “salt(s)" as used herein.
  • Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps, which may be employed during preparation.
  • Salts of the compounds of the active agent may be formed, for example, by reacting a compound of the active agent with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. When the compounds are in the forms of salts, they may comprise pharmaceutically acceptable salts.
  • Such salts may include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts.
  • Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like.
  • suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates,
  • hydroxynaphthoates examples include lithium, sodium, potassium, magnesium salts and the like.
  • metal salts include lithium, sodium, potassium, magnesium salts and the like.
  • ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium,
  • organic bases include lysine, arginine, guanidine, diethanolamine, choline and the like.
  • Table 1-11 Coghlan, M.P. et al. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol 7, 793-803 (2000). [0051] Table 1-12. Cross, D.A. et al. Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 77, 94-102 (2001).
  • Table 1-13 Gaisina, I.N. et al. From a natural product lead to the identification of potent and selective benzofuran-3-yl-(indol-3-yl)maleimides as glycogen synthase kinase 3beta inhibitors that suppress proliferation and survival of pancreatic cancer cells. J Med Chem 52, 1853-1863 (2009).
  • Table 1-19 Gould, T.D., Einat, H., Bhat, R. & Manji, H.K. AR-A014418, a selective GSK-3 inhibitor, produces antidepressant-like effects in the forced swim test. Int J Neuropsychopharmacol 7, 387-390 (2004).
  • Table 1-20 Chen, S., Sun, K.X., Liu, B.L., Zong, Z.H. & Zhao, Y.
  • a method of treating an individual having a T-cell mediated disease comprising the step of administering to an individual in need thereof, an effective amount of an inhibitor of glycogen synthase kinase-3 (for example, GSK3 or GSK3a).
  • the T-cell mediated disease may be associated with a loss of Treg immunosuppressive function.
  • the T-cell mediated disease may be an immune disease or an autoimmune disease.
  • the T cell mediated disease may be selected from an immunodeficiency, primary immunodeficiency disease (PID), type I diabetes, systemic lupus erythematosus (SLE), asthma, colitis, psoriasis, bronchiectasis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP) or combinations thereof.
  • the disclosed methods may be used to ameliorate, prevent, or reduce the severity and/or progression of a disease as described herein.
  • the methods may further include the step of identifying an individual having a loss of function (LOF) mutation in at least one Gimap5 allele.
  • LEF loss of function
  • the LOF mutation may be a heterozygous mutation, while in other instances the LOF mutation may be a homozygous mutation.
  • the LOF mutation may be a mutation that causes one or more of a loss of GIMAP5 protein expression, impairment of RNA stability, impairment of transcription factor (TF) binding sites, or a combination thereof.
  • the TF may be one that is a direct target of GSK3 ⁇ or GSK3a regulation.
  • a method of identifying an individual likely to benefit from administration of an inhibitor of glycogen synthase kinase-3 may comprise the step of determining whether a LOF mutation is present in the genome of the individual, wherein detection of an LOF may be performed on a sample isolated from the individual. If a LOF mutation is present, the individual may be administered an effective amount of an inhibitor of glycogen synthase kinase-3 (for example, GSK3 or GSK3a), for example, any inhibitor as described herein.
  • a method of maintaining immune homeostasis in an individual having, or suspected of having, a T-cell mediated disease associated with a loss of Treg immunosuppressive function may comprise the step of administering an inhibitor of glycogen synthase kinase-3 (for example, GSK3 or GSK3a), for example, a GSK3 inhibitor of Table 1 or as otherwise described herein.
  • the inhibitor may be LiCl and may be administered in a dose of about 100 to about 150 mg/kg/day to the individual.
  • the administration step may occur at a time period selected from within a day, within a week, within two weeks, within three weeks, within a month, within two months, within three months, within four months, within five months, within six months, or within a year of diagnosis. In one aspect, the administration step may occur prior to the disease state being characterized as late-stage, such that late-stage disease may be delayed or avoided entirely.
  • active agents provided herein may be administered in a dosage form selected from intravenous or subcutaneous unit dosage form, oral, parenteral, intravenous, and subcutaneous.
  • active agents provided herein may be formulated into liquid preparations for, e.g., oral administration. Suitable forms include suspensions, syrups, elixirs, and the like.
  • unit dosage forms for oral administration include tablets and capsules. Unit dosage forms configured for administration once a day; however, in certain embodiments it may be desirable to configure the unit dosage form for administration twice a day, or more.
  • compositions are isotonic with the blood or other body fluid of the recipient.
  • the isotonicity of the compositions may be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • An example includes sodium chloride.
  • Buffering agents may be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts.
  • Parenteral vehicles include sodium chloride solution, Ringer' s dextrose, dextrose and sodium chloride, lactated Ringer' s or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Viscosity of the pharmaceutical compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent.
  • Methylcellulose is useful because it is readily and economically available and is easy to work with.
  • suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose,
  • the concentration of the thickener will depend upon the thickening agent selected. An amount may be used that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.
  • a pharmaceutically acceptable preservative may be employed to increase the shelf life of the pharmaceutical compositions.
  • Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed.
  • a suitable concentration of the preservative is typically from about 0.02% to about 2% based on the total weight of the composition, although larger or smaller amounts may be desirable depending upon the agent selected. Reducing agents, as described above, may be advantageously used to maintain good shelf life of the formulation.
  • active agents provided herein may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like, and may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • a suitable carrier diluent, or excipient
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • Such preparations may include complexing agents, metal ions, polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components may influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.
  • compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and may include one or more of the following agents: sweeteners, flavoring agents, coloring agents and preservatives.
  • Aqueous suspensions may contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • Formulations for oral use may also be provided as hard gelatin capsules, wherein the active ingredient(s) are mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules.
  • an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin
  • the active agents may be dissolved or suspended in suitable liquids, such as water or an oil medium, such as peanut oil, olive oil, fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • Stabilizers and microspheres formulated for oral administration may also be used.
  • Capsules may include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredient in admixture with fillers such as lactose, binders such as starches, and/or lubrimayts such as talc or magnesium stearate and, optionally, stabilizers.
  • fillers such as lactose, binders such as starches, and/or lubrimayts such as talc or magnesium stearate and, optionally, stabilizers.
  • Tablets may be uncoated or coated by known methods to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time.
  • a time delay material such as glyceryl monostearate may be used.
  • the solid form typically comprises from about 0.001 wt. % or less to about 50 wt. % or more of active ingredient(s), for example, from about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 wt. %.
  • Tablets may contain the active ingredients in admixture with non-toxic pharmaceutically acceptable excipients including inert materials.
  • a tablet may be prepared by compression or molding, optionally, with one or more additional ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubrimayt, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered active agent moistened with an inert liquid diluent.
  • each tablet or capsule contains from about 1 mg or less to about 1,000 mg or more of an active agent provided herein, for example, from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 mg.
  • tablets or capsules are provided in a range of dosages to permit divided dosages to be administered. A dosage appropriate to the patient and the number of doses to be administered daily may thus be conveniently selected.
  • two or more of the therapeutic agents may be incorporated to be administered into a single tablet or other dosage form (e.g., in a combination therapy);
  • Suitable inert materials include diluents, such as carbohydrates, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, starch, and the like, or inorganic salts such as calcium triphosphate, calcium phosphate, sodium phosphate, calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride.
  • Disintegrants or granulating agents may be included in the formulation, for example, starches such as corn starch, alginic acid, sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite, insoluble cationic exchange resins, powdered gums such as agar, karaya or tragamayth, or alginic acid or salts thereof.
  • starches such as corn starch, alginic acid, sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite, insoluble cationic exchange resins, powdered gums such as agar, karaya or tragamayth, or alginic acid or salts thereof.
  • Binders may be used to form a hard tablet. Binders include materials from natural products such as acacia, tragamayth, starch and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, and the like.
  • Lubrimayts such as stearic acid or magnesium or calcium salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol, starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like, may be included in tablet formulations.
  • Surfactants may also be employed, for example, anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, cationic such as benzalkonium chloride or benzethonium chloride, or nonionic detergents such as polyoxy ethylene hydrogenated castor oil, glycerol monostearate, polysorbates, sucrose fatty acid ester, methyl cellulose, or carboxymethyl cellulose.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate
  • cationic such as benzalkonium chloride or benzethonium chloride
  • nonionic detergents such as polyoxy ethylene hydrogenated castor oil, glycerol monostearate, polysorbates, sucrose fatty acid ester, methyl cellulose, or carboxymethyl cellulose.
  • Controlled release formulations may be employed wherein the active agent or analog(s) thereof is incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms. Slowly degenerating matrices may also be incorporated into the formulation. Other delivery systems may include timed release, delayed release, or sustained release delivery systems.
  • Coatings may be used, for example, nonenteric materials such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols, or enteric materials such as phthalic acid esters.
  • Dyestuffs or pigments may be added for identification or to characterize different combinations of active agent doses.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added to the active ingredient(s).
  • Physiological saline solution, dextrose, or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol are also suitable liquid carriers.
  • the pharmaceutical compositions may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, such as olive or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof.
  • Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragamayth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as
  • polyoxyethylene sorbitan mono-oleate polyoxyethylene sorbitan mono-oleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Pulmonary delivery of the active agent may also be employed.
  • the active agent may be delivered to the lungs while inhaling and traverses across the lung epithelial lining to the blood stream.
  • a wide range of mechanical devices designed for pulmonary delivery of therapeutic products may be employed, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
  • These devices employ formulations suitable for the dispensing of active agent. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy.
  • the active ingredients may be prepared for pulmonary delivery in particulate form with an average particle size of from 0.1 um or less to 10 um or more, for example, from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 ⁇ to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 ⁇ .
  • Pharmaceutically acceptable carriers for pulmonary delivery of active agent include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol.
  • ingredients for use in formulations may include DPPC, DOPE, DSPC, and DOPC.
  • Natural or synthetic surfactants may be used, including polyethylene glycol and dextrans, such as cyclodextran.
  • Bile salts and other related enhancers, as well as cellulose and cellulose derivatives, and amino acids may also be used.
  • Liposomes, microcapsules, microspheres, inclusion complexes, and other types of carriers may also be employed.
  • compositions suitable for use with a nebulizer typically comprise the active agent dissolved or suspended in water at a concentration of about 0.01 or less to 100 mg or more of active agent per mL of solution, for example, from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution.
  • the formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure).
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the active agent caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the active ingredients suspended in a propellant with the aid of a surfactant.
  • the propellant may include conventional propellants, such as
  • Example propellants include trichlorofluoromethane, dichlorodifluoromethane,
  • Suitable surfactants include sorbitan trioleate, soya lecithin, and oleic acid.
  • Formulations for dispensing from a powder inhaler device typically comprise a finely divided dry powder containing active agent, optionally including a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in an amount that facilitates dispersal of the powder from the device, typically from about 1 wt. % or less to 99 wt. % or more of the formulation, for example, from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. % of the formulation.
  • a bulking agent such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in an amount that facilitates dispersal of the powder from the device, typically from about 1 wt. % or less to 99 wt. % or more of the formulation, for example, from about
  • an active agent provided herein may be administered by intravenous, parenteral, or other injection, in the form of a pyrogen-free, parenterally acceptable aqueous solution or oleaginous suspension.
  • Suspensions may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The preparation of acceptable aqueous solutions with suitable pH, isotonicity, stability, and the like, is within the skill in the art.
  • a pharmaceutical composition for injection may include an isotonic vehicle such as 1,3- butanediol, water, isotonic sodium chloride solution, Ringer' s solution, dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or other vehicles as are known in the art.
  • an isotonic vehicle such as 1,3- butanediol, water, isotonic sodium chloride solution, Ringer' s solution, dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or other vehicles as are known in the art.
  • sterile fixed oils may be employed conventionally as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono or diglycerides.
  • fatty acids such as oleic acid may likewise be used in the formation of injectable preparations.
  • the pharmaceutical compositions may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the
  • the duration of the injection may be adjusted depending upon various factors, and may comprise a single injection administered over the course of a few seconds or less, to 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more of continuous intravenous administration.
  • the active agents provided herein may be provided to an administering physician or other health care professional in the form of a kit.
  • the kit is a package which houses a container which contains the active agent(s) in a suitable pharmaceutical composition, and instructions for administering the pharmaceutical composition to a subject.
  • the kit may optionally also contain one or more additional therapeutic agents currently employed for treating one or more disease states as described herein.
  • a kit containing one or more compositions comprising active agents provided herein in combination with one or more additional active agents may be provided, or separate pharmaceutical compositions containing an active agent as provided herein and additional therapeutic agents may be provided.
  • the kit may also contain separate doses of an active agent provided herein for serial or sequential administration.
  • the kit may optionally contain one or more diagnostic tools and instructions for use.
  • the kit may contain suitable delivery devices, e.g., syringes, and the like, along with instructions for administering the active agent(s) and any other therapeutic agent.
  • the kit may optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included.
  • the kits may include a plurality of containers reflecting the number of
  • GTPase of immunity-associated protein 5 (Gimap5) is linked with lymphocyte survival, autoimmunity and colitis, but its mechanisms of action are unclear.
  • Gimap5 is essential for inactivation of glycogen synthase kinase-3 (GSK3 ) following T cell activation.
  • GSK3 glycogen synthase kinase-3
  • constitutive GSK3 activity constrains c-Myc induction and NFATcl nuclear import, thereby limiting CD4 + T cells to complete cell cycle.
  • Gimap5 facilitates Ser389 phosphorylation and nuclear translocation of GSK3 thereby limiting DNA damage in CD4 + T cells.
  • GTPase of immunity-associated protein 5 (Gimap5) is linked with lymphocyte survival, immune homeostasis and (auto-)immune disease.
  • polymorphisms in human GIMAP5 are associated with increased risk of islet autoimmunity in type 1 diabetes (T1D), systemic lupus erythematosus (SLE) 1, 2, 3 , and asthma 4 .
  • Mice and rats with complete loss-of-function (LOF) mutations have reduced lymphocyte survival, loss of immunological tolerance predisposing to autoimmunity and colitis, and abnormal liver pathology resulting from persistent post-natal extramedullary hematopoiesis 5, 6 ⁇ 7 ⁇ 8 ⁇ 9 ⁇ 10, ⁇ ⁇ 12, 13 ' 14 .
  • LEF loss-of-function
  • Gimap proteins are predominantly expressed in lymphocytes and regulate lymphocyte survival during development, selection, and homeostasis 15 .
  • Members of this family share a GTP-binding AIG1 homology domain 16, 17 and seem to be localized to different subcellular compartments, with Gimap5 localizing in multivesicular bodies (MVB) and lysosomes 18 .
  • MVB multivesicular bodies
  • sphinx mice which have a missense mutation in Gimap5 that results in what is essentially a null allele 6 .
  • Gimap5 sph/sph mice progressively lose CD4+ T cells and B cells, an effect that is associated with reduced regulatory T (Treg) cell function, while remaining CD4+ T cells have an activated phenotype, but have an impaired capacity to proliferate 5, 6 .
  • These immunologic defects result in spontaneous and lethal colitis that is preventable with CD4+ T cell depletion, Treg cell transplantation, or antibiotic therapy 5, 6 .
  • the cell-intrinsic defects in Gimap5 sph/sph CD4+ T cells persist.
  • livers from Gimap5 sph/sph mice have an abnormal morphology with extramedullary hematopoiesis and associated foci of hematopoietic cells and hepatocyte apoptosis 6, 7 ⁇ 8 .
  • the family of glycogen synthase kinases-3 includes constitutively active protein serine/threonine kinases encoded by two genes, Gsk3a and Gsk3b.
  • GSK3 has an essential function in T cell differentiation and proliferation 19, 20, 21, 22 .
  • GSK3 phosphorylates a variety of substrates, often within the phosphodegron domains, thereby regulating protein ubiquitination/degradation and their activity 23 .
  • the substrates targeted directly by GSK3 are c-Myc, NFATcl, Mcl-1, and ⁇ -catenin 24 .
  • Studies have established that upon antigen- specific activation of T cells, GSK3 activity is inhibited 19 ' 20 ' 21 ' 22 , thereby facilitating T cell activation and proliferation.
  • GSK3 inhibition 25 ' 26 Phosphorylation of GSK3 at residues Ser9 or Ser389 and physical separation of GSK3 from its target proteins through vesicle association have been proposed as mechanisms of GSK3 inhibition 25 ' 26 .
  • Inhibition of GSK3 by phosphorylation of Ser389 is essential for lymphocyte viability upon double-stranded DNA breaks (DSB) evident during V(D)J recombination in thymic T cell development or in B cells undergoing immunoglobulin class switch recombination (CSR) 27 .
  • DSB double-stranded DNA breaks
  • CSR immunoglobulin class switch recombination
  • Gimap5 is a critical inhibitor of GSK3 in both human and mouse CD4+ T cells, affecting c-Myc, and Mcl-lexpression, NFATcl nuclear translocation, and T cell fitness by controlling the DNA damage response occurring during T cell proliferation.
  • Gimap5 sph/sph mice have normal thymic output of CD4+ T cells
  • Applicant next assessed if reduced thymic output of CD4+ T cells might contribute to lymphopenia in Gimap5 sph/sph mice, and quantified the presence of recent thymic emigrants (RTE) 32 in spleen of WT and Gimap5 sph/sph mice. Importantly, Applicant found no marked differences in the frequency of splenic RTE as defined by CD24hi CD4+ T cells between 3-week-old WT or Gimap5 sph/sph mice (FIG 10B). These data are in line with Applicant's previous studies showing Gimap5 sph/sph mice have a relatively normal thymic development of CD4+ T cells 6 .
  • Applicant next focused on the peripheral survival of CD4+ T cells in Gimap5 sph/sph mice. Applicant considered that either post-thymic survival of Gimap5 sph/sph CD4+ T cells or TCR-induced activation contributes to the loss of CD4+ T cells in the periphery. The latter would be consistent with Applicant' s previous studies showing that Gimap5 sph/sph T cells failed to proliferate after TCR stimulation with aCD3/aCD28 +/- IL-2 6 . Moreover, a progressive loss of CD4+ T cells is observed post-weaning— a period in which the CD4+ T cell compartment has to cope with marked changes in gut microbial antigens.
  • Applicant next sought to identify whether the cell-intrinsic apoptosis pathways contributed to the reduced CD4+ T cell survival in Gimap5 sph/sph mice.
  • Applicant crossed the Gimap5 sph/sph allele onto a Bim _/ ⁇ deficient background.
  • Gimap5 sph/sph CD4+ T cells compared to WT controls after 24h of stimulation with aCD3/aCD28 (FIG HE).
  • aCD3/aCD28 aCD3/aCD28
  • Gimap5 sph/sph CD4+ T cells have abnormal GSK3 activity
  • TF transcription factor
  • c-Myc is a TF necessary for the metabolic programming of T cells following activation and is required for optimal T cell proliferation 34 ' 35 .
  • Gimap5 sph/sph CD4+ T cells were comparable to WT controls, suggesting that reduction in protein levels was the result of changes in post-translational regulation (Fig.2B).
  • Post- translational regulation of c-Myc is mediated in part by the family of GSK3 proteins, which is comprised of constitutively active protein serine/threonine kinase paralogs GSK3a and GSK3 .
  • GSIGa/ ⁇ phosphorylate c-Myc at Thr58, priming it for ubiquitination and subsequent proteasomal degradation 36 .
  • GSK3 plays an essential role in T cell differentiation and proliferation. Specifically, upon antigen-specific activation of T cells, GSK3 activity is inhibited 19, 20, 21 ' 22, 37 , thereby facilitating T cell activation and proliferation. GSK3 phosphorylates a variety of substrates, regulating protein ubiquitination/degradation and/or their activity 23 . Among the substrates targeted by GSK3 are c-Myc, Mcl-1, NFATcl, and ⁇ - catenin 24 ' 38 ' 39 ' 40 - 41 .
  • Elevated GSK3 activity in stimulated Gimap5 s P h/s P h CD4+ T cells would account for the reduced protein levels of Mcl-1(FIG 1 IE), which is targeted for proteasomal degradation upon phosphorylation by GSK3 39 ' 40 .
  • aCD3/aCD28-stimulated Gimap5 sph/sph CD4+ T cells also showed a slight reduction in ⁇ -catenin protein expression, but normal ⁇ -catenin mRNA levels (FIG 13A, 13B), consistent with an elevated GSK3 activity.
  • CD4+ T cells from Gimap5 sph/sph mice completely regained their proliferative capacity in vitro and showed comparable survival to WT CD4+ T cells (Fig.2E).
  • NFATcl nuclear translocation of NFATcl, a transcription factor necessary for productive T cell activation (FIG 13D) and regulated by GSIGa/ ⁇ 42 ⁇ 43 .
  • GSK3 phosphorylation of NFATcl regulates its localization rather than stability 38 41 .
  • Applicant stimulated WT and Gimap5 sph/sph CD4+ T cells with aCD3/aCD28 and examined NFATcl nuclear localization by ImageStream analysis using the similarity dilate algorithm (see Methods section).
  • Applicant quantified vesicular GSK3 intensity, the number of vesicles, and the size of GSK3 -associating vesicles in WT and Gimap5 sph/sph CD4+ T cells at 0, 6, and 24 hours after CD4+ T cell stimulation.
  • GSK3 became associated with punctate vesicles in WT cells and GSK3 intensity, vesicle number, and vesicle size were markedly decreased in Gimap5 s P h/s P h CD4+ T cells after 24- hours stimulation (Fig.3A), indicating that physical segregation of GSK3 from cytosolic target proteins 25 ' 44 45 may provide a mechanism of inhibition.
  • studies showed limited association between Rab5, Rab7 or Lampl expression and Gimap5/GSK3 DP vesicles (FIG 14A-14E).
  • Applicant examined WT and Gimap5 sph/sph CD4+ T cells by high-resolution, three-dimensional confocal microscopy, enabling us to visualize Gimap5- GSK3 colocalization in the axial as well as xy planes. Consistent with the ImageStream analysis, GSK3 colocalized with Gimap5 in stimulated WT CD4+ T cells in punctate vesicles (Fig.3C).
  • GSK3 vesicular sequestration has predominantly been described in the context of Wnt signaling 25 .
  • Applicant incubated WT CD4+ T cells either directly with Wnt3a or examined blocking of Wnt secretion by incubating activated CD4+ T cells in the presence/absence of rWP-2 (a Wnt secretion inhibitor 46 47 ).
  • Applicant assessed GSK3 sequestration using ImageStream and determined the proliferation capacity of WT CD4+ T cells.
  • Neither Wnt3a nor IWP-2 affected the vesicular sequestration in WT CD4+ T cells or impacted T cell proliferation (FIG 15A-15D).
  • Phosphorylation at residue Ser9 26 and Ser389 27 have been proposed as mechanisms of GSK3 inhibition.
  • Phosphorylation of GSK3 at Ser9 is mediated by AKT (and possibly other kinases) following TCR signaling 48
  • inhibition of GSK3 by phosphorylation of Ser389 is linked with lymphocyte fitness in the context of DNA DSB observed either during V(D)J recombination in thymic T cells or in B cells undergoing immunoglobulin class switch recombination (CSR) 27 .
  • CSR immunoglobulin class switch recombination
  • Gimap5 sph/sph mice with LiCl 150 mg/kg in drinking water, ad libitum) starting at 3 weeks of age until 8 weeks of age, by which time severe colitis can be observed.
  • LiCl treatment of Gimap5 sph/sph mice effectively maintained the CD4+ T cell and B cell populations in
  • Applicant purified regulatory T cells from the spleens of vehicle- and LiCl- treated WT and Gimap5 sph/sph mice and cocultured them with aCD3-stimulated WT CD8+ T cells. Strikingly, while Treg cells from vehicle-treated Gimap5 sph/sph mice were incapable of suppressing CD8+ T cell proliferation in vitro, Treg cells from LiCl-treated Gimap5 sph/sph mice demonstrated normal suppressive capacity (Fig.5D). Importantly, LiCl treatment completely prevented colitis and significantly reduced crypt loss and leukocyte infiltration (Fig.5E-5F and FIG 17A).
  • GSK3 consists of two ubiquitously expressed paralogs, GSK3a and GSK3 ; to assess selectivity and importance of GSK3 specifically in the CD4+ T cell-mediated pathology, Applicant next examined whether genetic ablation of GSK3 in CD4+ T cells was sufficient to prevent loss of CD4+ T cells and immunopathology in Gimap5 sph/sph mice. To test this hypothesis, Applicant generated
  • WT and Gimap5 sph/sph on a GskS TM or Gsk3 WT/WT background were treated with tamoxifen starting at 3 weeks of age. At 8 weeks of age, mice were characterized for CD4+ T cell survival and their phenotype. Tamoxifen treatment resulted in an effective ablation of GSK3 expression in
  • CD4+ T cells (FIG 18 A) and increased survival of CD4+ T (but not B) cells in the periphery
  • GSK3-inhibitors offer therapeutic potential to correct the immunodeficiency and prevent immune pathology.
  • Applicant next determined whether activation of GIMAP5 "7" CD4+ T cells was associated with increased DNA damage, by comparing the level of ⁇ 2 ⁇ in CD4+ T cells from control and patient cells. After 2 days of re- stimulation with aCD3/aCD28, a significant increase in intensity of ⁇ 2 ⁇ was observed in patient CD4+ T cells compared to control T cells upon activation (Fig.8I,J). Together these data reveal a striking similarity between the molecular pathways in mouse and human T cells that are affected by loss of GIMAP5 function.
  • Applicant showed that pharmacological targeting of GSK3 or genetic deletion of Gsk3 corrects T lymphocyte survival and prevents severe early- onset colitis in Gimap5 -deficient mice. Moreover, Applicant describes a human patient with a GIMAP5 LOF mutation. The patient presents with an immunodeficiency strikingly similar to Gimap5 -deficient mice, including the development of lymphopenia, reduction in TFs such as c-Myc, as well as increased DNA damage and reduced survival upon T cell activation.
  • GSK3 inhibitors can restore accumulation of c-Myc protein and improve T cell survival during activation in vitro.
  • GSK3 is constitutive active kinase in resting T cells and has a large number of reported targets (>100) that include important signaling components and TFs important for growth and suvival 50 . These include but are not limited to c-Myc, ⁇ -catenin and NFATcl, key TFs required for growth and activation of the transcriptional program in T cells 34, 51 ' 52 .
  • Applicant's studies identify an important function for GSK3 in limiting DNA damage during CD4+ T cell proliferation. The latter could very well be mediated by direct effect of GSK3 ⁇ on components such as p53, Mcll but also Mdm2, a GSK3 ⁇ target that is an important negative regulator of p53 53 .
  • Gimap5 sph/sph CD4+ T cells after 2 days stimulation suggest that Gimap5 facilitates the subcellular localization of GSK3 and imply that the nuclear translocation of GSK3 is required for a productive T cell response.
  • GSK3 While dysregulation of GSK3 may be the most striking phenotype observed in the absence of Gimap5, it may not be the only effect of Gimap5 deficiency.
  • Gimap5's homology to dynamin raises the possibility that it may be more broadly involved in vesicular transport, rather than restricted solely to the sequestration of GSK3 .
  • GSK3 dysregulation has more subtle effects than activation-induced T cell death.
  • Applicants have previously shown that a subset of highly pathogenic memory-like CD4+ T cells survives and drive colitis in the Gimap5 sph/sph mouse. These remaining cells have a limited capacity to proliferate but robustly produce ⁇ ⁇ and IL-17a.
  • the accumulation of these cells and concurrent loss in number and function of regulatory T cells in Gimap5-deficient models results in an imbalance between Thl7 and Treg cells that promotes the development of autoimmune conditions, including severe colitis (mice), aggravated EAE (LEW rats), and T1D (BB rats) 5 - 6 ⁇ 10 ⁇ ⁇ ⁇ 63 .
  • Applicant showed that a dysregulation of GSK3 in lymphocytes can lead to a primary immune deficiency, as observed in Gimap5 sph/sph mice and the GIMAP5 LOF patient. Given its importance in maintaining CD4+ T cell homeostasis and
  • Applicant proposes that even a minor dysregulation of GSK3 within the T cell compartment could result in abnormal polarization of CD4+ T cells, leading to preferential differentiation of Thl7 cells over regulatory T cells.
  • a dysregulation may be occurring in autoimmune diseases associated with pathogenic Thl7 cells, such as Crohn's disease, T1D, and multiple sclerosis 70 ' 71 72 , or in Gimap5-associated diseases such as SLE, T1D, and allergic asthma 1 ' 2 ' 3 4 .
  • selective inhibition of GSK3 activity offers a new promising therapeutic approach for treating patients with immunopathogenic T cell responses.
  • T cells depend on their ability to undergo clonal expansion for an efficient immune response during infection or to maintain immune homeostasis in the gut.
  • Applicant's studies reveal a key role for Gimap5 in inactivating GSK3 during CD4+ T cell activation, a link that is critically required to maintain T cell fitness and allows for productive T cell proliferation.
  • Applicant proposes that the Gimap5-mediated inactivation of GSK3 is an essential molecular mechanism to support productive CD4+ T cell responses.
  • Applicant's studies point to a remarkable therapeutic potential for GSK3 inhibitors to improve CD4+ T cell survival/proliferation and prevent immunopathology.
  • GSK3 inhibitors have been used to treat a variety of diseases including Alzheimer's disease, mood disorders, cancer, and diabetes mellitus (for extensive reviews see 73, 74).
  • Applicant's current data reveal a new therapeutic application of GSK3 inhibitors specifically in the treatment of immunodeficient patients that have GIMAP5 LOF mutations. These patients present a strikingly similar phenotype to Gimap5 -deficient mice and suffer from recurrent (viral) infections most likely stemming from an overall lack of T cell fitness.
  • Applicant posits that GSK3-inhibitors will improve overall T cell survival and function and may
  • therapeutic targeting of this pathway may be relevant for the treatment of patients with SNPs in GIMAP5 linked to development of islet autoimmunity in type I diabetes, systemic lupus erythematosus 1, 2 ⁇ 3 , or asthma 4 .
  • ITP thrombocytopenic purpura
  • IVIG gammaglobulin
  • the patient was positive for a direct Coombs test, but negative for anti-neutrophil or anti -platelet antibodies.
  • a bone marrow biopsy demonstrated mild hypocellularity with low myeloid to erythroid ratio.
  • Past medical history includes chickenpox infection following varicella vaccine at the age of 11, and shingles at the age of 14. He has suffered and continuous to suffer from extensive warts since the age of 12, which requires frequent cryotherapy.
  • Lymphocyte subset revealed CD3: 273 (62%), CD4: 246 (56%), CD8: 30 (7%), CD19: 135 (31%), CD16/56: 20 (7%) cells/mcL.
  • CD4 to CD8 ratio was 8.2.
  • Serum IgG, A, E levels and IgG subgroups were normal, except low IgM (IgG: 839, IgA: 70, IgM: 54 mg/dl, IgE: 282 IU/ml).
  • Titers to protein antigens were normal and 2/14 pneumococcal titers were protective.
  • B cell panel demonstrated normal proportion of naive, transitional, and isotype switched CD27+ memory B cells.
  • naive CD4 cells (11.9, N: 33-73.5% for age) with a normal proportion of CD31+ T cells (indicative of recent thymic emigrants), increased memory CD4 cells (88.1, N: 26.3-66.3%) and increased CD4 T-effector memory cells (80.2, N: 33.4-74.1%).
  • Naive CD8 cells were normal and CD8 TEMRA cells were slightly increased (11.2, N:0-10.3%). The proportion of TCRo$ expression was slightly low on CD 8 cells.
  • telomere length measurement revealed low telomere length of total lymphocytes, granulocytes, naive and memory T cells, but normal telomere length of B cells and NK cells. The patient was given a working diagnosis of immunodeficiency with features of autoimmunity. Mutations in known SCID genes, as well as LRBA, PI3KCD, CTLA-4, and STAT3 genes were excluded.
  • Whole exome sequencing revealed a homozygous c.611T>C (p.Leu204Pro) variant in GIMAP5, where his parents were found to be heterozygous.
  • mice were used for both male and female mice. All strains of mice used were generated on a C57BL/6J background and confirmed by whole genome SNP analysis and Applicant anticipated limited genetic variation. To minimize confounding secondary factors arising from lymphopenia and other late-stage pathologies that develop in Gimap5 sph/sph mice, CD4+ T cells from 3 -week-old mice were used unless otherwise noted. At this age,
  • Gimap5 sph/sph mice have relatively normal numbers of CD4+ T cells with a normal frequency of naive T cells that have a quiescent phenotype comparable to WT mice. Since Applicants were interested in T cell proliferation/survival in WT vs Gimap5-deficient cells, Applicant expected to find strong differences in measurement (e.g. normal vs absence of T cell survival/proliferation). In cases where the differences in the observed phenomena were clear and distinct, a group size of 6 mice was suitable to give statistically significant (i.e. P ⁇ 0.05) data.
  • Applicant estimated that, with a sample size of 6, Applicant would have a 99% power to detect at least 25% reduction in the % of proliferating cells in stimulated Gimap5 sph/sph cells with a significance level (alpha) of 0.05 (two-tailed).
  • mice were obtained from Jackson. Gimap5 s P h/s P h mice were generated as described6 and bred in-house to generate WT or Gimap5 sph/sph ;Rag2 _/ ⁇ ;OT-II, WT or Gimap5 s P h/s P h ;Tg (Cd4-cre/ert2) 1 IGnri/J; Gsk3 fl/fl mice in the vivarium of Cincinnati Children's Hospital. All mice were maintained under specific pathogen- free conditions.
  • a-mouse-CD3 (17A2) and a-mouse-CD28 (37.51) antibodies were used for murine T cell activation.
  • purified aCD3 (OKT3) and aCD28 (CD28.2) antibodies were used.
  • 7-AAD was purchased from BD.
  • Ovalbumin, LiCl, 6-Bromoindirubin-3'-oxime (BIO), Ionomycin, and phytohemagglutinin-L (PHA-L) were obtained from Sigma. All antibodies used for flow cytometry were purchased from eBioscience or Biolegend unless otherwise noted.
  • Antibodies for ⁇ , pERKl/2, pJNK, pAKT, p-p38, and pGSK3 were purchased from Cell Signaling Technologies. Monoclonal antibody MAC421 was used to probe for Gimap5 as described6. GSK3 was probed with monoclonal antibody Clone 7/GSK-3b from BD.
  • lymphocytes from spleen and mesenteric lymph node were isolated and stained with fluorochrome- conjugated antibodies for mouse CD4, CD8, B220, CD24, CD44, CD62L, and Foxp3.
  • samples were stained with a fixable viability dye; analysis was restricted to live cells unless otherwise stated.
  • CD4+ T cells were stimulated with plate-bound aCD3 (1 ⁇ g/ml) + soluble aCD28 (2 ⁇ g/ml).
  • Cells were cultured in supplemented IMDM medium containing 10% FBS, 2% penicillin/streptomycin, 1% L-glutamine, and 50 ⁇ BME.
  • T cell proliferation was quantified by incubating CD4+ T cells in 5 ⁇ CFSE in 0.2% FBS for 5 min. Cells were either left unstimulated or stimulated in the presence of GSK3 inhibitors LiCl (2.5 mM) or BIO (100 nM).
  • CD4+ T cells Early phosphorylation of AKT, p38, ERK, and JNK, and the degradation of ⁇ was also evaluated in CD4+ T cells upon stimulation with PMA (50 ng/mL) and Ionomycin (750 ng/mL). Ex vivo survival of CD4+ T cells was evaluated by culturing Mojo-purified peripheral CD4+ T cells or total thymocytes with 5 ng/mL IL-7 for 0-7 days. The number of surviving cells was determined by staining for CD4 and viability and counted on a flow cytometer using counting beads (Biolegend).
  • Protein lysates from human T cells were prepared according to standard methods from resting cells or cells stimulated with 5 ⁇ g/mL aCD3 + 2 ⁇ g/mL aCD28 + 5mM LiCl for 24h.
  • Mojo-isolated (BioLegend) CD4+ T cells were stimulated with aCD3 and aCD28 + 2.5 mM LiCl for the indicated time periods prior to preparing protein lysates.
  • 10 ⁇ of proteasomal inhibitor MG132 was added 4h prior to lysis of the cells.
  • Lysates were separated using 10% Bis-Tris Gels, transferred to nitrocellulose, and immunoblotted with primary antibodies to GIMAP5 (CST 14108), phospho-c-Myc (T58) (MyBioSource), c-Myc, pGSK3 (S389) (EMD
  • CD4+ T cells were stimulated with aCD3 + aCD28. After 6, 24h, or 48h CD4+ T cells were stained with antibodies to CD4 (GK1.5), Gimap5 (MAC421), GSK3 (BD 610202), pGSK3 (S389) (EMD Millipore 07- 2275), DAPI, and a fixable viability dye. In indicated experiments, 200 ng/niL Wnt3a and 2 ⁇ IWP-2 were added. For NFATcl localizations studies, isolated CD4+ T cells were stimulated indicated times. Cells were stained with antibodies for CD4 and NFATcl (clone 7A6), DAPI, and a fixable viability dye.
  • Live CD4+ T cells were analyzed for NFATcl and DAPI localization by delineating regions of positive signal (i.e. a mask). Nuclear translocation was measured by similarity dilate, which represents the log transformed Pearson's Correlation Coefficient and is a measure of the degree to which two images are linearly correlated within a masked region.
  • CD4+ T cells were isolated from the PBMCs of healthy donors by MACS-purification (Miltenyi Biotec) and stimulated with aCD3 (5 ⁇ g/ml) and aCD28 (2 ⁇ g/ml).
  • T cells were stained with antibodies to CD4, GIMAP5 (CST 14108), GSK3 (BD 610202), CD107b (Miltenyi Biotec), and a fixable viability dye.
  • GSK3 vesicular localization in patient and control CD4+ T cells was measured upon re- stimulation of resting T cells after IL-2 expansion.
  • Colocalization, vesicular localization, and nuclear localization was evaluated using ImageStream Data Exploration and Analysis Software (IDEAS) 6.1 as previously described 75 . Specifically, colocalization was quantified using the Bright Detail Similarity representing the log transformed Pearson' s correlation coefficient of the localized bright spots with a radius of 3 pixels or less within the masked area in the two input images.
  • IDEAS ImageStream Data Exploration and Analysis Software
  • CD4+ T cells isolated from the spleen and lymph nodes of WT and Gimap5 sph/sph mice were stimulated with aCD3/aCD28. After 24h, cells were stained with antibodies for Gimap5 (MAC421) and GSK3 (BD61202, 0.7ng / lxlO 6 cells). Cells were counterstained with DAPI (1 ⁇ g/mL) and mounted in Prolong Gold anti-fade reagent (Cell Signaling Technology). Samples were imaged on a Nikon Al LUN-V inverted microscope using a lOOx objective with oil immersion.
  • Z-stacks were generated by taking images at a 0.125 ⁇ step. To refine localization, images were deconvolved using the Landweber algorithm (15 iterations) in NIS Elements v4.5 (Nikon). Z-stacks were assembled and GSK3 and Gimap5 localization assessed in Imaris Image Analysis software v8.3 (Bitplane).
  • Splenocytes isolated from individual WT or Gimap5 sph/sph mice were stained with Indo-1 (2 ⁇ g/mL) at lxlO 6 cells/mL for 30 min at 37°C. Cells were then stained for CD4 and rested for >lh in cell loading medium (HBSS + ⁇ CaC12 + ⁇ MgC12 + 1% FBS) at room temperature. Five minutes prior to analysis, cells were warmed to 37°C. To evaluate calcium flux, samples were acquired for ⁇ 30s without stimulation and an additional 270s after stimulation with aCD3/aCD28 (1.25 ⁇ g/mL) or lonomycin (800 ng/mL) for a total of 5 minutes.
  • HBSS + ⁇ CaC12 + ⁇ MgC12 + 1% FBS cell loading medium
  • CD4+ T cells were rested or stimulated with aCD3/aCD28 for 24h.
  • Cells were lysed with TRIzol (Thermo Fisher Sci), mRNA isolated, and reverse transcription performed using a High-capacity cDNA Reverse
  • cDNAs were amplified with LightCycler 480 SYBR Green I Master (Roche) and quantified by Light-Cycler 480-11 instrument (Roche).
  • the following primer pairs were used: myc, forward: 5'-ATGCCCCTCAACGTGAACTTC-3' (SEQ ID NO: 1), reverse: 5 ' -GTCGCAGATGAAATAGGGCTG-3 ' (SEQ ID NO: 2); wnt3a, forward: 5 ' -CTCCTCTCGGATACCTCTTAGTG-3 ' (SEQ ID NO: 3), reverse: 5'- CCAAGGACCACCAGATCGG-3 ' (SEQ ID NO: 4); ctnnbl ( ⁇ -catenin), forward: 5'- ATGGAGCCGGACAG AAAAGC-3 '(SEQ ID NO: 5), reverse: 5'- CTTGCCACTCAGGGAAGGA-3 ' (SEQ ID NO: 6); L32, forward: 5'- GAAACT
  • GGATCTGGCCCTTGAACCTT-3 ' (SEQ ID NO: 8). Expression of myc, wnt3a, and ctnnbl was normalized to L32 and set relative to unstimulated WT samples.
  • mice were administered 150 mg/kg LiCl in drinking water. After 4-5 weeks, liver damage, colitis, and lymphocyte populations were evaluated by histology and flow cytometry, respectively. Animals were assigned to treatment and vehicle groups randomly, with equal numbers of male and female mice assigned to each group. For these experiments, WT and Gimap5 sph/sph genotypes were co-housed during treatment. Investigators were blinded to mouse genotype and treatment status during analysis.
  • GSK3 hyperactivation in Gimap5 sph/sph CD4+ T cells was evaluated by crossing Gimap5 sph/sph ; Gsk3 fl/fl mice to Cd4cre-ert2 mice, allowing for the tamoxifen- inducible genetic deletion of Gsk3 in CD4+ T cells. Tamoxifen was administered in food to 3-week-old mice (40mg/kg body weight; Harlan Laboratories Teklad Diets). At 8 weeks, liver damage, colitis, and lymphocyte populations were evaluated by histology and flow cytometry respectively.
  • Colon tissue was collected and immediately fixed in 10% buffered formalin solution overnight, followed by routine paraffin embedding. Hematoxylin and eosin staining were performed on 4 ⁇ sections from the paraffin-embedded tissue blocks for conventional light microscopy analysis. Histological scoring was performed double-blind as described before (5, 6). Briefly, scoring parameters included quantitation of the area of distal colon involved, edema, erosion/ulceration of the epithelial monolayer, crypt loss/damage, and infiltration of immune cells into the mucosa. Severity for the area involved
  • Treg cell suppression assays were performed as described using Treg cells isolated from the spleens of WT and Gimap5 sph/sph mice treated with either LiCl or vehicle (5,6).
  • CD4+ T cells were enriched by magnetic separation (Mojo, Biolegend) and stained for viability, CD4, and CD25.
  • Live CD4+CD25+ regulatory T cells were isolated by FACS using a Beckman Coulter MoFlo XDP cell sorter. Sorted Treg cells were cocultured with the indicated ratios with 5xl0 4 CTV-labeled CD8+ T cells isolated from a naive mouse.
  • lxlO 5 T cell depleted, gamma-irradiated (1500 rad) splenocytes were also cocultured as bystander cells.
  • CD8+ T cells were stimulated with 0.5 ⁇ g/mL aCD3;
  • PBMCs were isolated from whole blood of a GIMAP5-deficient patient and a healthy parent by Ficoll-Paque Plus density gradient centrifugation. Isolated PBMCs were cultured in RPMI, 10% FBS, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 2mM L-glutamine, and 10 mM HEPES and were stimulated with 5 ⁇ g/mL phytohemagglutinin (PHA-L) + 5mM LiCl.
  • PHA-L phytohemagglutinin
  • T cells were selectively expanded in 150 U/mL rhIL-2 (Miltenyi) + 5mM LiCl for 8 days. Expanded T cells were rested for 2d in the absence of IL-2 and LiCl before use in reactivation experiments. Prior to expansion, patient's PBMCs were stained for CD 19, CD3, CD4, CD8, CD16, CD56, and Foxp3 and analyzed by flow cytometry for evaluation of circulating lymphocyte populations.
  • Rested patient T cells were restimulated with 5 ⁇ g/mL aCD3 and 2 ⁇ g/mL aCD28. After 24, 48, and 72h stimulation, cells were stained for viability, CD3, CD4, CD8, and ⁇ 2 ⁇ and analyzed by flow cytometry. At 24 and 48h, cells were stained with a- CD4, ⁇ - ⁇ 2 ⁇ , DRAQ5, and a fixable viability dye. Analysis was performed on live CD4+ T cells using ImageStream flow cytometry as detailed above.
  • Nitta T et al. IAN family critically regulates survival and development of T lymphocytes.
  • Poirier GM et al. Immune-associated nucleotide- 1 (IAN-1) is a thymic selection marker and defines a novel gene family conserved in plants. JImmunol 163, 4960-4969 (1999).
  • Glycogen synthase kinase-3beta (GSK3beta) binds to and promotes the actions of p53. J Biol Chem 278, 48872-48879 (2003).
  • Calcineurin inhibitors decrease DNA repair and apoptosis in human keratinocytes following ultraviolet B irradiation. J Invest Dermatol 125, 1020-1025 (2005).

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Abstract

L'invention concerne des inhibiteurs de glycogène synthase kinase-3 (par exemple, des inhibiteurs de GSKβ ou GSKα) destinés à être utilisés dans la thérapie d'une maladie médiée par les lymphocytes T chez un individu en ayant besoin. La maladie médiée par les lymphocytes T peut être une maladie associée à une perte de la fonction immunosuppressive des Treg, par exemple, une maladie immunitaire ou une maladie auto-immune telle que, par exemple, une immunodéficience, une maladie d'immunodéficience primaire (MIP), le diabète de type I, le lupus érythémateux disséminé (LED), l'asthme, la colite, le psoriasis, la bronchiectasie, l'anémie hémolytique auto-immune, le purpura thrombocytopénique idiopathique (PTI) ou des combinaisons de ceux-ci. Les compositions et/ou les méthodes de l'invention peuvent être utilisées pour améliorer, prévenir ou réduire la gravité et/ou la progression de toute maladie susmentionnée.
PCT/US2018/043835 2017-07-26 2018-07-26 Compositions et méthodes pour le traitement d'une réponse immunitaire anormale par l'inhibition de gsk WO2019023426A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115197167A (zh) * 2022-07-22 2022-10-18 中国药科大学 1,2,4-噻二唑烷-3,5-二酮化合物及其制备方法和应用
WO2023175177A1 (fr) * 2022-03-18 2023-09-21 Institut Curie Diagnostic différentiel de la maladie de crohn et de la colite ulcéreuse
WO2023230507A3 (fr) * 2022-05-27 2023-12-28 Yale University Exploitation de complexes ikzf:beta-caténine dans le traitement de maladies ou d'affections associées aux lymphocytes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090306045A1 (en) * 2005-12-22 2009-12-10 Ira Mellman Inhibition of Glycogen Synthase Kinase and Methods of Treating Autoimmune or Immune Inflammatory Disease
US20140163079A1 (en) * 2011-07-06 2014-06-12 President And Fellows Of Harvard College Diphosphate mimetics and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090306045A1 (en) * 2005-12-22 2009-12-10 Ira Mellman Inhibition of Glycogen Synthase Kinase and Methods of Treating Autoimmune or Immune Inflammatory Disease
US20140163079A1 (en) * 2011-07-06 2014-06-12 President And Fellows Of Harvard College Diphosphate mimetics and uses thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023175177A1 (fr) * 2022-03-18 2023-09-21 Institut Curie Diagnostic différentiel de la maladie de crohn et de la colite ulcéreuse
WO2023230507A3 (fr) * 2022-05-27 2023-12-28 Yale University Exploitation de complexes ikzf:beta-caténine dans le traitement de maladies ou d'affections associées aux lymphocytes
CN115197167A (zh) * 2022-07-22 2022-10-18 中国药科大学 1,2,4-噻二唑烷-3,5-二酮化合物及其制备方法和应用
CN115197167B (zh) * 2022-07-22 2023-07-28 中国药科大学 1,2,4-噻二唑烷-3,5-二酮化合物及其制备方法和应用

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