WO2010043630A2 - Système d'administration d'arn - Google Patents

Système d'administration d'arn Download PDF

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WO2010043630A2
WO2010043630A2 PCT/EP2009/063368 EP2009063368W WO2010043630A2 WO 2010043630 A2 WO2010043630 A2 WO 2010043630A2 EP 2009063368 W EP2009063368 W EP 2009063368W WO 2010043630 A2 WO2010043630 A2 WO 2010043630A2
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sirna
rna
composition
solid
sirnas
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WO2010043630A3 (fr
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Ivan Coulter
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Sigmoid Pharma Limited
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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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A61K9/5042—Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose
    • A61K9/5047—Cellulose ethers containing no ester groups, e.g. hydroxypropyl methylcellulose
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • 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
    • 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
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • RNA interference e.g. siRNAs.
  • the invention also relates to pharmaceutical compositions for therapeutic application of RNA interference and to other subject matter.
  • RNA interference is a powerful and specific method for silencing or reducing the expression of a target gene, mediated by small single-or double-stranded RNA molecules. These molecules include small interfering RNAs (siRNAs), microRNAs (miRNAs), small hairpin RNAs (shRNAs), and others. Although the mechanism by which RNAi functions is not fully elucidated, it is clear that RNAi is a promising method of treatment, e. g. by targeting specific mRNAs for elimination.
  • RNAi small interfering RNAs
  • miRNAs microRNAs
  • shRNAs small hairpin RNAs
  • RNA delivery differs according to the target tissue or cell type.
  • target tissue For intestinal tissue, it is particularly desireable to be able to deliver RNAs able to silence/knockdown the expression of genes in cells lining the intestine particularly when these cells are inflamed, for example in the case of autoimmune diseases such as ulcerative colitis and Crohn's disease and intestinal graft- versus-host-disease.
  • HIF hypoxia-inducible factor
  • HIF is hydroxylated (inactivated) by prolyl hydroxylase (PHD) implying that a PHD inhibitor should provide benefit to diseases of this type.
  • PHD prolyl hydroxylase
  • three PHD isoforms have been identified (PHD 1-3), and shown to hydroxylate HIF- ⁇ in vitro (see P. Jaakkola et al. (2001) Science 292:468-472 and WC Hon et al Nature 417:975-978).
  • PHD 1-3 prolyl hydroxylase
  • the three enzymes have different tissue distributions and, at least under conditions of overexpression, have distinct patterns of subcellular localization.
  • PHDl mRNA is expressed in many tissues, with especially high expression in the testis.
  • PHD2 mRNA is widely expressed, with particularly abundant expression in adipose tissue.
  • HIF-selective PHDs as central regulators of HIF expression has now provided the basis for potential development of PHD-based molecular tools and therapies.
  • pharmacological inactivation of the PHDs by 2-OG analogues eg DMOG or 1 -dimethyloxallyl glycine
  • DMOG 2-OG analogues
  • 1 -dimethyloxallyl glycine 2-OG analogues
  • PHD inhibitors include direct inhibitors of the PHDs, analogs of naturally occurring cyclic hydroxamates, as well as antagonists of ⁇ -ketoglutarate.
  • PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady-state levels of HIF-Ia in normoxia. They point out that PHD2 is upregulated by hypoxia, providing an HIF- 1 -dependent auto -regulatory mechanism driven by the oxygen tension.
  • Cummins et al. and Berra et al used a transfection technique in which siRNA duplexes of the relevant ribonucleic acid sequences in cationic liposomes were used to transfect (or co-transfect along with reporter plasmid combinations) mammalian including HeLa cells. This technique is described by Elbashir et al (2001) Nature vol 411 p494 the entirety of which is incorporated herein by reference.
  • RNA interference is emerging as an indispensable strategy for target-specific knockdown of gene expression.
  • second-generation technologies representing advances in RNAi design, efficiency, and efficacy, daunting obstacles remain, such as how to deliver RNAi compounds to the right targets in the right amounts.
  • Recent advances in RNAi delivery include utilizing tiny delivery vehicles called nanoparticles and applying epigenetics for changing DNA expression without altering the gene sequence.
  • RNAi RNAi compound
  • a critical question is whether the tissue targeted will be exposed to the RNAi. If the RNA is injected and delivered intraperitoneally or intravenously it enters the systemic circulation (bloodstream), where it is taken up by the kidneys and removed. As a result, standard injections are believed not to provide a likely avenue of RNA delivery.
  • Exposure issues also depend on the type of tissue targeted. For example, exposure can be enhanced if it is possible to achieve local delivery, such as injecting RNAi into the eye for treating age-related macular degeneration.
  • local delivery such as injecting RNAi into the eye for treating age-related macular degeneration.
  • the physiological structure of the eye permits retention of the injection fluid locally.
  • Local delivery has also been attempted by injection directly into the central nervous system or via inhalation or topically.
  • Nanotransporters are chemicals of defined size that are mixed with an RNAi compound to form minute particles for delivery into target tissues.
  • the nanotransporter has a core to which layers are added by chemical synthesis. The final layer has a positive charge allowing it to attract and bind negatively charged RNAi compounds.
  • ALS amyotrophic lateral sclerosis
  • SODl superoxide dismutasel
  • Magnetic nanoparticles with a lipid core have been used in MRI studies in which tags, such as fluorescence are combined with active RNAi (see http ://www . genovis . com/RNAi) .
  • tags such as fluorescence are combined with active RNAi (see http ://www . genovis . com/RNAi) .
  • This approach uses tiny superparamagnetic nanoparticles that feature iron oxide cores coated with a specific cationic lipid formulation. This facilitates particle solubility and cellular uptake and enables experiments to be conducted to establish and track the system (such as in a whole cell, endosome, or liposome) in which RNAi is working.
  • the mammalian RNAi pathway contains an enzyme known as dicer, a natural initiation point for the RNAi cascade. It is possible to create longer-than-natural RNA precursors adapted to be substrates of dicer and in some settings this leads to a more potent and longer- lasting variant of RNA interference (see http://www.dicerna.com/science-technology.html).
  • the current challenge for this dicer-based approach is to optimize the delivery modalities for the highest efficiency.
  • RNA for gene knockdown is not equivalent to DNA delivery with DNA delivery techniques having been largely unsuccessful.
  • Some groups are attempting to design synthetic delivery systems, including polymeric systems for efficient RNA delivery and gene knockdown.
  • Egen see http://www.egencorp.com/index.htm
  • TheraSilenceTM technology platform for delivery of therapeutic siRNA or shRNA Candidates are being tested for proof of concept in animal models of diseases.
  • RNAi delivery system must efficiently function to protect the therapeutic cargo from degradation, to promote uptake by target cells, and to facilitate intracellular trafficking and that multiple approaches may be needed depending on a variety of factors, such as the disease being treated, the tissue, mode of administration, dose, and the specific siRNA or short hairpin RNA (shRNA).
  • shRNA short hairpin RNA
  • RNAi may also have application in epigenetics which refers to stable changes in gene expression that do not involve altering the actual DNA sequence.
  • Epigenetic mechanisms can control or alter gene activity in several ways, such as RNAi, DNA methylation, and modification of histones that encase DNA. It may not be necessary to ensure the permanent presence of a therapeutic compound for a permanent effect. So, in some cases a transient transfection of a gene can produce a permanent change. This approach has been used in the transient transfection of mesenchymal stem cells.
  • RNAi knocks down the expression of the target by destroying its mRNA If the target is a regulatory protein, knocking it down would alter the expression of other genes at the transcriptional level and some of these transcriptional effects may in certain circumstances be locked in by epigenetic changes, such as DNA methylation, thereby allowing the RNAi effect to persist after the RNAi-inducing molecule is gone.
  • the primary functions of the gastrointestinal tract are the processing and absorption of ingested nutrients, waste removal, fluid homeostasis, and the development of oral tolerance to nonpathogenic luminal antigens.
  • the last of these functions involves the intestinal mucosa being unique among tissues as it is in a constant state of controlled inflammation. This occurs as the mucosal immune system is constantly exposed to new food-borne material in the lumen, which is processed to avoid inappropriate inflammatory reactions to harmless ingested antigens.
  • a critical cell type in the maintenance of intestinal homeostasis is the epithelial cell of the gastrointestinal tract (GIT).
  • the intestinal epithelium is a monolayer of cells that covers an area of approximately 250-300 m2 in an adult human and forms a critical barrier between the external (luminal) and internal (vascular) compartments. This dynamic barrier is maintained primarily by the existence of regulated intercellular tight junctions. As well as being a critical barrier, the epithelium is responsible for the absorption of approximately nine litres of fluid from consumed liquids and secreted digestive fluids per day. This fluid transport function is carried out through coordinated ion transport events and the subsequent regulation of salt and water transport between the lumen of the gut and the bloodstream. Importantly, both the barrier and absorptive functions of the intestinal epithelium can be physiologically regulated by oxygen.
  • IBD Inflammatory bowel disease
  • ulcerative colitis and Crohn's disease are characterized by a breakdown in the intestinal epithelial barrier with subsequent unregulated exposure of the mucosal immune system to luminal antigenic material leading to inflammation and further barrier breakdown.
  • a self- perpetuating cycle of inflammation is initiated leading to severe pathology.
  • treatment often ultimately resorts to surgical resection of significant amounts of chronically inflamed intestinal tissue.
  • the present invention is based, in part, upon the discovery of delivery methods for siRNA or an engineered RNA precursor using minicapsules or minispheres.
  • RNA or an engineered RNA precursor and “RNA” are used interchangeably unless the context requires otherwise and includes small interfering RNAs (siRNAs), microRNAs (miRNAs), small hairpin RNAs (shRNAs), and others, including conjugates, such as described below particularly in the detailed description. It is intended that any such mention of RNA includes molecules with or without backbone modification or conjugated variants thereof (where the conjugate is can be another nucleic acid or a molecule of a different type such as a lipid or peptide).
  • siRNAs small interfering RNAs
  • miRNAs microRNAs
  • shRNAs small hairpin RNAs
  • conjugates such as described below particularly in the detailed description. It is intended that any such mention of RNA includes molecules with or without backbone modification or conjugated variants thereof (where the conjugate is can be another nucleic acid or a molecule of a different type such as a lipid or peptide).
  • the present invention features a method for delivering an siRNA or engineered RNA precursor to a cell, particularly an epithelial cell of the GIT (gastrointestinal tract), by bringing a multiplicity of RNA-containing minicapsules into contact with the target cell, for example by oral administration of a pharmaceutical formulation comprising such minicapsules.
  • the target cell is an intestinal epithelial cell.
  • the RNA comprised in the minispheres is adapted to interfere, knockdown or inhibit the expression of specific genes or gene products or messenger products (eg mRNA) and/or expression of enzymes, especially those affecting the control of hypoxia in the cells of the GI tract.
  • RNAs which affect (in particular knockdown, inhibit or interfere with) enzymes which normally cause HIF to be upregulated or retained at beneficial levels.
  • One embodiment of the invention is the knock-down of a target gene or gene product (including messenger) for a transient period.
  • RNA be adapted to knockdown, silence or inhibit the expression of one or more PHDs (prolyl hydroxylases), including PHD 1, 2 and 3.
  • PHDs prolyl hydroxylases
  • Such RNAs are referred to in the Examples herein as siRNAs "for" PHDl, 2 and 3 respectively.
  • the present invention features a method for delivering an siRNA to a cell, preferably a GI cell, by obtaining, identifying or targeting a cell (or system of cells or tissue), forming a minisphere comprising an siRNA and contacting the cell (or system of cells or tissue) with the minisphere or a plurality thereof.
  • the invention provides an siRNA or engineered RNA precursor conjugated to a delivery peptide, the conjugate being encapsulated in a minisphere.
  • the invention features biconjugates of targeting peptides susceptible of enhancing uptake of siRNA and thus promote gene silencing in vivo.
  • a pharmaceutical composition comprising mini-beads of solid matrix material wherein the mini-beads comprise one or more siRNAs or engineered RNA precursors dispersed in said solid matrix.
  • the invention provides an siRNA or engineered RNA precursor associated with a polymer eg a cationic polymer such as chitosan, to form an aggregate or a complex, the aggregate or complex optionally being encapsulated in a minisphere e.g. a minibead with or without the presence of liposomal materials.
  • the invention features such aggregates susceptible of enhancing uptake of siRNA and thus promote gene silencing in vivo.
  • An example is a chitosan-siRNA aggregate which may be further associated with liposomal materials and then optionally encapsulated to form a minicapsule or mini-bead.
  • the invention includes a medicament for delivering active agent selected from an siRNA and engineered RNA precursors to a target cell in the gastrointestinal tract, the medicament comprising a multiplicity of RNA-containing minicapsules and being adapted for the active agent to be released and contact the target cell after administration of the medicament. Also included is a medicament for delivering active agent selected from an siRNA and engineered RNA precursors to a predetermined region of the gastrointestinal tract, the medicament comprising a multiplicity of RNA-containing minicapsules and being adapted for the active agent to be released in said region; in one embodiment, the medicament is adapted to release the active agent in the colon.
  • the invention also provides an oral composition comprising minicapsules wherein the minicapsules comprise one or more siRNAs or engineered RNA precursors in a core susceptible of maintaining such RNA in a stable, active form. While the core may be liquid, semi-solid, or solid core, a preferred embodiment of the invention is a liquid core.
  • the minicapsules have release profiles to release the siRNA or engineered RNA precursor in an active form at one or more sites along the gastrointestinal tract, for example where absorption (for local or systemic benefit) is maximized or therapeutic efficacy is maximized.
  • the siRNA or engineered RNA precursor regardless of its inherent physicochemical property, when released from the minicapsule is in a soluble form or is readily soluble in the aqueous GIT environment.
  • the minicapsule may have one layer e.g. a minibead and may be essentially solid throughout or be a solid comprising inclusions selected from liquid inclusions, semi-solid inclusions or combinations thereof. Some minibeads thus have a solid phase with semi-solid interior portions. Included, therefore, are single layer minicapsules which are solid throughout.
  • the minicapsule may have two layers comprising a solid outer shell layer encapsulating a liquid, semi-solid or solid core.
  • the minicapsule may have three layers comprising a solid outer shell layer; a solid, semi-solid or liquid middle buffer layer; and a liquid, semi-solid or liquid core.
  • the minicapsules may be modified to enable modified release of the siRNA or engineered RNA precursor(s).
  • a modified release coating may be applied to the outer shell layer of the minicapsule.
  • an outer shell layer of the minicapsule may be modified to achieve modified release.
  • the minicapsule core or entirety may control the rate of active compound release.
  • a buffer layer of the minicapsule may be modified to achieve modified release.
  • the liquid, semi-liquid or solid core of the minicapsule may be modified to achieve modified release.
  • polymeric materials may be used achieve modified release such as polymeric materials that are sensitive to one or more of pH, time, thickness, erosion, and bacterial breakdown.
  • the minicapsule may comprise of one layer containing one or more active pharmaceutical agents as well as delivery enhancing excipients in addition to the siRNA or engineered RNA precursor and that layer may control the release of the siRNA or engineered RNA precursor (s).
  • the invention includes as such minibeads, minicapsules and minispheres as described herein, e.g. a single minibead, minicapsule or minisphere as described herein.
  • Another aspect is a medicament for delivering an active agent selected from siRNAs and engineered RNA precursors to the colon, the medicament comprising a multiplicity of RNA-containing minicapsules and being adapted for the active agent to be released in the colon after administration of the medicament.
  • the invention includes a product selected from a minicapsule, a minibead or a minisphere and comprising a solid phase and an active agent selected from siRNAs and engineered RNA precursors (e.g. a combination of at least two such agents or a single such agent).
  • the product may have one layer being a solid phase comprising inclusions selected from liquid inclusions, semi-solid inclusions or combinations thereof or have at least two layers comprising a solid phase outer shell layer encapsulating a liquid, semi-solid or solid core.
  • the product has one layer and the inclusions comprise the active agent; in other embodiments the product has at least two layers and the core comprises the active agent.
  • a product selected from a minicapsule, a minibead or a minisphere comprises a solid phase and, dispersed in the solid phase, an active agent selected from siRNAs and engineered RNA precursors (e.g. a combination of at least two such agents or a single such agent).
  • the siRNA or engineered RNA precursor(s) may be released along the gastrointestinal tract in a form that maximises systemic absorption and/or local absorption.
  • the siRNA or engineered RNA precursor (s) may be released along the gastrointestinal tract in a form that maximises lymphatic absorption.
  • Such delivery is desirable for RNAi molecules targeting micro- metastatic or metastatic tumour cells that pass through the lymphatic system or for RNAi molecules designed to modulate immune cell function and response.
  • the siRNA or engineered RNA precursor (s) may be released along the gastrointestinal tract in a form that maximises blood brain barrier absorption.
  • the siRNA or engineered RNA precursor (s) may be released along the gastrointestinal tract in a form that maximises pre-systemic and/or local absorption, in particular by the epithelial cells lining the GIT.
  • the siRNA or engineered RNA precursor(s) may be released along the gastrointestinal tract in a form that maximises local gastrointestinal activity.
  • the siRNA or engineered RNA precursor(s) may be released along the gastrointestinal tract in a form that maximises gastrointestinal lumen activity.
  • the siRNA or engineered RNA precursor(s) may be released along the gastrointestinal tract in a form that maximises chronotherapy.
  • the RNA formulation or component(s) thereof is (are) released in such that it (they) is (are) in soluble when released or is (are) readily soluble in the local GIT environment.
  • Another aspect of the invention is the release of RNAi molecules, formulations thereof or components of such formulations to act within the lumen to inhibit bacterial or viral entities.
  • RNA In relation to the types of RNA which may be incorporated into the minicapsules of the present invention, they can be selected from sequences appropriate to a "target gene” ie. a gene whose expression is to be selectively inhibited, knocked down or “silenced” by such RNA.
  • This silencing is achieved by cleaving the mRNA of the target gene by an siRNA, e. g., an isolated siRNA or one that is created from an engineered RNA precursor.
  • siRNA e. g., an isolated siRNA or one that is created from an engineered RNA precursor.
  • One portion or segment of a duplex stem of the siRNA, RNA precursor, or one strand of the siRNA is an anti-sense strand that is complementary, e. g. fully complementary, to a section, e.g. about 16 to about 40 or more nucleotides, of the mRNA of the target gene.
  • isolated nucleic acid molecule or sequence is a nucleic acid molecule or sequence that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA or RNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e. g. a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences.
  • RNA precursor that is part of a hybrid gene encoding an additional polypeptide sequence.
  • engineered indicates that the precursor or molecule is not found in nature, in that all or a portion of the nucleic acid sequence of the precursor or molecule is created or selected by man. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by mechanisms within a cell.
  • an RNA precursor produced within a cell from an engineered nucleic acid molecule e. g. a transgene
  • Engineered RNA precursors are artificial constructs that are similar to naturally occurring precursors of small temporal RNAs (stRNAs) that are processed in the body to form siRNAs.
  • the engineered RNA precursors can be synthesized by standard methods known in the art, e. g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.) or encoded by nucleic acid molecules.
  • Figure 1 shows western blots showing knock-down of p65 NFkB by siRNA in a liposomal formulation.
  • the present invention provides compositions and methods for delivering siRNAs, or siRNA precursors, into cells, e. g. eukaryotic cells such as mammalian cells (for example, human cells). These methods are useful both in vivo and in vitro. Sequence-selective, post-transcriptional inactivation of expression of a target gene can be achieved in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene, a phenomenon termed RNA interference (RNAi).
  • RNAi RNA interference
  • siRNA can trigger the degradation of mRNA corresponding to the siRNA sequence.
  • the siRNA must not only enter the cell, but must also enter the cell in sufficient quantities to have a significant effect.
  • RNAi methodology has been extended to cultured mammalian cells, but its application in vivo has been limited due to a lack of efficient delivery systems with little or no toxicity. The present application provides such a system.
  • nucleic acid delivery mediated by cationic liposomes such as LIPOFECTAMINETM, LIPOFECT ⁇ NTM, CYTOFECT ⁇ NTM as well as transfection mediated by polymeric DNA-binding cations such as poly-L-lysine or polyethyleneimine are extensively used transfection techniques. These methods can be associated with cytotoxicity and sensitivity to serum, antibiotics and certain cell culture media. In addition, these methods are limited by low overall transfection efficiency and time-dependency. Other methods such as microinjection or electroporation are simply not suitable for large-scale delivery of nucleic acids into living tissues. The relevance of these approaches does not appear to have been assessed for oral delivery of siRNA.
  • RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA in animals and plant cells (Hutvagner and Zamore (2002), Curr. Opin. Genet. Dev. , 12,225-232 ; Sharp (2001), Genes Dev. , 15,485-490).
  • dsRNA double-stranded RNA
  • RNAi can be triggered by 21 - nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al. (2002), MoI. Cell. , 10, 549-561 ; Elbashir et al.
  • RNA polymerase III promoters Zeng et al. (2002), MoI. Cell, 9,1327-1333 ; Paddison et al. (2002), Genes Dev. , 16, 948-958 ; Lee et al. (2002), Nature Biotechnol. , 20,500-505 ; Paul et al. (2002), Nature Biotechnol. , 20,505-508 ; Tuschl, T.
  • the nucleic acid molecules or constructs used in the invention include dsRNA molecules comprising 16-30, e. g. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in each strand, wherein one of the strands is substantially complementary to, e. g. at least 80% identical (or more, e.g. 85%, 90%, 95%, or 100%) (for example, having 3, 2, 1, or 0 mismatched nucleotide (s) ), to a target region, such as a target region that differs by at least one base pair between the wild type and mutant allele of a nucleic acid sequence.
  • dsRNA molecules comprising 16-30, e. g. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in each strand, wherein one of the strands is substantially complementary to, e. g. at least 80% identical (or more, e.g. 85%, 90%, 95%, or 100%) (for example, having
  • the target region can comprise a gain-of- function mutation, and the other strand is identical or substantially identical to the first strand.
  • the dsRNA molecules of the invention can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from an engineered RNA precursor, e. g., shRNA.
  • the dsRNA molecules can be designed using any method known in the art, for instance, by using the following protocol:
  • each AA and the 3'adjacent 16 or more nucleotides are potential siRNA targets.
  • the siRNA should be specific for a target region that differs by at least one base pair between the wild type and mutant allele, e. g. a target region comprising the gain of function mutation.
  • the first strand should be complementary to this sequence, and the other strand is identical or substantially identical to the first strand.
  • the nucleic acid molecules are selected from a region of the target allele sequence beginning at least 50 to 100 nt downstream of the start codon, e. g. of the sequence of SODl .
  • siRNAs with lower G/C content may be more active than those with G/C content higher than 55%.
  • the invention includes nucleic acid molecules having 35-55% G/C content.
  • the strands of the siRNA can be paired in such a way as to have a 3' overhang of 1 to 4, e. g. 2, nucleotides.
  • the nucleic acid molecules may have a 3'overhang of 2 nucleotides, such as TT.
  • the overhanging nucleotides may be either RNA or DNA.
  • the overhang nucleotides are deoxythymi dines or other appropriate nucleotides or nucleotide analogs.
  • Other embodiments are also envisioned where the strands of the siRNA do not have a 3' overhang. As noted above, it is desirable to choose a target region wherein the mutant: wild type mismatch is a purine: purine mismatch.
  • BLAST is available at www. ncbi. nhn. nih. gov/BLAST.
  • siRNA User Guide available at www. mpibpc. gwdg. de/ en/100/105/sirna. html.
  • siRNA may be purchased commercially for example from suppliers such as Dharmacon.
  • Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls may be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.
  • siRNAs used in the invention include both siRNA and crosslinked siRNA derivatives as described in U. S. Provisional Patent Application 60/413,529, which is incorporated herein by reference in its entirety.
  • Crosslinking can be employed to alter the pharmacokinetics of the composition, for example, to increase half-life in the body.
  • other chemical backbone modifications such as phosphorothiolation can be employed for example to enhance stability.
  • covalent attachment of lipid-based (or other) moieties such as chemical attachment of amphiphilic oligomers to specific sites on the RNA to form a so-called conjugate can be employed as described in more detail below.
  • RNA may be employed to enhance stability against enzymic degradation eg in the GI tract in addition to allowing incorporation of the RNA into formulations discussed in more detail below that facilitate absorption across intestinal mucosal barriers. More detail of this technique is provided for example in the paper by J. Gordon Still (2002) in Diabetes/Metabolism Research and Reviews, 18 (Suppl 1): S29-S37 the entirety of which is incorporated herein by reference as well as elsewhere in this description.
  • the invention makes use of siRNA derivatives that include siRNA having two complementary strands of nucleic acid, such that the two strands are crosslinked.
  • a 3'OH terminus of one of the strands can be modified, or the two strands can be crosslinked and modified at the 3'OH terminus.
  • the siRNA derivative can contain a single crosslink (e.g. a psoralen crosslink).
  • the siRNA derivates has at its 3' terminus a biotin molecule (e.g. a photocleavable biotin), a peptide (e.g. a Tat peptide), a nonoparticle, a peptidomimetic, organic compounds (e.g.
  • siRNAs can also be delivered by mixing with such a delivery agent. Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.
  • nucleic acid molecules used in the present invention can also be labeled using any method known in the art; for instance, the nucleic acid compositions can be labeled with a fluorophore, e.g. Cy3, fluorescein, or rhodamine.
  • the labeling can be carried out using a kit, e.g. the SILENCER siRNA labeling kit (Ambion). Additionally, the siRNA can be radiolabeled, e. g., using 3H, 32p, or other appropriate isotope.
  • Nucleic acid molecules described or recited herein are intended to comprise nucleotide sequences with or without 3' overhangs, e. g., with or without 3'- deoxythymidines. Other embodiments are also envisioned in which the 3' overhangs comprise other nucleotides, e. g., UU or the like.
  • siRNA Conjugates The siRNA formulations of the present invention, as well as comprising an engineered RNA precursor or engineered nucleic acid molecules that encode the precursors, can comprise such a molecule conjugated to delivery peptides or other compounds to enhance the efficiency of transport of the siRNA into living cells compared to the efficiency of delivery to unmodified siRNA.
  • Such conjugates are described in more detail in US 2004/0204377 Al the entirety of which is incorporated herein by reference.
  • the siRNAs used in the present invention can be conjugated to delivery peptides or other compounds to enhance the efficiency of transport of the siRNA into living cells compared to the efficiency of delivery to unmodified siRNA.
  • delivery peptides can include peptides known in the art to have cell-penetrating properties.
  • the delivery peptide can be, but is not limited to: TAT derived short peptide from human immunodeficiency virus (HIV-I), such as TAT 47-57 and Cys (amino acid sequence: CYGRKKRRQRRR), and TAT 49- 60 and (Arg)9 (Tat) (amino acid sequence RKKRRQRRRPPQC), and substantially similar variants thereof, e.g. , a variant that is at least 65% identical thereto.
  • the percent identity can be higher, e.g.
  • peptides with substitutions at 1, 2, 3, 4 or more residues e.g. , amino acid sequence: CYQRKKRRQRRR.
  • substitutions are conservative substitutions.
  • the methods of making such peptides are routine in the art.
  • the above mentioned delivery peptides can also have modified backbones, e.g. oligocarbamate or oligourea backbones; see, e.g. , Wang et al., J. Am. Chem. Soc, Volume 119, pp.
  • an siRNA-peptide conjugate or siRNA delivery agent mixture e.g. , an siRNA-dendrimer mixture (i.e., an effective dosage) depends on the nucleic acid selected. For instance, if a plasmid encoding shRNA is selected, single dose amounts in the range of approximately 1 /ug to 1000 mg may be administered; in some embodiments, 10, 30, 100 or 1000 ⁇ g cna be administered. In some embodiments, 1-5 g of the compositions can be administered. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • the nucleic acid molecules employed according to the invention can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5- thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3' UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al, (2002).
  • shRNAs small hairpin RNAs
  • siRNA design and use may be found the following web sites: katahdin.cshl.org:9331/RNAi/docs/BseRI-Bam- HI_Strategy.pdf and at katahdin.cshl.org:9331/RNAi/docs/ Web_version_of_PCR_strategyl.pdf.
  • siRNAs can then be modified as described herein, e.g.
  • the expression constructs may be any construct suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art.
  • Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or HI RNA polymerase III promoters, or other promoters known in the art.
  • the constructs can include one or both strands of the siRNA.
  • Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct. (Tuschl (2002), supra). Linear constructs may be delivered either by conjugation with a delivery peptide or by mixing with PAMAM; non-linear constructs may be delivered by mixing with PAMAM. siRNA Complexes
  • the siRNA formulations of the present invention can comprise one or more siRNA molecules (the same or different) associated with polymers of the kind described elsewhere in this specification eg to enhance the efficiency of transport of the siRNA into living cells compared to the efficiency of delivery to uncomplexed siRNA.
  • Complexes of particular interest are those with cationic polymers such as chitosan. Such complexes or aggregates (also referred to as nanoparticles) are described in more detail in Andersen (2008) Volume 29, Issue 4, Biomaterials, Pages 506-512 and Liu et al (2007) Volume 28, Issue 6, Pages 1280-1288 the entirety of both of which is incorporated herein by reference.
  • Such complexes are made as described in the above cited references and involve simple mixing of chitosan with siRNA solutions in the desired ratio (see examples herein).
  • RNA complexed with a protein may be included in or associated with liposomes or liposomal material.
  • the protein is preferably cationic eg chitosan.
  • the present invention is particularly directed towards siRNAs able to knock down or silence or otherwise inhibit, in whole or in part, the propylhydroxylase (PHD) group of enzymes in mammals particularly man. All organisms possess mechanisms to maintain oxygen homeostasis, which are essential for survival.
  • the hypoxia- inducible factor- 1 (HIF-I) conserved during evolution from worms to flies to vertebrates, is central to adaptation to low oxygen availability. HIF-I in turn regulates transcription of many genes involved in cellular and systemic responses to hypoxia, including breathing, vasodilation, anaerobic metabolism, erythropoiesis and angiogenesis. Therefore, hif represents a 'master' gene in oxygen homeostasis during embryonic development and postnatal life in both physiological and pathophysiological processes such as tumour growth and metastasis (for a review, see Semenza, 1998).
  • HIF-I is a heterodimer consisting of one of three a-subunits (HIF-Ia, HIF-2a or HIF-3a) and the b-subunit (HIF-Ib, also called aryl hydrocarbon nuclear translocator, or ARNT) (Wang et al., 1995; Ema et al., 1997; Tian et al., 1997; Gu et al., 1998).
  • HIF-Ib is a constitutive nuclear protein, which also participates in the cellular response to environmental toxins such as aryl hydrocarbons, whereas HIF-a is specific to the response to hypoxia (Hoffman et al., 1991).
  • HIF-Ia Although oxygen availability regulates multiple steps on HIF-I transcriptional activation, the dominant control mechanism occurs through oxygen-dependent proteolysis of HIF-a (Huang et al., 1996). The most extensively studied isoform of the a-subunits is the ubiquitous HIF-Ia.
  • HIF-Ia is constitutively synthesized and sent to destruction by the ubiquitin ⁇ proteasome pathway (half- life ⁇ 5 min) (Salceda and Caro, 1997; Huang et al., 1998; Kallio et al., 1999).
  • This process is mediated by the specific binding of pVHL, the product of the von Hippel ⁇ Lindau tumour suppressor gene, which is mutated in most sporadic clear cell carcinomas and in VHL disease (Kaelin and Maher, 1998; Maxwell et al., 1999; Cockman et al., 2000; Kamura et al., 2000; Ohh et al., 2000).
  • pVHL is part of a multiprotein complex that includes elongin B, elongin C, Rbxl and Cul2 (Kamura et al., 1999; Lisztwan et al., 1999; Stebbins et al., 1999).
  • This complex functions as an E3 ubiquitin ligase which, only in the presence of oxygen, binds directly to and targets HIF- 1 a for polyubiquitylation and proteasome- dependent degradation. Decreased oxygen levels result in the stabilization of HIF-Ia and the activation of the transcriptional complex leading to the expression of target genes such as vegf, epo and glut-1 (Semenza, 1998).
  • prolyl hydroxylation and acetylation by controlling HIF- la ⁇ pVHL physical interaction, are critical in the regulation of HIF-Ia steady-state levels (Ivan et al., 2001; Jaakkola et al., 2001; Jeong et al., 2002).
  • the proline residues subjected to hydroxylation reside in the HIF-Ia oxygendependent degradation domain (ODDD) within an LXXLAP sequence motif, which is strongly conserved between the HIF-a isoforms.
  • ODDD oxygendependent degradation domain
  • Lys532 when acetylated by ARDl, cooperates with the hydroxyl group in the recruitment of pVHL and subsequent HIF-Ia degradation (Jeong et al., 2002).
  • PHDs are dioxygenases that utilize oxygen as co-substrate providing the molecular basis for the oxygen-sensing function of these enzymes. Indeed, the activity of the purified PHDs has been reported to be strikingly sensitive to graded levels of hypoxia in vitro, mirroring the progressive increases in HIF- 1 a protein and DNA binding activity that are observed when cells are exposed to gradual hypoxia in culture (Epstein et al., 2001).
  • the prolyl hydroxylation reaction requires 2-oxoglutarate and iron as cofactors, thereby accounting for the well known x hypoxia- mimic' effects of iron chelators (such as desferrioxamine) and transition metals (such as Co2+, Mn2+ and Ni2+) on HIF- 1 a induction.
  • iron chelators such as desferrioxamine
  • transition metals such as Co2+, Mn2+ and Ni2+
  • HIF-Ia steady-state upregulation was dependent upon the amount of PHD2 siRNA transfected. Interestingly, they detected HIF-Ia induction at a concentration as low as 0.5 nM siRNA; at 2 nM, the level of HIF- 1 a surpassed that achieved after 4 h of hypoxic stress, and slightly increased up to 200 nM. In addition, transfection of an siRNA targeting the human HIF-
  • Ia isoform completely abolished the signal they detected by immunoblotting using the group's anti-HIF-la antibody in normoxia as well as in hypoxia. This result also validates that the two immunoreactive species shown in the SDS ⁇ gel correspond to the human HIF-Ia isoform. In addition, it isimportant to note that none of the transfected siRNA had an impact on the expression of p42MAPK.
  • PHD2 silencing upregulates HIF-Ia in all the human cells they investigated. They analysed whether specific silencing of PHD2-induced HIF-Ia upregulation was a common mechanism. Thus, they investigated a battery of human cells of different origin.
  • CAL27 derived from a squamous cell carcinoma of the tongue
  • CAL51 derived from a breast cancer
  • HaCAT keratinocyte cell line
  • HT29 derived from a colon carcinoma
  • RCC4/pVHL derived from a clear cell renal carcinoma in which we have re-introduced wild-type pVHL
  • WM9 melanoma cell line
  • HIF-Ia induced in normoxia by PHD2 silencing is functionally active.
  • oxygen deprivation affects the subcellular localization, DNA-binding capacity and transcriptionalactivation function of HIF-Ia in addition to regulating its stability (Kallio et al., 1999).
  • Berra et al's results demonstrated that silencing of PHD2 also triggers HIF- 1 a nuclear accumulation.
  • HIF-Ia induced in normoxia by PHD2 silencing performed luciferase assays by using a hypoxia-sensitive reporter gene vector (pRE-Dtk-LUC) coding for the LUC gene under the control of a minimal promoter containing three copies of the hypoxia response element (HRE) from the erythropoietin gene.
  • pRE-Dtk-LUC hypoxia-sensitive reporter gene vector
  • HRE hypoxia response element
  • HIF-Ia HIF- 1 a proteasome targeting factor
  • HPTF proteasome targeting factor
  • One embodiment of the invention is a composition of siRNA susceptible of knocking-down the expression of one or more of PHD 1 , PHD2 and PHD3.
  • the nuclear factor- ⁇ B (NF- ⁇ B) family is composed of homodimers and heterodimers of the ReI family proteins, including p65 (ReIA), c-Rel, ReIB, p52 and p50.
  • ReIA nuclear factor- ⁇ B
  • c-Rel nuclear factor- ⁇ B
  • ReIB nuclear factor- ⁇ B
  • p52 nuclear factor- ⁇ B
  • p50 nuclear factor- ⁇ B
  • NF- ⁇ B The most abundant form of NF- ⁇ B is a heterodimer with two subunits: one p50 and one p65.
  • NF- KB is bound to inhibitory IKB proteins in the cytoplasm.
  • NF- ⁇ B After stimulation by a variety of stimuli, NF- ⁇ B is released and translocates to the nucleus where it binds to its coactivators, mainly CBP (CREB-Binding Protein), and activates expression of pro-inflammatory genes, including the mast cell growth factor stem cell factor (SCF).
  • CBP CREB-Binding Protein
  • SCF mast cell growth factor stem cell factor
  • NF- ⁇ B is activated by phosphorylation, which plays a key role in the regulation of its transcriptional activity, and is associated with nuclear translocation, CBP recruitment and DNA- binding activity.
  • phosphorylation plays a key role in the regulation of its transcriptional activity, and is associated with nuclear translocation, CBP recruitment and DNA- binding activity.
  • Phosphorylation of p65 occurs on several serine residues. For instance, upon treatment with TNF ⁇ , Ser529 is phosphorylated by casein kinase II, Ser536 by the IKB kinase (IKK) complex, Ser311 by protein kinase C (PKC)- ⁇ , and Ser276 by both PKA and mitogen- and stress-activated protein kinase 1 (MSKl).
  • IKK IKB kinase
  • MSKl mitogen- and stress-activated protein kinase 1
  • Suppression of NFkB eg by knock-down using a composition of the invention may increase the barrier function of the intestine and/or protect the epithelial barrier and/or decrease epithelial apoptosis and may therefore have medical utility in one or more intestinal diseases as described more fully below.
  • One embodiment of the invention is a composition comprising siRNA susceptible of knocking- down the expression of NFKB and/or the 65KDa sub-unit of NFKB.
  • a related embodiment is a composition which comprises a siRNA which knocks down p65.
  • Another embodiment is the transient knock-down of NFkB eg in the intestine.
  • siRNA is as described elsewhere herein or as known in the art - see for example Tao et al (2006) MoI Cell Biol 26(3) 1038-1050 or may be purchased commercially for example from suppliers such as Dharmacon.
  • RNA-containing Minispheres and Pharmaceutical Compositions The release, including controlled release, of siRNAs, or siRNA precursors at specific sites for cellular absorption is only truly useful if the RNA is released and available in an active form or in a form which becomes active on absorption by the target cell.
  • An aspect of the invention is a drug delivery format that enables the release therefrom of siRNAs or siRNA precursors in soluble or readily-soluble form and adapted to be absorbed by the target cells eg intestinal epithelial cells.
  • the invention enables the provision of compositions which permit the release of the siRNAs, or siRNA precursors in soluble or readily-soluble form and maintain them in an appropriate solvent protected from exogenous influences until release, it addresses the question of limited stability and/or a short half- life.
  • the invention also provides an oral drug delivery technology that permits the colon-specific release of pre- or readily-solubilised siRNAs, or siRNA precursors in tandem with a controlled release formulation that permits release of the RNA and absorption of the RNA by cells in the small intestine, ileum and/or colon.
  • Medicaments of the invention may therefore be adapted for the colon-specific release of active agents as described herein, e.g. siRNAs.
  • Colon delivery is particularly advantageous as an effective drug delivery mechanism for siRNAs, or siRNA precursors addressing diseases of the colon (ulcerative colitis, Crohn's disease, Gastro- Intestinal Graft-Versus-Host-Disease (GI-GVHD), Irritable Bowel Syndrome, constipation, diarrhoea, carcinomas and other infections) whereby high local concentration can be achieved while minimizing side effects that occur because of release of drugs in the upper GIT or unnecessary systemic absorption.
  • diseases of the colon ulcerative colitis, Crohn's disease, Gastro- Intestinal Graft-Versus-Host-Disease (GI-GVHD), Irritable Bowel Syndrome, constipation, diarrhoea, carcinomas and other infections
  • the colon is rich in lymphoid tissue and uptake of siRNAs, or siRNA precursors, into the mast cells of the colonic mucosa is intended to modulate the cells to enhance or decrease sensitivity.
  • RNAi molecules that inhibit the release of mediators from sensitized mast cells represent therefore a preferred embodiment of the invention and may be preferentially used to treat or prevent mastocytosis.
  • the colon is a site where a drug molecule, particularly but not exclusively hydrophilic, such as certain siRNAs, or siRNA precursors, that has limited intestinal absorption may have an improved bioavailability or local effect.
  • the colon is recognized as having a somewhat less hostile environment with less diversity and intensity of activity than the stomach and small intestine. Additionally, the colon has a longer retention time and appears highly responsive to agents that enhance the absorption of poorly absorbed drugs such as siRNAs, or siRNA precursors.
  • a reliable colonic drug delivery system is also important for the colonic absorption of perorally administered, undigested, unchanged and fully active molecules such as siRNAs, or siRNA precursors.
  • siRNAs, or siRNA precursors intact and in soluble as well as in a form able to permeate directly into the colon enhances absorption of the drug from the epithelial and other cells lining the colon.
  • siRNAs, or siRNA precursors are protected from release in the upper gastrointestinal tract (GIT) but are able to be abruptly and/or released in a sustained manner, starting at the ileum or proximal colon and throughout the length of the colon.
  • GIT upper gastrointestinal tract
  • Such colon targeting is particularly of value for the treatment of diseases of colon such as Crohn's diseases, ulcerative colitis, graft-versus-host-disease (GVHD), colorectal cancer, amebiasis and mastocytosis.
  • GVHD graft-versus-host-disease
  • the delivery of RNA according to the invention is also applicable to other intestinal conditions, including carcinomas, gastritis, pancreatitis etc, as well as viral infections, including rotavirus, and bacterial infections, including Clostridium difficile.
  • compositions of the invention are comprised of a multitude of separate minicapsules or minispheres, either containing liquid, semi-solid or solid RNA formulations
  • the invention enables the development of novel combination therapies in a single dosage form, each component of the combination (or each population of different minicapsules/minispheres) containing the same or different RNAs or non-RNA active principles and having distinct release profiles, the release being inherent to the core formulation, the shell or the entirety of the minicapsule or some additional polymer coating thereon eg as a membrane.
  • the polymeric material may comprise methacrylic acid co-polymers, ammonio methacrylate co-polymers, or mixtures thereof.
  • Methacrylic acid co-polymers such as EUDRAGITTM S and EUDRAGITTM L (Evonik) are suitable for use in the controlled release formulations of the present invention. These polymers are gastroresistant and enterosoluble polymers. Their polymer films are insoluble in pure water and diluted acids. They dissolve at higher pHs, depending on their content of carboxylic acid. EUDRAGITTM S and EUDRAGITTM L can be used as single components in the polymer coating or in combination in any ratio. By using a combination of the polymers, the polymeric material can exhibit solubility at a pH between the pHs at which EUDRAGITTM L and EUDRAGITTM S are separately soluble.
  • the membrane coating can comprise a polymeric material comprising a major proportion (i.e., greater than 50% of the total polymeric content) of at least one pharmaceutically acceptable water-soluble polymers, and optionally a minor proportion (i.e., less than 50% of the total polymeric content) of at least one pharmaceutically acceptable water insoluble polymers.
  • the membrane coating can comprise a polymeric material comprising a major proportion (i.e., greater than 50% of the total polymeric content) of at least one pharmaceutically acceptable water insoluble polymers, and optionally a minor proportion (i.e., less than 50% of the total polymeric content) of at least one pharmaceutically acceptable water-soluble polymer.
  • the membrane may comprise an amylose, especially a "glassy” amylose as described in US Patent 6534549 and/or 6743445.
  • Other so-called “pore-formers” are also contemplated by the present invention.
  • Ammonio methacrylate co-polymers such as EUDRAGITTM RS and EUDRAGITTM RL (Evonik) are suitable for use in the modified release formulations of the present invention. These polymers are insoluble in pure water, dilute acids, buffer solutions, or digestive fluids over the entire physiological pH range. The polymers swell in water and digestive fluids independently of pH. In the swollen state, they are then permeable to water and dissolved active agents. The permeability of the polymers depends on the ratio of ethylacrylate (EA), methyl methacrylate (MMA), and trimethylammonioethyl methacrylate chloride (TAMCl) groups in the polymer.
  • EA ethylacrylate
  • MMA methyl methacrylate
  • TAMCl trimethylammonioethyl methacrylate chloride
  • EUDRAGITTM RL Those polymers having EA:MMA:TAMC1 ratios of 1 :2:0.2 (EUDRAGITTM RL) are more permeable than those with ratios of 1:2:0.1 (EUDRAGITTM RS).
  • Polymers of EUDRAGITTM RL are insoluble polymers of high permeability.
  • Polymers of EUDRAGITTM RS are insoluble films of low permeability.
  • the amino methacrylate co-polymers can be combined in any desired ratio, and the ratio can be modified to modify the rate of drug release.
  • a ratio of EUDRAGITTM RS: EUDRAGITTM RL of 90: 10 can be used.
  • the ratio of EUDRAGITTM RS: EUDRAGITTM RL can be about 100:0 to about 80:20, or about 100:0 to about 90: 10, or any ratio in between.
  • the less permeable polymer EUDRAGITTM RS would generally comprise the majority of the polymeric material with the more soluble RL, when it dissolves, permitting creating gaps through which solutes can enter the core and dissolved pharmaceutical actives escape in a controlled manner.
  • the amino methacrylate co-polymers can be combined with the methacrylic acid co-polymers within the polymeric material in order to achieve the desired delay in the release of the drug. Ratios of ammonio methacrylate co-polymer (e.g. , EUDRAGITTM RS) to methacrylic acid copolymer in the range of about 99: 1 to about 20:80 can be used.
  • the two types of polymers can also be combined into the same polymeric material, or provided as separate coats that are applied to the core.
  • EudragitTM FS 30 D is an anionic aqueous-based acrylic polymeric dispersion consisting of methacrylic acid, methyl acrylate, and methyl methacrylate and is pH sensitive. This polymer contains fewer carboxyl groups and thus dissolves at a higher pH (> 6.5). The advantage of such a system is that it can be easily manufactured on a large scale in a reasonable processing time using conventional powder layering and fluidized bed coating techniques.
  • Eudragit FS 30 D demonstrated its potential for colonic delivery by resisting drug release up to pH 6.5 and the combination of EudragitTM RL and RS proved successful for the sustained delivery of 5-ASA at the pH of the colon.
  • EudragitTM FS 30 D alone or with other controlled release polymers holds great potential to enable delivery of minicapsule formulations specifically to the colon.
  • EUDRAGITTM polymers In addition to the EUDRAGITTM polymers described above, a number of other such copolymers can be used to control drug release. These include methacrylate ester co-polymers such as the EUDRAGITTM NE and EUDRAGITTM NM ranges. Further information on the EUDRAGITTM polymers can be found in "Chemistry and Application Properties of Polymethacrylate Coating Systems," in Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms, ed. James McGinity, Marcel Dekker Inc., New York, pg 109-114.
  • HPMC hydroxypropyl methylcellulose
  • HPMCP hydroxypropyl methylcellulose acetate succinate
  • HPMCP may alternatively or additionally be incorporated into the film- forming solution (eg gelatine) and thus, in the case of single layer minispheres such as beads, be a component of the solidified matrix.
  • a polmeric coating substance which is pH-independent in its dissolution profile and/or in its ability to release active principles incorporated in the mini-beads of the invention.
  • Examples have already been given (e.g. , Eudragit RS and RL).
  • a pH-independent polymeric coating substance is ethylcellulose, in particular a dispersion of ethylcellulose in a sub-micron to micron particle size range, e.g. from about 0.1 to 10 microns in size, homogeneously suspended in water with the aid of an emulsification agent, e.g. ammonium oleate.
  • the ethylcellulose dispersion may optionally and preferably contain a plasticizer, for example dibutyl sebacate or medium chain triglycerides.
  • a plasticizer for example dibutyl sebacate or medium chain triglycerides.
  • Such ethylcellulose dispersions may, for example, be manufactured according to U.S. Pat. No. 4,502,888, which is incorporated herein by reference.
  • One such ethylcellulose dispersion suitable for use in the present invention and available commercially is marketed under the trademark Surelease®, by Colorcon of West Point, Pa. USA.
  • the ethylcellulose particles are, e.g. , blended with oleic acid and a plasticizer, then optionally extruded and melted.
  • the molten plasticized ethylcellulose is then directly emulsified, for example in ammoniated water optionally in a high shear mixing device, e.g. under pressure.
  • Ammonium oleate can be formed in situ, for instance to stabilize and form the dispersion of plasticized ethylcellulose particles. Additional purified water can then be added to achieve the final solids content. See also U.S. Pat. No. 4,123,403, which is incorporated herein by reference.
  • Surelease® is used hereinafter to refer to ethylcellulose coating materials, for example a dispersion of ethylcellulose in a sub-micron to micron particle size range, e.g. from about 0.1 to 10 microns in size, homogeneously suspended in water with the aid of an emulsification agent, e.g. ammonium oleate.
  • an emulsification agent e.g. ammonium oleate
  • Surelease® dispersion is a unique combination of film-forming polymer, plasticizer and stabilizers.
  • Surelease is an easy- to-use, totally aqueous coating system using ethylcellulose as the release rate controlling polymer.
  • the dispersion provides the flexibility to adjust drug release rates with reproducible profiles that are relatively insensitive to pH.
  • the principal means of drug release is by diffusion through the Surelease dispersion membrane and is directly controlled by film thickness. Increasing or decreasing the quantity of Surelease® applied can easily modify the rate of release.
  • Surelease dispersion reproducible drug release profiles are consistent right through from development to scale-up and production processes. Pore formers, for example those described in US Patent 6534549 and/or 6743445 may alternatively or additionally be included.
  • polymers can include phthalate, butyrate, succinate, and/or mellitate groups.
  • Such polymers include, but are not limited to, cellulose acetate phthalate, cellulose acetate succinate, cellulose hydrogen phthalate, cellulose acetate trimellitate, hydroxypropyl-methylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, starch acetate phthalate, amylose acetate phthalate, polyvinyl acetate phthalate, and polyvinyl butyrate phthalate.
  • any combination of polymer may be blended to provide additional controlled- or targeted-release profiles.
  • the coating membrane can further comprise at least one soluble excipient to increase the permeability of the polymeric material.
  • the at least one soluble excipient is selected from among a soluble polymer, a surfactant, an alkali metal salt, an organic acid, a sugar, and a sugar alcohol.
  • Such soluble excipients include, but are not limited to, polyvinyl pyrrolidone, polyethylene glycol, sodium chloride, surfactants such as sodium lauryl sulfate and polysorbates, organic acids such as acetic acid, adipic acid, citric acid, fumaric acid, glutaric acid, malic acid, succinic acid, and tartaric acid, sugars such as dextrose, fructose, glucose, lactose, and sucrose, sugar alcohols such as lactitol, maltitol, mannitol, sorbitol, and xylitol, xanthan gum, dextrins, and maltodextrins.
  • polyvinyl pyrrolidone polyethylene glycol, sodium chloride
  • surfactants such as sodium lauryl sulfate and polysorbates
  • organic acids such as acetic acid, adipic acid, citric acid, fumaric acid, glutaric acid, malic
  • polyvinyl pyrrolidone, mannitol, and/or polyethylene glycol can be used as soluble excipients.
  • the at least one soluble excipient can be used in an amount ranging from about 1% to about 10% by weight, based on the total dry weight of the polymer.
  • the coating process can be carried out by any suitable means, for example, by using a perforated pan system such as the GLATT, ACCELACOTA, Vector, Diosna, O'Hara, HICOATER or other such coating process equipment.
  • Seamless minicapsules may be manufactured using the method described in US5, 882,680 (Freund), the entire contents of which are incorporated herein by reference. Solubilisation and Suspension of RNA
  • RNA for encapsulation such that the core of minicapsules according to the invention is a fluid, eg a solution or an emulsion with or without appropriate surfactant.
  • the RNA may be encapsulated in a water-in-oil emulsion either as the fluid core of a true minisphere with outer capsule or as a bead of semi-solid or solid RNA-containing emulsion optionally within a matrix forming the body of the bead.
  • the invention therefore includes a product selected from a minicapsule, a minibead or a minisphere and comprising a solid phase and an active agent selected from siRNAs and engineered RNA precursors, the product having one layer being a solid phase comprising inclusions selected from liquid inclusions, semi-solid inclusions or combinations thereof or having at least two layers comprising a solid phase outer shell layer encapsulating a liquid, semisolid or solid core.
  • the inclusions comprise, e.g. consist of, a water-in-oil emulsion or, as the case may be, the core comprises, e.g. is, a water-in-oil emulsion.
  • at least one such active agent is comprised in an aqueous phase dispersed in an oil phase
  • RNA or siRNA-protein complexes may be included in, or associated with, liposomes, particularly cationic or other liposomes suitable for transfection.
  • liposomes particularly cationic or other liposomes suitable for transfection.
  • examples include Lipofectamine or Lipofectamine 2000 which are established transfection reagents, produced and sold by Invitrogen for the introduction (transfection) of siRNA or plasmid DNA into cells by lipofection. Lipofectamine treatment alters the cellular plasma membrane, allowing nucleic acids to cross into the cytoplasm and other such membrane-altering reagents are contemplated by the present invention.
  • Variant cationic liposomes for nucleic acid delivery according to the invention include those generated by using cationic lipids such as LipofectinTM and CytofectinTM.
  • Lipofectin is a mixture of N-[I -(2, 3- dioleyloyx) propyl] -N-N-N-trimethyl ammonia chloride (DOTMA) and DOPE (phosphatidylethanolamine).
  • DOTMA N-[I -(2, 3- dioleyloyx) propyl] -N-N-N-trimethyl ammonia chloride
  • DOPE phosphatidylethanolamine
  • Another possible cationic lipid is DOTAP (dioleoyl trimethylammonium propane.).
  • transfection agents are those known in the DNA field eg derivatives of phosphatidyl choline including DOPC (dioleoyl- phosphatidylcholine) and EDOPC (dioleoyl-sn-glycero 1-3 -ethylphosphocho line or O-ethyldioleoylphosphatidylcholine).
  • DOPC dioleoyl- phosphatidylcholine
  • EDOPC dioleoyl-sn-glycero 1-3 -ethylphosphocho line or O-ethyldioleoylphosphatidylcholine
  • neutral phospholipids such as dipalmitoyl phosphatidyl choline (DPPC) may be used or negatively charged phospholipids such as dipalmitoyl-phosphatidylglycerol (DPPG) may be used in either case with or without phosphatidylethanolamine (DOPE).
  • the liposome suspension may be stabilized for example by addition of a stabilizing agent such as sorbitol and the thus stabilized suspension may optionally be lyophilised for further formulation as described elsewhere herein.
  • a stabilizing agent such as sorbitol
  • the liposomes eg after rehydration of a lyophilisate, may be converted into beads in the manner described in more detail elsewhere in this description.
  • the liposome to siRNA quantity ratio may be expressed as molar charge ratio ie the molar ratio of positive charge from the (phospho) lipids to the negative charge of the siRNA component.
  • the molar charge ratio (positive : negative) may range from 1 : 1 to 55 : 1 , for example from 1 : 1 to 25 : 1 or from 1 : 1 to 12 : 1 depending on choice of the (phospho) lipid(s).
  • it is preferable for the charge rato to be from 2 : 1 to 5 : 1.
  • liposome stock solution made eg from phosphate buffered saline and one or more of the above lipids or phospholipids to siRNA (associated or not with a polymer) may lead to a loss of unilamellar liposomal structure and the adoption of a more complex molecular arrangement of siRNA and liposomal material.
  • These molecular arrangements are nevertheless generally aqueously soluble and/or hydrophilic owing to constituent (phospho) lipids orientated as in the outside layer of standard liposomes (outward- facing hydrophilic head).
  • Such liposomal-siRNA structures are stable in aqueous media and may therefore be handled and further processed according to the invention as if they were standard liposomes.
  • the aqueous media which contains such structures may be dispersed in an oil phase (such as described elsewhere herein) to create a water-in-oil emulsion which itself may be dispersed in an aqueous solution of a polymer matrix such as a gelling agent (described elsewhere herein) to form a water-in-oil-in-water (w/o/w) emulsion.
  • This w/o/w emulsion may be extruded to form minispheres, minicapsules or minibeads as described elsewhere herein.
  • liposome as used herein, unless the context demands otherwise, therefore includes unilamellar and multilamellar liposomes and/or liposomal components, having aqueous RNA phases enclosed in the aqueous cores (concentric or non-concentric) as well as more complex liposomal structures eg of liposomal components such as flat lamellar structures (eg where RNA is "sandwiched” between lipid bilayers), hexagonal, cylindrical, rod and columnar structures possibly with hexagonal cross-sectional aspect. So called “inverted" liposomal structures behaving as lipophilic entities are also contemplated.
  • mini-beads may be generated directly (in the manner described below) without prior formation of liposomes by generating a (water-in-oil) emulsion or microemulsion and dispersing droplets of such emulsion or microemulsion (as if it were an oil phase) in an aqueous matrix such as gelatin or other such material described in more detail in the section herein describing the structure of the minispheres/minicapsules.
  • a (water-in-oil) emulsion or microemulsion and dispersing droplets of such emulsion or microemulsion (as if it were an oil phase) in an aqueous matrix such as gelatin or other such material described in more detail in the section herein describing the structure of the minispheres/minicapsules.
  • the emulsion containing liposomes or not, particularly a water-in-oil emulsion, may favourably contain 2-(2-ethoxyethoxy) ethanol which is available under the tradename Transcutol or Cellusolve.
  • the emulsion may contain a nonionic surfactant, especially of the kind from polyethoxylated sorbitan and oleic acid.
  • a nonionic surfactant especially of the kind from polyethoxylated sorbitan and oleic acid.
  • An example is the surfactant and emulsifier available under the tradename Tween. Tween 80 is particularly preferred.
  • the oil component of the emulsion or microemulsion in this aspect and in other aspects of the invention may be any kind of pharmaceutically appropriate oil for oral administration including for example oleoyl and linoleoyl macrogolglycerides (and other polyoxylglycerides) as commercialised by Gattefosse under the name LabrafilTM.
  • oleoyl and linoleoyl macrogolglycerides and other polyoxylglycerides
  • Alternative or additional oils are caprylocaproyl macrogolglycerides such as Labrasol by Gattefosse.
  • oils which may be included in the oil phase according to the invention are medium chain triglycerides such as for example LabrafacTM Lipophile manufactured by Gattefosse in particular product number WL1349.
  • Other oils which may alternatively or additionally be included as the oil phase of the emulsion include poly-unsaturated fatty acids such as omega-3 oils such as eicosapentanoic acid (EPA), docosohexaenoic acid (DHA), alpha- linoleic acid (ALA). Combinations of such components are also contemplated eg a mixture of EPA and DHA in a ratio of 1 :5 available commercially under the trade name Epax 6000.
  • oils which may alternatively or additionally be used as or included in the oil phase are natural triglyceride-based oils which include olive oil, sesame oil, coconut oil, palm kernel oil. Oils which are particularly preferred include saturated coconut and palm kernel oil-derived caprylic and capric fatty acids and glycerin eg as supplied under the trade name MiglyolTM a range of which are available and from which one or more components of the oil phase of the invention may be selected including MiglyolTM 810, 812 (caprylic/capric triglyceride);
  • MiglyolTM 818 (caprylic/capric/linoleic triglyceride); MiglyolTM 829: (caprylic/capric/succinic triglyceride; MiglyolTM 840: (propylene glycol dicaprylate/dicaprate).
  • MiglyolTM 810/812 differ only in C 8 /C 10 -ratio and because of its low C 10 -content, the viscosity and cloud point of MiglyolTM 810 are lower.
  • the MiglyolTM range is available commercially from Sasol Industries.
  • the vehicle and/or transfection agent may include cationic surfactants including the Montanide family of reagents available from Seppic, France.
  • Other transfection agents/techniques include use of polymeric DNA-binding cations such as poly- L-lysine or polyethyleneimine.
  • the fluid core of minicapsules or the fluid or semi-solid components of a bead including transfection agents may include modified cyclodextrins with cationic moieties.
  • a further vehicle which may be utilised in the present invention are nanolipid vesicles or nanolipids which are very small spherical bodies arising from the long chain fatty acids which may be used in the formulation of the preparation according to the invention.
  • Such encapsulating vesicles may be used to penetrate and/or are useful to facilitate and/or enhance siRNA penetration of the cell membrane and to migrate to cell organelles.
  • Another vehicle appropriate for use according to the invention are self-emulsifying drug delivery systems (SEDDS) and other (eg micro-emulsion) systems which enhance the permeability of cells to macromolecules.
  • SEDDS self-emulsifying drug delivery systems
  • other (eg micro-emulsion) systems which enhance the permeability of cells to macromolecules.
  • the size of the emulsion droplets constituting the above minispheres or minicapsules can be varied according to the invention and microemulsions are preferred for example in the targeting of intestinal epithelial cells as well as the lymphatic system which, according to the invention, represents a route of RNA delivery from the GI tract to other parts of the body. Where lymphatic delivery is desired, emulsifying with bile salts, may be preferable.
  • RNAs For water-soluble RNAs, it is preferred to adopt the liposome or water-in-oil microemulsion approach.
  • hydrophobic or water-insoluble RNAs and RNA derivatives such as lipid conjugates it is preferred either to disperse the RNA in an appropriate medium before proceeding to further formulation in accordance with the invention or to select an appropriate solvent eg an oil or lipid, as described elsewhere herein, in which to dissolve the RNA before proceeding to further formulation as described herein such as formation of a water-in-oil emulsion or microemulsion.
  • modified-release formulations are known in the art and are described, for example, in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566.
  • modified-release formulations include but are not limited to, membrane-modified, matrix, osmotic, and ion-exchange systems.
  • Another approach according to the invention is to have the core itself controlling release of RNA or RNA-containing beads, microemulsions or liposomes.
  • a semi-permeable membrane can surround the formulation containing the active substance of interest.
  • Semi-permeable membranes include those that are permeable to a greater or lesser extent to both water and solute.
  • This membrane can include water-insoluble and/or water-soluble polymers, and can exhibit pH- dependent and/or pH-independent solubility characteristics. Polymers of these types are described in detail below.
  • the characteristics of the polymeric membrane which may be determined by, e.g. , the composition of the membrane, will determine the nature of release from the dosage form.
  • the present invention provides for formulations of minicapsules or minispheres wherein the modified release is dependent upon, where appropriate, any one of the core formulation constituents, the shell composition or the shell coating.
  • the minicapsules or minispheres may be produced through the utilisation of surface tension of one or more different solutions or liquids which, when ejected through an orifice or nozzle with a certain diameter and subject to specific frequencies and gravitational flow, form into a spherical form and falls into a cooling air flow or into a cooling or hardening solution and the outer shell solution where it is gelled or solidified.
  • Minispheres, minispheres or minibeads of diameter between 500 and 5000 microns are preferred with a range from lmm to 2mm being particularly preferred.
  • a core liquid or solution and a shell liquid or solution are ejected through a nozzle.
  • a core solution of RNA may be a hydrophobic solution or suspension of RNA as described above depending on the physicochemical characteristics of the RNA being formulated, eg whether it is a conjugate eg lipid conjugate or a hydrophilic derivative.
  • the core solution can be pre-mixed with a shell solution eg emulsified such that the shell solution acts as a "matrix" within which the core solution is already dispersed (a shell/core mixed suspension) which may be extruded to form single layer minicapsules (beads) without further processing.
  • the outer shell or bead matrix solution can be any gel forming agent but is preferably gelatine- or alginate-based although pectin, carrageenan and others may be used.
  • RNA solutions can also be hydrophilic and one of the advantages of the present invention is that hydrophilic solutions can also be encapsulated in the manner described although preferably with the existence of an intermediate solution, which can avoid the direct contact of the hydrophilic core solution (which contains the RNA) with the outer shell.
  • the temperature of the gelatin solution during manufacture depends on the gelatin type and the amount of softener but is typically in the range of 60 0 C to 70 0 C eg around 65°C.
  • a minicapsule or a bead of shell/core mixed suspension can be processed and may further be processed using a melt-extrusion-like process with solidification of the matrix occurring shortly after extrusion eg by change in temperature or exposure to cross-linking agents to form mini-beads.
  • a hydrophobic solution can be encapsulated.
  • both the core and / or shell may be comprised of a material or material composites that have been processed by a wet- or dry-granulation mechanism, melt or otherwise fluidized prior to mixing or granulation.
  • RNA content and release consistency it is preferred that all processes result in fairly uniform morphologies with a relatively smooth surface to facilitate quite even coating layers to be added in a uniform manner.
  • seamless minicapsules for various applications can be processed using minicapsule processing equipment enabled by, but not limited to, Freund Spherex, ITAS/Lambo Globex or Inotech processing equipment.
  • the coating process can be carried out by any suitable means, for example, by using a perforated pan or fluidized-baed system such as the GLATT, Vector, ACCELACOTA, Diosna, O'Hara and/or HICOATER processing equipment.
  • the result is modified release compositions that in operation deliver siRNAs, or siRNA precursors optionally with one or more additional active ingredients in a unique (unimodal), bimodal or multimodal manner.
  • the present invention further relates to solid oral dosage forms, sachets or suppositories containing such multiple minicapsule or minisphere controlled release compositions of siRNA or RNA precursors as well as methods for delivering one or more active ingredients to a patient in a unimodal, bimodal or multimodal manner.
  • the invention permits targeted release of orally delivered formulations to specific regions of the gastrointestinal tract to maximize absorption, confer protection on the payload, to optimize treatment of diseased intestinal tissue or enhance oral bioavailability.
  • the invention enables one or more RNAs to be administered sequentially or concomitantly to improve disease treatment and management and to benefit from the body's natural circadian rhythms.
  • the invention also permits the release of siRNAs, or siRNA precursors along with, optionally, other pharmaceutical actives into the ileum and colon for the enhanced treatment of local intestinal diseases or to facilitate the absorption of active pharmaceutical agents.
  • the other pharmaceutical actives may be small molecules or biopharmaceuticals such as peptides or proteins. Examples of drugs that have demonstrated limited colonic absorption that could be combined wth the siRNAs, or siRNA precursors in a combination formulation include Tacrolimus, Cyclosporine, ASA etc, Budesonide and Celecoxib.
  • the invention enables a siRNAs, or siRNA precursors, optionally co-released alongside a small molecule or macromolecule, to be clinically effective, to reach its intended target cell, cell system or tissue in an active form.
  • enteric polymer coatings protects the contents of minicapsules from gastric acid degradation while other colon-specific coatings permit release of minicapsule contents only in the colon where the proteolytic enzyme content is significantly less than in the small intestine.
  • the invention provides formulations that ensure that the siRNAs, or siRNA precursors and other optional active contents are released intact at sites where absorption or therapeutic activity is optimal.
  • the invention includes RNA delivery in the colon which has been largely overlooked from a drug delivery perspective.
  • the colon is the site of significant flow of water from the colonic lumen into the body.
  • the colon is home to a natural bacterial flora to degrade complex carbohydrates to ensure effective excretion, provide much needed fibre and some nutrient absorption.
  • proteolytic and other enzymes populated in the colon, it is a much more benign environment for nucleic acids, including RNAs and DNAs, proteins and peptides as well as other biological entities such as carbohydrates.
  • the colon presents a number of interesting possibilities: the bacteria can be harnessed to break down controlled release coatings that are resistant to acidic breakdown as well as pH differentials; the benign environment ensure than active pharmaceuticals, including biopharmaceuticals, are less likely to be degraded if released locally into the colon; the almost continuous flow of fluids from the colonic lumen to the bloodstream may be harnessed to carry hydrophilic entities from the intestine to the lumen. Finally, the long transit time in the colon, ranging form 10-20 hours provides greater residence and potential for interaction with the colonic mucus and epithelial cells leading to enhanced absorption.
  • this invention is based on various modifications of basic one- or multi-layered minicapsules or minispheres, modulating core, shell or coating to permit enhanced solubility and permeability of the siRNAs, or siRNA precursors or other active or non-active entity as well as conferring protection on siRNAs, or siRNA precursors or entities that are susceptible to various forms of intestinal, mucosal or systemic degradation and targeted release of the therapeutically- active or -inactive entities to predetermined regions of the gastrointestinal tract.
  • the minicapsules or minispheres may be solid drug-containing formulations or they may be encapsulated solid, semi-solid or liquid drug-containing formulations.
  • the present invention provides the coating of minicapsules or minispheres with a muco- or bio-adhesive entity which will ensure that they first adhere to the mucosa prior to releasing the fragile payload.
  • the advantages thus enabled include further protection of the active entities but also release of the actives proximal to the site of absorption.
  • absorption is, in part, related to the surface area exposed to the active as well as the concentration gradient from intestinal luminal side to the intestinal basal side, the higher local yet dispersed concentration has greater potential to ensure enhanced absorption, not only of siRNAs, or siRNA precursors but also of other hydrophilic, lipophilic or hydrophobic drugs.
  • a barrier to effective colonic delivery of hydrophobic and lipophilic drugs is that the colon did not evolve to solubilize foodstuffs and other entities but rather to ensure electrolyte balance and maximize fibre breakdown and fermentation.
  • the colon remains very porous to hydrophilic entities.
  • hydrophobic or lipophilic drugs to the colon in a pre-solubilised or readily soluble format and releasing such in the colon, the potential for absorption is enhanced significantly.
  • the present invention permits the encapsulation of pre-solubilized or readily soluble drugs in liquid or hydrolysable semi-solids or solids into the minicapsule core (especially RNA) and then modulation of the shell to include intestinal- or colon-controlled release polymers or coating the shell with same. The result is release of optimized formulations at specific sites along the intestinal tract for maximal therapeutic efficacy or systemic absorption..
  • chitosan can be directly associated with siRNA to form an aggregate or a complex which may be further formulated eg with liposomal materials before incorporation or encapsulation in a single layer minicapsule, such as a bead, or a multi-layered minicapsule.
  • the formulations of the present invention can exist as multi-unit or single-unit formulations.
  • multi-unit as used herein means a plurality of discrete or aggregated minicapsules, minispheres, particles, beads, pellets, granules, tablets, or mixtures thereof, for example, without regard to their size, shape, or morphology.
  • Single-unit formulations include, for example, tablets, hard gelatin capsules, caplets, and pills.
  • a formulation and/or method of the invention can contain components that exhibit extended-release and immediate-release properties, or both delayed-release and immediate- release properties, or both extended-release and delayed-release properties, or a combination of all three properties.
  • a multi-minicapsule or multi-minisphere formulation including both immediate-release and extended-release components can be combined in a capsule, which is then coated with an enteric coat to provide a delayed-release effect.
  • a delayed- and extended-release caplet may comprise a plurality of discrete extended-release particles held together with a binder in the caplet, which is coated with an enteric coating to create a delay in dissolution.
  • modified-release formulation or dosage form includes pharmaceutical preparations that achieve a desired release of the siRNAs, or siRNA precursors from the formulation.
  • a modified-release formulation can be designed to modify the manner in which the siRNAs, or siRNA precursors is exposed to the desired target, preferably intestinal epithelial cells.
  • a modified-release formulation can be designed to focus the delivery of the siRNAs, or siRNA precursors entirely in the distal large intestine, beginning at the cecum, and continuing through the ascending, transverse, and descending colon, and ending in the sigmoid colon.
  • a modified-release composition can be designed to focus the delivery of the siRNAs, or siRNA precursors in the proximal small intestine, beginning at the duodenum and ending at the ileum.
  • the modified-release formulations can be designed to begin releasing active agent in the jejunum and end their release in the transverse colon. The possibilities and combinations are numerous, and are clearly not limited to these examples.
  • modified-release encompasses "extended-release” and “delayed-release” formulations, as well as formulations having both extended-release and delayed-release characteristics.
  • An "extended-release” formulation can extend the period over which drug is released or targeted to the desired site.
  • a “delayed-release” formulation can be designed to delay the release of the siRNAs, or siRNA precursors for a specified period. Such formulations are referred to herein as “delayed-release” or “delayed-onset” formulations or dosage forms.
  • Modified-release formulations of the present invention include those that exhibit both a delayed- and extended-release, for example, formulations that only begin releasing after a fixed period of time or after a physicochemical change has occurred, for example, then continue releasing over an extended period.
  • immediate-release formulation is meant to describe those formulations in which more than about 50% of active ingredient is released from the dosage form in less than about 2 hours. Such formulations are also referred to herein as “conventional formulations.”
  • drug-release profile that is independent of surrounding pH means effectively a drug composition comprising a polymeric system that is non-enteric or whose permeability and solubility properties do not change with environmental, i.e., external, pH.
  • a drug composition having release characteristics such as dissolution is substantially unaffected by pH or regardless of pH-changes in the environment. This is in comparison to a release profile that is pH-dependent where the release characteristics vary according to the pH of the environment.
  • medium and long-chain fatty acids exert an intestinal epithelial effect which leads to an increased permeability of intestinal membranes to entities that may otherwise be impermeable or exhibit limited permeability.
  • the medium chain triglycerides including but not limited to sodium caprate, enhance absorption to a greater extent in the small intestine than in the ileum or colon.
  • medium chain triglycerides enhance intestinal permeability while DHA is a possible means of facilitating the intestinal absorption of insulin and possibly other macromolecules, nucleic acids, peptides and proteins included, without inducing any serious damage to epithelial cells.
  • DHA dihydroxyacetyl hydroxyacetyl acetate
  • Combining poorly permeable entities with medium- or long-chain fatty acids and targeted delivery to local regions of the intestine or colon has the potential to enhance absorption of otherwise poorly permeable entities.
  • the current invention seeks to enable such delivery through the encapsulation of entities formulated with medium or long chain polyunsaturated fatty acids using a gelling agent, including, but not limited to one or a mixture of gelatine, pectin, alginate or chitosan, with or without an additional colon-specific coating.
  • a gelling agent including, but not limited to one or a mixture of gelatine, pectin, alginate or chitosan, with or without an additional colon-specific coating.
  • this mixture of gelling agents may be gelatine and chitosan.
  • the chitosan may be first associated with siRNA to form an aggregate, complex or a nanoparticle which may then be further formulated eg with liposomal materials.
  • the resulting aqueous phase may then be dispersed in a medium or long chain triglyceride oil phase (water-in-oil emulsion) of the kind described above.
  • This w/o emulsion may then itself be dispersed (emulsified) in an aqueous solution of gelatine as the second gelling agent (although alternative second gelling agents such as alginate are possible as described elsewhere herein) to form a w/o/w emulsion.
  • This w/o/w emulsion may be converted eg into minibeads, minispheres or minicapsules (dependent on extrusion nozzle geometry described elsewhere herein) by extrusion followed by solidification by temperature reduction.
  • Inflammatory bowel diseases which genus encompass a range of diseases including Crohn's disease and ulcerative colitis, affect nearly 1 million people in the United States each year.
  • the two most common inflammatory conditions of the intestine ulcerative colitis (UC) and Crohn's disease (CD), are collectively known as inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • These conditions are diseases of the distal gut (lower small intestine, large intestine, and rectum) rather than the proximal gut (stomach and upper small intestine).
  • ulcerative colitis primarily affects the colon
  • Crohn's disease affects the distal small intestine as well.
  • IBD Inflammatory Bowel Disease
  • Drugs commonly used in their treatment include steroids (e.g. , budesonide and other corticosteroids, and adrenal steroids such as prednisone and hydrocortisone); cytokines such as interleukin-10; antibiotics; immunomodulating agents such as azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, and anti-tumor necrosis factor (TNF) agents such as soluble TNF receptor and antibodies raised to TNF; and also antinflammatory agents such as zinc.
  • steroids e.g. , budesonide and other corticosteroids, and adrenal steroids such as prednisone and hydrocortisone
  • cytokines such as interleukin-10
  • antibiotics e.g. , budesonide and other corticosteroids, and adrenal steroids such as prednisone and hydrocortisone
  • immunomodulating agents such as azathioprine, 6-mercaptopurine, methotrexate, cycl
  • sulfasalazine salicyl-azo-sulfapyridine, or "SASP"
  • 5-ASA 5-aminosalicylic acid
  • mesalazine mesalazine.
  • Inflammation of the ileum due to Crohn's disease is known as iletis.
  • Crohn's enterocolitis or ileocolitis.
  • Other descriptive terms may be used as well.
  • Diagnosis is commonly made by x-ray or colonoscopy. Treatment includes medications that are anti-inflammatories, immune suppressors, or antibiotics. Surgery can be necessary in severe cases. Crohn's disease is an area of active research around the world and new treatment approaches are being investigated which have promise to improve the lives of affected patients.
  • G-GVHD Gastrointestinal Graft- Versus-Host-Disease
  • GI GVHD is a life-threatening condition and one of the most common causes for bone marrow and stem cell transplant failure. These procedures are being increasingly used to treat patients with leukemia and other cancers to eliminate residual disease and reduce the likelihood of relapse. Unlike solid organ transplants where the patient's body may reject the organ, in GVHD it is the donor cells that begin to attack the patient's body - most frequently the gut, liver and skin. Patients with mild-to-moderate GI GVHD typically develop symptoms of anorexia, nausea, vomiting and diarrhea. If left untreated, GI GVHD can progress to ulcerations in the lining of the GI tract, and in its most severe form, can be fatal.
  • Systemic immunosuppressive agents such as prednisone, which are the current standard treatments for GI GVHD, are associated with high mortality rates due to infection and debility. Further, these drugs have not been approved for treating GI GVHD in the U.S. or European Union, but rather are used off-label as investigational therapies for this indication.
  • Minicapsule-enabled colon-targeted immunosuppressant therapy delivering agents such as cyclosporine A to the colon is a novel oral, locally acting active therapy which will reduce the need for systemic immunosuppressive drugs such as prednisone, which is currently used to prevent and control GI GVHD.
  • Minicapsule-enabled colon-targeted immunosuppressant therapy is designed to reduce the need for systemic immunosuppressive drugs and thereby improve the outcome of bone marrow and stem cell transplantation. Therefore, it is possible that delivery of intact peptides or proteins to the colon may be achieved.
  • the invention is directed to, among other things, a pharmaceutical composition for administration to a subject in need thereof comprising a dose of RNA, and at least one pharmaceutically acceptable excipient, wherein the composition exhibits localized release and exhibits:
  • This invention relates to formulations and methods for treating inflammatory bowel disease.
  • inflammatory bowel disease includes, but is not limited to, ulcerative colitis, Crohn's disease and GI-GVHD.
  • Other diseases contemplated for treatment or prevention by the present invention include non-ulcerative colitis, and carcinomas, polyps, and/or cysts of the colon and/or rectum. All of these diseases fall within the scope of the term "inflammatory bowel disease” as used in this specification, yet the invention does not require the inclusion of each recited member.
  • the invention may be directed to the treatment of Crohn's disease, to the exclusion of all the other members; or to ulcerative colitis, to the exclusion of all the other members; or to any single disease or condition, or combination of diseases or conditions, to the exclusion of any other single disease or condition, or combination of diseases or conditions.
  • TJ tight junctions
  • paracellular permeability molecules that transiently and reversibly open the TJs of epithelial and endothelial tissues such as the intestinal mucosa, blood brain barrier and pulmonary epithelia.
  • TJ regulatory pathways As increased paracellular permeability is implicated as a causal factor in many disease states, modulation of permeability by TJ regulatory pathways represents a very important therapeutic opportunity.
  • Potential applications range from the treatment of diseases involving tight junction dysfunction and autoimmunity to vaccine and drug delivery.
  • Certain TJ modulators such, but not limited to, parozotide acetate, have potential in the treatment of gastrointestinal disorders, including Celiac Disease and Inflammatory Bowel Disease.
  • the current invention permits the local delivery of tight junction modulators simultaneously with local delivery of siRNAs, or siRNA precursors thus further enhancing absorption by target tissue.
  • RNA handling Synthesis of RNA is as described above in the detailed disclosure of the invention. Annealing of siRNAs is generally performed as previously described by Tuschl and co-workers (Elbashir et al., 2001 the entirety of which is incorporated herein by reference). A particular annealing buffer is: 100 mM NaCl in 20 mM sodium phosphate buffer, pH 6.8.
  • Procedures for preparing RNA for formulation include one or more of the following steps:
  • siRNA stock solutions at -80 0 C (long term) or at -20 0 C for up to 6 months. 5. Do not keep solutions of highly diluted siRNA for more than an hour, as the double- stranded molecules tend to dissociate
  • siRNA stability depends on its sequence and the composition of the solvent (e. g. serum-free medium)
  • the pH and salt composition of the solvent may accelerate RNA decay in solution.
  • EXAMPLES The following materials, methods, and examples are illustrative only and not intended to be limiting.
  • the appropriate siRNA (see following examples) is incorporated into liposomes as follows.
  • An appropriate amount of lipid such as DPPC, DPPG with or without addition of DOPE or positively charged agents
  • chloroform or chloroform-methanol is dissolved in chloroform or chloroform-methanol and the solution is placed in a 50-ml round-bottomed flask.
  • the chloroform is evaporated by gently heating so that a thin film of the lipid is formed on the walls of the flask.
  • siRNA dissolved in a suitable volume of hydrophilic solvent like RNase-free sterile water
  • glass beads were put, which is mixed at room temperature for blending and hydration. The mixture is well shaken for 10-15 min to produce an almost homogeneous liquid of multilamellar liposomes
  • the sorbitol is added to the suspension as the membrane stabilizing agent in an amount of up 1 % wt/v of the suspension.
  • the lyophilisation of the liposomal suspension is preferably carried out by cooling the suspension to a temperature of about -25C.
  • the freeze dried composition of the present invention upon rehydratation, forms a suspension of MLVs which substantially maintains its native size distibution, siRNA/lipid ratio and the morphology of the vesicles.
  • an appropriate volume of liposomes were mixed with gelatin (90% gelatin, 10% sorbitol), pectin 2-4% or alginate 2-4% solution using Spherex Labo (single nozzle operation) to form beads.
  • the 21 -nucleotide siRNA targeting PHDl corresponding to the coding region 538 ⁇ 558 and 835 ⁇ 855 relative to the start codon is chemically synthesized by methods described above in the body of the description (annealing of siRNAs is also performed as described above) and is incorporated into liposomes in accordance with example 1.
  • the 21 -nucleotide siRNA targeting PHD2 corresponding corresponding to regions 885 ⁇ 905 and 1250 ⁇ 1270 relative to the start codon is chemically synthesized by methods described above in the body of the description (annealing of siRNAs is also performed as described above) and is incorporated into liposomes in accordance with example 1.
  • the 21 -nucleotide siRNA targeting PHD3 corresponding to the coding regions 351 ⁇ 371 and 389 ⁇ 409 relative to the start codon is chemically synthesized by methods described above in the body of the description (annealing of siRNAs is is also performed as described above) and is incorporated into liposomes in accordance with example 1.
  • RNA is prepared in a suitable siRNA solubility system as follows: siRNA is dissolved in water in the proper amount, then siRNA solution is mixed with a pre-blended mixture of Transcutol HP, Tween 80 and Labrafil M 1944 CS to form a w/o (water- in-oil) microemulsion (w/o ME) (Table 1);
  • Preparation of Gelatin Solution appropriate amounts of gelatine and D-sorbitol are dissolved in water at up to 70 degrees C until in solution;
  • Preparation of Beads appropriate quantities of w/o ME and Gelatin Solution are mixed at up to 70 degrees C (using a siRNA derivative stable at this temperature) to form a stable mixture, which is then processed using a Spherex Labo to produce a single layer bead.
  • siRNA for PHDl (as described in Example 2) stable to at most 70 0 C is synthesised as described above in the body of the description.
  • Single layer beads of this siRNA derivative are produced in accordance with Example 5.
  • siRNA for PHD2 (as described in Example 3) stable to at most 70 0 C is synthesised as described above in the body of the description.
  • Single layer beads of this siRNA derivative are produced in accordance with Example 5.
  • siRNA for PHD3 (as described in Example 4) stable to at most 70 0 C is synthesised as described above in the body of the description.
  • Single layer beads of this siRNA derivative are produced in accordance with Example 5.
  • siRNA (see following examples) is prepared in a suitable solubility system as follows: an appropriate quantity of siRNA is dissolved in water, then D-sorbitol and gelatine are added and dissolved at up to 70 degrees C.
  • a hydrophilic or hydrophobic derivative of siRNA for PHDl (as described in Example 2) stable to at most 70 0 C is synthesised as described above in the body of the description. The derivative is then used in accordance with Example 9 to generate beads.
  • a hydrophilic or hydrophobic derivative of siRNA for PHD2 (as described in Example 3) stable to at least 70 0 C is synthesised as described above in the body of the description. Single layer beads of this siRNA derivative are produced in accordance with Example 5.
  • Example 12 A hydrophilic or hydrophobic derivative of siRNA for PHD3 (as described in Example 4) stable to at least 70 0 C is synthesised as described above in the body of the description. Single layer beads of this siRNA derivative are produced in accordance with Example 5.
  • Example 13 - siRNA minicapsules This example is suitable for siRNAs or siRNA derivatives which are dispersible or soluble in the exemplified core materials.
  • An appropriate amount of 80°- stable siRNA (or appropriate derivative) is dispersed in a solid gelling agent as follows to prepare solid minicapsules (minispheres):: Appropriate quantities of siRNA gelatine and sorbitol are added to water and heated to 80 0 C, continually stirring until in a homogeneous solution. The solution is then processed into solid minispheres at an appropriate flow rate and vibrational frequency. The resulting minispheres are cooled in oil. The cooled minispheres are harvested and centrifuged to remove residual oil and dried overnight.
  • Table 7 Single-Layer siRNA Minicapsules (Minispheres) To enable the development of a once-daily or an ileum- and colon-specific product, the minicapsules are coated with a range of sustained release polymers, namely differing weight gains of Surelease®, ranging from 0 to 30% weight gain, or variable weight gains of Surelease® plus variable concentrations of pectin.
  • Example 14 Minicapsules prepared as per Example 13 are coated with 10% weight gain Surelease®.
  • Minicapsules prepared as per Example 13 are, coated with 15% weight gain Surelease®.
  • Minicapsules prepared as per Example 13 are coated with 25% weight gain Surelease®.
  • Example 18 Minicapsules prepared as per Example 13 are coated with 30% weight gain Surelease®.
  • Example 19
  • Minicapsules containing derivatives of siRNA for PHDl, PHD2 and PHD3 are prepared according to Example 13 and are coated according to Examples 14 to 19.
  • Example 20
  • Mammalian HeLa cells are transfected with siRNA duplexes of the ribonucleic acid of Examples 2, 3 and 4 in cationic liposomes following the technique of Elbashir et al (2001).
  • Transfection is conducted according to Example 20 but with the addition of MONTANIDETM ISA 50 V which is an oily adjuvant composition of mannide oleate and mineral oil.
  • Mammalian HeLa cells are transfected with siRNA duplexes of the ribonucleic acid of Examples 13 using uncoated minicapsules comprising siRNA for PHDl, PHD2 and PHD3 following the technique of Elbashir et al (2001).
  • Examples 23-28 are transfected with siRNA duplexes of the ribonucleic acid of Examples 13 using uncoated minicapsules comprising siRNA for PHDl, PHD2 and PHD3 following the technique of Elbashir et al (2001). Examples 23-28
  • lipids in chloroform eg DOTAP, DOPE, cholesterol etc
  • chloroform eg DOTAP, DOPE, cholesterol etc
  • Evaporation of the solvent (chloroform) was achieved by standard methods known in the art. However, gently applying a flow of nitrogen helped in the formation of a thin film of lipid on the walls of the flask.
  • Rehydration of the lipidic film was obtained by introducing buffer (PBS at pH7) previously warmed to 37°C, after which sample was vortexed for 15 minutes for liposome formation.
  • PBS buffer
  • Liposome stock solutions were kept in the freezer till further use. The stock solution concentration was 0.2 mg/ml.
  • siRNA formulations were prepared as follows (siRNA supplier was Dharmacon). Using a siRNA stock solution kept in PBS buffer, pH 7.0, at -80 0 C till use, the appropriate amount of sip65 (siRNA for p65 of NFkB) was added to the liposome stock solution (above) to meet the required positive to negative charge molar ratio defined per formulation described below. After an incubation period of 5 minutes, the samples were ready for use.
  • the chitosan was from NovaMatrix, named PROTASAN UP CL 113 which is based on a chitosan where between 75-90 percent of the acetyl groups are deacetylated.
  • the cationic polymer is a highly purified and well- characterized water-soluble chloride salt.
  • the molecular weight for PROTASAN UP CL 113 is in the 50000-150000 g/mol range. It has ultra low levels of endotoxins and proteins allowing for a wide variety of in vitro and in vivo applications.
  • the appropriate amount of siRNA was added to the chitosan solution, to meet the required positive to negative charge molar ratio defined per formulation. After an incubation period of 5 minutes the remaining amount of liposome solution was added to complete the sample volume. After another incubation period of 5 minutes the sample was ready for use.
  • siRNA with chitosan at a molar ratio of chitosan(+)/siRNA(-) of 50: 1 was made by simple mixing of l ⁇ L of chitosan with 13 ⁇ L of siRNA.
  • the chitosan-siRNA complex was then added to the liposomes used in Example 23 (containing DOTAP:DOPE:Cholesterol at the molar ratios of 6:2:2) to yield a formulation having the following components by weight %:
  • siRNA with chitosan at a molar ratio of chitosan(+)/siRNA(-) of 50: 1 was made by simple mixing of l ⁇ L of chitosan with 13 ⁇ L of siRNA to the liposomes used in Example 24 (containing DOTAP:DOPC:cholesterol at the molar ratios of 6:2:2) to yield a formulation having the following components by weight %:
  • siRNA with chitosan at a molar ratio of chitosan(+)/siRNA(-) of 50: 1 was made by simple mixing of l ⁇ L of chitosan with 13 ⁇ L siRNA to the liposomes used in Example 25 (containing DOTAP: cholesterol at the molar ratios of 6:4) to yield a formulation having the following components by weight %:
  • Figure 1 shows scanned western blots of the samples prepared.
  • the bands represent the proteins p65 and beta-actin.
  • the intensity of one band is proportional to the amount of the protein in the sample. Knock down of the target protein (p65) therefore leads to a lighter band (lower intensity) than in the control.
  • the control panel 1 of Figure 1 shows samples resulting from transfection with siRNA mixed with lipofectamine.
  • the potential gene knock-down effect of two siRNAs was tested namely a "non-target" (NT) siRNA or siNT which is a siRNA for which there is no target protein in the cells.
  • the second siRNA is for the p65 portion of NFkB referred to as sip65.
  • Beta-actin is a protein known not be affected by siRNA. Its inclusion in the experiment is to confirm the system is working and that proteins are detectable. It also acts as a negative control for which therefore no change in intensity between samples is expected. Also, use of this negative control demonstrates that the different intensities of p65 in Figure 1 are not due to loading of different amounts of protein.
  • the p65 protein is obviously the protein whose expression it is desired to inhibit by gene knock-down using siRNA.
  • Two samples of sip65 were used in the control (Panel A of Figure 1) namely a laboratory stock (kept in optimal conditions) and an ex-laboratory stock which had intentionally be exposed to movement and ambient temperature to test stability.
  • Panel A of Figure 1 shows that siRNA from lab stock as well as siRNA from ex-lab stock were able to knock down the protein (lighter bands).
  • Panel B of Figure 1 shows the results for the formulations of this Example. Two different concentrations of the formulated sip65 namely 2 and 20 nM were tested. Controls used the same formulations without siRNA (with PBS instead) in the same volumes as were used of the formulated siRNA (20 or 200 ul, respectively).
  • Panel B of Figure 1 shows that Ll and PLl show a knock down of the p65 protein especially with 2 nM. Examples 31-36
  • mini-beads were produced from a oil-in-water emulsion which can be referred to also as a water-in-oil-in-water (w/o/w) emulsion in which the oil phase was prepared by dispersing the siRNA- liposome formulations of Examples 23-25 and the siRNA- liposome-protein complex of Examples 26-28 in an oil phase to create a w/o emulsion.
  • This oil phase (in fact a w/o phase) and the outer aqueous phase were then mixed in a proportion in the range 1 :6-10, preferably approximately 1 :7 or 1 :8 with gentle continuous stirring of the components using a Magnetic Stirrer (manufactured by Stuart).
  • the outer aqueous phase (gelatin with sorbitol) was prepared by adding the appropriate quantities of sorbitol (optionally with surfactant eg SDS) to water, heating to approximately 60-75° C until in solution and then adding gelatin.
  • the "gelatin solution” comprised 15-25% (preferably 17- 18%) of gelatin; 75%-85% (preferably 77-82%) of water plus from 1-5% (preferably 1.5 to 3%) sorbitol.
  • the gelatin solution was maintained at 60°C-70°C to maintain it in a fluid state.
  • SDS was added to the aqueous phase at the same time the other components are added ie. gelatin and sorbitol at the beginning of the processing session.
  • SDS surfactant
  • the oil phase was made at room temperature with stirring until clear.
  • the w/o/w emulsion was formed by addition of the oil phase (or w/o phase) to the heated aqueous phase with stirring as described above.
  • the resultant emulsion then had the composition of the future solidified mini-beads but with water still present.
  • the beading step was begun without delay by using a pipette and dropping the fluid emulsion manually into MCT (cooling fluid) maintained in the range 8-12°C which effected solidification.
  • the beads of these Examples were produced initially as for Examples 31-36 but instead of pipetting the emulsion, the mini-beads were produced through ejection of the fluid w/o/w emulsion through a vibrating 3mm diameter single lumen nozzle applied to the Freund Spherex machine. Operation of the Spherex machine manufactured by Freund was in accordance with the manufacturer's instructions. The lines to the orifice/nozzle were maintained at 65-85°C to maintain the fluidity of the solution. The resulting beads had the following composition:
  • a method for delivering an siRNA or engineered RNA precursor to a target cell by bringing a multiplicity of RNA-containing minicapsules into contact with the target cell.
  • RNA is adapted to knockdown, silence or inhibit the expression of one or more PHDs, including PHD 1 , 2 and 3.
  • a method for delivering an siRNA to a cell by obtaining, identifying or targeting a cell (or system of cells or tissue), forming a minisphere comprising an siRNA and contacting the cell
  • An oral composition comprising minicapsules wherein the minicapsules comprise one or more siRNAs or engineered RNA precursors in a core susceptible of maintaining such RNA in a stable, active form.
  • the core is liquid, semi-solid, or solid.
  • composition of clause 12 or 13 wherein the minicapsules have release profiles to release the siRNA or engineered RNA precursor in an active form at one or more sites along the gastrointestinal tract.
  • composition of clauses 12 to 15 wherein the minicapsule has one layer and is solid throughout. 17. The composition of clauses 12-15 wherein the minicapsule has two layers comprising a solid outer shell layer encapsulating a liquid, semi-solid or solid core.
  • a pharmaceutical composition comprising mini-beads of solidified matrix material wherein the mini-beads comprise one or more siRNAs or engineered RNA precursors dispersed in said solidified matrix.
  • composition of clause 21 wherein the protein is cationic and is preferably chitosan.
  • composition of clauses 19-24 wherein the composition is adapted for oral administration.

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Abstract

L'invention porte sur un procédé d'administration d'un ARNsi ou d'un précurseur d'ARN synthétisé par génie génétique vers une cellule cible en amenant une pluralité de mini-capsules contenant de l'ARN en contact avec la cellule cible. L'invention porte également sur un produit choisi parmi une mini-capsule, une mini-bille ou une mini-sphère et comprenant une phase solide et un agent actif choisi parmi des ARNsi et des précurseurs d'ARN synthétisés par génie génétique.
PCT/EP2009/063368 2008-10-13 2009-10-13 Système d'administration d'arn WO2010043630A2 (fr)

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WO2014057432A3 (fr) * 2012-10-09 2014-07-24 Universita' Degli Studi Di Roma "La Sapienza" Nanoparticules lipidiques à multicomposant et leurs procédés de préparation
WO2014134509A3 (fr) * 2013-02-28 2015-03-12 University Of Massachusetts Particules de glucane modifiées par un peptide et une amine pour la délivrance de charges thérapeutiques
WO2015067763A1 (fr) * 2013-11-08 2015-05-14 Sigmoid Pharma Limited Formulations
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EP3474826A4 (fr) * 2016-06-27 2020-03-04 Tamarisk Technologies Group, LLC Préparation pharmaceutique pour l'administration de peptides et de protéines
US10993987B2 (en) 2014-11-07 2021-05-04 Sublimity Therapeutics Limited Compositions comprising Cyclosporin
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WO2004042024A2 (fr) * 2002-11-01 2004-05-21 The Trustees Of The University Of Pennsylvania Compositions et procedes destines a l'inhibition par arnsi des hif-1 alpha
WO2005072088A2 (fr) * 2003-12-11 2005-08-11 Sciperio, Inc. Compositions d'immunotherapie, methodes de preparation et d'utilisation de ces dernieres
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Cited By (15)

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WO2010133609A3 (fr) * 2009-05-18 2011-12-01 Sigmoid Pharma Limited Composition comprenant des gouttes d'huile
GB2483815A (en) * 2009-05-18 2012-03-21 Sigmoid Pharma Ltd Composition comprising oil drops
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WO2014057432A3 (fr) * 2012-10-09 2014-07-24 Universita' Degli Studi Di Roma "La Sapienza" Nanoparticules lipidiques à multicomposant et leurs procédés de préparation
WO2014134509A3 (fr) * 2013-02-28 2015-03-12 University Of Massachusetts Particules de glucane modifiées par un peptide et une amine pour la délivrance de charges thérapeutiques
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JP2016535055A (ja) * 2013-11-08 2016-11-10 シグモイド・ファーマ・リミテッドＳｉｇｍｏｉｄ Ｐｈａｒｍａ Ｌｉｍｉｔｅｄ 製剤
WO2015067763A1 (fr) * 2013-11-08 2015-05-14 Sigmoid Pharma Limited Formulations
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US10993987B2 (en) 2014-11-07 2021-05-04 Sublimity Therapeutics Limited Compositions comprising Cyclosporin
EP3474826A4 (fr) * 2016-06-27 2020-03-04 Tamarisk Technologies Group, LLC Préparation pharmaceutique pour l'administration de peptides et de protéines
US10034951B1 (en) 2017-06-21 2018-07-31 New England Biolabs, Inc. Use of thermostable RNA polymerases to produce RNAs having reduced immunogenicity
US11376338B2 (en) 2017-06-21 2022-07-05 New England Biolabs, Inc. Use of thermostable RNA polymerases to produce RNAs having reduced immunogenicity
WO2022064274A1 (fr) * 2020-09-28 2022-03-31 Immunovaccine Technologies Inc. Compositions lipidiques comprenant des antigènes polynucléotidiques

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