Compositions and methods for the treatment of cancer

Abstract

Compositions and methods for treating diseases associated with dysregulation of miR-21, particularly cancer, are disclosed.

Description

Technical Field

[0001] The present invention relates to the fields of oncology and medicinal chemistry. More specifically, it provides compositions comprising complementary microRNA-21 (miR-21) oligonucleotide analogs that inhibit the binding of miR-21 and its isomiRs to miR-21 binding sites within target mRNAs (including, but not limited to, mRNAs encoding tumor suppressor proteins and other mRNAs encoding proteins involved in cell proliferation, migration, metastasis, stress, and inflammation). The oligonucleotide analogs complementary to miR-21 are conjugated to ligands that induce uptake into cancer cells. Thereby, methods for regulating the expression of such proteins and providing therapeutic effects are disclosed.

[0002] Cross-reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 490,184, filed Mar. 14, 2023, the entire content of which is incorporated herein by reference as if fully set forth. Statement Regarding Federally Sponsored Research and Development

[0003] The present invention was made under government support in accordance with Grant No. 1 R41 CA 235707-01A1 awarded by the NIH. The government has certain rights in the invention. Incorporation by Reference of Materials Submitted in Electronic Format

[0004] The content of the electronic sequence listing (BDT-102.xml; size: 23,436 bytes; date: Feb. 14, 2023) is incorporated herein by reference in its entirety.

Background Art

[0005] In this specification, a number of documents and patent documents are cited to explain the state of the art related to the present invention. Each of these cited documents is incorporated herein by reference as if its full text were set forth.

[0006] Triple-negative breast cancer (TNBC) is a rare disease affecting 46,000 women in the United States each year, primarily young women of African American, Asian, Hispanic, or BRCA1 mutation ancestry. TNBC cells lack the human estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor 2 (Her2), which are targets for many existing drugs. Recent treatment options for TNBC include anti-angiogenic agents, poly(ADP-ribose) polymerase (PARP) inhibitors, checkpoint inhibitors, and antibody-drug conjugates (ADCs). However, the FDA revoked approval for the anti-angiogenic agent bevacizumab for breast cancer due to poor clinical results and safety concerns. Similarly, Roche voluntarily recalled its anti-PD-L1 antibody Tecentriq for TNBC due to poor treatment outcomes. The recently approved antibody-drug conjugate Troderbi has only extended overall survival for TNBC by a mere three months. Furthermore, only a small fraction of patients with BRCA1 / 2 mutations benefit from PARP inhibitors, and the effectiveness of checkpoint inhibitors in TNBC patients with elevated PD-L1 expression is limited. Therefore, despite numerous side effects and a median survival of 4 years, chemotherapy and radiotherapy remain the standard of treatment.

[0007] MicroRNAs (miRNAs) and their isomias are small, non-coding RNA molecules, approximately 22 base pairs long, that regulate gene translation through the silencing and degradation of target mRNA. They are involved in diverse biological processes such as differentiation, proliferation, metabolism, hemostasis, apoptosis, and inflammation, and are also involved in the pathophysiology of many diseases. Numerous studies have suggested that circulating miRNAs are promising diagnostic and prognostic biomarkers for various diseases. Triple-negative breast cancer (TNBC) cells express high levels of oncogenic miRNAs (oncomiRs). These are 18-25 nucleotide (nt) non-protein-coding RNAs that form base pairs with specific sequences within mRNA. They suppress mRNA expression through steric effect-mediated mRNA translation inhibition or Ago2-mediated mRNA degradation induction. All miRNA biosynthesis is initiated in the nucleus, and primary miRNAs are transcribed by RNA polymerase II or RNA polymerase III. The primary miRNA transcript is then processed by Drosha and its cofactor DGCR8 to produce a short precursor miRNA hairpin of approximately 70 nt. This precursor miRNA hairpin is exported to the cytoplasm by export-in 5 and cleaved by Dicer to produce a double-stranded miRNA. The guide strand of the double-stranded miRNA is thought to form a weak hydrogen bond at its 5' end, facilitating binding to Ago2 within the RNA-induced silencing complex (RISC), allowing the guide strand to be active against complementary mRNA. Therapeutic targeting of such oncomia can circumvent the heterogeneity of cancer cells because a single miRNA can simultaneously regulate different target mRNA molecules, thereby modulating the expression and function of multiple gene networks.

[0008] Existing treatment options have limitations in scope and various drawbacks, so there remains a technical need for compositions, methods, protocols, and kits that offer higher efficacy, specificity, and safety in molecular targeted therapy for triple-negative breast cancer (TNBC). This invention addresses this need. [Overview of the Initiative]

[0009] The present invention provides compositions and methods for the treatment of triple-negative breast cancer. In one embodiment, a miR-21 inhibitory nucleic acid-peptide analog is provided that is sequence-complementary to miRNA-21-5p and isolates miR-21-5p and its isomia from binding to a regulatory site present in mRNA. In a particular embodiment, the miR-21 inhibitory nucleic acid-peptide analog comprises modifications selected from BNA, LNA, FANA, PNA, 2'-fluoro, 2'-O-alkyl, morpholino, piperazine, phosphorothioate, boranophosphate, and boranophosphate combined with a phosphodiester bond, phosphorodithioate bond, or methylphosphonate bond. In another embodiment, the miR-21 inhibitory nucleic acid-peptide analog comprises at least one inhibitory sequence shown in Figure 2B or 2C, e.g., SEQ ID NOs. 6-25, and the 9mer and 8mer sequences shown. In a preferred embodiment, the miR-21 inhibitory peptide comprises SEQ ID NO. 5. In preferred embodiments, the inhibitory sequence includes at least one, preferably two, 5' and 3' BNA modifications, as shown in Figure 15. In some embodiments, the antisense sequence includes a deoxyribonucleic acid (DNA) moiety and an aminomethyl BNA 2'4'-BNA NC The antisense sequence includes, but is not limited to, at least one cross-linked nucleic acid (BNA) moiety. For example, in some embodiments, the antisense sequence is a gapmer having a BNA-DNA-BNA structure. In some embodiments, each of the BNA moiety and the DNA moiety contains the same number of nucleotides. For example, in one embodiment, the antisense sequence contains 15 nucleotides having a 5-5-5 gapmer structure. Alternatively, in some embodiments, the DNA moiety contains a different number of nucleotides compared to at least one of the BNA moieties. For example, in one embodiment, the antisense sequence contains 13 nucleotides having a 4-5-4 gapmer structure.

[0010] miR-21 inhibitory peptide analogs may also include cyclic peptides selected from CSKC, CRKC, CVKC, CGKC, CKGC, CFKC, CDKC, CHRC, CRVC, CGRC, CIRC, CQRC, CTRC, CRHC, CRGC, CRSC, CRKC, CSRC, or CERC, where all residues are D-amino acids and a disulfide bond is formed between the N-terminal cysteine ​​and the C-terminal cysteine.

[0011] miR-21 inhibitory peptide analogs may also include CSKC, a peptide ligand for the insulin-like growth factor 1 receptor (SEQ ID NO: 26).

[0012] Methods are also provided for inhibiting the binding of miR-21 to one or more binding sites in mRNA encoding proteins that regulate cell proliferation, migration, metastasis, stress, or inflammation. An exemplary method comprises contacting miR-21-5p with an inhibitor described herein, wherein the inhibitor binds to and sequestrates the miR-21-5p, thereby inhibiting the binding of miR-21-5p to regulatory sites present in mRNA encoding proteins that regulate the proliferation and / or metastasis of malignant cells. In a preferred embodiment, a method for treating triple-negative breast cancer (TNBC) in a patient requiring it is disclosed. An exemplary method comprises administering an effective amount of a miR-21 inhibitory peptide analog as described above, wherein the analog induces stasis or cell death in the TNBC. In a preferred embodiment, the analog is shown in Figure 15.

[0013] The methods of the present invention also include rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, sorafenib, bementinib, crizotinib, bosutinib, gilteritinib, amvatinib, and sunitinib, cabozantinib, foretinib, levastinib, cerastrol, dihydroartemisinin, PD-1 inhibitor, PD-L1 inhibitor, CTLA4 inhibitor, cyclophosphamide, ifosfamide, and thio This includes the administration of chemotherapeutic agents selected from tepa, methotrexate, mercaptopurine, fluorouracil and cytarabine, bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin, mitoxantrone, vincristine, etoposide, teniposide, paclitaxel and docetaxel, cisplatin, carboplatin and oxaliplatin, uprolide, tamoxifen, flutamide and formestane, and arsenic trioxide.

[0014] The present invention also provides compositions and methods for effectively reducing or increasing disease-driven protein levels to provide therapeutic benefits to treated patients (see Figure 1). In other embodiments, the compositions and methods target the expression of tumor suppressor proteins. In other embodiments, the tumor suppressor genes are selected from PTEN and PDCD4. [Brief explanation of the drawing]

[0015] [Figure 1] Figures 1A-1J: Figure 1A) Schematic diagrams showing many different signaling and disease pathways that provide targets for the miR-21 targeted drugs described below. Figures 1B-1J) Schematic diagrams of various ligands for the delivery of complementary miR-21 oligonucleotide analogs described in the present invention. [Figure 2]Figures 2A-2D: (Figure 1A) miR-21 pre-miRNA hairpin structure (SEQ ID NO: 1). (Figures 2B-2C) miR-21 oligonucleotide sequences. The pre-miRNA hairpin structure includes a miR-21-5p guide strand (upper magenta sequence) and a miR-21-3p passenger strand (lower magenta sequence). Most nucleotides in the miR-21-5p guide strand are complementary to the miR-21-3p passenger strand. Nucleosides to be excluded in the passenger strand seed sequence are shown in blue in SEQ ID NO: 4. Either the guide strand or the passenger strand may be selected as the active miRNA in the RNA-induced silencing complex (RISC). SEQ ID NOs: 5-25 and three 9mers and three 8mers provide further anti-miR-21-5p sequences of 8mer-17mer length, which should efficiently hybridize to the target. SEQ ID NO: 5, which is a 15-mer, is shown in bold in Figure 2B. (Figure 2D) Schematic diagram of the miR-21 oligonucleotide-peptide blockade mechanism. miR-21 inhibitory nucleic acids bind to the mature miR-21-5p guide strand, preventing binding to its regulatory site in the 3'UTR of the target mRNA. [Figure 3] Figure 3: Schematic diagrams of DNA / RNA analogs that enhance stability, binding affinity, and specificity. DNA phosphorothioates (PS), methylphosphonates (MP), 2'-4'-locked nucleic acids (LNA), 2'-aminomethylene-bridged nucleic acids (NC-BNA), and polyamide nucleic acids (PNA). [Figure 4] Figures 4A-4B: (Figure 4A) Structure of BND5412, a 15nt miR-21 blocker BNA-DNA-BNA phosphorothioate. The short BNA sequence avoids mimicking the passenger strand because it lacks the corresponding miR-21 passenger strand seed sequence. (Figure 4B) IC50 of the miR-21 blocker BND5412 transfected into human MDA-MB-231 TNBC cells to induce disinhibition of a sea urchin luciferase reporter vector with a miR-21 binding site inserted into the 3'UTR of the luciferase gene. Error bars, sd [Figure 5]Figures 5A-5B: (Figure 5A) Western blot analysis of protein expression ± sd after transfection of human MDA-MB-231 TNBC cells with 50 nM miR-21 blocker BND5412 for 48 hours (PDCD4) or 72 hours (other proteins). (Figure 5B) Western blot of immune checkpoint proteins 72 hours after transfection of human HCC1806 TNBC cells with 50 nM miR-21 blocker BND5412. [Figure 6] Figure 6: Cell titer Glo assay ± sem after transfecting seven different TNBC cell lines with 50 nM miR-21 blocker BND5412 on day 0. [Figure 7] Figure 7: Graph comparing FANA oligonucleotides with three known BNA oligonucleotide sequences. FANA sequences 1-7 did not show significant inhibition compared to scrambled BNA or vehicle control. Cell viability was measured by Cell-Titer Glo assay. Error bars, sd, n=3. [Figure 8] Figure 8: Correlation between miR-21 blocker BND5412 cell proliferation IC50 and miR-21 copies / cell in seven human TNBC cell lines transfected with a concentration gradient of miR-21 blocker BND5412 versus non-tumorogenic mammary epithelial cell lines. [Figure 9] Figure 9: LDH assay ± sd 72 hours after transfection with 50 nM miR-21 blocker BND5412 to investigate apoptosis in human MDA-MB-231 TNBC cells. [Figure 10] Figure 10: qPCR mRNA levels, 3 biological replications, ±sem of PD-L1, PD-L2, CD47, and JAK2 mRNA against GAPDH, extracted from human HCC1806 TNBC cells transfected with vehicle, 50 nM scrambled blocker (purple), or 50 nM miR-21 blocker BND5412 (blue). [*=p<0.05, **=p<0.01, vs vehicle control, 1-way t-test]. [Figure 11]Figures 11A - 11B: (Figure 11A) Cumulative frequency distribution of miR-21 target genes (red) and all other genes with significant differential expression (blue) in RNA-seq analysis from 6 biological replicates of human HCC1806 TNBC cells transfected with miR-21 blocker BND5412 at the IC90 concentration, [Kolmogorov-Smirnov test, p < 0.0001]. (Figure 11B) Top 3 pathways of genes with different expression from samples treated with miR-21 blocker. [Figure 12] Figure 12: Design of conjugate miR-21 blocker of IGF1 peptide and BNA-DNA-BNA gapmer targeting endocytosis by cell surface IGF1R, which is highly expressed in TNBC cells. [Figure 13] Figure 13: Structure of BND7673, conjugate of AF647-labeled miR-21 blocker BNA-DNA-BNA phosphorothioate and IGF1 peptide for IGF1R-mediated endocytosis. The short BNA sequences lack the seed sequences of the corresponding miR-21 passenger strands, thus avoiding mimicking the passenger strands. [Figure 14] Figure 14: Confocal fluorescence images of live human HCC1806 TNBC cells after 4 hours in 100 nM Cal560-miR-21 BNA-IGF1 peptide. Green: LysoTracker. Red: Cal560. Yellow: Overlap of Cal560-LysoTracker. [Figure 15] Figure 15: Structure of miR-21 blocker BNA-DNA-phosphorothioate-IGF1 peptide, BND6482, for endocytosis via IGF1R. The short BNA sequences lack the seed sequences of the corresponding miR-21 passenger strands, thus avoiding mimicking the passenger strands. [Figure 16]Figures 16A - 16B: Dose - dependent inhibition of miR - 21 activity by 50 nM miR - 21 blocker BND6482 without lipofection (blue) compared to miR - 21 blocker BND6482 transfected at 50 nM (red) in high IGF1R - expressing HCC1806 cells (Figure 16A) and low IGF1R - expressing MDA - MB - 157 cells (Figure 16B) was measured by miR - 21 luciferase reporter assay. Error bars, s.d. [Figure 17] Figure 17: Distribution of fluorescent miR - 21 blocker BND767 over 2 - 96 hours after IP injection at 5 mg / kg into orthotopic grafts in the mammary fat pads of immunodeficient female Balb / c mice generated using mouse EMT6 TNBC cells. [Figure 18] Figure 18: In the TBC mouse model described in Figure 17, tumor growth was inhibited as seen in the white - light image (left) or fluorescent image (right) of dissected tumors after IP injection of fluorescent anti - miR - 21 - peptide BND7673 at 5 mg / kg once a day for 3 days. [Figure 19] Figure 19: Tumor growth was inhibited over 4 days in the TBC mouse model described in Figure 17 after IP injection of fluorescent anti - miR - 21 blocker peptide BND7673 at 5 mg / kg once a day for 3 days. Individual tumor volumes are shown as mean values and S.E.M. [Figure 20] Figure 20: miR - 21 and PDCD4 mRNA levels in tumors of female Balb / c mice with syngeneic mouse EMT6 TNBC grafts after administration of 5 mg / kg of fluorescent miR - 21 blocker peptide BND7673 three times a day. Error bars indicate S.E.M. [Figure 21] Figure 21: Tumor volumes of mouse EMT6 TNBC orthotopic grafts remained small after IP injection of 5 mg / kg of miR - 21 blocker peptide BND6482 twice a week for 13 days. Growth continued with vehicle, scrambled drug, and toradelbine. Error bars indicate mean and SEM. *p < 0.05 (by one - way ANOVA, Dunnett's multiple - comparison test). [Figure 22]Figure 22: After intravenous injection of 5 mg / kg of miR-21 blocker peptide BND6482 twice weekly for 13 days, the tumor volume of mouse EMT6 TNBC orthotopic grafts was significantly reduced. Error bars indicate mean and SEM. *p<0.05 (one-way ANOVA, Dunnett's multiple comparison test). [Figure 23] Figure 23: Toxicity markers in serum samples taken from mice with tumors after receiving intravenous injections of 5 mg / kg of miR-21 blocker peptide BND6482 twice a week for 13 days, or three intravenous injections of 6.25 mg / kg. Error bars indicate mean and SEM. *p<0.05 (one-way ANOVA, Dunnett's multiple comparison test). Detailed description of the invention

[0016] This specification describes the design and synthesis of miR-21-targeted therapeutics. miR-21 is a 22nt single-stranded RNA that is elevated in TNBC compared to adjacent normal tissue (Radojicic et al. 2011). Overexpression of miR-21 is universally observed in breast cancer cell lines and tissues (Ozgun et al. 2013) and affects cell proliferation, cell cycle checkpoints, and metastasis (Anastasov et al. 2012). miR-21 expression in TNBC is also correlated with poor clinical outcomes (Dong et al. 2014). The passenger strand, miR-21-3p, is upexpressed in TNBC and associated with chemotherapy resistance (Ouyang et al. 2014). Previously, 19mer anti-miR-21 nucleoside analogs with specific scaffold modifications were patented for use in the treatment of liver disease. Its use in the treatment of breast cancer is not disclosed (Bhat and Marcusson 2015).

[0017] definition Before further describing the present invention in general terms and in terms of various non-limiting specific embodiments, the following terms are used in the context describing the present invention. Unless otherwise indicated, the following terms have the following meanings when used in this specification and the appended claims. Terms not defined below or elsewhere in this specification have the meanings recognized in the art.

[0018] In this invention, the term "pharmacological activity" refers to the inherent physical properties of a peptide or polypeptide. These properties include, but are not limited to, half-life, solubility, stability, and other pharmacokinetic properties.

[0019] The words "high," "higher," "increase," "rise," or "rise" mean, for example, an increase above the baseline level compared to a control. The words "low," "decrease," "decrease," or "decrease" mean, for example, a decrease below the baseline level compared to a control.

[0020] As used in this invention, the term “modulate” refers to the ability of a compound to alter its activity in some measurable way compared to a suitable control. As a result of the presence of a compound in an assay, the activity may increase or decrease compared to a control in the absence of the compound. Preferably, the increase in activity is at least 25%, more preferably at least 50%, and most preferably at least 100%, compared to the activity level in the absence of the compound. Similarly, the decrease in activity is preferably at least 25%, more preferably at least 50%, and most preferably at least 100%, compared to the activity level in the absence of the compound. A compound that increases known activity is an “agonist.” A compound that decreases or inhibits known activity is an “antagonist.”

[0021] The word "inhibit" means to reduce or decrease activity or expression. This can be complete inhibition or partial inhibition. Inhibition can be compared to a control or standard level. Inhibition is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 It could be 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0022] As used in this invention, the term "prevent" refers to administering a compound before the clinical symptoms of a disease or condition appear, in order to prevent the physical manifestation of abnormalities associated with the disease or condition.

[0023] As used in this invention, "treatment is necessary" means that a caregiver (for example, a physician, nurse, nurse practitioner, or individual in the case of a human; or a veterinarian in the case of an animal, including non-human mammals) determines that the subject needs treatment or would benefit from treatment. This determination is within the scope of the caregiver's expertise and is based on various factors, including knowledge of whether the subject is ill or will become ill as a result of a condition treatable by the disclosed compound.

[0024] "Treatment" and "to treat" mean the medical management of a subject for the purpose of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward the improvement of a disease, pathological condition, or disorder, and also causal treatment, i.e., treatment directed toward the removal of the cause of the associated disease, pathological condition, or disorder. Furthermore, this term includes palliative treatment, i.e., treatment aimed at alleviating symptoms rather than curing the disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing, or partially or completely suppressing, the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy aimed at improving the associated disease, pathological condition, or disorder. The effects of treatment can be measured or evaluated as described herein and as known in the art, as appropriate for cancer and related conditions.

[0025] A "cell" can be the cell of any living organism, including but not limited to bacteria. A cell may be in vitro, or it may be in vivo and found in the object.

[0026] "Pharmacologically acceptable" means a material that can be administered to a subject together with a selected compound without causing any biological or otherwise undesirable material, i.e., without causing any undesirable biological effects or harmful interactions with other components of the pharmaceutical composition in which it is contained.

[0027] "Percent (%) sequence identity" and "homology" for nucleic acid sequences, peptides, polypeptides, or antibody sequences are defined as the percentage of nucleic acid residues or amino acid residues in a candidate sequence that are identical to a given nucleic acid residue or amino acid residue in a given nucleic acid sequence or polypeptide sequence, after the sequences have been aligned and gaps introduced, if necessary, to achieve maximum percentage sequence identity, and conservative substitutions are not considered as part of sequence identity. Alignment for determining the sequence identity percentage can be achieved in various ways within the scope of the art using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0028] In the present invention, the terms "component," "composition," "compound composition," "compound," "agent," "pharmacological activator," "activator," "therapeutic agent," "therapy," "treatment," or "pharmaceutical product" are interchangeable in the present invention and refer to compounds or compound or substance compositions that, when administered to a subject (human or animal), induce desired pharmacological and / or physiological effects through local and / or systemic effects. The terms "agent" and "test compound" refer to chemical compounds, mixtures of chemical compounds, biological polymers, or extracts made from biological materials such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues.

[0029] "Administering," "administering," or "to administer" means providing a substance to a subject in a pharmacologically useful manner. The compounds of the present invention can be administered via any acceptable route, such as intravenously, intrapulmonaryly, or orally, transdermally, or systemically.

[0030] In the present invention, the term “combination therapy” is intended to define a therapy consisting of the use of a combination of two or more compounds / agents (as defined above). Therefore, references to “combination therapy,” “combination,” and the “combination” use of materials / agents in this application may refer to materials / agents administered as part of the same overall treatment regimen. Thus, each of the two or more materials / agents may be different: they may be administered simultaneously or at different times. Therefore, it will be understood that the materials / agents of a combination may be administered sequentially (e.g., before or after) or simultaneously in the same pharmaceutical formulation (i.e., separately) or in different pharmaceutical formulations. Simultaneous administration in the same formulation constitutes a unified formulation, while simultaneous administration in different pharmaceutical formulations constitutes a non-unified formulation.

[0031] "In conjunction" means administering two or more substances / agents in a time-related manner, preferably with sufficient time relevance to result in the modulation of a physiological or immunological response, and more preferably by combining two or more substances / agents. In embodiments, co-administration includes administering two or more substances / agents within a predetermined time, preferably within one month, more preferably within one week, even more preferably within one day, and most preferably within one hour. In embodiments, the substances / agents may be administered repeatedly and simultaneously, i.e., multiple co-administrations may be performed, as may be provided in the examples.

[0032] The term "disease-promoting protein level" refers to the amount of intracellular proteins that promote cell division, such as receptors, kinases, transcription factors, or components of growth signaling pathways.

[0033] The term "disease-limiting protein level" refers to the amount of intracellular proteins that inhibit cell division, and includes receptors, phosphatases, proteases, tumor suppressors, transcription repressors, or components of stalled or apoptotic signaling pathways.

[0034] The terms “effective dose” or “therapeutic dose” refer to a sufficient amount of drug to produce a beneficial or desired result. A therapeutic dose may vary depending on one or more of the following: the patient and disease state, the patient’s weight and age, the severity of the disease state, the method of administration, etc. These are matters readily apparent to those skilled in the art. This term also applies to doses that provide an image for detection by any of the imaging methods described herein. Specific doses may vary depending on one or more factors, including the chosen drug, the administration plan to be followed, whether or not other compounds are used concomitantly, the timing of administration, the tissue to be imaged, and the physical delivery system used to transport the drug.

[0035] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is a pharmacologically inert substance used in combination with a pharmacologically active substance to prepare a pharmaceutical formulation. Pharmaceutically acceptable excipients include, but are not limited to, a variety of substances known in the art, such as sugars (glucose, lactose, etc.), preservatives such as antibacterial agents, reconstitution aids, colorants, physiological salines (phosphate-buffered saline, etc.), and buffers.

[0036] In the present invention, the terms “subject,” “individual,” and “patient” are used interchangeably and refer to an animal, such as a human, that is treated (including prophylactic treatment) with the pharmaceutical composition according to the present invention. In this specification, the term “subject” refers to both humans and non-human animals. In the present invention, the terms “non-human animal” and “non-human mammal” are used interchangeably and include all vertebrates, such as mammals (non-human primates (especially higher primates)), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, horses, and non-mammals such as reptiles, amphibians, chickens, and turkeys.

[0037] The terms "polynucleotide," "nucleotide sequence," "nucleic acid," and "nucleotide" are used interchangeably. These refer to polymers of nucleotides of any length, consisting of deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. If present, modifications to the nucleotide structure are imparted before or after the assembly of the polymer. Nucleotide sequences may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by binding with labeling components.

[0038] MicroRNA (miRNA) refers to small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. They are present in a wide variety of organisms and some viruses, and are involved in RNA silencing and post-transcriptional regulation of gene expression.

[0039] "isomiR" refers to a miRNA sequence that has specific mutations relative to a reference miRNA sequence but can bind to the Ago protein and play a similar role in gene expression regulation as a regular miRNA. See, for example, Kuchenbauer, et al. (2008) Genome Research 18:1787-1797.

[0040] An "antisense oligonucleotide or chain" is an oligonucleotide that is complementary to the sense oligonucleotide, premRNA, RNA, or sense strand of a particular gene and binds to such gene and gene product by base pairing. Examples of base-pairing nucleotides include, but are not limited to, uracil, thymine, adenine, cytosine, guanine, and hypoxanthine, each amino group protected by an acyl protecting group, pyrimidine analogs such as 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pseudoisocytosine, pseudouracil, and other modified nucleic acid bases such as 8-substituted purines, xanthine, or hypoxanthine. Further examples of base-pairing nucleotides include, but are not limited to, expanded-size nucleobases with one or more benzene rings. When an antisense oligonucleotide binds to a sense oligonucleotide, it is not necessary for it to base-pair with all nucleotides in the sense oligonucleotide.

[0041] "Tm," or melting temperature, is the midpoint of the temperature range at which an oligonucleotide separates from its target nucleotide sequence. At this temperature, 50% of oligonucleotides exist as helical (hybridized) and coiled (non-hybridized) forms. Tm is measured using UV absorption spectroscopy to determine the formation and degradation (melting) of hybridization. Tm can be measured using techniques well known in the art. There are also formulas for estimating Tm based on the sequence and common chemical modifications.

[0042] A "gene target" or "target gene" refers to a gene having an RNA transcript (processed or unprocessed) containing a nucleic acid sequence including miR-21, and therefore, the expression of a protein encoded by mRNA containing a miR-21 binding site, which is conjugated by the miR-21 inhibitory RNA-peptide analog described in the present invention, can be regulated.

[0043] In the present invention, "modified nucleotide" refers to a non-naturally occurring moiety that, compared to polynucleotides that differ from inhibitory nucleic acids solely by having a native nucleotide instead of the modified nucleotide, confers increased nuclease resistance or thermodynamic stability during hybridization. In some embodiments, the ribose moiety of the nucleotide is modified with an extra crosslink linking the 2' oxygen and 4' carbon. Numerous chemical modifications are commonly used in the synthesis of oligonucleotides for various reasons. For example, to increase the stability of the phosphate backbone, to regulate the stability of the double helix, to alter the conformation of the oligonucleotide, or to enhance its ability to penetrate the lipid bilayer. Modified sugar moieties are also incorporated into therapeutic oligonucleotides. By altering the sugar chain moiety, nuclease resistance and binding affinity to complementary targets are generally improved.

[0044] "Cross-linked nucleic acid" ("BNA") refers to 2'-O,4'-C-methylene-modified nucleic acid.

[0045] "Locked nucleic acid nucleotides" ("LNA nucleotides"), as used in this invention, refer to modified RNA nucleotides that, compared to polynucleotides that differ from LNAs only by having native ribonucleotides instead of modified RNA nucleotides, provide greater thermodynamic stability to polynucleotides during hybridization.

[0046] In this invention, the term "wild type" is a term understood by those skilled in the art and refers to a typical form of an organism, strain, gene, or trait found in nature, distinct from a variant or cultivar. In use in this invention, the term "variant" should be interpreted as meaning exhibiting a pattern of properties that deviates from the wild type, or containing components that do not exist in nature.

[0047] The term "peptide" refers to a compound containing multiple linked amino acids. The amino acids used in the compounds provided in the present invention (e.g., peptides and proteins) may be one of the 20 genetically encoded amino acids, naturally occurring non-genetically encoded amino acids, or synthetic amino acids. Both L- and D-enantiomers of any of the above can be used in the compounds. In one embodiment, all amino acids are D-enantiomers. In the present invention, the following abbreviations may be used for the following genetically encoded amino acids (and their residues): alanine (Ala, A); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D); cysteine ​​(Cys, C); glycine (Gly, G); glutamic acid (Glu, E); glutamine (Gln, Q); histidine (His, H); isoleucine (Ile, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (Val, V). In some embodiments, the residues of a protein or peptide are continuous without interrupting the sequence of amino acid residues by non-genetically encoded or synthetic amino acids. In one embodiment, the sequence may include one or more non-genetically encoded amino acids or synthetic amino acid moieties. In one embodiment, the sequence of peptide residues may be interrupted by one or more non-genetically encoded amino acids or synthetic amino acid moieties, including but not limited to those shown in Table 1.

[0048] [Table 1] JPEG2026510461000003.jpg171170

[0049] Amino acids that are mutually substitutable generally belong to similar classes or subclasses. As those skilled in the art know, amino acids can be classified into different classes, primarily based on the chemical and physical properties of their side chains. For example, some amino acids are generally considered hydrophilic or polar amino acids, while others are considered hydrophobic or nonpolar amino acids. Polar amino acids include those with acidic, basic, or hydrophilic side chains, while nonpolar amino acids include those with aromatic or hydrophobic side chains. Nonpolar amino acids are further subdivided, including aliphatic amino acids, etc. The definitions of the classes of amino acids used in this invention are as follows:

[0050] "Nonpolar amino acids" refer to amino acids that do not carry an electric charge at physiological pH, are nonpolar, and generally have side chains that are repelled in aqueous solutions. Examples of genetically encoded hydrophobic amino acids include Ala, Ile, Leu, Met, Trp, Tyr, and Val. Examples of non-genetically encoded nonpolar amino acids include t-BuA, Cha, and Nle.

[0051] "Aromatic amino acids" refer to nonpolar amino acids that have at least one conjugated n-electron system (aromatic group) in their side chain. The aromatic group may be further substituted with substituents such as alkyl groups, alkenyl groups, alkynyl groups, hydroxyl groups, sulfonyl groups, nitro groups, and amino groups. Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine, and tryptophan. Commonly encountered non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, β-2-thienylalanine, 3-benzothiazole-2-ylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine.

[0052] "Aliphatic amino acids" refer to nonpolar amino acids that have saturated or unsaturated linear, branched, or cyclic hydrocarbon side chains. Examples of genetically encoded aliphatic amino acids include Ala, Leu, Val, and Ile. An example of a non-coding aliphatic amino acid is Nle.

[0053] "Polar amino acids" refer to hydrophilic amino acids whose side chains are charged or uncharged at physiological pH, and which have a bond in which the electron pair shared between two atoms is more strongly held by one atom. Polar amino acids generally mean amino acids that are hydrophilic, i.e., have side chains that are attracted to aqueous solutions. Examples of genetically encoded polar amino acids include asparagine, cysteine, glutamine, lysine, and serine. Examples of non-genetically encoded polar amino acids include citrulline, homocysteine, N-acetyllysine, and methionine sulfoxide.

[0054] "Acidic amino acids" refer to hydrophilic amino acids with a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains under physiological pH conditions due to hydrogen ion loss. Examples of gene-encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).

[0055] "Basic amino acids" refer to hydrophilic amino acids whose side chain pK value is greater than 7. Under physiological pH conditions, basic amino acids are usually positively charged in their side chains due to binding with hydronium ions. Examples of genetically encoded basic amino acids include arginine, lysine, and histidine. Examples of non-genetically encoded basic amino acids include ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, and homoarginine.

[0056] "Ionizable amino acids" refer to amino acids that can acquire an electric charge at physiological pH. Such ionizable amino acids include acidic amino acids and basic amino acids. Examples include D-aspartic acid, D-glutamic acid, D-histidine, D-arginine, D-lysine, D-hydroxylysine, D-ornithine, L-aspartic acid, L-glutamic acid, L-histidine, L-arginine, L-lysine, L-hydroxylysine, or L-ornithine.

[0057] As those skilled in the art will understand, the above classification is not absolute. Some amino acids exhibit more than one property and can therefore be placed in multiple categories. For example, tyrosine has both a nonpolar aromatic ring and a polar hydroxyl group. Thus, tyrosine has several properties that can be described as nonpolar, aromatic, and polar. However, because the nonpolar ring is dominant, tyrosine is generally considered nonpolar. Similarly, cysteine ​​is also nonpolar, in addition to being able to form disulfide bonds. Therefore, although it is not strictly classified as a hydrophobic or nonpolar amino acid, cysteine ​​can often be used to confer hydrophobicity or nonpolarity to peptides.

[0058] In some embodiments, the polar amino acids intended by the present invention include, for example, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine, hydroxylysine, ornithine, serine, threonine, and structurally related amino acids. In one embodiment, the polar amino acid is an ionizable amino acid such as arginine, aspartic acid, glutamic acid, histidine, hydroxylysine, lysine, or ornithine.

[0059] Examples of available polar or nonpolar amino acid residues include, for example, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tryptophan, and tyrosine.

[0060] The terms “non-natural origin” or “manipulated” are used interchangeably and indicate human involvement. When referring to nucleic acid molecules or polypeptides, this means that the nucleic acid molecule or polypeptide does not contain, at least substantially, at least one other component that is naturally related to and found in nature. Nucleic acid molecules that inhibit the expression of genes or nucleic acids may be called “inhibitory nucleic acids” (referring to their composition). Inhibitory nucleic acid technologies are known in this field and include, but are not limited to, small nucleic acid molecules that can mediate RNA interference (RNAi), such as antisense oligonucleotides, catalytic nucleic acids such as ribozymes and deoxyribozymes, aptamers, triple-stranded nucleic acids, external guide sequences, and RNA interference molecules (RNAi), particularly short-chain interfering nucleic acids (siNA), deoxyribozymes, aptamers, triple-stranded nucleic acids, external guide sequences, and RNA interference molecules (RNAi), particularly small nucleic acid molecules that can mediate RNA interference (RNAi), such as short-chain interfering nucleic acids (siNA), short-chain interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miR), and short hairpin RNA (shRNA).

[0061] "miR-21 inhibitory nucleic acids" or "miR-21 inhibitory nucleic acid analogs" hybridize to miR-21 or its isomi R, inhibit its activity, and thereby collectively reduce the expression of proteins encoded by mRNA or mRNA variants that possess a miR-21 binding site.

[0062] As used in this invention, the term "analog" refers to a chemical compound that is structurally similar but has a slightly different composition (such as one atom being replaced by another, or the presence or absence of a specific functional group).

[0063] In this invention, the term "bioavailability" refers to the extent or rate to which a drug or other substance is absorbed or made available at its biologically active site after administration. This property depends on many factors, including the solubility of the compound, the rate of absorption in the intestinal tract, protein binding, and the degree of metabolism. This invention describes various bioavailability tests that are familiar to those skilled in the art.

[0064] As used in this application, the term "water-soluble" refers to solubility in aqueous media, such as phosphate-buffered saline (PBS) at pH 7.4, 0.9% saline, or 5% glucose. The water-soluble test is shown below as the "water-soluble assay" in the examples.

[0065] Consideration The present invention provides miR-21 inhibitory nucleic acid-peptide analogs. In some embodiments, the inhibitor is an antisense oligonucleotide. An antisense nucleic acid sequence (antisense oligonucleotide) typically comprises a nucleotide sequence complementary to a sense nucleic acid that codes for a regulatory RNA or protein, for example, complementary to the coding strand of a double-stranded cDNA molecule, or complementary to a target RNA. In such embodiments, the antisense inhibitory nucleic acid sequence binds to one or more binding sites present on the target miR-21.

[0066] The inhibitory nucleic acid contains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity with at least a portion of miR-21. In some embodiments, the inhibitory nucleic acid does not contain a portion of the nucleotide that mirrors the seed region of the miR-21 passenger strand. In such embodiments, the complementarity of the inhibitory nucleic acid relates to the remaining portion of the target miR-21 (i.e., the sense strand) corresponding to the portion of the miR-21 passenger strand that lacks the seed region. For example, in one embodiment, the inhibitory nucleic acid has 100% sequence complementarity with at least a portion of the miR-21 portion corresponding to the portion of the passenger strand that lacks the seed region. In other embodiments, the inhibitory nucleic acid has at least 70% sequence complementarity with at least a portion of the miR-21 portion corresponding to the portion of the passenger strand that lacks the seed region. Suitable inhibitory nucleic acids include, but are not limited to, any of the sequences provided in the present invention. As those skilled in the art will understand, sequence variations expected due to genetic variation, strain polymorphism, or evolutionary divergence can be tolerated by the inhibitory nucleic acids disclosed in the present invention.

[0067] In some embodiments, the inhibitory nucleic acid-peptide analog comprises 8-17 nucleotides, 8-15 nucleotides, 10-17 nucleotides, 10-15 nucleotides, or any combination, subcombination, range, or subrange thereof. Furthermore, or alternatively, in some embodiments, the inhibitory nucleic acid is a gapmer. The gapmer comprises any suitable number of parts, each part comprising any suitable number of nucleotides according to the total length of the inhibitory nucleic acid. In one embodiment, for example, the inhibitory nucleic acid comprises a 15-nucleotide gapmer having a 5-5-5 structure. In other embodiments, it comprises any suitable variation of the number of nucleotides in any part, each of which is expressly invoked in the present invention.

[0068] In some embodiments, the gapmer comprises a deoxyribonucleic acid (DNA) moiety and at least one crosslinking nucleic acid (BNA). In one embodiment, for example, the inhibitory nucleic acid comprises a gapmer having three moies having a BNA-DNA-BNA structure. In another embodiment, the inhibitory nucleic acid comprises an 8-nucleotide gapmer having a BNA-DNA-BNA moiety containing at least the sequence ATAAGC (Figure 2C). In some embodiments, the sequence of the DNA moiety is modified while retaining its ability to bind to the target miR-21.

[0069] In some embodiments, the BNA moiety includes 2' oxygen and 4' carbon crosslinked by methylene groups. Another example of BNA is 2',4'-BNA NC [NH], 2',4'-BNA NC [NMe] and 2',4'-BNANC[NBn] may be included, but are not limited to, these. In some embodiments, one or more portions of the gapmer comprise 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), where the 2' oxygen and 4' carbon are bridged by an ethylene group. Furthermore, or alternatively, in some embodiments, (s)-cEt (S-restricted ethyl) and / or tcDNA (tricycloDNA) modifications may be used to restrict the nucleotides.

[0070] In some embodiments, the ribose moiety of a modified RNA nucleotide is modified with an extra crosslink linking the 2'-oxygen and 4'-carbon. LNA nucleotides may contain any type of extra crosslink between the 2'-O and 4'-C of the RNA, increasing the thermodynamic stability of the double helix between LNA and its complement. Other 2'-O-modified nucleotides, such as 2'-O-Me, similarly exhibit greater stability. In some embodiments, inhibitory nucleic acids include oligonucleotide backbone configurations that exhibit particularly high binding affinity (measured by melting temperature or Tm) to the target in order to implement steric hindrance mechanisms. Examples of such backbones include, but are not limited to, LNA, FANA, 2'-fluoro, 2'-O-methoxyethyl (2'-MOE), 2'-NH2, 2'-F-RNA, morpholino, and piperazine backbones. Other modifications on oligonucleotide ribose include, but are not limited to, FHNA (fluorohexitol nucleic acid), (s)-5'-C-methyl, UNA (unlocked nucleic acid), 4'-thio-RNA, and cyclohexene nucleic acid.

[0071] To minimize the degradation of oligonucleotides by nucleases, modified skeletal bonds may be used instead of phosphodiester bonds. Examples include, but are not limited to, phosphorothioates, boranephosphonates, phosphoramides, methylphosphonates, (SC5'Rp)-a,b-CNA (dioxaphospholinane-restricted nucleic acid), PNA (peptide nucleic acid), PMO (phosphodiamide morpholino oligonucleotide), and phosphorylguanidine. Examples of 5' modifications that increase phosphate stability include, but are not limited to, E-VP ((E)-vinylphosphonate), 5'-methylphosphonate, 5'-phosphorothioate, (s)-5'-methyl phosphate, and 5'-methoxy. Examples of 3' modifications that improve phosphate stability include, but are not limited to, 2-hydroxyethyl phosphate, 3'-ddc (dideoxycytosine), and 3'-amino. Examples of base modifications that improve 3' stability include, but are not limited to, 2'-thio-dT.

[0072] The synthesis of oligonucleotides with mixed bonds, such as boranophosphate bonds or phosphate bonds, has been achieved by several solid-phase methods, including one using bis(trimethylsiloxy)cyclodecyloxysilyl as the 5'-O-protecting group (Brummel and Caruthers, Tetrahedron Lett 43: 749, 2002). In another example, the 5'-hydroxyl group is first protected with a benzhydroxybis-(trimethylsilyloxy)silyl group and then deblocked with Et3N:HF before the next cycle (McCuen et al., J Am Chem Soc 128: 8138, 2006). This method yields a 99% coupling yield and can be applied to the synthesis of oligonucleotides with pure boranophosphate bonds or boranophosphates mixed with phosphodiester, phosphorothioate, phosphorodithioate, or methylphosphonate bonds. In a further example, boranophosphate-linked oligoribonucleotides can be prepared using the boranophosphorylation reagent 2-(4-nitrophenyl)ethyl ester of boranophosphoramidates. This reagent readily reacts with the hydroxyl group on the nucleoside in the presence of 1H-tetrazole as a catalyst. The 2-(4-nitrophenyl)ethyl group can be removed by β-elimination with 1,4-diazabicyclo[5.4.0]undeca-7-ene (DBU), yielding the corresponding nucleoside boranomonophosphate (NMPB) in good yield.

[0073] In some embodiments, the inhibitory nucleic acid sequence includes one or more nucleic acid base modifications to increase binding affinity. Suitable nucleic acid base modifications to increase binding affinity include, but are not limited to, 5'-methylcytidine, 5-methyluridine (ribothymidine), and abasic RNA.

[0074] Despite having a reduced length (i.e., number of nucleotides), the antisense sequences disclosed in this invention bind sufficiently strongly to miR-21 to specifically block the activation of mRNA translation. In addition or alternatively, and without wishing to be bound by theory, BNA is thought to reduce or eliminate hybridization-dependent and hybridization-independent toxicity while simultaneously providing improved hybridization affinity compared to existing backbone modifications (e.g., lock nucleotides (LNA)). Furthermore, and without wishing to be bound by theory, the elimination of the passenger strand seed region is thought to reduce or eliminate passenger strand mimicry by the antisense sequence.

[0075] Furthermore, in some embodiments of the present invention, methods are provided for treating or improving symptoms associated with cancer and / or other types of overproliferative disorders regulated by microRNA21 activity. In some embodiments, the method involves administering one or more inhibitory nucleic acid sequences and analogs disclosed in the present invention, and / or one or more sequences that are at least 80%, 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto and that retain the ability to treat or improve cancer-related symptoms, to a subject in need. In one embodiment, for example, the method involves administering one or more sequences disclosed in the present invention to a subject having cancer such as triple-negative breast cancer (TNBC), but is not limited to these.

[0076] Useful delivery methods for administering inhibitory nucleic acids are known in the art. For example, (Goodchild, Curr. Opin. Mol. Ther., 6(2):120-128 (2004); Clawson, et al., Gene Ther., 11(17):1331-1341 (2004); Durymanov M., et al. Front. Pharmacol. 9:971 (2018); Kulkarni et al., Nature Nanotechnology 16:630 (2021)) are incorporated in their entirety by reference. Antisense nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (e.g., antisense oligonucleotides) can be chemically synthesized using naturally occurring nucleotides, or modified nucleotides designed to increase the biological stability of the molecule or the physical stability of the double helix formed between the antisense nucleic acid and the sense nucleic acid, such as phosphorothioate derivatives and modifications to the 3' and 5' ends or synthetic nucleotides, can be used. The nucleic acid sequences provided in the present invention are not limited to those in the sequence list, but are intended to encompass nucleic acids including any combination of natural or modified RNA and / or DNA, including such nucleic acids having modified nucleic acid bases. In some embodiments, the oligonucleotides provided in the present invention may include one or more modifications to nucleic acid bases, sugars, and / or nucleoside linkages, and such are modified oligonucleotides.

[0077] Antisense nucleic acids can also be biologically produced using expression vectors in which the nucleic acid is subcloned in the antisense direction (i.e., the RNA transcribed from the inserted nucleic acid is in the antisense direction relative to the target nucleic acid). In a particularly preferred embodiment, the miR-21 inhibitory nucleic acid is functionally linked to a peptide ligand of a receptor protein overexpressed on cancer cells (e.g., IGF1R ligand), thereby promoting endocytosis of the inhibitor to target cells. The peptide ligand may be covalently linked to either the 5' or 3' end of the inhibitory nucleic acid.

[0078] In certain embodiments, the delivery of miR-21 inhibitory nucleic acids may be enhanced by covalently linking the inhibitory nucleic acid to a lipid molecule, including but not limited to cholesterol, α-tocopherol, or long-chain fatty acids. In another embodiment, the delivery of miR-21 inhibitory nucleic acids may be enhanced by covalently linking the inhibitory nucleic acid to GalNAc. In yet another embodiment, the delivery of miR-21 inhibitory nucleic acids may be enhanced by covalently linking the inhibitory nucleic acid to an antibody. In yet another embodiment, the delivery of miR-21 inhibitory nucleic acids may be enhanced by covalently linking the inhibitory nucleic acid to an aptamer. In yet another embodiment, the delivery of miR-21 inhibitory nucleic acids may be enhanced by covalently linking the inhibitory nucleic acid to a protein or peptide, including but not limited to polybasic amino acids, cell entry peptides, cell targeting peptides, or receptor-binding proteins. In yet another embodiment, the delivery of miR-21 inhibitory nucleic acids may be enhanced by associating the inhibitory nucleic acid with another cell-permeable molecule via non-covalent interactions. In another embodiment, delivery of miR-21 inhibitory nucleic acids can be enhanced by packaging the inhibitory nucleic acids in nanocarriers. In another embodiment, delivery of miR-21 inhibitory nucleic acids can be enhanced by packaging the inhibitory nucleic acids in liposomes, including but not limited to functionalized lipid nanoparticles having PEGylated lipids, or other ligands associated with lipid nanoparticles for cell-targeted delivery. In another embodiment, delivery of miR-21 inhibitory nucleic acids can be enhanced by loading the inhibitory nucleic acids into exosomes. In another embodiment, delivery of miR-21 inhibitory nucleic acids can be enhanced by chemically linking the inhibitory nucleic acids to the surface of spherical nanoparticles. In another embodiment, delivery of miR-21 inhibitory nucleic acids can be enhanced by incorporating the inhibitory nucleic acids into stimulus-sensitive nanostructures, including but not limited to DNA origami, scaffold molecules bound to multiple delivery sites.

[0079] In some embodiments, miR-21 inhibitory nucleic acid-peptide analogs may contain an external guide sequence (EGS). The EGS is a molecule that binds to a target nucleic acid molecule to form a complex, which is recognized by RNase P and cleaves the target molecule. EGS can be designed to specifically target a chosen RNA molecule. RNase P assists in the processing of transfer RNA (tRNA) within cells. Bacterial RNase P can be made capable of cleaving virtually any RNA sequence by using an EGS such that the target RNA:EGS complex mimics a native tRNA substrate. Similarly, eukaryotic EGS / RNase P-targeted RNA cleavage can be used to cleave desired targets within eukaryotic cells. Examples of methods for constructing and using EGS molecules that facilitate the cleavage of various different target molecules are known in the art.

[0080] The implementation of the present invention utilizes prior art in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, unless otherwise specified, but these are within the scope of the art of those skilled in the art. In some embodiments, the vector can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the regulatory function of the expression vector is usually provided by one or more regulatory elements. For example, commonly used promoters are derived from polyomas, adenovirus 2, cytomegalovirus, Simianvirus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0081] The pharmaceutically acceptable salts of the compounds of the present invention include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases, as well as quaternary ammonium acid addition salts. More specific examples of suitable salts include hydrochloride, hydrobromide, sulfate, phosphate, nitrate, perchlorate, fumarate, acetate, propionate, succinate, glycolate, formate, lactate, maleate, tartrate, citrate, palmate, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumarate, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, steric acid, tannic acid, and the like. Hydrochloride salts are particularly targeted. Other acids, such as oxalic acid, are not pharmaceutically acceptable themselves, but are useful in preparing salts that serve as intermediates for obtaining the compounds of the present invention and their pharmaceutically acceptable salts. More specific examples of suitable basic salts include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine salts.

[0082] formulation The novel miR-21 inhibitory nucleic acid-peptide analogs described in the present invention can be formulated for enteral, parenteral, topical, or pulmonary administration. The compounds can be combined with one or more pharmaceutically acceptable carriers and / or excipients that are considered safe and effective and can be administered to an individual without causing undesirable biological side effects or interactions. A carrier is any component present in a pharmaceutical formulation other than the active ingredient. See, for example, Remington's Pharmaceutical Sciences, latest edition, by EW Martin Mack Pub. Co., Easton, PA. This document discloses conventional methods for preparing typical carriers and pharmaceutical compositions that may be used in conjunction with the preparation of formulations of the compounds described in the present invention, and is incorporated by reference into the present invention. The most typical of these would be standard carriers for administration of compositions to humans and non-humans, including solutions such as sterile water, physiological saline, and physiological pH buffers. Other compounds are administered according to standard procedures used by those skilled in the art.

[0083] These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0084] Parenteral formulations The compounds described in the present invention can be formulated for parenteral administration. For example, parenteral administration may include administration to a patient by intravenous, intradermal, intra-arterial, intraperitoneal, intrapenile, intracranial, intra-articular, intrathoracic, intratracheal, intravitreous, intratumoral, intramuscular, subcutaneous, subconjunctival, intrauveal, intrapericardial, intraumbilical, injection, and intravenous infusion.

[0085] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, such as solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions by adding a reconstitution medium before injection; water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and their microemulsions, liposomes, or emulsions.

[0086] For intravenous administration, the composition is packaged in a sterile isotonic aqueous buffer solution. The composition may, if necessary, contain a solubilizer. The components of the composition are supplied, for example, as dried lyophilized powder or concentrated solution, separately or mixed in unit dose form in sealed containers such as ampoules or pouches indicating the amount of activator. When the composition is administered by infusion, it can be prepared using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline can be provided so that the components can be mixed before injection.

[0087] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and / or by using a surfactant. In many cases, it would be preferable to include an isotonic agent, such as sugars or sodium chloride.

[0088] Solutions and dispersions of active compounds as free acids or bases or pharmaceutically acceptable salts thereof can be prepared in water or other solvents or dispersion media by appropriately mixing with one or more pharmaceutically acceptable excipients, including but not limited to surfactants, dispersants, emulsifiers, pH adjusters, viscosity modifiers, and combinations thereof.

[0089] Suitable surfactants are anionic, cationic, amphoteric, or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate ions, sulfonate ions, and sulfate ions. Examples of anionic surfactants include sodium, potassium, and ammonium long-chain alkyl sulfonates and alkylaryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates such as sodium bis-(2-ethylthioxy)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyldimethylbenzylammonium chloride, polyoxyethylene, and quaternary ammonium compounds such as coconutamine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl 4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polyoxyethylene monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0090] The formulation may contain preservatives to prevent microbial growth. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain antioxidants to prevent degradation of the activator. The formulation is usually buffered to pH 3-8 for parenteral administration during reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0091] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0092] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent or dispersion medium, along with one or more of the excipients listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary components from the above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques to obtain powders of the active ingredient and any additional desired components from a pre-sterile filtered solution. The powders can be prepared so that the particles are porous, thereby increasing the solubility of the particles. Methods for producing porous particles are well known in the art.

[0093] Controlled-release formulations The parenteral formulations described in the present invention can be formulated for release control, including immediate release, delayed release, sustained release, pulsatile release, and combinations thereof.

[0094] Nanoparticles and microparticles For parenteral administration, one or more compounds and any one or more additional activators may be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the compounds and / or the one or more additional activators. In a formulation comprising two or more drugs, the drugs may be formulated for the same type of controlled release (e.g., delayed, prolonged, immediate, or pulsatile), or the drugs may be formulated independently for different types of release (e.g., immediate and delayed, immediate and prolonged, delayed and prolonged, delayed and pulsatile, etc.).

[0095] For example, compounds and / or one or more additional activators can be incorporated into polymer microparticles that provide controlled release of a drug. Drug release is controlled by the diffusion of the drug from the microparticles and / or by the degradation of the polymer microparticles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0096] Polymers that dissolve slowly and form gels in an aqueous environment, such as hydroxypropyl methylcellulose and polyethylene oxide, are also suitable as materials for drug-containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyric acid (PHB) and its copolymers, poly-4-hydroxybutyric acid (P4HB) and its copolymers, polycaprolactone and its copolymers, and combinations thereof.

[0097] Alternatively, the drug may be insoluble in aqueous solution or slowly soluble in aqueous solution, but can be incorporated into microparticles prepared from materials that are biodegradable in the digestive tract by means including enzymatic hydrolysis, the surfactant action of bile acids, and / or mechanical erosion. In this invention, the term "slowly soluble in water" refers to a substance that does not dissolve in water within 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances, and mixtures thereof. Suitable fats and fatty substances include, but are not limited to, fatty alcohols (such as lauryl, myristylstearyl, cetyl, or cetostearyl alcohol), fatty acids and derivatives (fatty acid esters, fatty acid glycers (monoglycers, diglycers, and triglycerides), and hydrogenated fats). Specific examples include, but are not limited to, hydrogenated vegetable oils, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and wax-like substances include natural or synthetic waxes, hydrocarbons, and ordinary waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffin, and candelilla wax. In this invention, a wax-like substance is defined as any substance that is normally solid at room temperature and has a melting point of approximately 30°C to 300°C.

[0098] In some cases, it is desirable to alter the rate of water penetration into the microparticles. For this purpose, a rate-controlling (wicking) agent can be incorporated with the fats or waxes mentioned above. Examples of rate-controlling substances include certain starch derivatives (e.g., waxy maltodextrin and drum-dried corn starch), cellulose derivatives (e.g., hydroxypropyl methylcellulose, hydroxypropylcellulose, methylcellulose, and carboxymethylcellulose), alginic acid, lactose, and talc. Furthermore, a pharmaceutically acceptable surfactant (e.g., lecithin) may be added to promote the degradation of such microparticles. Water-insoluble proteins can also be used as materials for forming drug-containing microparticles. In addition, water-soluble proteins, polysaccharides, and combinations thereof can be formulated into microparticles with drugs and then crosslinked to form an insoluble network. For example, cyclodextrin can be complexed with individual drug molecules and then crosslinked.

[0099] Methods for producing nanoparticles and microparticles The production of therapeutic microparticles by encapsulating or incorporating the miR-21 inhibitory nucleic acid-peptide analogs described in the present invention into a carrier material can be achieved by known pharmaceutical formulation techniques. In the case of formulation into fats, waxes, or wax-like substances (including, optionally, the peptide ligand itself bound to a hydrophobic tail), the carrier material is typically heated above its melting point, and an imitation or drug is added to form a mixture containing drug particles suspended in the carrier material, a drug dissolved in the carrier material, or a mixture thereof. The microparticles can then be formulated by several methods, including but not limited to coagulation, extrusion, spray cooling, or aqueous dispersion. In a preferred process, the wax is heated above its melting point, the drug is added, and the mixture of molten wax and drug is coagulated under constant stirring while cooling. Alternatively, the mixture of molten wax and drug can be extruded and spheroidized to form pellets or beads. These processes are known in the art.

[0100] Depending on the carrier material, it may be desirable to produce drug-containing microparticles using solvent evaporation. In this case, the drug and carrier material (including the peptide ligand itself, which may be bound to the hydrophobic tail) are co-dissolved in a mutual solvent, and the material can then be produced by several techniques, including, but not limited to, forming an emulsion in water or another suitable medium, spray drying, or evaporating the solvent from the bulk solution and grinding the resulting material.

[0101] In some embodiments, particulate drugs are slowly and uniformly dispersed in a water-insoluble or water-soluble material. To minimize the size of drug particles in the composition, the drug powder itself may be pulverized before formulation to produce fine particles. The jet milling process known in the pharmaceutical art can be used for this purpose. In some embodiments, particulate drugs are uniformly dispersed in a wax or similar substance, which may contain the peptide ligand itself, possibly bound to a hydrophobic tail, by heating the wax or similar substance above its melting point and adding the drug particles while stirring the mixture. In this case, a pharmaceutically acceptable surfactant may be added to the mixture to facilitate the dispersion of the drug particles.

[0102] Particles can also be coated with one or more modified release coatings. Solid esters of fatty acids hydrolyzed by lipase can be spray-coated onto microparticles or drug particles. Natural water-insoluble proteins can be coated onto drug-containing microparticles or drug particles by spray coating or wet granulation techniques. In addition to natural water-insoluble substances, crosslinking can be performed depending on the substrate of digestive enzymes, resulting in the formation of a water-insoluble network. Numerous methods for crosslinking proteins have been reported by chemical and physical methods. One of the most common methods for obtaining crosslinking is the use of chemical crosslinking agents. Examples of chemical crosslinking agents include aldehydes (glutaraldehyde, formaldehyde), epoxy compounds, carbodiimides, and genipin. In addition to these crosslinking agents, oxidized sugars and native sugars have been used to crosslink gelatin. For example, transglutaminase is approved as a GRAS substance for crosslinking seafood. Finally, crosslinking can be initiated by physical means such as heat treatment, UV irradiation, and gamma irradiation.

[0103] To create drug-containing microparticles or a coating layer of cross-linked protein surrounding drug particles, a water-soluble protein can be spray-coated onto the microparticles and then cross-linked by one of the methods described above. Alternatively, drug-containing microparticles can be microencapsulated within a protein by coacervation-phase separation (e.g., salt addition) and then cross-linked. Suitable proteins for this purpose include gelatin, albumin, casein, and gluten.

[0104] Polysaccharides can also crosslink to form water-insoluble networks. In many polysaccharides, this is achieved by reaction with calcium salts or polyvalent cations, crosslinking the main polymer chains. Pectin, alginic acid, dextran, amylose, and guar gum, for example, are crosslinked in the presence of polyvalent cations. Pectin and chitosan, for example, complex together through electrostatic interactions.

[0105] Injectable / implantable formulation The miR-21 inhibitory nucleic acid-peptide analogs described in this invention can be incorporated into injectable / implantable solid or semi-solid implants, such as polymer implants. In some forms, these compounds are incorporated into polymers that are liquid or paste-like at room temperature but increase in viscosity upon contact with an aqueous medium, such as physiological fluids, to form a semi-solid or solid material. Typical polymers include, but are not limited to, hydroxyalkanoate polyesters obtained by copolymerization of at least one unsaturated hydroxy fatty acid with a hydroxyalkanoate. The polymer can be melted, mixed with the active ingredient, and molded into a device by casting or injection molding. Such melt molding requires a polymer with a melting point lower than the temperature at which the target substance and the polymer decompose or react. Alternatively, the device can be prepared by solvent casting, in which the polymer is dissolved in a solvent, the drug is dissolved or dispersed in the polymer solution, and then the solvent is evaporated. In the solvent process, the polymer must be soluble in organic solvents. Another method involves compression molding of a mixed powder of polymer and drug, or polymer particles filled with the active ingredient.

[0106] Alternatively, the compound can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is solid at room temperature. For example, the compound can be incorporated into biodegradable polymers such as polyanhydrides, polyhydroalkanoates (PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, collagen, hyaluronic acid, albumin, gelatin, and other proteins and polysaccharides, as well as combinations thereof, and then compressed into a solid device such as a disc or extruded into a device such as a rod.

[0107] The release of one or more compounds from an implant can be altered by the selection of the polymer, the molecular weight of the polymer, and / or the modification of the polymer to promote degradation (e.g., pore formation and / or incorporation of hydrolyzable linkages). Methods for modifying the properties of biodegradable polymers to alter the compound release profile from implants are well known in the art.

[0108] Enteral preparations Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, sodium saccharin, starch, magnesium stearate, cellulose, and magnesium carbonate. Such compositions contain a therapeutically effective amount of the compound and / or antibiotic, along with an appropriate amount of carrier, to provide the patient with an appropriate form based on the mode of administration used.

[0109] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be manufactured using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared using techniques well known in the art as rigid or flexible capsule shells capable of enclosing liquid, solid, and semi-solid fillers.

[0110] The formulations can be prepared using pharmaceutically acceptable carriers. The “carriers” commonly used in this invention include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof. Carriers also include the entirety of a coating composition that may contain plasticizers, pigments, colorants, stabilizers, and lubricants.

[0111] Examples of suitable coating materials include, but are not limited to, cellulose polymers (e.g., cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate, acrylic polymers and copolymers, and methacrylic resins, zein, shellac, and polysaccharides commercially available under the trademark name EUDRAGIT® (Roth Pharma, Westerstadt, Germany). Furthermore, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, lubricants, stabilizers, pore-forming agents, and surfactants.

[0112] Diluents, also known as fillers, are typically needed to increase the volume of solid dosage forms and provide a practical size suitable for compressing tablets or forming beads or granules. Suitable diluents include, but are not limited to, dicalcium dihydrate phosphate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dried starch, hydrolyzed starch, gelatinized starch, silicon dioxide, titanium dioxide, magnesium aluminum silicate, and powdered sugar.

[0113] A "binder" is used to impart cohesiveness to a solid dosage form and maintains its shape after the formation of the dosage form, such as tablets, beads, or granules. Suitable binder materials include, but are not limited to, starch, pregelled starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, wax, natural and synthetic gums such as acacia gum, tragacanth gum, and sodium alginate, cellulose (natural and synthetic gums such as hydroxypropyl methylcellulose, hydroxypropylcellulose, ethylcellulose, and vee gum), and synthetic polymers such as acrylic acid / methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid, and polyvinylpyrrolidone.

[0114] Lubricants are used to facilitate the manufacturing of tablets. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, behenyl glyceryl, polyethylene glycol, talc, and mineral oil.

[0115] "Disintegrants" are used to promote the disintegration or "decomposition" of a dosage form after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropylcellulose, gelatinized starch, clay, cellulose, alginic acid, gum, or cross-linked polymers (such as cross-linked PVP (Polyplasdon® XL, manufactured by GAF Chemicals)).

[0116] "Stabilizers" are used to suppress or delay the decomposition reactions of drugs, including oxidation reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT), ascorbic acid, its salts and esters, vitamin E, tocopherol and its salts, sulfites (e.g., sodium metabisulfite), cysteine ​​and its derivatives, citric acid, propyl gallate, and butylated hydroxyanisole (BHA).

[0117] Controlled-release enteral preparations Oral dosage forms such as capsules, tablets, solutions, and suspensions can be formulated for controlled release. For example, one or more compounds and one or more additional activators can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in soft or hard gelatin or non-gelatin capsules, or dispersed in a dispersion medium to form an oral suspension or syrup. The particles can be formed with the drug and a controlled-release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled-release coatings before being incorporated into the final dosage form.

[0118] In another form, one or more compounds and one or more additional activators are dispersed in a matrix material, which gels or emulsifies upon contact with an aqueous medium such as a physiological fluid. In the case of a gel, the matrix swells, encapsulating the activators, and they are slowly released over time by diffusion and / or decomposition of the matrix material. Such matrices can be formulated as tablets or as fillers for hard or soft capsules.

[0119] In yet another form, one or more compounds and any one or more additional activators are formulated into commercially available oral dosage forms such as tablets or capsules, and the solid dosage forms are coated with one or more controlled-release coatings such as delayed-release coatings or sustained-release coatings. The coating or film may also contain the compounds and / or additional activators.

[0120] Sustained-release dosage form Sustained-release formulations are generally prepared as diffusion systems or osmotic systems, which are well known in the art. Diffusion systems generally consist of two types of devices: a reservoir and a matrix, which are well known and described in the art. Matrix devices are generally prepared by compressing the drug into tablet form together with a polymer carrier that slowly dissolves the drug. The three main types of materials used in the preparation of matrix devices are insoluble polymers, hydrophilic polymers, and fatty compounds. Examples of polymer matrices include, but are not limited to, methyl acrylate / methyl methacrylate, polyvinyl chloride, and polyethylene. Examples of hydrophilic polymers include, but are not limited to, cellulosic polymers such as methylcellulose and ethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, hydroxyalkylcellulose such as Carbopol® 934, polyethylene oxide, and mixtures thereof. Examples of fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate, waxy substances such as hydrogenated castor oil and hydrogenated vegetable oil, or mixtures thereof.

[0121] In certain preferred embodiments, the polymer material is a pharmaceutically acceptable acrylic polymer, and includes, but is not limited to, copolymers of acrylic acid and methacrylic acid, methyl methacrylate, methyl methacrylate copolymer, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), alkylamine methacrylate copolymer, poly(methyl methacrylate), poly(methacrylic acid) (anhydride), polymethacrylate, polyacrylamide, poly(methacrylic anhydride), and glycidyl methacrylate copolymer.

[0122] In certain preferred embodiments, the acrylic polymer comprises one or more ammonia methacrylate copolymers. Ammonia methacrylate copolymers are well known to those skilled in the art as complete polymerization copolymers of acrylic acid esters and methacrylate esters with a low quaternary ammonium group content.

[0123] In one preferred form, the acrylic polymer is an acrylic resin lacquer, such as those commercially available from Rohm Pharma under the trademark name EUDRAGIT®. In a more preferred form, the acrylic polymer includes a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the trademarks EUDRAGIT®RL30D and EUDRAGIT®RS30D, respectively. EUDRAGIT®RL30D and EUDRAGIT®RS30D are copolymers of acrylic acid esters and methacrylic acid esters with a low quaternary ammonium group content, where the molar ratio of ammonium groups to the remaining neutral (meth)acrylic acid ester is 1:20 for EUDRAGIT®RL30D and 1:40 for EUDRAGIT®RS30D. The average molecular weight is approximately 150,000. EUDRAGIT®S-100 and EUDRAGIT®L-100 are also preferred. The code designations RL (high permeability) and RS (low permeability) indicate the permeability characteristics of these drugs. EUDRAGIT® RL / RS mixtures are insoluble in water and digestate. However, multi-particulate systems formed to contain these mixtures exhibit swelling and permeability in aqueous solutions and digestate.

[0124] The polymers described above, such as EUDRAGIT®RL / RS, can be mixed in any ratio to obtain a sustained-release formulation with a desired solubility profile. For example, a desirable sustained-release multi-particle system can be obtained from 100% EUDRAGIT®RL, 50% EUDRAGIT®RL, and 50% EUDRAGIT®RS, as well as from 10% EUDRAGIT®RL and 90% EUDRAGIT®RS. Those skilled in the art will understand that other acrylic polymers, such as EUDRAGIT®L, can also be used.

[0125] Alternatively, sustained-release formulations can be prepared using an osmotic pressure system or by applying a semipermeable membrane coating to the formulation. In the latter case, the desired drug release profile can be achieved by combining low-permeability and high-permeability coating materials in appropriate ratios.

[0126] The devices with different drug release mechanisms described above can be used in combination with a final dosage form containing one or more units. Examples of multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules. To add the immediate-release portion to a sustained-release system, one can form an immediate-release layer on a sustained-release core using a coating or compression process, or use a multi-unit system such as sustained-release beads and a capsule containing the immediate-release beads.

[0127] Sustained-release tablets containing hydrophilic polymers are prepared using techniques commonly known in this field, such as direct compression, wet granulation, and dry granulation. These formulations typically contain the active ingredient, polymer, excipients, binders, and lubricants. Common excipients include inert powders such as starch, powdered cellulose (especially crystalline and microcrystalline cellulose), sugars like fructose, mannitol, and sucrose, and grain flour and similar edible powders. Typical excipients include various starches, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts like sodium chloride, and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include sugars such as starch, gelatin, lactose, fructose, and glucose. Natural and synthetic gums such as acacia gum, alginate, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose, and waxes also function as binders. In tablet formulations, lubricants are necessary to prevent the tablet and punch from adhering to the mold. The lubricant is selected from lubricating solids such as talc, magnesium calcium stearate, stearic acid, and hydrogenated vegetable oil.

[0128] Sustained-release tablets containing wax materials are generally prepared using conventional methods such as direct mixing, coagulation, and aqueous dispersion. In the coagulation method, the drug is mixed with the wax material and prepared through either spray coagulation or coagulation, sieving, and processing.

[0129] Delayed-release dosage form Delayed-release formulations can be produced by coating a solid dosage form with a polymer film that does not dissolve in the acidic environment of the stomach but dissolves in the neutral environment of the small intestine.

[0130] Delayed-release dosing units can be prepared, for example, by coating a drug or drug-containing composition with a selected coating material. The drug-containing composition may be, for example, a tablet for incorporation into a capsule, a tablet for use as the core of a "coated core" dosage form, or a plurality of drug-containing beads, particles, or granules for incorporation into either a tablet or a capsule. Preferred coating materials include bioerosive, slow hydrolyzable, slow water-soluble, and / or enzymatically degradable polymers, which may be conventional "enteric-coated" polymers. Enteric-coated polymers become soluble in the high pH environment of the lower gastrointestinal tract, or are gradually eroded as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly the colon.Suitable coating materials for delayed release include cellulose-based polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, and sodium carboxymethylcellulose; acrylic acid polymers and copolymers, preferably acrylic. Eudragit® (Rohm) includes methacrylic resin formed from lylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate, and EUDRAGIT® L30D-55 and L100-55 (soluble at pH 5.5 or higher), EUDRAGIT® L-100 (soluble at pH 6.0 or higher), EUDRAGIT® S (soluble at pH 7.0 or higher due to high degree of esterification), EUDRAGITS® NE, RL, RS (water-insoluble polymers with different permeability and swelling properties). Other methacrylic resins marketed under the trade name Pharma (Westerstadt, Germany); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinyl phthalate acetate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; azopolymers, pectin, chitosan, amylose, and guar gum; zein and shellac are examples, but are not limited to these. Different coating materials can also be used in combination. Multilayer coatings using different polymers are also possible.

[0131] The preferred coating weight of a particular coating material can be easily determined by those skilled in the art by evaluating the individual release profiles of tablets, beads, and granules prepared with different amounts of various coating materials. The combination of materials, application method, and form are what produce the desired release characteristics, and these can be determined solely from clinical trials.

[0132] The coating composition may contain conventional additives, such as plasticizers, pigments, colorants, stabilizers, and lubricants. Plasticizers are typically present to reduce the brittleness of the coating. Plasticizers generally account for about 10 wt% to 50 wt% of the dry weight of the polymer. Typical examples of plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, acetyl triethyl citrate, castor oil, and acetylated monoglycerides. Stabilizers are preferably used to stabilize the particles in the dispersion. Typical stabilizers are nonionic emulsifiers such as sorbitan esters, polysorbates, and polyvinylpyrrolidone. Lubricants are recommended to reduce the tackiness during film formation and drying, and generally account for about 25 wt% to 100 wt% of the polymer weight in the coating solution. One effective lubricant is talc. Other lubricants such as magnesium stearate and glycerol monostearate can also be used. Pigments such as titanium dioxide can also be used. A small amount of defoaming agent such as silicone (e.g., simethicone) may be added to the coating composition.

[0133] Inhalation preparations The inhibitors of the present invention can be delivered topically to the respiratory system, such as the nose, sinuses, sinus membranes, or lungs. The miR-21 inhibitory nucleic acid-peptide analogs of the present invention, or a pharmaceutical composition comprising one or more miR-21 inhibitory nucleic acid-peptide analogs, can be delivered to the respiratory system by any suitable method, such as oral inhalation or intranasal inhalation. The compositions can be formulated as powder or liquid nasal sprays, suspensions, nasal drops, gels, or ointments, through tubes or catheters, by syringes, by packtails, by pre-jets, or by submucosal injection. Compounds of preferred embodiments of the present invention can be conveniently delivered in the form of aerosol sprays using a pressurized pack or nebulizer and a suitable propellant, such as, but not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of pressurized aerosols, the administration unit can be controlled by providing a valve to supply a metered amount. For use in inhalers or inhalers, for example, gelatin capsules and cartridges can be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch. Propellants for aerosol formulations include compressed air, nitrogen, carbon dioxide, or hydrocarbon-based low-boiling solvents. The compounds of the present invention can be delivered in the form of an aerosol spray from a nebulizer or the like. In some embodiments, the active ingredient is preferably pulverized to allow substantially all of the active ingredient to be inhaled into the lungs when the dry powder formulation is administered, and therefore the active ingredient has a particle size in the range of less than 100 microns, preferably less than 20 microns, and more preferably between 1 and 10 microns.

[0134] The following examples are provided to illustrate specific embodiments of the present invention. They are not intended to limit the invention in any way. [Examples]

[0135] Example 1 Targeted, low-toxicity molecular therapies to extend survival represent a significant unmet need for TNBC, with a projected market size of $7 billion annually in the United States. As mentioned above, miR-21 inhibitory nucleoside-peptide analogs contain IGF1 receptor ligands, and TNBC cells exhibit strong IGF1R signaling activation, which correlates with reduced survival. However, signaling feedback between miR-21 and IGF1R is unknown (Dobre, et al. Cells 10(8):1856, 2021).

[0136] miR-21 inhibitory nucleoside peptide analogs offer a unique approach to inhibiting miR-21-mediated gene expression by targeting and delivering the inhibitory nucleoside peptide analog to TNBC cells via the IGF1R ligand. Administration of the miR-21 inhibitory nucleoside peptide analogs described here elevated tumor suppressor proteins, suppressed immune checkpoint gene expression, slowed migration, slowed proliferation, and increased apoptosis in multiple TNBC strains. Intraperitoneal administration of 5 mg / kg of the miR-21 inhibitory nucleoside peptide analog twice weekly in sterile saline inhibited the proliferation of mouse EMT6 TNBC orthotopic grafts by 74% after 14 days compared to vehicle or scrambled controls. Therefore, novel TNBC therapeutics and their variants are provided to treat this devastating disease and significantly extend the survival time of TNBC patients.

[0137] The design of miR-21 inhibitory nucleoside-peptide analogs was brought about by previous research that created miR-17-5p blockers that actively mimic the passenger strand of miR-17-3p, undesirably downregulating PDCD4 and PTEN tumor suppressor proteins, thereby producing previously unknown off-target effects. See Figure 4A and Jin, Y.-Y., et al. PLoS One 10(12): e0142574 (2015).

[0138] The newly constructed miR-21 inhibitory nucleic acid-peptide analogs described in this invention contain a 15nt sequence designed to specifically block miR-21-5p (identical in mouse and human) without mimicking the counterchain (Figures 2A-2C, Figure 15). In the pre-miR-21 hairpin structure (Figure 2A), most of the nucleotides of the miR-21-5p guide strand form base pairs with most of the nucleotides of the miR-21-3p passenger strand. Either the guide strand or the passenger strand can be selected as the active mature miRNA in the RNA-induced silencing complex (RISC). The 15nt miR-21-5p guide strand inhibitory nucleic acid sequence in the 5'-to-3' direction shows high sequence homology to the continuous sequence region in the 5'-to-3' direction of the miR-21-3p passenger strand, excluding the seed region (nt1-9 in the 5'-to-3' direction) (Figures 2B-2C). Particularly preferred to exclude are at least 2, 3, 4, 5, 6, 7, or 8 nucleosides, shown in blue in Sequence ID No. 4. In one embodiment, 3 nucleosides are omitted or excluded. In another embodiment, 4 nucleosides are omitted. In another embodiment, 5 nucleosides are omitted. In another embodiment, 6 nucleosides are omitted. In another embodiment, 7 nucleosides are omitted. In another embodiment, 8 nucleosides are omitted.

[0139] Since miRNAs primarily function through complementary base pairing between the seed region and regulatory binding sites on target mRNA, nucleic acid inhibitory sequences in the miR-21-5p guide strand that exclude or partially exclude the miR-21-3p passenger strand seed region are expected to specifically inhibit the miR-21-5p guide without causing further side effects by mimicking the miR-21-3p passenger strand.

[0140] The miR-21 inhibitory nucleic acid moiety of the miR-21 blocker of the present invention contains 8 to 17 linked nucleosides, as shown in Figures 2B to 2C. As described below, certain parts of the nucleic acid moiety are more resistant to modification than other parts. For example, the first 4 to 5 consecutive nucleosides may consist of BNA with phosphorothioate bonds, and the last 4 to 5 consecutive nucleosides may consist of BNA with 5' to 3' oriented phosphorothioate bonds. A ligation nucleoside between consecutive BNAs at the 5' and 3' ends may contain one or more of the following modifications: phosphorothioate, boranophosphonate, (SC5'Rp)-a,b-dioxaphospholinane-restricted nucleic acid, (E)-vinylphosphonate, 5'-methylphosphonate, 5'-phosphorothioate, (s)-5'-methylphosphate, 5'-methoxy, 2-hydroxyethylphosphate, 3'-dideoxycytosine, 3'-amino, 2'-thio-dT, 2'-O-methyl, 2'-O-methoxy, 2'-NH2, 2'-F-RNA, 2'-F-ANA, LNA, 2'-O,4'-C-ethylene-bridged nucleic acid, (s)-cEt, fluorohexitol nucleic acid, (s)-5'-C-methyl, unlocked nucleic acid, 4'-thio-RNA. As described above, such modifications are preferably inserted outside the minimal binding region.

[0141] As described above, in order to achieve excellent hybridization affinity while minimizing hybridization-dependent and independent toxicity, the RNA analog 2'4'-BNA NC (BNA) skeletal modification was introduced (Figure 3). 2'4'-BNA NC (BNA) is a nuclease-resistant derivative of 2'O,4'-C-methylene-bridged nucleic acid (LNA) (Figure 15).

[0142] The efficacy of miR-21 blockers in cells. Transfection of human MDA-MB-231 triple-negative breast cancer cells resulted in an IC50 value of 22 nM for the 15-nucleotide-length BNA miR-21 gapmer BND5412, which inhibits the desuppression of miR-21 against luciferase expression from the luciferase-3'-UTR vector (Figure 4A). Transfection with this miR-21 blocker significantly increased the expression of the miR-21 target protein PDCD4, followed by suppression of the CD47, PD-L1, PD-L2, and Jak2 immune checkpoints (Figures 5A-5B). Furthermore, it showed up to an 8-fold inhibitory effect on cell proliferation in MDA-MB-231, HCC1806, BT-20, MDA-MB-157, MDA-MB-436, BT-549, and HCC1937 cells (Figure 6).

[0143] As a control to BNA drugs, human MDA-MB-231 triple-negative breast cancer cells were also tested after being transfected with 50 nM FANA oligonucleotides for 48 hours. FANA sequences 1-7 did not show significant inhibition compared to scrambled BNA or the solvent control (Figure 7). However, miR-21 and MYCC BNA suppressed proliferation by more than 50% after 48 hours. Therefore, BNA oligonucleotides were unexpectedly more effective than FANA oligonucleotides, which are different scaffold derivatives being developed for therapeutic use in other laboratories.

[0144] In seven human triple-negative breast cancer cell lines, anti-miR-21-mediated inhibition of cell proliferation was observed in IC. 50 A significant correlation was observed between the value and the miR-21 copy number / cell count, consistent with our hypothesized mechanism of action. The non-tumor-forming human mammary epithelial cell line MCF-10A was significantly less sensitive to anti-miR-21 treatment (Figure 8).

[0145] The 50 nM miR-21 blocker BND5412 also significantly induced apoptosis in human MDA-MB-231 triple-negative breast cancer cells as measured by the LDH assay (Figure 9). Similar results were observed in human HCC1937, HCC1806, BT-549, and BT-20 breast cancer cells. Furthermore, treatment with the 50 nM miR-21 blocker BND5412 reduced the number of MDA-MB-231 cells migrating to the wound area compared to the randomly sequenced control.

[0146] Furthermore, the 50 nM miR-21 blocker BND5412 significantly reduced the immune checkpoint mRNAs of the immune checkpoint molecules PD-L1, PD-L2, CD47, and JAK2 (Figure 10), and also reduced the corresponding PD-L1, PD-L2, CD47, and JAK2 immune checkpoint proteins (Figures 5A-5B) in human HCC1806 triple-negative breast cancer (TNBC) cells. This suggests that inhibition of miR-21 may induce T cell recognition of TNBC cells.

[0147] RNA expression profile after miR-21 blockade. RNA-seq analysis was performed on RNA samples from human HCC1806 TNBC cells transfected with the miR-21 blocker BND5412 at IC90 concentration, and from vehicle-treated cells. To globally assess changes in miR-21-regulated transcripts, a list of predicted miR-21 target genes was obtained from TargetScan (https: / / www.targetscan.org / vert_80 / ). The Kolmogorov-Smirnov test, which compares the cumulative distribution of transcripts between miR-21 target genes and all other genes, showed a significant difference (Figure 11A), indicating that treatment with the miR-21 blocker BND5412 has a global on-target effect on miR-21-regulated transcripts.

[0148] Off-target effects in treated HCC1806 cells were evaluated using GGGenome (a web-based program at gggenome.dbcls.jp) for human splicing RNA containing 0-2 mismatches, insertions, or deletions from the miR-21 blocker BND5412 sequence. Over 300 RNA targets were predicted from the search results. Among these, transcripts containing 0-1 mismatches were not significantly downregulated by at least twofold. Eight genes containing two mismatches relative to the miR-21 blocker BND5412 were reduced at least twofold. Among these, SH3PXD2A, DIAPH2, PTPRK, MGAT5, and NLGN4X were identified as oncogenes. The remaining three genes, ERC1, ATRN, and FHOD3, were found not to be associated with any disease in the wild type when downregulated. No off-target effects with RNA levels changing more than fourfold were observed.

[0149] Gene set enrichment analysis using HallMark genes from the MSigDB dataset (GSEA, https: / / www.gsea-msigdb.org / gsea / index.jsp) identified the top three enrichment pathways for differentially expressed genes. The enrichment results showed that miR-21 blockade activates interferon-α and interferon-γ responses (Figure 11B), both of which have a positive effect on antitumor immunity (Jorgovanovic, et al. 2020, Biomark Res 8:49; Vidal, P., 2020, Scand J Immunol 91(5):e12863). In addition, the KRAS inhibition pathway was also significantly enriched in anti-miR-21 BND5412 treated samples (Figure 11B). Disease enrichment analysis using the iDEP platform with the Jensen Disease database (http: / / bioinformatics.sdstate.edu / idep96 / ) showed that breast cancer was significantly enriched among the downregulated genes. Furthermore, cancers that were highly enriched in the downregulated genes included kidney cancer, liver cancer, melanoma, pancreatic cancer, and malignant glioma. Other diseases that were significantly enriched included Alzheimer's disease, schizophrenia, cardiac conduction disorders, obesity, and acquired metabolic disorders such as type 2 diabetes. Upregulated genes did not show a strong association with disease. These results suggest that miR-21 blockade may be effective for a wider range of indications.

[0150] Cell-specific delivery Direct targeting of therapeutic agents is a major challenge in RNA analog therapy. While current delivery approaches using liposomes and nanoparticles are available, they suffer from low delivery efficiency, toxicity, and primary in vivo distribution to the liver and kidneys, making them unsuitable for long-term clinical use. Our design provides safe and efficient extrahepatic delivery of nucleic acid therapeutics. By conjugating the RNA analog to a receptor-targeting peptide (Figure 12), selective delivery to cell type becomes possible. IGF1R is elevated in aggressive breast cancer, including TNBC. Most importantly, TNBC cells exhibit strong IGF1R signaling activation, which correlates with low survival rates. However, signaling feedback between miR-21 and IGF1R is unknown (Dobre, et al. Cells 10(8):1856, 2021).

[0151] In previous research, we explored polyamide nucleic acid (PNA) oligomers with a protease-resistant retroinverso-cyclized D(CSKC) tetrapeptide IGF1 analog at the C-terminus of PNA to induce endocytosis into IGF1R-overexpressing cells. See, for example, Tian, ​​X et al. Journal of Nuclear Medicine vol. 48 (10): 1699-1707, 2007. We found that radiolabeled PNA 12mers with cyclo-D(CSKC) at the C-terminus remain in circulation by complexing with IGF1BP (Opitz, et al., Oligonucleotides 20(3):117-25, 2010), extending the drug's lifespan in the body and reducing the required dose. 99mUrine from mice injected with Tc]MYCC PNA-cyclo-D (CSKC) showed 83% of the radioactivity in intact probe peaks, demonstrating in vivo stability. We observed receptor-mediated knockdown of cyclin D1 protein by PNA-cyclo-D (CSKC) in MCF7 breast cancer xenografts. We also used cyclo-D (CSKC) for tissue-specific delivery of various radioimaging agents. In mice with ER+ breast cancer xenografts, HER2+ breast cancer xenografts, pancreatic cancer xenografts, and transgenic mice with spontaneously occurring mammary or lung tumors, we were able to image CCND1, MYCC, HER2, or KRAS2 mRNA using radionuclide-PNA-cyclo-D (CSKC) PET agents and demonstrate a DOX response due to a decrease in HER2 mRNA PET SUV. Importantly, specific PET imaging was inhibited by excess IGF1, which is consistent with our hypothesis of intracellular uptake via IGF1R-mediated endocytosis. When HER2-positive breast cancer xenografts were treated with doxorubicin (DOX), the HER2 mRNA SUV decreased to 54±17% one week after DOX treatment. This is consistent with a decrease of 80±47% in CT volume at 7 weeks. In contrast, HER2 mRNA SUV increased to 145±82% one week after untreated patients, and the CT volume increased to 213±78% at 7 weeks.

[0152] The newly designed miRNA inhibitor (Figure 13) minimizes and avoids passenger strand mimicry by removing the seed region of the counterchain. Thus, unlike the LNA inhibitors described in previous studies, the miR inhibitory nucleic acid-peptide composition effectively blocks the target miRNA without exerting counterchain function. As shown in Figure 13, human HCC1806 TNBC cells took up fluorescent AF647-miR-21 BNA-d(CSKC)BND7673 without transfection and transported it to the cytoplasm, demonstrating efficient IGF1R-mediated endocytosis and cytoplasmic delivery (Figure 14).

[0153] Cell-specific activity of non-fluorescent drugs. A non-fluorescent anti-miR-21 RNA peptide analog, BND6482 (Figure 15), was synthesized and taken up by TNBC cells via endocytosis. Similar to the fluorescent BND7673, the internalization and function of this inhibitory blocking molecule effectively increased the expression of desirable tumor suppressor proteins, thereby inhibiting TNBC cell proliferation. BND6482 dose-dependently released miR-21 luciferase reporter vector expression in high-IGF1R-expressing human HCC1806 TNBC cells without transfection (Figure 16A). In contrast, low-IGF1R-expressing human MDA-MB-157 TNBC cells were largely unresponsive to the miR-21 blocker BND6482 without lipofection (Figure 16B).

[0154] Example 2 Uptake of miR-21 RNA-peptide analog in EMT6 grafts. When immunodeficient female Balb / c mice carrying a mouse EMT6 TNBC graft were administered a single intraperitoneal dose of 5 mg / kg of the fluorescent miR-21 blocker BND7673, efficient distribution of the fluorescent drug to the tumor was evident within 24 hours and persisted for at least 96 hours (Figure 17).

[0155] Three-day response to TNBC grafts. Tumor suppression with the fluorescent miR-21 blocker BND7673 at 5 mg / kg was observed after daily intraperitoneal administration for 3 days (Figure 18). Several treated tumors were clearly significantly smaller than vehicle tumors. Fluorescence imaging showed drug concentrations in treated tumors extracted from each animal. Tumor images (Figure 18) showed differences between treated tumors and vehicle tumors, but widespread scattering of measured mass after only 3 days of treatment (Figure 19) indicated only a mean 28% reduction in tumor mass. Consistent with our hypothesis, BND7673 treatment reduced miR-21 and increased miR-21 target transcript PDCD4 mRNA in the tumors (Figure 20).

[0156] TNBC graft response on day 13. Subsequently, a 2-week long-term study was conducted using the non-fluorescent anti-miR-21 BNA peptide BND6482 (Figure 15). After the mouse EMT6 TNBC allograft reached a diameter of approximately 5 mm, scrambled BNA peptide BND6372 or anti-miR-21 BNA peptide BND6482 was administered intraperitoneally at a dose of 5 mg / kg twice a week for 2 weeks on days 0, 3, 7, and 10 in a sterile saline vehicle. Tumor volume was measured simultaneously with the injection. The tumor was excised on day 13 and its weight was measured.

[0157] Tumors treated with the miR-21 blocker BND6482 remained small with no apparent tumor progression over a two-week period (Figure 21). After two weeks, tumors treated with the miR-21 blocker showed a significantly reduced tumor volume (mean 74% reduction) compared to tumors treated with the vehicle or scramble (Figure 22).

[0158] Toxicity markers in serum samples of treated tumor-bearing mice were indistinguishable from each other by one-way ANOVA using Dunnett's multiple comparison test, after receiving 5 mg / kg of miR-21 blocker-peptide BND6482 or scrambled BND6372 twice weekly for 13 days, or after receiving 6.25 mg / kg three times via ANOVA. See Figure 23. Error bars indicate mean and SEM values. This result means that no hepatic or renal toxicity was detected as a result of RNA-peptide analog treatment.

[0159] As can be seen from the above, new molecular therapeutics and their variants that act alone or in combination with chemotherapy with higher efficacy, specificity, and safety are now available for targeted and effective TNBC therapy.

[0160] Example 3 Compositions and methods for improving symptoms associated with miR-21 regulated disorders, particularly TNBC. To treat individuals with miR-21 regulated disorders, particularly TNBC and other cancers, and to alleviate, for example, the signs or symptoms of the disease, the miR-21 inhibitory nucleoside-peptide analog inhibitor described in Example 1 can be administered alone or in combination with other agents useful for various symptoms associated with malignancies to provide therapeutic benefits to the patient. Such agents are administered in effective doses to modulate cancer cell proliferation, cell cycle checkpoints, cell migration, and metastasis. Those skilled in the art will understand that the therapeutic references in this invention extend not only to the treatment of established tumors or symptoms but also to prevention.

[0161] In one embodiment, a biological sample is obtained from a patient to confirm the presence and number of miR-21 copies / cell and IGF1R copies / cell in the tumor.

[0162] In some embodiments, this information may already be available. The total therapeutic dose can be administered to a subject as a single dose, or it can be administered using a divided therapy protocol, where multiple / separate doses are administered over a longer period, for example, over a period of one day to allow for the administration of a daily dose, or over a longer period to administer doses over a desired period. Those skilled in the art will know that the amount of miR-21 inhibitory nucleoside-peptide analog inhibitor required to obtain an effective and minimally toxic dose in a subject depends on many factors, including the subject's age, weight, and general health, as well as the route of administration and the number of treatments to be administered. Taking these factors into account, those skilled in the art will adjust the specific dose to obtain an effective dose for treating individuals with cancer treatable with the miR-21 inhibitory nucleoside-peptide analog inhibitor described herein.

[0163] In individuals suffering from cancer, particularly more severe and advanced cancer, administration of the miR-21 inhibitory nucleic acid-peptide analog inhibitors described in the present invention, when administered in combination with other modified nucleic acid oligonucleotides or nucleotide peptide analogs, is particularly useful and includes, but is not limited to, compounds targeting mRNA encoding c-Myc transcription factor, tropomyosin kinase receptor (TRK), MAPK pathway, androgen receptor (AR) pathway, growth factor receptor pathway, PI3K-AKT pathway, immune checkpoints, DNA damage repair pathway, CDK4 / 6, CHK1, CHK2, WEE1, and ATR, and / or cancer symptoms are reduced compared to a control. Therefore, in some embodiments, the method of the present invention includes the administration of further chemotherapeutic agents or chemotherapy. In some embodiments, additional therapies include surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, and / or hormone therapy. In some embodiments, the chemotherapeutic agent is an alkylating agent, an antimetabolic antineoplastic agent, an antitumor antibiotic, an antitumor plant agent, a platinum compound antineoplastic agent, a hormone-balancing antineoplastic agent, and a miscellaneous antineoplastic agent. For example, the inhibitors described may act additively or synergistically with chemotherapeutic agents for treating and inhibiting the proliferation of cancer cells. Such drugs include, but are not limited to, rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, sorafenib, bementinib, crizotinib, bosutinib, gilteritinib, amvatinib, sunitinib, cabozantinib, foretinib, levastinib, cerastrol, dihydroartemisinin, PD-1 inhibitor, PD-L1 inhibitor, CTLA4 inhibitor, PARP inhibitor, cyclophosphamide, ifosfamide, thiotepa, methotrexate, mercaptopurine, fluorouracil / cytarabine, bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin, mitoxantrone, These include vincristine, etoposide, teniposide, paclitaxel and docetaxel, cisplatin, carboplatin and oxaliplatin, uprolide, tamoxifen, flutamide and hormistan, and arsenic trioxide. In some embodiments, the tumor shrinks or is eradicated.

[0164] The inhibitors described may act additively or synergistically with PARP inhibitors to treat and inhibit the proliferation of cancer cells. Examples of PARP inhibitors include, but are not limited to, orlaparib, talazoparib, veliparib, lucaparib, niraparib, pamiparib, and fluzoparib.

[0165] The inhibitors described may act additively or synergistically with antibody-drug conjugates (ADCs) for treating and inhibiting the growth of cancer cells. Examples of ADCs include, but are not limited to, sacituzumab govitecan, radilatuzumab vedotin, patrizumab deruxtecan, trastuzumab deluxtecan, datapotamab deruxtecan, enfortamab vedotin, SKB264, MGC018, and PTK7-ADC.

[0166] The inhibitors described may act additively or synergistically with PI3K-ACT pathway inhibitors for the treatment and inhibition of cancer cell proliferation. Examples of PI3K-ACT pathway inhibitors include, but are not limited to, alpelisib, taselicib, samotricib, copanlicib, eganericib, and gedatricib.

[0167] The inhibitors described may act additively or synergistically with androgen receptor inhibitors to treat and inhibit the proliferation of cancer cells. Examples of androgen receptor inhibitors include, but are not limited to, bicalutamide, enzalutamide, abiraterone, enobotherm, and darolutamide.

[0168] The inhibitors described may act additively or synergistically with TRK inhibitors for the treatment and inhibition of cancer cell proliferation. Examples of TRK inhibitors include, but are not limited to, larotrectinib, ceritrectinib, and repotrectinib.

[0169] The inhibitors described may act additively or synergistically with mutant Her2 inhibitors for the treatment and inhibition of cancer cell proliferation. Examples of mutant Her2 inhibitors include, but are not limited to, neratinib.

[0170] The inhibitors described may act additively or synergistically with immune checkpoint inhibitors to treat and inhibit the proliferation of cancer cells. Examples of immune checkpoint inhibitors include, but are not limited to, pembrolizumab, atezolizumab, avelumab, JS001, nivolumab, and duramab.

[0171] The inhibitors described may act additively or synergistically with CDK4 / 6 inhibitors for the treatment and inhibition of cancer cell proliferation. Examples of CDK4 / 6 inhibitors include, but are not limited to, palbociclib, abemaciclib, and ribociclib.

[0172] The inhibitors described may act additively or synergistically with CHK1 inhibitors to treat and inhibit the proliferation of cancer cells. Examples of CHK1 inhibitors include, but are not limited to, LY2880070 and prexasertib.

[0173] The inhibitors described may act additively or synergistically with WEE1 inhibitors for treating and inhibiting the proliferation of cancer cells. Examples of WEE1 inhibitors include, but are not limited to, AZD1175 and ZN-c3.

[0174] The inhibitors described may act additively or synergistically with CHK2 inhibitors to treat and inhibit the proliferation of cancer cells. Examples of CHK2 inhibitors include, but are not limited to, LY2606368.

[0175] The inhibitors described may act additively or synergistically with ATR inhibitors for treating and inhibiting the proliferation of cancer cells. Examples of ATR inhibitors include, but are not limited to, ceracertib.

[0176] The inhibitors described may act additively or synergistically with RAD51 inhibitors to treat and inhibit the growth of cancer cells. Examples of RAD51 inhibitors include, but are not limited to, CYT-0851.

[0177] Those skilled in the art would administer the miR-21 inhibitor alone or in combination with at least one chemotherapeutic agent and monitor the effectiveness of such treatment using routine methods such as radiological, immunological assays, or, if instructed, histopathological methods.

[0178] The administration of a pharmaceutical preparation is preferably an "effective dose," which is a sufficient amount to show a benefit to the individual. This dose prevents, alleviates, reduces or otherwise diminishes the severity of cancer in the patient.

[0179] In a preferred embodiment of the present invention, a method for treating cancer is provided that uses the therapeutic agents disclosed in this embodiment in a combinatorial approach. Advantageously, the additive or preferably synergistic methods of this invention reduce cancer progression or alleviate cancer symptoms in mammalian hosts. Importantly, the information provided in this invention will guide clinicians in new therapies for the management of this disease.

[0180] The present invention also encompasses pharmaceutical compositions useful for the treatment of cancer, comprising administering one or more of the above-mentioned mimetics in a therapeutically effective amount in a pharmaceutically acceptable carrier or diluent. Compositions comprising the miR-21 inhibitor of the present invention can be administered orally or parenterally, including via intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, and topical administration routes.

[0181] Generally, the above compounds do not need to be administered in the same pharmaceutical composition, and may need to be administered via different routes due to their differing physical and chemical properties. For example, the first compound can be administered orally to produce and maintain a good blood concentration, while the second compound can be administered intravenously. The determination of the administration method and, if possible, whether to administer them in the same pharmaceutical composition is within the scope of the knowledge of those skilled in the art. The initial administration can be carried out according to established protocols known in the art, and thereafter, the dosage, mode of administration, and administration time can be modified by clinicians skilled in the art based on the observed effects.

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[0183] While specific preferred embodiments of the present invention have been described and illustrated above, the present invention is not intended to be limited to such embodiments. Various modifications can be made without departing from the scope and spirit of the invention, as set forth in the following claims.

Claims

1. A miR-21 inhibitory nucleic acid-peptide analog having a sequence complementary to miR-21-5p, wherein miR-21-5p and its isomia are isolated from binding to regulatory sites present in mRNA.

2. The miR-21 inhibitory nucleic acid-peptide analog according to claim 1, wherein the nucleic acid has modifications selected from BNA, LNA, FANA, PNA, 2'-fluoro, 2'-O-alkyl, morpholino, piperazine, phosphorothioate, boranophosphate, and boranophosphate combined with a phosphodiester bond, phosphorodithioate bond, or methylphosphonate bond.

3. A miR-21 inhibitory nucleic acid-peptide analog according to claim 1 or claim 2, comprising at least one inhibitory sequence shown in Figures 2B to 2C.

4. The miR-21 inhibitory nucleic acid-peptide analog according to claim 3, wherein at least one inhibitory nucleic acid sequence of (SEQ ID NO: 5) reduces undesirable side effects associated with mimicking miR-21-3p passenger strand function by substantially eliminating the miR-21-3p passenger strand seed region.

5. The miR-21 inhibitory nucleic acid-peptide analog according to claim 3, wherein at least nucleoside 3, 4, 5, 6, 7, 8, or 9 of SEQ ID NO: 4 is removed.

6. A miR-21 inhibitory nucleic acid-peptide analog according to any one of claims 1 to 5, comprising the 5' and 3' BNA modifications shown in Figure 15.

7. The miR-21 inhibitory nucleic acid-peptide analog according to claim 1, wherein the cyclic peptide is selected from CSKC, CRKC, CVKC, CGKC, CKGC, CFKC, CDKC, CHRC, CRVC, CGRC, CIRC, CQRC, CTRC, CRHC, CRGC, CRSC, CRKC, CSRC, and CERC, wherein all residues are D-amino acids and a disulfide bond is formed between the N-terminal cysteine ​​and the C-terminal cysteine.

8. The miR-21 inhibitory nucleic acid-peptide analog according to claim 1, wherein the peptide is CSKC, which is a ligand for the insulin-like growth factor 1 receptor of SEQ ID NO: 26, and the inhibitory nucleic acid sequence is selected from SEQ ID NOs: 5-25, GATAAGCTA, TGATAAGCT, CTGATAAGC, ATAAGCTA, GATAAGCT, and TGATAAGC.

9. The miR-21 inhibitory nucleic acid-peptide analog according to claim 1, wherein the nucleic acid comprises 8 to 17 nucleotides.

10. The miR-21 inhibitory nucleic acid-peptide analog according to claim 1, wherein the nucleic acid is a gapmer.

11. The miR-21 inhibitory nucleic acid-peptide analog according to claim 10, wherein the gapmer comprises three parts.

12. The miR-21 inhibitory nucleic acid-peptide analog according to claim 11, wherein the three parts comprise a first BNA part, a DNA part, and a second BNA part.

13. A method for inhibiting the binding of miR-21-5p to one or more binding sites in mRNA encoding proteins that regulate cell proliferation, migration, metastasis, stress, or inflammation, comprising contacting miR-21-5p with an inhibitor according to any one of claims 1 to 12, wherein the inhibitor binds to and sequestrates the miR-21-5p, thereby inhibiting or preventing the binding of miR-21-5p to regulatory sites present in mRNA encoding proteins that regulate the proliferation and / or metastasis of malignant cells.

14. A method for treating triple-negative breast cancer (TNBC) in a patient requiring such treatment, comprising administering an effective amount of a miR-21 inhibitory nucleic acid-peptide analog according to any one of claims 1 to 12, wherein the analog causes stasis or cell death of the TNBC.

15. The method according to any one of claims 13 or 14, wherein the analog is shown in Figure 15.

16. Rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, sorafenib, bementinib, crizotinib, bosutinib, gilteritinib, amvatinib, and sunitinib, cabozantinib, foretinib, levatinib, cerastrol, dihydroartemisinin, PD-1 inhibitor, PD-L1 inhibitor, CTLA4 inhibitor, cyclophosphamide, ifosfamide, thiotepa, methotrexate, mercaptopurine The method according to any one of claims 13, 14, or 15, further comprising the administration of a chemotherapeutic agent selected from fluorouracil and cytarabine, bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin, mitoxantrone, vincristine, etoposide, teniposide, paclitaxel and docetaxel, cisplatin, carboplatin and oxaliplatin, uprolide, tamoxifen, flutamide and formestane, and arsenic trioxide.

17. The method according to claim 13, 14, or 15, wherein the inhibitor treatment reduces disease-driven protein levels.

18. The method according to any one of claims 13, 14, or 15, wherein the inhibitor treatment increases the disease-limiting protein level.

19. The method according to any one of claims 13, 14, or 15, wherein the disease restriction mRNA encodes a tumor suppressor protein.

20. The method according to any one of claims 13, 14, or 15, wherein the disease-driven or disease-restricting mRNA encodes the protein shown in Figure 1.

21. The method according to claim 19, wherein the tumor suppressor protein is selected from PTEN and PDCD4.

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