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WO2025076082A1 - Nanoéponges peptidiques pour l'administration de médicaments - Google Patents

Nanoéponges peptidiques pour l'administration de médicaments Download PDF

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Publication number
WO2025076082A1
WO2025076082A1 PCT/US2024/049592 US2024049592W WO2025076082A1 WO 2025076082 A1 WO2025076082 A1 WO 2025076082A1 US 2024049592 W US2024049592 W US 2024049592W WO 2025076082 A1 WO2025076082 A1 WO 2025076082A1
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peptide
block
nanosponge
cancer
seq
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WO2025076082A9 (fr
Inventor
Stefan Bossmann
Raul NERI-SIERRA
Obdulia COVARRUBIAS-ZAMBRANO
Prasad DANDAWATE
Greg GAN
Robin MASER
Thais Motria Sielecki-Dzurdz
Michael VANSAUN
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University of Kansas
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University of Kansas
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/641—Branched, dendritic or hypercomb peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom

Definitions

  • PEPTIDE NANOSPONGES FOR DRUG DELIVERY CROSS-REFERENCE TO RELATED APPLICATIONS [0001]
  • the present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63/542,052, filed October 2, 2023, entitled PEPTIDE NANOSPONGES FOR DRUG DELIVERY, incorporated by reference in its entirety herein.
  • FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with government support under 75N91023C00008 awarded by the National Institutes of Health. The government has certain rights in the invention.
  • peptide nanosponges comprising a plurality of peptide-based building blocks that each comprise a core; a peptide block; a linking amino acid or peptide covalently linking the peptide block to the core; a capping moiety covalently attached to an N-terminal amino acid of the peptide block; a therapeutically active compound covalently attached to one or more amino acids of the peptide block; and a signaling sequence.
  • IPF Idiopathic pulmonary fibrosis
  • Immunosuppression therapy for IPF was examined in a Phase III trial (PANTHER) using combinatorial therapy (prednisone, azathioprine, and N-acetylcysteine) and demonstrated no clinical survival benefit and was surprisingly associated with increased treatment-related serious adverse events. Additional therapies such as Interferon gamma-1 ⁇ therapy and sildenafil have similarly failed to demonstrate improvement in lung function, overall survival benefit, or improvement in functional outcome studies when examined in randomized, placebo-controlled trials. While the pathobiology of IPF remains poorly understood, the current understanding is that repeated subclinical injury (i.e., smoking, trauma) directly impacts alveolar epithelium leading to cell death, persistent cell turnover, inflammation, and fibroblast recruitment in genetically susceptible individuals.
  • a peri-vascular niche indicates that expression of CXCR7 on pulmonary capillary epithelial cells can lead to epithelial cell regeneration, reduced inflammation, and suppression of tissue fibrosis. Furthermore, this process leads to an imbalance between the epithelial-to-mesenchymal ratio of cells leading to tissue fibrosis through increased extracellular matrix deposition.
  • This biology has led to development of new agents that target epithelial fibroblast microenvironmental interactions with a new class of drugs (pirfenidone, nintedanib). While these treatments represent a step forward, issues with patient drug tolerability, lack of significant improvement in lung function, and no improvement in quality of life limit their utility. Therefore, the need to develop novel agents that improve the therapeutic index is necessary.
  • OS osteosarcoma
  • OS has a US incidence around 1,000.
  • OS is commonly found in children and adolescents with 60% of cases occurring in patients who are between 10 and 20 years of age, accounting for 2% of childhood cancers, and cases are increasing, presumably due to increases in the pediatric population.
  • Survival of OS remains low (5-year survival of 70% for non- metastasized disease, 30% for metastasized), primarily as a result of its highly metastatic and drug- resistant nature and demonstrates the high unmet need for these patients.
  • OS is characterized by malignant mesenchymal cells producing osteoid or immature bone which arises from the medullary cavity and grows toward the cortex and adjacent soft tissue.
  • a peptide nanosponge comprising: a core; a peptide block; a linking amino acid or peptide covalently linking the peptide block to the core; a capping moiety covalently attached to a terminal amino acid of the peptide block; a therapeutically active compound covalently attached to one or more amino acids of the peptide block; and optionally, a signaling sequence.
  • the disclosure concerns peptide nanosponges comprising a plurality of self-assembled peptide building blocks, wherein said plurality of self-assembled peptide building blocks comprises a first peptide building block comprising: a branched polymeric core comprising at least three arms; a first block co-peptide of 50 amino acids or less covalently linked via its C-terminus to one of said arms, wherein said first block co-peptide comprises a first peptide block and a second peptide block which is different from the first peptide block; a lipid- based capping moiety having a molecular weight of 400 Da or less covalently attached to an N- terminal amino acid of the first block co-peptide; and a therapeutically active compound covalently attached via a cleavable linkage to one or more amino acids of the first block co-peptide.
  • the block co-peptide is covalently attached to the core such that it is resistant to enzymatic cleavage from the core.
  • the disclosure is also directed to a peptide building blocks for nanosponges comprising: a core; a peptide block; a linking amino acid or peptide covalently linking the peptide block to the core; a capping moiety covalently attached to a terminal amino acid of the peptide block; a therapeutic peptide; and a “don’t eat me” sequence.
  • the disclosure is directed to a peptide building blocks for nanosponges comprising: a core; a peptide block; a linking amino acid or peptide covalently linking the peptide block to the core; a capping moiety covalently attached to a terminal amino acid of the peptide block.
  • the disclosure also is directed to methods of treating a disease or disorder in a patient in need thereof comprising administering the peptide nanosponge described herein in a therapeutic dose to the patient, as well as pharmaceutical compositions, medicaments and uses of the peptide nanosponges to treat diseases or disorders.
  • Other objects and features will be in part apparent and in part pointed out hereinafter.
  • FIG. 1A is a cartoon illustration of one exemplary embodiment of the general components of a peptide-based building block for use in forming supramolecular aggregates (nanosponges).
  • FIG. 1B is a cartoon illustration of a further exemplary embodiment of the general components of a peptide-based building block for use in forming supramolecular aggregates (nanosponges).
  • FIG. 1A is a cartoon illustration of one exemplary embodiment of the general components of a peptide-based building block for use in forming supramolecular aggregates (nanosponges).
  • FIG. 1B is a cartoon illustration of a further exemplary embodiment of the general components of a peptide-based building block for use in forming supramolecular aggregates (nanosponges).
  • FIG. 1C is a cartoon illustration of a further exemplary embodiment of the general components of a peptide-based building block for use in forming supramolecular aggregates (nanosponges).
  • FIG.2 is a schematic illustration of a peptide-based building block that could be used for delivering a DCKL1 inhibitor compound.
  • FIG.3 is a schematic illustration of a peptide-based building block that could be used for delivering a MK2 inhibitor compound.
  • FIG.4 is a schematic illustration of a peptide-based building block that could be used for delivering a cytotoxic agent against p53 deficient cells.
  • FIG.5 shows TEM images of nanosponges assembled.
  • FIG.6 shows (Top) Representative colony formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 for 48h with IC50 and 1 ⁇ 2 IC50 as previously determined by hexosaminidase assay. (Bottom) Quantification of colony number and size.
  • FIG.7 shows (Top) Representative colony formation images of HUH28 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 for 48h with IC50 and 1 ⁇ 2 IC50 as previously determined by hexosaminidase assay. (Bottom) Quantification of colony number and size. Data are represented as mean number and size from 3 independent experiments, * p ⁇ 0.05.
  • FIG. 8 shows (Left) Representative spheroid formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 at IC50, 1 ⁇ 2 IC50 and 1 ⁇ 4 IC50 doses, as previously determined by hexosaminidase assay. (Right) Quantification of spheroid number. Data are represented as the mean from 3 independent experiments, * p ⁇ 0.05 [0027]
  • FIG. 9 is an immunoblot showing that TGF ⁇ 1 can stimulate collagen 1 protein expression in BJ(hTERT).
  • FIG.10 is an immunoblot showing that a nanosponge carrier without any conjugated small molecule drug can induce p38 MAPK pathway activation and is cytotoxic at 50 ⁇ M and that the nanosponge backbone can induce collagen production.
  • FIG.11 is an immunoblot showing the PF-3655022 (naked drug, without attachment to a nanosponge) and PF-3655022-nanosponge dose response curve.
  • FIG. 12 is an immunoblot showing HSP27 phosphorylation in BJ(hTERT) and CAF 067 following MK2 inhibitor treatment.
  • FIG.10 is an immunoblot showing that a nanosponge carrier without any conjugated small molecule drug can induce p38 MAPK pathway activation and is cytotoxic at 50 ⁇ M and that the nanosponge backbone can induce collagen production.
  • FIG.11 is an immunoblot showing the PF-3655022 (naked drug, without attachment to a nanosponge) and PF-3655022-nanosponge
  • FIG. 13 is an immunoblot showing MK-Inh-1 small molecule drug conjugate or by itself does not inhibit MK2 phosphorylation in a dose response fashion, in vitro.
  • FIG.14 is a TEM image of a D10K20 (SEQ ID NO:21) nanosponge where water-filled nanovesicles are discernible as darker spots within the nanosponge.
  • FIG. 15 shows a graph of percent calculated of PCZ loaded in nanosponge (marked with star).
  • FIG. 16 is a graph showing the concentration versus percent proliferation of PDAC cells for prochlorperazine (PCZ) and prochlorperazine-nanosponge agents.
  • PCZ prochlorperazine
  • FIG. 16 is a graph showing the concentration versus percent proliferation of PDAC cells for prochlorperazine (PCZ) and prochlorperazine-nanosponge agents.
  • FIG. 17 shows images that are representative of a cell uptake assay showing PCZ- nanosponge uptake in MiaPaCa-2 and S2-007 cells where the white arrows show uptake.
  • FIG. 18 show images of PCZ-nanosponge uptake in KPCC-orthotopic tumors in C57BL/6 mice (Ex-Vivo IVIS imaging).
  • FIG.19 shows graphs and blot images for the activity of peptides tested in HEK293T (wildtype) and HEK293T-PKD1-KO-c4 (CRISPR-generated) cell lines.
  • FIG. 19 shows graphs and blot images for the activity of peptides tested in HEK293T (wildtype) and HEK293T-PKD1-KO-c4 (CRISPR-generated) cell lines.
  • FIG. 20 shows images of organs in mice treated after plectin-nanosponge uptake in PDAC-bearing mice after overnight incubation.
  • FIG.21 shows images and heat map from ex vivo IVIS imaging (fluorescent) imaging confirming specific uptake of plectin-nanosponge in PDAC tumor only.
  • Corresponding reference characters indicate corresponding parts throughout the drawings. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS [0041] One aspect of this disclosure is focused on developing a widely applicable peptide nanosponge drug delivery system.
  • the peptide nanosponges are characterized as water-soluble sponge-like supramolecular assemblies or aggregates comprising a plurality of individual peptide- based building blocks (e.g., 1,000-50,000 building blocks) that can dynamically self-assemble, disassemble, and reassemble into small clusters to facilitate site-selective delivery and uptake of their therapeutic payload into cells.
  • the nanosponges can be used to deliver a variety of active agents covalently bound via cleavable linkages to the peptide-based building blocks and/or noncovalently sequestered within the interstitial spaces of the assemblies.
  • the assemblies are rationally designed, but unlike previous peptide nanosponge versions do not contain cleavable linkages throughout the assembly network, such as consensus sequences that can be readily cleaved by various enzymes within the body.
  • the assemblies are designed to associate with or be preferentially taken up the targeted cell type into the early endosome, and further are designed to escape the endosome and be released into the cytoplasm where enzymes, such as esterases or proteases, cleave the linkage via which the active agent is tethered thereby quickly releasing the active agent in a targeted vicinity.
  • the remainder of the nanosponge matrix will be more slowly broken down (biodegraded) via normal physiological processes, such as phagocytosis by macrophages or enzymatic degradation, etc.
  • the peptide nanosponge is generally nontoxic to tissues and can be developed to target particular cells or tissues that are the object of the particular treatment.
  • a therapeutic small molecule or peptide can be covalently bound to the peptide nanosponge via cleavable linkages, and then the bond broken once the peptide nanosponge reaches the target cells or tissue. In this way, an effective treatment for various conditions can be delivered with fewer side effects that can result from systemic administration of the same therapeutic compound.
  • CCA cholangiocarcinoma
  • cholangiocarcinoma a rare, heterogenous, and aggressive malignancy of the bile ducts that presents at a late stage with poor outcomes.
  • CCA cholangiocarcinoma
  • a 5-Year survival of CCA is 13% (median survival 7 months), primarily due to non-resectable tumors and advanced disease at the time of initial diagnosis.
  • Incidence of CCA is increasing and has been estimated at more than 10,865 US incidence in 2022. Cytotoxic chemotherapy is currently the standard of care for CCA patients treated perioperatively.
  • Doublecortin Calmodulin-like Kinase 1 (DCLK1) is a serine-threonine kinase and is a novel therapeutic target in oncology.
  • Our team has shown that both knockdown of DCLK1 expression and small molecule DCLK1 inhibitors suppress xenograft tumor growth in mice.
  • the inhibitors will be delivered to the site of the tumors in the biliary tract using a rationally designed peptide nanosponge technology.
  • the peptide nanosponge comprises a plurality of peptide-based building blocks 10.
  • the specific components used in each building block are variable, however, each building block 10 will generally comprise a multi-armed core 12; a linear peptide sequence 14 having its C-terminal end 14a covalently attached to each arm of the core 12, directly or via a cysteine residue as a spacer, and its N-terminal end 14b typically capped with a capping moiety 16 (but could be some other kind of terminal moiety, as discussed below).
  • the peptide sequence 14 is a block co-peptide comprising two or more distinct peptide blocks covalently attached to the core 12 via a linking amino acid (C) and spacer residue (G) covalently linking the peptide block 14 at its C-terminus 14a to the core 12.
  • the linear peptide sequence 14 is preferably free of any enzymatic consensus sequences and also is preferably not attached to the core 12 via any cleavable consensus sequences. That is, the peptide building block itself is preferably free any cleavable linkages or enzymatic consensus sequences, and is therefore resistant to enzymatic degradation.
  • the block co-peptide sequence 14 includes a capping moiety 16 covalently attached to a terminal amino acid of the peptide block 14.
  • a capping moiety 16 covalently attached to a terminal amino acid of the peptide block 14.
  • the building block arms in the assembly composition should comprise lipid-based capping moieties 16.
  • at least half of the arms include lipid-based capping moieties 16 to sufficient drive building block assembly into the supramolecular aggregates the solution.
  • the block co-peptide sequence 14 includes a therapeutically active compound or other active agent 18 covalently attached to one or more amino acids of the peptide block 14 (and preferably the second N-terminal block of the block co-peptide sequence) via a cleavable linkage. That is, the cleavable linkages in these novel peptide-based building blocks are used only for attaching the active agent 18 to the peptide block 14, and preferably present nowhere else in the nanosponge or building block.
  • Cleavable linkages for attaching the active agent 18 are preferably esterase cleavable bonds and/or short (8 residues or less) enzymatic consensus sequences, preferably esterase consensus sequences.
  • each peptide block 14 can have two or more therapeutically active compounds 18 attached thereto, as described in more detail below.
  • the building block can include a signaling sequence or targeting moiety 20 covalently attached to the core 12 via a respective linkage 22 on one or more of the arms (depending on the number of arms).
  • the linkage 22 that connects the signaling sequence or targeting moiety 20 is different from the peptide sequence used for the block co-peptide 14 used to attach the lipid capping moiety 16 and active agent 18 to the core.
  • the signaling sequence 20 is a “don’t eat me” sequence, an “eat me” sequence, or a targeting moiety.
  • the signaling sequence 20 may also be itself a detectable moiety (e.g., fluorescent label) or it may further include a detectable label conjugated to a secondary signaling sequence.
  • the signaling sequence 20 may itself be a therapeutic agent. That is, the signaling sequence could be a targeting moiety that preferentially hones the nanosponge to a particular cell type or target receptor, and then also is able to block, bind, or otherwise interact with the target in a therapeutic manner.
  • MK2 inhibitors Such embodiments are exemplified in the working examples using MK2 inhibitors.
  • the individual building blocks can be designed and synthesized to have the same or different sequence characteristics and moieties on each arm of the core.
  • the exemplary building block depicted in FIG.1B comprises (consists of) a trigonal core on which all three arms are attached the same block co-peptide 14, active agent 18, and capping moiety 16.
  • the exemplary building block comprises (consists of) a trigonal core having two arms from which peptide blocks 14 with active agents 18 coupled thereto extend, and one arm coupled with a signaling sequence 20 via its respective linker 22.
  • the peptide nanosponges are a composite comprising two or more different types of peptide-based building blocks.
  • the peptide nanosponges can be assembled from a plurality of peptide-based building blocks having therapeutic moieties coupled thereto in combination with a plurality of peptide-based building blocks having signaling sequences.
  • the resulting nanosponge assembly matrix is characterized by a plurality of individual branched building blocks associating with one another to form a three-dimensional network that defines open spaces (i.e., cavities, voids, and pores) throughout the matrix (i.e., internal and external open spaces).
  • the core is a branched structure that can have three or more “arms” terminating in functional groups that can covalently link the core to a respective peptide sequence.
  • the cores are preferably dendrimer-like branched or hyperbranched polymeric structures of 800 Da or less in size.
  • the core can comprise three or more maleimide groups, three or more carboxylate groups, three or more amide groups, or three or more hydroxyl groups as illustrated by Formula A or B: , where n is 2 or -OH. In some cases, Z itself represents a nitrogen with further branching.
  • the peptide nanosponge building blocks can have the core be derived from a trimaleimide compound or analogs thereof: O , such as:
  • hyperbranched polymeric cores include, without limitation:
  • the terminal -COOH group of the peptide sequence can be linked to the terminal -NH2 to form a very stable amide bond at each arm of the core.
  • the -NH2 groups will be converted into amides with attached peptide chains.
  • the branched polymeric cores can be synthesized with arms terminating in other suitable functional groups, including COOH as also illustrated, or maleimide groups, amide groups, or hydroxyl groups, for covalent bonding to the peptide sequences.
  • the peptide sequences can be directly attached to the core and/or can be attached via short spacers of one to two amino acids.
  • the peptide building blocks can have a linking amino acid or peptide comprising cysteine.
  • the peptide building blocks described herein can have the linking peptide comprise cysteine with a glycine spacer residue at the C-terminus.
  • a glycine residue will be included at the C-terminus of the entire peptide sequence, including the linker cysteine, as an artifact from peptide synthesis.
  • linking amino acids or peptides include glutamate, aspartate, serine, threonine, tyrosine, lysine, histidine, and the like, provided they are capable of forming a covalent bond with the C- terminal end of block co-peptide. That is, the block co-peptide is preferably directly or indirectly attached to the core in a manner that is not specifically cleavable, i.e., is not an enzyme consensus sequence, and is preferably resistant to enzymatic cleavage and not readily cleaved or broken down in vivo at least until after the active agent (which is attached via a readily cleavable sequence) is released.
  • the peptide sequence comprises a block co-peptide comprising a first peptide block and a second peptide block which is different from the first peptide block.
  • the first peptide block can be covalently attached to the linking amino acid or spacer peptide.
  • the second peptide block can be covalently attached to the first peptide block.
  • Block co-peptide sequences comprising a third peptide block can also be envisioned.
  • the peptide sequence has an overall length of 50 amino acids or less, preferably 5 to 40 amino acids, and preferably 10 to 35 amino acids.
  • the first block which makes up the C-terminal end of the sequence and is attached to the core is selected from amino acids having positively charged side chains, such as lysine, histidine, arginine, and the like.
  • the first block is a homopeptide of 10 to 25 amino acid residues, preferably 15 to 20 amino acid residues, and more preferably consists of lysine residues.
  • a peptide building block has two or more arms comprising a block co-peptide
  • the same peptide sequence be used for both block co-peptides, and more preferably that each peptide sequence is positioned the same distance (e.g., via a linker or peptide spacer) from the core to enhance stability of the structure.
  • the second block which makes up the N-terminal end of the sequence (and is connected to the first block) is selected from amino acids having side chains terminating in - COOH or -OH groups, namely serine, aspartic acid (or aspartate), tyrosine, glutamic acid (or glutamate), threonine, as well as non-natural amino acids having amine, hydroxyl or carboxylic acid functions, and the like.
  • the second block is a homopeptide of 5 to 15 amino acid residues, more preferably 5 to 10 amino acid residues, and more preferably consists of serine or aspartic acid.
  • the second block consists of the same type of amino acid residue having side chains terminating in -COOH or -OH groups, namely serine, aspartic acid (or aspartate), tyrosine, glutamic acid (or glutamate), threonine, as well as non-natural amino acids having amine, hydroxyl or carboxylic acid functions (i.e., all serines, all tyrosines, etc.), but the selected amino acids are separated by inert spacer residues (e.g., alanines) to facilitate spacing of the placement of the active agent along the chain.
  • inert spacer residues e.g., alanines
  • the peptide sequence is designed such that it does not include and is preferably free of an enzyme consensus sequence (whether in the middle of or at the N-terminal or C-terminal end of the peptide block).
  • the only cleavable linkages in the building block are the linkages tethering the active agent to the block co-peptide.
  • Suitable consensus sequences for attaching the active agent preferably consist of 2 to 8 amino acid residues. Consensus sequences are well known in the art for being selective for proteases (e.g., caspases, matrix metalloproteinases, cathepsins, calpains, and the like.
  • the active agent can be attached cleavable chemical linkages, such as ester linkages which are cleaved by esterases found throughout the body. Again, combinations of consensus sequences and chemical linkages can be designed into the building block to modulate how slowly or quickly the drug is released after administration.
  • the second peptide block preferably has a covalently attached capping moiety.
  • the capping moiety is a lipid moiety having a molecular weight of 400 Da or less.
  • the capping moiety comprises cholesterol (386 Da).
  • Other types of lipids include betulinic acid, saturated and unsaturated fatty acids, adamantane, and the like, of 400 Da or less.
  • the peptide nanosponges can have the capping moiety be cholic acid when the nanosponge is designed to treat cholangiocarcinoma.
  • the signaling sequence can be covalently attached directly or indirectly to the core via its own respective peptide linkage (that is different from the block co- peptide sequences described above).
  • Signaling sequences can be “eat me” sequences, “don’t eat me” sequences, targeting sequences, detectable labels, and/or combinations thereof.
  • the signaling sequence can, for example, be a signaling sequence for CD44 comprising sHPWSYLWTQQAs (SEQ ID NO:1); a plectin-targeting sequence comprising KTLLPTPG (SEQ ID NO:2); a signaling sequence for EGFR comprising sYHWYGYTPENVIGsG (SEQ ID NO:3); a signaling sequence for LDLR comprising sCMPRLRGCGAGsG (SEQ ID NO:4); a signaling sequence for CD46 comprising sLPGTICKRTMLDGLNDYCTGsG (SEQ ID NO:5); a signaling sequence for SIRP ⁇ “don’t eat me” comprising kGNYTCEVTELSREGKTVIELKsG (SEQ ID NO:6); a signaling sequence for SIRP ⁇ “don’t eat me” comprising kGNYTCEVTELSREGKTVIELKk (SEQ ID NO:7); a signaling sequence for SIRP ⁇ “eat
  • the peptide nanosponges described herein can be used to deliver therapeutic peptides for polycystic kidney disease comprise TAFGASLFVPPSHVQFIG (P17) (SEQ ID NO:12), TAFGASLFVPPSHVQFIGGKKKKKG (KP17) (SEQ ID NO:13), or TAFGASLFVPPSHVQFIGGDDDDDG (DP17) (SEQ ID NO:14).
  • the peptide building blocks can be used to deliver a wide variety of therapeutically active agents, which are bound via cleavable bonds to the block co-peptide sequence, and preferably to the second (N-terminal) block.
  • the peptide block can include spacers (e.g., alanines) between each serine residue and the block could carry 10 active agents attached to each serine.
  • the peptide nanosponges can be used to deliver a wide variety of therapeutically active agents including, without limitation, a DCLK1 inhibitor, a MK2 inhibitor, a cytotoxic agent against p53 deficient cells, a MIF inhibitor, a SMAD phosphorylation inhibitor, a dopamine receptor inhibitor, or a combination thereof.
  • the active agents can be attached to the peptide building blocks. Alternatively, active agents can also be sequestered within the interstitial spaces of the assembled nanosponge for controlled release and delivery as the nanosponge assembly is broken down. This is particularly advantageous for lipophilic active agents.
  • the peptide nanosponges can have the therapeutically active agent be covalently attached to one or more amino acid residues in the second peptide block.
  • Rationally designed peptide nanosponges in the working examples feature trigonal building blocks (e.g., tris-maleimides) to which other peptides can be tethered via cysteine- mediated Michael addition. This strategy provides a great flexibility when targeting cells in vitro and especially in vivo.
  • peptide-based building blocks then form supramolecular aggregates (nanosponges) with each other characterized by a flexible three-dimensional matrix in which the assembled building blocks and their respective branched structures cooperate to form the matrix network having interstitial open spaces.
  • This is driven by the interaction of the hydrophobic components of the peptide building blocks (e.g., cholesterol, cholesterol-derivatives (such as cholic acid), hydrophobic peptide sequences or hydrophobic dyes) in the reaction solution which comprises an aqueous or organic solvent system.
  • the assembled matrix can dynamically flex and bend and disassemble into smaller clusters and reassemble depending upon the environment and conditions being applied to it (e.g., in vivo through the cellular uptake process).
  • cleavage sequences for any of the approximately 600 human proteases can be used to tether the active agents to respective peptide-based building blocks described herein.
  • cleavage sequences for groups of proteases e.g. MMPs, cathepsins, etc.
  • the peptide nanosponges can be used to deliver a wide variety of therapeutically active agents, including small molecules, therapeutic peptides, and the like. In general, any active agent with a size of ⁇ 2nm (longest axis) or less could be delivered using the nanosponges.
  • DCLK1 Inhibitors [0069]
  • the peptide nanosponges described herein can have the therapeutically active agent comprise a DCLK1 inhibitor.
  • DCLK1 Doublecortin Like Kinase 1 (DCLK1) is considered a cancer stem cell (CSC) marker.
  • CSC cancer stem cell
  • the DCLK1-signaling pathway involves AKT phosphorylation at Thr308 which leads to Notch1 activation.
  • Notch1 activates transcription factor Pregnane X Receptor (PXR) expression.
  • PXR induces cancer cell proliferation and metastasis and regulates the expression of multidrug resistance protein 1 and other proteins involved in drug metabolism including cytochrome P4503A4 (one of six enzymes responsible for the metabolism of approximately 90% of all small molecule drugs).
  • DCLK1 Doublecortin-like kinase 1 (DCLK1).
  • DCLK1 is a cancer stem cell (CSC) marker. CSCs are rare and difficult to isolate and identify cells within the tumor mass.
  • DCLK1 is confirmed as a CRC and pancreatic cancer stem cell marker.
  • DCLK1 is a member of the calmodulin-like protein kinase superfamily.
  • the full-length encoded protein contains two N-terminal doublecortin (DCX) domains, which bind microtubules and regulate microtubule polymerization, a C-terminal serine/ threonine kinase domain that has homology to Ca2+/calmodulin-dependent protein kinase, and a serine/proline-rich domain in between the doublecortin and the protein kinase domains.
  • DCLK1 is predominantly transcribed from a second promoter in colon cancer cells.
  • AKT1 peptide surrounding Thr308 interacts with kinase domain of DCLK1 protein, a short form that lacks the N-terminal DCX domains.
  • DCLK1 knock out mouse models suppress colon polyps in APCmin/+ mice but do not affect normal intestine. These data suggest that DCLK1+ cells mark CSCs, not normal stem cells. An important consequence of this finding is that targeting DCLK1 will specifically target CSCs while sparing normal proliferating cells. [0072] DCLK1 has been shown to phosphorylate the microtubule-associated protein MAP7D1. Using in silico modeling, AKT1 was identified surrounding threonine at position 308 (IKDGATMKTFCGTP (SEQ ID NO:15)).
  • DCLK1 amino acids targeted for AKT1 interaction include ASP398, ASP475, GLU515, and THR552.
  • An in vitro assay using recombinant DCLK1 and the AKT peptide also confirmed significant phosphorylation of the AKT substrate by DCLK.
  • the DCLK1 inhibitor is gemcitabine.
  • lead candidate MRL16 inhibits colon cancer growth by inhibiting proliferation at the G2/M phase of the cell cycle, inducing apoptosis and suppressing stemness by inhibiting spheroid growth. MRL16 suppressed DCLK1-mediated phosphorylation of AKT1 at Thr308, resulting in downstream suppression of Notch signaling and PXR. In vivo mouse xenograft studies with MRL16 subsequently demonstrated antitumor activity providing evidence that DCLK1 is a viable CRC therapeutic target.
  • DCLK1 LLC aims to improve treatment outcomes by enhancing efficacy and reducing toxicity. Described herein is the synthesis of novel nanosponge-linked DCLK1 inhibitors and evaluation of the agents in vitro. This novel nanosponge drug delivery platform has not been previously employed for the treatment of CRC. [0075]
  • MRL marmelin (1-hydroxy-5,7- dimethoxy-2-naphthalene-carboxaldehyde, MRL) from Aegle marmelos, an Ayurveda treatment for gastrointestinal cancers.
  • MRL16 was selected for target validation studies based on its early ADMET properties summarized in Table 1. Despite limited aqueous solubility and moderate in vitro metabolic stability, the compounds proved excellent probes for proof of principle studies to validate DCLK1 as a therapeutic target. MRL16 significantly interacts with the kinase domain, 24KU009M-01 61012-PCT with a binding energy of -7.9 kcal/mol, with the key amino acid being VAL468.
  • DCLK1 is a protein that marks quiescent stem cells in colon cancer. Stem cells support spheroid growth. We performed spheroid assays with HCT116 cells where DCLK1 is knocked down using specific shRNA.
  • trimaleimide structure is the core that is attached to a first peptide block of 20 lysine residues and a second peptide block of 10 residues of serine or aspartic acid (although the second block could also be threonine, aspartate, or glutamic acid/glutamate).
  • two of the blocks are capped with a lipid cap (cholesterol) and the other position is attached to a signaling sequence that can be either a sequence targeting CD44 or targeting SIRP ⁇ (“don’t eat me” sequence).
  • the DLCK1 inhibitor can be covalently attached to the hydroxyl groups of serine or threonine residues to form a cleavable carbonate bond or covalently attached to the carboxylic acid groups of aspartic acid or glutamic acid groups to form cleavable ester bonds.
  • the DCLK1 inhibitors can have a structure corresponding to Formula 1 , wherein A is a 5- containing heterocycle, preferably a nitrogen- group; , , are independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyls (e.g., C1-C12, preferably C1-C6 alkyls), and alkaryls (e.g., C 1 -C 12 alkyl-substituted C 3 -C 6 aryls), or R 1 and R 2 together with the carbons they are attached to form a fused 5- or 6-membered ring with the adjacent 6-membered ring to which R1 and R2 are bound; R4 is H or -CH3; X is -C(O)-, -NHC(O)-, -C(O)NH-, or –NHC(O)NH-; and J 1 and J 2 are
  • the DCLK1 inhibitors can have the structure corresponding to Formula 1A , wherein A is a 5- containing heterocycle, preferably a nitrogen-containing heteroaryl group; R3 is independently selected from -H, -O, -N, - S, -F, -Cl, -OH, -OCH 3 , NO 2 , alkyls (e.g., C 1 -C 12 , preferably C 1 -C 6 alkyls), and alkaryls (e.g., C 1 - C 12 alkyl-substituted C 3 -C 6 aryls); R 4 is H or -CH 3 ; X is -C(O)-, -NHC(O)-, -C(O)NH-, or – NHC(O)NH-; and J1 and J2 are each independently -CH- or -N- and preferably with the proviso that at least one of J1 or J2 is -N-.
  • A is
  • the DCLK1 inhibitors having the structure corresponding to Formula 1 or Formula 1A wherein A is a 5- or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, having one of the following structures: . 24KU00099MM-0-101 61012- -PCT T a N a
  • MK2 Inhibition of MK2 in vitro leads to reduced TGF ⁇ 1-mediated myofibroblast development and extracellular matrix deposition. Furthermore, inhibition of MK2 could prevent bleomycin-induced pulmonary injury and inflammation in mice.
  • MK2 inhibitors there are no FDA-approved MK2 inhibitors and early generation small molecule MK2 inhibitors lack “druggability” because of lack of enzymatic ATP-pocket selectivity and poor water solubility lead to unrealistically high EC50 levels needed to achieve therapeutic benefit
  • Suppression of the MK2 pathway using a small molecule pharmaceutical linked to a nanosponge will allow delivery of the small molecule directly to the cell which will lead to downregulated activation via disruption of the p38 ⁇ -MK2 interaction which will in turn downregulate idiopathic pulmonary fibrosis inflammatory cytokine production and EMT activation thereby improving overall IPF progression.
  • Known MK2 inhibitors that have poor aqueous solubility or those that are easily degraded by endogenous plasma proteases can advantageously be conjugated to the peptide nanosponges described herein.
  • the nanosponge platform can allow for increased drug solubility; can be modified to prevent degradation (increasing drug access); or further modified to allow delivery of the payload to the target cell-tissue with localized drug release at the site of disease.
  • FIG. 3 illustrates design components of peptide nanosponges for the treatment of pulmonary fibrosis.
  • FIG.3A shows three MK2-blockers (PF-3644022, MK2-IN-1, and MK2-IN- 4).
  • FIG. 3B shows variable structure of the peptide nanosponges.
  • FIG.3C shows alternate chemical connections of the MK2-blockers via a tertiary amide bond to D10 of the block co-peptide ((SEQ ID NO:21) or a urethane bond to S 10 of the block co-peptide (SEQ ID NO:19).
  • Peptide nanosponges offer the opportunity to deliver covalently attached drugs to biologic targets. According to results from coarse-grained molecular dynamics (MD) simulations, the cholesterol units of the nanosponges form hydrophilic nanodomains. Water-solvent filled nanoholes are present in their structures as well.
  • the integration of fluorescent labels i.e., rhodamine B is feasible, because they can be covalently attached to the N-terminal end of the K20S10 (SEQ ID NO:19) building blocks. As shown in FIG.
  • MK2 inhibitors feature secondary amine groups that will be utilized to attach them either to S 10 blocks via esterase-cleavable urethane bonds, or to D 10 blocks by means of protease-cleavable amide bonds.
  • urethane bonds will be faster cleaved upon entering of the nanosponges into the cytoplasm than peptide bonds. This permits the optimization of pharmacokinetics.
  • Up to 10 MK2-inhibitors will be tethered to each nanosponge building block. Quantitation will be performed by means of 1 H-NMR.
  • Targeting of activated myofibroblasts will be achieved by utilizing a peptide that service as both a signaling and therapeutic sequence YARAAARQARAKALARQLGVAA (SEQ ID NO:16).
  • the “Don’t Eat Me” peptide sequence kGNYTCEVTELSREGKTVIELKk (SEQ ID NO:7) will be incorporated into the nanosponge structure.
  • This peptide sequence mimics the binding motif of regulatory protein alpha (SIRP ⁇ ) in phagocytes. This event triggers the auto- protective response of CD47.
  • Both peptide sequences bear D-amino acids at both terminal ends to prevent rapid proteolytic degradation while in circulation.
  • MK2 pathway inhibition using a small molecular pharmaceutical conjugated to the nanosponge will reduce radiation induced fibrosis by reducing inflammatory cytokine production and mesenchymal gene activation.
  • Apatanib or another MK2 inhibitor will be linked to the nanosponge moiety via amide and esterase-cleavable bonds.
  • MK2 pathway inhibition using a small molecular pharmaceutical conjugated to the nanosponge will reduce radiation induced fibrosis by reducing collagen-1 deposition and inflammatory cytokine production.
  • Apatanib or another MK2 inhibitor will be linked to different nanosponge backbones via amide and esterase cleavable bond.
  • Conjugated compounds will be iteratively developed and tested using in vitro cell and protein-based screens.
  • One lead compound will be advanced to in vivo biologic studies to determine a maximum tolerated dose and pharmacokinetic and pharmacodynamic distribution of drug at two doses (MTD and 1 ⁇ 2 MTD).
  • Cytotoxic agents effective against p53 deficient cells [0093]
  • the peptide nanosponges can have the therapeutically active agent comprise a cytotoxic agent against p53 deficient cells.
  • KU-D2 and KU-D2F comply with the Lipinski requirements for effective drug delivery, they feature three benzylic groups that are prone to effective oxidation, leading to poor pharmacokinetics.
  • FIG.4 shows the design components of nanosponges for osteosarcoma treatment.
  • FIG.4A shows four analogs of KU-D2F.
  • FIG. 4B shows variable structures of the nanosponges.
  • FIG.4C shows alternate chemical connections via ester bond to D 10 and carbonate bond to S 10 (SEQ ID NO:19 or 21).
  • KU-D2F* The synthesis of four p53 synthetic lethality inducers (KU-D2F* and three analogs) that will be linked to four types of nanosponges.
  • KU-D2F* differs from the parent KU-D2F by the presence of a phenolic group instead of a phenyl-methyl-ether.
  • the former will permit the attachment to either aspartic acid (D10) or serine (S10) blocks of the nanosponges.
  • D10 aspartic acid
  • S10 serine
  • Three analogs of KU-D2F* will be synthesized. The four analogs will be tethered to four conceptionally different nanosponges.
  • the synthesis of the nanosponge/small molecule assemblies will be followed by in vitro studies required to recognize the two best embodiments, which will then be tested in vivo.
  • the nanosponge-linked analogs will release the active small molecule to the cytoplasm of the tumor cells. Execution of the aims described in this proposal will generate novel nanosponge-linked compounds that maintain the active small molecule moieties.
  • MIF Inhibitors [0098]
  • the peptide nanosponges can have the therapeutically active agent comprise a MIF inhibitor.
  • the peptide nanosponges can have a trigonal linker (e.g., tris(maleimide)) covalently attached to a signaling sequence for EGFR comprising sYHWYGYTPENVIGsG (SEQ ID NO:3); a signaling sequence SIRP ⁇ “don’t eat me” comprising kGNYTCEVTELSREGKTVIELKsG (SEQ ID NO:6), and a S 10 K 20 C (SEQ ID NO:17) covalently attached to the trigonal linker through the cysteine residue and capped with cholesterol covalently attached to the serine peptide block and having a MIF inhibitor covalently attached to the amino acid residue(s) of the S10K20 (SEQ ID NO:19) peptide block.
  • a trigonal linker e.g., tris(maleimide)
  • the compounds of the present invention that can be used as MIF1/MIF2 inhibitors are shown as follows: (2-((5- yl)-N-(4-methylbenzo[d]thiazol-2- chloropyridin-2-yl)amino)thiazol-4- yl)acetamide yl)acetamide KU0180931; F5950-0872; Compound 2 KU0180903; F5950-0730; Compound 6 1H- ylsulfonyl)benzamide pyrazole-5-carboxylate KU0168339; F0725-0226; Compound 14 KU0171533; F2496-3431; Compound 30
  • compositions are described herein which comprise a nanosponge (or a plurality of nanosponges) dispersed in a suitable delivery vehicle or carrier for administration to a subject.
  • the therapeutic compositions comprise a therapeutically effective amount of peptide nanosponge with active agent dispersed or suspended in a pharmaceutically acceptable carrier.
  • the compositions can comprise a mixture of two or more different types of nanosponges and/or different types of active agents.
  • nanosponges carrying therapeutically active agents could be co-administered with nanosponges carrying detectable labels for monitoring and visualizing delivery of the nanosponges to the targeted area.
  • a pharmaceutically acceptable carrier or vehicle is any carrier suitable for in vivo administration.
  • the term “pharmaceutically acceptable” means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained.
  • a pharmaceutically-acceptable carrier would be selected to minimize any degradation of the nanosponge (at least during storage) and to minimize any adverse side effects in the subject.
  • Pharmaceutically-acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use.
  • pharmaceutically-acceptable carriers suitable for mucosal administration include aqueous solutions such as, water, buffered solutions, commercially available proprietarily formulated carriers and adjuvants, naturally occurring mono, di-, and polysaccharides, carbohydrates, sugars, polymers, proteins, and the like, including any of the following including mixtures thereof: celluloses, derivatives thereof, and microcrystalline forms thereof such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and the like; other naturally derived polysaccharides and polymers such as sodium alginate, gelatin, chitosan, collagen, hyaluronic acid, dextran; mono or oligosaccharides such as D-mannitol, sorbitol, glucose, lactose, fructose, inositol, sucrose, amylose, and the like; dextrins such as
  • Suitable carriers can further include proteins, such as serum proteins, casein, albumin, and the like.
  • Additional components of the compositions may suitably include excipients such as stabilizers, preservatives, diluents, emulsifiers, and lubricants.
  • excipients such as stabilizers, preservatives, diluents, emulsifiers, and lubricants.
  • flavoring agents or palatability enhancers can also be included in the compositions.
  • Other ingredients may be included in the composition, such as adjuvants, other active agents, preservatives, buffering agents (e.g., histidine, phosphates), salts, and other pharmaceutically-acceptable ingredients.
  • the pharmaceutically-acceptable carrier comprises a combination of one or more of the above-described vehicles, and preferably is configured for enhancing delivery via the desired route.
  • the formulation can include is enteric coated for protecting the nanosponge against premature gastrointestinal degradation.
  • the nanosponges can be formulated in a variety of solid or liquid dosage forms.
  • the nanosponges can advantageously be dried, e.g., lyophilized, spray-dried, etc., to create as shelf-stable powder. Powders can be used for inhalation, packaged in gel caps, compressed into tablets, and the like.
  • Such powders can include one or more inert carriers or vehicles to facilitate free-flowing powder and reduce aggregation or clumping. Powders can also be reconstituted into a liquid form for various routes of administration, e.g., via nebulizer, injection, IV, oral, ocular, otic, or nasal drops or sprays, oral gavage, and the like.
  • the nanosponges can be formulated as sublingual or buccal tablets, drops, lozenges, and the like.
  • the nanosponges can be formulated as topical creams, gels, ointments.
  • the nanosponges can be formulated as suppositories, bladder installations, and the like.
  • a “therapeutically effective” amount or “therapeutic dose” refers to the amount or dosage that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, such as to elicit some desired therapeutic or prophylactic effect as against the disease or condition depending upon the active agent delivered.
  • an amount may be considered therapeutically “effective” even if the disease, condition, or symptom is not totally eradicated or prevented, but it or its symptoms and/or effects are improved or alleviated partially in the subject (e.g., reduction in tumor volume, etc.).
  • the disclosure is also directed to a method of treating a disease or disorder in a subject in need thereof comprising administering the peptide nanosponges or compositions disclosed herein in a therapeutic dose to the patient.
  • the peptide nanosponges can be enterically coated in order to have the nanosponges pass without hydrolysis into the gastrointestinal tract.
  • the peptide nanosponses described herein can be administered via virtually any route of administration, depending on the condition to be treated, including direct application to the site to be treated or systemic administration.
  • Non-limiting routes of administration include intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, buccally, sublingual, topically, intranasally, rectally, intraosseously, or parenterally, and the like
  • the nanosponge composition may be administered by one or more dosages, which dosages can be administered, by the same or different route, to achieve the desired prophylactic or therapeutic effect.
  • the nanosponge composition can be provided in unit dosage form in a suitable container.
  • unit dosage form refers to a physically discrete unit suitable as a unitary dosage for human or animal use.
  • Each unit dosage form may contain a predetermined amount of the nanosponge (and/or other active agents) in the carrier calculated to produce the desired effect.
  • the nanosponge composition can be provided separate from the carrier (e.g., in its own vial, ampule, sachet, or other suitable container) for on- site mixing before administration to a subject.
  • a kit comprising the nanosponge composition is also disclosed herein. The kit further comprises instructions for administering the nanosponge composition to a subject, including reconstituting it if needed.
  • the nanosponge can be provided as part of a dosage unit, already dispersed in a pharmaceutically-acceptable carrier, or it can be provided separately from the carrier.
  • the kit can further comprise instructions for preparing the nanosponge for administration to a subject.
  • the phrase "and/or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed.
  • the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
  • the present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention.
  • Example 1A Preparation of MRL16 N O and isonicotinic hydrazide (1 eq.) followed by 0.5 mL of concentrated HCl and 5 mL of methanol. The reaction mixture is then heated to 60°C and stirred for 4 hours. During this time precipitate may form. If precipitate does not form after this time, the reaction flask is allowed to cool slowly to RT and then placed in the freezer to induce recrystallization of product.
  • Example 1B Preparation of MRL17 OH O O N H 2 N N 2 + 1 ′-
  • To a dry round bottom flask is added 1′-hydroxy- and nicotinic hydrazide (1 eq.) followed by 0.5 mL of concentrated HCl and 5 mL of methanol. The reaction mixture is then heated to 60°C and stirred for 4 hours. During this time precipitate may form.
  • reaction flask is allowed to cool slowly to RT and then placed in the freezer to induce recrystallization of product. After either the reaction time or recrystallization, the mixture is filtered and the solid is washed with DCM. The yellow solid is isolated, and no further purification was needed. [0122] It is important to note for the synthesis of both MRL compounds, it is likely that the Z isomer also forms but in trace amounts due to steric hindrance of the Z isomer. As such only the E isomer is being shown.
  • Example 1C Preparation of DCLK3 O O H OH O O O of NaOH (14.4 g, 36.0 mmol) and H 2 O (30 mL) was made and added dropwise. The mixture is then heated to 75°C. Once at the desired temperature, chloroform (6.5 mL, 80 mmol) was added dropwise, and the reaction is heated for 2-3 hours. Upon the completion of the reaction (monitored by TLC), the mixture was acidified with 50mL of 1N HCl, and extracted with DCM. The organic layer is washed with brine and dried over Na 2 SO 4 , filtered, concentrated, and purified via silica gel flash column chromatography using hexane: ethyl acetate gradient.
  • Step 2 To a mixture of DCLK3_Int1 (1 eq.) and K2CO3 (1 eq.) in 10 mL of THF: DMF (10:1) under argon is iodomethane (3 eq.) added dropwise at RT. the mixture is then stirred for overnight. After overnight, water (15 mL) was added to the mixture. The aqueous phase was extracted with ethyl acetate, washed with brine, dry with sodium sulfate. The organic phase was filtered and concentrated, and the crude product was purified via silica column chromatography using hexane: DCM gradient.
  • Step 3 To a mixture of DCLK3_Int2 (1 eq.) and potassium carbonate (1.4 eq.) in 5 mL of MeOH, under argon, is added dimethyl (1-diazo-2-oxopropyl) phosphonate (1 eq.) in one portion at RT. This mixture is stirred at RT for overnight, TLC to monitor reaction. After overnight, the reaction mixture is concentrated and purified via silica column chromatography using hexane: DCM gradient.
  • Step 4 To a mixture of DCLK3_Int3 (1 eq.) and ethyl 2-chloro-2-(hydroxyimino) acetate (2.2 eq) dissolved in 5 mL of ethyl acetate is KHCO3 (2 eq.) added. The mixture is flushed under argon and stirred at RT for overnight. After overnight, heptane (5 mL) was added and allowed to stir for an additional 5 minutes. The mixture was filtered using heptane to aid in the filtration. The filtrate is concentrated and purified via silica column chromatography using hexane: DCM gradient.
  • Step 5 To a mixture of DCLK3_Int4 (1 eq) in MeOH (48 eq) was added 1N NaOH (79 eq). The mixture was stirred at RT for overnight. After overnight, the reaction mixture was further diluted with water ( ⁇ 25 mL) and adjusted the pH to 2 ⁇ 3 using 1M HCl and stirred for an additional hour. Within the hour, a white precipitate was generated. The mixture was then filtered and washed with water. The precipitate was collected and dried under reduced pressure at 50°C.
  • Step 6 DCLK_Int5 (1.0 eq.), 4-aminopyridine (2.0 eq.) and DIPEA (5.0 eq.) were dissolved in 1 mL of DMF, flushed under argon and placed in a dry ice: acetonitrile bath in the dark. To this was added a solution of T3P in EA (50 wt%, 2.0 eq.) dropwise while keeping the internal temperature between -30°C and -10 °C. Following the dropwise addition, the resulting mixture was warmed to -10°C and stirred for an hour. After, the reaction mixture was warmed to 0°C, and water was added. Ethyl acetate was added to the resulting suspension.
  • Step 7 DCLK_Int6 (1 eq.) was dissolved in 1 mL of DCM, placed in an ice bath, and flushed under argon. While at 0°C, 1M BCl3 in DCM (68 eq.) is added dropwise. Post dropwise addition, the reaction mixture is kept in the ice bath for overnight. After overnight, the reaction mixture is quenched via the addition of ice water (5 mL) and stirring for an additional hour.
  • Example 1D Preparation of DCLK3a O O H OH O O O
  • Step 6 A mixture of DCLK3a_Int5 (1 eq.), 3-aminopyridine (1.5 eq.), triethylamine (3.4 eq.) in 3 mL of DCM is flushed under argon and placed in an ice bath in the dark. While at 0°C, a solution of T 3 P in ethyl acetate (50 wt%, 1.7 eq.) is added dropwise while stirring.
  • reaction mixture Post dropwise addition the reaction mixture is allowed to stir at 0°C for an additional hour. This is then allowed to warm to RT and left to stir for 3-4 days in the dark. TLC (5: 95 Methanol: DCM) to monitor the reaction. After the reaction time, the mixture is further diluted with DCM and the organic layer is washed with water (x2), saturated sodium bicarbonate (x1) and brine (x1). The organic layer is isolated, dried over Na2SO4, filtered and concentrated. The crude residue was purified via silica column chromatography using DCM: Methanol gradient (99:1 ⁇ 90:10).
  • Step 7 DCLK_Int6 (1 eq.) was dissolved in 1 mL of DCM, placed in an ice bath, and flushed under argon. While at 0°C, 1M BCl3 in DCM (68 eq.) is added dropwise. Post dropwise addition, the reaction mixture is kept in the ice bath for overnight. After overnight, the reaction mixture is quenched via the addition of ice water (5 mL) and stirring for an additional hour. The reaction mixture is then further diluted with water and washed multiple times with DCM. The organic layer is isolated, dried over Na2SO4 and concentrated. The crude residue was purified via silica column chromatography using DCM: Methanol (99:1) to yield DCLK3a.
  • Example 1E Preparation of DCLK4 O O H OH O O O O , (1.5 eq.), triethylamine (3.4 eq.) in 3 mL of DCM is flushed under argon and placed in an ice bath in the dark. While at 0°C, a solution of T 3 P in ethyl acetate (50 wt%, 1.7 eq.) is added dropwise while stirring. Post dropwise addition the reaction mixture is allowed to stir at 0°C for an additional hour. This is then allowed to warm to RT and left to stir for 3-4 days in the dark. TLC (5: 95 Methanol: DCM) to monitor the reaction.
  • DCLK4_Int6 is light sensitive and must be kept in the dark during synthesis and storage. This will quickly degrade if not handled properly. Purification efforts of this all resulted in degradation. As such, DCLK4_Int6 is used crude.
  • Step 7 To a suspension of 4-pyridylmagnesiurn bromide in THF, which was freshly prepared by treating 4- iodopyridine (16.6 mmol, 6.5 eq) in 55 mL of THF with 16.6 mL of 1.0M ethyl magnesium bromide at room temperature, was added DCLK4_Int6 (1 eq.) in 3.5 mL of THF dropwise. After 2 hours, the reaction was quenched with 1.0 mL of acetic acid and sat. NH4Cl, and then extracted with 2 x ethyl acetate at 0-5 °C.
  • the organic layer extracts were dried over MgSO 4 , filtered, concentrated by rotary evaporator. The residue was suspended in ethyl acetate, filtered, and washed the filter cake with ethyl acetate, to give a light-yellow solid as the first crop of product. The filtrate was concentrated with silica gel to dryness, providing a free-flowing solid, which was dry loaded onto a column of silica gel wet with heptane.
  • Step 3 suspended in 10 mL of toluene. This mixture is heated to reflux (115°C) for overnight. After, the reaction mixture is concentrated, and the product is isolated and further dried in vacuo overnight. After the product is collected and no further purification is necessary.
  • EXAMPLE 3 SOLID PHASE PEPTIDE SYNTHESIS PROTOCOL [0143] All peptides mentioned in Table 1 and 2 below were synthesized following the Fmoc (N- (9-fluorenyl)methoxycarbonyl) solid phase peptide synthesis procedure (Wang H., Udukala D. N., Samarakoon T. N., Basel M.
  • a solution containing Fmoc-protected amino acid (resin:amino acid, 1:3 molar ratio) and O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU) as a coupling agent (resin:HBTU, 1:29 molar ratio) was prepared in a 1:23 diisopropylethylamine (DIEA):DMF solution and was added to the resin and allowed to swirled for 30 minutes for the coupling reaction. Each amino acid was added two times to enhance addition 24KU009M-01 61012-PCT of amino acid to the peptide chain.
  • DIEA diisopropylethylamine
  • the last Fmoc-protected amino acid was deprotected using a 20% diethylamine solution in DMF, followed by 5 washes with DMF to remove any excess diethylamine present.
  • Each deprotection and addition of an amino acid is a repeated cycle during peptide synthesis until all amino acids on the peptide sequence chain have been added.
  • some peptides were labeled with cholesterol, cholic acid, and cleaved unlabeled. For cholesterol and cholic acid additions, these were coupled on the N-terminal end of the peptide using 1,1'-Carbonyldiimidazole (CDI) as the coupling reagent.
  • CDI 1,1'-Carbonyldiimidazole
  • S 10 K 20 (SEQ ID NO: 19) and D 10 K 20 (SEQ ID NO:21) block co-peptides were prepared that included a C-terminal linking amino acid (cysteine) and spacer residue (glycine, from resin synthesis).
  • Table 1 Peptide sequences synthesized for DCLK1 nanosponges Calculated molecular weight Peptide sequence/name Table 2.
  • Peptide sequences synthesized for MK2 nanosponges Peptide sequence/name Calculated molecular weight (g/mol) 24KU009M-01 61012-PCT Rhodamine B – EGFR * * 2394.108 s YHWYGYTPENVIGs ** CG EXAMPLE 4 NANOSPONGE ASSEMBLY AND COUPLING OF SMALL MOLECULE PROTOCOLS [0144]
  • the trimaleimide linker used to assemble nanosponges contains three -NH 2 groups, serving as the anchor to link up to 3 different peptides, which determined the molar ratio to use for assembly.
  • Block co-peptides S/D10K20CG were used (SEQ ID NO:18 or SEQ ID NO:20). Nanosponges assembled in the presence of the “Don’t eat me peptide” sequence consisted in a 1:2:1 ratio of linker : cholesterol-block co-peptide : “Don’t eat me peptide”. On the other hand, for nanosponges assembled without the “Don’t eat me peptide”, the ration consisted in 1:3 linker : cholesterol/cholic acid- block co-peptide. And lastly, when 3 different peptides were coupled to the same central linker (MK2 nanosponges), the ratio was kept at a 1:1:1:1 for all.
  • FIG. 5A shows TEM images of assembled S10K20CG-based (SEQ ID NO:18) nanosponge labeled with cholesterol; size average around 96 nm.
  • FIG. 5B shows TEM image for assembled D10K20CG-based (SEQ ID NO:20) nanosponge labeled with cholesterol; size average around 77nm.
  • 24KU009M-01 61012-PCT Small molecules were coupled to the nanosponges using CDI or HBTU (only for D 10 K 20 CG nanosponges (SEQ ID NO:20)) as the coupling reagents.
  • Gemcitabine has also been linked to the nanosponge and shown in vitro and in vivo activity. Using the nanosponge technology to allow for local exposure will enable higher doses of Gemcitabine to be administered to the site of the tumor with lower risk of toxicity and limiting systemic exposure and thus slowing resistance. Scale-up synthesis of the nanosponge linked Gemcitabine and will be optimized to complete in vivo efficacy and release characterization of the material. Table 3 and 4 summarize all compounds assembled for each project. Table 3.
  • Cells were then treated with test articles at IC50, and 1 ⁇ 2 IC50 doses for 48h. Media is then aspirated and replaced with complete media without drug. Colonies are then allowed to grow for 2 weeks before staining, counting, and size determination using ImageJ software. [0150] For spheroid formation, cells were plated in 24-well low attachment plates. Spheroids were allowed to form for 72 hours before the addition of test articles at IC50, 1 ⁇ 2 IC50, and 1 ⁇ 4 IC50 doses. Spheroids were allowed to grow for an additional 96 hours before imaging and quantification.
  • FIG.6 shows representative colony formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 for 48h with IC50 and 1 ⁇ 2 IC50 as previously determined by hexosaminidase assay.
  • bottom Quantification of colony number and size. Data are represented as mean number and size from 3 independent experiments, * p ⁇ 0.05.
  • FIG. 7 shows representative colony formation images of HUH28 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 for 48h with IC50 and 1 ⁇ 2 IC50 as previously determined by hexosaminidase assay.
  • Nanosponges have been synthesized or are proposed for synthesis (Table 7). Table 7. Additional nanosponges.
  • Small molecule Nanosponge e Therapeutic peptide KP17 (TAFGASLFVPPSHVQFIGGKKKKKG) (SEQ ID NO:13) NANOSPONGE-MK2 INHIBITOR DATA
  • BJ(hTERT) and patient-derived cancer associated fibroblasts (CAF) 036 and 067 were obtained from the University of Colorado Denver Anschutz Cancer Pavillion from Dr. Antonio Jimeno. BJhTERT cells were cultured and maintained in DMEM (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. All cells tested negative for mycoplasma.
  • Cell confluency was measured using Countess II FL hemacytometer.
  • Cells were treated with a log dilution of MK2 inhibitor (PF-3644022), MK2 inhibitor 1 (MK2i1), MK2i+nanosponge, or nothing (control).
  • Cells were stimulated with 2ng/mL TGF ⁇ 1.
  • MK2i and MK2i+nanosponge were added 2 h prior to stimulation with TGF ⁇ 1.
  • Cells were grown an additional 48 h from stimulation at which time they were harvested for protein and mRNA analysis.
  • Our protocol was adapted from an established IPF study [PMID: 23470623].
  • CAF 036 and 067 were treated in a similar fashion to BJ(hTERT). [0157] Immunoblotting.
  • MK2 Cell Signaling Technologies #12155; clone D1E11; 1:10,000 dilution
  • pMK2 Cell Signaling Technologies #3007; Thr334; Clone 27B7; 1:10,000 dilution
  • p-MK2 ThermoFischer; PA5-12619, 1:10,000 dilution
  • HSP27 Cell Signaling technologies; #2402; clone G31, 1:10,000 dilution
  • pHSP27 Cell Signaling Technologies; #9707; Ser82, clone D1H2F6, 1:3,000 dilution.
  • PF-3644022 had MK2 phosphorylation better than the PF-3644022- nanosponge conjugate (PF conjugate) (FIG. 11).
  • the nanosponge conjugate drug only reduced collagen expression at 50 ⁇ M and was also found to be cytotoxic (FIG.11).
  • PF-3644022 reduced BJ(hTERT) HSP27 phosphorylation 1 order of magnitude lower concentration compared to PF-conjugate.
  • PF-3644022 is the more effective MK2 inhibitor, but increased inhibitory efficacy of conjugation with the nanosponge is inconclusive. As PF-3644022 is extremely hydrophobic, the nanosponge carrier has improved drug solubilization compared to the naked drug alone.
  • PCZ Nanosponge-prochlorperazine
  • the peptide-based nanosponges for advanced drug delivery comprised of a trigonal tris-maleimide linker, a D10K20CG (SEQ ID NO:20) peptide building block label with cholesterol and CD44- targeting peptide sequence (sHPWSYLWTQQAs (SEQ ID NO:1)) labeled with rhodamine B for drug delivery to PDAC.
  • the “Don’t eat me” peptide sequence kGNYTCEVTELSREGKTVIELKk (SEQ ID NO:7) (bearing D-lysines at both terminals ends to prevent rapid proteolytic degradation) was included in order to enhance the circulation time of the nanosponges in blood.
  • the molar mass used for assembly of nanosponges was as follows, 5 (cholesterol labeled D10K20CG, (SEQ ID NO:20)) : 1 (trimalimide linker): 1 (rhodamine B labeled CD44): 1 (“Don’t eat me” peptide”).
  • PCZ nanosponges Upon CD44 receptor- mediated endocytosis, the nanosponges can quickly escape into the cytoplasm. After assembling nanosponges, these were loaded with prochlorperazine dimeleate salt (PCZ), 10% per weight.
  • PCZ nanosponges were characterized using TEM, and an exemplary TEM image is shown in FIG.14. Percent loaded of PCZ in nanosponge was also determined. A calibration curve was developed for PCZ drug alone, and then a known concentration of PCZ- nanosponge was measured using 320nm absorbance and plotted into the calibration curve to calculate amount and percentage of PCZ loaded in nanosponge. Results demonstrated that nanosponge was loaded with 10.12% of PCZ, as shown in FIG.15.
  • Nanosponges were designed for polycystic kidney disease (PKD) treatment by using modified peptide fragments from the stalk sequence in the polycystin-1 (PC1) C-terminal fragment, which are, P17 (TAFGASLFVPPSHVQFIG (SEQ ID NO:12)), KP17 (TAFGASLFVPPSHVQFIGGKKKKKG (SEQ ID NO:13)), DP17 (TAFGASLFVPPSHVQFIGGDDDDDG (SEQ ID NO:14)).
  • P17 TAFGASLFVPPSHVQFIG (SEQ ID NO:12)
  • KP17 TAFGASLFVPPSHVQFIGGKKKKKG
  • DP17 TAFGASLFVPPSHVQFIGGDDDDDG
  • nanosponges consisted of a trigonal tris-maleimide linker, a D10K20CG (SEQ ID NO:20) peptide building block labeled with cholesterol, the short PC1 CTF fragment (P17, KP17, and RP17), and a “don’t eat me” peptide sequence (kGNYTCEVTELSREGKTVIELKk (SEQ ID NO:7)), with a molar ratio of 1:2:1:1 respectively.
  • Characterization Characterization.
  • KP17 peptide demonstrated to have the highest activity in comparison to all other peptides and controls tested in both cell lines, followed by P17 fragment peptide. The results are shown in FIG.19.
  • EXAMPLE 9 APATANIB AS MK2 INHIBITOR [0169] Multiple models demonstrate that TGF ⁇ 1-mediated fibrosis is caused by activated myofibroblast that signals through the TGF ⁇ 1R-p38-MK2 signaling axis and direct inhibition of MK2 can prevent myofibroblast development and subsequent fibrotic injury in idiopathic pulmonary fibrosis and cardiac fibrosis injury models.
  • Plectin nanosponge consisted of a trigonal tris-maleimide linker, a S 10 K 20 CG (SEQ ID NO:18) peptide building block label with cholesterol and plectin-targeting peptide sequence (sHPWSYLWTQQAs, (SEQ ID NO:1)) labeled with rhodamine B to track and confirm delivery into the tumor site.
  • sHPWSYLWTQQAs SEQ ID NO:1
  • the “Don’t eat me” peptide sequence kGNYTCEVTELSREGKTVIELKk SEQ ID NO:7.
  • the molar mass used for assembly of nanosponges was as follows, 2 (cholesterol-labeled S10K20CG, (SEQ ID NO:18)) : 1 (trimaleimide linker): 1 (rhodamine B labeled plectin): 1 (“Don’t eat me” peptide”). [0172] Characterization. Characterization of S10K20CG (SEQ ID NO:18) using DLS and zeta potential Nanosponge DLS (nm) PDI Zeta potential (mV) [0173] Cell and animal experiments. PDAC bearing mice, were treated with plectin-nanosponge after tumor was developed and had grown for over 3 weeks.
  • mice After IP injecting plectin-nanospnoges in mice and incubating overnight, mice were sacrificed and various organs and tumor tissues were removed and imaged ex vivo to determine localization of plectin-nanosponge. IVIS imaging confirmed specific uptake of plectin-nanosponge in tumor site only, with no uptake in other healthy organs. The results are shown in FIG.20 and FIG.21.
  • the articles "a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements.
  • the terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

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Abstract

La présente divulgation concerne des nanoéponges peptidiques comprenant une pluralité de blocs de construction peptidiques auto-assemblés. La pluralité de blocs de construction peptidiques auto-assemblés comprend un premier bloc de construction peptidique comportant : un noyau polymère ramifié comprenant au moins trois bras ; un premier co-peptide bloc lié de manière covalente à l'un desdits bras, ledit premier co-peptide bloc comprenant un premier bloc peptidique et un second bloc peptidique qui est différent du premier bloc peptidique ; une fraction de coiffage à base de lipide fixée de manière covalente au premier co-peptide bloc ; et un composé thérapeutiquement actif lié de manière covalente par l'intermédiaire d'une liaison clivable au premier co-peptide bloc. Avantageusement, le co-peptide bloc est fixé de manière covalente au coeur de telle sorte qu'il est résistant au clivage enzymatique du coeur.
PCT/US2024/049592 2023-10-02 2024-10-02 Nanoéponges peptidiques pour l'administration de médicaments Pending WO2025076082A1 (fr)

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Publication number Priority date Publication date Assignee Title
US20160324977A1 (en) * 2014-01-08 2016-11-10 Haemostatix Limited Peptide dendrimers comprising fibrinogen-binding peptides
US20190070216A1 (en) * 2016-09-02 2019-03-07 Drexel University Methods of Treating Osteoarthritis
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US20160324977A1 (en) * 2014-01-08 2016-11-10 Haemostatix Limited Peptide dendrimers comprising fibrinogen-binding peptides
US20220047728A1 (en) * 2015-04-17 2022-02-17 University Of Kentucky Research Foundation Rna nanoparticles and method of use thereof
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WANG HONGWANG; YAPA ASANKA S.; KARIYAWASAM NILUSHA L.; SHRESTHA TEJ B.; KALUBOWILAGE MADUMALI; WENDEL SEBASTIAN O.; YU JING; PYLE : "Rationally designed peptide nanosponges for cell-based cancer therapy", NANOMEDICINE: NANOTECHNOLOGY, BIOLOGY, AND MEDICINE, ELSEVIER, AMSTERDAM, NL, vol. 13, no. 8, 1 January 1900 (1900-01-01), AMSTERDAM, NL, pages 2555 - 2564, XP085257024, ISSN: 1549-9634, DOI: 10.1016/j.nano.2017.07.004 *

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