Leukocyte-specific cell permeability molecule

Abstract

Disclosed herein is a leukocyte-targeting molecule, a pharmaceutical composition comprising the leukocyte-targeting molecule and any agent, and a method of using them.In some embodiments, the leukocyte-targeting molecule is a peptide having the formula: X1X2AAX3AX4X5X17AX6X7X8AX9X10A(P)nX11X12(X13)n, or a pharmaceutically acceptable salt thereof, wherein X1, X2, X11, and X12 are each independently lysine, arginine, or ornithine, X3, X4, X5, X6, X7, X8, X9, and X10 are each independently valine, leucine, isoleucine, or norleucine, X13 is tyrosine, X17 is praline or alanine, and n is 0 or 1.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 236,187, filed August 23, 2021, and U.S. Provisional Patent Application No. 63 / 341,253, filed May 12, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The claimed invention relates to leukocyte-targeting molecules and pharmaceutical compositions containing same. Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein are leukocyte-targeting molecules, and compositions comprising the leukocyte-targeting molecules and an optional agent.

[0004] In some embodiments disclosed herein, the compound has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein X 1 , X 2 , X 11 , and X 12 are each independently lysine, arginine, or ornithine; X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , and X 10 are each independently valine, leucine, isoleucine, or norleucine, and X 13 is tyrosine, and X 17 is proline or alanine, and n is 0 or 1. In some embodiments, X 3and X 6 is valine. In some embodiments, X 4 , X 5 , X 7 , X 8 , X 9 , and X 10 are each independently leucine, isoleucine, or norleucine.

[0005] In some embodiments, the leukocyte-targeting molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0006] In some embodiments, the leukocyte-targeting molecule comprises at least one of ornithine, isoleucine, or norleucine.

[0007] In some embodiments, the leukocyte-targeting molecule comprises an additional 1-5 amino acids at either or both the C-terminus and N-terminus of the peptide, hi some embodiments, the additional amino acids are basic amino acids.

[0008] In some embodiments, the leukocyte-targeting molecule has an amino acid sequence selected from KKAAVALLPAVLLALLAKK (SEQ ID NO:5), RRAAVALLPAVLLALLARR (SEQ ID NO:6), RRAAVALLPAVLLALLARK (SEQ ID NO:7), RKAAVALLPAVLLALLARKY (SEQ ID NO:8), AAVALLPAVLLALLAPCVQRKRQKLMPC (SEQ ID NO:9), AAVALLPAVLLALLAPVQRKRQKLMP (SEQ ID NO:13), KKAAVALLPAVLLALLAPKK (SEQ ID NO:39), RRAAVALLPAVLLALLAPRR (SEQ ID NO:40), RRAAVALLPAVLLALLAPRK (SEQ ID NO:41), or RKAAVALLPAVLLALLAPRKY (SEQ ID NO:42).

[0009] In some embodiments, the peptide further comprises the amino acid sequence CVQRKRQKLMPC (SEQ ID NO: 38) at the carboxy terminus. [ka] or a pharma- ceutically acceptable salt thereof.

[0010] In some embodiments, the compound comprises at least one radioisotope of iodine. In some embodiments, the peptide is iodinated. In some embodiments, the at least one radioisotope of iodine is independently 123 I, 124 I, 125 I, or 131 I, or the peptide is independently selected from 123 I, 124 I, 125 I or 131 It is iodized with I.

[0011] In some embodiments, the peptide comprises at least one D-amino acid.

[0012] Also disclosed herein are compositions comprising the compounds disclosed herein. In some embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable excipient.

[0013] Also provided herein are methods for targeting leukocytes, CD34, and / or leukocyte-targeting molecules in a subject in need thereof, comprising administering a leukocyte-targeting molecule or composition disclosed herein. + Also disclosed are methods of delivering active agents to stem cells, or both. In some embodiments, the leukocytes are eosinophils, basophils, neutrophils, or monocytes.

[0014] Also disclosed herein is a method for treating a disease in a subject, comprising administering a therapeutically effective amount of a leukocyte-targeting molecule or composition disclosed herein. In some embodiments, the disease is an inflammatory disease. In some embodiments, the disease is an autoimmune disease.

[0015] In some embodiments, the disease is a skin disorder. In some embodiments, the skin disorder is atopic dermatitis, psoriasis, rosacea, or acne. In some embodiments, the skin disorder is atopic dermatitis.

[0016] In some embodiments, the disease is chronic cutaneous lupus or systemic lupus erythematosus. In some embodiments, the disease is a viral disease. In some embodiments, the disease is shingles, herpes simplex type 1 or type 2, or severe acute respiratory virus type 2 (SARS-CoV-2).

[0017] In some embodiments, the autoimmune disease is rheumatoid arthritis, inflammatory bowel disease, asthma, or diabetes mellitus type 1. In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0018] In some embodiments, the disease is atherosclerosis, nonalcoholic steatohepatitis, or hypercholesterolemia. In some embodiments, the disease is age-related macular degeneration, diabetic retinopathy, conjunctivitis, uveitis, or chronic sinusitis.

[0019] In some embodiments, the leukocyte-targeting molecule or composition is administered topically, orally, or by injection. In some embodiments, topical administration includes topical creams, controlled release topical patches, eye drops, and nasal sprays.

[0020] Also disclosed herein are methods of reducing aberrant cytokine signaling in a subject in need thereof comprising administering a leukocyte-targeting molecule or composition disclosed herein. In some embodiments, the active pharmaceutical ingredient targets leukocytes, CD34 rather than red blood cells. + In some embodiments, the leukocytes are preferentially delivered to stem cells, leukocytes, or both. In some embodiments, the leukocytes are eosinophils, basophils, neutrophils, or monocytes. [Brief description of the drawings]

[0021] [Figure 1A] Figure 1A-B show a comparison of radioactivity concentrations (expressed as μg equivalents / mL) following a single subcutaneous (Figure 1A) and intravenous (Figure 1B) administration of [3H]-AMTX-100 at a target dose of 5 mg / kg in male albino rats. Plasma refers to whole blood from which red blood cells have been removed. [Figure 1B] Figure 1A-B show a comparison of radioactivity concentrations (expressed as μg equivalents / mL) following a single subcutaneous (Figure 1A) and intravenous (Figure 1B) administration of [3H]-AMTX-100 at a target dose of 5 mg / kg in male albino rats. Plasma refers to whole blood from which red blood cells have been removed. [Diagram 2] Figure 2 shows the plasma levels of (3H)-AMTX-100 in plasma following administration by intravenous, subcutaneous, intratracheal, intraduodenal, esophageal, or topical (dermal) routes. All studies use the L-amino acid form of (3H)-AMTX-100, except for intraduodenal administration, which used a D-amino acid. [Figure 3A] Figure 3A-B show the median AMTX-100-FITC fluorescence intensity in mouse leukocytes on two scales: Figure 3A shows the intensity on a scale of 0 to 6,000 to provide the intensity of eosinophils; [Figure 3B] Figure 3A-B show the median AMTX-100-FITC fluorescence intensity in mouse leukocytes on two scales: Figure 3B shows the intensity on a scale of 0 to 3,000 to provide the intensity of other leukocyte populations. [Figure 4A]Figure 4A-B show the median AMTX-100-FITC fluorescence intensity in human leukocytes on two scales: Figure 4A shows the intensity on a scale of 0 to 300,000 to provide the intensity of eosinophils; [Figure 4B] Figure 4A-B show the median AMTX-100-FITC fluorescence intensity in human leukocytes on two scales: Figure 4B shows the intensity on a scale of 0 to 10,000 to provide intensities for other leukocyte populations; [Figure 5A] 5A-B show the distribution of radioactivity in rat tissues 2 hours (FIG. 5A) and 12 hours (FIG. 5B) after a single intravenous administration of [3H]-AMTX-100 at a dose of 5 mg / kg. [Figure 5B] 5A-B show the distribution of radioactivity in rat tissues 2 hours (FIG. 5A) and 12 hours (FIG. 5B) after a single intravenous administration of [3H]-AMTX-100 at a dose of 5 mg / kg. [Figure 6] FIG. 6 shows a comparison of radioactivity concentrations (expressed as ng equivalents / mL) following a single local administration of [3H]-AMTX-100 at a target dose of 5 mg / kg in male albino rats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] AMTX-100 is a small peptide as disclosed in WO2001 / 37821, which is incorporated herein by reference in its entirety. It is a chimera not found in nature with a cell-penetrating amino terminus (sometimes called a signal sequence hydrophobic region or SSHR). The SSHR binds to a stress-responsive transcription factor (SRTF) at the carboxy terminus to generate AMTX-100. The SRTF contains a nuclear localization sequence (NLS) from NF-κB that allows binding to the importin α / importin β nuclear transporter complex. The amino terminus also contains an NLS found in carbohydrate and lipid metabolism promoting transcription factors. Molecules with the SSHR can penetrate cells, but deleting the SSHR abolishes cell penetration. In other words, the NF-κB sequence alone cannot penetrate cells. AMTX-100 has three important properties: 1) cell penetration, 2) regulation of SPTFs involved in inflammatory pathways by nuclear transporters, and 3) regulation of large transcription factors involved in metabolic pathways by nuclear transporters. This results in a competition between fully functional transcription factors with their NLS and the similar NLS contained in AMTX-100, which regulates the amount of transcription factor that is transported into the nucleus to initiate transcription.

[0023] The transporter of AMTX-100 is importin-α, which forms a complex with importin-β (Impα5 and Impβ1, respectively). Impα5 is an adaptor protein that has docking sites for the NLS of many SRTFs and recognizes a docking site on Impβ1, a functional element that allows passage through the nuclear membrane. The interaction between Impα5 and Impβ1 allows SRTFs bound to Impα5 to be transported through the nuclear pore.

[0024] In addition to binding and transporting SRTFs bound to Impα5, Impβ1 has a second function: Impβ1 also recognizes NLSs of TFs required for lipid (SREBP) and carbohydrate (ChREBP) metabolism that are distinct from the NLSs of SRTFs. Impβ1 can transport these TFs to the nucleus without adaptor proteins.

[0025] Cells that are activated to become proinflammatory and produce SRTF also require energy and lipids to proliferate and perform their functions (production of cytokines, chemokines and growth factors). Thus, the AMTX-100 sequence can competitively regulate the NLS-binding domains of SRTF, ChRFBP and SREBP in a natural way, acting as a decoy for TF docking sites at two important checkpoints in inflammation and metabolism.

[0026] The amino terminus of AMTX-100 is derived from the fibroblast growth factor (FGF)-4 leader sequence, which was thought to allow FGF-4 to enter the endoplasmic reticulum for processing and extrusion of FGF-4 from the cell. Extracellular FGF-4 binds indiscriminately to various FGF receptors (1-4), which is facilitated by heparin. Binding of FGF-4 to its cell surface receptor triggers a tyrosine kinase cascade that triggers signal transduction within the cell.

[0027] Initially, it was believed that SSHR on AMTX-100 could enter cells without receptors, energy, or endocytosis. However, in our study, an AMTX-100-related peptide (FITC-SMTX-100) with a double null knockout of importin β obtained by amino acid substitution and containing a FITC molecule was incubated with human blood and analyzed by flow cytometry. This FITC-conjugated, AMTX-100-related peptide only enters leukocytes (white blood cells [WBCs]) and not red blood cells (RBCs); this is considered the first sign of cell specificity. In a pharmacokinetic (PK) study performed in mice, we were unable to detect AMTX-100 in RBC- and WBC-depleted plasma using LC-MS with a sensitivity of 2 ng / ml. This therefore suggested that AMTX-100 could travel within WBCs and be transmitted to other cells or indirectly affect other cells through a by-product of its interaction with leukocytes.

[0028] Intraperitoneal injection of AMTX-100 has shown efficacy in many animal models of lung, liver, sepsis, and metabolic syndrome-induced inflammation (see WO 2001 / 037821, WO 2013 / 052813, WO 2018 / 232383, and WO 2020 / 056250, all of which are incorporated by reference herein for all that they disclose regarding AMTX-100 and inflammation). It is known that leukocytes are present in the peritoneal region. Most leukocytes migrate in the blood or lymph, but some leukocytes are fixed to certain tissues.

[0029] Pharmacokinetic / biodistribution studies were performed in rats administering radioactive forms of AMTX-100 ([ 3 The T of AMTX-100 in blood was 1 / 2 The time to radiation exposure was 45-51 hours, and radiation was distributed to the WBCs. 3 [H]-AMTX-100 localized in organs where immune cells are known to reside and pass through (spleen, lung, liver, lymph nodes, kidneys, adrenal glands, bone marrow, pancreas, small intestine, etc.). Surprisingly, there was no lack of localization of radioactivity in cardiac and other muscle cells. This suggests that AMTX-100 does not enter all organs and cells, which is contrary to published information.

[0030] Since concentrations of radioactivity are likely to be present in organs with immune cells, we began a series of flow cytometry experiments with human blood. We first stained all cells with FITC-AMTX-100 and then incubated with immunotyping reagents that separate hematopoietic cells into broad categories of RBCs, granulocytes, monocytes, B and T lymphocytes, and NK cells. No double staining of RBCs was observed, confirming what was observed in human blood incubated with FITC-AMTX-100. In contrast, subsets of granulocytes and monocytes were observed to be double stained, indicating specificity within the leukocyte markers tested. A small subset of CD34+ hematopoietic stem cells was also double stained. Again, a highly unexpected finding for a peptide that is "purported" to be a promiscuous cell-permeable leader sequence.

[0031] Fibroblast growth factors are very important molecules in the development of limb formation and cell proliferation in organisms and have been well studied. There is no literature on FGF leader sequences that show cell permeability and specificity to cells, especially these properties of FGF-4. Not all FGFs have leader sequences. The inventors realized that the discovery of this specificity for the FGF-4 leader sequence has important medical and industrial applications as a highly specific binding or encapsulation drug delivery vehicle to target important immune cells.

[0032] Also disclosed herein are non-naturally occurring forms of the leader sequence of FGF-4 that conjugate with peptides, proteins, small molecule drugs, RNA, DNA, and radioisotopes. As seen in the FGF-4 leader sequence, there are additional FGFs with leader sequences that may have their own biological activities and cell specificities.

[0033] (composition) As used herein, white blood cells (WBCs, leukocytes) and CD34 +Compositions are disclosed that include molecules capable of targeting stem cells and therapeutic, diagnostic, or prophylactic agents. White blood cells are a type of blood cell made in the bone marrow and present in blood and lymphatic tissues and are part of the body's immune system. They help the body fight infections and other diseases. Types of white blood cells (leukocytes) include granulocytes (neutrophils, eosinophils, basophils), monocytes, and lymphocytes (T cells, B cells, NK cells).

[0034] In some embodiments, the composition comprises a compound provided herein, such as a leukocyte-targeting molecule, and an agent, such as a therapeutic, diagnostic, or prophylactic agent, and the compound and agent may be covalently or non-covalently linked. The compositions disclosed herein can be tailored to deliver the agent to the cytoplasm or nucleus of a cell. The compositions disclosed herein can also comprise a leukocyte-targeting molecule fused to a protein or peptide agent.

[0035] The leukocyte-targeting molecules disclosed herein comprise a cell-permeable amino terminus derived from a fibroblast growth factor sequence (FGF signal sequence, also referred to as signal sequence hydrophobic region or SSHR). In some embodiments, the FGF signal sequence is linked at the carboxy terminus to a stress response transcription factor (SRTF) nuclear localization sequence (NLS). In some embodiments, the SRTF comprises an NLS derived from NF-κB that allows binding to the importin α / importin β nuclear transporter complex. The amino terminus also comprises an NLS found in carbohydrate and lipid metabolism facilitating transcription factors. In some embodiments, the leukocyte-targeting molecules comprise a cell-permeable amino terminus linked to a peptide, a nucleic acid, or a radioisotope drug.

[0036] In some embodiments, the leukocyte-targeting molecule is derived from a fibroblast growth factor (FGF) signal sequence, for example based on the underlined FGF sequence of SEQ ID NO: 1. Variants of the FGF4 signal sequence are also useful in the claimed compositions, as shown in Table 1. In some embodiments, the leukocyte-targeting molecule sequence includes a modification to knock out importin beta activity. In some embodiments, the leukocyte-targeting molecule sequence maintains importin beta activity.

[0037] FGF4 (signal sequence underlined): [ka]

[0038] [Table 1-1] [Table 1-2] [Table 1-3]

[0039] In some embodiments, the peptides in Table 1 are linear. In some embodiments, SEQ ID NOs: 9, 10, 12, 14, and 37 are optionally cyclized at the cysteine ​​residue. In some embodiments, the sequences are cyclized.

[0040] In some embodiments, the leukocyte-targeting molecule is [ka] or a salt thereof.

[0041] In some embodiments, the agent is a peptide, a protein, a nucleic acid, a small molecule, a heavy metal, an imaging agent, or a radiopharmaceutical. In some embodiments, the agent is an enzyme, including but not limited to, horseradish peroxidase or alkaline phosphatase. In some embodiments, the heavy metal includes but is not limited to, colloidal gold or gadolinium. In some embodiments, the radiopharmaceutical is 3 H-, 14 C-, 125 I-, 18 F-, or 131 In some embodiments, the imaging agent is a radioactive agent or a fluorescent label, including, but not limited to, fluorescein, rhodamine, or green fluorescent protein, or derivatives thereof.

[0042] In some embodiments, the leukocyte-targeting molecule and agent are encapsulated in a micelle or liposome. In some embodiments, the leukocyte-targeting molecule is incorporated into a micelle or liposome such that it is available for interaction with leukocytes on the surface of the micelle or liposome.

[0043] In some embodiments, the leukocyte-targeting molecule specifically targets eosinophils, basophils, neutrophils, and monocytes, hi some embodiments, the leukocyte-targeting molecule does not target T lymphocytes, B lymphocytes, or NK cells.

[0044] The compositions disclosed herein can optionally include conjugating leukocyte-targeting molecules to antibodies to allow bispecific targeting. These antibody-conjugated molecules would target leukocytes via the disclosed leukocyte-targeting molecules, and the second specificity would be antibody-mediated targeting. These bispecific molecules would disrupt signaling pathways involved in tumor development and redirect or recruit immune cells to the target tissue, depending on the specificity of the antibody.

[0045] (How to use) The compositions disclosed herein are useful for targeting diagnostic, therapeutic, and prophylactic agents to leukocytes, in some embodiments, the leukocytes are targeted to subjects in need of treatment for autoimmune, inflammatory, or neurodegenerative diseases, or in need of reduction in aberrant cytokine production.

[0046] As disclosed herein, inflammatory diseases include acute disseminated encephalomyelitis (ADEM), Addison's disease, allergies, allergic rhinitis, Alzheimer's disease, antiphospholipid syndrome (APS), arthritis, e.g., monoarthritis, oligoarthritis, or polyarthritis, such as osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, septic arthritis, spondyloarthropathy, gout, pseudogout, Still's disease, asthma, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, celiac disease, Sharp's disease, and the like. gastrointestinal disorders such as bronchitis, chronic obstructive pulmonary disease (COPD), type 1 diabetes mellitus (IDDM), endometriosis, irritable bowel disease, or inflammatory bowel disease such as Crohn's disease or ulcerative colitis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, such as discoid lupus erythematosus, drug-induced lupus erythematosus, lupus nephritis, neonatal lupus, subacute cutaneous lupus erythematosus, or systemic lupus erythematosus Lupus such as thyroiditis, morphea, multiple sclerosis (MS), myasthenia gravis, myopathies such as dermatomyositis, inclusion body myositis or polymyositis, myositis, narcolepsy, neuromyotonia, Parkinson's disease, pemphigus vulgaris, pernicious anemia, primary biliary cirrhosis, psoriasis, relapsing disseminated encephalomyelitis, rheumatic fever, scleroderma, Sjogren's syndrome, skin disorders such as dermatitis, eczema, stasis electrodermatitis, atopic dermatitis, hidradenitis suppurativa, psoriasis, rosacea or scleroderma, tendonitis, buboes, Diseases or disorders including, but not limited to, uveitis, vasculitis such as Buerger's disease, cerebral vasculitis, Churg-Strauss arteritis, cryoglobulinemia, essential cryoglobulinemic vasculitis, giant cell arteritis, golfer's vasculitis, Henoch-Schonlein purpura, hypersensitivity vasculitis, Kawasaki disease, microscopic polyarteritis / polyangiitis, polyarteritis nodosa, polymyalgia rheumatica (PMR), rheumatic vasculitis, Takayasu's arteritis, Wegener's granulomatosis, alopecia areata, or vitiligo.

[0047] In some embodiments, the autoimmune or inflammatory disease is a skin disorder. In some embodiments, the skin disorder is psoriasis. In some embodiments, the skin disorder is atopic dermatitis. In some embodiments, the skin disorder is treated by topical administration of the compounds, compositions, or leukocyte-targeting molecules disclosed herein.

[0048] As disclosed herein, neurodegenerative disease includes, but is not limited to, Parkinson's disease, Alzheimer's disease, multiple sclerosis, optic neuritis, stroke, CNS trauma, amyotrophic lateral sclerosis, neuropathy, nervous system hypoxia, CNS toxicity, dementia, retinopathy, Huntington's disease, synucleinopathy, epilepsy, autism, and age-related CNS degeneration or disorder.In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0049] In some embodiments, the abnormal cytokine production is cytokine release syndrome (CRS). CRS is the abnormal overproduction of proinflammatory cytokines such as INF-γ, IL-1α, IL-1β, IL-6, IL-8, GM-CSF, M-CSF, TNF-α, etc., which causes high concentrations of systemic circulating cytokines. In some embodiments disclosed herein, the compositions disclosed herein can reduce the production of proinflammatory cytokines.

[0050] Administration of the compositions disclosed herein may result in the induction of white blood cells or CD34 +The administration of the composition may be by any suitable means that results in the incorporation of the composition into the stem cells. The composition may be included in any suitable amount in any suitable carrier material, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form suitable for local or systemic administration (e.g., parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal). Pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, for example, (Gennaro, A R Ed. (2000) Remington: The Science and Practice of Pharmacy (20th ed.), Lippincott Williams & Wilkins, Baltimore, Md.; Swarbrick, J. and Boylan, J C eds. (1988-1999) Encyclopedia of Pharmaceutical Technology, Marcel Dekker, New York).

[0051] The compositions described herein may be administered parenterally by injection, infusion, topical application, or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) in a dosage form, formulation, or via a suitable delivery device or implant containing conventional non-toxic pharmaceutically acceptable carriers and adjuvants. In one embodiment, the compositions described herein are administered via an osmotic pump. The compositions may be administered orally in sublingual form or with a coating that protects the composition from gastrointestinal peptidases. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations are described in Gennaro, supra.

[0052] Compositions for parenteral use may be provided in unit dosage form (e.g., single-dose ampoules) or in vials containing several doses and to which suitable preservatives may be added. The compositions may be in the form of a solution, suspension, emulsion, infusion device, or implantable delivery device, or may be provided as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent for treating or preventing inflammation, for example, the composition may include suitable parenterally acceptable carriers and / or excipients. The active therapeutic agent may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, etc. for controlled release. In addition, the composition may include suspending agents, solubilizing agents, stabilizing agents, pH adjusting agents, tonicity agents, and / or dispersing agents.

[0053] As indicated above, the pharmaceutical compositions described herein may be in a form suitable for sterile injection. To prepare such compositions, the appropriate active therapeutic agent is dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that can be used include water, water adjusted to an appropriate pH by adding an appropriate amount of hydrochloric acid, sodium hydroxide or an appropriate buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution or glucose solution. Aqueous formulations may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). If one of the compounds is poorly or sparingly soluble in water, a dissolution enhancer or solubilizer can be added, or the solvent may contain 10-60% w / w propylene glycol, or the like.

[0054] Materials used in the preparation of microspheres and / or microcapsules are biodegradable / bioerodible polymers such as, for example, polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid). Biocompatible carriers that can be used when formulating controlled release parenteral formulations are carbohydrates (e.g., dextran), proteins (e.g., albumin), lipoproteins, or antibodies. Materials used for implants can be non-biodegradable (e.g., polydimethylsiloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(orthoesters), or combinations thereof).

[0055] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharma- ceutically acceptable excipients, and such formulations are known to those skilled in the art. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, glidants, and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharma- ceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0056] The tablets may be uncoated or may be optionally coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. The coating may be adapted to release the active ingredient (e.g., drug) in a predetermined pattern (e.g., to achieve a controlled release formulation) or may be adapted not to release the active ingredient until after passage through the stomach (enteric coating). The coating may be sugar coating, film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycol and / or polyvinylpyrrolidone), or enteric coating (e.g., based on methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and / or ethylcellulose). Additionally, a time-delay material, such as, for example, glyceryl monostearate or glyceryl distearate, may be used.

[0057] The solid tablet composition may include a coating adapted to protect the composition from undesired chemical changes (e.g., chemical degradation before the release of the therapeutically active substance). The coating may be applied onto the solid dosage form in a manner similar to that described in Swarbrick J. and Boylan, JC, supra. Two or more compounds may be mixed together in the tablet or may be divided. In one example, a first active therapeutic agent is included inside the tablet and a second active therapeutic agent is included outside, such that a substantial portion of the second active therapeutic agent is released before the release of the first active therapeutic agent. For example, therapeutic combinations that reduce the level of inflammation have been identified as useful in the compositions, methods, and kits described herein.

[0058] Formulations for oral use may be provided as chewable tablets or hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil. Powders and granules may be prepared in a conventional manner, for example using a mixer, fluid bed apparatus, or spray drying apparatus, using the ingredients previously mentioned under tablets and capsules.

[0059] The compositions described herein can also be formulated for inhalation, topical application, and intravitreal injection.Combinations are expected to have beneficial synergistic effects.Topical formulations include creams or ointments, and controlled release patches that deliver the compositions disclosed herein through the skin.

[0060] The therapeutic methods described herein generally involve administering a therapeutically effective amount of a composition described herein to a subject (e.g., an animal) in need thereof, including a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. Such treatment would be appropriately administered to a subject in need thereof. The determination of a subject "in need thereof" may be made by objective or subjective determination by diagnostic testing, or by the opinion of the subject or a health care provider.

[0061] (Example) Example 1 Pharmacokinetics / biodistribution of AMTX-100 Formulation Analysis [ 3 The radiochemical purity of [H]-AMTX-100 was assessed using high performance liquid chromatography (HPLC) with radiochemical detection before and after dose administration, which was performed by injecting a 50 μL aliquot of the dose formulation into the HPLC system.

[0062] Radioactivity concentration and homogeneity checks were performed on each formulation before and after dose administration by direct quantitative radiochemical analysis.

[0063] Drug Administration Each rat was weighed prior to dose administration and the individual dose to be administered was calculated based on body weight, target dose volume, and radioactive concentration of the dose formulation.

[0064] Administration was accomplished using butterfly needles, tubing, and syringes. Formulations were administered directly into the tail vein. A dose volume (2.5 mL / kg) was used to achieve a dose level of 5 mg / kg, and radioactive dose levels of 100 μCi / kg (Phase 1) and 500 μCi / kg (Phase 2). Dose formulations were stirred continuously throughout the dosing procedure.

[0065] Volumetric Dosing In phases 1 and 2, an appropriate volume of dose formulation, including an excess, was loaded into the dosing device for each dose. When possible, a new dosing device was used for each animal. Entrapped air and excess formulation were evacuated, leaving the dosing device filled with the appropriate volume. Doses were administered and the radioactive dose received was calculated by the volume of dose dispensed and the calculated radioactive concentration of the dose formulation.

[0066] [ 3 H]-NSP label The main phase of the study consisted of preparing the test protein and labeling it with N-succinimidyl-[2,3- 3 H]-propionate ([ 3 The antibodies were radiolabeled with [H]-NSP and purified as described below.

[0067] 1.6mL [ 3Three aliquots of the [H]-NSP solution were dispensed into appropriate vials. The solvent was allowed to evaporate under a gentle stream of nitrogen gas at ambient temperature. Once sufficient solvent had evaporated, the three aliquots were combined into one vial and the remaining solvent was allowed to evaporate to dryness. To ensure complete mixing of the protein and labeling reagent, the residue in the vial was resuspended in 0.6 mL of 1 mg / mL AMTX-100 by gentle vortex mixing. The solution was allowed to stand at ambient temperature for approximately 1 hour.

[0068] next,[ 3 The [H]-NSP-protein solution was purified by reverse phase chromatography technique, and the resulting solution was frozen and lyophilized using a freeze dryer. The resulting material was dissolved in 2 mL of ethanol:water (50:50) and stored at approximately -20°C until formulation for dose preparation.

[0069] After purification, reverse-phase HPLC and quantitative radiochemical analysis (QRA) were used to determine 3 The radiochemical purity and specific activity of the [H]-labeled test proteins were determined.

[0070] During the development of a method for radiolabeling AMTX-100, the radiolabeling of AMTX-100 and [ 3 Additional analysis by LC-RAD-MS / MS of [H]-AMTX-100 (5 min) was also performed to confirm labeling of the full-length peptide.

[0071] The specific activity of the radiolabeled protein was calculated according to liquid scintillation counting (LSC) of weighed aliquots of the diluted samples. Specific activity was determined as μCi / mg protein.

[0072] formulation The required amount of each radiolabeled test item was combined with the corresponding non-labeled test item as necessary to achieve sufficient radioactivity concentration to meet the study objectives. Dose solutions were prepared in PBS buffer at pH 7.4 with the aim of administering a total dose volume of 5 mg / kg (2.5 mL / kg, 100 μCi / kg-Phase 1 and 2.5 mL / kg, 500 μCi / kg-Phase 2).

[0073] Quantitative Whole-Body Autoradiography (QWBA) Frozen carcasses were subjected to QWBA. Sections were presented at up to five different levels of the rat body to include 30–40 tissues (provided sufficient radioactivity was present).

[0074] Lyophilized whole-body autoradiography sections were exposed to phosphor storage imaging plates and incubated for 7 days at ambient temperature in the dark. Calibrated autoradiography blood standards containing known amounts of radioactivity (nCi / g) were included with each exposure.

[0075] Organization and 3 The distribution of radioactivity in [H]-blood standards (prepared and validated at Pharmaron UK Ltd.) was determined and quantified using a Fuji FLA-5100 fluorescent image analysis system and associated Tina (version 2.09) and SeeScan (version 2.0) software. For each exposure, data from the autoradiographic blood standards were used to generate a standard curve in SeeScan, from which tissue concentrations of radioactivity (μg equivalents / g) were determined.

[0076] Representative background radioactivity measurements were also taken for each exposure plate used. The number of measurements for each specific tissue was determined by the number of levels of occurrence for that tissue. Thus, multiple measurements were performed for larger tissues (e.g., liver) that appeared at multiple levels, and only one measurement was obtained for smaller tissues (e.g., thyroid). The limit of accurate quantification was the lowest visible [ 3 H]-blood standard.

[0077] Prior to analysis, samples were stored at approximately −20°C (carcasses) or ambient temperature (sections after freeze-drying). After analysis, carcass remains were stored at approximately −20°C until disposal, and sections were stored at ambient temperature.

[0078] Phase 1: Total radioactivity in plasma

[0079] Eight male Sprague Dawley rats were each 3 A single IV dose of [H]-AMTX-100 was administered, and terminal samples of whole blood (approximately 5–10 mL each) were collected from one rat by cardiac puncture under isofluorane anesthesia at the following time points: 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, and 12 h after administration.

[0080] Animals were sacrificed by exsanguination via cardiac puncture and carcasses were immediately flash frozen in a hexane / dry ice mixture (approximately -70°C) after terminal blood collection. Each carcass was stored at approximately -20°C pending analysis by the QWBA.

[0081] Whole blood was collected into tubes containing K2-EDTA as an anticoagulant. After collection, an aliquot of whole blood (approximately 4 mL) was retained for analysis of whole blood and white blood cell (WBC) concentrations. The remaining whole blood sample was processed to plasma. Plasma samples were not leukocyte-removed.

[0082] Phase 2: Tissue distribution of radioactivity

[0083] [ 3 Three carcasses from eight male Sprague Dawley rats that had been subjected to whole blood collection and analysis after a single intravenous dose of [H]-AMTX-100 were selected for QWBA analysis at each of the following time points: 10 min, 2 h, and 12 h after administration.

[0084] Each carcass was flash frozen by immersion in a hexane / dry ice mixture immediately after collection and then stored at approximately −20°C until analysis by the QWBA.

[0085] [Table 2]

[0086] [Table 3]

[0087] [Table 4]

[0088] At a target dose of 5 mg / kg, 3 The distribution of radioactivity in rats after a single intravenous dose of [H]-AMTX-100 is shown in Figure 5A (2 hours) and Figure 5B (12 hours) and in Table 5. 3 The distribution of radioactivity in rats after a single local administration of [H]-AMTX-100 is shown in Figure 6.

[0089] [Table 5-1] [Table 5-2]

[0090] Example 2: Timeline of plasma appearance of AMTX-100 following multiple routes of administration AMTX-100 at the lysine residue 3 The drugs were radiolabeled with 3H and injected into Sprague-Dawley rats at a single dose by seven different routes (intravenous, subcutaneous, topical [dermal], intratracheal, intraduodenal, esophageal, and oral). The rats were evaluated at various time points up to 120 hours by a) taking blood samples for pharmacokinetics (PK) by cardiac puncture, b) freezing and sectioning for QWBA and / or microautoradiography and histology, and c) making frozen sections, lyophilizing them, exposing them to phosphor-accumulating imaging plates, and quantifying tissue radioactivity in the images as tissue:blood ratios.

[0091] For all PK studies, the following definitions were used: WB = whole blood; plasma = whole blood with red blood cells removed; WBCD = whole blood with white blood cells removed and passed through a Pall Acrodisc filter (efficiency approximately 60%); WBC = cells that bind and are washed off the Pall Acrodisc filter.

[0092] [ 3 [H]-AMTX-100 enters the bloodstream by all routes of administration, and PK studies show extremely long plasma residence times (Sub-Q>672 hours or 28 days) that are unexpected for a peptide by several routes. 3 The specific target of [H]-AMTX-100 is WBCs, not RBCs. 3 Esophageal administration of [H]-AMTX-100 in the L-amino acid form was resistant to degradation, whereas duodenal administration was in the D-amino acid form. We hypothesize that the peptide enters WBCs very rapidly via its receptor, protecting them from extracellular enzymes. 3 The persistence of radioactivity in [H]-AMTX-100 was 3 H]-AMTX-100 remained relatively constant at 1 μg equivalent / ml for more than 120 hours after a single dose, and tissues where WBCs congregate contained large amounts of 3 H]-AMTX-100 is present in IL-1 cells but not in cardiac or skeletal muscle tissue, and the biological effects are expected to persist for a very long time in WBCs.

[0093] [ 3 H]-AMTX-100 was administered IV and showed a half-life (T 1 / 2 ) is very long (25-51 hours).

[0094] Example 3: Flow cytometry studies mouse Leukocytes from mouse whole blood (n = 4 C57BI / 6 mice) were pooled and treated with [FITC]-AMTX-100 for 5 min at 37 °C. The results show that [FITC]-AMTX-100 targets eosinophils, neutrophils, and monocytes, but not lymphocytes (Figure 3A-B).

[0095] Human Human whole blood (WB) was obtained from multiple normal donors and incubated with μg / mL amounts of [FITC]-AMTX-100 in replicate ex vivo experiments.

[0096] Using a panel of monoclonal antibodies labeled with fluorescent dyes, we phenotypically identified [FITC]-AMTX-100-permeabilized WB cells, including classical monocytes, non-classical monocytes, neutrophils, basophils, eosinophils, and T and B lymphocytes as well as NK cells (Figure 4A-B). Classical monocytes are characterized by high-level expression of the CD14 cell surface receptor (CD14++CD16- monocytes). Non-classical monocytes show low-level expression of CD14 and additional co-expression of the CD16 receptor (CD14+CD16++ monocytes).

[0097] RBCs were excluded because they were not penetrated by [FITC]-AMTX-100, and the fluorescence was determined to be present in the WBC leukocyte population, which is consistent with the [FITC]-AMTX-100 in the WB population estimated from various analytical methods. 3 H]-AMTX-100.

[0098] The specificity seen in PK / biodistribution studies in rats coupled with flow cytometry studies using human WB supports the hypothesis that AMTX-100 acts through receptors found specifically on leukocytes (classical and non-classical monocytes, neutrophils, basophils and eosinophils) (Figure 4A-B).

[0099] Example 4: Staining of RAW 264.7 cells with labeled peptides FITC-labeled peptides (SEQ ID NOs: 17-20, 22, and 24-26) were constructed to contain the SSHR region (or different versions) with a FITC group attached to a lysine at the amino terminus, an arginine added at the amino terminus, and a lysine added at the carboxy terminus, as shown in Table 1. [ka]

[0100] These peptides were used for fluorescent labeling of RAW 264.7 cells (Table 6).

[0101] [Table 6]

[0102] Previous work (U.S. Pat. No. 11,026,992) disclosed a peptide containing a membrane translocation motif (MTM) with the sequence AAVLPVLLAL (SEQ ID NO: 27). The results in Table 6 indicate that the proline in the prior art sequence is not required for binding to RAW 264.7 cells, as changing the proline to alanine did not alter the binding activity.

[0103] Example 5: Safety and Tolerability of Topically Applied AMTX-100CF in Adult Patients with Mild-to-Moderate Atopic Dermatitis Atopic dermatitis (AD) is a chronic, relapsing, pruritic, inflammatory skin disease of unknown etiology with an eczematous morphology that usually begins in early infancy but also affects a significant number of adults. The prevalence of AD in US adults is 7.3% and 15%, 15.1% and 14.5% in children aged 5, 9 and 15 years, respectively. For the majority of patients, the disease is cured by adulthood, but 10-30% of the affected population do not. A smaller percentage of patients develop symptoms in adulthood. AD is often associated with a personal or family history of type I allergy with elevated serum immunoglobulin E (IgE) levels and proinflammatory mediators such as interleukin 4 (IL-4), IL-13, IL-22, IL-31, interferon gamma (IFN-γ), and thymic stromal lymphopoietin (TSLP), which signal through the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. AD is usually associated with pruritus, xerosis, lichenification, and eczematous lesions. Excoriation and crusting are common, and some patients may present with nodular prurigo-like lesions. The mainstay of treatment is moisturization (e.g., frequent warm baths and use of petrolatum or aquaphor) in combination with topical steroids. In more severe cases, other treatment options are also used, such as immunomodulators (e.g., tacrolimus and pimecrolimus), biologics (e.g., dupilumab and omalizumab), topical phosphodiesterase-4 (PDE-4) inhibitors (e.g., crisaborole), probiotics, and phototherapy. Many of these treatments are expensive and are believed to have various side effects when used chronically. Therefore, patients with AD are in need of new treatment options that are effective and safe for the long-term management of the disease.

[0104] Several nonclinical safety studies have been conducted with AMTX-100CF to evaluate its toxicological profile (Liu XY, J Biol Chem 275:16774-8, 2000; Liu DLX, J Biol Chem 279:19239-46, 2004; Veach RA, et al. J Biol Chem 279:11425-31, 2004). This first-in-human, open-label, dose-escalating, Phase I clinical trial aimed to determine the tolerability and safety of topically applied AMTX-100CF as well as to evaluate exploratory efficacy endpoints in adult patients with mild to moderate AD.

[0105] (method) Study design The AMTX100-AD-01 study (NCT04313400) was designed as an adaptive phase I / II clinical trial. Only the phase I portion of the study is described below. The phase I study was a 6-week, multicenter, open-label, dose-escalation clinical trial evaluating the safety, tolerability, and efficacy of AMTX-100CF in adults with mild to moderate AD. The study included a screening period of up to 21 days, a treatment period of 7 days, and a follow-up period of 14 days. A central Institutional Review Board (IRB) approved the study protocol, informed consent forms, study sites, and recruitment materials prior to patient enrollment. This clinical trial was conducted in accordance with the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines, applicable regulations, and the Declaration of Helsinki. Patients were provided with written informed consent prior to the start of screening and study-related procedures.

[0106] Clinical Trial Procedures Twenty-six patients were enrolled sequentially into five cohort dose levels (per AD-affected body surface area [BSA]) by escalating application of AMTX-100CF 1.1%. Enrolled patients were assigned to receive AMTX-100CF twice daily for 7 days starting from the baseline visit.

[0107] The dose of AMTX-100CF applied was assessed by the investigator and dependent on the percentage of BSA affected by AD, as indicated by the protocol (400 cm 2 Approximately 1 g of cream per dose). The first dose of study treatment was administered topically in the study clinic during the baseline visit by trained site staff. Subjects were trained on administration techniques, including the appropriate amount of topical use, how to measure the assigned dose, and how to record study treatment compliance in a patient diary. All other doses were self-administered by the subject at home. Subjects were required to apply AMTX-100CF topical cream to all AD lesions (excluding scalp, face, eyes, eyelids, neck, hands, palms, feet, groin, genitals, or axillae) for 7 consecutive days (14 total applications), regardless of whether the lesions became clinically clear during the 7-day treatment period. Rescue therapy was defined as any topical or systemic immunomodulatory treatment initiated for AD and could occur at any time at the investigator's discretion. If rescue therapy is administered, the subject will be considered a treatment failure for analytical purposes.

[0108] The decision to escalate the cohort (dose level) (to enroll patients with higher BSA involved in AD) was made by the Safety Assessment Committee (SAC) after reviewing the safety data of the previous cohort. If a dose-limiting toxicity (DLT) was observed, the Study Data and Safety Monitoring Committee (DSMC) was required to perform an independent review of the data and make a final recommendation regarding dose escalation to the next cohort.

[0109] Safety parameters The primary safety outcome measure was to determine the maximum tolerated dose (MTD) of AMTX-100CF (1.1%) (according to the highest percentage of BSA treated) by evaluation of DLTs. Other safety outcome measures were treatment-emergent adverse events (TEAEs), clinically significant changes and shifts in laboratory measurements, and vital signs in all patients who received at least one dose of study drug during the follow-up period. TEAEs were defined as adverse events (AEs) that began or worsened in severity after initiation of AMTX-100CF. All AEs presented were treatment-emergent unless otherwise stated.

[0110] Efficacy parameters Efficacy outcome measures included change from baseline in percentage of BSA treated at Day 7 (end of treatment) and Day 21 (end of follow-up), and change from baseline in vIGA-AD™.

[0111] statistical analysis Statistical analyses were performed on the safety population, defined as subjects receiving study treatment. Statistical analyses were performed using SAS for Windows, version 9.4. Descriptive statistics (n, mean, standard deviation, median, minimum, maximum) were calculated for continuous variables. Frequencies and percentages for categorical variables were presented.

[0112] (result) A total of 26 subjects were enrolled in part 1 (phase I) of the study to receive AMTX-100CF 1.1%. All subjects completed the full course of treatment, with follow-up available in five subjects each in cohorts 1, 2, and 3, four subjects in cohort 4, and seven subjects in cohort 5 (Table 7). Baseline demographics and disease characteristics of the subjects are shown in Table 8.

[0113] [Table 7]

[0114] Of the 26 subjects enrolled, 14 (54%) were male. The mean age was 48.7 years, with a range of 20 to 75 years. Safety was assessed by evaluating treatment-emergent adverse events (TEAEs) in all 26 subjects. AEs were classified by system organ class (SOC) and preferred term (PT) according to the MedDRA dictionary (version 23.1).

[0115] [Table 8]

[0116] Safety Results Eight of the 26 subjects experienced TEAEs. The most frequent TEAE term reported was headache (mostly mild), experienced by five subjects. Three AEs with moderate severity (viral upper respiratory tract infection and urinary tract infection, headache) were reported by three subjects. No significant changes or shifts in laboratory measurements and vital signs were detected for any of the patients enrolled during the study.

[0117] Study treatment application site reactions were defined as symptoms potentially related to the application of the topical study treatment, including dryness, erythema / skin irritation, burning / stinging, erosion / ulceration, edema / swelling, itching, pain in the treatment area, scabbing, blistering / pustulation, peeling / scaling, and bleeding, assessed at baseline (pre-dose, post-dose), end-of-treatment, and follow-up visits. Symptoms similar to application site reactions were present in the AD lesions (due to the nature of the AD lesion) before the first application of study medication, but mostly improved or resolved during the study after study medication administration. Treatment application site reactions (burning / stinging, dryness, and itching) after baseline testing occurred in 4 of 26 (15.5%) subjects. Reactions in all of these subjects were mild to moderate, and in 3 of the subjects, they improved during the study. Only one subject experienced mild itching at the end of the treatment visit, but itching continued during follow-up.

[0118] No TEAEs were considered related to study treatment. All AEs were resolved. No AEs led to discontinuation of study drug, and no SAEs or deaths were reported in this study.

[0119] The decision to cohort escalate to higher dose levels (based on higher BSA treated with higher AMTX-100CF 1.1%) was based on the absence of dose-limiting toxicities (DLTs). No DLTs occurred in any cohort, so dose escalation continued safely until the last cohort (cohort 5). The maximum daily dose of AMTX-100CF assigned to subjects was 60 g / day in the highest cohort (48-70% treatable BSA).

[0120] Efficacy Results Compared to baseline, the mean percentage of BSA affected by AD decreased by 37.5% and 37.9% at the day 7 visit (end of treatment, all cohorts) and follow-up visits (cohorts 4 and 5 only), respectively (Table 4). The reductions by cohort at day 7 compared to baseline were 46.7% (cohort 1), 44.3% (cohort 2), 43.3% (cohort 3), 38.1% (cohort 4), and 21.7% (cohort 5).

[0121] BSA affected by AD was not assessed at the follow-up visit for cohorts 1–3. The reduction in cohorts 4 and 5 at the follow-up visit compared with baseline was 42.1% (cohort 4) and 35.1% (cohort 5).

[0122] Across the study cohort, validated Investigator Global Assessment for Atopic Dermatitis (vIGA-AD™) scores improved following AMTX-100CF intervention. At baseline, 11 (42.3%) and 15 (57.7%) subjects had vIGA scores of Grade 2 (mild) and Grade 3 (moderate).

[0123] By day 7 (end of treatment), three subjects (11.5%) had a grade 0 (clear) score, 10 subjects (38.5%) had a grade 1 (almost clear) score, five subjects (19.2%) had a grade 2 (mild) score, and eight subjects (30.8%) had a grade 3 (moderate) score.

[0124] At the day 21 (follow-up) visit, four subjects (15.4%) achieved a grade 0 (clear) score, 10 subjects (38.5%) had a grade 1 (almost clear) score, seven subjects (26.9%) had a grade 2 (mild) score, and five subjects (19.2%) had a grade 3 (moderate) score (Table 9).

[0125] [Table 9]

[0126] (Conclusion) The currently approved topical therapies, corticosteroids and calcineurin inhibitors, have been shown to be effective in improving mild to moderate AD, but long-term use of these agents may carry safety risks, including striae, increased risk of adrenal suppression, and slower linear growth with long-term use of glucocorticoids.

[0127] This specification discloses a new approach to treat AD, which involves the inhibition of the main mediator of inflammation. The competitive binding of AMTX-100 to Impα / β complex or Impβ leads to a decrease in the nuclear translocation of SRTF in inflammation and ChREBP or SHREBP in metabolic syndrome, resulting in a decrease in the production of inflammatory cytokines / chemokines or lipid metabolites, respectively. AMTX-100 has a good potential to suppress inflammatory response and alleviate the associated symptoms.

[0128] AMTX-100CF 1.1% topical cream was generally well tolerated during the study. The most frequently reported TEAE was mild-to-moderate headache, which occurred in 5 of 26 subjects and was not associated with a specific cohort. All resolved after dosing and were considered unrelated to study treatment. There were no serious adverse events (SAEs). No adverse events led to discontinuation of study treatment, and no deaths occurred. No DLTs occurred in any cohort of the study. Study treatment was administered at a maximum of 60 grams / day to subjects in cohort 5 (48-70%), who had the highest amount of BSA implicated in AD.

[0129] In terms of efficacy, AMTX-100CF 1.1% topical cream improved disease severity and intensity in all cohorts, and also reduced the area affected by AD lesions.

[0130] Example 6: Conjugation of methotrexate to AMTX-100 As shown in FIG. 7, methotrexate was conjugated to AMTX-100.

[0131] Unless otherwise indicated, all numbers used in the specification and claims expressing the amounts of ingredients, properties such as molecular weights, reaction conditions, and the like, should be understood in all instances to be modified by the term "about". As used herein, the terms "about" and "approximately" mean within 10-15%, preferably within 5-10%. Thus, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations and may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and without limiting the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed by applying ordinary rounding techniques in light of at least the number of reported significant digits. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0132] The terms "a", "an", "the" and similar referents used in the context of describing the present invention (particularly in the context of the claims below) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely for the purpose of better understanding the invention and is not intended to impose limitations on the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0133] Grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. The elements of each group may be referenced and claimed individually or in any combination with other elements of the group or other elements described herein. It is anticipated that one or more elements of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When such inclusion or deletion is made, the specification is deemed to include the modified group and thus fulfills the written description of all Markush groups used in the appended claims.

[0134] Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be adopted by those skilled in the art as appropriate, and intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, this invention encompasses any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

[0135] Certain embodiments disclosed herein may be further limited in the claims using the phrases "consisting of" or "consisting essentially of." When used in the claims, whether as filed or added by amendment, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps, and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are essentially or specifically described and enabled herein.

[0136] Additionally, throughout this specification, numerous references have been made to patents and printed publications. Each of the above cited references and printed publications is herein individually incorporated by reference in its entirety.

[0137] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely as shown and described.

[0138] (Additional Note) (Appendix 1) formula: [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, X 1 , X 2 , X 11 , and X 12 are each independently lysine, arginine, or ornithine; X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , and X 10 are each independently valine, leucine, isoleucine, or norleucine; X 13 is tyrosine, X 17 is proline or alanine, and n is 0 or 1; Leukocyte-targeting molecules.

[0139] (Appendix 2) X 3 and X 6 is valine.

[0140] (Appendix 3) X 4 , X 5 , X 7 , X 8 , X9 , and X 10 and x are each independently leucine, isoleucine, or norleucine.

[0141] (Appendix 4) The peptide has the formula: [ka] or a pharma-ceutically acceptable salt thereof.

[0142] (Appendix 5) 5. The leukocyte-targeting molecule of any one of claims 1 to 4, wherein the peptide comprises at least one of ornithine, isoleucine, or norleucine.

[0143] (Appendix 6) 6. The leukocyte-targeting molecule of any one of claims 1 to 5, wherein the peptide has a formula selected from KKAAVALLPAVLLALLAKK (SEQ ID NO:5), RRAAVALLPAVLLALLARR (SEQ ID NO:6), RRAAVALLPAVLLALLARK (SEQ ID NO:7), RKAAVALLPAVLLALLARKY (SEQ ID NO:8), AAVALLPAVLLALLAPCVQRKRQKLMPC (SEQ ID NO:9), AAVALLPAVLLALLAPVQRKRQKLMP (SEQ ID NO:13), KKAAVALLPAVLLALLAPKK (SEQ ID NO:39), RRAAVALLPAVLLALLAPRR (SEQ ID NO:40), RRAAVALLPAVLLALLAPRK (SEQ ID NO:41), or RKAAVALLPAVLLALLAPRKY (SEQ ID NO:42).

[0144] (Appendix 7) 7. The leukocyte-targeting molecule of any one of claims 1 to 6, further comprising the amino acid sequence CVQRKRQKLMPC (SEQ ID NO: 38) at the C-terminus.

[0145] (Appendix 8) The peptide has the formula: [ka] or a pharma-ceutically acceptable salt thereof.

[0146] (Appendix 9) The leukocyte-targeting molecule of claim 7 or 8, wherein the peptide is cyclized at a cysteine ​​residue.

[0147] (Appendix 10) 9. The leukocyte-targeting molecule of any one of claims 1 to 8, wherein the peptide is linear.

[0148] (Appendix 11) 11. The leukocyte-targeting molecule of any one of claims 1 to 10, wherein the peptide further comprises one or two C-terminal lysines.

[0149] (Appendix 12) 12. The leukocyte-targeting molecule of any one of claims 1 to 11, wherein the peptide further comprises an N-terminal lysine.

[0150] (Appendix 13) The peptide is a peptide having a lysine residue at the N-terminus and a lysine residue at the X 1 13. The leukocyte-targeting molecule of any one of claims 1 to 12, further comprising an arginine residue disposed between:

[0151] (Appendix 14) 14. The leukocyte-targeting molecule of any one of claims 1 to 13, wherein the compound comprises at least one radioactive isotope of iodine.

[0152] (Appendix 15) The at least one radioisotope of iodine is independently 123 I, 124 I, 125 I, or 131 15. The leukocyte-targeting molecule of claim 14, selected from I.

[0153] (Appendix 16) 16. The leukocyte-targeting molecule of any one of claims 1 to 15, wherein the peptide comprises at least one D-amino acid.

[0154] (Appendix 17) 17. The leukocyte-targeting molecule of any one of claims 1 to 16, wherein the leukocyte is an eosinophil, a basophil, a neutrophil, or a monocyte.

[0155] (Appendix 18) A composition comprising the leukocyte-targeting molecule described in any one of appendix 1 to 17.

[0156] (Appendix 19) 19. The composition of claim 18, wherein the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable excipient.

[0157] (Appendix 20) A method for targeting leukocytes, CD34, or leukocytes comprising administering to a subject a leukocyte-targeting molecule according to any one of claims 1 to 17 or a composition according to claim 18 or 19. + leukocytes, CD34 in a subject requiring delivery of an active agent to stem cells, or both. + A method for delivering an active agent to the stem cells, or both.

[0158] (Appendix 21) 21. The method of claim 20, wherein the leukocytes are eosinophils, basophils, neutrophils, or monocytes.

[0159] (Appendix 22) A method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a leukocyte-targeting molecule according to any one of appendices 1 to 17, or a composition according to appendices 18 or 19.

[0160] (Appendix 23) 23. The method of claim 22, wherein the disease is an inflammatory disease.

[0161] (Appendix 24) 23. The method of claim 22, wherein the disease is an autoimmune disease.

[0162] (Appendix 25) 23. The method of claim 22, wherein the disease is a skin disorder.

[0163] (Appendix 26) 26. The method of claim 25, wherein the skin disorder is atopic dermatitis, psoriasis, rosacea, or acne.

[0164] (Appendix 27) 27. The method of claim 26, wherein the skin disorder is atopic dermatitis.

[0165] (Appendix 28) 23. The method of claim 22, wherein the disease is chronic cutaneous lupus or systemic lupus erythematosus.

[0166] (Appendix 29) 23. The method of claim 22, wherein the disease is a viral disease.

[0167] (Appendix 30) 30. The method of claim 29, wherein the disease is shingles, herpes simplex type 1 or 2, or severe acute respiratory virus type 2 (SARS-CoV-2).

[0168] (Appendix 31) 25. The method of claim 24, wherein the autoimmune disease is rheumatoid arthritis, inflammatory bowel disease, asthma, or type 1 diabetes.

[0169] (Appendix 32) 32. The method of claim 31, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0170] (Appendix 33) 23. The method of claim 22, wherein the disease is atherosclerosis, nonalcoholic steatohepatitis, or hypercholesterolemia.

[0171] (Appendix 34) 23. The method of claim 22, wherein the disease is age-related macular degeneration, diabetic retinopathy, conjunctivitis, uveitis, or chronic sinusitis.

[0172] (Appendix 35) 35. The method of any one of claims 22 to 34, wherein the leukocyte-targeting molecule or composition is administered topically, orally, or by injection.

[0173] (Appendix 36) The method of claim 35, wherein topical administration includes topical creams, controlled release topical patches, eye drops, and nasal sprays.

[0174] (Appendix 37) A method of reducing aberrant cytokine signaling in a subject in need thereof comprising administering to the subject a leukocyte-targeting molecule of any of appendices 1-17 or a composition of appendices 18 or 19.

[0175] (Appendix 38) The active pharmaceutical ingredient is more effective in inhibiting white blood cells, CD34, than red blood cells. + 38. The method of any one of claims 20 to 37, wherein the IL-16 receptor agonist, ...

[0176] (Appendix 39) 39. The method of claim 38, wherein the leukocytes are eosinophils, basophils, neutrophils, or monocytes.

Claims

1. formula: 【Chemical 1】 or a pharmaceutically acceptable salt thereof, During the ceremony, X １ , X ２ , X １１ , and X １２ are each independently lysine, arginine, or ornithine; X ３ , X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , and X １０ are each independently valine, leucine, isoleucine, or norleucine; X １３ is tyrosine, X １７ is proline or alanine, and n is 0 or 1; Leukocyte-targeting molecules.

2. X ３ and X ６ The leukocyte-targeting molecule of claim 1, wherein is valine.

3. X ４ , X ５ , X ７ , X ８ , X ９ , and X １０ The leukocyte-targeting molecule of claim 1 , wherein each of is independently leucine, isoleucine, or norleucine.

4. The peptide has the formula: 【Chemistry 2】 2. The leukocyte-targeting molecule of claim 1, which is a peptide having the formula:

5. 2. The leukocyte-targeting molecule of claim 1, wherein the peptide comprises at least one of ornithine, isoleucine, or norleucine.

6. The peptides are KKAAVALLPAVLLALLAKK (SEQ ID NO: 5), RRAAVALLPAVLLALLARR (SEQ ID NO: 6), RRAAVALLPAVLLALLARK (SEQ ID NO: 7), RKAAVALLPAVLLALLARKY (SEQ ID NO: 8), AAVALLLPAVLLALLAPCVQRKRQKLMPC (SEQ ID NO: 9), AAVALLLPAVLLALLAPVQ 2. The leukocyte-targeting molecule of claim 1, having a formula selected from RKRQKLMP (SEQ ID NO: 13), KKAAVALLPAVLLALLAPKK (SEQ ID NO: 39), RRAAVALLPAVLLALLAPRR (SEQ ID NO: 40), RRAAVALLPAVLLALLAPRK (SEQ ID NO: 41), or RKAAVALLPAVLLALLAPRKY (SEQ ID NO: 42).

7. 2. The leukocyte-targeting molecule of claim 1, further comprising the amino acid sequence CVQRKRQKLMPC (SEQ ID NO: 38) at the C-terminus.

8. The peptide has the formula: 【Chemistry 3】 2. The leukocyte-targeting molecule of claim 1, which is a peptide having the formula:

9. 8. The leukocyte-targeting molecule of claim 7, wherein the peptide is cyclized at a cysteine ​​residue.

10. The leukocyte-targeting molecule of claim 1, wherein the peptide is linear.

11. The leukocyte-targeting molecule of claim 1, wherein the peptide further comprises one or two C-terminal lysines.

12. The leukocyte-targeting molecule of claim 1, wherein the peptide further comprises an N-terminal lysine.

13. The peptide has a structure in which the N-terminal lysine and X １ The leukocyte-targeting molecule of claim 12, further comprising an arginine residue located between

14. 2. The leukocyte-targeting molecule of claim 1, wherein said peptide comprises at least one radioactive isotope of iodine.

15. The at least one radioisotope of iodine is independently １２３ I, １２４ I, １２５ I, or １３１ The leukocyte-targeting molecule of claim 14, selected from I.

16. The leukocyte-targeting molecule of claim 1, wherein the peptide comprises at least one D-amino acid.

17. The leukocyte-targeting molecule of claim 1 , wherein the leukocyte is an eosinophil, a basophil, a neutrophil, or a monocyte.

18. A composition comprising the leukocyte-targeting molecule of any one of claims 1 to 17.

19. 20. The composition of claim 18, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

20. A leukocyte-targeting molecule or composition comprising the leukocyte-targeting molecule of any of claims 1 to 17, used to deliver an active agent to leukocytes, CD34 + stem cells, or both, in a subject in need of delivery of the active agent to leukocytes, CD34 + stem cells, or both, wherein the molecule or composition is administered to the subject.

21. A leukocyte-targeting molecule or composition comprising the leukocyte-targeting molecule of any one of claims 1 to 17 for use in treating a disease in a subject, wherein a therapeutically effective amount of the molecule or composition is administered to the subject.

22. 22. The leukocyte-targeting molecule or composition of claim 21, wherein the disease is an inflammatory disease.

23. 22. The leukocyte-targeting molecule or composition of claim 21, wherein the disease is an autoimmune disease.

24. 22. The leukocyte-targeting molecule or composition of claim 21, wherein the disease is a skin disorder.

25. 25. The leukocyte-targeting molecule or composition of claim 24, wherein the skin disorder is atopic dermatitis, psoriasis, rosacea, or acne.

26. 26. The leukocyte-targeting molecule or composition of claim 25, wherein the skin disorder is atopic dermatitis.

27. 22. The leukocyte-targeting molecule or composition of claim 21, wherein the disease is chronic cutaneous lupus or systemic lupus erythematosus.

28. 22. The leukocyte-targeting molecule or composition of claim 21, wherein the disease is a viral disease.

29. 29. The leukocyte-targeting molecule or composition of claim 28, wherein the disease is shingles, herpes simplex type 1 or 2, or severe acute respiratory virus type 2 (SARS-CoV-2).

30. 24. The leukocyte-targeting molecule or composition of claim 23, wherein the autoimmune disease is rheumatoid arthritis, inflammatory bowel disease, asthma, or type 1 diabetes.

31. 31. The leukocyte-targeting molecule or composition of claim 30, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

32. 22. The leukocyte-targeting molecule or composition of claim 21, wherein the disease is atherosclerosis, non-alcoholic steatohepatitis, or hypercholesterolemia.

33. 22. The leukocyte-targeting molecule or composition of claim 21, wherein the disease is age-related macular degeneration, diabetic retinopathy, conjunctivitis, uveitis, or chronic sinusitis.

34. 22. The leukocyte-targeting molecule or composition of claim 21, wherein the leukocyte-targeting molecule or composition is administered topically, orally, or by injection.

35. 35. The leukocyte-targeting molecule or composition of claim 34, wherein topical administration includes topical creams, controlled release topical patches, eye drops, and nasal sprays.

36. A leukocyte-targeting molecule or composition comprising the leukocyte-targeting molecule of any of claims 1 to 17, used to reduce abnormal cytokine signaling in a subject in need of such reduction, wherein the molecule or composition is administered to the subject.

37. The active agent binds to white blood cells, CD34, rather than red blood cells. ＋ 21. The leukocyte-targeting molecule or composition of claim 20, which is preferentially delivered to stem cells, or both.

38. The leukocyte-targeting molecule or composition of claim 21, wherein the active agent is preferentially delivered to leukocytes, CD34 + stem cells, or both, over erythrocytes.

39. The leukocyte-targeting molecule or composition of claim 36, wherein the active agent is preferentially delivered to leukocytes, CD34 + stem cells, or both, over red blood cells.

Images