WO2017019523A1 - Compositions de mir-124 pour agents thérapeutiques immunomodulateurs - Google Patents

Compositions de mir-124 pour agents thérapeutiques immunomodulateurs Download PDF

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WO2017019523A1
WO2017019523A1 PCT/US2016/043607 US2016043607W WO2017019523A1 WO 2017019523 A1 WO2017019523 A1 WO 2017019523A1 US 2016043607 W US2016043607 W US 2016043607W WO 2017019523 A1 WO2017019523 A1 WO 2017019523A1
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mir
glioma
composition
nanoparticles
cells
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PCT/US2016/043607
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Amy Heimberger
Jun Wei
Padmanabh Chivukula
Joseph E. Payne
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Arcturus Therapeutics, Inc.
Board Of Regents, The University Of Texas System
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Publication of WO2017019523A1 publication Critical patent/WO2017019523A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • This invention relates to the fields of biopharmaceuticals and therapeutics having at least in part active ingredients comprising one or more micro-RNA structures for gene regulation and modulation, including immune system modulation.
  • the invention also embraces micro-RNA compositions and their methods of use and delivery to cells, tissues and organisms using lipids or lipid nanoparticles.
  • micro-RNAs also known as miRNAs and miRs
  • therapeutics are appealing owing to robust preclinical data demonstrating their therapeutic efficacy, broad biological activity, and ability to shut down signaling networks at multiple nodes along various pathways, including alternative ligand activation.
  • Therapeutic translation of miRNAs has been limited by technical challenges concerning delivery, stability of the miRNAs, and avoidance of activating immune responses.
  • endogenous microRNAs remain stable when secreted into the circulation if they are enclosed in microvesicles, such as exosomes (Camussi et al., 2010, Kidney Int. 78:838-48, 2010).
  • a drawback of nanoparticle chemotherapeutics is that none has been devised to modulate the immune system. For example, nanoparticle delivery of micro-RNAs for intracellular immune targeting has not been achieved.
  • tumor-mediated immunosuppression is a major barrier to therapeutic approaches.
  • Tumors can inhibit immune responses by several mechanisms.
  • a tumor can express various antigens and/or attract immunosuppressive lymphocytes (Chen et al., PLoS One. 6:e24671 2011).
  • tumors can secrete immunosuppressive cytokines (Orleans-Lindsay et al., Clin Exp Immunol.
  • a tumor can express surface molecules, which inhibit immune responses (Mann et al., Br J Cancer. 79: 1262-69, 1999).
  • Tumors can establish an immunosuppressive environment acting like an invisibility cloak, allowing the tumor to grow while the immune system remains "blind" to the threat.
  • microRNAs miRs
  • compositions for selective delivery of microRNAs to various compartments of the immune system such as immune system cells.
  • compositions and methods for exploiting the immune system to mediate antitumor immune effects There is a long-standing need for nanoparticle formulations of miRNAs which can be used to overcome tumor-mediated immunosuppression.
  • This invention provides nanoparticle formulations of miRNAs, which can be used to treat solid malignancies and other tumors.
  • the compositions of the invention can be used as active pharmaceutical formulations for ameliorating, preventing or treating solid malignancies and tumors, particularly gliomas or glioblastomas, in various anatomical locations, e.g., cells, tissues and organs.
  • compositions of this invention may reduce the growth of tumors or
  • compositions of this invention may cause regression of a malignancy or tumor size.
  • Embodiments of this invention include compositions that advantageously provide enhanced effectiveness against tumors or malignancies, and which can provide clinical agents.
  • this invention provides nanoparticle-encapsulated miR- 124 mimics, whether single or double stranded.
  • the nanoparticle-encapsulated miR-124 mimic compositions provide surprisingly enhanced delivery of an effective amount of miR-124 to the immune system that thereby prevents glioma growth.
  • compositions of this invention provide increased therapeutic efficacy and immune compartment delivery of nanoparticle-encapsulated miR-124 mimics.
  • a nanoparticle-encapsulated miR-124 mimic composition of this invention can provide increased immunomodulatory effects, including reversal of tumor-mediated immunosuppression.
  • this invention can provide miR-124 mimic compositions that can induce immunological memory.
  • compositions of this invention can induce the immune system to mediate antitumor immune effects over long time periods.
  • the present invention includes methods for preventing, ameliorating or treating a disease associated with a glioma in a subject by administering to the subject an effective amount of a nanoparticle composition comprising miR-124 mimics.
  • the administration can reduce the growth of a glioma, or induce regression of a glioma.
  • the glioma can be located in the brain or spine of the subject.
  • administration may be intravenous.
  • the nanoparticles encapsulate the miR-124 mimic.
  • the nanoparticles can comprise one or more cationic lipids and one or more helper lipids.
  • Lipid nanoparticles for use in this invention can be any lipid nanoparticles known in the art.
  • the nanoparticles may include one or more cationic lipids and one or more helper lipids, as are known in the art.
  • lipid nanoparticles can include one or more lipids selected from cationic lipids, anionic lipids, cholesterol, sterols, pegylated lipids, and any combination of the foregoing.
  • This invention further contemplates methods for activating immune system cells against glioma.
  • the immune system cells can be contacted with a nanoparticle composition containing miR-124 mimics.
  • the immune system cells can be contacted with the nanoparticle composition in vivo or ex vivo.
  • the nanoparticles can encapsulate the miR-124 mimics.
  • Embodiments of this invention further include compositions containing immune system cells that are activated against a glioma.
  • the cells may express activated STAT3 (signal transducer and activator of transcription 3), as compared to cells that are not activated.
  • STAT3 signal transducer and activator of transcription 3
  • the cells may have increased activity against glioma when the STAT3 activation is reduced with miR-124.
  • This invention includes methods for activating immune response against glioma in a subject, by administering to the subject a nanoparticle composition containing miR- 124 mimics.
  • the nanoparticle composition may selectively deliver the miR-124 mimic to an immune system compartment in the subject.
  • the immune system compartment can be peripheral blood mononuclear cells (PBMCs).
  • the immune system compartment can be monocytes and macrophages.
  • the level of miR-124 in the monocytes and macrophages can be at least five-fold greater than in the monocytes and macrophages of a subject to which the composition was not administered.
  • the immune compartment can be B cells.
  • the level of miR-124 in the B cells can be at least five-fold greater than in the B cells of a subject to which the composition was not administered.
  • a glioma can be located in the brain or spine of a subject.
  • the administration of a nanoparticle composition can be intravenous.
  • Additional embodiments of this invention contemplate methods for inducing immunological memory against a glioma in a subject.
  • the nanoparticle formulation can be administered to the subject containing an effective amount of miR- 124 mimic.
  • the immunological memory can include persistence of anti-glioma immune effects over a period of at least 5 days, or at least 15 days, or at least 50 days, or at least 100 days.
  • FIG. 1 shows that nanoparticle formulations can enhance the effect of miR-124 mimic.
  • Four lipid-based nanoparticle formulations encapsulating miR-124 mimic were designed and tested in the same murine GL261 glioma model.
  • nanoparticles in NB5-55-2 nanoparticle formulation LUNAR-301 showed the greatest effect relative to LIPOFECTAMINE, and produced a median survival time that could not be calculated because >60% of mice were long-term survivors.
  • LUNAR-301 comprises ATX-002:DSPC:Chol:DMG- PEG at a molar ratio of 55: 10:33.5: 1.5 with an N/P ratio of 6; where, ATX-002 is an ionizable lipid; DSPC is l,2-distearoyl-sn-glycero-3-phosphocholine; Choi is
  • N is the ionizable lipid Nitrogens (N) and P is the Phosphate groups (P) in the miRNA or mimic.
  • FIG. 2 shows that nanoparticle formulations can be selectively delivered to the immune compartment.
  • Samples from peripheral blood mononuclear cells (PBMCs), serum, and liver were taken at 0 and 15 minutes, as well as at 1, 4, 8, and 24 hours. Quantitative PCR was used to assess the miR-124 level in each compartment.
  • Nanoparticle formulation LUNAR-301 showed surprisingly more effective delivery of miR-124 to the PBMC compartment than LIPOFECTAMINE.
  • FIG. 3 shows that nanoparticle formulations can be selectively delivered to the immune compartment.
  • Circulating PBMCs were isolated from C57BL/6 mice after a single treatment with nanoparticle formulation LUNAR-301 at the peak distribution window of 15-20 minutes post injection.
  • T cells CD3+
  • B cells CD 19+
  • NK cells NK1.1+
  • monocytes/macrophages CD1 lb+
  • FIG. 4 shows that nanoparticle formulations can inhibit immune suppression, an immunomodulatory property.
  • C57BL/6 mice with subcutaneously implanted GL261 gliomas were treated 5 times with either miR-124 mimic + LIPOFECTAMINE(TM) or nanoparticle formulation LUNAR-301. Thereafter, the glioma-infiltrating T cells were isolated and assessed for intracellular p-STAT3 and FoxP3 (forkhead box P3).
  • Two sample t-test was used for pair- wise comparison. Error bars, mean ⁇ SD.
  • FIG. 5 shows that nanoparticle formulations can induce immunological memory.
  • FIG. 6 shows that nanoparticle formulations can induce long term immunological memory.
  • Long-term surviving rechallenged mice in FIG. 5 were subsequently rechallenged a third time with intracerebrally implanted gliomas in the original brain hemisphere, again with no further treatment.
  • All but one of the mice previously treated with nanoparticle formulation LUNAR-301 were protected from tumor recurrence, and the mouse that had a recurrence survived for longer than the median survival time of untreated control mice (33 days).
  • FIG. 7 shows that LUNAR-301 does not cause weight loss.
  • Non-tumor- bearing C57BL/6 mice were treated with either nanoparticle formulation LUNAR-301 (LUN), seed sequence control (SSC), or PBS for a total of 9 doses. Their body weights were measured throughout the course of treatment and did not differ among treatment groups. Error bars, mean ⁇ SD.
  • FIG. 8 shows that LUNAR-301 does not induce clinically significant hepatomegaly or splenomegaly.
  • FIG. 9 shows that LUNAR-301 does not induce clinically significant increases in liver transaminases.
  • LFTs liver function tests
  • AST aspartate aminotransferase
  • Canines were treated with a single escalating dose of nanoparticle formulation LUNAR- 301 (301) or with nine doses at 0.5 mg/kg. Blood samples were drawn pretreatment and at various time points up to 72 hours post treatment. Sera and PBMCs were isolated, and miR-124 was quantified by PCR for each compartment. In all cases, miR-124 was elevated in the sera and PBMCs of canines treated with nanoparticle formulation
  • LUNAR-301 relative to control canines receiving only empty nanoparticles.
  • canines receiving higher doses of nanoparticle formulation LUNAR-301 had greater levels of miR-124 quantified, and the increases in miR-124 levels were sustained with multi-dosing.
  • FIG. 1 1 shows that nanoparticle formulations can be selectively delivered to the immune compartment for activity in a canine model. Looking at the multi-dose study in greater resolution over the time course, miR-124 was quantified at doses 1, 4, 7, and 9. Up to a 50-fold increase in miR-124 in the serum and 14-fold increase in miR-124 within the PBMCs was observed relative to pretreatment baselines. Additionally, in the PBMC compartment, there was a dose accumulation effect with each subsequent dosing over the treatment period.
  • FIG. 12 shows that PBMCs from canines were also isolated from fresh blood and evaluated for effector cytokine production. There appeared to be a dose- dependent response in the production of IL-2, INF a, and INFy after nanoparticle formulation LUNAR-301 treatment; the highest INFy production was achieved with multi-dosing.
  • FIG. 13 shows a schematic of a proposed mechanism of nanoparticle formulation LUNAR-301 action. While not wishing to be bound by any one particular theory, nanoparticle formulation LUNAR-301 (miR-124 mimic within lipid
  • nanoparticles is delivered to the patient intravenously and immediately come in contact with circulating PBMCs, most notably monocytes/macrophages and B cells.
  • PBMCs most notably monocytes/macrophages and B cells.
  • the nanoparticles are engulfed and rapidly degraded via a pH-mediated process within the endosome, thereby delivering the miR-124 mimic payload inside the cell.
  • MiR-124 subsequently inhibits the STAT3 pathway, facilitating reversal of tumor-mediated immune suppression and resultant immune activation, allowing resident and peripheral immune cells to target the CNS tumor.
  • FIG. 14 shows modifications of nanoparticle formulation dosing and schedule.
  • C57BL/6 mice with established intracerebrally implanted gliomas were treated with either LIPOFECTAMINE alone, empty nanoparticles, or miR-124 mimic +
  • Mice treated with nanoparticle formulation LUNAR-301 at 2.5 mg/kg had a median survival time (27 days) comparable with the LIPOFECTAMINE and empty nanoparticle controls (22 days
  • FIG. 15 shows in Table 1 : Murine in vivo pharmacokinetics of nanoparticle formulation LUNAR-301 relative to miR-124 mimic + LIPOFECTAMINE. Pharmacokinetic parameters were calculated for nanoparticle formulation LUNAR-301 and miR-124 mimic + LIPOFECTAMINE using the non-compartmental model (NCA).
  • NCA non-compartmental model
  • Cmax the observed maximal concentration
  • Tmax time at observed maximal concentration
  • Tmax apparent elimination half-life estimated from the terminal time-concentration curve
  • ⁇ 1 ⁇ 2 ⁇ apparent elimination half-life estimated from the terminal time-concentration curve
  • ⁇ first-order rate constant associated with the terminal portion of the time-concentration curve
  • AUClast area under the curve from time zero to the last sample time
  • AUCinf obs percent AUC extrapolated from last sample time to infinity
  • AUC % extrap area under the moment curve from time zero to last sample time
  • AUMClast area under the moment curve from time zero extrapolated to infinity
  • MRTlast mean residence time based on time zero to last sample time
  • MRTinf obs mean residence time based on time zero extrapolated to infinity
  • This invention provides a range of novel agents and compositions to be used as therapeutics against glioma in a subject.
  • this invention encompasses microRNAs (miRNAs) directed to preventing or treating glioma in a subject.
  • miRNAs microRNAs
  • miRNAs are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs.
  • miRNAs can be transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding.
  • the primary transcript is cleaved by the Drosha ribonuclease III enzyme, which can produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA).
  • pre-miRNA the Drosha ribonuclease III enzyme
  • RISC RNA-induced silencing complex
  • MIR124-1 which is microRNA 124-1 ⁇ Homo sapiens, human
  • NC_000008.11 (9903388..9903472, complement)
  • This invention provides a range of compositions containing miR-124 that are useful for providing therapeutic effects because of their activity in modulating expression of a gene, as well as immune effects.
  • the compositions of this invention provides gene regulating or gene silencing activity in vitro and in vivo.
  • Embodiments of this invention provide compositions for use as therapeutic agents against glioma.
  • the compositions can be used as pharmaceutical agents for ameliorating, preventing or treating a glioma in a subject.
  • a microRNA of this invention can be modified with non-natural nucleotides, or modified nucleotides, or chemically-modified nucleotides, including any such nucleotides known in the art.
  • non-natural, modified, and chemically-modified nucleotide monomers include any such nucleotides known in the art, for example, 2'-0-methyl ribonucleotides, 2'-0-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'- deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl-nucleotides, and inverted
  • non-natural, modified, and chemically-modified nucleotide monomers include 3 '-end stabilized nucleotides, 3 '-glyceryl nucleotides, 3 '-inverted abasic nucleotides, and 3'-inverted thymidine.
  • non-natural, modified, and chemically-modified nucleotide monomers include locked nucleic acid nucleotides, 2'-0,4'-C-methylene-(D- ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, and 2'-0-methyl nucleotides.
  • locked nucleic acid nucleotides 2'-0,4'-C-methylene-(D- ribofuranosyl) nucleotides
  • MOE methoxyethoxy
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 2'-amino nucleotides, 2'-0-amino nucleotides, 2'-C-allyl nucleotides, and 2'-0-allyl nucleotides.
  • non-natural, modified, and chemically-modified nucleotide monomers include N 6 -methyladenosine nucleotides.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include nucleotide monomers with modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 2'-0-aminopropyl substituted nucleotides.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R, a 2'-OR, a 2'- halogen, a 2'-SR, or a 2'-amino, where R can be H, alkyl, alkenyl, or alkynyl.
  • LNA locked nucleic acid monomers
  • this invention provides methods for preventing, ameliorating or treating a disease associated with glioma in a subject.
  • the methods can include intravenous administration to the subject an effective amount of a nanoparticle composition comprising miR-124 mimic.
  • the nanoparticle composition can deliver miR- 124 mimic for therapeutic effects in reducing the growth of a glioma, or inducing regression of a glioma.
  • the nanoparticles can encapsulate the miR-124 mimic and provide endosomolytic access of the miR-124 mimic to cells.
  • methods of this invention include activating immune system cells against glioma.
  • the immune system cells can be contacted with a nanoparticle composition containing miR-124 mimic.
  • the contacting of cells with the nanoparticle composition can be performed in vivo or ex vivo.
  • compositions containing immune system cells that are activated against glioma may have immune system cells that have reduced levels of STAT3 with the miR-124 as compared to cells that are not activated. Such compositions can have increased activity against glioma.
  • this invention provides methods for activating an immune response against glioma in a subject.
  • a nanoparticle composition of this invention can selectively deliver miR-124 mimic to an immune system compartment in the subject.
  • Immune system compartments can include myeloid lineage cells, lymphocytes, monocytes, peripheral blood mononuclear cells, or macrophage thereof.
  • the immune system compartment can be PBMCs, or can be monocytes and macrophages.
  • the level of miR-124 in the monocytes and macrophages can be at least five-fold greater than in the monocytes and macrophages of a subject to which the composition was not administered.
  • the immune system can be PBMCs, or can be monocytes and macrophages.
  • the compartment can include B cells.
  • the level of miR-124 in the B cells can be at least five-fold greater than in the B cells of a subject to which the composition was not administered.
  • Additional modalities of this invention include methods for inducing immunological memory against a glioma in a subject.
  • administering to the subject an effective amount of a nanoparticle composition containing miR-124 mimic provide immune system cells that are activated against a glioma and create immunological memory against a glioma.
  • the immunological memory and persistent immunotherapeutic effects in vivo can include persistence of anti-glioma immune effects over a long period of time, from days, to weeks, to months, and longer.
  • Methods and compositions of this invention can be used for treatment, amelioration, or prevention of diseases in mammalian subjects associated with glioma.
  • a subject can be a human or mammal.
  • a subject in need of treatment or prevention can be administered an effective amount of a nanoparticle composition of this invention.
  • the nanoparticles can encapsulate a nucleic acid agent, such as miR-124 mimic.
  • nucleic acid agent and “nucleic acid” refer to any nucleic acid polymer composed of either polydeoxyribonucleotides (containing 2-deoxy-D-ribose), or polyribonucleotides (containing D-ribose), or any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases.
  • An effective amount of miR-124, single or double-stranded can be a dose ranging from 0.001 mg/kg to 50.0 mg/kg.
  • a dose of double- stranded miR-124 of 1 mg/kg can be administered.
  • the dose can be administered once per week, or 2 times per week, or three times per week, or daily, or twice daily, or 3-5 times per day.
  • this invention provides pharmaceutical compositions containing an nucleic acid agent and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition can be capable of local or systemic
  • a pharmaceutical composition can be capable of any modality of administration.
  • the administration can be intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, or nasal administration.
  • Embodiments of this invention include pharmaceutical compositions containing miR-124 in a lipid or lipid nanoparticle formulation.
  • Lipid nanoparticles for use in this invention can be any lipid nanoparticles known in the art.
  • a pharmaceutical composition may comprise one or more lipids selected from cationic lipids, anionic lipids, sterols, pegylated lipids, and any combination of the foregoing.
  • a pharmaceutical composition can be substantially free of liposomes.
  • a pharmaceutical composition can include liposomes, lipid nanoparticles or nanoparticles.
  • lipids and lipid compositions for delivery of an active molecule of this invention are given in WO/2015/074085, which is hereby incorporated by reference in its entirety.
  • this invention includes nanoparticle compositions that can encapsulate and deliver miR-124 to cells with surprisingly advantageous potency.
  • the nanoparticles can be formed with lipid molecules, for example, any one or more of the compounds ATX-001 to ATX-032 disclosed in WO/2015/074085, the contents of which are incorporated herein by reference in their entirety.
  • lipid nanoparticles of this invention can be formed with compound ATX-002 (Compound I), as disclosed in WO/2015/074085, and the formulation is referred to herein as LUNAR- 301, which encapsulates miR-124.
  • a pharmaceutical composition of this disclosure may include carriers, diluents or excipients as are known in the art. Examples of pharmaceutical compositions are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. 1985).
  • excipients for a pharmaceutical composition include antioxidants, suspending agents, dispersing agents, preservatives, buffering agents, tonicity agents, and surfactants.
  • Example 1 Preparation of nanoparticles.
  • DOTAP cholesterol nanoparticles with miR-124 were created by Dr. Jack Roth (MD Anderson, Houston, TX). Chitosan miR-124 nanoparticles were prepared on an ionic gelation of anionic tripolyphosphate by Dr. Anil Sood (MD Anderson, Houston, TX).
  • a bi-lipid delivery nanoparticle formulation was used to make four test articles containing miR-124: B5-55-1, B5-55-2 (using compound ATX-002 and designated LUNAR-301), NB5-55-3, and B5-55-4.
  • the lipid nanoparticles (LNPs) were prepared by mixing appropriate volumes of lipids in ethanol with an aqueous phase containing miRNA duplexes, employing a NANOESSEMBLER microfluidic device, followed by downstream processing.
  • RNA For the encapsulation of miRNA, the desired amount of double-stranded RNA was dissolved in 5 mM citric acid buffer, pH 3.5. Lipids at the desired molar ratio were dissolved in ethanol. The mol% ratio for the constituent lipids is 58% ATX-002
  • the total combined flow rate was 12 mL/min, per microfluidics chip. From one to four microfluidics chips were utilized, in a custom unit for parallelization (Precision NanoSystems), allowing a variable throughput for different batch sizes.
  • the microfluidics chips employed a herringbone micromixer for extremely quick mixing times, yielding high encapsulation and narrow particle size distribution.
  • the mixed material was then diluted 3x with deionized water after leaving the micromixer outlet, reducing the ethanol content to 6.25%.
  • the diluted LNP slurry was concentrated on a TFF with hollow fiber membranes (mPES Kros membranes, Spectrum Laboratories, Inc., Collinso Dominguez, California), and then diafiltration was performed with modified DPBS, without magnesium or calcium (HyClone, Logan, Utah). A total of 10 diavolumes were exchanged, effectively removing the ethanol.
  • Particle size was determined by dynamic light scattering (ZEN3600, Malvern Instruments, UK). Encapsulation efficiency was calculated by determining
  • the miR-124 duplex used for all formulations comprised the following sense and anti-sense strands:
  • sense 5'-UAAGGCACGCGGUGAAUGCCA-3' (SEQ ID NO: 1)
  • antisense 3'-UAAUUCCGUGCGCCACUUACG-5' (SEQ ID NO: 2)
  • Example 2 Intracerebral glioma murine model.
  • a GL261 glioma is a carcinogen-induced mouse syngeneic glioma model, which is a brain tumor model developed in immunocompetent animals that has growth characteristics similar to human glioblastoma multiform (GBM).
  • Example 3 Murine pharmacokinetic analysis.
  • the pharmacokinetics of nanoparticle formulation LUNAR-301 comprising the miR-124 duplex in vivo were compared with those of double-stranded miR-124 + LIPOFECTAMINE in non-tumor-bearing C57BL/6 mice at a 1 mg/kg i.v. dose.
  • the serum, liver, and PBMCs were fractioned for total RNA extraction.
  • the miR-124 concentration in each compartment was assessed by quantitative RT-PCR using a standard curve containing a series of miR-124 duplex dilutions.
  • a noncompartmental analysis was performed in mice using industry-standard software (WinNonLin 6.3, Pharsight) to estimate the pharmacokinetic parameters for each individual animal, using drug concentrations observed in serum, liver, and PBMCs.
  • the following parameters were estimated for each animal and tissue: time of peak serum drug concentration (Tmax), peak drug concentration (Cmax), apparent elimination half-life (Tl/2, calculated as ln(2)/lambda z, lambda z being the first order rate constant associated with the terminal portion of the time-concentration curve, as estimated by linear regression of time vs.
  • mice were subcutaneously implanted with 2 x 10 6 GL261 cells in matrigel to have a tumor sufficiently large for analysis of the infiltrating immune population. Two weeks after implantation, mice were randomized by tumor size and treated with empty nanoparticles (with exactly the same composition as LUNAR-301, but without the double-stranded miR-124 double-stranded miR-124 mimic +
  • subcutaneous gliomas were homogenized in cold MACS buffer (lx PBS, 2% FBS, 2nM EDTA) to make a single-cell suspension.
  • Splenocytes were also harvested and homogenized in cold MACS buffer. Red blood cells were removed with red blood cell lysing buffer (Sigma- Aldrich) to form a single-cell suspension of splenocytes that were cocultured with Dynabeads CD3/CD28 (Life Technologies) and supplemental IL-2 for seven days to activate T cells.
  • glioma-infiltrating T cells were fixed and
  • Flow cytometry acquisition was performed with a FACS Calibur (Becton Dickinson, San Diego, CA) and data analyzed using FlowJo software (TreeStar, Ashland, OR).
  • 11-99-01 is comprised of ATX-002: DSPC: Choi: DMG-PEG at a molar ratios of 58:7:33.5: 1.5 and a non-targeting control siRNA at an siRNA to total lipid ration of 0.062.
  • ATX-002 is an ionizable lipid
  • DSPC is l,2-distearoyl-sn-glycero-3- phosphocholine
  • Choi is Cholesterol
  • DMG-PEG is l,2-Dimyristoyl-rac-glycero-3- methyl-polyoxy ethylene.
  • 11-99-02 is an empty lipid nanoparticle (meaning no RNA payload) comprised of ATX-002: DSPC: Choi: DMG-PEG at a molar ratios of 58:7:33.5: 1.5, where, ATX- 002 is an ionizable lipid; DSPC is l,2-distearoyl-sn-glycero-3-phosphocholine; Choi is Cholesterol and DMG-PEG is l,2-Dimyristoyl-rac-glycero-3-methyl-polyoxyethylene.
  • mice Male and female CD-I mice were treated once on Day 0 by injection into the left lateral tail vein at doses of 5, 10, 20 or 30 mg/kg at a volume of approximately 6 mL/kg. Control groups received only the vehicle, sterile PBS. Body weights of the mice were monitored on Day 0 and at the time of sacrifice (Day 2, approximately 48 hours post treatment), and terminal blood samples were collected for assessment of clinical chemistry and hematology parameters after an overnight fast. The wet weights of the spleen and liver were also measured and recorded on Day 2 (i.e., 48 hours post treatment).
  • Example 6 Canine model.
  • Dogs were allowed to acclimate to the surroundings for 14 days prior to initiating the treatment. Serial physical examinations as well as measurement of body temperature, pulse and respiratory rate were performed. Blood sampling in the dogs was performed via jugular venipuncture. Three days before (Day -3) and immediately prior to the delivery of nanoparticle formulation LUNAR-301 (Day 1) baseline physical examination and clinical observations were performed, and blood samples (12 mL) were collected for complete blood counts (CBCs), serum chemistry profiles, and coagulation profiles.
  • CBCs complete blood counts
  • Blood samples (3 mL) for analysis of the native wild type single stranded miR- 124 concentration in serum and in PBMCs were collected at 10, 20, 30, 45, and 60 minutes, and at 2, 4, 6, 12, 24, 48, and 72 hours post-LUNAR-301 delivery.
  • Immune activation status was sampled at 0 and 24 hours. At 72 hours, blood was obtained for CBC, serum chemistry profile, and the coagulation profile. RT-PCR was utilized to quantify wild type miR-124 in canine PBMCs and sera. Nanoparticle formulation LUNAR-301 and empty nanoparticle administration (i.v.) was through the cephalic vein. Canines were subsequently euthanized and received a complete necropsy, with gross and histological examination. Portions of the drug delivery site, heart, liver, spleen, brain, lung, intestine, kidney, lymph node, thymus, and bone marrow were fixed in formalin and then paraffin embedded. Four-micrometer sections were mounted on glass slides and stained with H&E. Slides were microscopically evaluated by an anatomic pathologist who was blinded to the treatment regimen.
  • Example 7 Canine flow cytometry.
  • PMBCs were isolated from fresh blood using Histopaque (Sigma).
  • PBMCs were transferred to 96-well round-bottom plates and washed with FACS buffer (lx PBS, 2% FBS, 0.09% NaN3), then fixed and permeabilized (eBioscience).
  • FACS buffer lx PBS, 2% FBS, 0.09% NaN3
  • PBMCs were stained for thirty minutes at 4°C with 1 :30 Anti -Mouse/Rat FoxP3 APC (eBioscience).
  • PBMCs were transferred to 96-well flat-bottom plates and cultured for six hours in RMPI 1640 (Gibco) complete medium with 50 ng/mL PMA (Sigma-Aldrich), 500 ng/mL ionomycin (Sigma-Aldrich), and 1 :500 Protein Transport Inhibitor Cocktail (eBioscience) for T-cell activation.
  • Activated PBMCs were transferred to 96-well round-bottom plates and washed, fixed, and permeabilized. Cells were incubated with 1 : 100 Biotin-conjugated anti-canine IL-2, INFy, or T Fa antibody (RD Systems) for 20 minutes at 4°C.
  • APC-conjugated Streptavidin (BD) 1 :50 was used as the secondary antibody.
  • Flow cytometry acquisition was performed with a FACS Calibur (Becton Dickinson, San Diego, CA), and data were analyzed using FlowJo software (TreeStar, Ashland, OR).
  • Example 8 Statistical analysis.
  • OS Overall survival
  • the log-rank test and a stratified Cox proportional hazards regression model were used to explore the survival differences between individual treatment groups.
  • a one-sided Fisher's exact test was used for comparison of the cure rates between nanoparticle formulation LUNAR-301 and other treatment groups.
  • the longitudinal mixed-effects model ENREF 16 was employed to assess the tumor growth rate (on a natural logarithmic scale).
  • Statistical software R V-3.0.2 (with packages survival v2.37-4 and nlme v3.1-111) was used for data analysis. P values less than or equal to 0.05 were considered statistically significant.
  • Nanoparticle formulations can enhance the therapeutic effect of a double-stranded miR-124 mimic, and it can be delivered to the immune
  • Double stranded miR-124 mimic was encapsulated with an efficiency of >95% and a polydispersity index ⁇ 0.05 using a GMP-ready process of a NANOESSEMBLER microfluidic device, followed by downstream processing. This produced an 80 nm particle with long-term stability (>4 months) and no variation in particle size.
  • Four test articles nanoparticles containing double-stranded miR-124 mimics with modifications of the lipid formulation were devised.
  • NB5-55-2 (formulation LUNAR-301) demonstrated superiority, with a median survival time that could not be calculated because >60% of mice were long-term survivors.
  • the three remaining double-stranded miR-124 mimic- nanoparticle formulations produced median survivals of 30.5, 43.5, and 6 days for NB5- 55-1, B5-55-3, and B5-55-4, respectively. Significant toxicity was observed in the B5-55-4 group, and necropsy showed the deaths did not result from intracranial tumors.
  • Example 10 Frequency of drug administration.
  • formulation LUNAR-301 dosed at 1 mg/kg or 2.5 mg/kg of the double- stranded miR-124 mimic inside the formulation was evaluated on a Monday and
  • Example 11 Nanoparticle formulations can be selectively delivered to the immune compartment.
  • miR-124 kinetics were further analyzed in the PBMC compartment because this is the desired delivery compartment (e.g., immune cells). As shown in FIG. 15, Table 1, after single dosing, peak concentrations were achieved at 15 minutes, and miR-124 persisted for ⁇ 4 hours. The PBMC immune subsets were fractionated, and analyzed for the delivery of miR-124. It was found that formulation LUNAR-301 preferentially targeted monocytes, as denoted by CD1 lb+ in FIG. 3, consistent with their biological function of phagocytosis and representing an important site of STAT3 regulation, given the innate monocyte/macrophage interface with adaptive immune responses.
  • LUNAR-301 preferentially targeted monocytes, as denoted by CD1 lb+ in FIG. 3, consistent with their biological function of phagocytosis and representing an important site of STAT3 regulation, given the innate monocyte/macrophage interface with adaptive immune responses.
  • Example 12 Nanoparticle formulation immunomodulatory properties.
  • mice surviving initial GL261 glioma implantation after treatment with double-stranded miR- 124 mimic + LIPOFECTAMINE or formulation LUNAR-301 were rechallenged with a second intracerebral glioma implanted in the contralateral hemisphere. As shown in FIG. 5, subsequently, only one mouse died in the LUNAR-301 group (survival >200 days post initial tumor implantation). These mice were again rechallenged with a third intracerebral tumor, with no subsequent treatment with formulation LUNAR-301.
  • immunological memory may not be a general property of anti- STAT3 agents, because small molecular inhibitors of STAT3 may not generate immunological memory in murine melanoma models.
  • Example 13 Nanoparticle formulation toxicity.
  • Toxicity was tested by treating mice for two weeks with either formulation LUNAR-301 or empty nanoparticles, followed by necropsy. Microscopic analysis of spleen, thymus, lung, heart, kidney, brain, liver, and GI tract, showed no treatment- related abnormalities. Sporadic vascular/perivascular inflammation was observed in the lungs, probably from foreign-body (i.e., hair) injection during i.v. administration.
  • Example 14 Immune activation resulting from miR-124.
  • the duplex resulting from annealing these two oligonucleotides was packaged in the LUNAR nanoparticle formulation and designated seed sequence control (SSC), which was verified to not exert any therapeutic activity in mice harboring intracerebral GL261 (median survival, 16 days).
  • SSC seed sequence control
  • Example 15 Nanoparticle formulation delivery in canines.
  • LUNAR-301 In a second species, purpose-bred non-tumor- bearing beagles were utilized. Single-dose escalations of LUNAR-301 (0.25, 0.5, and 1 mg/kg, i.v.) or multiple doses of LUNAR-301 (0.5 mg/kg x 9) were given intravenously to the dogs. Pre-delivery (0 h) physical exams were normal (including pulse and respiration) in all dogs and were not altered post administration. But occasionally, mild sedation and emesis were observed (Veterinary Cooperative Oncology Group Common Terminology Criteria for Adverse Event: grade I) that were not dose related.
  • Nanoparticle formulations can be selectively delivered to the immune compartment for activity in a canine model.
  • FIG. 13 shows a schematic of a proposed mechanism of formulation LUNAR- 301 action. While not wishing to be bound by any one particular theory, LUNAR-301 (miR-124 within lipid) nanoparticles are delivered to the patient intravenously and immediately come in contact with circulating PBMCs, most notably
  • the nanoparticles are engulfed and rapidly degraded via a pH-mediated process within the endosome, thereby delivering the double- stranded miR-124 mimic payload inside the cell.
  • MiR-124 subsequently inhibits the STAT3 pathway, facilitating reversal of tumor-mediated immune suppression and resultant immune activation, allowing resident and peripheral immune cells to target the CNS tumor.
  • LUNAR nanoparticles have several advantages over conventional methods of delivery, including: 1) higher dose-limiting toxicity; 2) long-term stability (>4 months) with minimal variation in particle size; 3) lack of immunogenicity (e.g., cytokine storm), thereby negating the requirement for antihistamine therapy; and 4) biodegradability (without accumulation after multiple dosing).
  • Initial dose escalations of LUNAR-301 in canines and the delivery of double-stranded miR-124 mimic to the immune effector compartment suggest that allometric scaling may not apply.
  • Formulation LUNAR-301 has characteristics that make it similar to drugs that have successfully navigated the FDA approval system, is safe in multiple species, is GBM-ready, and can be rapidly translated to patients, because human and mouse miR- 124 are 100% homologous.
  • Example 19 Combination of STAT3 inhibition and radiation enhances the therapeutic effect against murine brain metastasis.
  • C57BL/6 mice were intracerebrally implanted with B 16 melanoma, irradiated with 7.5 Gy, and treated with LUNAR-301 which inhibits the STAT3 pathway, or WP1066, a cell permeable AG 490 tyrphostin analog small molecule inhibitor of STAT3. Mice surviving 130+ days after tumor implantation were subcutaneously rechallenged with tumor.
  • Monotherapy demonstrated longer survival relative to untreated control mice; combination therapies further extended survival compared to single treatment agents.
  • Median survival for mice receiving radiation combined with LUNAR-301 or WP1066 was 65 days and 45.5 days, respectively, compared to 15 days for untreated mice and 19 - 40 days for monotherapy. While no untreated mice (control) survived over 20 days, the survival rate at day 130 for mice receiving combination therapy was almost 40%.
  • Flow cytometry analysis showed higher IL-2 production in T cells from tumor-draining lymph nodes in mice treated with radiation plus STAT3 inhibitor.
  • Example 20 Serum cytokine analysis of formulation LUNAR-301.
  • Non-tumor-bearing C57BL/6 mice were treated with nine doses of LUNAR-301 (LUN) or SSC at the therapeutic dose of 1 mg/kg (based on the weight of the double- stranded RNA), or treated with the same volume of PBS.
  • Serum samples were obtained five hours after both the first dose and the 9th dose for evaluation of serum cytokine changes to identify potential nonspecific immune activation secondary to i.v. nucleic acid treatment.
  • the serum cytokines were measured using the Bio-Rad Bio-Plex Pro assay.
  • MCP-1 Monocyte chemoattractant protein- 1
  • G-CSF G-CSF
  • Spontaneous brain tumors are reported commonly in canines, where malignancy types are similar to those seen in humans but incidence is more frequent, especially in certain breeds.
  • In vivo analysis can be performed in a canine model using pet dogs with naturally occurring glioma to validate therapies that are promising in murine models prior to initiation of human clinical trials.
  • LUNAR-301 is administered to pet dogs with naturally occurring gliomas diagnosed by brain biopsy with owner consent. Tumor volume is measured by using magnetic resonance imaging (MRI) before therapy and at various time points after drug treatment to determine if the drug is shrinking the tumors. Additionally, blood is obtained to ensure that dogs are tolerating the drug and developing expected immune responses. Animals receive supportive medical care for brain tumors (for example, steroids and anti-seizure medications) in addition to the experimental drug LUNAR-301.
  • MRI magnetic resonance imaging
  • Results of administration of LUNAR-301 are used to assess the safety of drug delivery in glioma-bearing dogs; evaluate immunological responses; determine response to treatment based on lesion volume as measured by magnetic resonance imaging (MRI); and establish the overall survival time in dogs treated with this therapeutic strategy.
  • MRI magnetic resonance imaging
  • Eligible subjects include dogs with: 1) either a pathological diagnosis of high- grade glioma or imaging features consistent with a high-grade glioma (See: Young et al Vet Radiol Ultrasound 2011); 2) measurable parenchymal contrast enhancement of tumor detected on brain MRI; 3) age greater than 1 year; 4) a modified Glasgow coma score greater than 14 that are ambulatory at the time of enrollment; 5) normal organ and bone marrow function; and 6) owner understanding and willing to sign a written informed consent document.
  • Exclusion criteria include dogs: 1) currently receiving any other type of anticancer therapy (except corticosteroids at the lowest tolerable amount) and 2) having leptomeningeal disease only or imaging/ histopathologic evidence of gliomatosis cerebri. The following clinical observations are monitored: 1) complete blood count, serum biochemistry, coagulation parameters, and immune assays (effector function, Treg inhibition, microRNA quantification etc.); 2) daily physical and neurological
  • Phase I Phase I - three glioma bearing dogs are used for an inter-canine dose escalation study, and Phase II - up to 21 dogs with glioma are used for a clinical efficacy study.
  • Phase II Phase II of the trial proceeds according to an accelerated intercanine dose escalation design.
  • a three dog cohort is chosen to proceed with dose escalation after a one week delay following the first treatment.
  • Each dog receives a single test dose of LUNAR-301 at 0.5 mg/kg (doses used in healthy dogs ranged from 0.25-1 mg/kg) delivered through an intravenous catheter followed by a one week observation period.
  • PBMCs peripheral blood mononuclear cells
  • Dogs in Phase II that had an MRI performed at another institution are required to have an MRI to permit accurate comparison of pre- and post-treatment volume measurement.
  • the dog is placed under general anesthesia by using a combination of standard premedications and utilizing inhalant gas anesthesia for maintenance of anesthesia, and then placing the dog in a 3T Siemens MRI located at a diagnostic imaging center.
  • Time of anesthesia is typically about 1 hour to acquire standard Tl, T2, T2 FLAIR, T2*, and post-contrast images. All dogs in this trial arm are identically monitored to that described for Phase I dogs and also have immune responses assayed in an identical manner.
  • blood (6 mL) is banked immediately prior to the first LUNAR-301 dose and 10 minutes following drug delivery for doses 3, 6, and 9.
  • This banked blood is used for miR-124 quantification and potentially molecular profiling.
  • the 10 minutes post-delivery time point was selected as this is when miR-124 peak concentrations were found in the blood and PBMC
  • the purpose of this small cohort is to obtain additional safety and dose optimization data from dogs with glioma.
  • a 21 dog (maximum number of enrollees) efficacy study (Phase II) follows the Phase I study.
  • Phase II detection of an adverse event of greater than or equal to grade 3, which are severe conditions requiring prolonged hospitalization but are not life-threatening, that were attributed to the drug results in cessation of drug delivery to that animal.
  • a 25% dose reduction was made at the principal investigator's discretion in the setting of grade 3 toxicities.
  • Dogs had LUNAR-301 administered within 24 hours after missing a dose; however, if more than 24 hours elapsed, then that dose was held and the next dose resumed on the designated schedule. Dogs requiring a delay of more than 2 weeks or dogs requiring more than 2 dose reductions were withdrawn from the study. If posterior probability of a greater than or equal grade 3 event greater than 30% was above 90%, as determined by Bayesian model of averaging continual reassessment methods (B MA-CRM), a dose reduction of 25% was applied. Due to the unique study population (i.e., dogs with CNS malignancy), there are several toxicities that are anticipated in this study population based on the underlying disease. A toxicity grade greater than or equal to 3 in severity was considered a dose- limiting toxicity with the following exceptions: seizure, thromboembolism, syndrome of inappropriate antidiuretic hormone (SIADH), and tumor progression.
  • SIADH inappropriate antidiuretic hormone
  • Dogs in the Phase II study have an MRI follow-up at 6-8 weeks (optional time point observed if clinical worsening/progression was evident), 3 months, 6 months, 9 months, and 12 months following initiation of LUNAR-301 delivery using protocols identical to those outlined for MRI obtained pre-delivery.
  • Response and progression is evaluated in this study using the new international criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (vl . l) and/or volumetric software.
  • the enhancing lesion is measured bi-dimensionally on pre- and post-treatment studies. Lesion size is the product of its two largest perpendicular diameters. For multi-centric lesions with discrete foci of enhancement, each enhancing lesion is measured and then the sum is calculated.
  • a non-measurable lesion includes foci of enhancement that are less than 5 mm, non-enhancing lesions seen on T2 -weighted or T2 FLAIR images, hemorrhagic lesions, predominantly cystic lesions, necrotic lesions, and leptomeningeal disease. Hemorrhagic lesions often have intrinsic Tl-weighted hyperintensity that could be misinterpreted as enhancing tumor, and for this reason, the pre-contrast Tl-weighted image is examined to exclude baseline or interval sub-acute hemorrhage.
  • Dogs included in the Phase II component are serially tracked following drug delivery to determine survival time.
  • the progression free survival (PFS) is defined based on MRI as less than a 25% increase in tumor volume on Tl-weighted post-contrast MRI scans compared with the MRI obtained prior to treatment. If progression is suspected but there is uncertainty regarding whether imaging changes represent true tumor progression, imaging is repeated in 6-8 weeks or the subject is withdrawn from the study, at which time additional diagnostics or intervention are pursued as indicated. If the repeat imaging or additional procedures show progression, then the PFS is declared at the time progression is first suspected. At the time of death/euthanasia, samples from the CNS are obtained to evaluate immune infiltration, tumor response, and RNA expression to ascertain mechanisms of treatment resistance/failure.
  • T Fa, IL-2 and IFNy percent of PBMCs expressing STAT3/Foxp3 and T cell effector responses
  • GLMMs generalized linear mixed-effects models
  • Blood is collected from living animals by IV puncture in 6 mL aliquots for CBC/CHEM/COAG three times: prior to dose, 24 hours following 1st dose, and 24 hours after final dose. Blood is also collected by IV puncture in 6 mL aliquots for FACS staining 4 times: enrollment, 24 hours after 1st dose, 24 hours before last dose, and 24 hours after last dose. Lastly, blood is collected by IV puncture in 6 mL aliquots for miR- 124 quantification during Phase II only: prior to 1st dose and 10 minutes after doses 3, 6, and 9. A canine brain biopsy is performed to diagnose dogs as having glioma prior to inclusion in study and drug delivery. Canine urine cystocentesis is performed in 5mL aliquots at enrollment and 24 hours after 1st and last dose.
  • the treatment protocol was altered to include pre-treatment with diphenhydramine and famotidine to minimize the risk of anaphylaxis. While additional steroids have not been given for this purpose, all 3 enrolled patients have been receiving 0.5-1.0mg/kg prednisone daily for treatment of tumor-associated cerebral edema.
  • Canine PBMCs were isolated by density gradient centrifugation and then stained with antibodies for cytokine expression or cell surface markers and analyzed by flow cytometry as taught herein.
  • LUNAR-301 lead to an increase in CD4+ T cells of 0.9 fold over baseline just after dose 1 and a 1.2 fold increase after dose 7; an increase in CD8+ T cells of 3.5 fold over baseline just after dose 1 and a 2.2 fold increase after dose 7; and a decrease in CD11+ cells of 2.5 fold below baseline just after dose 1 and a 2.0 fold decrease after dose 7.
  • effector cytokines There was an increase in effector cytokines in all three cell types.

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Abstract

La présente invention concerne des méthodes permettant de prévenir, d'atténuer ou de traiter une maladie associée à un gliome chez un sujet qui en a besoin, ladite méthode consistant à administrer au sujet une quantité efficace d'une composition de nanoparticules comprenant le miR-124. Lesdites méthodes permettent d'activer des cellules du système immunitaire contre le gliome, afin de préparer des compositions comprenant des cellules activées du système immunitaire. L'invention concerne également des méthodes d'activation d'une réponse immunitaire contre le gliome chez un sujet, et d'administration de manière sélective de miR-124 à un compartiment du système immunitaire. De plus, lesdites méthodes permettent d'induire une mémoire immunologique contre le gliome.
PCT/US2016/043607 2015-07-24 2016-07-22 Compositions de mir-124 pour agents thérapeutiques immunomodulateurs WO2017019523A1 (fr)

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