Compositions and methods for treating cancer

Abstract

Compositions and methods for the treatment of conditions associated with miR-21 disorder, in particular cancer, are disclosed.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 490,184, filed March 14, 2023, the entire contents of which are incorporated herein by reference as if fully set forth herein.

[0003] Statement regarding federally funded research or development

[0004] This invention was made with government support under NIH license 1 R41 CA 235707-01A1. The government owns certain rights to this invention.

[0005] Materials submitted electronically and incorporated by reference

[0006] The contents of the electronic serial number (BDT-102.xml; size: 23,436 bytes; dated February 14, 2023) are incorporated herein by reference in their entirety. Technical Field

[0007] This invention relates to the fields of oncology and medicinal chemistry. More specifically, it provides compositions comprising complementary microRNA-21 (miR-21) oligonucleotide analogs, said oligonucleotide analogs inhibiting the binding of miR-21 and its isomiR to miR-21 binding sites in 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 miR-21 complementary oligonucleotide analogs are conjugated to ligands that guide cancer cell uptake. Methods for modulating the expression of such proteins to provide therapeutic benefits are disclosed. Background Technology

[0008] Throughout this specification, numerous publications and patent documents have been cited to describe the current state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as if fully explained.

[0009] Triple-negative breast cancer (TNBC) is a rare disease that attacks 46,000 U.S. women annually, primarily in young women of African, Asian, Hispanic, or BRCA1-mutated ancestry. TNBC cells lack human estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor 2 (Her2), all of which are targets of many existing drugs. Recently explored treatment options for TNBC include anti-angiogenic agents, poly(ADP-ribose) polymerase (PARP) inhibitors, checkpoint inhibitors, and antibody-drug conjugates. However, due to poor clinical outcomes and safety concerns, the FDA withdrew approval for the anti-angiogenic agent bevacizumab for breast cancer. Similarly, Roche voluntarily withdrew Tecentriq, an anti-PD-L1 antibody for TNBC, due to unsatisfactory results. Trodelvy, a recently approved antibody-drug conjugate, only increased overall survival in TNBC by 3 months. Furthermore, only a small percentage of patients with BRCA1 / 2 mutations benefit from PARP inhibitors, while checkpoint inhibitors show only limited efficacy in TNBC patients with elevated PD-L1 levels. Therefore, despite being fraught with side effects and offering a median survival of 4 years, chemotherapy and radiation remain the standard of care.

[0010] MicroRNAs (miRNAs) and their isoforms miRs are small non-coding RNA molecules, approximately 22 nucleotides in length, that regulate gene translation by silencing or degrading target mRNAs. They participate in a variety of biological processes, including differentiation and proliferation, metabolism, hemostasis, apoptosis, and inflammation, and are involved in the pathophysiology of many diseases. Numerous studies have shown that circulating miRNAs are promising biomarkers for the diagnosis and prognosis of many diseases. TNBC cells exhibit high levels of oncogenic miRNAs, or oncomiRs, which are 18-25 nucleotide (nt) non-protein-coding RNAs that form base pairs with specific sequences in the mRNA. They inhibit mRNA translation spatially or by inducing mRNA degradation with Ago2. The biogenesis of all miRNAs begins in the cell nucleus, where primary miRNAs are transcribed by RNA polymerase II or RNA polymerase III. The primary miRNA transcripts are then processed by Drosha and its cofactor DGCR8 to produce shorter precursor miRNA hairpins of approximately 70 nt. The pre-miRNA hairpin is exported to the cytoplasm via exportin 5, and then cleaved by Dicer to produce a double-stranded miRNA. The guide strand of the double-stranded miRNA is thought to exhibit a weak hydrogen bond at its 5' end, which facilitates its binding to Ago2 in the RNA-induced silencing complex (RISC), thereby making the guide strand active against complementary mRNAs. Therapeutic targeting of this oncomiR can bypass cancer heterogeneity because a single miRNA can simultaneously regulate different target mRNA molecules, thereby modulating the expression and function of multiple gene networks.

[0011] Due to limited applicability and various drawbacks associated with existing treatment options, there remains a need in the art for compositions, methods, regimens, and kits that provide greater potency, specificity, and safety for molecularly targeted therapies for TNBC. This invention addresses this need. Summary of the Invention

[0012] This invention provides compositions and methods for treating triple-negative breast cancer. In one embodiment, a miR-21 repressive nucleic acid-peptide analog with sequence complementarity to miRNA-21-5p is provided, which sequesters the binding of miR-21-5p and its isomeric miRs to regulatory sites present in mRNA. In some embodiments, the miR-21 repressive nucleic acid-peptide analog comprises a modification selected from BNA, LNA, FANA, PNA, 2'-fluoro, 2'-O-alkyl, morpholino, piperazine, thiophosphate, borophosphate, and borophosphate mixed with a phosphodiester bond, dithiophosphate bond, or methylphosphonate bond. In other embodiments, the miR-21 repressive nucleic acid-peptide analog comprises at least one of the following: Figure 2BOr the inhibitory sequence shown in 2C, such as SEQ ID NO: 6-25 and the shown 9-base (9 mer) and 8-base sequences. In a preferred embodiment, the miR-21 inhibitory peptide analog comprises SEQ ID NO: 5. In a preferred embodiment, the inhibitory sequence comprises at least one, preferably two, 5' and 3' BNA modifications, such as Figure 15 As shown. In some embodiments, the antisense sequence includes a deoxyribonucleic acid (DNA) portion and at least one bridging nucleic acid (BNA) portion, such as, but not limited to, aminomethyl BNA 2'4'-BNA. NC For example, in some embodiments, the antisense sequence is a gapmer having a BNA-DNA-BNA structure. In some embodiments, each of the BNA portions and the DNA portion comprises the same number of nucleotides. For example, in one embodiment, the antisense sequence comprises 15 nucleotides having a 5-5-5 gapmer structure. In another embodiment, the gapmer comprises SEQ ID NO: 5. Alternatively, in some embodiments, the DNA portion comprises a different number of nucleotides compared to at least one BNA portion. For example, in one embodiment, the antisense sequence comprises 13 nucleotides having a 4-5-4 gapmer structure.

[0013] miR-21 inhibitory peptide analogs may also contain cyclic peptides selected from CSKC, CRKC, CVKC, CGKC, CKGC, CFKC, CDKC, CHRC, CRVC, CGRC, CIRC, CQRC, CTRC, CRHC, CRGC, CRSC, CRKC, CSRC, or CERC, wherein all residues are D-amino acids and a disulfide bond is formed between the N-terminal cysteine ​​and the C-terminal cysteine.

[0014] miR-21 inhibitory peptide analogs may also include the peptide ligand CSKC targeting the insulin-like growth factor 1 receptor, SEQ ID NO: 26.

[0015] Methods for inhibiting miR-21 binding to one or more binding sites in mRNA encoding proteins that regulate cell proliferation, migration, metastasis, stress, or inflammation are also provided. An exemplary method includes contacting miR-21-5p with an inhibitor described herein, wherein the inhibitor binds to and isolates the miR-21-5p, thereby preventing miR-21-5p from binding to regulatory sites present in mRNA encoding proteins that regulate malignant cell growth and / or metastasis. In a preferred embodiment, methods for treating triple-negative breast cancer (TNBC) in patients of need are disclosed. An exemplary method includes administering an effective amount of the miR-21 inhibitory peptide analog described above, wherein the analog causes TNBC arrest or cell death. In a preferred embodiment, the analog is as follows: Figure 15 As shown.

[0016] The method of the present invention may further include administering a chemotherapy agent selected from the following: rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, sorafenib, bemcentinib, crizotinib, bosutinib, gilteritinib, amuvatinib, sunitinib, cabozantinib, and freundilamide. Foretinib, rebastinib, celastrol, dihydroartemisinin, PD-1 inhibitors, PD-L1 inhibitors, CTLA4 inhibitors, cyclophosphamide, ifosfamide, thiotepa, methotrexate, mercaptopurine, fluorouracil, cytarabine, bleomycin, daunorubicin, actinomycin D (actinomycin D), mitomycin, doxorubicin, mitoxantrone, vincristine, docetaxel, teniposide, paclitaxel and docetaxel, cisplatin, carboplatin and oxaliplatin, euprolide, tamoxifen, flutamide and formestane, and arsenic trioxide.

[0017] This invention also provides compositions and methods for effectively reducing or increasing levels of disease-driving proteins (see Figure 1), thereby providing therapeutic benefits to patients receiving treatment. In other embodiments, the compositions and methods target the expression of tumor suppressor proteins. In other embodiments, the tumor suppressor gene is selected from PTEN and PDCD4. Attached Figure Description

[0018] Figure 1A-1J :( Figure 1A The diagram illustrates numerous different signal transduction and disease pathways that provide targets for the miR-21-directing agents described below. Figure 1B-1J (A schematic diagram of the different ligands used to deliver the complementary miR-21 oligonucleotide analogues described herein.)

[0019] Figure 2A-2D :( Figure 1A The hairpin structure of the pre-miRNA miR-21 (SEQ ID NO: 1). Figure 2B-2C The image shows the miR-21 oligonucleotide sequence. The pre-miRNA hairpin structure comprises a miR-21-5p guide strand (top magenta sequence) and a miR-21-3p guest strand (bottom magenta sequence). Most nucleotides in the miR-21-5p guide strand are complementary to the miR-21-3p guest strand. Nucleosides to be excluded in the guest strand seed sequence are shown in blue in SEQ ID NO: 4. Either the guide strand or the guest strand can be selected as the active miRNA in the RNA-induced silencing complex (RISC). SEQ ID NO: 5 to 25, along with three 9-base sequences and three 8-base sequences, provide additional anti-miR-21-5p sequences ranging in length from 8 to 17 bases, which should effectively hybridize to the target. SEQ ID NO: 5 is a 15-base sequence, in Figure 2B The text is in bold. Figure 2D A schematic diagram of the miR-21 oligonucleotide-peptide blocking mechanism. The miR-21 inhibitory nucleic acid binds to the mature miR-21-5p guide strand, preventing it from binding to the regulatory site in the 3'UTR of the target mRNA.

[0020] Figure 3 DNA / RNA analogs used to increase stability, binding affinity, and specificity. Schematic diagram of DNA phosphate thioester (PS), methylphosphonate (MP), 2'-4'-locked nucleic acid (LNA), 2'-aminomethylene-bridging nucleic acid (NC-BNA), and polyamide nucleic acid (PNA).

[0021] Figures 4A-4B :( Figure 4A The structure of BND5412 is described; BND5412 is a 15 nt miR-21 inhibitor, BNA-DNA-BNA phosphate thioester. The short BNA sequence lacks the seed sequence for the corresponding miR-21 transit chain, thus avoiding transit chain simulation. Figure 4B IC of miR-21 blocker BND5412 50BND5412 was transfected into human MDA-MB-231 TNBC cells to induce derepression of the Renilla luciferase reporter vector containing a miR-21 binding site inserted into the 3'UTR of the luciferase gene. Error bar, sd.

[0022] Figures 5A-5B :( Figure 5A Western blot analysis of protein expression ± SD was performed after transfecting human MDA-MB-231 TNBC cells with 50 nM miR-21 inhibitor BND5412 for 48 h (PDCD4) or 72 h (other proteins). Figure 5B Western blot of immune checkpoint proteins in human HCC1806 TNBC cells 72 h after transfection with 50 nM miR-21 inhibitor BND5412.

[0023] Figure 6 Cell Titer Glo assay ± sem was performed on day 0 after transfection of 50 nM miR-21 inhibitor BND5412 into seven different TNBC cell lines.

[0024] Figure 7 Comparison of FANA oligonucleotides with three BNA oligonucleotides of known sequences. FANA sequences 1-7 did not show significant inhibitory effects compared to scrambled BNA or vehicle controls. Cell viability was measured using Cell-Titer Glo assay. Error bars, sd, n = 3.

[0025] Figure 8 In seven human TNBC cell lines and non-tumorigenic mammary epithelial cell lines transfected with the miR-21 inhibitor BND5412 at varying concentrations, the cell proliferation IC50 of the miR-21 inhibitor BND5412 was [not specified]. 50 Correlation between miR-21 copy number / cell.

[0026] Figure 9 LDH ± sd was measured 72 h after transfection with the 50 nM miR-21 inhibitor BND5412 to test apoptosis in human MDA-MB-231 TNBC cells.

[0027] Figure 10PD-L1, PD-L2, CD47, and JAK2 mRNA levels relative to GAPDH qPCR mRNA levels ± sem. These mRNAs were extracted from human HCC1806 TNBC cells transfected with the vector, 50 nM out-of-order inhibitor (purple), or 50 nM miR-21 inhibitor BND5412 (blue). Results were from 3 biological replicates, ± sem. Relative expression levels were normalized relative to the vector control. [* = p < 0.05, ** = p < 0.01, compared with the vector control by one-way t-test].

[0028] Figure 11A-11B :( Figure 11A In RNA-seq analysis of six biological replicates from human HCC1806TNBC cells transfected with the miR-21 inhibitor BND5412 at an IC90 concentration, the cumulative frequency distribution of miR-21 target genes (red) and all other differentially expressed genes (blue) [Kolmogorov-Smirnov test, p < 0.0001]. Figure 11B The top three enrichment pathways for differentially expressed genes from samples treated with miR-21 blockers.

[0029] Figure 12 The design of a miR-21 blocking conjugate of BNA-DNA-BNA gapmer and IGF1 peptide for the endocytosis of IGF1R on the cell surface, which is highly expressed on TNBC cells.

[0030] Figure 13 The structure of BND7673: BND7673 is an AF647-labeled miR-21 blocker, BNA-DNA-BNA phosphate thioester, coupled with an IGF1 peptide, used for IGF1R-mediated endocytosis. The short BNA sequence lacks the seed sequence for the corresponding miR-21 passchain, thus avoiding passchain mimicry.

[0031] Figure 14 Confocal fluorescence image of live human HCC1806 TNBC cells after 4 h in 100 nM Cal560-miR-21 BNA-IGF1 peptide. Green: LysoTracker. Red: Cal560. Yellow: Cal560-LysoTracker overlap.

[0032] Figure 15The structure of BND6482: BND6482 is a miR-21 blocker, BNA-DNA-BNA phosphate thioester-IGF1 peptide, used for IGF1R-mediated endocytosis. It lacks a fluorescent dye for distribution. The short BNA sequence lacks the seed sequence for the corresponding miR-21 transit chain, thus avoiding transit chain simulation.

[0033] Figures 16A-16B In the miR-21 luciferase reporter gene assay, in HCC1806 cells with high IGF1R expression ( Figure 16A ) and MDA-MB-157 cells with low IGF1R expression ( Figure 16B In the study, dose-dependent inhibition of miR-21 activity by lipid-free 50 nM miR-21 blocker BND6482 (blue) was measured compared to that by the 50 nM transfected miR-21 blocker BND6482 (red). Error bars, sd.

[0034] Figure 17 Distribution of the fluorescent miR-21 inhibitor BND767 over 2–96 hours in orthotopic allogeneic grafts of immunocompetent female Balb / c mice generated from syngeneic EMT6 TNBC cells. The distribution of the fluorescent miR-21 inhibitor BND767 over 2–96 hours following a single intraperitoneal injection of 5 mg / kg.

[0035] Figure 18 :exist Figure 17 Imaging of tumor growth inhibition in the TBC mouse model described in the article after intraperitoneal injection of fluorescent anti-miR-21-peptide BND7673 at 5 mg / kg once daily for 3 days, as seen in white light images (left) or fluorescent images (right) of the dissected tumor.

[0036] Figure 19 :exist Figure 17 In the TBC mouse model described, tumor growth was inhibited by intraperitoneal injection of the fluorescent antimiR-21 inhibitor peptide BND7673 at a daily dose of 5 mg / kg for 3 days. Individual tumor weights are shown as mean and by SEM.

[0037] Figure 20 The levels of miR-21 and PDCD4 mRNA in tumors from female Balb / c mice carrying syngeneic EMT6 TNBC allogeneic grafts were measured after three daily doses of the fluorescent miR-21 inhibitor peptide BND7673 at a dose of 5 mg / kg. Error bars indicate SEM.

[0038] Figure 21Following intraperitoneal injections of the miR-21 blocker peptide BND6482 twice weekly for 13 days, tumor volume in mouse EMT6TNBC orthotopic allogeneic grafts remained small. The mediator, out-of-order drug, and trodelvy allowed continued growth. Error bars show the mean versus SEM. *p<0.05, by one-way ANOVA using Dunnett's multiple comparison test.

[0039] Figure 22 Following intraperitoneal injection of 5 mg / kg miR-21 blocker peptide BND6482 twice weekly over 13 days, tumor mass of mouse EMT6TNBC orthotopic allogeneic grafts was significantly reduced. Error bars show the mean versus SEM. *p<0.05, by one-way ANOVA using Dunnett's multiple comparison test.

[0040] Figure 23 Toxicity markers in serum samples from treated tumor-bearing mice following intraperitoneal injections of the miR-21 blocker peptide BND6482 twice weekly for 13 days or three intraperitoneal injections of 6.25 mg / kg. Error bars show the mean versus SEM. *p<0.05, by one-way ANOVA using Dunnett's multiple comparison test. Detailed Implementation

[0041] This article describes the design and synthesis of miR-21-guided therapeutics. miR-21 is a 22 nt single-stranded RNA that is elevated in TNBC compared to adjacent normal tissue (Radojicic et al. 2011). Overexpression of miR-21 is commonly observed in breast cancer cell lines and tissues (Ozgun et al. 2013), affecting cell proliferation, cell cycle checkpoints, and metastasis (Anastasov et al. 2012). miR-21 expression in TNBC is also associated with poor clinical outcomes (Dong et al. 2014). The permachain miR-21-3p is upregulated in TNBC and is associated with chemotherapy resistance (Ouyang et al. 2014). Previously, a 19-base anti-miR-21 nucleic acid analog with specific backbone modifications has been patented for the treatment of liver disease. No use for the treatment of breast cancer has been disclosed (Bhat and Marcusson 2015).

[0042] definition

[0043] Before further describing the invention in a general sense and in various non-limiting specific embodiments, certain terms used in the context of describing the invention are set forth. Unless otherwise stated, the following terms have the following meanings when used herein and in the appended claims. Those terms not defined below or elsewhere in the specification should have their generally accepted meanings in the art.

[0044] As used herein, 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.

[0045] The terms “high,” “higher,” “increase,” “elevate,” or “elevation” refer to an increase above baseline levels, for example, compared to a control. The terms “low,” “lower,” “reduce,” or “reduction” refer to a decrease below baseline levels, for example, compared to a control.

[0046] As used herein, the term "modulation" refers to the ability of a compound to alter its activity in a measurable manner compared to a suitable control. Due to the presence of the compound in the assay, the activity may increase or decrease compared to a control in the absence of these compounds. Preferably, the activity increases by at least 25% compared to the activity level in the absence of the compound, more preferably at least 50%, and most preferably at least 100%. Similarly, the activity preferably decreases by at least 25% compared to the activity level in the absence of the compound, more preferably at least 50%, and most preferably at least 100%. Compounds that increase known activity are "agonists." Substances that decrease or inhibit known activity are "antagonists."

[0047] The term "inhibition" refers to a reduction or decrease in activity or expression. This can be complete inhibition of activity or expression, or partial inhibition. Inhibition can be compared to a control or standard level. Inhibition can be 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, 53, 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%.

[0048] The term “prevention” as used in this article refers to the administration of a compound prior to the onset of clinical symptoms of a disease or condition in order to prevent abnormal physical manifestations associated with the disease or condition.

[0049] As used herein, the term “requires treatment” refers to a judgment made by a caregiver (e.g., in the case of humans, a physician, nurse, registered nurse, or individual; in the case of animals (including non-human mammals), a veterinarian) that a subject requires or will benefit from treatment. This judgment is based on a number of factors within the caregiver’s area of ​​expertise, but includes knowledge that the subject is already ill or will become ill as a result of a condition that can be treated with the disclosed compound.

[0050] "Treatment" refers to the medical administration of a subject to cure, alleviate, stabilize, or prevent a disease, pathological condition, or symptom. This term includes active treatment, which is treatment specifically aimed at improving a disease, pathological condition, or symptom, and etiological treatment, which is treatment aimed at eliminating the cause of the associated disease, pathological condition, or symptom. Furthermore, this term includes palliative treatment, which aims to relieve symptoms rather than cure a disease, pathological condition, or symptom; preventive treatment, which aims to minimize or partially or completely suppress the development of an associated disease, pathological condition, or symptom; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving an associated disease, pathological condition, or symptom. Treatment effects can be measured or assessed as described herein and as known in the art, provided that it is applicable to cancer and its related pathologies.

[0051] "Cell" can be a cell from any organism, including but not limited to bacteria. Cells can be in vitro. Alternatively, cells can be in vivo and can be found in the subject.

[0052] "Pharmaceutical acceptable" means that the substance is not biologically or otherwise undesirable, meaning that the substance can be administered to a subject together with the selected compound without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition containing it.

[0053] After aligning sequences and introducing gaps (if necessary) to obtain the maximum percentage of sequence identity, and disregarding any conserved substitutions as part of sequence identity, the "percentage of sequence identity (%)" and "homology" for nucleic acid, peptide, polypeptide, or antibody sequences are defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to those in a specific nucleic acid or polypeptide sequence. Alignment for the purpose of determining the percentage of sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared.

[0054] As used herein, the terms “component,” “composition,” “composition of compounds,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” “therapeutic agent,” “treatment,” “treatment,” or “medicine” are used interchangeably to refer to a composition of one or more compounds or substances that, when administered to a subject (human or animal), induce a desired pharmacological and / or physiological effect through local and / or systemic action. The terms “agent” and “test compound” mean a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.

[0055] "Administration" 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 intravenous, intrapulmonary, oral, skin, or systemic administration.

[0056] As used herein, the term "combination therapy" is intended to define a therapy that includes the use of a combination of two or more compounds / pharmaceuticals (as defined above). Therefore, the terms "combination therapy," "combination," and "use of substances / pharmaceuticals in combination" as used in this application can refer to substances / pharmaceuticals administered as part of the same overall treatment regimen. Thus, each of the two or more substances / pharmaceuticals can be different: each can be administered at the same time or at different times. Therefore, it should be understood that substances / pharmaceuticals in a combination can be administered sequentially (e.g., before or after) or simultaneously in the same pharmaceutical formulation (i.e., together) or in different pharmaceutical formulations (i.e., individually). The simultaneous presence in the same formulation is a single formulation, while the simultaneous presence in different pharmaceutical formulations is a non-single formulation.

[0057] "Concomitant administration" refers to the administration of two or more substances / pharmaceuticals to a subject in a time-related manner, preferably in a sufficiently time-related manner, to provide modulation of a physiological or immune response, and even more preferably, the administration of two or more substances / pharmaceuticals in combination. In embodiments, concomitant administration may include the administration of two or more substances / pharmaceuticals within a specific time period, preferably within one month, more preferably within one week, even more preferably within one day, and even more preferably within one hour. In embodiments, the substances / pharmaceuticals may be repeatedly administered concomitantly, i.e., administered more than once, as may be provided in the examples.

[0058] The phrase “disease-driving protein level” refers to the amount of cellular proteins that accelerate cell division, such as receptors, kinases, transcription factors, or any member of growth signaling pathways.

[0059] The phrase “disease-limiting protein levels” refers to the amount of cellular proteins that inhibit cell division, such as receptors, phosphatases, proteases, tumor suppressor factors, transcriptional repressors, or any member of arrest or apoptosis signaling pathways.

[0060] The phrase "effective amount" or "therapeutic effective amount" refers to the amount of a drug agent sufficient to achieve a beneficial or desired outcome. Therapeutic effective amounts can vary depending on one or more of the following factors: the treated subject and disease condition, the subject's weight and age, the severity of the disease condition, the method of administration, etc., factors that can be readily determined by one of ordinary skill in the art. The term also applies to the dose used to provide an image for detection via any of the imaging methods described herein. The specific dose can vary depending on one or more of the following factors: the particular drug agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system carrying the drug.

[0061] "Pharmaceutically acceptable excipients" or "pharmaceutically acceptable carriers" refer to pharmacologically inactive substances used together with pharmacologically active substances to formulate compositions. Pharmaceutically acceptable excipients include a variety of substances known in the art, including but not limited to sugars (such as glucose, lactose, etc.), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline solutions (such as phosphate-buffered saline), and buffer solutions.

[0062] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to an animal, such as a human, to which the pharmaceutical compositions of the present invention are provided for treatment (including preventative treatment). The term “subject” as used herein refers to both human and non-human animals. The terms “non-human animal” and “non-human mammal” are used interchangeably herein and include all vertebrates, such as mammals like 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.

[0063] The terms "polynucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

[0064] The term "microRNA (miRNA)" refers to small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in many species and some viruses, miRNAs are involved in post-transcriptional regulation of RNA silencing and gene expression.

[0065] "Heterogeneous miRs" are miRNA sequences that have certain variations relative to the reference miRNA sequence, but which can bind to Ago proteins and play a similar role in gene expression regulation as their standard miRNAs. See, for example, Kuchenbauer, et al. (2008) Genome Research 18:1787–1797.

[0066] An "antisense oligonucleotide or strand" is an oligonucleotide that is complementary to a sense oligonucleotide, pre-mRNA, RNA, or sense strand of a specific gene and binds to these genes and gene products through base pairing. Examples of base-paired nucleotides include, but are not limited to: uracil, thymine, adenine, cytosine, guanine, and hypoxanthine, whose respective amino groups are protected by acyl protecting groups; 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil; and other modified nucleobases such as 8-substituted purines, xanthines, or hypoxanthines. Further examples of base-paired nucleotides include, but are not limited to, nucleobases with one or more enlarged benzene rings. When bound to a sense oligonucleotide, the antisense oligonucleotide does not need to pair with every nucleotide base in the sense oligonucleotide.

[0067] "Tm," or melting temperature, is the midpoint of the temperature range at which the oligonucleotide separates from the target nucleotide sequence. At this temperature, 50% of the helical (hybridization) pairs exist in coiled (unhybridized) form. Tm is measured by determining the formation and breakdown (melting) of hybridization using ultraviolet absorption spectroscopy. Techniques well-known in the art can be used to determine Tm. Alternative formulas are also available for estimating Tm based on sequence and common chemical modifications, if any.

[0068] "Gene target" or "target gene" refers to a gene that has an RNA transcript (processed or unprocessed) containing a nucleic acid sequence including miR-21, and is therefore capable of being bound by the miR-21 repressive RNA-peptide analog described herein, and regulating the expression of proteins encoded by mRNA containing a miR-21 binding site.

[0069] As used herein, a "modified nucleotide" refers to a non-naturally occurring portion that, compared to a polynucleotide that differs from a repressive nucleic acid simply by replacing the modified nucleotide with a natural nucleotide, confers increased nuclease resistance or thermodynamic stability during hybridization. In some embodiments, the ribose moiety of the nucleotide is modified with an additional bridge linking the 2' oxygen and 4' carbon. Many chemical modifications are commonly used in the synthesis of oligonucleotides for various reasons. For example, increasing the stability of the phosphate backbone, modulating the stability of the duplex, altering the conformation of the oligonucleotide, or increasing its ability to penetrate the lipid bilayer. Modified sugar moieties are also being incorporated into therapeutic oligonucleotides. Modifying the sugar moieties typically increases nuclease resistance and binding affinity to complementary targets.

[0070] "Bridging nucleic acid" ("BNA") refers to nucleic acid modified with 2'-O,4'-C-methylene.

[0071] The term "locked nucleonucleotide" ("LNA nucleotide") as used in this article refers to modified RNA nucleotides that provide greater thermodynamic stability to polynucleotides during hybridization compared to polynucleotides that differ from LNAs simply by replacing the modified RNA nucleotides with natural ribonucleotides.

[0072] As used herein, the term "wildtype" is a term understood by those skilled in the art and refers to the typical form of an organism, strain, gene, or trait that exists in nature, as distinct from mutant or variant forms. As used herein, the term "variant" should be understood to mean a pattern that deviates from the wildtype or contains components not naturally present.

[0073] The term "peptide" refers to a compound comprising a plurality of linked amino acids. The amino acids used in the compounds (e.g., peptides and proteins) provided herein can be any of the 20 genetically encoded amino acids, naturally occurring non-genetically encoded amino acids, or synthetic amino acids. L- and D-enantiomers of any of the above can be used in the compound. In some embodiments, all amino acids are D-enantiomers. The following abbreviations may be used herein 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 protein or peptide residues are continuous, without any non-genetically encoded amino acids or amino acids that interrupt the sequence of amino acid residues in the synthesis of amino acids. In other embodiments, the sequence may comprise one or more non-genetically encoded or synthetic amino acid motifs. In particular embodiments, the residue sequence of the peptide may be interrupted by one or more non-genetically encoded or synthetic amino acid motifs, including but not limited to those shown in Table 1.

[0074]

[0075]

[0076]

[0077] Interchangeable amino acids typically belong to similar classes or subclasses. As those skilled in the art know, amino acids can be classified into different categories primarily based on the chemical and physical properties of their side chains. For example, some amino acids are generally considered hydrophilic or polar, while others are considered hydrophobic or nonpolar. 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 can be further subdivided to include, among other things, aliphatic amino acids. The definitions of amino acid categories used herein are as follows:

[0078] "Nonpolar amino acids" are amino acids with side chains that are uncharged at physiological pH. They are not polar and are generally repelled by 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.

[0079] "Aromatic amino acids" are nonpolar amino acids having a side chain containing at least one ring (aromatic group) with a conjugated n-electron system. The aromatic group can be further substituted by substituents such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro, and amino, as well as other substituents. Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine, and tryptophan. Common non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, β-2-thienylalanine, 3-benzothiazol-2-yl-alanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine.

[0080] Aliphatic amino acids are nonpolar amino acids with saturated or unsaturated straight-chain, branched, or cyclic hydrocarbon side chains. Examples of genetically encoded aliphatic amino acids include Ala, Leu, Val, and Ile. Examples of non-coding aliphatic amino acids include Nle.

[0081] "Polar amino acids" are hydrophilic amino acids with side chains that are charged or uncharged at physiological pH, and in which the shared electron pair between the two atoms in the side chain bond is held more tightly by one of the atoms. Polar amino acids are typically hydrophilic, meaning that their amino acids 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.

[0082] "Acidic amino acids" are hydrophilic amino acids with side chains having a pK value of less than 7. Due to the loss of hydrogen ions, acidic amino acids typically have negatively charged side chains at physiological pH. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).

[0083] "Basic amino acids" are hydrophilic amino acids with side chains having a pK value greater than 7. Due to their binding to hydrated hydrogen ions, basic amino acids typically have positively charged side chains at physiological pH. 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.

[0084] "Ionizable amino acids" are amino acids that can carry a charge at physiological pH. These ionizable amino acids include acidic and basic amino acids, such as 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.

[0085] As those skilled in the art will understand, the above classification is not absolute. Several amino acids exhibit more than one property and can therefore be included in more than one category. For example, tyrosine has both a nonpolar aromatic ring and a polar hydroxyl group. Therefore, tyrosine has several characteristics that can be described as nonpolar, aromatic, and polar. However, the nonpolar ring is predominant, so tyrosine is generally considered nonpolar. Similarly, cysteine ​​also has a nonpolar characteristic in addition to its ability to form disulfide bonds. Therefore, although there is no strict classification as a hydrophobic or nonpolar amino acid, in many cases, cysteine ​​can be used to endow peptides with hydrophobicity or nonpolarity.

[0086] In some embodiments, the polar amino acids considered in this invention include, for example, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine, hydroxylysine, ornithine, serine, threonine, and structure-related amino acids. In one embodiment, the polar amino group is an ionizable amino acid, such as arginine, aspartic acid, glutamic acid, histidine, hydroxylysine, lysine, or ornithine.

[0087] Examples of available polar or nonpolar amino acid residues include, for example, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tryptophan, tyrosine, etc.

[0088] The terms “non-naturally occurring” and “engineered” are used interchangeably and indicate human intervention. When referring to nucleic acid molecules or peptides, these terms mean that the nucleic acid molecule or peptide is at least substantially free of at least one other component naturally associated with them in nature and found in nature. Nucleic acid molecules that suppress gene or nucleic acid expression may be called “repressive nucleic acids” (referring to combinations thereof). Repressive nucleic acid technologies are known in the art and include, but are not limited to, antisense oligonucleotides, catalytic nucleic acids such as ribozymes and deoxyribozymes, aptamers, triplet-forming nucleic acids, external guide sequences, and RNA interference molecules (RNAi), particularly small nucleic acid molecules such as short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miR), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi).

[0089] “miR-21 inhibitory nucleic acid” or “miR-21 inhibitory nucleic acid analog” can hybridize with miR-21 or its isoform miR, blocking its activity, thereby jointly reducing the expression of proteins encoded by mRNAs or their mRNA variants that have miR-21 binding sites.

[0090] As used herein, the term “analog” refers to a compound that is structurally similar to another compound but differs slightly in composition (e.g., one atom is replaced by another, or a particular functional group is present or absent).

[0091] As used herein, the term "bioavailability" refers to the extent or rate at which a drug or other substance is absorbed or becomes 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 intestine, the extent of protein binding, and metabolism. Various bioavailability tests familiar to those skilled in the art are described herein.

[0092] As used in this application, the term "water-soluble" refers to solubility in an aqueous medium, such as phosphate-buffered saline (PBS) at approximately pH 7.4, 0.9% saline, or approximately 5% glucose. Tests for water solubility are given as "water solubility determination" in the examples below.

[0093] discuss

[0094] This article provides miR-21 repressive 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 encoding a regulatory RNA or protein, such as complementary to the coding strand of a double-stranded cDNA molecule or to a target RNA. In such embodiments, the antisense repressive nucleic acid sequence binds to one or more binding sites present in the target miR-21.

[0095] The repressive nucleic acid comprises 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 repressive nucleic acid does not include a nucleotide segment reflecting the seed region of the miR-21 transit chain. In such embodiments, the complementarity of the repressive nucleic acid relates to any remaining portion (i.e., sense strand) of the target miR-21 that corresponds to a segment of the miR-21 transit chain without a seed region. For example, in one embodiment, the repressive nucleic acid has 100% sequence complementarity with at least a portion of the miR-21 portion corresponding to a transit chain segment without a seed region. In other embodiments, the repressive nucleic acid has at least 70% sequence complementarity with at least a portion of the miR-21 portion corresponding to a transit chain segment without a seed region. Suitable repressive nucleic acids include, but are not limited to, any sequences provided herein. As will be understood by those skilled in the art, sequence variations that may be expected due to genetic mutations, strain polymorphism, or evolutionary differences can be tolerated by the repressive nucleic acids disclosed herein.

[0096] In some embodiments, the repressive nucleic acid-peptide analog comprises 8 to 17 nucleotides, 8 to 15 nucleotides, 10 to 17 nucleotides, 10 to 15 nucleotides, or any combination, subcombination, range, or subrange thereof. Additionally, or alternatively, in some embodiments, the repressive nucleic acid is a gapmer. A gapmer comprises any suitable number of portions, each portion comprising any suitable number of nucleotides depending on the total length of the repressive nucleic acid. For example, in one embodiment, the repressive nucleic acid comprises a 15-nucleotide gapmer having a 5-5-5 structure. Other embodiments include any suitable variations in the number of nucleotides in any portion, each of which is explicitly covered herein.

[0097] In some embodiments, the gapmer includes a deoxyribonucleic acid (DNA) portion and at least one bridging nucleic acid (BNA). For example, in one embodiment, the repressive nucleic acid includes a gapmer having a three-part BNA-DNA-BNA structure. In another embodiment, the repressive nucleic acid includes an 8-nucleotide gapmer having a BNA-DNA-BNA portion, said BNA-DNA-BNA portion including at least the sequence ATAAGC (…). Figure 2C In some implementations, the sequence in the DNA portion is modified while retaining the ability to bind to the target miR-21.

[0098] In some implementations, the BNA moiety comprises a 2' oxygen and a 4' carbon bridged by a methylene group. Other examples of BNA may include, but are not limited to, 2',4'-BNA. NC [NH]、2',4'-BNA NC [NMe] and 2',4'-BNA NC [NBn]. In some embodiments, one or more portions of the gapmer include a 2'-O,4'-C-ethylidene-bridged nucleic acid (ENA), wherein the 2' oxygen and 4' carbon are bridged by the ethylidene group. Additionally, or alternatively, in some embodiments, (s)-cEt (S-bound ethyl) and / or tcDNA (tricyclic DNA) modifications may be used to bind the nucleotide.

[0099] In some embodiments, the ribose portion of the modified RNA nucleotide is modified with an additional bridge connecting the 2' oxygen and 4' carbon. The LNA nucleotide can contain any type of additional bridge between the 2'-O and 4'-C of the RNA, which increases the thermodynamic stability of the duplex between the LNA and its complement. Other 2'-O-modified nucleotides, such as 2'-O-Me, also exhibit greater stability. In some embodiments, the repressive nucleic acid comprises an oligonucleotide backbone architecture that exhibits a particularly high binding affinity to the target (measured by melting temperature or Tm) to achieve a steric hindrance mechanism. BNA, such a backbone includes, but is not limited to, backbones containing LNA, FANA, 2'-fluorine, 2'-O-methoxyethyl (2'-MOE), 2'-NH2, 2'-F-RNA, morpholino, and piperazine. Other modifications to the oligonucleotide ribose include, but are not limited to, FHNA (fluorohexetol nucleic acid), (s)-5'-C-methyl, UNA (unlocked nucleic acid), 4'-thio-RNA, and cyclohexene nucleic acid.

[0100] Sometimes, modified backbone links are used instead of phosphodiester bonds to minimize nuclease degradation of oligonucleotides. Examples include, but are not limited to, thiophosphates, borophosphonates, aminophosphates, methylphosphonates, (SC5' Rp)-α,β-CNA (dioxocyclohexane-bound nucleic acids), PNA (peptide nucleic acids), PMO (phosphodiamidomorpholino oligonucleotides), and phosphorylguanidine. 5' modifications that increase phosphate stability include, but are not limited to, E-VP ((E)-vinylphosphonate), 5'-methylphosphonates, 5'-thiophosphates, (s)-5'-methylphosphates, and 5'-methoxy. 3' modifications that increase phosphate stability include, but are not limited to, 2-hydroxyethyl phosphates and 3'-ddc (dideoxycytosine), and 3'-amino. Base modifications that improve 3' stability include, but are not limited to, 2'-thio-dT.

[0101] Several solid-phase methods, including those involving the use of di(trimethylsiloxy)cyclododecyloxysilyl as a 5'-O-protecting group (Brummel and Caruthers, Tetrahedron Lett 43: 749, 2002), have been used to generate oligonucleotides with mixed bonds such as borophosphate and phosphate bonds. In another example, the 5'-hydroxyl group is initially protected with benzylhydroxydi-(trimethylsiloxy)silyl 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 oligomers with pure borophosphate bonds or with borophosphate bonds mixed with phosphate diesters, thiophosphates, dithiophosphates, or methylphosphonates. In another example, the boron phosphorylation agent 2-(4-nitrophenyl)ethyl ester of boron phosphoramide can be used to generate boron phosphate-linked oligonucleotides. This agent 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]undec-7-ene (DBU) to produce the corresponding nucleoside boron monophosphate (NMPB) in good yield.

[0102] In some implementations, the repressive nucleic acid sequence includes one or more nucleotide modifications to increase binding affinity. Suitable nucleotide modifications to increase binding affinity include, but are not limited to, 5'-methylcytidine, 5-methyluridine (ribothymidine), and abase-free RNA.

[0103] Despite its reduced length (i.e., number of nucleotides), the antisense sequence disclosed herein binds to miR-21 with sufficient strength to specifically block activation of mRNA translation. Additionally or alternatively, without being bound by theory, it is believed that BNA reduces or eliminates hybridization-dependent and hybridization-independent toxicity, while also providing improved hybridization affinity compared to existing backbone modifications (e.g., locked nucleic acids (LNAs)). Furthermore, again without being bound by theory, it is believed that excluding the transit chain in the seed region reduces or eliminates the mimicry of the transit chain by the antisense sequence.

[0104] In some embodiments, this document also provides methods for treating or improving symptoms associated with cancer and / or other types of hyperproliferative disorders regulated by microRNA 21 activity. In some embodiments, the methods include administering to a subject in need one or more inhibitory nucleic acid sequences and analogues disclosed herein, 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 retain the ability to treat or improve cancer-related symptoms. In one embodiment, for example, the method includes administering to a subject with cancer, such as, but not limited to, triple-negative breast cancer (TNBC).

[0105] Delivery methods for administering inhibitory nucleic acids are known in the art. See, 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)), which are incorporated herein by reference in their entirety. Antisense nucleic acids can be constructed using procedures known in the art through chemical synthesis and enzymatic ligation reactions. For example, antisense nucleic acids (e.g., antisense oligonucleotides) can be chemically synthesized using naturally occurring nucleotides and / or modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, for example, using phosphate thioester derivatives and nucleotides modified at the 3' and 5' ends or synthetic nucleotides. The nucleic acid sequences provided herein, including but not limited to those in the sequence listing, are intended to cover nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to nucleic acids having modified nucleotides. In some embodiments, the oligonucleotides provided herein may contain one or more modifications to nucleotides, sugars, and / or nucleotide internucleotides, and are therefore modified oligonucleotides.

[0106] Antisense nucleic acids can also be biologically generated using an expression vector, wherein the nucleic acid has been subcloned into the expression vector in the antisense direction (i.e., the RNA transcribed from the inserted nucleic acid will be in the antisense direction of the target nucleic acid). In a particularly preferred embodiment, the miR-21 repressive nucleic acid is operatively linked to a peptide ligand (e.g., an IGF1R ligand) that promotes the internalization of the inhibitor into the target cells of a receptor protein overexpressed on cancer cells. The peptide ligand may be covalently coupled to the 5' or 3' end of the repressive nucleic acid.

[0107] In some embodiments, delivery of the miR-21 repressive nucleic acid can be enhanced by covalently conjugating the repressive nucleic acid to a lipid molecule, including but not limited to cholesterol, α-tocopherol, or long-chain fatty acids. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by covalently conjugating the repressive nucleic acid to GalNAc. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by covalently conjugating the repressive nucleic acid to an antibody. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by covalently conjugating the repressive nucleic acid to an aptamer. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by covalently conjugating the repressive nucleic acid to a protein or peptide, including but not limited to multiple amino acids, cell-penetrating peptides, cell-targeting peptides, or receptor-binding proteins. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by non-covalently associating the repressive nucleic acid with another cell-penetrating molecule. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by packaging the repressive nucleic acid into a nanocarrier. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by packaging the repressive nucleic acid into liposomes, said liposomes including, but not limited to, functionalized lipid nanoparticles with PEGylated lipids or other ligands associated with lipid nanoparticles for cell-targeted delivery. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by loading the repressive nucleic acid into exosomes. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by chemically attaching the repressive nucleic acid to the surface of spherical nanoparticles. In another embodiment, delivery of the miR-21 repressive nucleic acid can be enhanced by incorporating the repressive nucleic acid into stimulus-sensitive nanostructures, said stimulus-sensitive nanostructures including, but not limited to, DNA origami, scaffold molecules conjugated to multiple delivery moieties.

[0108] In some forms, miR-21 repressive nucleic acid-peptide analogs may contain an external guide sequence (EGS). An EGS is a molecule that binds to a target nucleic acid molecule to form a complex, which is recognized by an RNAse P that cleaves the target molecule. EGS can be programmed to specifically target selected RNA molecules. RNAse P facilitates the processing of intracellular transfer RNA (tRNA). Bacterial RNAse P can be recruited to cleave virtually any RNA sequence using EGS, resulting in a target RNA:EGS complex mimicking the native tRNA substrate. Similarly, eukaryotic EGS / RNAse P-guided RNA cleavage can be used to cleave desired targets within eukaryotic cells. Examples of how EGS molecules are prepared and used to facilitate the cleavage of a variety of different target molecules are known in the art.

[0109] Unless otherwise stated, the practice of this invention employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, all of which are within the scope of this art. In some embodiments, the vector is capable of driving 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 control function of the expression vector is typically provided by one or more regulatory elements. Commonly used promoters are derived from polyomaviruses, adenovirus 2, cytomegaloviruses, simian virus 40, and other promoters disclosed herein and known in the art. For other suitable expression systems for prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0110] Pharmaceutically acceptable salts of the compounds of this 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 acid salts include those of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, palmitic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthalic acid, hydroiodic acid, malic acid, stearic acid, tannic acid, etc. Hydrochloride salts are of particular interest. Other acids, such as oxalic acid, although not pharmaceutically acceptable on their own, can be used to prepare salts that can be used as intermediates to obtain the compounds of this 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-methylglucosamine, and procaine salts.

[0111] preparation

[0112] The novel miR-21 inhibitory nucleic acid-peptide analogs described herein can be formulated for enteral, parenteral, topical, or pulmonary administration. These 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 individuals without inducing undesirable biological side effects or unwanted interactions. A carrier is any component present in a pharmaceutical formulation other than the active ingredient. For example, see the latest edition of Remington's Pharmaceutical Sciences, published by EW Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods for preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds described herein, and this literature is incorporated herein by reference. These are most typically standard carriers used for administering compositions to humans and non-humans, and include solutions such as sterile water, saline, and buffered solutions at physiological pH. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0113] These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0114] Parenteral preparations

[0115] The compounds described herein can be formulated for parenteral administration. For example, parenteral administration may include administration to patients via intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intravitreal, intratumoral, intratumoral, intramuscular, subcutaneous, subconjunctival, intravesical, intraperitoneal, intraumbilical, injection, and infusion.

[0116] Parenteral formulations can be prepared into 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 when added to a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions and their microemulsions, liposomes, or emulsions.

[0117] If intended for intravenous administration, the composition is packaged in a sterile isotonic buffer solution. If necessary, the composition may also contain a solubilizer. The components of the composition are provided individually or mixed together in unit dosage forms, for example, as lyophilized powders or concentrated solutions in sealed containers (such as ampoules or sachets indicating the amount of active agent). If 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 to allow for mixing of the components prior to injection.

[0118] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. For example, in the case of dispersions, the desired particle size can be maintained by using a coating such as lecithin, and / or by using a surfactant, appropriate flowability can be maintained. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred.

[0119] Solutions and dispersions of active compounds in the form of free acids or bases or their pharmaceutically acceptable salts can be prepared in water or another solvent or dispersion medium suitably mixed with one or more pharmaceutically acceptable excipients, including but not limited to surfactants, dispersants, emulsifiers, pH adjusters, viscosity modifiers, and combinations thereof.

[0120] Suitable surfactants can be anionic, cationic, amphoteric, or nonionic. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include sodium, potassium, and ammonium salts of long-chain alkyl sulfonic acids and alkyl aryl sulfonic acids, such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinate, such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinate, such as bis-(2-ethylhexyl)-sulfosuccinate; and alkyl sulfates such as sodium dodecyl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzyl chloride, cetrimonium bromide, stearoyl dimethyl benzyl ammonium chloride, polyoxyethylene, and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglycerol-4-oleate, sorbitol ester, sucrose ester, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 hexadecyl 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, myristoyl amphoteric acetate, lauryl betaine, and lauryl sulfobetaine.

[0121] The formulation may contain preservatives to prevent microbial growth. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and mercuric chloride. The formulation may also contain antioxidants to prevent degradation of the active agent. The formulation is typically buffered to a pH of 3-8 for parenteral administration during reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0122] Water-soluble polymers are frequently used in parenteral formulations. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethyl cellulose, and polyethylene glycol.

[0123] Sterile injectable solutions can be prepared by incorporating the desired amount of an active compound with one or more of the excipients listed above (if desired) into a suitable solvent or dispersion medium, followed by filtration and sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium containing a basic dispersion medium and other desired components listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient plus any other desired components from their previously sterile filtered solutions. Powders can be prepared in such a way that the particles are inherently porous, which increases particle solubility. Methods for preparing porous particles are well known in the art.

[0124] Controlled-release formulations

[0125] The parenteral formulations described herein can be formulated for controlled release, including immediate release, delayed release, extended release, pulsatile release, and combinations thereof.

[0126] Nanoparticles and microparticles

[0127] For parenteral administration, one or more compounds and optionally one or more additional active agents may be incorporated into microparticles, nanoparticles, or combinations thereof to provide controlled release of said compound and / or one or more additional active agents. In formulations containing two or more drugs, said drugs may be formulated for the same type of controlled release (e.g., delayed, prolonged, immediate, or pulsatile), or said 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.).

[0128] For example, the compound and / or one or more additional active agents can be incorporated into polymeric microparticles, providing controlled drug release. Drug release is controlled by diffusion of the drug from the microparticles and / or by degradation of the polymer particles through hydrolysis and / or enzymatic degradation. Suitable polymers include ethyl cellulose and other natural or synthetic cellulose derivatives.

[0129] Polymers that slowly dissolve and form gels in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, are also suitable as substances containing drug particles. Other polymers include, but are not limited to, polyanhydrides, poly(acetic anhydrides), polyhydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA), poly(lactide-co-glycolic acid) (PLGA), poly-3-hydroxybutyrate (PHB) and its copolymers, poly-4-hydroxybutyrate (P4HB) and its copolymers, polycaprolactone and its copolymers, and combinations thereof.

[0130] Alternatively, the drug may be incorporated into microparticles prepared from substances that are insoluble in aqueous solutions or slowly soluble in aqueous solutions but capable of degradation in the gastrointestinal tract by means including enzymatic degradation, surfactant action of bile acids, and / or mechanical erosion. As used herein, the term "slowly soluble in water" refers to a substance that is insoluble in water over a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, waxy substances, and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl alcohol, myristearyl alcohol, cetyl alcohol, or cetearyl alcohol); fatty acids and derivatives, including but not limited to fatty acid esters, fatty acid glycerides (monoglycerides, diglycerides, 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 waxy substances include natural or synthetic waxes, hydrocarbons, and common waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffin wax, and candelilla wax. As used herein, waxy substances are defined as any substance that is normally solid at room temperature and has a melting point of about 30°C to 300°C.

[0131] In some cases, it may be necessary to modify the rate at which water permeates into the microparticles. For this purpose, rate-controlling (wicking) agents can be formulated with the fats or waxes listed 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, hydroxypropyl cellulose, methylcellulose, and carboxymethyl cellulose), alginate, lactose, and talc. Furthermore, pharmaceutically acceptable surfactants (e.g., lecithin) can be added to promote the degradation of these microparticles. Water-insoluble proteins can also be used as substances for forming drug-containing microparticles. Additionally, water-soluble proteins, polysaccharides, and combinations thereof can be formulated with drugs into microparticles, which are then cross-linked to form an insoluble network. For example, cyclodextrin can be complexed with a single drug molecule and then cross-linked.

[0132] Methods for preparing nanoparticles and microparticles

[0133] The encapsulation or incorporation of the miR-21 inhibitory nucleic acid-peptide analog described herein into a carrier material to produce therapeutic microparticles can be achieved using known pharmaceutical formulation techniques. In cases where the formulation is located in a fatty, waxy, or waxy substance, including in some cases where the peptide ligand itself is conjugated with a hydrophobic tail, the carrier material is typically heated above its melting temperature, and an analog or drug is added to form a mixture comprising 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 processes of coagulation, extrusion, spray cooling, or aqueous dispersion. In a preferred process, the wax is heated above its melting temperature, the drug is added, and the molten wax-drug mixture coagulates under continuous agitation as the mixture cools. Alternatively, the molten wax-drug mixture can be extruded and spheroidized to form pellets or beads. These processes are known in the art.

[0134] For some carrier substances, solvent evaporation techniques may be required to produce drug-containing microparticles. In such cases, including situations where the peptide ligand itself is conjugated with a hydrophobic tail, the drug and carrier substance are co-soluble in a mutual solvent, and microparticles can then be prepared using several techniques, including but not limited to forming an emulsion in water or other suitable media, spray drying, or removing the solvent from the bulk solution by evaporation and milling the resulting substance.

[0135] In some formulations, the drug in particulate form is uniformly dispersed in a water-insoluble or slowly water-soluble substance. To minimize the size of the drug particles in the composition, the drug powder itself can be ground to produce fine particles before formulation. Jet milling processes known in the pharmaceutical industry can be used for this purpose. In some formulations, including in certain cases where the peptide ligand itself is conjugated with a hydrophobic tail, the drug in particulate form is uniformly dispersed in the wax or waxy substance by heating the wax or waxy substance above its melting point and adding the drug particles while stirring the mixture. In this case, a pharmaceutically acceptable surfactant can be added to the mixture to promote the dispersion of the drug particles.

[0136] Particles can also be coated with one or more modified release coatings. Solid fatty acid esters hydrolyzed by lipases can be sprayed onto microparticles or drug particles. Natural water-insoluble proteins can be coated onto drug-containing microparticles or drug particles via spray coating or wet granulation techniques. In addition to naturally water-insoluble substances, some substrates of digestive enzymes can be treated with cross-linking methods, resulting in the formation of insoluble networks. Many methods for cross-linking proteins, initiated by chemical and physical means, have been reported. One of the most common methods for obtaining cross-links is the use of chemical cross-linking agents. Examples of chemical cross-linking agents include aldehydes (glutaraldehyde and formaldehyde), epoxides, carbodiimides, and genipins. In addition to these cross-linking agents, oxidized sugars and natural sugars have also been used to cross-link gelatin. Cross-linking can also be accomplished enzymatically; for example, transglutaminase has been approved as a GRAS material for cross-linking seafood products. Finally, cross-linking can be initiated by physical means such as heat treatment, ultraviolet irradiation, and gamma irradiation.

[0137] To create a cross-linked protein coating layer around a drug containing microparticles or drug particles, a water-soluble protein can be sprayed onto the microparticles and subsequently cross-linked using one of the methods described above. Alternatively, drug-containing microparticles can be microencapsulated within a protein via condensed phase separation (e.g., by adding salt) and subsequently cross-linked. Some suitable proteins for this purpose include gelatin, albumin, casein, and gluten.

[0138] Polysaccharides can also crosslink to form water-insoluble networks. For many polysaccharides, this can be achieved by reacting with calcium salts or polyvalent cations, thereby crosslinking the host polymer chain. Pectin, alginate, dextran, amylose, and guar gum crosslink in the presence of polyvalent cations. Complexes can also be formed between polysaccharides with opposite charges; for example, pectin and chitosan can complex through electrostatic interactions.

[0139] Injectable / Implantable Formulations

[0140] The miR-21 inhibitory nucleic acid-peptide analogs described herein can be incorporated into injectable / implantable solid or semi-solid implants, such as polymer implants. In some forms, the compound is incorporated into a polymer that is a liquid or paste at room temperature but exhibits increased viscosity upon contact with an aqueous medium, such as a physiological fluid, forming a semi-solid or solid material. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from copolymerization of at least one unsaturated hydroxy fatty acid with a hydroxyalkanoic acid. The polymer can be melted, mixed with the active substance, and cast or injection molded into a device. This melt manufacturing requires the polymer to have a melting point below the temperature at which the substance to be delivered and the polymer degrades or is readily reactive. The device can also 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. Solvent processes require the polymer to be soluble in organic solvents. Another method is to compress a mixture of polymer and drug or polymer particles carrying the active agent into a powder.

[0141] Alternatively, the compound can be incorporated into a polymer matrix and molded, pressed, or extruded into a device that is solid at room temperature. For example, the compound can be incorporated into biodegradable polymers such as polyanhydrides, polyhydroxyalkanoates (PHA), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins, and polysaccharides such as collagen, hyaluronic acid, albumin, and gelatin, as well as combinations thereof, and pressed into a solid device, such as a disc, or extruded into a device, such as a rod.

[0142] The release of one or more compounds from an implant can be altered by selecting the polymer, the molecular weight of the polymer, and / or modifying the polymer to increase degradation, such as pore formation and / or the incorporation of hydrolyzable bonds. Methods for modifying the properties of biodegradable polymers to alter the release profile of compounds from implants are well known in the art.

[0143] Intestinal preparations

[0144] Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, sodium saccharin, starch, magnesium stearate, cellulose, magnesium carbonate, etc. This composition will contain a therapeutically effective amount of the compound and / or antibiotic, as well as an appropriate amount of carrier, to provide a suitable form to the patient based on the method of administration used.

[0145] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be prepared using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared into hard or soft capsule shells using techniques well known in the art, which can encapsulate liquid, solid, and semi-solid filler materials.

[0146] Pharmaceutically acceptable carriers can be used to prepare formulations. The term "carrier" as used herein generally includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof. Carriers also include all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizers, and gliding agents.

[0147] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate; polyvinyl acetate phthalates, acrylic polymers and copolymers, and commercially available methacrylic resins, zein, shellac, and polysaccharides under the trade name EUDRAGIT® (Roth Pharma, Weststadt, Germany). Furthermore, coating materials may contain conventional carriers such as plasticizers, pigments, colorants, flow aids, stabilizers, pore-forming agents, and surfactants.

[0148] "Filling agents," also known as "fillers," are generally necessary to increase the volume of solid dosage forms in order to provide a practical size for the compression of tablets or the formation of beads and granules. Suitable fillers include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starch, pregelatinized starch, silica, titanium dioxide, magnesium aluminum silicate, and powdered sugar.

[0149] "Binders" are used to impart adhesiveness to solid dosage forms, thereby ensuring that tablets, beads, or granules remain intact after the dosage form is formed. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums (such as gum arabic, gum tragali, and sodium alginate), cellulose (including hydroxypropyl methylcellulose, hydroxypropyl cellulose, and ethyl cellulose), and veegum, as well as synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid, and polyvinylpyrrolidone.

[0150] "Lubricant" is used to facilitate tablet manufacturing. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, polyethylene glycol, talc, and mineral oil.

[0151] "Disintegrants" are used to promote the disintegration or "decomposition" of a formulation after application and are generally including, but not limited to, starch, sodium starch hydroxyacetate, sodium carboxymethyl starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, pregelatinized starch, clay, cellulose, alginate, gum, or cross-linked polymers such as cross-linked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0152] "Stabilizers" are used to inhibit or delay drug degradation reactions, including those via oxidation. 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, such as sodium metabisulfite; cysteine ​​and its derivatives; citric acid; propyl gallate and butylated hydroxyanisole (BHA).

[0153] Controlled release of intestinal preparations

[0154] Oral dosage forms, such as capsules, tablets, solutions, and suspensions, can be formulated for controlled release. For example, one or more compounds and optionally one or more additional active agents can be formulated into nanoparticles, microparticles, or combinations thereof, and encapsulated in soft or hard gelatin or non-gelatin capsules, or dispersed in a dispersion medium to form oral suspensions or syrups. The particles can be formed from the drug and a controlled-release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled-release coatings before incorporation into the final dosage form.

[0155] In another form, one or more compounds and optionally one or more additional active agents are dispersed in a matrix material that gels or emulsifies upon contact with an aqueous medium such as a physiological fluid. In the case of gelation, the matrix swells, encapsulating the active agent, which is slowly released over time through diffusion and / or degradation of the matrix material. This matrix can be formulated as a filler material for tablets or hard and soft capsules.

[0156] In another form, one or more compounds and optionally one or more additional active agents are formulated into a solid oral dosage form, such as tablets or capsules, and the solid dosage form is coated with one or more controlled-release coatings (such as delayed-release coatings or extended-release coatings). The coating may also contain the said compound and / or additional active agents.

[0157] Extended release formulation

[0158] Extended-release formulations are typically prepared as diffusion or permeation systems 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 typically prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three main types of materials used to prepare matrix devices are insoluble polymers, hydrophilic polymers, and aliphatic compounds. Polymer matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulose polymers such as methyl and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and Carbopol® 934, polyethylene oxide, and mixtures thereof. Aliphatic compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate, and waxy substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.

[0159] In certain preferred forms, the polymeric material is a pharmaceutically acceptable acrylic polymer, including but not limited to copolymers of acrylic acid and methacrylic acid, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), alkylamine methacrylate copolymers, poly(methyl methacrylate), poly(methacrylic acid) (anhydride), polymethacrylate, polyacrylamide, poly(methacrylic anhydride), and glycidyl methacrylate copolymers.

[0160] In certain preferred forms, the acrylic polymer comprises one or more ammonium methacrylate copolymers. Ammonium methacrylate copolymers are well known in the art as copolymers of fully polymerized acrylates and methacrylates having a low content of quaternary ammonium groups.

[0161] In a preferred form, the acrylic polymer is an acrylic resin paint, such as the acrylic resin paint commercially available from Rohm Pharma under the trade name EUDRAGIT®. In a further preferred form, the acrylic polymer comprises a mixture of two acrylic resin paints commercially available from Rohm Pharma under the trade names EUDRAGIT® RL30D and EUDRAGIT® RS30D. EUDRAGIT® RL30D and EUDRAGIT® RS30D are copolymers of acrylates and methacrylates with low contents of quaternary ammonium groups, wherein the molar ratio of ammonium groups to the remaining neutral (meth)acrylate is 1:20 in EUDRAGIT® RL30D and 1:40 in EUDRAGIT® RS30D. The average molecular weight is about 150,000. EUDRAGIT® S-100 and EUDRAGIT® L-100 are also preferred. The code designations RL (high permeability) and RS (low permeability) refer to the permeability of these reagents. EUDRAGIT® RL / RS mixtures are insoluble in water and digestion solutions. However, multi-particle systems formed to include them are swellable and permeable in aqueous solutions and digestion solutions.

[0162] The polymers described above, such as EUDRAGIT® RL / RS, can be mixed together in any desired proportion to ultimately obtain a sustained-release formulation with the desired dissolution profile. For example, the desired sustained-release multi-particle system can be obtained from 100% EUDRAGIT® RL, 50% EUDRAGIT® RL and 50% EUDRAGIT® RS, and 10% EUDRAGIT® RL and 90% EUDRAGIT® RS. Those skilled in the art will recognize that other acrylic polymers, such as EUDRAGIT® L, can also be used.

[0163] Alternatively, extended-release formulations can be prepared using a permeation system or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low-permeability and high-permeability coating materials in an appropriate ratio.

[0164] The aforementioned devices with different drug release mechanisms can be combined into a final dosage form comprising one or more units. Examples of multi-unit systems include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules. An immediate-release component can be added to a prolonged-release system by applying an immediate-release layer on top of a prolonged-release core using a coating or compression process, or by including both prolonged-release and immediate-release beads in a multi-unit system such as a capsule.

[0165] Extended-release tablets containing hydrophilic polymers are prepared using techniques known in the art, such as direct compression, wet granulation, or dry granulation. Their formulations typically incorporate polymers, fillers, binders, lubricants, and the active pharmaceutical ingredient. Commonly used fillers include inert powdered substances such as starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol, and sucrose, cereal flours, and similar edible powders. Typical fillers include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts such as sodium chloride, and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin, and sugars (such as lactose, fructose, and glucose). Natural and synthetic gums, including gum arabic, alginate, methylcellulose, and polyvinylpyrrolidone, can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose, and waxes can also be used as binders. In tablet formulations, lubricants are necessary to prevent the tablet and punch from sticking to the mold. The lubricant is selected from such smooth solids, such as talc, magnesium stearate and calcium stearate, stearic acid and hydrogenated vegetable oil.

[0166] Extended-release tablets containing waxy substances are typically prepared using methods known in the art, such as direct mixing, coagulation, and aqueous dispersion. In the coagulation method, the drug is mixed with a waxy substance and then subjected to spray coagulation or condensation, as well as screening and processing.

[0167] Delayed-release formulation

[0168] Delayed-release formulations can be prepared by coating solid dosage forms with polymer films that are insoluble in the acidic environment of the stomach but soluble in the neutral environment of the small intestine.

[0169] Delayed-release dose units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition can be, for example, a tablet for incorporation into a capsule, a tablet used as a core in a "coated core" dosage form, or a variety of drug-containing beads, granules, or particles for incorporation into a tablet or capsule. Preferred coating materials include biodegradable, gradually hydrolyzable, gradually water-soluble, and / or enzymatically degradable polymers, and can be conventional "enteric" polymers. As understood by those skilled in the art, enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract, or are slowly 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 achieving delayed release include, but are not limited to, cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, and sodium carboxymethyl cellulose; acrylic polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate; and other commercially available methacrylic resins under the trade name Eudragit® (Rohm Pharma; Westerstadt, Germany), including EUDRAGIT® L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT® L-100 (soluble at pH 6.0 and above), EUDRAGIT® S (soluble at pH 7.0 and above due to a higher degree of esterification), and EUDRAGITS® NE, RL, and RS (water-insoluble polymers with varying degrees of permeability and swelling); vinyl polymers and copolymers, such as polyvinylpyrrolidone, vinyl acetate, vinyl phthalate, vinyl acetate-crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers, such as azo polymers, pectin, chitosan, amylose, and guar gum; corn gluten and shellac. Combinations of different coating materials can also be used. Multilayer coatings using different polymers can also be applied.

[0170] Those skilled in the art can readily determine the preferred coating weight of a particular coating material by evaluating the individual release profiles of tablets, beads, and granules prepared with varying amounts of different coating materials. It is the combination of substance, method, and application form that produces the desired release characteristics, which can only be determined through clinical studies.

[0171] Coating compositions may include conventional additives such as plasticizers, pigments, colorants, stabilizers, and flow aids. Plasticizers are typically used to reduce the brittleness of the coating. Plasticizers typically comprise about 10 wt.% to 50 wt.% of the polymer by dry weight. Examples of typical plasticizers include polyethylene glycol, propylene glycol, glyceryl triacetate, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, acetylated triethyl citrate, castor oil, and acetylated monoglycerides. Stabilizers are preferably used to stabilize particles in the dispersion. Typical stabilizers are nonionic emulsifiers such as sorbitan esters, polysorbates, and polyvinylpyrrolidone. Flow aids are recommended to reduce adhesion effects during film formation and drying; flow aids typically comprise about 25 wt.% to 100 wt.% of the polymer by weight in the coating solution. A suitable flow aid is talc. Other flow aids, such as magnesium stearate and glyceryl monostearate, can also be used. Pigments such as titanium dioxide can also be used. Small amounts of defoamers, such as silicones (e.g., simethicone), can also be added to the coating composition.

[0172] Inhalation formulations

[0173] The inhibitors of the present invention can be delivered locally to the respiratory system, such as to the nose, sinuses, sinus membranes, or lungs. The miR-21 inhibitory nucleic acid-peptide analogs of the present invention, or pharmaceutical compositions containing one or more miR-21 inhibitory nucleic acid-peptide analogs, can be delivered to the respiratory system in any suitable manner, such as by oral or intranasal inhalation. The compositions of the present invention can be dispensed in the form of powder or liquid nasal sprays, suspensions, drops, gels, or ointments via tubes or catheters, via syringes, via packtails, via cotton swabs, or via submucosal infusion. The compounds of preferred embodiments of the present invention can be conveniently delivered in the form of an aerosol spray using pressurized packaging or nebulizers and suitable propellants, such as, but not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of pressurized aerosols, the dosage unit can be controlled by providing a valve to deliver a measured amount. Capsules and cartridges, such as gelatin, for use in inhalers or blowpipes can be formulated as powder mixtures containing the compound and a suitable powder matrix such as lactose or starch. The propellant for the aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a low-boiling-point hydrocarbon-based solvent. The compounds of the present invention can be delivered in the form of an aerosol spray via a nebulizer or the like. In some embodiments, the active ingredient is suitably micronized to allow substantially all of the active ingredient to be inhaled into the lungs upon application of the dry powder formulation; thus, the active ingredient will have a particle size of less than 100 micrometers, ideally less than 20 micrometers, and preferably in the range of 1 to 10 micrometers.

[0174] The following examples are provided to illustrate certain embodiments of the present invention. They are not intended to limit the invention in any way.

[0175] Example I

[0176] For TNBC, targeted, low-toxicity molecular therapies to prolong survival represent a key unmet need, with a potential US market size of $7 billion per year. As mentioned above, miR-21 inhibitory nucleic acid-peptide analogs include IGF1 receptor ligands because TNBC cells exhibit strong IGF1R signaling activation, which is associated with poor survival. However, there is no known signaling feedback between miR-21 and IGF1R (Dobre, et al. Cells 10(8):1856, 2021).

[0177] miR-21 inhibitory nucleic acid-peptide analogs offer a unique approach to inhibiting miR-21-mediated gene expression by targeting and delivering the inhibitory nucleic acid-peptide analogs into TNBC cells via an IGF1R ligand. The administration of miR-21 inhibitory nucleic acid-peptide analogs described in this article increased tumor suppressor protein expression, inhibited immune checkpoint gene expression, slowed migration, reduced proliferation, and increased apoptosis in multiple TNBC lineages. Twice-weekly intraperitoneal administration of 5 mg / kg miR-21 inhibitory nucleic acid-peptide analogs in sterile saline for 14 days inhibited the growth of mouse EMT6 TNBC orthotopic allogeneic grafts by 74% compared to the mediator or scrambled control. Therefore, novel TNBC therapeutics and their variants are provided for treating this devastating disease and significantly increasing patient survival for TNBC.

[0178] Our design of miR-21 inhibitory nucleic acid-peptide analogs was inspired by previous work in which we generated a miR-17-5p blocker that actively mimicked the full-length miR-17-3p transit chain, thereby producing previously unknown off-target effects by unexpectedly downregulating the tumor suppressor proteins PDCD4 and PTEN. See [link to previous work]. Figure 4A and Jin, Y.-Y., et al. PLoS One 10(12): e0142574 (2015).

[0179] The novel miR-21 inhibitory nucleic acid-peptide analog described in this article comprises a 15 nt sequence designed to specifically block miR-21-5p (identical in mice and humans) without mimicking the opposite strand ( Figure 2A-2C , Figure 15 In the former miR-21 hairpin structure ( Figure 2AMost nucleotides in the miR-21-5p guide strand pair with most nucleotide bases in the miR-21-3p guest strand. Both the guide strand and the guest strand can be selected as the active mature miRNA in the RNA-induced silencing complex (RISC). The 15 nt repressive nucleic acid sequence of the miR-21-5p guide strand in the 5'-3' direction has high sequence homology with the continuous sequence region on the miR-21-3p guest strand in the 5'-3' direction (excluding the seed region of the miR-21-3p guest strand (nts 1–9 in the 5'-3' direction)). Figure 2B-2C Particularly preferred are at least 2, 3, 4, 5, 6, 7, or 8 nucleosides shown in blue in SEQ ID NO: 4. In some embodiments, 3 nucleosides are omitted or excluded. In other embodiments, 4 nucleosides are omitted. In other embodiments, 5 nucleosides are omitted. In other embodiments, 6 nucleosides are omitted. In other embodiments, 7 nucleosides are omitted. In other embodiments, 8 nucleosides are omitted.

[0180] Since miRNAs primarily function through complementary base pairing between the seed region and the regulatory binding site on the target mRNA, it is expected that nucleic acid inhibitor sequences of the miR-21-5p guide strand that exclude or partially exclude the miR-21-3p transit strand seed region will specifically inhibit the miR-21-5p guide strand without producing additional side effects due to mimicking the miR-21-3p transit strand.

[0181] like Figure 2B-2C As shown, the miR-21 inhibitory nucleic acid moiety of the miR-21 blocker of the present invention comprises 8 to 17 linked nucleosides. As explained below, certain segments of the nucleic acid moiety are more resistant to modification than other segments. For example, the first 4 to 5 consecutive nucleosides may comprise a BNA having a phosphate thioester bond, and the last 4 to 5 consecutive nucleosides may comprise a BNA having a phosphate thioester bond in the 5' to 3' direction. The nucleoside linked between the continuous BNA at the 5' and 3' ends may contain one or more of the following modifications: thiophosphate, borophosphonate, (SC5' Rp)-α,β-dioxophosphazenecyclohexane-bound nucleic acid, (E)-vinylphosphonate, 5'-methylphosphonate, 5'-thiophosphate, (s)-5'-methylphosphate, 5'-methoxy, 2-hydroxyethylphosphate, 3'-ddeoxycytosine, 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-bridging nucleic acid, (s)-cEt, fluorohexetol nucleic acid, (s)-5'-C-methyl, non-locked nucleic acid, 4'-thio-RNA. As described above, it is preferred to insert such modifications outside the minimal binding region.

[0182] As mentioned above, we incorporated the RNA analog 2'4'-BNA. NC (BNA) Skeletal modification ( Figure 3 To minimize hybridization-dependent and hybridization-independent toxicity, and to exhibit excellent hybridization affinity. 2'4'-BNA NC (BNA) is an anti-nuclease derivative of 2'O,4'-C-methylene-bridged nucleic acid (LNA). Figure 15 ).

[0183] The efficacy of miR-21 blockers in cells

[0184] We observed a 22 nM IC of the 15-base BNA miR-21 gapmer BND5412. 50 ( Figure 4A When transfected into human MDA-MB-231 TNBC cells, it blocked miR-21 derepression of luciferase expression from the luciferase-3'-UTR vector. Figure 4B Transfection with the same miR-21 blocker significantly increased the expression of the miR-21 target protein PDCD4, followed by a decrease in the expression of CD47, PD-L1, PD-L2, and Jak2 immune checkpoints. Figures 5A-5B ), and reduced cell proliferation in MDA-MB-231, HCC1806, BT-20, MDA-MB-157, MDA-MB-436, BT-549, and HCC1937 by up to 8-fold ( Figure 6 ).

[0185] As a comparison with the BNA drug, we also tested 50 nM FANA oligonucleotides transfected into human MDA-MB-231 TNBC cells for 48 hours. Compared with the disordered BNA or the mediator control, FANA sequences 1-7 did not show significant inhibitory activity. Figure 7 However, miR-21 and MYCC BNA slowed proliferation by approximately 50% within 48 hours. Therefore, BNA oligonucleotides were unexpectedly more effective than FANA oligonucleotides, which are different scaffold derivatives developed by other laboratories for therapeutic purposes.

[0186] We observed anti-miR-21 IC50 in seven human TNBC cell lines to inhibit cell proliferation. 50 A significant correlation was observed between miR-21 copy number / cell ratio, consistent with our hypothesized mechanism of action. The non-tumorigenic human breast epithelial cell line MCF-10A showed significantly reduced sensitivity to anti-miR-21 treatment. Figure 8 ).

[0187] The 50 nM miR-21 inhibitor BND5412 also induced significant apoptosis in human MDA-MB-231 TNBC cells, as measured by LDH assay. Figure 9 Similar results were observed in human HCC1937, HCC1806, BT-549, and BT-20 TNBC cells. Furthermore, treatment with the 50 nM miR-21 inhibitor BND5412 showed fewer MDA-MB-231 cells migrating to the wound area compared to a randomized sequence control.

[0188] Furthermore, in human HCC1806 TNBC cells, the 50 nM miR-21 blocker BND5412 significantly reduced (p<0.01) the levels of immune checkpoint mRNAs of PD-L1, PD-L2, CD47, and JAK2. Figure 10 ), and the corresponding PD-L1, PD-L2, CD47 and JAK2 immune checkpoint proteins ( Figures 5A-5B This indicates that miR-21 blockade can induce T cell recognition in TNBC cells.

[0189] RNA expression profile after miR-21 blockade

[0190] We performed RNA-seq analysis on RNA samples from human HCC1806 TNBC cells transfected with the miR-21 inhibitor BND5412 at an IC90 concentration, as well as from cells treated with the vector. To globally evaluate 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 assay compared the cumulative distribution of transcripts between miR-21 targets and all other genes, showing significant differences. Figure 11A This indicates that treatment with the miR-21 blocker BND5412 has global on-target effects on miR-21-regulated transcripts.

[0191] Off-target effects in treated HCC1806 cells were assessed using GGGenome (a program on the World Wide Web, located at gggenome.dbcls.jp) containing 0-2 mismatches, insertions, or deletions of human spliced ​​RNA from the miR-21 blocker BND5412. More than 300 RNA targets were predicted from the search results. None of the transcripts containing 0-1 mismatches were significantly downregulated by at least 2-fold. Eight genes containing two mismatches with the miR-21 blocker BND5412 were downregulated by at least 2-fold. Of these, SH3PXD2A, DIAPH2, PTPRK, MGAT5, and NLGN4X were identified as oncogenes. The remaining three genes, ERC1, ATRN, and FHOD3, were not found to be associated with any disease in the wild type when downregulated. No off-target effects with RNA level changes exceeding 4-fold were observed.

[0192] Gene set enrichment analysis of 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 activated interferon α and interferon γ responses. Figure 11B Both of these have positive effects on anti-tumor immunity (Jorgovanovic, et al. 2020, Biomark Res8:49; Vidal, P., 2020, Scand J Immunol 91(5):e12863). Furthermore, the KRAS inhibition pathway was also significantly enriched in samples treated with anti-miR-21 BND5412. Figure 11B Disease enrichment analysis using the Jensen Disease Database on the iDEP platform (http: / / bioinformatics.sdstate.edu / idep96 / ) showed significant enrichment in downregulated genes in breast cancer. Furthermore, the cancers with the most enriched downregulated genes included kidney cancer, liver cancer, melanoma, pancreatic cancer, and malignant glioma. Significant enrichment in other diseases included Alzheimer's disease, schizophrenia, cardiac conduction disorders, obesity, and acquired metabolic diseases such as type 2 diabetes. No disease-related enrichment was found in upregulated genes. These results suggest that miR-21 blockade may be effective for many other indications.

[0193] Cell-specific delivery

[0194] Targeted delivery of therapeutic agents is the biggest challenge in RNA analog therapy. Currently, delivery methods using liposomes or nanoparticles are available, but these are not ideal for long-term clinical use due to low delivery efficiency, toxicity, and limited biodistribution to the liver and kidneys. Our design provides a safe and efficient extrahepatic delivery of nucleic acid therapeutic agents. This involves conjugating RNA analogs with receptor-targeting peptides (…). Figure 12 IGF1R provides cell type-selective delivery. It is elevated in aggressive breast cancer, including TNBC. Most importantly, TNBC cells exhibit strong IGF1R signaling activation, which is associated with low survival. However, there is no known signaling feedback between miR-21 and IGF1R (Dobre, et al. Cells 10(8):1856,2021).

[0195] In earlier studies, we pioneered the direct internalization of polyamide nucleic acid (PNA) oligomers with a protease-resistant reverse cyclization D (CSKC) tetrapeptide IGF1 analog at the C-terminus of PNA into IGF1R-overexpressing cells. For example, see Tian, ​​Xet al. Journal of Nuclear Medicine vol. 48 (10): 1699-1707, 2007. We found that the radiolabeled 12-base sequence of PNA with C-terminal loop-D (CSKC) remained in circulation by complexing with IGF1BP (Opitz, et al., Oligonucleotides 20(3):117-25, 2010), prolonging the lifespan of the drug in vivo, which reduced the required dose. 99mThe urine of mice with Tc]MYCC PNA-loop-D (CSKC) showed 83% radioactivity in the intact probe peak, indicating in vivo stability. We observed receptor-mediated knockdown of cyclin D1 protein by PNA-loop-D (CSKC) in MCF7 breast cancer xenografts. We also used loop-D (CSKC) for tissue-specific delivery of various radioimaging agents. Thus, we were able to image CCND1, MYCC, HER2, or KRAS2 mRNA with the radionuclide-PNA-loop-D (CSKC) PET agent in mice carrying ER+ breast cancer xenografts, Her2+ breast cancer xenografts, demonstrating a DOX response by reducing HER2 mRNA PET SUV, in mice carrying pancreatic cancer xenografts, and in transgenic mice with spontaneous breast or lung tumors, demonstrating a response to cisplatin with single mismatch specificity. Importantly, specific PET imaging was blocked by excessive IGF1, consistent with our hypothesized cellular uptake mechanism via IGF1R-mediated endocytosis. In treatment of Her2+ breast cancer xenografts with doxorubicin (DOX), we observed a decrease in HER2 mRNA SUV to 54 ± 17% after 1 week of DOX therapy, consistent with a decrease in CT volume to 80 ± 47% at 7 weeks, and an increase in HER2 mRNA SUV to 145 ± 82% after 1 week without therapy, consistent with an increase in CT volume to 213 ± 78% at 7 weeks.

[0196] Newly designed miRNA blockers ( Figure 13 This minimizes and avoids guest chain mimicry by excluding the seed region of the opposing chain. Therefore, unlike the LNA inhibitors described in our earlier work, the miR repressive nucleic acid-peptide composition effectively blocks the target miRNA without influencing the function of the opposing chain. Figure 13 As shown, human HCC1806 TNBC cells, without transfection, take up fluorescent AF647-miR-21 BNA-D(CSKC) BND7673 and transport it into the cytoplasm, demonstrating efficient IGF1R-mediated endocytosis and cytoplasmic delivery. Figure 14 ).

[0197] Cell-specific activity of non-fluorescent drugs

[0198] A non-fluorescent anti-miR-21 RNA-peptide analog, BND6482, was synthesized. Figure 15And it is taken up into TNBC cells via endocytosis. Like fluorescent BND7673, the internalization and function of the inhibitory blocking molecule effectively increases the expression of the desired tumor suppressor protein, thereby inhibiting the growth of TNBC cells. BND6482 releases miR-21 luciferase reporter gene vector expression in human HCC1806 TNBC cells with high IGF1R expression in a dose-dependent manner. Figure 16A No transfection is required. Conversely, human MDA-MB-157 TNBC cells with low IGF1R expression showed little response to the miR-21 blocker BND6482 without lipid transfection. Figure 16B ).

[0199] Example II

[0200] Uptake of miR-21 RNA-peptide analogs in EMT6 allogeneic grafts

[0201] In immunocompetent female Balb / c mice carrying mouse EMT6 TNBC allogeneic grafts, a single intraperitoneal administration of the fluorescent miR-21 blocker BND7673 at 5 mg / kg resulted in effective fluorescent drug distribution to the tumor, which was evident at 24 h and persisted for at least 96 h. Figure 17 ).

[0202] 3-day response to TNBC allogeneic graft

[0203] After daily intraperitoneal administration for 3 days, tumor-suppressive effects of the fluorescent miR-21 blocker BND7673 were observed at a dose of 5 mg / kg. Figure 18 Clearly, several treated tumors were significantly smaller than control tumors. Fluorescence imaging showed the drug concentration in the treated tumors extracted from each animal. Although tumor images ( Figure 18 This showed the difference between treated and control tumors, but also revealed a wide dispersion in quality measured only 3 days after treatment. Figure 19 The results showed that tumor mass decreased by only 28% on average. Consistent with the hypothesis, BND7673 treatment reduced miR-21 in tumors and increased miR-21 target transcript PDCD4 mRNA (…). Figure 20 ).

[0204] 13-day response to TNBC allogeneic graft

[0205] Subsequently, we conducted a 2-week experiment using the non-fluorescent anti-miR-21 BNA-peptide BND6482. Figure 15After the mouse EMT6 TNBC allogeneic grafts reached approximately 5 mm in diameter, 5 mg / kg of randomized BNA-peptide BND6372 or anti-miR-21 BNA-peptide BND6482 in sterile saline medium was injected intraperitoneally twice weekly on days 0, 3, 7, and 10 for 2 weeks. Tumor volume was measured at the time of injection. On day 13, the tumor was harvested and weighed.

[0206] Tumors treated with the miR-21 blocker BND6482 remained small over two weeks with no significant tumor progression. Figure 21 At the end of 2 weeks, tumors treated with miR-21 blockade agents showed a significant reduction in tumor mass (mean reduction of 74%) compared to tumors treated with mediators or in disordered therapy. Figure 22 ).

[0207] Following twice-weekly intraperitoneal injections (IP) of 5 mg / kg of the miR-21 blocker peptide BND6482 or the disordered BND6372 over 13 days, or three IP injections of 6.25 mg / kg, toxicity markers in serum samples from treated tumor-bearing mice were indistinguishable from each other by one-way ANOVA and Dunnett's multiple comparison test. See also Figure 23 The error bars show the mean versus SEM. This result means that no liver or kidney toxicity was detected as a result of RNA-peptide analog treatment.

[0208] As can be seen from the above, a new molecular therapeutic agent and its variants are now available, which can be used alone or in combination with chemotherapy, with higher efficacy, specificity and safety, for effective targeted TNBC therapy.

[0209] Example III

[0210] Compositions and methods for improving symptoms associated with miR-21-modulatory disorders, particularly TNBC.

[0211] To treat individuals with miR-21-regulated disorders, particularly TNBC and other cancers, and to alleviate signs or symptoms of the disease, miR-21 inhibitory nucleic acid-peptide analogue inhibitors, such as those described in Example I, can be administered alone or in combination with other agents used for various symptoms associated with malignancies to provide therapeutic benefit to the patient. These agents are administered at effective doses to regulate cancer cell proliferation, cell cycle checkpoints, cell migration, and metastasis. Those skilled in the art will understand that the treatments mentioned herein extend to prevention and treatment of identified tumors or symptoms.

[0212] In some implementations, biological samples are obtained from the patient to confirm the presence and quantity of miR-21 copy number / cell and IGF1R copy number / cell in the tumor.

[0213] In other embodiments, this information is already available. The total therapeutic dose may be administered to the subject as a single dose, or it may be administered using a fractionated treatment regimen, wherein multiple / separate doses are administered over a longer period of time, for example, a daily dose may be administered over a period of day, or a dose for a desired period of time may be administered over a longer period of time. Those skilled in the art will recognize that the amount of miR-21 inhibitory nucleic acid-peptide analogue inhibitor required to obtain an effective, minimally toxic dose in a subject depends on many factors, including the subject's age, weight, and overall health condition, as well as the route of administration and the number of treatments to be administered. In view of these factors, those skilled in the art will adjust the specific dose to obtain an effective dose for treating an individual with cancer that can be treated with the miR-21 inhibitory nucleic acid-peptide analogue inhibitor described herein.

[0214] Compared to controls, in individuals with cancer, particularly more severe advanced cancer, administration of the miR-21 inhibitory nucleic acid-peptide analogue inhibitors described herein may be particularly useful when combined with other modified nucleic acid oligonucleotides or nucleotide peptide analogues, including but not limited to compounds targeting mRNAs encoding c-Myc transcription factor, tropomyosin kinase receptor (TRK), the MAPK pathway, the androgen receptor (AR) pathway, the growth factor receptor pathway, the PI3K-AKT pathway, immune checkpoints, DNA damage repair pathways, CDK4 / 6, CHK1, CHK2, WEE1, ATR, and / or cancer symptom relief. Therefore, in some embodiments, the method of the present invention includes administration of additional chemotherapeutic agents or chemotherapy. In some embodiments, the additional therapies are surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, and / or hormone therapy. In some embodiments, the chemotherapeutic agents are alkylating agents, antimetabolite antitumor agents, antitumor antibiotics, antitumor herbal medicines, platinum-based antitumor agents, hormone-balanced antitumor agents, and various antitumor agents. For example, these inhibitors can act additively or synergistically with chemotherapeutic agents to treat and inhibit cancer cell growth. These agents include, but are not limited to, rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, sorafenib, besentinib, crizotinib, bosutinib, giratetinib, amvatinib, and sunitinib, cabozantinib, veritinib, rebastinib, tripterygium wilfordii, dihydroartemisinin, PD-1 inhibitors, PD-L1 inhibitors, and CTL inhibitors. A4 inhibitors, PARP inhibitors, cyclophosphamide, ifosfamide, thiotepa, methotrexate, mercaptopurine, fluorouracil and cytarabine, bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin, mitoxantrone, vincristine, etoposide, teniposide, paclitaxel and docetaxel, cisplatin, carboplatin and oxaliplatin, euprolide, tamoxifen, flutamide and formestan, and arsenic trioxide. In some implementations, the tumor shrinks or is eradicated.

[0215] The described inhibitors can act additively or synergistically with PARP inhibitors to treat and inhibit cancer cell growth. Examples of PARP inhibitors include, but are not limited to, orlaparib, talazoparib, veliparib, rucaparib, niraparib, pamiparib, and fluzoparib.

[0216] The described inhibitors can act additively or synergistically with antibody-drug conjugates (ADCs) to treat and inhibit cancer cell growth. Examples of ADCs include, but are not limited to, sacituzumab govitecan, ladiratuzumab vedotin, patritumab deruxtecan, trastuzumab deruxtecan, datopotamab deruxtecan, enfortumab vedotin, SKB264, MGC018, and PTK7-ADC.

[0217] The described inhibitors can be used additively or synergistically with PI3K-AKT pathway inhibitors to treat and inhibit cancer cell growth. Examples of PI3K-AKT pathway inhibitors include, but are not limited to, alpelisib, taselisib, samotolisib, copanlisib, eganelisib, and gedatolisib.

[0218] The described inhibitors can act additively or synergistically with androgen receptor inhibitors to treat and inhibit cancer cell growth. Examples of androgen receptor inhibitors include, but are not limited to, bicalutamide, enzalutamide, abiraterone, embosarm, and darolutamide.

[0219] The described inhibitors can act additively or synergistically with TRK inhibitors to treat and inhibit cancer cell growth. Examples of TRK inhibitors include, but are not limited to, larotrectinib, selitrectinib, and repotrectinib.

[0220] The described inhibitors can be used additively or synergistically with mutant Her2 inhibitors to treat and inhibit cancer cell growth. Examples of mutant Her2 inhibitors include, but are not limited to, neratinib.

[0221] The described inhibitors can act additively or synergistically with immune checkpoint inhibitors to treat and inhibit cancer cell growth. Examples of immune checkpoint inhibitors include, but are not limited to, pembrolizumab, atezolizumab, avelumab, JS001, nivolumab, and duralumab.

[0222] The described inhibitors can act additively or synergistically with CDK4 / 6 inhibitors to treat and inhibit cancer cell growth. Examples of CDK4 / 6 inhibitors include, but are not limited to, palbociclib, abemaciclib, and ribociclib.

[0223] The described inhibitors can act additively or synergistically with CHK1 inhibitors to treat and inhibit cancer cell growth. Examples of CHK1 inhibitors include, but are not limited to, LY2880070 and prexasertib.

[0224] The described inhibitors can act additively or synergistically with WEE1 inhibitors to treat and inhibit cancer cell growth. Examples of WEE1 inhibitors include, but are not limited to, AZD1175 and ZN-c3.

[0225] The described inhibitors can act additively or synergistically with CHK2 inhibitors to treat and inhibit cancer cell growth. Examples of CHK2 inhibitors include, but are not limited to, LY2606368.

[0226] The described inhibitors can act additively or synergistically with ATR inhibitors to treat and inhibit cancer cell growth. Examples of ATR inhibitors include, but are not limited to, ceralasertib.

[0227] The described inhibitors can act additively or synergistically with RAD51 inhibitors to treat and inhibit cancer cell growth. Examples of RAD51 inhibitors include, but are not limited to, CYT-0851.

[0228] Skilled technicians will administer miR-21 inhibitors alone or in combination with at least one chemotherapeutic agent, and will monitor the effectiveness of such treatment using conventional methods such as radiological, immunological assays, or (where indicated) histopathological methods.

[0229] The administration of pharmaceutical preparations is preferably at an "effective amount" sufficient to demonstrate benefit to the individual. This amount can prevent, alleviate, reduce, or decrease the severity of the patient's cancer.

[0230] In a preferred embodiment of the invention, a method for treating cancer using the therapeutic agents disclosed in this embodiment in a combination approach is provided. Advantageously, the additive or preferably synergistic approach of the invention reduces cancer progression in mammalian hosts or reduces cancer symptoms. Importantly, the information provided herein guides clinicians in adopting new treatment methods to manage this disease.

[0231] The present invention also includes pharmaceutical compositions for treating cancer, comprising, by administering a therapeutically effective amount of one or more pharmaceutically acceptable carriers or fillers containing the miR-21 inhibitor of the present invention. Compositions containing the miR-21 inhibitor of the present invention can be administered orally or parenterally, including via intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, and local routes of administration.

[0232] Generally, the compounds listed above do not need to be administered in the same pharmaceutical composition, and may need to be administered via different routes due to their different physical and chemical properties. For example, the first compound may be administered orally to produce and maintain good blood levels, while the second compound may be administered intravenously. Determining the appropriate mode of administration and its rationality (where possible) within the same pharmaceutical composition is within the knowledge of a skilled clinician. Initial administration can be performed according to established protocols known in the art, and then, based on observed effects, an experienced clinician may modify the dosage, mode of administration, and frequency of administration.

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[0305] While certain preferred embodiments of the invention have been described and specifically illustrated above, this does not mean that the invention is limited to these embodiments. Various modifications can be made thereto without departing from the scope and spirit of the invention, as set forth in the appended claims.

Claims

1. A miR-21 inhibitory nucleic acid-peptide analog, wherein the miR-21 inhibitory nucleic acid-peptide analog has sequence complementarity with miRNA-21-5p to isolate miR-21-5p and its isoform miR 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 a modification selected from: BNA, LNA, FANA, PNA, 2'-fluoro, 2'-O-alkyl, morpholino, piperazine, thiophosphate, borophosphate, and borophosphate mixed with a phosphate diester bond, dithiophosphate bond, or methylphosphonate bond.

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

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

5. The miR-21 inhibitory peptide analog of claim 3, wherein at least 3, 4, 5, 6, 7, 8 or 9 nucleosides of SEQ ID NO: 4 are excluded.

6. The miR-21 inhibitory peptide analog according to any one of claims 1 to 5, comprising 5' and 3' BNA modifications as shown in FIG15.

7. The miR-21 inhibitory 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, 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 peptide analog of claim 1, wherein the peptide is the ligand CSKC of SEQ ID NO: 26 for the insulin-like growth factor 1 receptor, and the inhibitor nucleic acid sequence is selected from SEQ ID NO: 5 to 25, GATAGCTA, TGATAAGCT, CTGATAAGC, ATAAGCT, GATAAGCT, and TGATAAGC.

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

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

11. The miR-21 inhibitory peptide analog of claim 10, wherein the gapmer comprises three parts.

12. The miR-21 inhibitory peptide analog of claim 11, wherein the three portions comprise a first BNA portion, a DNA portion, and a second BNA portion.

13. A method for inhibiting miR-21-5p from binding 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 of any of the preceding claims, wherein the inhibitor binds to and sequesters the miR-21-5p, thereby inhibiting or preventing miR-21-5p from binding to regulatory sites present in mRNA encoding proteins that regulate the growth and / or metastasis of malignant cells.

14. A method for treating triple-negative breast cancer (TNBC) in patients in need, comprising: Administering an effective amount of the miR-21 inhibitory peptide analog according to any one of claims 1 to 12, the analog causing TNBC arrest or cell death.

15. The method according to any one of claims 13 or 14, wherein the analogue is shown in FIG15.

16. The method according to any one of claims 13, 14, or 15, further comprising administering a chemotherapeutic agent selected from: rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, sorafenib, besentinib, crizotinib, bosutinib, gidatetinib, amvatinib, sunitinib, cabozantinib, veritinib, rebastinib, tripterygium wilfordii, dihydroartemisinin. PD-1 inhibitors, PD-L1 inhibitors, CTLA4 inhibitors, cyclophosphamide, ifosfamide, thiotepa, methotrexate, mercaptopurine, fluorouracil and cytarabine, bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin, mitoxantrone, vincristine, etoposide, teniposide, paclitaxel and docetaxel, cisplatin, carboplatin and oxaliplatin, euprolide, tamoxifen, flutamide and formestan, and arsenic trioxide.

17. The method of claim 13, 14 or 15, wherein the level of the disease-driving protein is reduced by treatment with the inhibitor.

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

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

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

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

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