Peptide nanosponges for drug delivery

Abstract

This disclosure describes a peptide nanosponge comprising a plurality of self-assembling peptide building blocks. The plurality of self-assembling peptide building blocks comprises a branched polymer core having at least three arms, a first block copeptide covalently bonded to one of the arms, the first block copeptide comprising a first peptide block and a second peptide block distinct from the first peptide block, a lipid-based capping moiety covalently bonded to the first block copeptide, and a therapeutically active compound covalently bonded to the first block copeptide via a cleavable linkage. Advantageously, the block copeptide is covalently bonded to the core to be resistant to enzymatic cleavage from the core.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 542,052, filed on 2 October 2023, entitled "PEPTIDE NANOSPONGES FOR DRUG DELIVERY," which is incorporated herein by reference in its entirety.

[0002] (Research and development supported by the federal government) This invention was developed with government support under 75N91023C00008, awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] (Sequence Listing) The following application includes a sequence listing submitted electronically as a standard ST.26 compliant XML file titled "SequenceListing_61012.xml" created on 2 October 2024, with a size of 27,342 bytes, the contents of which are incorporated herein by reference.

[0004] (Field of Invention) This disclosure describes a peptide nanosponge, which comprises a plurality of peptide-based building blocks, each comprising a core, a peptide block, a linked amino acid or peptide covalently bonded to the core the peptide block, a capping portion covalently bonded to the N-terminal amino acid of the peptide block, a therapeutically active compound covalently bonded to one or more amino acids of the peptide block, and a signaling sequence. [Background technology]

[0005] The following background explanation of this technology is provided solely to aid in understanding this technology and is not intended to describe or constitute prior art in relation to this technology.

[0006] Many cancer treatments are administered systemically and have systemic effects, leading to serious side effects, including damage to healthy tissue. These systemic effects can sometimes cause pain and discomfort that necessitates reducing the dose of the treatment or even discontinuing the treatment altogether. Therefore, there is a great need for more targeted treatments, particularly those targeting the affected tissue.

[0007] Colon cancer is the second leading cause of cancer-related death in the United States. Recent advances in early diagnosis and treatment are not curative in patients with advanced disease. Standard treatment involves three different combination regimens of chemotherapy, each of which has significant toxicity. At least 50% of colon cancer patients receiving these treatments develop metastases. Other combination therapies include immunotherapy, but these suffer from the development of resistance and increased side effects. Therefore, there remains a high and unmet medical need to improve the treatment of colon cancer.

[0008] Idiopathic pulmonary fibrosis (IPF) is a rare disease with adjusted incidence estimates (per 10,000 people in the population) ranging from 0.35 to 1.30 in Asia-Pacific countries, 0.09 to 0.49 in Europe, and 0.75 to 0.93 in North America. IPF is a devastating disease with a 5-year survival rate of 20-40% (chronic and irreversible). There is no curative treatment for clinical IPF. Immunosuppressive therapy for IPF has been tested in a Phase III trial (PANTHER) using combination therapy (prednisone, azathioprine, and N-acetylcysteine) and has not shown any clinical survival benefit, and, surprisingly, has been associated with an increased incidence of treatment-related serious adverse events. Further therapies such as interferon-gamma-1β therapy and sildenafil have similarly failed to demonstrate improvement in lung function, overall survival benefit, or functional outcome studies when tested in randomized, placebo-controlled trials. The pathobiology of IPF remains poorly understood, but current understanding suggests that repeated asymptomatic injuries (i.e., smoking, trauma) directly affect the alveolar epithelium, leading to cell death, persistent cell turnover, inflammation, and fibroblast recruitment in genetically susceptible individuals. Identification of the perivascular niche indicates that CXCR7 expression on pulmonary capillary epithelial cells may lead to epithelial cell regeneration, reduced inflammation, and suppression of histofibrosis. Furthermore, this process leads to an imbalance between the epithelial-to-mesenchymal ratio of cells, resulting in histofibrosis via increased extracellular matrix deposition. This biology has led to the development of novel drugs targeting the interaction between the epithelial fibroblast microenvironment and a new class of drugs (pirfenidone, nintedanib). While these treatments represent a step forward, their usefulness is limited by issues such as patient drug tolerance, lack of significant improvement in lung function, and no improvement in quality of life. Therefore, the need to develop novel drugs that improve the therapeutic index is essential.

[0009] Cancer cells with deletions or mutations in the tumor suppressor p53 (p53 deficiency) are associated with osteosarcoma (OS). OS has an incidence of approximately 1,000 in the United States. OS is commonly found in childhood and adolescence, with 60% of cases occurring in patients aged 10-20 years, accounting for 2% of childhood cancers, and the number of cases is increasing, likely due to the growing pediatric population. Survival for OS remains low, mainly as a result of its highly metastatic and drug-resistant nature (70% 5-year survival rate for non-metastatic disease, 30% for metastatic disease), indicating a high unmet need for these patients. OS is characterized by malignant mesenchymal cells that originate from the bone marrow cavity and produce osteoid or immature bone that grows toward the cortex and adjacent soft tissue. This bone formation results in swelling and severe pain, which is severe enough to awaken a sleeping patient. Unfortunately, for more than 30 years, there has been little progress in the development of treatments to address the unmet needs of these patients. [Overview of the project]

[0010] This disclosure relates to a peptide nanosponge comprising a core, a peptide block, linked amino acids or peptides covalently bonded to the core the peptide block, a capping portion covalently bonded to the terminal amino acids of the peptide block, a therapeutically active compound covalently bonded to one or more amino acids of the peptide block, and optionally, a signaling sequence.

[0011] In one or more embodiments, the present disclosure relates to peptide nanosponges comprising a plurality of self-assembling peptide building blocks, wherein the plurality of self-assembling peptide building blocks comprises a branched polymer core containing at least 3 arms, a first block copolypeptide of no more than 50 amino acids, which is covalently bonded to one of the arms via its C-terminus, and the first block copolypeptide comprises a first peptide block and a second peptide block different from the first peptide block, a lipid-based capping moiety with a molecular weight of 400 Da or less covalently bonded to the N-terminal amino acid of the first block copolypeptide, and a therapeutically active compound covalently bonded to one or more amino acids of the first block copolypeptide via a cleavable linkage. Advantageously, the block copolypeptide is covalently bonded to the core so as to be resistant to enzymatic cleavage from the core.

[0012] The present disclosure also relates to peptide building blocks for nanosponges, comprising a core, a peptide block, a linking amino acid or peptide that covalently bonds the peptide block to the core, a capping moiety covalently bonded to the terminal amino acid of the peptide block, a therapeutic peptide, and a "don't eat me" sequence.

[0013] Additionally, the present disclosure relates to peptide building blocks for nanosponges, comprising a core, a peptide block, a linking amino acid or peptide that covalently bonds the peptide block to the core, and a capping moiety covalently bonded to the terminal amino acid of the peptide block.

[0014] The present disclosure also relates to methods of treating a disease or disorder in a patient in need of treatment of the disease or disorder, comprising administering a therapeutic dose of the peptide nanosponge described herein to the patient, as well as the use of the peptide nanosponges, pharmaceutical compositions, and medicaments for treating the disease or disorder.

[0015] Other objects and features are in part apparent hereinafter and in part pointed out. [Brief explanation of the drawing]

[0016] A patent or application file shall include at least one drawing performed in color. A copy of the published patent or patent application containing the color drawing shall be provided by the Patent Office upon request and payment of the necessary fees. [Figure 1A] This is a schematic diagram illustrating one exemplary embodiment of a typical component of a peptide-based building block for use in the formation of supramolecular aggregates (nanosponges). [Figure 1B] This is a schematic diagram illustrating a further exemplary embodiment of a common component of peptide-based building blocks for use in the formation of supramolecular aggregates (nanosponges). [Figure 1C] This is a schematic diagram illustrating a further exemplary embodiment of a common component of peptide-based building blocks for use in the formation of supramolecular aggregates (nanosponges). [Figure 2] This is a schematic diagram illustrating peptide-based building blocks that can be used to deliver DCKL1 inhibitory compounds. [Figure 3] This is a schematic diagram illustrating peptide-based building blocks that can be used to deliver MK2 inhibitory compounds. [Figure 4] This is a schematic diagram illustrating peptide-based building blocks that can be used to deliver cytotoxic agents to p53-deficient cells. [Figure 5] TEM images of organized nanosponges are shown. A) TEM image of organized S10K20CG-based (SEQ ID NO: 18) nanosponge labeled with cholesterol, average size approximately 96 nm, and B) TEM image of organized D10K20CG-based (SEQ ID NO: 20) nanosponge labeled with cholesterol, average size approximately 77 nm. [Figure 6](Top) Representative colony formation images of HUCCT1 cells treated for 48 hours with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50 and 1 / 2 IC50 previously determined by hexosaminidase assay. (Bottom) Quantification of colony number and size. Data are expressed as the average number and size from three independent experiments, *p<0.05. [Figure 7] (Top) Representative colony formation images of HUH28 cells treated for 48 hours with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50 and 1 / 2 IC50 previously determined by hexosaminidase assay. (Bottom) Quantification of colony number and size. Data are expressed as the average number and size from three independent experiments, *p<0.05. [Figure 8] (Left) Representative spheroid formation images of HUCCT1 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50, 1 / 2IC50, and 1 / 4IC50 doses previously determined by hexosaminidase assay. (Right) Quantification of spheroid count. Data are expressed as the mean from three independent experiments, *p<0.05 [Figure 9] This immunoblot shows that TGFβ1 can stimulate collagen 1 protein expression in BJ(hTERT). [Figure 10] This immunoblot shows that a nanosponge support without any conjugated small molecule drug can induce p38MAPK pathway activation, is cytotoxic at 50 μM, and that the nanosponge backbone can induce collagen production. [Figure 11] This is an immunoblot showing the dose-response curves of PF-3655022 (the drug not bound to the nanosponge) and PF-3655022-nanosponge. [Figure 12] This immunoblot shows HSP27 phosphorylation in BJ(hTERT) and CAF067 after treatment with an MK2 inhibitor. [Figure 13] This immunoblot shows that, in vitro, MK-Inh-1 small molecule drug conjugates or the drug itself do not inhibit MK2 phosphorylation in a dose-response manner. [Figure 14] This is a TEM image of the D10K20 (SEQ ID NO: 21) nanosponge, where water-filled nanovesicles are identifiable as darker spots within the nanosponge. [Figure 15] The graph shows the calculated percentage of PCZ filled into the nanosponge (marked with asterisks). [Figure 16] This graph shows the concentration versus proliferation percentage of PDAC cells for prochlorperazine (PCZ) and prochlorperazine-nanosponge. [Figure 17] The image shows a cell uptake assay demonstrating the uptake of PCZ-nanosponge in MiaPaCa-2 and S2-007 cells, with white arrows indicating uptake. [Figure 18] This image shows the uptake of PCZ-nanosponge in orthotopic KPCC tumors in C57BL / 6 mice (ex vivo IVIS imaging). [Figure 19] Graphs and blot images showing the activity of peptides tested in HEK293T (wild-type) and HEK293T-PKD1-KO-c4 (CRISPR-producing) cell lines are shown. [Figure 20] Images of organs in mice treated after plectin-nanosponge incorporation in PDAC-supported mice following overnight incubation are shown. [Figure 21] Images and heatmaps from ex vivo IVIS imaging (fluorescence) confirming the specific uptake of plectin-nanosponge only in PDAC tumors are shown.

[0017] Throughout the drawing, corresponding reference numerals indicate corresponding parts. [Modes for carrying out the invention]

[0018] One aspect of this disclosure focuses on developing a widely applicable peptide nanosponge drug delivery system. Peptide nanosponges are characterized as water-soluble sponge-like supramolecular tissues or aggregates containing a plurality of individual peptide-based building blocks (e.g., 1,000 to 50,000 building blocks), which can dynamically self-assemble, degrade, and reorganize into smaller clusters to facilitate site-selective delivery and uptake of their therapeutic payloads into cells. Using nanosponges, various activators can be delivered covalently bonded to the peptide-based building blocks via cleavable linkages and / or non-covalently sequestered within the interstitial spaces of the tissue. The tissues are rationally designed but, unlike previous peptide nanosponge versions, do not contain cleavable linkages throughout the tissue network, such as consensus sequences that can be readily cleaved by various enzymes in the body. The tissue is designed to associate with targeted cell types or to be preferentially taken up by early endosomes, and further designed to escape endosomes and be released into the cytoplasm, where enzymes such as esterases or proteases cleave the ligation links to which the activator is anchored, thereby rapidly releasing the activator to the targeted vicinity. The remainder of the nanosponge matrix is ​​more slowly degraded (biodegraded) through normal physiological processes such as macrophage phagocytosis or enzymatic degradation, which naturally remove foreign substances and cellular debris in vivo.

[0019] Peptide nanosponges are generally non-toxic to tissues and can be developed to target specific cells or tissues that are the target of particular treatment. In some cases, therapeutic small molecules or peptides can be covalently bonded to the peptide nanosponge via cleavable linkages, and then, when the peptide nanosponge reaches the target cells or tissue, the linkage is broken. In this way, effective treatment for various conditions can be delivered with fewer side effects that may result from systemic administration of the same therapeutic compound.

[0020] Another aspect of this disclosure focuses on the development of a novel “nanosponge” linked small molecule DCLK1 inhibitor for the treatment of cholangiocarcinoma (CCA), a rare, heterogeneous, invasive malignant tumor of the bile duct that appears late with poor outcomes. The incidence of CCA increases with age, with a median age of onset of 70–72 years. The 5-year survival rate for CCA is 13% (median survival time of 7 months), mainly due to unresectable tumors at initial diagnosis and advanced disease. The incidence of CCA is increasing, with an estimated US incidence of over 10,865 in 2022. Cytotoxic chemotherapy is currently the standard of care for CCA patients treated perioperatively. The first-line standard of care is based on gemcitabine + cisplatin, and recently approved targeted agents have done little to improve the high unmet need for transformative treatments for CCA patients, for whom surgery is considered the only effective treatment, but is often not possible.

[0021] Doublecortin Calmodulin-like Kinase 1 (DCLK1) is a serine-threonine kinase and a novel therapeutic target in oncology. Our team has shown that both DCLK1 expression knockdown and small molecule DCLK1 inhibitors suppress xenograft tumor growth in mice. To maximize the efficacy and safety of the DCLK1 inhibitor, it is delivered to the tumor site in the bile duct using a rationally designed peptide nanosponge technology. Combining these two unique technologies offers the potential to deliver a groundbreaking, direct treatment to the liver, potentially changing the treatment paradigm for CCA patients.

[0022] Generally, referring to Figure 1A, the peptide nanosponge comprises a plurality of peptide-based building blocks 10. Although the specific components used in each building block are variable, each building block 10 generally comprises a multi-arm core 12, a linear peptide sequence 14 having a C-terminus 14a covalently bonded to each arm of the core 12 directly or via cysteine ​​residues as spacers, and an N-terminus 14b typically capped with a capping portion 16 (however, it may be some other kind of terminal portion, as will be considered below). The peptide sequence 14 is a block copeptide comprising two or more distinct peptide blocks covalently bonded to the core 12 via linking amino acids (C) and spacer residues (G) that covalently bond the peptide block 14 to the core 12 at its C-terminus 14a. The linear peptide sequence 14 preferably does not contain any enzymatic consensus sequences and preferably does not bind to the core 12 via any cleavable consensus sequences. In other words, the peptide building block itself preferably does not contain any cleavable bonds or enzymatic consensus sequences, and is therefore resistant to enzymatic degradation.

[0023] More specifically, as shown in Figure 1B, the block copeptide sequence 14 includes a capping moiety 16 covalently bonded to the terminal amino acids of the peptide block 14. In the case of a nanosponge structure, it is preferable that 50% or more of the building block arms in the structure composition contain lipid-based capping moieties 16. In other words, of all the arms of all peptide-based building blocks present in the nanosponge, at least half of the arms contain lipid-based capping moieties 16 to sufficiently drive the building block organization of the solution into a supramolecular aggregate. As also shown in Figure 1B, the block copeptide sequence 14 includes a therapeutic compound or other activator 18 covalently bonded to one or more amino acids of the peptide block 14 (and preferably the second N-terminal block of the block copeptide sequence) via cleavable links. That is, the cleavable links in these novel peptide-based building blocks are used solely to bond the activator 18 to the peptide block 14 and are preferably not present elsewhere in the nanosponge or building block. The cleavable linkage for binding the activator 18 is preferably an esterase-cleavable linkage and / or a short (8 residues or less) enzyme consensus sequence, preferably an esterase consensus sequence.

[0024] Each peptide block 14 may have two or more therapeutically active compounds 18 bound thereto, as described in more detail below. In some embodiments, as shown in Figure 1C, the building block may include a signaling sequence or targeting moiety 20 covalently bound to the core 12 via each linkage 22 on one or more of the arms (depending on the number of arms). Preferably, the linkage 22 connecting the signaling sequence or targeting moiety 20 is different from the peptide sequence used for the lipid capping moiety 16 and the block copeptide 14 used to bind the activator 18 to the core.

[0025] In some embodiments, 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 a detectable moiety (e.g., a fluorescent label) or may further include a detectable label conjugated to a secondary signaling sequence. In some embodiments, the signaling sequence 20 may be a therapeutic agent itself. That is, the signaling sequence may be a targeting moiety that preferentially binds the nanosponge to a particular cell type or target receptor, and then block, bind to, or otherwise interact with the target in a therapeutic manner. Such embodiments are exemplified in examples using MK2 inhibitors.

[0026] Individual building blocks can be designed and synthesized to have the same or different sequence features and portions on each arm of the core. For example, the exemplary building block depicted in Figure 1B comprises a triangular planar core in which all three arms are coupled to the same block copolymer 14, activator 18, and capping portion 16. Alternatively, as illustrated in Figure 1C, the exemplary building block comprises a triangular planar core having two arms from which the peptide block 14 coupled with the activator 18 extends, and one arm coupled to a signaling sequence 20 via their respective linkers 22. In one or more embodiments, the peptide nanosponge is a complex comprising two or more different types of peptide-based building blocks. For example, the peptide nanosponge can be organized from multiple peptide-based building blocks having coupled therapeutic portions, in combination with multiple peptide-based building blocks having signaling sequences. Thus, the resulting nanosponge structure matrix is ​​characterized by a plurality of individual branched building blocks that associate with one another to form a three-dimensional network structure (i.e., internal and external open spaces) that defines open spaces (i.e., cavities, gaps, and pores) throughout the matrix. The branched arms and peptide sequences of the organized building blocks together form an interconnected network of the matrix, which defines the open spaces of the matrix, and the pores are open spaces or holes that construct the gaps between the branched arms and peptide sequences.

[0027] In one or more embodiments, the core is a branched structure that may have three or more “arms” terminated with functional groups that can covalently bond the core to each peptide sequence. Generally, the core is preferably a dendrimer-like branched or superbranched polymer structure with a size of 800 Da or less. In one or more embodiments, the core may contain 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.

[0028] [ka] In the formula, n is 2 to 5, preferably 2 to 3, preferably 3, and Z is maleimide, -NH2, -COOH, or -OH. In some cases, Z itself represents nitrogen with further branching.

[0029] In one or more embodiments, the peptide nanosponge building block may have a core derived from a trimaleimide compound or an analog thereof.

[0030] [ka] In the formula, n is as defined above. Suitable core linkers are:

[0031] [ka] Other analogues include the following.

[0032] Superbranched dendrimers with repeating units including the following are also conceivable:

[0033] [ka] In the formula, n and Z are defined as above, and m is 1 to 5. Examples of such superbranched polymer cores include, but are not limited to,

[0034] [ka]

[0035] [ka] These are some examples.

[0036] As illustrated above, various branched polymer cores having three or more terminal amine groups can be envisioned. In such embodiments, the terminal -COOH group of the peptide sequence can be linked to the terminal -NH2 to form a very stable amide bond in each arm of the core. Those skilled in the art will recognize that the -NH2 group is converted into an amide to which the peptide chain is bound. Again, branched polymer cores can be synthesized using arms terminated with COOH, as illustrated, or other suitable functional groups including maleimide, amide, or hydroxyl groups for covalent bonding to the peptide sequence.

[0037] Peptide sequences can be directly bound to the core and / or bound via short spacers of one or two amino acids. For example, a peptide building block may have a linked amino acid or peptide containing cysteine. In yet another aspect of this disclosure, the peptide building block described herein may have a linked peptide containing cysteine ​​having a glycine spacer residue at its C-terminus. Generally, the glycine residue is included at the C-terminus of the entire peptide sequence containing the linker cysteine ​​as an artifact from peptide synthesis. Other linked amino acids or peptides include glutamate, aspartate, serine, threonine, tyrosine, lysine, histidine, etc., provided that they are capable of forming a covalent bond with the C-terminus of the block copeptide. That is, the block copeptide is preferably not specifically cleavable, i.e., not an enzymatic consensus sequence, preferably resistant to enzymatic cleavage, and is bound directly or indirectly to the core in a manner that it is not readily cleaved or degraded in vivo until at least after the release of an activator (bound via a readily cleavable sequence). However, like most biodegradable matrices, it will be understood that the nanosponge network will eventually be destroyed through normal physiological processes used by the body to remove dead cells, debris, and foreign matter in vivo.

[0038] The peptide sequence includes a block copeptide comprising a first peptide block and a second peptide block distinct from the first peptide block. The first peptide block can be covalently bonded to linked amino acids or spacer peptides. The second peptide block can be covalently bonded to the first peptide block. Block copeptide sequences including a third peptide block can also be conceivable. Generally, the peptide sequence has a total length of 50 amino acids or less, preferably 5 to 40 amino acids, and preferably 10 to 35 amino acids.

[0039] Preferably, the first block constituting the C-terminus of the sequence and bound to the core (directly or via a linker or spacer) is selected from amino acids having positively charged side chains, such as lysine, histidine, and arginine. Preferably, the first block is a homopeptide of 10 to 25 amino acid residues, preferably 15 to 20 amino acid residues, and more preferably consisting of lysine residues. If the peptide building block has two or more arms containing block copeptides, it is preferable that the same peptide sequence is used for both block copeptides, and more preferably, each peptide sequence is positioned at the same distance from the core (e.g., via a linker or peptide spacer) to enhance structural stability.

[0040] Preferably, the second block constituting the N-terminus of the sequence (and linked to the first block) is selected from amino acids having side chains ending with a -COOH group or an -OH group, i.e., serine, aspartic acid (or aspartate), tyrosine, glutamic acid (or glutamate), threonine, and unnatural amino acids having amine, hydroxyl, or carboxylic acid functional groups. Preferably, the second block is a homopeptide of 5 to 15 amino acid residues, more preferably 5 to 10 amino acid residues, and more preferably serine or aspartic acid. In some embodiments, the second block consists of the same type of amino acid residues having side chains terminated with -COOH or -OH groups, i.e., serine, aspartic acid (or aspartate), tyrosine, glutamic acid (or glutamate), threonine, and unnatural amino acids having amine, hydroxyl, or carboxylic acid functional groups (i.e., all serine, all tyrosine, etc.), but the selected amino acids are separated by inactive spacer residues (e.g., alanine) to facilitate spacing of activators along the chain.

[0041] The peptide sequence is designed to be free from enzyme consensus sequences, preferably (whether in the middle, N-terminus, or C-terminus of the peptide block).

[0042] In other words, the only cleavable bond in the building block is a linkage that anchors the activator to the block copeptide. A suitable consensus sequence for binding the activator preferably consists of 2 to 8 amino acid residues. It is well known in the art that consensus sequences are selective for proteases (e.g., caspases, matrix metalloproteinases, cathepsins, calpain, etc.). Alternatively or additionally, the activator can be bound to a cleavable chemical linkage, such as an ester bond that is cleaved by esterases found throughout the body. Again, the combination of consensus sequences and chemical links can be designed in the building block to control how slowly or rapidly the drug is released after administration.

[0043] The second peptide block preferably has a covalently bonded capping moiety. Preferably, the capping moiety is a lipid moiety having a molecular weight of 400 Da or less. In one or more embodiments, the capping moiety includes cholesterol (386 Da). Other types of lipids include betulinic acid, saturated and unsaturated fatty acids, and adamantane, with a molecular weight of 400 Da or less. Alternatively, the peptide nanosponge may have a capping moiety that is cholic acid, if the nanosponge is designed to treat cholangiocarcinoma.

[0044] In one or more embodiments, the signaling sequence can be covalently bonded to the core directly or indirectly via its own peptide bonds (different from the block copeptide sequences described above).

[0045] The signaling sequences can be "eat me" sequences, "don't eat me" sequences, targeting sequences, detectable labels, and / or combinations thereof. Examples of signaling sequences include: a CD44 signaling sequence containing sHPWSYLWTQQAs (SEQ ID NO: 1), a plectin targeting sequence containing KTLLPTPG (SEQ ID NO: 2), an EGFR signaling sequence containing sYHWYGYTPENVIGsG (SEQ ID NO: 3), an LDLR signaling sequence containing sCMPRLRGCGAGsG (SEQ ID NO: 4), a CD46 signaling sequence containing sLPGTICKRTMLDGLNDYCTGsG (SEQ ID NO: 5), and a SIRPα "don't eat me" sequence containing kGNYTCEVTELSREGKTVIELKsG (SEQ ID NO: 6). The signaling sequence may include the SIRPα "Don't Eat Me" signaling sequence containing the nal signaling sequence kGNYTCEVTELSREGKTVIELKk (SEQ ID NO: 7), the SIRPα "Eat Me" signaling sequence containing 4N1:RFYVVMWK (SEQ ID NO: 8) and 7N3:FIRVVMYEGKK (SEQ ID NO: 9), the CD206 "Shutdown M2 Polarization" (rp-182) signaling sequence containing KFRKAFKRFFGsG (SEQ ID NO: 10), and the mucosal signaling sequence: PLKWPGKKKKGKPGKRKEQEKKKRRTRG (SEQ ID NO: 11) and WTAS. As described, certain types of sequences can be considered both signaling sequences and therapeutic sequences. Using the peptide nanosponges described herein, therapeutic peptides for polycystic kidney disease, including TAFGASLFVPPSHVQFIG(P17) (SEQ ID NO: 12), TAFGASLFVPPSHVQFIGGKKKKKG(KP17) (SEQ ID NO: 13), and TAFGASLFVPPSHVQFIGGDDDDDG(DP17) (SEQ ID NO: 14), can be delivered.

[0046] Peptide building blocks can be used to deliver a wide variety of therapeutic activators to a block copeptide sequence via cleavable bonds, preferably bound to a second (N-terminal) block. Generally, depending on the length of the block, multiple activators can be bound. For example, in the case of a second block consisting of 10 amino acid residues, up to five activators can be bound, for example, to every other serine in the block. Alternatively, the peptide block may contain spacers (e.g., alanine) between each serine residue, and the block may carry 10 activators bound to each serine.

[0047] Using peptide nanosponges, a wide variety of therapeutic activators can be delivered, including but not limited to DCLK1 inhibitors, MK2 inhibitors, cytotoxic agents for p53-deficient cells, MIF inhibitors, SMAD phosphorylation inhibitors, dopamine receptor inhibitors, or combinations thereof. The activators can be bound to peptide building blocks. Alternatively, the activators can also be sequestered within the interstitial spaces of the organized nanosponges for controlled release and delivery when the nanosponge tissue is degraded. This is particularly advantageous for lipophilic activators.

[0048] The peptide nanosponge may have a therapeutic activator covalently bonded to one or more amino acid residues in a second peptide block.

[0049] The rationally designed peptide nanosponges in the examples feature a three-sided planar building block (e.g., tris-maleimide) that can anchor other peptides via cysteine-mediated Michael addition. This strategy offers great flexibility when targeting cells in vitro, and especially in vivo.

[0050] Therefore, these peptide-based building blocks form supramolecular aggregates (nanosponges) of each other characterized by a flexible three-dimensional matrix, within which the organized building blocks and their respective branched structures cooperate to form a matrix network with open spaces between them. This is driven by the interaction of the hydrophobic components of the peptide building blocks (e.g., cholesterol, cholesterol derivatives (e.g., cholic acid), hydrophobic peptide sequences, or hydrophobic dyes) in reaction solutions containing aqueous or organic solvent systems. The organized matrix can dynamically bend and curve, decompose into smaller clusters, and reorganize depending on the environment and conditions applied to it (e.g., in vivo via cellular uptake processes).

[0051] The flexible structure of peptide nanosponges allows for their customized design.

[0052] Additionally, cleavage sequences from approximately 600 human proteases can be used to anchor the activators to each of the peptide-based building blocks described herein. Furthermore, cleavage sequences from a group of proteases (e.g., MMPs, cathepsins, etc.) can be incorporated into the nanosponge depending on the desired target.

[0053] Peptide nanosponges can be used to deliver a wide variety of therapeutic activators, including small molecules and therapeutic peptides. Generally, any activator with a size of less than 2 nm (longest axis) can be delivered using nanosponges.

[0054] DCLK1 inhibitors The peptide nanosponges described herein may contain a therapeutic activator including a DCLK1 inhibitor.

[0055] Doublecortin-like kinase 1 (DCLK1) is considered a cancer stem cell (CSC) marker. The DCLK1 signaling pathway involves AKT phosphorylation at Thr308, which leads to Notch1 activation. Notch1 activates the transcription factor pregnane X receptor (PXR) expression. PXR induces cancer cell proliferation and metastasis and regulates the expression of other proteins involved in drug metabolism, including multidrug resistance protein 1 and cytochrome P4503A4 (one of six enzymes responsible for approximately 90% of the metabolism of all small molecule drugs). Oral administration of representative small molecule DCLK1 inhibitors showed in vivo antitumor activity in a mouse colon cancer xenograft model. While we were able to demonstrate that DCLK1 inhibition is associated with an antitumor response, the chemical scaffolds from which lead candidates were generated are pharmaceutically unmanageable. Nanosponges linked to small molecule DCLK1 inhibitors can be directly delivered to tumor sites in the colon, allowing for enhanced antitumor activity while "protecting" the small molecule DCLK1 inhibitors from potential metabolism.

[0056] Doublecortin-like kinase 1 (DCLK1). DCLK1 is a cancer stem cell (CSC) marker. CSCs are rare, and isolating and identifying cells within tumor masses is difficult. Several methods used to identify CSCs include globular ability, dynamolytic elimination by overexpression of efflux pumps, intracellular enzymatic activity, and expression of cell surface markers (3, 4). Gordon and colleagues first identified DCLK1 in the gut. DCLK1 has been identified 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 that bind to microtubules and regulate microtubule polymerization, a C-terminal serine / threonine kinase domain homologous to Ca2+ / calmodulin-dependent protein kinases, and a serine / proline-rich domain between the doublecortin and protein kinase domains. DCLK1 is preferentially transcribed from a second promoter in colon cancer cells. The AKT1 peptide around Thr308 interacts with the kinase domain of the DCLK1 protein, which is a shorter form lacking the N-terminal DCX domain. A DCLK1 knockout mouse model suppresses colon polyps in APCmin / + mice but does not affect the normal gut. These data suggest that DCLK1+ cells are CSCs, not normal stem cells. A key consequence of this finding is that targeting DCLK1 specifically targets CSCs while leaving normal proliferating cells intact.

[0057] DCLK1 has been shown to phosphorylate the microtubule-associated protein MAP7D1. Using in silico modeling, AKT1 was identified around the threonine at position 308 (IKDGATMKTFCGTP (SEQ ID NO: 15)). Using AutoDock Vina and the crystal structure of the DCLK1 kinase domain (PDB ID 5JZN), it was observed that the AKT1 peptide around Thr308 interacts with the DCLK1 kinase domain (binding energy of -5.7 kcal / mol). DCLK1 amino acids targeted by AKT1 interaction include ASP398, ASP475, GLU515, and THR552. The inventors confirmed the interaction using magnetic relaxation assays and immunoprecipitation-coupled Western blot assays. In vitro assays using recombinant DCLK1 and AKT peptides also confirmed significant phosphorylation of AKT substrates by DCLK. This phosphorylation was lost when DCLK1 was denatured.

[0058] In one or more embodiments, the DCLK1 inhibitor is gemcitabine.

[0059] Lead candidate MRL16 has been demonstrated to inhibit colon cancer growth by inhibiting proliferation during the G2 / M phase of the cell cycle, inducing apoptosis, and suppressing stem cell characteristics by inhibiting spheroid proliferation. MRL16 suppressed DCLK1-mediated phosphorylation of AKT1 at Thr308, resulting in downstream inhibition of Notch signaling and PXR. Subsequently, in vivo mouse xenograft studies using MRL16 demonstrated antitumor activity, providing evidence that DCLK1 is a viable therapeutic target for CRC. By combining inhibition of this novel drug target with a novel approach for selectively delivering DCLK1 inhibitors to CRC tumors, DCLK1 LLC aims to improve therapeutic outcomes by enhancing efficacy and reducing toxicity. This specification describes the synthesis of a novel nanosponge-linked DCLK1 inhibitor and its in vitro evaluation. This novel nanosponge drug delivery platform has not been previously used for the treatment of CRC.

[0060] Researchers at the University of Kansas Medical Center isolated marmelin (1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxyaldehyde, marmelin, MRL) from Aegle marmelos, an Ayurvedic treatment for gastrointestinal cancer.

[0061] [ka]

[0062] Based on this scaffolding, MRL16 was selected for target validation studies based on its initial ADMET properties summarized in Table 1. Despite limited water solubility and moderate in vitro metabolic stability, the compound demonstrated excellent probe potential for proof-of-principle studies to validate DCLK1 as a therapeutic target. MRL16 significantly interacts with the kinase domain with a binding energy of -7.9 kcal / mol, and the key amino acid is VAL468.

[0063] [ka]

[0064] DCLK1 is a protein that indicates quiescent stem cells in colon cancer. Stem cells support spheroid growth. The inventors performed a spheroid assay using HCT116 cells in which DCLK1 was knocked down using specific shRNA. 500 cells were seeded in a serum-deficient medium containing factors essential for spheroid proliferation in a very low-adhesion 12-well dish. A significant reduction in spheroid formation was observed compared to the control. The cells were then treated with MRL16 (0.5 μM), showing a significant reduction in primary and secondary colonosphere growth. Secondary spheroids arise from primary spheroids. No additional drugs were added to secondary spheroids, depending on the number of surviving CSCs when the primary spheroids were treated. MRL16 did not cause a significant reduction in DCLK1-deficient cells, confirming that its analogues inhibit spheroid proliferation by suppressing DCLK1+ CSCs.

[0065] Figure 2 shows one embodiment intended for a nanosponge for the delivery of a DCLK1 inhibitor, where the trimareimide structure is a core that binds to a first peptide block of 20 lysine residues and a second peptide block of 10 serine or aspartic acid residues (the second block can also be threonine, aspartate, or glutamic acid / glutamate). Additionally, two of the blocks are capped with lipid caps (cholesterol), and the other position binds to a signaling sequence that can be either a CD44-targeting sequence or a SIRPα-targeting sequence ("don't eat me" sequence). Depending on the peptide used in the second 10-residue block, the DLCK1 inhibitor can be covalently bonded to the hydroxyl group of the serine or threonine residue to form a cleavable carbonate bond, or covalently bonded to the carboxylic acid group of the aspartic acid or glutamic acid group to form a cleavable ester bond.

[0066] DCLK1 inhibitors can have a structure corresponding to Formula 1,

[0067] [ka] In the formula, A is a 5 or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, preferably a nitrogen-containing heteroaryl group, and R1, R2, and R3 are each independently -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C) 12 , preferably C1-C6 alkyl, and alkaryl (for example, C1-C 12 R1 and R2 are selected from alkyl-substituted C3-C6 aryl groups, or R1 and R2, together with the carbons to which they are bonded, form a 5 or 6-membered ring fused with the adjacent 6-membered ring to which R1 and R2 are bonded, R4 is H or -CH3, X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(O)NH-, and J1 and J2 are each independently -CH- or -N-, preferably at least one of J1 and J2 is -N-.

[0068] Additionally, DCLK1 inhibitors may have a structure corresponding to formula 1A.

[0069] [ka] In the formula, A is a 5 or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, preferably a nitrogen-containing heteroaryl group, and R3 is independently -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C) 12 , preferably C1-C6 alkyl, and alkaryl (for example, C1-C 12 Selected from alkyl-substituted C3-C6 aryls, R4 is H or -CH3, X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(O)NH-, and J1 and J2 are each independently -CH- or -N-, preferably at least one of J1 or J2 is -N-.

[0070] A DCLK1 inhibitor having a structure corresponding to formula 1 or formula 1A, wherein A is a 5- or 6-membered heterocycle having one of the following structures, preferably a nitrogen-containing heterocycle:

[0071] [ka]

[0072] In one or more embodiments, the DCLK1 inhibitor also has the following structure

[0073] [ka]

[0074] [ka] It can have.

[0075] MK2 inhibitors Furthermore, therapeutic activators, including MK2 inhibitors, can be delivered using peptide nanosponges.

[0076] The MK2 pathway is crucial for inflammatory cytokine production and mesenchymal gene expression. Preclinical studies demonstrate higher levels of phosphorylated MK2 (p-MK2) (active MK2) in IPF patients compared to patients with normal lung function. Inhibition of MK2 in vitro results in reduced TGFβ1-mediated myofibroblast development and extracellular matrix deposition. Furthermore, MK2 inhibition can prevent bleomycin-induced lung injury and inflammation in mice. However, there are no FDA-approved MK2 inhibitors, and early-generation small molecule MK2 inhibitors lack "drugability" due to a lack of enzymatic ATP pocket selectivity and low water solubility, resulting in unrealistically high EC50 levels required to achieve therapeutic benefits.

[0077] Suppression of the MK2 pathway using small molecule drugs linked to nanosponges allows for direct delivery of small molecules to cells, which results in downregulated activation via disruption of the p38α-MK2 interaction. This then downregulates idiopathic pulmonary fibrosis inflammatory cytokine production and EMT activation, thereby improving overall IPF progression.

[0078] Known MK2 inhibitors with low water solubility or those readily degraded by endogenous plasma proteases can be favorably conjugated to the peptide nanosponges described herein. The nanosponge platform can be modified to allow for increased drug solubility, prevent degradation (increasing drug access), or further modified to allow for delivery of the payload to target cell tissue with localized drug release at the disease site.

[0079] The inventors acknowledge that IPF and fibrotic lung injury are already established in their clinical patients. They also acknowledge that current bleomycin lung injury models do not assess the ability to prevent acute pneumonia or whether established soft tissue fibrosis can be reversed. The inventors propose to specifically investigate whether delivery of their MK2 inhibitor drug conjugate can reverse / reduce established fibrotic lung injury in a cohort of mice that maintain initial bleomycin injury (more than 60 days after bleomycin treatment). This represents an innovative experimental approach that more uniquely addresses the clinical paradigm being proposed for study.

[0080] Using known MK2 inhibitors, the inventors synthesized the first three nanosponge-linked MK2 inhibitor conjugates ("drug conjugates") to demonstrate proof of principle that the inventors' drug development platform is feasible. Subsequently, the inventors showed that these first-generation compounds can inhibit TGFβ1-induced myofibroblast development by reducing the expression levels of p-MK2, αSMA, collagen, and fibronectin proteins. Based on these results, the inventors performed iterative improvements and modifications of the drug conjugates and retested their ability to inhibit myofibroblast development and activation. After the inventors identified the best drug conjugate, they proceeded to in vivo animal studies. First, the inventors established the maximum tolerated dose of the drug conjugate. Next, they determined the optimal dose delivery schedule (1-week vs. 2-week drug delivery). Finally, the inventors investigated the effects of the best drug conjugate using both an acute inflammatory pneumonia model and a subsequent chronic fibrous injury model.

[0081] Figure 3 illustrates the design components of peptide nanosponges for the treatment of pulmonary fibrosis. Figure 3A shows three MK2-blockers (PF-3644022, MK2-IN-1, and MK2-IN-4). Figure 3B shows the variable structure of the peptide nanosponge. Signaling and therapeutic sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 16) and CD47-targeted "Don't Eat Me" sequence (on / on, on / off, off / on), three low molecular weight MK2 blockers, and D 10 and S 10 By alternating the blocks, 18 novel nanosponges were obtained. Figure 3C shows the block copolymer ((SEQ ID NO: 21) D 10 A tertiary amide bond or S of a block copolymer (SEQ ID NO: 19) 10 This demonstrates an alternative chemical connection of MK2-blocker via a urethane bond.

[0082] Peptide nanosponges offer the opportunity to deliver covalently bound drugs to biological targets. According to the results of coarse-grained molecular dynamics (MD) simulations, the cholesterol units of the nanosponges form hydrophilic nanodomains. Nanoholes filled with aqueous solvent are also present in their structures. Incorporation of a fluorescent label (i.e., rhodamine B) is feasible as they can covalently bind to the N-terminus of the K 20 S 10 (SEQ ID NO: 19) building block. As shown in Figure 3, all three MK2 inhibitors (PF-3644022, MK2-IN-1, and MK2-IN-4) feature a secondary amine group that is utilized either to bind them to the S 10 block via an esterase-cleavable urethane bond, or to bind them to the D 10 block via a protease-cleavable amide bond. The present inventors expect that the urethane bond will be cleaved faster than the peptide bond once the nanosponge enters the cytoplasm. This allows for optimization of pharmacokinetics. Up to 10 MK2 inhibitors are tethered to each nanosponge building block. Quantitation is 1This is performed by 1H-NMR. Targeting of activated myofibroblasts is achieved by utilizing the peptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 16), which functions as both a signaling sequence and a therapeutic sequence. To enhance the circulation time of the nanosponge, the "Don't Eat Me" peptide sequence kGNYTCEVTELSREGKTVIELKk (SEQ ID NO: 7) is incorporated into the nanosponge structure. This peptide sequence mimics the binding motif of regulatory protein alpha (SIRPα) in phagocytic cells. This event triggers a self-defense response of CD47. Both peptide sequences have D-amino acids at both ends to prevent rapid proteolysis during circulation. The inventors envision synthesizing 12 to 15 repeatable novel nanosponges featuring three MK2 inhibitors anchored via urethane or amide bonds. The nanosponges will incorporate either a signaling sequence or the "Don't Eat Me" sequence, or both, to optimize drug delivery. The inventors synthesize 500 mg of each nanosponge.

[0083] In certain embodiments, inhibition of the MK2 pathway using small molecule drugs conjugated to nanosponges reduces radiation-induced fibrosis by reducing inflammatory cytokine production and mesenchymal gene activation. In one or more embodiments, apatanib or another MK2 inhibitor is conjugated to the nanosponge portion via amide and esterase-cleavable bonds. Compounds are synthesized in an iterative process and evaluated by in vitro cell-based screening. A lead compound is then evaluated in an in vivo lung disease pulmonary fibrosis model, and the inventors determine the maximum tolerated dose, the optimal drug delivery schedule, and its ability to reduce acute pneumonia lung injury and chronic late fibrotic lung injury.

[0084] In certain embodiments, MK2 pathway inhibition using small molecule drugs conjugated to nanosponges reduces radiation-induced fibrosis by reducing collagen-1 deposition and inflammatory cytokine production. In one or more embodiments, apatanib or another MK2 inhibitor is conjugated to different nanosponge backbones via amide and esterase-cleavable bonds. Conjugated compounds are iteratively developed and tested using in vitro cell and protein-based screening. One lead compound is advanced to in vivo biological studies to determine the maximum tolerated dose and the pharmacokinetic and pharmacodynamic distribution of the drug at two doses (MTD and 1 / 2MTD).

[0085] Effective cytotoxic agents for p53-deficient cells Furthermore, peptide nanosponges can contain therapeutic activators, including cytotoxic agents against p53-deficient cells. While KU-D2 and KU-D2F meet the Lipinski requirements for effective drug delivery, they are characterized by three benzyl groups that are susceptible to effective oxidation, leading to poor pharmacokinetics. Binding to rationally designed peptide nanosponges protects the drug from oxidation due to their supramolecular structure. Moreover, targeted peptide sequences have been proven to successfully facilitate targeted drug delivery.

[0086] The development of small molecule-nanosponges has linked compounds that selectively release their active site with high potency and selectivity in tumor cells. One analogue has already demonstrated proof of principle in an animal model of osteosarcoma (OS), showing no detectable adverse effects when administered intraperitoneally, indicating that appropriate delivery of this compound and analogue may be a viable strategy for the treatment of OS. Although it exhibits excellent activity, systemic exposure to chemotherapy is a concern, especially with long-term administration in pediatric populations. By using "nanosponge" technology to enable local exposure, it becomes possible to deliver higher doses of small molecules to the tumor site while lowering the risk of toxicity. The synthesis of a series of 16 combinations of the lead compound KU-D2F and three analogues links them to four different nanosponges via esterase-cleavable bonds. The compounds are synthesized in an iterative process and evaluated by in vitro screening. Two lead nanosponge / small molecule assemblies are then evaluated in an in vivo OS mouse model.

[0087] Figure 4 shows the design components of a nanosponge for osteosarcoma treatment. Figure 4A shows four analogues of KU-D2F. Figure 4B shows the variable structure of the nanosponge. Signaling sequences derived from CD47 and CD49, and D 10 and S 10 The blocks (sequences 19 or 21) are alternated, and the d8 signaling sequence remains in the indicated position. Figure 4C shows D 10 Ester bond and S 10 This shows an alternative chemical bond via a carbonate bond (SEQ ID NO: 19 or 21).

[0088] Four types of nanosponges linked to four p53 synthetic lethal inducers (KU-D2F) * Synthesis of (and three analogues). KU-D2F * This differs from the parent KU-D2F in the presence of a phenol group instead of a phenyl-methyl ether. The former allows the nanosponge to bond to either the aspartic acid (D10) or serine (S10) block. KU-D2F *Three analogues are synthesized. The four analogues are anchored to four conceptually distinct nanosponges. The inventors combine two DEM signaling sequences derived from CD47 and CD49 with two different chemical bonds (ester bond vs. carbonate bond) of the four analogues. This enables optimized drug delivery to OS and subsequent release of DEM inducers utilizing esterase activity in the cytoplasm.

[0089] Following the synthesis of the nanosponge / small molecule assembly, in vitro studies necessary to identify two best embodiments will be conducted, and then tested in vivo. The nanosponge-bound analogs release active small molecules into the cytoplasm of tumor cells. The execution of the objectives described in this proposal will generate novel nanosponge-bound compounds that maintain the active small molecule moiety.

[0090] MIF inhibitors Peptide nanosponges may contain therapeutic activators, including MIF inhibitors.

[0091] In one example, the peptide nanosponge contains a trilateral planar linker (e.g., Tris(maleimide)) covalently bound to the EGFR signaling sequence containing sYHWYGYTPENVIGsG (SEQ ID NO: 3), and kGNYTCEVTELSREGKTVIELKsG (SEQ ID NO: 6) and S covalently bound to the trilateral planar linker via cysteine ​​residues. 10 K 20 It contains C (SEQ ID NO: 17), is capped with cholesterol covalently bonded to a serine peptide block, and S 10 K 20 (SEQ ID NO: 19) A signaling sequence SIRPα "Don't Eat Me" can have an MIF inhibitor covalently bonded to an amino acid residue of the peptide block. Certain nanosponges can have several combinations of these elements, as long as there are amino acid residues that enable the covalent bonding of the peptide block and the MIF inhibitor compound, which allows for the formation of a supramolecular structure.

[0092] In some embodiments, compounds of the present invention that can be used as MIF1 / MIF2 inhibitors are shown below:

[0093] [ka]

[0094] Composition and treatment method Depending on the activator and the condition being treated, nanosponges can be formulated for various delivery routes and modes of treatment. Therapeutic compositions comprising nanosponges (or multiple nanosponges) dispersed in a suitable delivery vehicle or carrier for administration to a subject are described herein. Therapeutic compositions comprise a therapeutically effective amount of peptide nanosponges having an activator dispersed or suspended in a pharmaceutically acceptable carrier. In one or more embodiments, the composition may include a mixture of two or more different types of nanosponges and / or different types of activators. For example, it is conceivable that nanosponges carrying a therapeutic activator can be co-administered with nanosponges carrying a detectable label for monitoring and visualizing the delivery of the nanosponges to a target region.

[0095] A pharmaceutically acceptable carrier or vehicle is any carrier suitable for in vivo administration. As used herein, the term “pharmaceutically acceptable” means that it can be administered to a subject without excessive toxicity, irritation, or allergic reaction, does not cause an unacceptable biological effect, or interacts in a harmful manner with any of the other components of the composition in which it is contained, and is not biologically or otherwise undesirable. A pharmaceutically acceptable carrier is selected to minimize any degradation of the nanosponge (at least during storage) and to minimize any adverse side effects in the subject.

[0096] Medicinally acceptable ingredients include those acceptable for veterinary use and for human medicinal use. For example, pharmaceutically acceptable carriers suitable for mucosal administration include aqueous solutions such as water and buffer solutions, commercially available specifically formulated carriers and adjuvants, naturally occurring monosaccharides, disaccharides and polysaccharides, carbohydrates, sugars, polymers, and proteins, which include any of the following, and mixtures thereof: cellulose such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose, their derivatives, and their microcrystalline forms; other naturally derived polysaccharides and polymers such as sodium alginate, gelatin, chitosan, collagen, hyaluronic acid, and dextran; monosaccharides or oligosaccharides such as D-mannitol, sorbitol, glucose, lactose, fructose, inositol, sucrose, and amylose; dextrins such as α-, β-, or γ-cyclodextrin, dimethyl-β-cyclodextrin, and dextrin; natural starches such as hydroxyethyl or hydroxypropyl starch and carboxymethyl starch and their derivatives; and gums such as gum arabic, gum tragacanth, and glucomannan. A suitable carrier may further contain proteins such as serum proteins, casein, and albumin.

[0097] Additional components of the composition may preferably include excipients such as stabilizers, preservatives, diluents, emulsifiers, and lubricants. For oral delivery, flavoring agents or palatability enhancers may also be included in the composition.

[0098] Other components may be included in the composition, such as adjuvants, other activators, preservatives, buffers (e.g., histidine, phosphates), salts, and other pharmaceutically acceptable ingredients.

[0099] In one or more embodiments, a pharmaceutically acceptable carrier comprises a combination of one or more of the above-described vehicles and is preferably configured to enhance delivery via the desired route. For example, for mucosal delivery, the formulation may include one that is enterically coated to protect the nanosponge from premature gastrointestinal degradation. The nanosponge can be formulated in various solid or liquid dosage forms. For example, the nanosponge can be advantageously dried, for example, by freeze-drying, spray-drying, etc., to produce a storage-stable powder. The powder can be used for inhalation, packaged in a gel cap, compressed into a tablet, etc. Such a powder may contain one or more inert carriers or vehicles to promote free-flowing powder and reduce aggregation or clamping. The powder can also be reconstituted into liquid form for various administration routes, for example, via a nebulizer, injection, IV, oral, ocular, ear, or nasal droplets or spray, or oral gastric tube feeding. The nanosponge can be formulated as sublingual or buccal tablets, drops, lozenges, etc. The nanosponge can be formulated as topical creams, gels, ointments, etc. Nanosponges can be formulated into suppositories, bladder implants, and the like.

[0100] As used herein, “therapeutic effective” amount or “therapeutic dose” means an amount or dosage that elicits a biological or medical response in a tissue, system, or subject, as sought by a researcher or clinician, such as to induce some desired therapeutic or preventive effect against a disease or condition, depending on the activator delivered. A person skilled in the art will recognize that a dose may be considered therapeutic “effective” even if the disease, condition, or symptom is not completely eradicated or prevented, but it or its symptoms and / or effect is only partially improved or mitigated in the subject (e.g., reduction of tumor volume).

[0101] This disclosure also relates to a method for treating a disease or disorder in a subject requiring treatment of the disease or disorder, which comprises administering a peptide nanosponge or composition disclosed herein to a patient in a therapeutic dose.

[0102] A method for treating a disease or disorder described herein, wherein the disease or disorder is colon cancer, bile duct cancer, genitourinary cancer, gynecological cancer, gastrointestinal cancer, lymphoma, melanoma or skin cancer, head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain tumor, glioma, idiopathic pulmonary fibrosis (IPF), head and neck fibrosis, osteosarcoma, or bladder cancer, and further wherein the disease or disorder is colon cancer, and additionally, the disease or disorder is idiopathic pulmonary fibrosis (IPF), and the disease or disorder is osteosarcoma.

[0103] As described, peptide nanosponges can be enterically coated to allow them to pass through the gastrointestinal tract without hydrolysis.

[0104] The peptide nanosponses described herein may be administered via substantially any route of administration, including direct application to the treatment site or systemic administration, depending on the condition being treated. Non-limited routes of administration include intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, oral cavity, sublingual, topical, intranasal, rectal, intraosseous, or parenteral administration.

[0105] The nanosponge composition can be administered in one or more doses, and these doses can be administered via the same or different routes to achieve the desired preventive or therapeutic effect. In some embodiments, the nanosponge composition can be provided in unit dosage forms in a suitable container. The term "unit dosage form" refers to a physically separated unit suitable as a unit dose for use in humans or animals. Each unit dosage form may contain a predetermined amount of nanosponge (and / or other activators) in a carrier, calculated to produce the desired effect. In other embodiments, the nanosponge composition can be provided separately from the carrier (e.g., in its own vial, ampoule, sachet, or other suitable container) for mixing in situ before administration to a subject. Kits containing the nanosponge composition are also disclosed herein. The kit further includes instructions for administering the nanosponge composition to a subject, including reconfiguration as necessary. The nanosponge can be provided as part of a dose unit already dispersed in a pharmaceutically acceptable carrier, or it can be provided separately from the carrier. The kit may further include instructions for preparing the nanosponge for administration to a subject.

[0106] The additional advantages of various embodiments of the present invention will become apparent to those skilled in the art by examining the disclosure herein and the following examples. It will be understood that the various embodiments described herein are not necessarily mutually exclusive unless otherwise stated herein. For example, features described or depicted in one embodiment may, but do not necessarily, be included in other embodiments. Accordingly, the present invention encompasses various combinations and / or integrations of the specific embodiments described herein.

[0107] As used herein, the phrase "and / or" means that, when used in a list of two or more items, any one of the listed items may be used alone, or any combination of two or more of the listed items may be used. For example, if it is stated that a composition contains or excludes components A, B, and / or C, the composition may contain or exclude only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0108] This description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. When numerical ranges are provided, it should be understood that such ranges are to be interpreted as providing literal support to claims that specify only the lower limit of a range, and to claims that specify only the upper limit of a range. For example, the disclosed numerical range of about 10 to about 100 provides literal support to claims that specify "about 10 or more" (no upper limit) and to claims that specify "less than about 100" (no lower limit).

[0109] Having described this disclosure in detail, it will be clear that modifications and variations are possible without departing from the scope of the invention as defined in the attached claims. [Examples]

[0110] The following examples illustrate methods according to the present invention. However, it should be understood that the following non-limiting examples are provided for illustrative purposes only and do not limit the overall scope of the invention.

[0111] Example 1 Synthesis Procedure for DCLK and MRL Compounds Example 1A: Preparation of MRL16

[0112] [ka]

[0113] In a dry round-bottom flask, add 1'-hydroxy-2'-acetonaphthone (1 equivalent) and isonicotinic acid hydrazide (1 equivalent), followed by the addition of 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, a precipitate may form. If no precipitate forms after this time, slowly cool the reaction flask to room temperature, then place it in the freezer to induce recrystallization of the product. After either the reaction time or recrystallization, filter the mixture and wash the solid with DCM. The yellow solid was isolated, and no further purification was required.

[0114] Example 1B: Preparation of MRL17

[0115] [ka]

[0116] In a dry round-bottom flask, add 1'-hydroxy-2'-acetonaphthone (1 equivalent) and nicotinic acid hydrazide (1 equivalent), followed by the addition of 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, a precipitate may form. If no precipitate forms after this time, slowly cool the reaction flask to room temperature, then place it in the freezer to induce recrystallization of the product. After either the reaction time or recrystallization, filter the mixture and wash the solid with DCM. The yellow solid was isolated, and no further purification was required.

[0117] Regarding the synthesis of both MRL compounds, it is important to note that while Z isomers are also formed, they are likely to be present only in trace amounts due to steric hindrance. Therefore, only the E isomer is shown.

[0118] Example 1C: Preparation of DCLK3

[0119] [ka]

[0120] Step 1: Dissolve naphthalene-1-ol (7.2 g, 50 mmol) in EtOH (28 mL) to prepare a mixture of NaOH (14.4 g, 36.0 mmol) and H2O (30 mL), and add it dropwise. Then heat the mixture to 75°C. Once the desired temperature is reached, add chloroform (6.5 mL, 80 mmol) dropwise and heat the reaction mixture for 2-3 hours. Once the reaction is complete (monitored by TLC), acidify the mixture with 50 mL of 1N HCl and extract with DCM. Wash the organic layer with brine, dry over Na2SO4, filter, concentrate, and purify via silica gel flash column chromatography using a hexane:ethyl acetate gradient.

[0121] Step 2: Iodomethane (3 equivalents) was added dropwise to a mixture of DCLK3_Int1 (1 equivalent) and K2CO3 (1 equivalent) in 10 mL of THF:DMF (10:1) under argon at room temperature. The mixture was then stirred overnight. After overnight, water (15 mL) was added to the mixture. The aqueous phase was extracted with ethyl acetate, washed with brine, and dried over sodium sulfate. The organic phase was filtered and concentrated, and the crude product was purified by silica column chromatography using a hexane:DCM gradient.

[0122] Step 3: Add dimethyl(1-diazo-2-oxopropyl)phosphonate (1 equivalent) all at once at room temperature to a mixture of DCLK3_Int2 (1 equivalent) and potassium carbonate (1.4 equivalents) in 5 mL of MeOH under argon. Stir the mixture overnight at room temperature and monitor the reaction by TLC. After overnight, concentrate the reaction mixture and purify it via silica column chromatography using a hexane:DCM gradient.

[0123] Step 4: Add KHCO3 (2 equivalents) to a mixture of DCLK3_Int3 (1 equivalent) and ethyl 2-chloro-2-(hydroxyimino)acetate (2.2 equivalents) dissolved in 5 mL of ethyl acetate. Flash the mixture under argon and stir overnight at room temperature. After overnight, add heptane (5 mL) and stir for a further 5 minutes. Filter the mixture using heptane to aid filtration. Concentrate the filtrate and purify it via silica column chromatography using a hexane:DCM gradient.

[0124] Step 5: 1N NaOH (79 equivalents) was added to a mixture of DCLK3_Int4 (1 equivalent) in MeOH (48 equivalents). The mixture was stirred overnight at room temperature. After overnight, the reaction mixture was further diluted with water (approximately 25 mL), the pH was adjusted to 2-3 using 1M HCl, and the mixture was stirred for another hour. Within 1 hour, a white precipitate formed. The mixture was then filtered and washed with water. The precipitate was collected and dried under reduced pressure at 50°C.

[0125] Step 6: Dissolve DCLK_Int5 (1.0 equivalent), 4-aminopyridine (2.0 equivalents), and DIPEA (5.0 equivalents) in 1 mL of DMF, flush under argon, and place in a dry ice:acetonitrile bath in the dark. Add a solution of T3P in EA (50% by weight, 2.0 equivalents) dropwise while maintaining the internal temperature at -30°C to -10°C. After addition, heat to -10°C and stir for 1 hour. Then, heat the reaction mixture to 0°C and add water. Add ethyl acetate to the resulting suspension. Separate the layers, wash the organic layer with an aqueous K2CO3 solution (5% by weight), dry over magnesium sulfate, then filter and concentrate to obtain the product. This procedure was modified for DCLK3a and DCLK4.

[0126] Step 7: Dissolve DCLK_Int6 (1 equivalent) in 1 mL of DCM, place in an ice bath, and flush under argon. While maintaining 0°C, add 1 M BCl3 in DCM (68 equivalents) dropwise. After addition, keep the reaction mixture in the ice bath overnight. After overnight, quench the reaction mixture by adding ice water (5 mL) and stir for a further 1 hour. Then, further dilute the reaction mixture with water and wash multiple times with DCM. Isolate the organic layer, dry it over Na2SO4, and concentrate it to obtain DCLK3. No further purification was necessary.

[0127] Example 1D: Preparation of DCLK3a

[0128] [ka]

[0129] Steps 1-5 of DCLK3a are the same as steps of DCLK3.

[0130] Step 6: A mixture of DCLK3a_Int5 (1 equivalent), 3-aminopyridine (1.5 equivalents), and triethylamine (3.4 equivalents) in 3 mL of DCM is flushed under argon and placed in an ice bath in the dark. While maintaining 0°C, a solution of T3P in ethyl acetate (50% by weight, 1.7 equivalents) is added dropwise with stirring. After addition, the reaction mixture is stirred at 0°C for a further 1 hour. Then, it is warmed to room temperature and stirred in the dark for 3-4 days. The reaction is monitored by TLC (5:95 methanol:DCM). After the reaction time, the mixture is further diluted with DCM and the organic layer is washed with water (×2), saturated sodium bicarbonate (×1), and brine (×1). The organic layer is isolated, dried over Na2SO4, filtered, and concentrated. The crude residue was purified by silica column chromatography using a DCM:methanol gradient (99:1 → 90:10).

[0131] Step 7: Dissolve DCLK_Int6 (1 equivalent) in 1 mL of DCM, place in an ice bath, and flush under argon. While maintaining 0°C, add 1 M BCl3 in DCM (68 equivalents) dropwise. After addition, keep the reaction mixture in the ice bath overnight. After overnight, quench the reaction mixture by adding ice water (5 mL) and stir for a further 1 hour. Then, further dilute the reaction mixture with water and wash multiple times with DCM. Isolate the organic layer, dry it over Na2SO4, and concentrate it. The crude residue was purified by silica column chromatography using a DCM:methanol gradient (99:1) to obtain DCLK3a.

[0132] Example 1E: Preparation of DCLK4

[0133] [ka]

[0134] Steps 1-5 are the same as for DCLK3.

[0135] Step 6: A mixture of DCLK4_Int5 (1 equivalent), N,O-dimethylhydroxylamine hydrochloride (1.5 equivalents), and triethylamine (3.4 equivalents) in 3 mL of DCM is flushed under argon and placed in an ice bath in the dark. While maintaining 0°C, a solution of T3P in ethyl acetate (50% by weight, 1.7 equivalents) is added dropwise with stirring. After the addition, the reaction mixture is stirred for a further 1 hour at 0°C. Then, it is warmed to room temperature and stirred in the dark for 3-4 days. The reaction is monitored by TLC (5:95 methanol:DCM). After the reaction time, the mixture is further diluted with DCM and the organic layer is washed with water (×2), saturated sodium bicarbonate (×1), and brine (×1). The organic layer is isolated, dried over Na2SO4, filtered, and concentrated to obtain the crude product. It is important to note that DCLK4_Int6 is photosensitive and must be kept in the dark during synthesis and storage. If not handled properly, it degrades rapidly. All attempts to purify it have resulted in decomposition. Therefore, DCLK4_Int6 is used as is.

[0136] Step 7: A suspension of 4-pyridylmagnesium bromide in freshly prepared THF was prepared by treating 4-iodopyridine (16.6 mmol, 6.5 equivalents) in 55 mL of THF with 16.6 mL of 1.0 M ethylmagnesium bromide. To this suspension, 1 equivalent of DCLK4_Int6 in 3.5 mL of THF was added dropwise. After 2 hours, the reaction was quenched with 1.0 mL of acetic acid and saturated NH4Cl, and then extracted with 2× ethyl acetate at 0–5°C. The organic layer extract was dried over MgSO4, filtered, and concentrated using a rotary evaporator. The residue was suspended in ethyl acetate, filtered, and the filtration cake was washed with ethyl acetate to obtain a pale yellow solid as the first harvest of the product. The filtrate was concentrated to dryness on silica gel to obtain a free-flowing solid, which was dried and packed onto a heptane-moistened silica gel column. The column was sequentially eluted with ethyl acetate:heptane (1:3, 1:2, and 1:1), and the resulting product fractions were combined and distilled under vacuum at 35°C to obtain a second harvest of the product as a white solid.

[0137] Example 2 Trimaleimide linker (CORE) synthesis

[0138] [ka]

[0139] Step 1: Add maleic anhydride (1 equivalent) to a dry round-bottom flask and dissolve in 5-10 mL of tetrahydrofuran. Add furan (1 equivalent) dropwise to this mixture at room temperature while stirring. After the addition, stir the reaction mixture overnight in RT. A precipitate may form during this time. After overnight, filter the reaction mixture, wash the precipitate with hexane (3 × 5 mL), collect it, and dry it under vacuum. Heat the filtrate, cool it to room temperature, and place it in the freezer to induce recrystallization of the product.

[0140] If no precipitate forms overnight, add 15 mL of hexane to the reaction mixture. The change in polarity may cause the product to precipitate. If a precipitate forms, filter the mixture, collect the precipitate, and dry it under vacuum. If no precipitate forms, recrystallize the mixture as described above. Collect the recrystallized product via vacuum filtration as described above. No further purification is necessary.

[0141] [ka]

[0142] Step 2: Add intermediate A (5.2 equivalents) to a dry round-bottom flask and dissolve in 10 mL of methanol. Place this mixture in an ice bath and stir. Prepare a solution of tris(2-aminoethyl)amine (1 equivalent) in methanol (3 mL) and add it dropwise to the cold mixture. After addition, stir the resulting mixture at 0°C for 5 minutes, then warm to room temperature and stir for a further 30 minutes. After this, heat the reaction mixture under reflux for 4 hours (70°C). Then, slowly cool it to room temperature and leave it in the freezer overnight to induce recrystallization of the product. Then, filter it, wash the solid with cold methanol (3 × 5 mL), isolate it, and dry it under vacuum. No further purification is necessary.

[0143] [ka]

[0144] Step 3: Add 1 equivalent of intermediate B to a dry round-bottom flask and suspend in 10 mL of toluene. Heat the mixture under reflux overnight (115°C). Then concentrate the reaction mixture, isolate the product, and dry it under vacuum overnight. No further purification is required after collecting the product.

[0145] Example 3 Solid-phase peptide synthesis protocol All peptides listed in Tables 1 and 2 below were synthesized according to the Fmoc(N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis procedure (Wang H., Udukala DN, Samarakoon TN, Basel MT, Kalita M., Abayaweera G., Manawadu H., Malalasekera A., Robinson C., Villanueva D., Maynez P., Bossmann L., Riedy E., Barriga J., Wang N., Li P., Higgins DA, Zhu G., Troyer DL, and Bossmann, SH Nanoplatforms for Highly Sensitive Fluorescence Detection of Cancer-Related Proteases. Photochemistry & Photobiology Sciences. 2014, 13:231-240 and Duro-Castano A., Conejos-Sanchez I., and Vicent M. Peptide-Based Polymer). (Therapeutics.Polymers.2014,6(2):515-551). Briefly, a 2-ClTrt (2-chlorotrityl) resin containing the first amino acid in the peptide sequence (in this case, Gly) was first swollen in dichloromethane (DCM) for 20 minutes, and then washed with N,N-dimethylformamide (DMF) for 1 minute. After washing, a solution containing Fmoc-protecting amino acids (resin:amino acid, 1:3 molar ratio) and O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HBTU) (resin:HBTU, 1:29 molar ratio) as a coupling agent was prepared in a 1:23 diisopropylethylamine (DIEA):DMF solution and added to the resin, and stirred for 30 minutes for the coupling reaction. Each amino acid was added twice to enhance the addition of amino acids to the peptide chain.Before moving to the next amino acid, the last Fmoc-protected amino acid was deprotected using a 20% diethylamine solution in DMF, followed by five washes with DMF to remove any excess diethylamine present. Each deprotection and addition of an amino acid is a repeating cycle during peptide synthesis until all amino acids on the peptide sequence chain have been added. After synthesis was complete, some peptides were labeled with cholesterol and cholic acid, and cleaved unlabeled. In the case of cholesterol and cholic acid addition, these were coupled to the N-terminus of the peptide using 1,1'-carbonyldiimidazole (CDI) as the coupling reagent. Cholesterol was coupled after five additions within a 24-hour reaction period, while cholic acid was coupled after a total of seven additions within a 24-hour reaction period. Cleavage of the completed sequences (labeled or unlabeled) was performed using trifluoroacetic acid incubated for 3 hours. After a 3-hour cleavage reaction, the peptide was precipitated in cold ether, collected by centrifugation (4,000 × g, 5 minutes), and washed a total of 4-6 times with cold ether. The cleaved peptide was then dried and kept under argon at -20°C. It contained the C-terminal linked amino acid (cysteine) and spacer residue (glycine, from resin synthesis). 10 K 20 (Sequence ID 19) and D 10 K 20 (SEQ ID NO: 21) Block Copeptide was prepared.

[0146] [Table 1] k * :D-Lys amino acids

[0147] [Table 2] s ** : D-Ser amino acid

[0148] Example 4 Nanosponge organization and small molecule protocol coupling The trimalimide linker used to organize the nanosponges contained three -NH2 groups, acting as anchors for linking up to three different peptides, which determined the molar ratio used for organization. Block Copeptide S / D 10 K 20 CG was used (SEQ ID NO: 18 or SEQ ID NO: 20). Nanosponges assembled in the presence of the "Don't Eat Me Peptide" sequence consisted of a linker:cholesterol block copolymer:"Don't Eat Me Peptide" in a 1:2:1 ratio. On the other hand, nanosponges assembled without the "Don't Eat Me Peptide" had a linker:cholesterol / cholic acid-block copolymer ratio of 1:3. Finally, when three different peptides were coupled to the same central linker (MK2 nanosponges), the ratio was maintained at 1:1:1:1 for all of them. In detail, before assembly, 1× phosphate-buffered saline (PBS, pH 7.2) and anhydrous DMF were degassed with argon. In separate vials, the tris-maleimide linker was dissolved in 10-15 μL of degassed DMF, and the peptides were dissolved together in 1-2 mL of degassed PBS. The two solutions were then combined and stirred at room temperature under an argon atmosphere (deoxygenated) for 24 hours. After 24 hours, to remove unbound components, the nanosponges were transferred to a dialysis membrane tube (3,500 Da MWCO) and dialyzed in water for 1-2 hours. The nanosponges were then freeze-dried overnight and stored at -20°C. Figure 5A shows a TEM image of the cholesterol-labeled, organized S10K20CG-based (SEQ ID NO: 18) nanosponge. Average size approximately 96 nm. Figure 5B shows a TEM image of the cholesterol-labeled, organized D10K20CG-based (SEQ ID NO: 20) nanosponge. Average size approximately 77 nm.

[0149] The nanosponges were compared to previous constructs made from cholesterol-(peptide)3-trimaleimide units consisting of homopeptides of various lengths (n=5, 10, 15, 20) (all either K or D), SEQ ID NO: 23 or 24, and a trilateral planar maleimide linker to which the consensus sequence of the executioner caspase (DEVDGC, SEQ ID NO: 26) was added via Michael addition.

[0150] CDI or HBTU (D) are used as coupling reagents. 10 K 20 Small molecules were coupled to nanosponges using CG nanosponges (only in the case of SEQ ID NO: 20). Briefly, for the DCLK1 small molecule, 10% (by weight) of each small molecule and CDI were dissolved in anhydrous DMF and stirred at room temperature for 10 minutes. In a separate vial, the nanosponges were dissolved in DMF, the solution was transferred to the small molecule-CDI reaction solution, and the reactants were stirred at room temperature for 24 hours. Similarly, 10% of each MK2 inhibitor (PF3644022, MK2-IN-1, and MK2-IN-3) and CDI were dissolved in DMSO and reacted for 10 minutes, then the nanosponges dissolved in DMF were added and reacted at room temperature for 24 hours. Dialysis of the nanosponge-small molecule complexes was repeated for 1 hour to remove any unbound components, and then freeze-dried overnight. Gemcitabine also bound to nanosponges, and in vitro and in vivo activity has been demonstrated. By using nanosponge technology to enable localized exposure, it becomes possible to administer higher doses of gemcitabine to tumor sites while reducing the risk of toxicity, limiting systemic exposure, and thus delaying tolerance. The scale-up synthesis of nanosponge-bound gemcitabine will be optimized to complete the in vivo efficacy and release characterization of the material. Tables 3 and 4 summarize all organized compounds for each project.

[0151] [Table 3]

[0152] [Table 4]

[0153] Example 5 Nanosponge IC50 and its properties The compounds were screened using a colorimetric hexosaminidase assay. Briefly, cells were seeded in a 96-well plate. The cells were then treated with escalating doses of the compound for 48 hours. The medium was aspirated, and the cells were incubated with a substrate buffer containing p-nitrophenol-N-acetyl-β-D-glucosaminidase. The plates were read at 405 nm absorbance.

[0154] The survival rate was calculated as the percentage of absorbance of the treated organism compared to the control. The obtained IC50 data are shown in Table 5.

[0155] [Table 5]

[0156] Cells were seeded in 6-well plates for colony formation. The cells were then treated with test substances at IC50 and 1 / 2 IC50 doses for 48 hours. The medium was then aspirated and replaced with complete medium free of the drug. Colonies were then grown for 2 weeks, stained, counted, and their size determined using ImageJ software.

[0157] Cells were seeded in 24-well low-adhesion plates for spheroid formation. After 72 hours of spheroid formation, test substances were added at IC50, 1 / 2IC50, and 1 / 4IC50 doses. The spheroids were grown for a further 96 hours, after which imaging and quantification were performed.

[0158] Figure 6 (top) shows representative colony formation images of HUCCT1 cells treated for 48 hours with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50 and 1 / 2 IC50 previously determined by hexosaminidase assay. (bottom) Quantification of colony number and size. Data are expressed as the average number and size from three independent experiments. * p<0.05.

[0159] Figure 7 (top) shows representative colony formation images of HUH28 cells treated for 48 hours with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50 and 1 / 2 IC50 previously determined by hexosaminidase assay. (bottom) Quantification of colony number and size. Data are expressed as the average number and size from three independent experiments. * p<0.05.

[0160] Figure 8 shows (left) representative spheroid formation images of HUCCT1 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50, 1 / 2IC50, and 1 / 4IC50 doses previously determined by hexosaminidase assay. (right) Quantification of spheroid count. Data are expressed as the average from three independent experiments. * p<0.05.

[0161] Table 6 shows the calculated molecular weight (g / mol), logP, calculated solubility (mol / L), experimental solubility (mg / mL), measured dynamic light scattering (DLS) in nm units, polydispersity index (PDI), and measured zeta potential (mV) for the selected nanosponges.

[0162] [Table 6] The calculated solubility and logP were performed using SWISSADME (www.swissadme.ch / ) via a segmentation approach. * The consensus sequence of the executioner caspase (DEVDGC, SEQ ID NO: 26).

[0163] Further nanosponges have been synthesized or proposed for synthesis (Table 7).

[0164] [Table 7]

[0165] Example 6 Nanosponge-MK2 inhibitor data Cell culture. BJ(hTERT) and patient-derived cancer-associated fibroblasts (CAF) 036 and 067 were obtained from Antonio Jimeno at the University of Colorado Denver Anschutz Cancer Pavilion. BJhTERT cells were cultured and maintained in DMEM (Invitrogen) supplemented with 10% FBS and 1% penicillin / streptomycin. All cells tested were negative for mycoplasma. Cell density was measured using a Countess II FL hemocytometer. Cells were treated with a logarithmic dilution of MK2 inhibitor (PF-3644022), MK2 inhibitor 1 (MK2i1), or MK2i+ nanosponge, or untreated (control). Cells were stimulated with 2 ng / mL TGFβ1. MK2i and MK2i+ nanosponge were added 2 hours prior to TGFβ1 stimulation. The cells were allowed to grow for a further 48 hours after stimulation and then harvested for protein and mRNA analysis. Our protocol was adapted from an established IPF study [PMID:23470623]. CAF036 and 067 were processed in a manner similar to that of BJ(hTERT).

[0166] Immunoblotting. After 48 hours, cells were lysed in ice-cold RIPA buffer (ThermoFisher) containing 1× concentration of protease and phosphatase inhibitors (Pierce). Protein concentrations were determined using a standard BCA protein assay (ThermoFisher). Proteins were separated by gradient SDS-PAGE, with 25 μg of total protein loaded into each lane. The gel was transferred to a PVDF membrane using iBlot2 (Invitrogen). The following antibodies were used: 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), and pHSP27 (Cell Signaling Technologies, #9707, Ser82, clone D1H2F6, 1:3,000 dilution). Standard horseradish peroxidase conjugate secondary antibodies were used for detection using Super Signal West Femto (ThermoFischer).

[0167] The inventors observed a time-dependent increase in collagen 1 and steady-state expression by immunoblotting after constant TGFβ1 stimulation (Figure 9). The inventors attempted to immunoblot α-SMA. However, this protein could not be detected because TERT-mediated immortalization of these cells interfered with its expression (data not shown). After treatment with the nanosponge backbone alone, it was concluded that higher concentrations (50 μM) were cytotoxic and also induced the p38-MK2 pathway. It was also observed that the nanosponge alone induced collagen production (Figure 10). The inventors conducted a drug dose-response study, testing both the MK2 inhibitor and PF-3644022, and showed that PF-3644022 could reduce MK2 phosphorylation better than the PF-3644022-nanosponge conjugate (PF conjugate) (Figure 11). The nanosponge conjugate drug only reduced collagen expression at 50 μM and was also found to be cytotoxic (Figure 11). Next, the inventors conducted another drug dose-response study to evaluate the effects of PF-3644022 versus PF conjugate and immunoblotted for p-HSP27, a known downstream phosphorylation target of MK2. PF-3644022 reduced BJ(hTERT)HSP27 phosphorylation at a concentration an order of magnitude lower than that of PF conjugate. In contrast, the inventors also examined patient CAF067, who showed a dose-response effect with PF conjugate and was better able to suppress HSP27 phosphorylation than PF-3644022 alone (Figure 12).

[0168] MK2i1, whether as a naked drug or a conjugate, was not shown to reduce MK2 phosphorylation or affect downstream targets (Figure 13). In conclusion, PF-3644022 is a more effective MK2 inhibitor, but the increased inhibitory efficacy from conjugation with nanosponge is not conclusive. Because PF-3644022 is highly hydrophobic, the nanosponge support improved drug solubilization compared to the naked drug alone.

[0169] Example 7 Nanosponge-Prochlorperazine Data Nanosponge-Prochlorperazine (PCZ) Synthesis. Nanosponges were designed and filled with PCZ to increase the potency of PCZ for drug accumulation and delivery to the clinic. Peptide-based nanosponges for advanced drug delivery were labeled with a trilateral planar trismaleimide linker and cholesterol. 10 K 20 It consisted of a CG (SEQ ID NO: 20) peptide building block and a CD44-targeted peptide sequence (sHPWSYLWTQQAs (SEQ ID NO: 1)) labeled with rhodamine B for drug delivery to PDAC. For in vivo experiments, the "Don't Eat Me" peptide sequence kGNYTCEVTELSREGKTVIELKk (SEQ ID NO: 7) (with D-lysine at both ends to prevent rapid proteolysis) was included to enhance the circulation time of the nanosponge in the blood. For the in vivo designed nanosponge, the molecular mass used for organizing the nanosponge was as follows: 5 (cholesterol-labeled D 10 K 20 CG, (SEQ ID NO: 20):1 (Trimarimid Linker):1 (Rhodamine B-labeled CD44):1 ("Don't Eat Me" peptide). During CD44 receptor-mediated endocytosis, the nanosponges can rapidly escape into the cytoplasm. After organizing the nanosponges, they were filled with 10% by weight of prochlorperazine dimerylate (PCZ).

[0170] Characterization. PCZ nanosponges were characterized using TEM, and exemplary TEM images are shown in Figure 14. The percentage of PCZ filling in the nanosponges was also determined. Calibration curves were created for PCZ drug alone, and then PCZ-nanosponges of known concentrations were measured using 320 nm absorbance and plotted on the calibration curve to calculate the amount and percentage of PCZ filling in the nanosponges. The results showed that the nanosponges were filled with 10.12% PCZ, as shown in Figure 15.

[0171] Results from cell and animal experiments. The effect of PCZ nanosponges on the proliferation of pancreatic cancer cells. The inventors treated MiaPaCa-2 pancreatic cancer cells with prochlorperazine and its nanoparticles at concentrations of 0-30 μM. The inventors recorded cell viability using hexosaminidase activity at 24, 48, and 72 hours. The inventors observed that both prochlorperazine and its nanosponges inhibited the proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner (Figure 16).

[0172] Cell uptake assay of prochlorperazine nanosponges in pancreatic cancer cells. To confirm uptake, the inventors treated MiaPaCa-2 and S2-007 cells with PCZ nanosponges, fixed them, stained them with DAPI (blue nucleus) and phalloidin (green cytoplasm), and imaged them using confocal microscopy. Rhodamine B is tagged to the nanoparticles, which are imaged in red. The inventors found significant uptake of nanoparticles in PDAC cells (Figure 17).

[0173] In vivo uptake. To confirm the uptake of nanosponges in mice by tracking the accumulation of nanoparticles in mouse PDAC tumors using IVIS, we intravenously injected rhodamine B-labeled PCZ nanosponges at a dose of 10 mg / kg into KPCC orthotopic tumor-carrying C57BL / 6 mice, and imaged ex vivo 24 hours later. We observed significant uptake of PCZ nanosponges in the tumors (Figure 18).

[0174] Example 8 Nanosponge for PKD treatment Synthesis. Nanosponges were designed for the treatment of polycystic kidney disease (PKD) by using modified peptide fragments from the axial sequence in the C-terminal fragment of polycystin-1 (PC1), namely P17(TAFGASLFVPPSHVQFIG(SEQ ID NO: 12)), KP17(TAFGASLFVPPSHVQFIGGKKKKKG(SEQ ID NO: 13)), and DP17(TAFGASLFVPPSHVQFIGGDDDDDG(SEQ ID NO: 14)). These nanosponges consisted of a trilateral planar trismaleimide linker, a cholesterol-labeled D10K20CG(SEQ ID NO: 20) peptide building block, short PC1 CTF fragments (P17, KP17, and RP17), and the "Don't Eat Me" peptide sequence (kGNYTCEVTELSREGKTVIELKk(SEQ ID NO: 7)), each in a molar ratio of 1:2:1:1.

[0175] Characterization. Each of the PC1 CTF peptides was characterized using LC-MS, which showed characteristic peaks confirming P17 (calculated MW 1875.16 g / mol, 96% purity), KP17 (calculated MW 2630.12 g / mol, 98% purity), and DP17 peptide (calculated MW 2564.71 g / mol, 95% purity) respectively (graph not shown).

[0176] Cellular experiments. P17, KP17, and DP17 peptides were tested at a concentration of 1 mM in HEK293T (wild-type) and HEK293T-PKD1-KO-c4 (CRISPR-producing) cell lines, and incubated overnight for approximately 20 hours. The KP17 peptide showed the highest activity in both cell lines compared to all other peptides tested and the control, followed by the P17 fragment peptide. The results are shown in Figure 19.

[0177] Example 9 Apatanib as an MK2 inhibitor Multiple models demonstrate that TGFβ1-mediated fibrosis is caused by activated myofibroblasts that signal via the TGFβ1R-p38-MK2 signaling axis, and that direct inhibition of MK2 can prevent myofibroblast development and subsequent fibrous injury in idiopathic pulmonary fibrosis and cardiac fibrosis injury models. Furthermore, activation of pro-angiogenic signaling processes has similarly been shown to promote soft tissue fibrosis in in vivo liver, intestine, and lung injury models.

[0178] While screening for novel MK2 inhibitors using our p38α-MK2 high-throughput screening assay, we identified apatanib, a VEGFR2 inhibitor. Our preliminary findings showed that apatanib can inhibit MK2 phosphorylation (prevent MK2 activation), reduce HSP27 phosphorylation (a downstream target of MK2), and subsequently reduce collagen-1 protein production. However, there are no FDA-approved MK2 inhibitors, and early-generation small molecule MK2 inhibitors lack "drugability" due to a lack of enzymatic ATP pocket selectivity and low water solubility, resulting in unrealistically high EC50 levels required to achieve therapeutic benefits.

[0179] Example 10 Nanosponge for Plectin-Targeted PDAC Therapy Synthesis. To increase nanosponge accumulation in pancreatic ductal adenocarcinoma (PDAC), we designed plectin-targeted nanosponges. The plectin nanosponges were triangular planar trismaleimide linkers, cholesterol-labeled S 10 K 20The CG (SEQ ID NO: 18) peptide building block consisted of a rhodamine B-labeled plectin-targeted peptide sequence (sHPWSYLWTQQAs, (SEQ ID NO: 1)) to track and confirm delivery to the tumor site. For in vivo experiments, the "Don't Eat Me" peptide sequence kGNYTCEVTELSREGKTVIELKk (SEQ ID NO: 7) was used. For in vivo designed nanosponges, the molecular mass used for organizing the nanosponges was as follows: 2 (cholesterol-labeled S 10 K 20 CG, (SEQ ID NO: 18):1 (trimaleimide linker):1 (rhodamine B-labeled plectin):1 ("Don't Eat Me" peptide).

[0180] Characterization. Characterization of S10K20CG (SEQ ID NO: 18) using DLS and zeta potential.

[0181] [Table 8]

[0182] Cell and animal experiments. PDAC-carrying mice were treated with plectin-nanosponge after tumor development and growth over three weeks. Plectin-nanosponge was intravenously injected into the mice, incubated overnight, and then sacrificed. Various organs and tumor tissues were removed and imaged ex vivo to determine the localization of plectin-nanosponge. IVIS imaging confirmed specific uptake of plectin-nanosponge only in tumor sites, with no uptake in other healthy organs. The results are shown in Figures 20 and 21.

[0183] When describing elements of this disclosure or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to indicate that there is one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be comprehensive and mean that there may be additional elements other than those listed.

[0184] Considering the above, it will be evident that some of the objectives of this disclosure have been achieved and other favorable results have been obtained.

[0185] Because various modifications can be made in the manner described above without departing from the scope of this disclosure, all matters contained herein are intended to be interpreted as illustrative and not restrictive.

Claims

1. A peptide nanosponge comprising a plurality of self-assembling peptide building blocks, wherein the plurality of self-assembling peptide building blocks comprises a first peptide building block, and the first peptide building block is A branched polymer core including at least three arms, A first block copeptide having 50 amino acids or less, covalently bonded to one of the arms via its C-terminus, wherein the first block copeptide comprises a first peptide block and a second peptide block different from the first peptide block, A lipid-based capping moiety having a molecular weight of 400 Da or less, covalently bonded to the N-terminal amino acid of the first block copolymer, A peptide nanosponge comprising a therapeutically active compound covalently bonded to one or more amino acids of the first block copolymer via cleavable links.

2. The peptide nanosponge according to claim 1, wherein the branched polymer core has a dendrimer-like branched or superbranched polymer structure with a density of 800 Da or less.

3. The peptide nanosponge according to claim 1 or 2, wherein the arms of the branched polymer core are terminated with functional groups for peptide bonding selected from the group consisting of maleimide groups, carboxylate groups, amide groups, and hydroxyl groups, and the first block copolymer is resistant to cleavage from the branched polymer core.

4. The peptide nanosponge according to any one of claims 1 to 3, wherein a cysteine ​​and / or glycine spacer residue is included between the block copolymer and the arm.

5. The peptide nanosponge according to any one of claims 1 to 4, wherein the core is derived from a tris(maleimide) compound or an analog thereof.

6. The peptide nanosponge according to any one of claims 1 to 5, wherein the first peptide block consists of an amino acid having a positively charged side chain.

7. The peptide nanosponge according to claim 6, wherein the first peptide block is composed of an amino acid selected from the group consisting of lysine, histidine, and arginine.

8. The peptide nanosponge according to claim 6, wherein the first peptide block is a homopeptide consisting of 10 to 25 lysine residues.

9. The peptide nanosponge according to any one of claims 1 to 8, wherein the second peptide block comprises natural or unnatural amino acids having side chains terminated with an amine, hydroxyl, or carboxylic acid group.

10. The peptide nanosponge according to claim 9, wherein the second peptide block is composed of an amino acid selected from the group consisting of serine, aspartic acid (or aspartate), tyrosine, glutamic acid (or glutamate), and threonine.

11. The peptide nanosponge according to claim 9, wherein the second peptide block is a homopeptide consisting of 5 to 15 amino acids.

12. The peptide nanosponge according to any one of claims 1 to 11, wherein the second peptide block is covalently bonded to the first block peptide.

13. The peptide nanosponge according to any one of claims 1 to 12, wherein the first peptide building block further comprises a signaling sequence covalently bonded to an arm of the branched polymer core different from the block copolymer.

14. The peptide nanosponge according to claim 13, wherein the signal transduction sequence is bound to the different arms via each peptide bond, and the signal transduction sequence is selected from the group consisting of a targeting moiety, a detectable label, a "Don't Eat Me" sequence, a "Eat Me" sequence, and combinations thereof.

15. The aforementioned signaling sequences include a CD44 signaling sequence containing sHPWSYLWTQQAs (SEQ ID NO: 1), a plectin targeting sequence containing KTLLPTPG (SEQ ID NO: 2), an EGFR signaling sequence containing sYHWYGYTPENVIGsG (SEQ ID NO: 3), an LDLR signaling sequence containing sCMPRRRGCGAGsG (SEQ ID NO: 4), a CD46 signaling sequence containing sLPGTICKRTMLDGLNDYCTGsG (SEQ ID NO: 5), a SIRPα "Don't Eat Me" signaling sequence containing kGNYTCEVTELSREGKTVIELKsG (SEQ ID NO: 6), and kGN The peptide nanosponge according to claim 13 or 14, comprising a signaling sequence for SIRPα "Don't Eat Me" containing YTCEVTELSREGKTVIELKk (SEQ ID NO: 7), a signaling sequence for SIRPα "Eat Me" containing 4N1:RFYVVMWK (SEQ ID NO: 8) or 7N3:FIRVVMYEGKK (SEQ ID NO: 9), a signaling sequence for CD206 "Shutdown M2 Polarization" (rp-182) containing KFRKAFKRFFGsG (SEQ ID NO: 10), and a WTAS containing the mucosal signaling sequence: PLKWPGKKKKKGKPGKRKEQKKKRRTRRG (SEQ ID NO: 11).

16. The peptide nanosponge according to any one of claims 1 to 15, wherein the lipid-based capping portion contains cholesterol.

17. The peptide nanosponge according to any one of claims 1 to 15, wherein the lipid-based capping portion contains cholic acid.

18. The peptide nanosponge according to any one of claims 1 to 17, wherein the therapeutic activator is selected from the group consisting of a DCLK1 inhibitor, an MK2 inhibitor, a cytotoxic agent for p53-deficient cells, an MIF inhibitor, or a combination thereof.

19. The plurality of self-assembling peptide building blocks differ from the first peptide building block, A branched polymer core including at least three arms, A second block copeptide having 50 amino acids or less, covalently bonded to one of the arms via its C-terminus, wherein the second block copeptide comprises a first peptide block and a second peptide block different from the first peptide block, A lipid-based capping moiety having a molecular weight of 400 Da or less, covalently bonded to the N-terminal amino acid of the first block copolymer, The peptide nanosponge according to any one of claims 1 to 18, further comprising a second peptide building block, which includes a signaling sequence covalently bonded to an arm different from the block copolymer of the branched polymer core.

20. The peptide nanosponge according to claim 19, wherein the signal transduction sequence is bound to the different arms via peptide bonds that are not block copolymers.

21. A pharmaceutical composition comprising a plurality of peptide nanosponges according to any one of claims 1 to 20, dispersed in a pharmaceutically acceptable carrier.

22. A method for treating a disease or disorder in a subject, comprising administering a therapeutically effective amount of a peptide nanosponge or a pharmaceutical composition thereof according to any one of claims 1 to 20 to a subject in need thereof.

23. The method according to claim 22, wherein the disease or disorder is colon cancer, bile duct cancer, genitourinary cancer, gynecological cancer, gastrointestinal cancer, lymphoma, melanoma or skin cancer, head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain tumor, glioma, idiopathic pulmonary fibrosis (IPF), head and neck fibrosis, osteosarcoma, or bladder cancer.

24. The method according to claim 23, wherein the cancer is colon cancer or bile duct cancer.

25. A pharmaceutical product for treating a disease or disorder in a subject requiring treatment of a disease or disorder, comprising a therapeutically effective amount of the peptide nanosponge described in any one of claims 1 to 20.

26. Use of the peptide nanosponge according to any one of claims 1 to 20 for treating a disease or disorder in a subject requiring treatment of a disease or disorder.

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