Nanostructured delivery system for tumor therapy

Abstract

The present invention relates to the use of a nanostructured delivery system comprising at least one polymer and / or at least one lipid and at least one polymethine dye for treating a subject suffering from an adenocarcinoma, including adenocarcinoma cells having an altered gene expression in one or more SLC0 genes. The at least one polymethine dye mediates the targeted transport of the nanostructured delivery system to the adenocarcinoma cells. The present invention also relates to a pharmaceutical composition comprising the nanostructured delivery system. The present invention also relates to a method of using the nanostructured delivery system for targeted transport into tumor tissue and the use of the nanostructured delivery system or pharmaceutical composition for fluorescence detection based on the accumulation of the system or composition in tumor tissue or cells.

Description

Technical Field

[0001] This invention relates to the use of a nanostructured delivery system comprising at least one polymer and / or at least one lipid and at least one polymethine dye in the treatment of subjects with adenocarcinoma, said adenocarcinoma including adenocarcinoma cells with altered gene expression in one or more SLCO genes, wherein said at least one polymethine dye mediates the targeted delivery of the nanostructured delivery system to said adenocarcinoma cells. The invention also relates to a pharmaceutical composition comprising said nanostructured delivery system, to a method of targeted delivery of said nanostructured delivery system to tumor tissue, and to the use of the nanostructured delivery system or pharmaceutical composition of the invention for fluorescence detection based on the accumulation of said system or composition in tumor tissue or cells. Background Technology

[0002] There is a growing recognition of the importance of organ-specific drug delivery as a way to optimize drug efficacy. In the case of cancer treatment, the ability to target specific organs or tissues allows for improved efficacy and reduced nonspecific side effects of anticancer drugs. Furthermore, targeted drug delivery can avoid subject-to-subject variations in drug expression and activity along pathways, manifested as significant changes in the pharmacokinetic characteristics of such drugs. The use of nanoparticles to deliver active ingredients to specific tissues in a targeted manner is known in the art (C. Sheridan, Proof of concept for next-generation nanoparticle drugs in humans. Nat Biotechnol, 2012 30(6):pp.471-3; SEGratton et al., The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci USA, 2008 105(33):pp.11613-8). These nanoparticles are used in tumor therapy and function based on the following mechanism: the nanoparticles have a shell or antibody. If coated with an aqueous shell, the nanoparticles will not be recognized by the immune system. If these nanoparticles are injected without being attacked by the immune system, they diffuse through porous blood vessels, which have significantly larger pores (openings) in tumor tissue compared to normal blood vessels. The nanoparticles are then absorbed by surrounding cells, characterized by increased permeability compared to healthy cells. This passive enrichment process of nanoparticles, liposomes, or macromolecules, as described above, is known as the EPR effect ("enhanced permeability and retention"), and thus refers to passive drug targeting. However, a drawback is that not only tumor cells absorb the nanoparticles, but healthy cells also absorb them—the nanoparticles are non-specifically transported to healthy cells via blood vessels. This can lead to serious adverse reactions. If the nanoparticles are modified with antibodies on their surface after synthesis, they target cells expressing surface antigens that bind to these antibodies. This transport mechanism is also passive and non-selective.

[0003] Adenocarcinoma is a tumor originating from glands and / or epithelial tissue with glandular characteristics. Several of the most common forms of cancer are adenocarcinomas, which can be found in common sites such as the breast, lung, prostate, and gastrointestinal tract (colon, rectum, pancreas, stomach, esophagus). Adenocarcinomas can be histologically diagnosed using light microscopy, where diagnosis is typically based on the identification of glandular structures. In poorly differentiated adenocarcinomas, where only minimal or no glandular formation is observed under a light microscope, immunohistochemistry (IHC) is an important tool for diagnosing the type of adenocarcinoma. Diagnostic IHC (see, e.g., Dabbs(ed): Diagnostic Immunohistochemistry) is a well-known tool in the differential diagnosis of adenocarcinomas: for example, in diagnosing lung adenocarcinoma, antibodies against cytokeratins, such as cytokeratin 7 (CK-7), can be used to identify the cancer. These adenocarcinomas often exhibit mutations in epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and c-ROS oncogene 1 (ROS-1). In advanced lung cancer, PD-L1 (programmed cell death 1-ligand 1) IHC assays are performed. For example, positive IHC staining for CK-7, GATA-binding protein 3 (GATA-3), and giant cystic disease liquid protein 15 suggests breast adenocarcinoma. For example, positive IHC staining for prostate-specific antigen (PSA) is specific for prostate adenocarcinoma, while nuclear basal protein P63 is used to differentiate normal prostate tissue from prostate adenocarcinoma. For example, positive IHC staining for tail-type homeobox 2 (CDX-2) or cytokeratin-20 (CK-20) combined with negative CK-7 staining suggests colorectal adenocarcinoma. For example, positive IHC staining for CK-7, pairing cassette protein 8 (PAX 8), and Wilm tumor protein 1 (WT-1) suggests ovarian adenocarcinoma. For example, positive IHC staining for thyroglobulin and thyroid transcription factor-1 (TTF-1) suggests thyroid adenocarcinoma.

[0004] Extensive evidence suggests that the metabolism of both exogenous and endogenous substances influences the growth characteristics of many cancer tissues. These metabolic changes can be categorized based on the metabolic alterations involved, namely changes in glucose, amino acid, and lipid processing pathways. Nutrient transporters (such as glucose and amino acid transporters) are typically differentially regulated at both the transcriptional and post-translational levels in cancer cells, supporting high levels of cell proliferation and pathological growth (Zhang et al., 2018). The SLCO gene (a solute carrier of the organic anion transport subfamily) encodes an organic anion transport polypeptide (OATP), which is currently believed to play a role in cancer development and treatment (Obaidat et al., 2012). In particular, studies have found that OATP expression may be altered in different types of cancer. Therefore, it is speculated that OATP expression may play a potential role in cancer development and progression, as well as in the management of anticancer drugs.

[0005] Nanoparticles for transporting active ingredients are known from existing technologies and are categorized into different types: simple delivery systems (such as micelles, nanoemulsions, nanoliposomes, solid lipid microparticles, polymers, and hydrogel particles), and composite systems created by applying structural design principles to modify the properties and functions of simple delivery systems. For example, such composite nanoparticles are liposomes carrying a protective hydrophilic layer composed of a flexible hydrophilic polymer (such as polyethylene glycol (PEG)). By providing this coating, clearance of the composite nanoparticles from the reticuloendothelial system (RES) is reduced, resulting in prolonged cycle times and improved pharmacokinetic characteristics. Another example of composite nanoparticles is polymer nanoparticles based on polylactic-glycolic acid copolymer (PLGA), which, compared to other nanocarriers (such as liposomes and micelles), offer improved stability, high drug loading, sustained drug release, non-immunogenicity, reduced drug toxicity, improved bioavailability, biocompatibility, biodegradability, enhanced efficacy of encapsulated drugs, and versatile surface modifications. PLGA-based nanoparticles can be endowed with desired surface properties (i.e., zeta potential and hydrophilicity) using surfactants and / or other surface modifications to improve absorption into solid tissues or tumors, for example, through adsorbent transcytosis. Press et al. demonstrated that PLGA nanoparticles functionalized with polyacetylenic dyes possess the ability to be selectively absorbed by the liver and kidneys via clathrin-mediated endocytosis due to their affinity for transmembrane carrier proteins.

[0006] In the context of cancer therapy, there is a need for an active and selective transport and / or delivery system, wherein the active transport occurs selectively into tumor tissue via a specific targeting unit, allowing the active pharmaceutical ingredient to be simultaneously transported into the tissue. This invention provides, for example, a selective transport and / or delivery system to adenocarcinoma tissue characterized by altered expression of one or more SLCO genes. Summary of the Invention

[0007] In a first aspect, the present invention relates to the use of a nanostructure delivery system comprising at least one polymer and / or at least one lipid and at least one polymethystine dye in the treatment of a subject with adenocarcinoma, said adenocarcinoma including adenocarcinoma cells having altered gene expression in one or more SLCO genes, wherein said at least one polymethystine dye mediates the targeted delivery of the nanostructure delivery system to said adenocarcinoma cells.

[0008] As used herein, a “nanostructure delivery system” refers to a system comprising at least one or more polymers and / or lipids; if it comprises one or more polymers, it is referred to herein as a “nanoparticle”; if it comprises one or more lipids, it is referred to herein as a “liposome.” If the nanostructure delivery system according to the invention comprises polymers and lipids, it is referred to herein as a “nanoparticle” or “liposome.” Therefore, according to the invention, the terms “nanoparticle” and “liposome” are used synonymously, and also relate to nanostructure delivery systems comprising polymers and lipids. Nanoparticles are structures with a size less than 1 μm and can be composed of multiple molecules. Generally, they are characterized by a high surface area to volume ratio, thus providing great chemical reactivity. These nanoparticles can be composed of polymers, wherein these polymers are characterized by certain units (monomers) being repeating units. The polymers are covalently bonded to each other through the chemical reaction (polymerization) of these monomers. If some of these polymers are hydrophobic, they may form nanoscale structures (e.g., nanoparticles, micelles, vesicles) in an aqueous environment. Due to their hydrophobicity, lipids can also be used to form nanoparticles (micelles, liposomes).

[0009] According to the present invention, the term "adenocarcinoma" refers to a tumor of glandular origin and / or of epithelial tissue with glandular characteristics. In this document, the subjects suffering from adenocarcinoma are mammals, preferably humans.

[0010] According to the present invention, the term "gene expression" refers to the process of using information from a gene to synthesize a functional gene product, wherein the gene product can be a peptide, protein, or any type of RNA. In the context of the present invention, the gene product produced by the gene expression process is a functional protein. The term "altered gene expression" refers to an alteration in the protein synthesis of a corresponding gene, present in tumor cells of a tissue but not present or present to less than the same extent in normal non-tumor cells of said tissue. One example of such altered gene expression is the upregulation or downregulation of a protein in tumor tissue, wherein the expression level is increased or decreased compared to normal tissue. Another example of altered gene expression involves differential cellular distribution of a protein in tumor cells, such as a shift from regular localization on the cell membrane to intracellular compartments in tumor cells. Another example involves alterations affecting gene function, resulting in proteins with enhanced (gain) or diminished (loss) function.

[0011] As described above, the SLCO gene (a solute carrier of the organic anion transport subfamily) encodes organic anion transport polypeptides (OATP). To date, 11 human OATPs are known, classified into six families based on amino acid sequence identity (SLCO1A2, SLCO1B1, SLCO1B3, SLCO1C1; SLCO2A1, SLCO2B1; SLCO3A1; SLCO4A1, SLCO4C1; SLCO5A1; and SLCO6A1). All OATPs possess 12 transmembrane domains, with their respective amino and carboxyl terms facing the intracellular space. OATP mediates the transport of its substrates through an electroneutrally neutral process, in which the influx of extracellular OATP substrates is accompanied by the efflux of intracellular anions (such as bicarbonate).

[0012] As described above, the use of nanoparticles for targeted delivery of active ingredients to specific tissues (“targeted delivery”) is known from the prior art. “Targeted delivery” (or cell-specific delivery) is understood to refer to the targeted and selective accumulation of a carrier at a desired site of action; in practice, targeted delivery is used to deliver active ingredients to specific tissues / cell populations (“targeted drug delivery”), enabling the active ingredient to be released at the desired site of action, thereby enhancing the efficacy of the active ingredient and reducing its systemic side effects. The delivered active ingredients typically include antibodies, peptides, or small molecules such as oligonucleotides or nucleic acids.

[0013] In a preferred embodiment, at least one polyacetylenic dye included in the nanostructure delivery system can mediate the targeted delivery of the nanostructure delivery system to the adenocarcinoma cells via the expression products of one or more SLCO genes. In another preferred embodiment, the adenocarcinoma may include adenocarcinoma cells in which the gene expression alteration affects the SLCO1 gene; more preferably, the adenocarcinoma may include adenocarcinoma cells in which the gene expression alteration affects the SLCO1B1 or SCLO1B3 genes. Of all OATPs, the respective roles of OATP1A2, OATP1B1, OATP1B3, and OATP2B1 in normal tissues have been most extensively characterized, particularly their roles in the absorption of pharmacological substances. In these tissues, OATP1B1 and OATP1B3 are expressed in the basolateral membrane of sinusoidal hepatocytes, exhibiting an expression gradient from the perivenous to the periportal venous region, while OATP1A2 is primarily expressed in the blood-brain barrier and duodenal enterocytes.

[0014] In another preferred embodiment, the adenocarcinoma treated using the nanostructure delivery system of the present invention can be a cancer selected from the group including gastrointestinal cancer, lung cancer, prostate cancer, and breast cancer. As used in this invention, the term "gastrointestinal cancer" refers to cancer originating from gastrointestinal tumor cells, such as stomach cancer, colon cancer, colorectal cancer, rectal cancer, pancreatic cancer, gallbladder cancer, and liver cancer.

[0015] In another preferred embodiment, at least one polyacetylenic dye contained in the nanostructure delivery system can mediate targeted delivery of the nanostructure delivery system to adenocarcinoma stem cells. As used herein, the term "adenocarcinoma stem cell" refers to a cell derived from normal stem cells or mutated progenitor cells. One or more mutations can occur at any point in the normal developmental process from stem cell to progenitor cell to mature cell. Thus, the mutated cell produces a functionally defined entity, namely, an adenocarcinoma stem cell: a stem cell that can differentiate into any cell carrying the defective corresponding adenocarcinoma lineage. Such stem cells have been described in the context of lung cancer, gastrointestinal cancer, and prostate cancer.

[0016] In a preferred embodiment, the adenocarcinoma treated using the nanostructure delivery system of the present invention can be prostate cancer or colon cancer.

[0017] In a preferred embodiment, the nanostructure delivery system can be used according to the purpose of the invention, wherein the at least one polyacetylene dye can be a symmetrical or asymmetric polyacetylene with the following general formula.

[0018]

[0019]

[0020] an represents the value 1, 2, or 3;

[0021] b. R1-R19 can be the same or different, and can be hydrogen or deuterium, one or more alkyl, tertiary alkyl, cycloalkyl (“alkyl” and “cycloalkyl” radicals also include olefin structures) or aryl, carboxyaryl, dicarboxyaryl, heteroaryl or heterocyclic aliphatic radicals, alkoxy, acyloxy, alkyl mercapto, aryloxy, aryl mercapto, heteroaryloxy, heteroaryl mercapto, hydroxyl, halogen, nitro, amino, aminoalkyl, aminoaryl, aminoacyl, Halogenated alkyl, formyl, azide, thio(iso)cyano, (iso)cyano, carbamate, thiocarbamate, urea, thiourea, guanidine, sulfonamide or cyano, alkyl-substituted or cyclic amine functional groups and / or two ortho radicals, such as R3 and R4, R13 and R14, and / or R1 and R2, and R11 and R12, and / or R7 and R9 together can form additional aromatic, heteroaromatic, aliphatic or heteroaliphatic rings.

[0022] c. At least one of the R1-R19 substituents has a solubilizing and / or ionizable or ionizable substituent, such as SO3 - , (-SO3H), PO3 2- COOH, OH or NR3 +The hydrophilicity of these polyacetylenic dyes is determined by cyclodextrins or sugars, where the substituent can also bind to the polyacetylenic dye via spacer groups.

[0023] d. At least one of the substituents R1-R19 has a reactive group (linker), such as isocyanate, isothiocyanate, hydrazine, amine, monochlorotriazine and dichlorotriazine or monobromotriazine and dibromotriazine, aziridine, epoxide, sulfonyl halide, acyl halide, carboxylic anhydride, N-hydroxysuccinimide ester, imide ester, carboxylic acid, glyoxal, acetaldehyde, maleimide or iodoacetamide and phosphoramidide derivative or azide, alkyne or olefin, wherein the substituent may also be linked to the polyacetylenic dye via a spacer group.

[0024] e. Aromatic, heteroaromatic, aliphatic, or heteroaliphatic spacers consisting of structural elements such as [(CH2)]. a -Y-(CH2) b ] c Or [(C6H4)] a -Y-(C6H4) b ] c The composition, wherein Y can be the same or different and includes CR2-, O-, S-, -SO2, SO2NH-, NR-, COO-, or CONR functional groups, wherein it is bonded to one of the R1-R19 substituents, and a.) and b.) can be the same or different and have values ​​of 0-18, and c has a value of 0-18.

[0025] f. R7, R8, and R9 substituents and / or R17 and R18 may also be present two, three, four, or five times, and these may be the same or different.

[0026] g. R15 and R16 and / or R5 and R6 can form additional alicyclic or heterocyclic systems.

[0027] Particularly preferred is if at least one of the R1-R19 substituents has a solubilizing and / or ionizable or ionizable substituent, such as SO3. - , (-SO3H), PO3 2- The hydrophilicity of these polyacetylenic dyes is determined by the presence of substituents such as COOH, OH, cyclodextrin, or sugars, where the substituent can also bind to the polyacetylenic dye via spacer groups. Furthermore, it is particularly preferred if the aromatic, heteroaromatic, aliphatic, or heteroaliphatic spacer group is composed of structural elements such as [(CH2)]. a -Y-(CH2) b ] c Or [(C6H4)] a -Y-(C6H4) b ]c The composition, wherein Y may be the same or different and includes CH2-, O-, S-, -SO2, SO2NH-, NH-, COO- or CONH or alkylated analogues, wherein the H atom is substituted by an alkyl chain C-alk2, O-, S-, -SO2, SO2N-alk, N-alk, COO- or CON-alk functional group, wherein it is bonded to one of the R1-R19 substituents, and a.) and b.) may be the same or different and have values ​​of 0-18, and c has a value of 0-18.

[0028] In a preferred embodiment, at least one polyacetylenic dye contained in the nanostructure delivery system can be characterized by at least one molecular descriptor. A “molecular descriptor” (or “molecular predictor”) is a value or coefficient relating to the defined properties of a molecule of interest, where the value is determined based on the symbolic representation of the molecule. Thus, molecular descriptors allow the representation of a molecule’s chemical information in a computer-interpretable numerical vector. In contrast, the actual physicochemical properties of a given molecule are derived from experimental measurements. In the context of this invention, the at least one molecular descriptor can be selected from the group comprising topological polar surface area, logP value, number of atoms, molecular weight, number of oxygen and nitrogen atoms, number of OH and NH (H bond donors), number of sulfonyl residues or other functional groups constituting a negative charge at pH 7.4, number of rotatable bonds, and molecular volume. Preferably, the topological polar surface area, defined as the sum of the surfaces of all polar atoms or molecules, primarily oxygen and nitrogen (including the hydrogen atoms they are connected to), can be 50 to Between, preferably between 100 and Between, or even more preferably, about As is well known, the logP coefficient is one of the main parameters for estimating the lipophilicity of a compound (determining its pharmacokinetic properties), and it can be between -3 and 7, preferably between 4 and 6.5. The number of atoms can be between 30 and 70, preferably between 40 and 60. The molecular weight can be between 400 and 1100 g / mol, preferably between 500 and 800, and most preferably between 650 and 750 g / mol. The number of oxygen and nitrogen atoms can be between 4 and 20, preferably between 7 and 12, while the number of OH and NH (H bond donors) can be between 2 and 8, preferably between 3 and 6. The number of sulfonyl residues or other functional groups that constitute a negative charge at pH 7.4 can be between 0 and 5, preferably between 0 and 3, and most preferably between 1 and 2. The number of rotatable bonds, i.e., the number of bonds that allow free rotation around themselves (defined as any single bond not in the ring bonded to a non-terminal heavy atom), can be between 5 and 30, preferably between 10 and 20. Molecular volume is the volume occupied by one mole of a substance (chemical element or compound) at a given temperature and pressure (equal to molar mass M divided by mass density ρ), and can range from 500 to... Between, preferably between 600 and between.

[0029] In a preferred embodiment, the polyacetylenic dye is a class II dye, n=1, and R1, R2, and R3 are heterocyclic groups fused with octahydro-2H-quinazine to form the 2,3,6,7-tetrahydro-1H,5H-benzo(ij)quinazine (also known as Juulolidine) moiety, and R6 is tert-butyl, R15 and R16 are methyl groups, R10 is a hexanoic acid residue, and R13 is a sulfonic acid residue. DY635:

[0030] In a preferred embodiment, the polyacetylenic dye is a class II dye, n=1, and wherein R1, R2, and R3 are heterocyclic groups fused with octahydro-2H-quinazine to form the 2,3,6,7-tetrahydro-1H,5H-benzo(ij)quinazine (also known as julonidine) moiety, and R6 is tert-butyl, R15 is methyl, R16 is a butyric acid residue, R10 is propane-1-sulfonic acid, and R13 is a sulfonic acid residue. 636

[0031] In a preferred embodiment, the polyacetylenic dye is a class II dye, n=1, and R2 is NH2, R6 is tert-butyl, R15 and R16 are methyl, R10 is a hexanoic acid residue, and R13 is a sulfonic acid residue. 615

[0032] In a preferred embodiment, the polyacetylenic dye is a class IA dye, n=1, and wherein R3 is diethylamino, R6 is tert-butyl, R15 and R16 are methyl, R10 is hexanoic acid residue, and R13 is sulfonic acid residue.

[0033] In a preferred form, the polyacetylenic dye is a Class I dye, n=1, and wherein R2 is diethylamino, R6 is tert-butyl, R15 and R16 are methyl, R10 is hexanoic acid residue, and R13 is sulfonic acid residue.

[0034] In a preferred form, the polyacetylenic dye is a Class II dye, n=1, and wherein R2 and R3 are heterocyclic groups fused with 3-[2,2-dimethyl-4-(sulfomethyl)pyridin-1(2H)-yl]propane-1-sulfonic acid to form the 3-[2,2-dimethyl-4-(sulfomethyl))quinoline-1(2H)-yl]propane-1-sulfonic acid moiety, R6 is phenyl, R15 is methyl, R16 is butyric acid residue, R10 is propane-1-sulfonic acid, and R13 is sulfonic acid residue.

[0035] In a preferred form, the polyacetylenic dye is a Class IIa dye, n=1, and wherein R2 is diethylamino, R6 is tert-butyl, R15 and R16 are methyl, R10 is hexanoic acid residue, and R13 is sulfonic acid residue.

[0036] In a preferred form, the polyacetylenic dye is a class IIa dye, n=1, and wherein R2 and R3 are heterocyclic groups fused with 1-ethyl-2,2,4-trimethyl-1,2-dihydropyridine to form the 1-ethyl-2,2,4-trimethyl-1,2-dihydroquinoline moiety, R15 and R16 are methyl groups, R6 is tert-butyl, R10 is a hexanoic acid residue, and R13 is a sulfonic acid residue.

[0037] In a preferred form, the polyacetylenic dye is a class IIa dye, n=1, and R1, R2 and R3 are heterocyclic groups fused with octahydro-2H-quinazine to form the 2,3,6,7-tetrahydro-1H,5H-benzo(ij)quinazine (also known as julonidine) moiety, and R6 is tert-butyl, R15 is methyl, R16 is butyric acid, R10 is propane-1-sulfonic acid, and R13 is a sulfonic acid residue.

[0038] In a preferred form, the polyacetylenic dye is a Class II dye, wherein R2 is diethylamino, R6 is tert-butyl, R15 is methyl, R10 is hexanoic acid, and R13 is a sulfonic acid residue.

[0039] In a preferred form, the polyacetylenic dye is a Class II dye, n=1, and wherein R2 is diethylamino, R6 is tert-butyl, R15 is methyl, R16 is butyric acid, R10 is propane-1-sulfonic acid, and R13 is a sulfonic acid residue.

[0040] In a preferred form, the polyacetylenic dye is a Class IIIa dye, n=1, and wherein R3 and R13 are sulfonic acid residues, R5, R6, R15 and R16 are methyl groups, R10 is a 2-methoxyethyl residue, and R17 is hexanoic acid.

[0041] In a preferred form, the polyacetylenic dye is a Class IIIa dye, n=1, and wherein R3 and R13 are sulfonic acid residues, R5, R6 and R15 are methyl groups, R16 is propane-1-sulfonic acid, R10 is a 2-methoxyethyl residue, and R17 is hexanoic acid.

[0042] In a preferred form, the polyacetylenic dye is a Class IIIa dye, n=1, and wherein R5, R6, R15 and R16 are methyl groups, R10 is a 2-methoxyethyl residue, R17 is hexanoic acid, and R13 is a sulfonic acid residue.

[0043] In a preferred embodiment, the polyacetylenic dye is a Class IIIa dye, n=1, wherein R3, R4 and R13, R14 are benzofused, R5, R6, R15, R16 and R10 are methyl, and R17 is hexanoic acid.

[0044] In a preferred form, the polyacetylenic dye is a Class IIIa dye, n=2, and wherein R7 and R9 are partially fused with cyclohexene, R8 is phenoxy, R3 and R4 are benzofused, R15 and R16 are methyl, R10 is hexanoic acid, and R17 is butane-1-sulfonic acid.

[0045] In a preferred form, the polyacetylenic dye is a class IIa dye, n=1, wherein R2 and R3 are heterocyclic groups fused with 3-[2,2-dimethyl-4-(sulfonylmethyl)pyridin-1(2H)-yl]propane-1-sulfonic acid to form the 3-[2,2-dimethyl-4-(sulfonylmethyl)quinoline-1(2H)-yl]propane-1-sulfonic acid moiety, R6 is phenyl, R15 is methyl, R16 is butyric acid residue, R10 is propane-1-sulfonic acid, and R13 is sulfonic acid residue.

[0046] In another embodiment, at least one polyacetylenic dye in the nanostructure delivery system of the present invention may be selected from the group consisting of: DY635, DY780, DY730, DY750, DY736, DY615, DY636, DY731, DY647P1, DY648P1, CY-E10 derivatives, CY5.5 derivatives, DY630, IRDye800; DY778.

[0047] In a preferred embodiment, the nanostructure delivery system may additionally comprise at least one active pharmaceutical ingredient; in a particularly preferred embodiment, the at least one active pharmaceutical ingredient may be selected from the group consisting of: nucleic acids, such as small RNA (e.g., miRNA, siRNA) or DNA (e.g., plasmids), oligonucleotides, aptamers, DNases; proteins; protacs (proteolytic targeting chimeras), corticosteroids; cell inhibitors, antimetabolites; intercalation substances; antibodies; interferons; contrast agents; protein kinase inhibitors, such as tyrosine kinase inhibitors or phosphatidylinositol-3-kinase inhibitors; antibiotics; antifungals; antivirals; anti-inflammatory drugs, such as COX inhibitors; glucocorticoids; cell growth inhibitors; mitochondrial uncoupling agents; protein biosynthesis inhibitors; energy metabolism inhibitors; respiratory chain inhibitors; growth factor inhibitors.

[0048] In a second aspect, the present invention relates to a pharmaceutical composition comprising the nanostructure delivery system described above, as well as suitable excipients and additives. In a preferred embodiment of the pharmaceutical composition, the composition is specifically adapted for inhalation.

[0049] In a third aspect, the present invention relates to a method for targeted delivery to adenocarcinoma tissue and / or adenocarcinoma cells, comprising contacting the adenocarcinoma tissue or adenocarcinoma cells in vitro with a nanostructure delivery system as described above. In a preferred embodiment of the method of the present invention, the tumor tissue may be prostate tissue.

[0050] In a fourth aspect, the present invention relates to the use of a nanostructure delivery system as described above or the use of a pharmaceutical composition as described above, wherein the accumulation of the nanostructure delivery system and / or its components can be detected in adenocarcinoma tissue and / or adenocarcinoma cells by the fluorescent properties of the at least one polymethystine dye.

[0051] Specific preferred embodiments of the present invention will become apparent from the following more detailed description of certain preferred embodiments. Attached Figure Description

[0052] Figure 1 An overview of polyacetylenic dyes classified into different categories based on their structure, and an overview of their physicochemical properties.

[0053] Figure 2 . Figure 1 The selected molecular descriptors for polyacetylenic dye amines are shown. Molecular descriptors were determined at pH 7.4 with deprotonation of sulfonic acid residues and protonation of primary aliphatic amines. (Counterions are negligible).

[0054] Figure 3 Different dyes can be coupled to PLGa using the procedure described in Example 2. Specifically, the polymer can be covalently bound to an amine-functionalized dye based on its active carboxylic acid group. Two Class I dyes (DY 680, DY 780) and four Class II dyes (DY 630, DY 635, DY 654, DY 778) are used as examples. The coupling of each dye moiety was monitored by size exclusion chromatography (SEC) using UV and reflectance index (RI) detection.

[0055] Figure 4 Nanoparticles of PLGA (e.g., DY635-PLGA) conjugated with corresponding polyacetylenic dyes were prepared under constant parameters and characterized using various experiments involving, for example, the size and charge of the synthesized particles. Dynamic light scattering was used to determine the size. A Zeta-Sizer was used to measure the electrophoretic signal in a charge measurement assay to determine the surface charge (zeta potential).

[0056] Figure 5. Four adherent tumor cell lines, DLD-1, HCC-78, DU-145, and MKN-45, and 293T cells as a negative control (a). OATP1B3 mRNA expression and protein levels were detected by RT-qPCR and Western blot, respectively (b, c). Cells were trypsinized and washed twice with PBS / 1% FCS. NP stock solution was diluted to 50 μg / mL in PBS / 1% FCS. Cells and NP were preheated at 37°C for at least 10 min. Then 5 x 10⁻⁶ cells were cultured.5 Cells were incubated with 200 μL of NP at 37°C for 5 min, then fixed with 4% paraformaldehyde and washed twice with PBS. Cells were then measured on a BD LSR Fortessa. NP uptake was determined by the emission of NP's cargo dye 5(6) carboxyfluorescein (FAM) relative to non-functionalized NP (NP[FAM]) (d, e, f). Cells were incubated with the dye at a concentration of 100 nM according to the NP protocol, and then measured on a BD LSR Fortessa (e, g). Detailed Implementation

[0057] The details shown herein are merely examples and are intended for the purpose of illustrative discussion of preferred embodiments of the invention, and are set forth to provide the most useful and readily understood description of the principles and concepts that are believed to be various embodiments of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a basic understanding of the invention; the accompanying drawings and / or the description of the examples enable those skilled in the art to understand how several forms of the invention are embodied in practice.

[0058] The following definitions and interpretations are intended to govern any future structures unless clearly and explicitly modified in the following examples, or when the application of such meaning renders any structure meaningless or substantially meaningless. If the structure of a term renders it meaningless or substantially meaningless, the definition shall be taken from the third edition of Webster's Dictionary or a dictionary known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

[0059] Example 1: Synthesis of Functionalized Polymers

[0060] The synthesized nanoparticles are based on the hydrophobic polymer polylactic-glycolic acid copolymer (PLGA), which is biocompatible and biodegradable. This polymer can be covalently bonded to amine-functionalized dyes, as previously described (EP2848262A1). In short, coupling agents such as EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) can be used due to the acid-terminated groups of the dye molecules. In this paper, polyacetylenic dyes DY635, DY630, DY615, and DY736 were used for most experiments (see [link to original text]). Figure 1). Each 100 polymer chains are functionalized. Specific polyacetylenic dyes are selected according to predetermined molecular descriptors; in Figure 2 In the middle, it is described Figure 1 Molecular descriptors for polyacetylenic dye amines. Molecular descriptors were determined at pH 7.4 with deprotonation of sulfonic acid residues and protonation of primary aliphatic amines. Counterions were neglected. Molecular descriptors were selected from a group including logP value, topological polar surface area, number of atoms, molecular weight, number of oxygen and nitrogen atoms, number of OH and NH (H bond donors), number of sulfonyl residues or other functional groups constituting a negative charge at pH 7.4, number of rotatable bonds, and molecular volume. Topological polar surface area was defined as the total surface area of ​​all polar atoms or molecules, primarily oxygen and nitrogen (including the hydrogen atoms they are bonded to). The logP coefficient is well known as one of the main parameters for estimating the lipophilicity of a compound (determining its pharmacokinetic properties). The number of rotatable bonds is the number of bonds that allow free rotation around themselves (defined as any single bond not in a ring bonded to a non-terminal heavy atom). Molecular volume is the volume occupied by one mole of a substance (chemical element or compound) at a given temperature and pressure (equal to the molar mass M divided by the mass density ρ).

[0061] Example 2: Production of Nanoparticles

[0062] Following polymer functionalization, nanoparticles were prepared using simple (A) and bis (B) emulsions, as previously described (EP2848262A1). In short, high-frequency ultrasound was used to form nanoparticles in the presence of a surfactant. A hydrophobic polymer was dissolved in ethyl acetate. The polymer suspension, along with the surfactant, was added to water, and nanoparticles (A) were formed by ultrasound. If a hydrophilic substance was to be included, it was dissolved in water, added to the polymer in ethyl acetate, and sonicated. Subsequently, water mixed with the surfactant was added, and nanoparticles (B) were formed by ultrasound. The resulting nanoparticles were then stirred under an airflow until all the organic solvent (ethyl acetate) evaporated and the particles were thus stable in water. To remove excess surfactant, the nanoparticles were thoroughly washed at least twice with ultrapure water. Finally, the particles were lyophilized and their mass was determined.

[0063] Example 3: Characterization of nanoparticles

[0064] Nanoparticles of various DY-conjugated PLGA particles were prepared, such as DY635-conjugated PLGA (DY635-PLGA-NP), DY654-conjugated PLGA (DY654-PLGA-NP), DY678-conjugated PLGA (DY678-PLGA-NP), DY680-conjugated PLGA (DY680-PLGA-NP), DY778-conjugated PLGA (DY778-PLGA-NP), DY780-conjugated PLGA (DY780-PLGA-NP), Cy5-conjugated PLGA (Cy5-PLGA-NP), CyE10-conjugated PLGA (CyE10-PLGA-NP), and DY647P1-conjugated PLGA (DY647P1-PLGA-NP), and were replicated with constant parameters. The determination methods used are as follows:

[0065] Size: by dynamic light scattering (e.g., Zetasizer (Malvern Instruments GmbH)) or electron micrographs (see...) Figure 4 The size of various nanostructured carrier systems dissolved in deionized water was measured. Alternatively, the size was determined by size exclusion chromatography (SEC) combining UV and reflectance index (RI) detection. The SEC elution plots of the exemplary dyes described above are shown below. Figure 3 As shown.

[0066] Shape: The shape was determined using electron microscopy.

[0067] Charge: Various nanostructured carrier systems dissolved in deionized water were measured by measuring electrophoretic signals (ζ-potential, surface charge) at Zetasizer (Malvern Instruments GmbH). See [link to Zetasizer documentation]. Figure 4 .

[0068] Endotoxin: Endotoxin was determined by LAL colorimetric assay (Guilfoyle, DE et al., Evaluation of achromogenic procedure for use with the Limulus lysate assay of bacterial endotoxin in drug products. J Parenter Sci Technol, 1985, 39(6): pp. 233-6).

[0069] Hemolysis: The hemoglobin concentration of red blood cells incubated with particles in physiological buffer for 1 hour was measured. If the red blood cell membrane is damaged, the measurable hemoglobin concentration in the supernatant will increase.

[0070] Aggregation: The uptake of erythrocytes incubated with polymers in physiological buffer was measured. Cell aggregate samples showed lower uptake than uniformly distributed non-aggregated cells.

[0071] Example 4: RNA isolation

[0072] RNA was isolated from cultured cell lines using the InnuPREP RNA Mini Kit (Analytik Jena AG, Jena, Germany). This step was performed following the manufacturer's instructions. In short, after removing cells from the culture flask, they were washed once with 10 ml of D-PBS and then centrifuged at 1000 rpm for 5 minutes at room temperature. Cells were lysed with 450 μl of lysine solution containing guanidine thiocyanate at a maximum of 5 x 10⁻⁵ ppm. 6 The lysate was collected from individual cells. The lysate was then transferred to a SpinFilter D in a 2 ml receiving tube and centrifuged at 12,000 rpm for 2 minutes at room temperature for selective removal of gDNA. 400 μl of 70% ethanol was added to the homogenate and thoroughly mixed by repeated pipetting. The entire volume was then transferred to a SpinFilter R in a 2 ml receiving tube and centrifuged at 12,000 rpm for 2 minutes at room temperature for selective RNA binding on the filter. This tube was discarded, and SpinFilter R was placed in a new 2 ml receiving tube. 500 μl of wash solution HS was added to the filter and centrifuged at 12,000 rpm for 1 minute at room temperature. SpinFilter R was then placed back into a new 2 ml receiving tube, and 700 μl of wash solution LS was added to the filter and centrifuged at 12,000 rpm for 3 minutes at room temperature. The RNeasy spin column was then placed in a 1.5 ml capped elution tube. For elution, 30 μl of RNase-free water was transferred to filter column R, incubated at room temperature for 1 minute, and centrifuged at 8,000 rpm for 1 minute. The RNA concentration in the elution buffer was then determined using NanoDrop, and cDNA synthesis was performed directly.

[0073] Example 5: cDNA Synthesis

[0074] For efficient cDNA synthesis, the RNA was initially prepared to a concentration of 500 ng / μL by diluting it with RNase-free water (Analytik Jena AG). -1RNA was extracted. In each case, 7.7 μL of RNA elution buffer was pipetted into a 1.5 mL Eppendorf tube with 4 μL of dNTP mixture (Fermentas) and 1 μL of random hexameric primers (ThermoScientific), and then incubated at 65 °C for 5 min in a thermal cycler (Eppendorf, Thermomixercomfort). Subsequently, 4 μL of reaction buffer (5×First Strand Buffer, Invitrogen), 2 μL of 0.1 M DTT (Invitrogen), and 0.5 μL of RNaseOut were pipetted into the tube. TM Recombinant (40 U / ml) -1 A ribonuclease inhibitor was added to the sample, and the sample was incubated in a thermal cycler at 37°C for 2 minutes. Then, 0.8 μL of M-MLV reverse transcriptase (200 U μL) was added to the reaction. -1 The mixture was thoroughly mixed by vortexing and briefly centrifuged (Eppendorf 5415D benchtop centrifuge). cDNA synthesis was then performed in a hot mixer (Eppendorf). Initially annealed at 25°C for 10 minutes, followed by extension at 37°C for 60 minutes, and inactivated at 70°C for 15 minutes. The resulting cDNA was then stored at -20°C.

[0075] Table E5: Reagents used for cDNA synthesis

[0076] reagents concentration company M-MLV reverse transcriptase <![CDATA[200UμL -1 ]]> Invitrogen, Karlsruhe, Germany Random hexamer primers <![CDATA[0.2μgμL -1 ]]> Thermo Scientific, Darmstadt, Germany dNTP mixture 2.5mM Fermentas, St. Leon-Rot, Germany reaction buffer 5 times Invitrogen DTT 0.1M Invitrogen <![CDATA[RNaseOUT TM ]]> <![CDATA[40UμL -1 ]]> Invitrogen

[0077] Example 6: Real-time quantitative PCR (qRT-PCR)

[0078] To reliably detect altered gene expression, qRT-PCR can be used, as described below. For vector protein expression analysis using the density map shown in Figure 5b, all operations were performed under UV sterilization on a PCR bench (Peqlab, Biotechnology GmbH). A total reaction solution volume of 20 μL (10 μL) was prepared. 480 μL of SYBR Green I reaction mixture (Roche Diagnostics) + 8 μL of sterile PCR water + 1 μL of primer mixture diluted 1:10 + 1 μL of cDNA was added to a 96-well plate (Twin.tec PCR Plate 96, half-skirt, blue, Eppendorf). The wells were then sealed with flat cap strips (Eppendorf) and the 96-well plate was briefly centrifuged (centrifuge 5810R, Eppendorff). PCR was performed on an epgradation S realplex4 (Eppendorf AG, Hamburg, Germany). Between final extension and cooling, the products were slowly melted to 95°C to assess PCR product quality using melting profiles. Samples were run in triplicate on 96-well plates, and ΔC was used. t The gene expression of the housekeeping gene β-glucuronidase (β-GUS) was standardized using a method. 2 -△△Ct The method compares gene expression levels in suspension cell lines with those in HepaRG cells.

[0079] Table E6.1: Parameters for PCR Steps

[0080]

[0081] Table E7.2: Sequences of PCR Primers

[0082]

[0083] Example 7: Dye and Nanoparticle Experiments

[0084] Four adherent tumor cell lines—DLD-1, HCC-78, DU-145, and MKN-45—and 293T cells were used as a negative control. A 100 nM dilution of each dye was prepared by diluting the stock solution with D-PBS. Subsequently, 5 × 10⁶ cells were subjected to... 5 Cells were trypsinized and pipetted 100 μl into 1.5 ml tubes, and stored at 37°C in a thermal cycler (Thermomixer Comfort, Eppendorf) and on ice at 4°C for at least 10 min. Cells were then simultaneously incubated in the dark at 37°C and 4°C with 100 μl of different concentrations of dye for 5 min, followed by fixation with 4% paraformaldehyde for 15 min. Samples were centrifuged at 1000 rpm for 5 min (Eppendorf centrifuge 5415C), and washed twice with 1 ml D-PBS + 1% BSA after discarding the supernatant. The pellet was then resuspended in 500 μl D-PBS + 1% BSA, transferred to FACS tubes, and measured after short vortexing in a BDL-SFR Ortessa. For nanoparticle (NP) experiments, 5 × 10⁶ cells per 100 μl were prepared with preheated RPMI + 10% FCS. 5 Cells were seeded at 100 μl per well in two 24-well plates to allow for incubation of nanoparticles at 4°C and 37°C. The nanoparticles were then incubated by adding 100 μg of water per ml. -1 MilliQ aqueous dilution, from NP stock solution (10 mg / ml each) -1Prepared in [a specific process], so that after applying 100 μl of a cell suspension to each well, 50 μg / ml can be achieved. -1 The incubation concentration was determined by incubating cells on ice at 4°C and in the dark at 37°C in a heat mixer for 5 minutes. Cells were then transferred to FACS tubes containing 250 μl of D-PBS and immediately measured in BD LSR Fortessa. sequence list <110> Smart Deliverley LLC <120> Nanostructure delivery systems for tumor therapy <130> 3141-2-PCT <140> EP 20217124.5 <141> 2020-12-23 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 1 gtccagtcat tggctttgca 20 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 2 caacccaacg agagtcctta gg 22 <210> 3 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 3 agaaacgatt gcagggtttc ac 22 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 4 ccgagtgaag atcccctttt ta 22

Claims

1. Use of a nanostructure delivery system in the preparation of a medicament for treating a subject with adenocarcinoma, the nanostructure delivery system comprising: at least one polymer and / or at least one lipid; at least one polymethystyl dye; and at least one active pharmaceutical ingredient, wherein the adenocarcinoma is a cancer selected from the group consisting of gastrointestinal cancer, lung cancer, prostate cancer, breast cancer, and colon cancer and includes adenocarcinoma cells having altered gene expression in one or more SLCO1B3 genes, wherein the altered gene expression refers to changes in protein synthesis of one or more SLCO1B3 genes present in adenocarcinoma cells of the tissue, but not present or present to less than the same extent in normal non-adenocarcinoma cells of the tissue, wherein the at least one polymethystyl dye mediates the targeted delivery of the nanostructure delivery system to the adenocarcinoma cells via the expression products of the one or more SLCO1B3 genes, and The at least one polyacetylenic dye is selected from the group consisting of: DY635, DY730, DY736, DY615, DY636, DY731, DY647P1, DY648P1, E10-07066, CY5 amine, DY630, IRDye800 and DY778, and The at least one active pharmaceutical ingredient is selected from the group consisting of: nucleic acids, proteins, proteolytic targeted chimeras, corticosteroids, cell inhibitors, antimetabolites, intercalation substances, antibodies, interferons, contrast agents, protein kinase inhibitors, antibiotics, antifungal drugs, antiviral drugs, anti-inflammatory drugs, glucocorticoids, cell growth inhibitors, mitochondrial uncoupling agents, protein biosynthesis inhibitors, energy metabolism inhibitors, respiratory chain inhibitors, and growth factor inhibitors.

2. The use according to claim 1, wherein the at least one polyacetylene dye mediates the targeted delivery of the nanostructure delivery system to adenocarcinoma stem cells.

3. Use of the pharmaceutical composition in the preparation of a medicament for treating a subject with adenocarcinoma, said pharmaceutical composition comprising the nanostructure delivery system used in claim 1, and suitable excipients and additives, wherein said adenocarcinoma is a cancer selected from the group consisting of gastrointestinal cancer, lung cancer, prostate cancer, breast cancer, and colon cancer and includes adenocarcinoma cells having altered gene expression in one or more SLCO1B3 genes, wherein said altered gene expression refers to changes in protein synthesis in one or more SLCO1B3 genes present in adenocarcinoma cells of said tissue, but not present or present to less than the same extent in normal non-adenocarcinoma cells of said tissue.

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