WO2006130949A1 - Molecules contre des maladies liees a l'amyloide et leur utilisation - Google Patents

Molecules contre des maladies liees a l'amyloide et leur utilisation Download PDF

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Publication number
WO2006130949A1
WO2006130949A1 PCT/CA2006/000702 CA2006000702W WO2006130949A1 WO 2006130949 A1 WO2006130949 A1 WO 2006130949A1 CA 2006000702 W CA2006000702 W CA 2006000702W WO 2006130949 A1 WO2006130949 A1 WO 2006130949A1
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oligonucleotide
formulation
nucleotides
amyloid
sequence
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PCT/CA2006/000702
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Andrew Vaillant
Jean-Marc Juteau
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Replicor Inc.
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Publication of WO2006130949A1 publication Critical patent/WO2006130949A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0312—Animal model for Alzheimer's disease

Definitions

  • the present invention relates to oligonucleotides having an anti amyloid-related disease activity that act by a sequence independent mechanism.
  • the anti amyloid-related disease activity is greater for large oligonucleotides and for oligonucleotides with sulfur modification.
  • amyloid-related disease or amyloidosis refers to a pathological condition characterized by the presence of amyloid fibrils.
  • Amyloid is a generic term referring to a group of diverse protein deposits (intracellular or extracellular) which are seen in a number of different diseases. Amyloid-related diseases can either be restricted to one organ or spread to several organs. The first instance is referred to as “localized amyloidosis” while the second is referred to as “systemic amyloidosis.”
  • Some amyloid diseases can be idiopathic, but most of these diseases appear as a complication of a previously existing disorder.
  • primary amyloidosis can appear without any other pathology or can follow plasma cell dyscrasia or multiple myeloma.
  • Secondary amyloidosis is usually seen associated with chronic infection (such as tuberculosis) or chronic inflammation (such as rheumatoid arthritis).
  • a familial form of secondary amyloidosis is also seen in other types of familial amyloidosis, e.g., Familial Mediterranean Fever (FMF). This familial type of amyloidosis is genetically inherited and is found in specific population groups. In both primary and secondary amyloidosis, deposits are found in several organs and are thus considered systemic amyloid diseases.
  • FMF Familial Mediterranean Fever
  • “Localized amyloidoses” are those that tend to involve a single organ system. Different amyloids are also characterized by the type of protein present in the deposit. For example, neurodegenerative diseases such as scrapie, bovine spongiform encephalitis, Creutzfeldt- Jakob disease, and the like are characterized by the appearance and accumulation of a protease-resistant form of a prion protein (referred to as AScr or PrP-27) in the central nervous system. Similarly, Alzheimer's disease, another neurodegenerative disorder, is characterized by neuritic plaques and neurofibrillary tangles.
  • amyloid plaques found in the parenchyma and the blood vessel is formed by the deposition of fibrillar A beta amyloid protein.
  • Other diseases such as adult- onset diabetes (type II diabetes) are characterized by the localized accumulation of amyloid fibrils in the pancreas.
  • Another type of amyloidosis is related to beta 2 microglobulin and is found in long-term hemodialysis patients.
  • Reactive or secondary amyloidosis is a manifestation of a number of diseases that provoke a sustained acute phase response. Such diseases include chronic inflammatory disorders, chronic local or systemic microbial infections, and malignant neoplasms. The most common form of reactive or secondary amyloidosis is seen as the result of long-standing inflammatory conditions. For example, patients with Rheumatoid Arthritis or Familial Mediterranean Fever (which is a genetic disease) can develop AA amyloidosis.
  • the terms "AA amyloidosis” and "secondary AA amyloidosis” are used interchangeably.
  • Deposition of AA fibrils can be widespread in the body, with a preference for parenchymal organs. The kidneys are usually a deposition site, and the liver and the spleen may also be affected. Deposition is also seen in the heart, gastrointestinal tract, and the skin.
  • oligonucleotides mainly antisense ONs
  • ODNs antisense oligodeoxynucleotides
  • APP amyloid precursor protein
  • Kumar et al. tested antisense phosphorothioate-oligodeoxynucleotide (PS- ODN) in a mouse model of Alzheimer's and showed that antisense directed at the Abeta region of APP can reduce APP levels in the brain and reverse deficits in learning and memory (Kumar et al. (2000) Peptides 21 :1769-1775).
  • PS- ODN antisense phosphorothioate-oligodeoxynucleotide
  • One aim of the present invention is to provide a sequence independent ONs that could be used to treat amyloid-related diseases.
  • an anti amyloid-related disease oligonucleotide formulation comprising at least one anti amyloid-related disease oligonucleotide and an excipient.
  • the oligonucleotide has an activity against amyloid- related disease and the activity occurs principally by a sequence independent mode of action.
  • Another object of the present invention is to provide an oligonucleotide formulation, wherein said oligonucleotide is at least 15, 20, 25, 30, 35, 40, 45, 50, 60 or 80 nucleotides in length.
  • the oligonucleotide formulation of contains an oligonucleotide which is 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nucleotides in length.
  • the oligonucleotide formulation has an oligonucleotide which is not complementary to any equal length portion of a genomic sequence.
  • the genomic sequence is a genomic sequence of a human or non human animal.
  • the oligonucleotide used in the formulation comprises at least 10 contiguous nucleotides of randomer sequence, more preferably 20 nucleotides of randomer sequence, 30 nucleotides of randomer sequence, 40 nucleotides of randomer sequence or more preferably is a randomer oligonucleotide.
  • the oligonucleotide comprises a homopolymer sequence of at least 10 contiguous A nucleotides, or 10 contiguous T nucleotides, or 10 contiguous U nucleotides, or 10 contiguous G nucleotides, or 10 contiguous I nucleotide analogs or 10 contiguous C nucleotides.
  • the oligonucleotide comprised in the oligonucleotide formulation is a homopolymer of C nucleotides.
  • the oligonucleotide formulation comprises an oligonucleotide having a poly AT sequence at least 10 nucleotides in length; a poly AC sequence at least 10 nucleotides in length; a polyAG sequence at least 10 nucleotides in length; a poly AU sequence at least 10 nucleotides in length; a polyAI sequence at least 10 nucleotides in length; a polyGC sequence at least 10 nucleotides in length; a polyGT sequence at least 10 nucleotides in length; a polyGU sequence at least 10 nucleotides in length; a polyGI sequence at least 10 nucleotides in length; a polyCT sequence at least 10 nucleotides in length; a polyCU sequence at least 10 nucleotides in length; a polyCI sequence at least 10 nucleotides in length; a polyTI sequence at least 10 nucleotides in length; a polyTU sequence at least 10 nucleotides
  • the oligonucleotide formulation comprises an oligonucleotide having at least one ribonucleotide.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one modification to its chemical structure, more preferably at least two different modifications to its chemical structure.
  • an oligonucleotide formulation comprising an oligonucleotide having at least one sulfur modification
  • the oligonucleotide formulation comprises an oligonucleotide having at least one phosphorothioated linkage; at least one phosphorodithioated linkage; or at least one boranophosphate linkage.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one sulfur modified nucleobase moiety such as one sulfur modified ribose moiety, one 2' modification to the ribose moiety, one 2'-0 alkyl modified ribose moiety, one 2'-0 methyl modified ribose, one 2'-methoxyethyl modified ribose, or one 2'-FANA modified ribose.
  • one sulfur modified nucleobase moiety such as one sulfur modified ribose moiety, one 2' modification to the ribose moiety, one 2'-0 alkyl modified ribose moiety, one 2'-0 methyl modified ribose, one 2'-methoxyethyl modified ribose, or one 2'-FANA modified ribose.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one methylphosphonate linkage.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphorothioate backbone.
  • GAA glycol nucleic acid
  • the oligonucleotide formulation comprises an oligonucleotide having at least one locked nucleic acid moiety.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one phosphorodiamidate morpholino moiety. [0029] In another embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one abasic nucleic acid.
  • the oligonucleotide formulation comprises an oligonucleotide having a linker to form a concatemer of two or more oligonucleotide sequences.
  • the oligonucleotide formulation of the present invention comprises an oligonucleotide linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, an improved ability to be formulated, a detectable signal, higher anti amyloid-related disease activity, better pharmacokinetic properties, specific tissue distribution and lower toxicity.
  • the oligonucleotide formulation comprises an oligonucleotide linked or conjugated to a PEG molecule; or linked or conjugated to a cholesterol molecule.
  • the oligonucleotide formulation comprises a double stranded oligonucleotide.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one base which is capable of hybridizing via non- Watson- Crick interactions.
  • the oligonucleotide formulation comprises an oligonucleotide having a portion complementary to a genome.
  • the oligonucleotide of the present invention has at most 90%, preferably 80%, more preferably 75% identity with the genomic sequence.
  • the oligonucleotide has at most 60%, preferably 50%, more preferably 40% identity with a genomic sequence.
  • the oligonucleotide formulation comprises an oligonucleotide that binds to one or more amyloid protein.
  • the oligonucleotide formulation comprises an oligonucleotide that interacts with one or more cellular components, wherein said interaction resulting in inhibition of a protein activity or expression.
  • the oligonucleotide formulation comprises an oligonucleotide wherein at least a portion of the sequence of said oligonucleotide is derived from a genome.
  • the oligonucleotide formulation comprises an oligonucleotide having an activity against amyloid-related disease, such as Alzeimer's disease.
  • the targeted Alzheimer's disease is sporadic (non-hereditary) Alzheimer's disease, familial (hereditary) Alzheimer's disease, or early Alzheimer's disease.
  • the targeted amyloid-related disease is Mild Cognitive Impairment or Mild-to-Moderate Cognitive Impairment.
  • the targeted amyloid-related disease is Beta 2-microglobulin-related (dialysis-related) amyloidosis.
  • the targeted amyloid-related disease is Type II diabetes related amyloidosis. More preferably, the amyloid-related disease is cerebral amyloid angiopathy or hereditary cerebral hemorrhage. More preferably, the amyloid-related disease is senile dementia, Down's syndrome, inclusion body myositis, or age-related macular degeneration.
  • an oligonucleotide mixture comprising a mixture of at least two different anti amyloid-related disease oligonucleotide formulation according to the present invention.
  • the oligonucleotide mixture comprises a mixture oligonucleotide having of at least ten different anti amyloid-related disease oligonucleotide formulation according to the present invention. More preferably, the oligonucleotide mixture of the present invention comprises a mixture of at least 100 different anti amyloid-related disease oligonucleotide formulation; or at least 1000 different anti amyloid-related disease oligonucleotide formulation; or at least 10 6 different anti amyloid-related disease oligonucleotide formulation according to the present invention.
  • the oligonucleotide mixture comprises oligonucleotides which are at least 10 nucleotides in length; at least 20 nucleotides in length; at least 30 nucleotides in length; at least 40 nucleotides in length; at least 50 nucleotides in length; at least 60 nucleotides in length; at least 70 nucleotides in length; are at least 80 nucleotides in length; or at least 120 nucleotides in length.
  • an anti amyloid-related disease pharmaceutical composition comprising a therapeutically effective amount of at least one pharmacologically acceptable, anti amyloid-related disease oligonucleotide formulation according to the present invention, and a pharmaceutically acceptable carrier.
  • the anti amyloid-related disease pharmaceutical composition of the present invention is adapted for delivery by a mode selected from the group consisting of ocular administration, eye drop administration, oral ingestion, inhalation, subcutaneous injection, intramuscular injection, intrathecal, intracerebral, and intravenous injection.
  • the anti amyloid-related disease pharmaceutical composition further comprises a delivery system.
  • the anti amyloid-related disease pharmaceutical composition further comprises at least one other anti amyloid-related disease drug in combination.
  • the anti amyloid-related disease pharmaceutical composition further comprises a non-nucleotidic anti amyloid-related disease drug in combination.
  • the anti amyloid-related disease pharmaceutical composition further comprises an anti amyloid-related disease antisense oligonucleotide in combination.
  • the anti amyloid-related disease pharmaceutical composition further comprises an anti amyloid-related disease RNAi-inducing oligonucleotide.
  • the present invention involves the identification and the use of anti amyloid- related disease ONs that act by a sequence independent mechanism, and includes the discovery that the anti amyloid-related disease activity is greater for larger ONs and for ONs with sulfur modification.
  • the invention relates to ONs acting predominantly by a sequence independent mode of action for the treatment of amyloid-related diseases.
  • the invention also relates to ONs and their use as therapeutic agents, and more particularly for their use and formulations for the treatment of amyloid-related diseases.
  • oligonucleotide in the context of the present invention refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) and/or analogs thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotide in the context of the present invention refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) and/or analogs thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions which function similarly.
  • Oligonucleotides that include backbone and/or others modifications can also be referred to as oligonucleosides. Except otherwise specified, oligonucleotide definition includes homopolymers, heteropolymers, randomers, random sequence oligonucleotides, genomic-derived sequence oligonucleotides and oligonucleotides purified from natural sources.
  • sequence independent activity indicates that the mechanism by which the material exhibits an anti amyloid- related disease effect is not due to hybridization of complementary nucleic acid sequences, e.g., an antisense effect nor it is due to a sequence-specific aptameric activity.
  • a “sequence dependant mode of action or activity” means that the anti amyloid-related disease effect of a material involves hybridization of complementary nucleic acid sequences or involves a sequence-specific aptameric interaction.
  • anti amyloid-related disease means inhibiting, reverting, curing, or preventing an amyloid-related disease.
  • the expression anti amyloid- related disease also means inhibiting, reverting or preventing the formation of amyloid plaque or deposit.
  • An anti amyloid-related disease compound can be used to treat a disease whose etiology is based on amyloid plaque formation.
  • amyloid-related disease when referring to the term disease, means a disease involving unwanted formation of amyloid plaque or amyloid deposits or amyloid fibril formation. Amyloid-related disease also means amyloidosis; both terms can be use interchangeably.
  • amyloid includes beta-amyloid, amylin and any other form of amyloid.
  • amyloid also refers to amyloidogenic proteins, peptides, or fragments thereof which can be soluble (e.g., monomeric or oligomeric) or insoluble (e.g., having fibrillary structure or in amyloid plaque).
  • anti amyloid-related disease refers to an effect due to the presence of ONs or other material inhibiting, stopping, reverting, curing or preventing an amyloid-related disease compared to untreated cells, systems or organisms.
  • anti amyloid-related disease ONs will have anti amyloid-related disease activity against multiple diseases.
  • anti amyloid-related disease oligonucleotide formulation refers to a preparation that includes at least one anti amyloid-related disease oligonucleotide that is adapted for use as an anti amyloid-related disease agent.
  • the formulation includes the ON or ONs, and can contain other materials that do not interfere with their use as an anti amyloid-related disease agents in vivo. Such other materials can include without restriction diluents, excipients, carrier materials, delivery systems and/or other anti amyloid-related disease materials.
  • amyloid-related disease targeted by the ONs of the present invention, formulation, pharmaceutical composition can be used to target for example Alzheimer's disease (e.g., sporadic, familial, or early AD), Down's syndrome, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, mild cognitive impairment, sporadic inclusion body myositis, age-related macular degeneration (drusen associated, islet amyloid polypeptide (IAPP)-related amyloidosis), Type II diabetes related amyloidosis, primary (AL) amyloidosis , secondary (AA) amyloidosis, Beta 2-microglobulin-related (dialysis-related) amyloidosis.
  • Alzheimer's disease e.g., sporadic, familial, or early AD
  • Down's syndrome cerebral amyloid angiopathy
  • hereditary cerebral hemorrhage hereditary cerebral hemorrhage
  • mild cognitive impairment sporadic inclusion body myositis
  • amyloid-related disease targeting ONs, formulation, pharmaceutical composition or method of treatment can also target secondary amyloidosis caused, without limitation, by rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, Adult Still's disease, Behcet's syndrome, Crohn's disease, chronic microbial infections (such as leprosy, tuberculosis), bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis, Whipple's disease, Castleman's disease, Schnitzler's syndrome, malignant neoplasms (including Hodgkin's lymphoma, renal carcinoma, carcinomas of gut, lung and urogenital tract, basal cell carcinoma, and hairy cell leukemia) and hereditary systemic amyloidoses.
  • rheumatoid arthritis juvenile chronic arthritis, an
  • diseases or conditions related to amyloid targeted by ON of this invention are nodular AL amyloid, cutaneous amyloid, endocrine amyloid, tumor-related amyloid, familial amyloid polyneuropathy (FAP), senile systemic amyloidosis, tenosynovium, familial amyloidosis, Ostertag-type, non-neuropathic amyloidosis, cranial neuropathy, hereditary cerebral hemorrhage, familial dementia, chronic dialysis; Gerstmann-Strussler-Scheinker syndrome, familial Mediterranean fever, Muckle-Well's syndrome, nephropathy, deafness, urticaria, limb pain, cardiomyopathy, cutaneous deposits, multiple myeloma, benign monoclonal gammopathy, maccoglobulinaemia, myeloma associated amyloidosis, medullary carcinomas of the thyroid, isolated atrial amyloid, and diabetes.
  • FAP familial amyloid
  • the present invention involves the discovery that ONs, e.g., oligodeoxynucleotides (ODNs), including modified oligonucleotides, can have a therapeutic application through a sequence independent mode of action. It is not necessary for the oligonucleotide to be complementary to any sequence or to have a particular distribution of nucleotides in order to have activity. Such an oligonucleotide can even be prepared as a randomer, such that there will be at most a few copies of any particular sequence in a preparation, e.g., in a 15 micromol randomer preparation of an ON having 32 or more nucleotides in length.
  • ONs e.g., oligodeoxynucleotides (ODNs), including modified oligonucleotides
  • oligonucleotide has different activity.
  • present results indicate that the length of oligonucleotide that produces maximal effect when modified with sulfur linkages is typically in the range of 30-120 nucleotides but not restricted to these length.
  • another embodiment concerns the properties of oligonucleotides.
  • the present invention provides oligonucleotide agents that can have activity against diseases and conditions described herein. Such agents are particularly advantageous in view of the limited therapeutic options currently available.
  • the ONs, e.g., ODNs, of the present invention are useful in therapy for treating or preventing diseases and conditions described herein.
  • Such treatments are applicable to many types of patients and treatments, including, for example, the prophylaxis or treatment of diseases and conditions described herein
  • the activity of the oligonucleotides occurs principally by a sequence independent (e.g., non- sequence complementary or non-sequence dependant aptameric activity) mode of action, and formulations containing such oligonucleotides.
  • sequence independent e.g., non- sequence complementary or non-sequence dependant aptameric activity
  • Oligonucleotides useful in the present invention can be of various lengths, e.g., at least 6, 10, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or more nucleotides in length.
  • the length of the oligonucleotide can be within a range, e.g., a range defined by taking any two of the preceding listed values as inclusive end points of the range, for example 10-20, 20-30, 20-40, 30-40, 30-50, 40-50, 40-60, 40-80, 50-60, 50-70, 60-70, 70-80, 60-120, and 80-120 nucleotides.
  • the nucleotide can include various modifications, e.g., stabilizing modifications, and thus can include at least one modification in the phosphodiester linkage and/or on the sugar, and/or on the base.
  • the oligonucleotide can include one or more phosphorothioate linkages, phosphorodithioate linkages, and/or methylphosphonate linkages.
  • Different chemically compatible modified linkages can be combined, e.g., modifications where the synthesis conditions are chemically compatible.
  • the oligonucleotides can include phosphodiester linkages, e.g., include at least one phosphodiester linkage, or at least 5, 10, 20, 30% or more phosphodiester linkages. Additional useful modifications include, without restriction, modifications at the T- position of the sugar, such as 2'-O-alkyl modifications such as 2'-O-methyl modifications, 2'-amino modifications, 2'-halo modifications such as 2'-fluoro; acyclic nucleotide analogs.
  • the oligonucleotide has modified linkages throughout, e.g., phosphorothioate; has a 3'- and/or 5'-cap; includes a terminal 3 '-5' linkage; the oligonucleotide is or includes a concatemer consisting of two or more oligonucleotide sequences joined by a linker(s).
  • Such modified ONs include, for example, ONs containing modified backbones or non-natural internucleoside linkages.
  • ONs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Such modified ON backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, carboranyl phosphate and borano-phosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5 ' to 5' or 2' to 2' linkage.
  • Oligonucleotides having inverted polarity typically include a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts, mixed salts and free acid forms are also included.
  • Some exemplary modified ON backbones that do not include a phosphodiester linkage have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH 2 component parts.
  • Modified ONs may also contain one or more substituted sugar moieties.
  • such oligonucleotides can include one of the following 2 '-modifications: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S— or N-alkynyl; or O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C 2 Io Cio alkenyl and alkynyl, or 2'-O-(O-carboran-l-yl)methyl.
  • Particular examples are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) ⁇ OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON [(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to 10.
  • exemplary ONs include one of the following 2 '-modifications: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 .
  • OCF 3 SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an ON, or a group for improving the pharmacodynamic properties of an ON.
  • Examples include 2'-methoxyethoxy (2'-0 — CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al, HeIv. Chim.
  • LNAs Locked Nucleic Acids
  • the linkage can be a methelyne ( — CH 2 — ) ⁇ group bridging the T oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in international patent application publication Nos WO 98/39352 and WO 99/14226.
  • modifications include sulfur-nitrogen bridge modifications, such as locked nucleic acid as described in Orum et al. ((2001) Curr. Opin. MoI. Ther. 3:239-
  • the 2 '-modification may be in the arabino (up) position or ribo (down) position. Similar modifications may also be made at other positions on the ON, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. ONs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Exemplary U.S. patents describing the preparation of such modified sugar structures include, for example, U.S. patents Nos.
  • Still other modifications include an ON concatemer consisting of multiple ON sequences joined by a linker(s).
  • the linker may, for example, consist of modified nucleotides or non-nucleotide units.
  • the linker provides flexibility to the ON concatemer. Use of such ON concatemers can provide a facile method to synthesize a final molecule, by joining smaller ON building blocks to obtain the desired length.
  • a 12 carbon linker (C12 phosphoramidite) can be used to join two or more ON concatemers and provide length, stability, and flexibility.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). ONs may also include base modifications or substitutions.
  • Modified bases include other synthetic and naturally-occurring bases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl( — C ⁇ C — CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guan
  • Additional modified bases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4- b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4- b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those described in U.S. patent No.
  • Another modification includes phosphorodithioate linkages. Knowing that phosphorodithioate ONs (PS2-ONs) and PS-ONs have a similar binding affinity to proteins (Tonkinson et al. (1994) Antisense Res. Dev. 4:269-278) (Cheng et al. (1997) J. MoI. Recogn. 10:101-107) and knowing that a possible mechanism of action of ONs is binding to protein involved in amyloid-related disease, it could be desirable to include phosphorodithioate linkages on the anti amyloid-related disease ONs described in this invention.
  • ONs prepared by conventional methods consist of a mixture of diastereomers by virtue of the asymmetry around the phosphorus atom involved in the internucleotide linkage. This may affect the stability of the binding between ONs and targets such as proteins involved in amyloid-related disease. Previous data showed that protein binding is significantly stereo-dependent (Yu et al.). Thus, using stereodefmed or stereo-enriched ONs could improve their protein binding properties and improve their anti amyloid-related disease efficacy.
  • modified oligonucleotides containing phosphorothioate or dithioate linkages may also contain one or more substituted sugar moieties particularly modifications at the sugar moieties including, without restriction, 2'-ethyl, 2'-ethoxy, 2'-methoxy, 2'-aminopropoxy, 2'-allyl, 2'-fluoro, 2'-pentyl, 2'-propyl, 2'-dimethylaminooxyethoxy, and T- dimethylaminoethoxyethoxy.
  • the 2'-modification may be in the arabino (up) position or ribo (down) position.
  • a preferred 2'-arabino modification is 2'-fluoro. Similar modifications may also be made at other positions on the ON, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. ONs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Moreover ONs may have a structure of or comprise a portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone (Zhang L, et al. (2005) J. Am. Chem. Soc.
  • GAA glycol nucleic acid
  • Zhang L et al. (2005) J. Am. Chem. Soc.
  • Such GNA may comprise phosphorothioate linkages and may comprise only pyrimidine bases.
  • oligonucleotides having each linkage phosphorothioated and each ribonucleotide modified at the 2'-position of the ribose may have anti amyloid-related disease activity but do not trigger RNase H activity , a property desirable for traditional antisense ONs but completely dispensable for the activity described in this present invention.
  • results demonstrate that modifications at the 2'-position of each ribose of PS-ONs renders the ON more resistant to nucleases in comparison with a PS-ON comprising the same modifications but only at both ends (gapmer). Gapmers are preferentially used in the antisense technology. Nuclease resistance of PS-ONs including modifications at the T- position of each ribose could display beneficial properties, such as improved pharmakokinetics and/or oral availability.
  • PS-ONs that include modifications at the 2'-position of each ribose show the desirable properties
  • PS-ONs with substantial numbers of modifications at the 2'-position of ribose could also display the desirable properties, e.g., modification at least 50 % of the riboses and more preferably 80% or even more.
  • the present invention further provides an oligonucleotide, wherein said oligonucleotide is linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, higher protein interaction, an improved ability to be formulated for delivery, a detectable signal, higher activity, better pharmacokinetic properties, specific tissue distribution, lower toxicity.
  • the oligonucleotide includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% modified linkages, e.g., phosphorothioate, phosphorodithioate, and/or methylphosphonate.
  • At least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or all of the nucleotides are modified at the 2'-position of the ribose, e.g., 2'-OMe, T- fluoro, 2'-amino.
  • modified linkages are combined with 2 '-modifications in oligonucleotides, for example, at least 30% modified linkages and at least 30% T- modifications; or respectively at least 40% and 40%, at least 50% and 50%, at least 60% and 60%, at least 70% and 70%, at least 80% and 80%, at least 90% and 90%, or further 100% and 100%.
  • the oligonucleotide includes at least 30, 40, 50, 60, 70, 80, 90, or 100% modified linkages and at least 30, 40, 50, 60, 70, 80, 90, or 100% 2 '-modifications.
  • each combination of listed modified linkage percentage and 2 '-modification percentage e.g., at least 50% modified linkage and at least 80% 2 '-modifications, and at least 80% modified linkages and 100% 2 '-modifications.
  • the modified linkages are phosphorothioate linkages; the modified linkages are phosphorodithioate linkages; the T- modifications are 2'-OMe; the 2 '-modifications are 2'-fluoro; the 2 '-modifications are a combination of 2'-OMe and 2'-fluoro; the modified linkages are phosphorothioate linkages and the 2 '-modifications are 2'-OMe; the modified linkages are phosphorothioate linkages and the 2 '-modifications are 2'-fluoro; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'-OMe; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'-OMe; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'-fluoro; the modified linkages are phosphorodithioate linkages and the 2 '
  • the oligonucleotides described herein combine a particular percentage of modified linkages and a particular percentage of 2'-modifications, the oligonucleotide is at least 6, 10, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or more nucleotides in length, or is in a length range defined by taking any two of the specified lengths as inclusive endpoints of the range.
  • all but 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the internucleotidic linkages and/or 2'-positions of the ribose moiety are modified, e.g., with linkages modified with phosphorothioate, phosphorodithioate, or methylphosphonate linkages and/or 2'-0Me, 2'-fluoro, and/or 2'-amino modifications of the ribose moiety.
  • the oligonucleotide includes at least 1 , 2, 3, or 4 ribonucleotides, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90%, or even 100% ribonucleotides.
  • the oligonucleotide includes non-nucleotide groups in the chain (i.e., form part of the chain backbone) and/or as side chain moieties, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more, or up to 5, 10, 20% or more of the chain moieties and/or side chain moieties.
  • the oligonucleotide is free of self-complementary sequences longer than 5, 8, 10, 15, 20, 25, 30 nucleotides; the oligonucleotide is free of catalytic activity, e.g., cleavage activity against RNA; the oligonucleotide does not induce an RNA interference mechanism.
  • the oligonucleotide binds protein involved in a disease or condition described in the present invention ; the sequence of the oligonucleotide (or a portion thereof, e.g., at least 20, 30, 40, 50, 60, 70% or more) is derived from a genome; the activity of an oligonucleotide with a sequence derived from a genome is not superior to a randomer oligonucleotide or a random oligonucleotide of the same length; the oligonucleotide includes a portion complementary to a genome sequence and a portion not complementary to a genome sequence; unless otherwise indicated, the sequence of the oligonucleotide includes A(x), C(x), G(x), T(x), U(x), I(x), AC(x), AG(x), AT(x), AU(x), CG(x), CT(x), CU(x), GT(x), GU(x), TU(x), C(x), G(x
  • the oligonucleotide is at least 6, 10, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or more nucleotides in length or is in a range defined by taking any two of the listed values as inclusive endpoints, or the length of the specified repeat sequence is at least a length or in a length range just specified);
  • the oligonucleotide includes a combination of repeat sequences (e.g., repeat sequences as specified above), including, for example, each combination of the above monomer and/or dimer repeats taken 2, 3, or 4 at a time;
  • the oligonucleotide is single stranded (RNA or DNA);
  • the oligonucleotide is double stranded (RNA or DNA);
  • the oligonucleotide includes at least one Gquarte
  • phosphorothioated ONs containing only (or at least primarily) pyrimidine nucleotides, including cytosine and/or thymidine and/or other pyrimidines are resistant to low pH and polycytosine oligonucleotides showed increased resistance to a number of nucleases, thereby providing two important characteristics for oral administration of an ON.
  • the oligonucleotide has at least 80, 90, or 95, or 100% modified internucleotidic linkages (e.g., phosphorothioate or phosphorodithoiate) and the pyrimidine content is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100%, i.e.; is a pyrimidine oligonucleotide or the cytosine content is more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or 100% i.e. is a polycytosine oligonucleotide.
  • modified internucleotidic linkages e.g., phosphorothioate or phosphorodithoiate
  • the length is at least 29, 30, 32, 34, 36, 38, 40, 45, 50, 60, 70, or 80 nucleotides, or is in a range of 20-28, 25-35, 29-40, 30-40, 35-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, or 70-80, or is in a range defined by taking any two of the listed values as inclusive endpoints of the range.
  • the oligonucleotide is at least 50, 60, 70, 80, or 90% cytosine; at least 50, 60, 70, 80, or 90% thymidine (and may have a total pyrimidine content as listed above).
  • the oligonucleotide contains a listed percentage of either cytosine (C) or thymidine (T). Also in certain embodiments, the oligonucleotide includes at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, or more contiguous pyrimidine nucleotides, e.g., as C nucleotides, T nucleotides, or CT dinucleotide pairs.
  • the pyrimidine oligonucleotide consists only of pyrimidine nucleotides; includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-pyrimidine moieities; includes 1-5, 6-10, 11-15, or at least 16 non- pyrimidine backbone moieties; includes further at least one, 1-20, 1-5, 6-10, 11-15, or 16- 20 non-nucleotide moieties; includes at least one, 1-20, 1-5, 6-10, 11-15, or 16-20 purine nucleotides.
  • the linkages between such moieties or between such moieties and nucleotides are at least 25, 35, 50, 70, 90, or 100 % as resistant to acidic conditions as PS linkages in a 40-mer polyC oligonucleotide as evaluated by gel electrophoresis under conditions appropriate for the size and chemistry of the oligonucleotide.
  • oligonucleotides can also be used in combinations, e.g., as a mixture.
  • Such combinations or mixtures can include, for example, at least 2, 3, 4, 5, 10, 20, 50, 100, 1000, 10000, 100,000, 1,000,000, or more different oligonucleotides.
  • Such combinations or mixtures can, for example, be different sequences and/or different lengths and/or different modifications and/or different linked or conjugated molecules.
  • pluralities of oligonucleotides have a minimum length or are in a length range as specified above for oligonucleotides.
  • at least one, or a plurality, or each of the oligonucleotides can have any of the other properties specified herein for individual oligonucleoties (which can also be in any consistent combination).
  • the sequence of the oligonucleotide is not perfectly complementary to any equal length portion of the a genome sequence, or has less than 95, 90, 80, 70, 60, or 50% complementarity to any equal length portion of the genomic sequence, the oligonucleotide sequence does not consist essentially of polyA, polyC, polyG, polyT, Gquartet, or a TG-rich sequence.
  • TG-rich indicates that the sequence of the oligonucleotide consists of at least 50 percent T and G nucleotides, or if so specified, at least 60, 70, 80, 90, or 95% T and G, or even 100%.
  • the invention provides a mixture of oligonucleotides that includes at least two different oligonucleotides as described herein, e.g., at least 2, 3, 4, 5, 7, 10, 50, 100, 1000, 10,000, 100,000, 1,000,000, or even more.
  • oligonucleotide includes at least 20 linked nucleotides.
  • the oligonucleotide may also include additional, non-nucleotide moieties, which may form part of the backbone of the oligonucleotide chain. Unless otherwise indicated, when non-nucleotide moeities are present in the backbone, at least 10 of the linked nucleotides are contiguous.
  • sequence independent mode of action indicates that the particular biological activity is not dependent on a particular oligonucleotide sequence in the oligonucleotide.
  • the activity does not depend on sequence dependent hybridization such as with antisense activity, or a particular sequence resulting in a sequence dependent aptameric interaction.
  • non-sequence complementary mode of action indicates that the mechanism by which the material exhibits an effect is not due to hybridization of complementary nucleic acid sequences, e.g., an antisense effect.
  • sequence complementary mode of action means that the effect of a material involves hybridization of complementary nucleic acid sequences or sequence specific aptameric interaction.
  • sequence complementary mode of action means that the activity of the oligonucleotide satisfies at least one of the 4 tests provided herein.
  • the oligonucleotide satisfies test 1 or test 2, defined hereinafter; the oligonucleotide satisfies a combination two of the tests, i.e., tests 1 & 2.
  • a related aspect concerns an oligonucleotide randomer or randomer formulation that contains at least one randomer, where the activity of the randomer occurs principally by a sequence independent, e.g., non-sequence complementary mode of action.
  • a randomer formulation can, for example, include a mixture of randomers of different lengths, e.g., at least 2, 3, 5, 10, or more different lengths, or other mixtures as described herein.
  • the phrase "derived from a genome” indicates that a particular sequence has a nucleotide base sequence that has at least 70% identity to a genomic nucleotide sequence or its complement (e.g., is the same as or complementary to a genomic sequence), or is a corresponding RNA sequence.
  • the expression "derived from a genome” indicates that the sequence is at least 70% identical to a genomic sequence of a particular gene involved in the disease or condition against which the oligonucleotide is directed, or to its complementary sequence.
  • the identity is at least 80, 90, 95, 98, 99, or 100%.
  • Genome can be from an animal, e.g. a human, from a microorganism, e.g. a virus, a bacteria, a parasite, or from plant.
  • the invention provides a use for the prophylaxis or treatment in an acidic environment in a subject, comprising administering to a subject in need of such a treatment a therapeutically effective amount of at least one pharmacologically acceptable pharmaceutical composition of the invention, said composition being adapted for administration to an acidic in vivo site.
  • the oligonucleotide is a polyp yrimidine oligonucleotide (or a formulation or pharmaceutical composition containing such polypyrimidine oligonucleotide), which may be adapted for oral or vaginal administration, e.g., as described herein.
  • the oligonucleotide(s) having a sequence independent mode of action is not associated with a transfection agent; the oligonucleotide(s) having a sequence independent mode of action does not need to be, and thus is not encapsulated in liposomes and/or non-liposomal lipid particles.
  • the oligonucleotide(s) having a sequence independent mode of action is in a pharmaceutical composition or is administered in conjunction with (concurrently or sequentially) an oligonucleotide that acts principally by a sequence dependent mode of action, e.g., antisense oligonucleotide or siRNA, where the oligonucleotide(s) having a sequence dependent mode of action can be associated with a transfection agent and/or encapsulated in liposomes and/or non- liposomal lipid particles.
  • a sequence dependent mode of action e.g., antisense oligonucleotide or siRNA
  • the invention provides a polymer mix that includes at least one oligonucleotide and at least one non-nucleotide polymer.
  • the invention provides an oligonucleotide randomer, where the randomer is at least 6 nucleotides in length.
  • the randomer has a length as specified above for oligonucleotides;
  • the randomer includes at least one phosphorothioate linkage, the randomer includes at least one phosphorodithioate linkage or other modification as listed herein;
  • the randomer oligonucleotides include at least one non-randomer segment (such as a segment complementary to a selected nucleic acid sequence), which can have a length as specified above for oligonucleotides;
  • the randomer is in a preparation or pool of preparations containing at least 5, 10, 15, 20, 50, 100, 200, 500, or 700 micromol, 1, 5, 7, 10, 20, 50, 100, 200, 500, or 700 mmol, or 1 mole of randomer, or a range defined by taking any two different values from the preceding as inclusive end points, or is synthesized at
  • ONs have been tested for potential broad-spectrum activity against many types of diseases involving amyloidosis.
  • Several ON randomers of different sizes and chemical type were selected to be tested in vitro for binding to the amyloid proteins, amyloid-beta and amylin. It is disclosed in the present invention that ONs interact with beta-amyloid and amylin and that this interaction is sequence independent, dependent on size, and chemical (i.e. sulfur) modification.
  • the results suggest that ONs have an anti amyloid-related disease activity by binding to the amyloid and amylin and therefore preventing, inhibiting or reversing amyloid plaque formation, deposition, polymerization or aggregation.
  • ONs have a therapeutic activity in slowing the progression of plaque formation in a mouse model of Alzheimer's disease.
  • This data shows that ONs of this invention can be used in a treatment for an amyloidal- related disease such as, but without restriction, Alzheimer's disease.
  • ONs with different chemical modifications.
  • a modification of the ON such as, but not limited to, a phosphorothioate modification or other sulfur modifications, appears to be beneficial for anti amyloid-related disease activity.
  • Such sulfur modifications may include without restriction mono and diphosphorothioation of the phosphodiester linkage, 4' or 5' thiolation of the uracil moiety, 5' thiolation of the cytidine moiety, 2' or 4' thiolation of the thymine moiety, 6' thiolation of the guanine moiety, sulfur modifications to any other nucleobase moiety and sulfur modifications to the ribose moiety of any nucleotide or combinations of any of the above mentioned modifications.
  • ONs may have more than one sulfur substitution on each nucleotide, which can potentially increase the activity.
  • any single or multiple sulfur substitution may be combined with other modifications known to improve properties of ONs.
  • ONs of this invention may also have chemical modifications including without restriction: any 2' ribose modification including 2'-0 methyl, 2'-fluorine, 2'-FANA, 2'-methoxyethyl, locked nucleic acids, methylphosphonates, boraophosphates and phosphorodiamidate morpholino oligomers.
  • ONs may have a structure of or comprise a portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone capable of forming stable antiparallel duplexes following the Watson-Crick base pairing rules (Zhang L, et al (2005) J. Am. Chem. Soc. 127(12):4174-5).
  • GNA glycol nucleic acid
  • Such GNA may comprise phosphorothioate linkages or other appropriate modifications as described above.
  • the invention also provides an pharmaceutical composition that includes a therapeutically effective amount of a pharmacologically acceptable, oligonucleotide or mixture of oligonucleotides as described herein, e.g., at least 15 nucleotides in length or other length as listed herein, where the activity of the oligonucleotide occurs principally by a sequence independent, e.g., non-sequence complementary or non-sequence dependent aptamer, mode of action, and a pharmaceutically acceptable carrier.
  • a sequence independent e.g., non-sequence complementary or non-sequence dependent aptamer, mode of action
  • the oligonucleotide or a combination or mixture of oligonucleotides is as specified above for individual oligonucleotides or combinations or mixtures of oligonucleotides.
  • the pharmaceutical compositions are approved for administration to a human, or a non-human animal such as a non-human primate.
  • composition refers to an anti amyloid-related disease ON formulation that includes a physiologically or pharmaceutically acceptable carrier or excipient. Such compositions can also include other components that do not make the composition unsuitable for administration to a desired subject, e.g., a human.
  • the phrase "adapted for use as an anti amyloid-related disease agent" indicates that the material exhibits an anti amyloid-related disease effect and does not include any component or material that makes it unsuitable for use in inhibiting such disease in an in vivo system, e.g., for administering to a subject such as a human subject.
  • the term "subject” refers to a living higher organism, including, for example, animals such as mammals, e.g., humans, non-human primates, non-human animals and plants, e.g., fruit trees.
  • the pharmaceutical composition can be formulated for delivery by a mode selected from the group consisting of but not restricted to oral ingestion, oral mucosal delivery, intranasal drops or spray, intraocular injection, subconjunctival injection, eye drops, ear drops, by inhalation, intratracheal injection or spray, intrabronchial injection or spray, intrapleural injection, intraperitoneal injection perfusion or irrigation, intrathecal injection or perfusion, intracranial injection or perfusion, intramuscular injection, intravenous injection or perfusion, intraarterial injection or perfusion, intralymphatic injection or perfusion, subcutaneous injection or perfusion, intradermal injection, topical skin application, by organ perfusion, by topical application during surgery, intratumoral injection, topical application, gastric injection perfusion or irrigation, enteral injection or perfusion, colonic injection perfusion or irrigation, rectal injection perfusion or irrigation, by rectal suppository or enema, by urethral suppository or injection, intravesical injection per
  • compositions and formulations for administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Other formulations include those in which the ONs of the invention are in mixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include neutral (e.g.
  • ONs may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, ONs may be complexed to lipids, in particular to cationic lipids.
  • Preferred fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, laurie acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, 1- dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a Ci.io alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
  • Ci.io alkyl ester e.g. isopropylmyristate IPM
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or nonaqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsif ⁇ ers, dispersing aids or binders may be desirable.
  • Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Exemplary surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Exemplary bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate.
  • DCA chenodeoxycholic acid
  • UDCA ursodeoxychenedeoxycholic acid
  • cholic acid dehydrocholic acid
  • deoxycholic acid deoxycholic acid
  • glucholic acid glycholic acid
  • glycodeoxycholic acid taurocholic acid
  • taurodeoxycholic acid sodium tauro-24,25-dihydro-fusidate
  • sodium glycodihydrofusidate sodium glycodihydrofusidate.
  • Exemplary fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 - monocaprate, 1 -dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium).
  • arachidonic acid arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
  • penetration enhancers for example, fatty acids/salts in combination with bile acids/salts.
  • a particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further exemplary penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • ON complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses, and starches.
  • Particularly advantageous complexing agents include chitosan, N-trimethytchitosan, poly-L-lysine, polyhistidine, polyorithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.
  • PEG polyethylene glycol
  • compositions and formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • the composition includes a delivery system, e.g., targeted to specific cells or tissues; a liposomal formulation, another drug, e.g., a non- nucleotide polymer, an antisense molecule, a siRNA, or a small molecule drug.
  • a delivery system e.g., targeted to specific cells or tissues
  • a liposomal formulation e.g., another drug, e.g., a non- nucleotide polymer, an antisense molecule, a siRNA, or a small molecule drug.
  • the term "delivery system” refers to a component or components that, when combined with an ON as described herein, facilitates the transfer of ONs inside cells, increases the amount of ONs that contact the intended location in vivo, and/or extends the duration of its presence at the target or increases its circulating lifetime in vivo, e.g., by at least 10, 20, 50, or 100%, or even more as compared to the amount and/or duration in the absence of the delivery system.
  • the term delivery system also means encapsulation system or encapsulation reagent.
  • To encapsulate ONs means to put in contact an ON with a delivery system or an encapsulation reagent.
  • An ON in contact with a delivery system can be referred to as an "encapsulated ON".
  • the oligonucleotide, oligonucleotide preparation, oligonucleotide formulation, or pharmaceutical composition has an in vitro IC 50 or EC 50 of 10, 5, 2, 1, 0.50, 0.20, 0.10, 0.09. 0.08, 0.07, 0.75, 0.06, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or 0.01 ⁇ M or less.
  • the pharmaceutical composition contains at least one polypyrimidine oligonucleotide as described herein.
  • a composition is adapted for delivery to an acidic in vivo site, e.g., oral delivery or vaginal delivery.
  • the term "acidic site” means a site that has a pH of less than 7. Examples include the stomach (pH generally 1-2), the vagina (pH generally 4-5 but may be lower), and to a lesser degree, the skin (pH generally 4-6).
  • the phrase "adapted for oral delivery" and like terms indicate that the composition is sufficiently resistant to acidic pH to allow oral administration without a clinically excessive loss of activity, e.g., an excessive first pass loss due to stomach acidity of less than 50% (or is indicated, less than 40%, 30%, 20%, 10%, or 5%).
  • small molecule means that the molecular weight of the molecule is 1500 daltons or less. In some cases, the molecular weight is 1000, 800, 600, 500, or 400 daltons or less.
  • the invention provides a kit that includes at least one oligonucleotide, oligonucleotide mixture, oligonucleotide formulation, or pharmaceutical composition that includes such oligonucleotide, oligonucleotide mixture, or oligonucleotide formulation in a labeled package, where the activity of the oligonucleotide occurs principally by a sequence independent e.g., non-sequence complementary or non-sequence dependent aptameric, mode of action and the label on the package indicates that the oligonucleotide can be used against at least one amyloid- related disease or condition.
  • a sequence independent e.g., non-sequence complementary or non-sequence dependent aptameric, mode of action indicates that the oligonucleotide can be used against at least one amyloid- related disease or condition.
  • the kit includes a pharmaceutical composition that includes at least one oligonucleotide as described herein.
  • the kit contains a mixture of at least two different oligonucleotides.
  • the oligonucleotide is adapted for in vivo use in an animal and/or the label indicates that the oligonucleotide or composition is acceptable and/or approved for use in an animal; wherein the animal is a mammal, such as human, or a non-human mammal such as bovine, porcine, a ruminant, ovine, or equine; the animal is a non-human animal; wherein the animal is a bird; the kit is approved by a regulatory agency such as the U.S.
  • the different random oligonucleotides comprises randomers of different lengths; the random oligonucleotides can have different sequences or can have sequence in common, such as the sequence of the shortest oligos of the plurality; and/or the different random oligonucleotides comprise a plurality of oligonucleotides comprising a randomer segment at least 5 nucleotides in length or the different random oligonucleotides include a plurality of randomers of different lengths.
  • Other oligonucleotides e.g., as described herein oligonucleotides, can be tested in a particular system.
  • the term "randomer” is intended to mean a single stranded nucleic acid polymer, modified or not, having degenerate sequences at every position, such as NNNNNNNNNN.
  • Each degenerate nucleotide position actually exists as a random population of the five naturally occurring bases on the nucleotide (adenine, guanine, cytosine, thymine, and uracil) at this particular position, resulting in a completely degenerate pool of ONs of the same size but having no sequence identity as a population.
  • Randomers can also include nucleobases which do not occur naturally including without restriction hypoxanthine, xanthosine, imidazole, 2-aminopurines or 5- nitroindole.
  • the term randomer can apply to a sequence or a portion of a sequence.
  • degenerate means that a sequence is made of a mix of nucleotides.
  • a completely degenerate sequence means that A, C, G, and T (or other nucleobases) are randomly used at each position of the sequence and nucleotide position are identified by N.
  • a degenerate sequence means also that at least two nucleobases are randomly used at each position of the sequence. Degenerate can apply to a sequence, a portion of a sequence or one nucleotide position in a sequence.
  • the invention provides a method for the prophylaxis or treatment in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as described herein, e.g., a sequence independent oligonucleotide of at least 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s).
  • oligonucleotide as described herein, e.g., a sequence independent oligonucleotide of at least 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s).
  • the invention provides a use for the prophylaxis or treatment in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as described herein, e.g., a sequence independent oligonucleotide of at least 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s).
  • oligonucleotide as described herein, e.g., a sequence independent oligonucleotide of at least 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s).
  • the disease or condition targeted be any of those listed herein as suitable for inhibition using the present invention
  • the subject is a type of subject as indicated herein, e.g., human, non-human animal, non-human mammal, bird, plant, and the like
  • the treatment is for a condition or a disease as intended in the present invention.
  • terapéuticaally effective amount refers to an amount that is sufficient to affect a therapeutically or prophylactically significant reduction amyloid- related disease when administered to a typical subject of the intended type.
  • an anti amyloid-related disease ON to a subject typically the ON, formulation, or composition should be administered in a therapeutically effective amount.
  • the anti-amyloid-related disease ON, ON formulation, ON pharmaceutical composition or use of ON for treatment described herein prevent, reverse or inhibit amyloid protein assembly into insoluble fibrils which, in vivo, are deposited in various organs, or it favors clearance of pre-formed deposits or slows deposition in patients already having deposits.
  • the ON may also prevent the amyloid protein, in its soluble, oligomeric form or in its fibrillar form, from binding or adhering to a cell surface and causing cell damage or toxicity.
  • the ON may block amyloid-induced cellular toxicity or macrophage activation.
  • the ON may block amyloid-induced neurotoxicity or microglial activation.
  • the ON protects cells from amyloid induced cytotoxicity of B-islet cells to protect against amyloidal induced renal diseases.
  • the ON may enhance clearance from a specific organ, e.g., the brain or it decreases concentration of the amyloid protein in such a way that amyloid fibril formation is prevented in the targeted organ.
  • the anti-amyloid-related disease ON, ON formulation, ON pharmaceutical composition or use of the ON for treatment described herein may be administered therapeutically or prophylactically to treat diseases associated with amyloid fibril formation, aggregation or deposition.
  • the ONs of the invention may act to ameliorate the course of an amyloid related disease using, without limitation, any of the following mechanisms: slowing the rate of amyloid fibril formation or deposition; lessening the degree of amyloid deposition; inhibiting, reducing, or preventing amyloid fibril formation; inhibiting neurodegeneration or cellular toxicity induced by amyloid; inhibiting amyloid induced inflammation; enhancing the clearance of amyloid; or favoring the degradation of amyloid protein prior to its organization in fibrils, and preventing the association of amyloid proteins and or fibrils with cells.
  • the anti-amyloid-related disease ON, ON formulation, ON pharmaceutical composition or use of the ON for treatment described herein may be administered therapeutically or prophylactically to treat diseases associated with amyloid-beta fibril formation, aggregation or deposition.
  • the ONs of the invention may act to ameliorate the course of an amyloid-beta related disease using, without limitation, any of the following mechanisms: slowing the rate of amyloid-beta fibril formation or deposition; lessening the degree of amyloid-beta deposition; inhibiting, reducing, or preventing amyloid-beta fibril formation; inhibiting neurodegeneration or cellular toxicity induced by amyloid-beta; inhibiting amyloid-beta induced inflammation; enhancing the clearance of amyloid-beta from the brain; or favoring the degradation of amyloid-beta protein prior to its organization in fibrils.
  • the present oligonucleotides can be prepared in an ON formulation or pharmaceutical composition.
  • the present ONs may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • Exemplary United States patents that describe the preparation of such uptake, distribution and/or absorption assisting formulations include, for example, U.S. patents Nos.
  • the ONs, formulations, and compositions of the invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • prodrug versions of the present oligonucleotides are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in Gosselin et al. (international patent application publication No WO 93/24510) and in Imbach et al. (international patent application publication No WO 94/26764) and U.S. patent No 5,770,713.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the present compounds: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Many such pharmaceutically acceptable salts are known and can be used in the present invention.
  • useful examples of pharmaceutically acceptable salts include but are not limited to salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and salts formed from element
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaking the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • the preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • the formulations and compositions of the present invention may be prepared and formulated as emulsions.
  • Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ m in diameter.
  • Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be either water-in-oil (w/o) or of the oil-in- water (o/w) variety.
  • Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed.
  • compositions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in- water-in-oil (o/w/o) and water-in-oil-in- water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.
  • Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N. Y., 1988, volume 1, p. 199).
  • Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion.
  • HLB hydrophile/lipophile balance
  • surfactants may be classified into different classes based on the nature of the hydrophilic group: non-ionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
  • Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations.
  • polar inorganic solids such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • a large variety of non-emulsifying materials is also included in emulsion formulations and contributes to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199).
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong inter- facial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and carb
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid, Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulf ⁇ te, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulf ⁇ te
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • the compositions of ONs are formulated as microemulsions.
  • a microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 245).
  • micro- emulsions are systems that are prepared by first dispersing oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system.
  • microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface- active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).
  • Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte.
  • microemulsion is of the water-in-oil (w/o) or an oil-in- water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
  • microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants.
  • ionic surfactants non-ionic surfactants
  • Brij 96 polyoxyethylene oleyl ethers
  • polyglycerol fatty acid esters tetraglycerol monolaurate (ML310),
  • the cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.
  • the aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions both o/w and w/o have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al, Pharmaceutical Research, 1994, 11, 1385-1390; Ritschet, Met/i. Find. Exp. Clin. PharmacoL, 1993, 13, 205).
  • Micro-emulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant- induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al. , Pharmaceutical Research, 1994, 11, 1385; Ho et al, J. Pharm. Set, 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of ONs and nucleic acids from the gastrointestinal tract.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention.
  • Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al. , Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).
  • liposome refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers, i.e., liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior.
  • the aqueous portion typically contains the composition to be delivered.
  • lipid vesicles In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores. Additional factors for liposomes include the lipid surface charge, and the aqueous volume of the liposomes.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 245).
  • liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target.
  • liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine.
  • Neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome include one or more glycolipids, such as monosialoganglioside G MI , or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • Patent No 4,837,028 and International Application Publication No WO 88/04924 (sphingomyelin and the ganglioside G M I or a galactocerebroside sulfate ester); Webb et al, U.S. patent No 5,543,152 (sphingomyelin); Lim et al, International Application Publication No WO 97/13499 (1,2-sn- dimyristoylphosphatidylcholine).
  • Liposomes that include lipids derivatized with one or more hydrophilic polymers, and methods of preparation are described, for example, in Sunamoto et al, Bull. Chem. Soc. Jpn., 1980, 53, 2778 (a nonionic detergent, 2C 12 15G, that contains a PEG moiety); Ilium et al, FEBS Lett., 1984, 167, 79 (hydrophilic coating of polystyrene particles with polymeric glycols); Sears, U.S. patents Nos.
  • EP 0 445 131 Bl and Internaltional application publication No WO 90/04384 (covalently bound PEG moieties on liposome external surface); Woodle et al, U.S. patents . Nos. 5,013,556 and 5,356,633, and Martin et al, U.S. patents. No. 5,213,804 and European Patent No. EP 0 496 813 Bl (liposome compositions containing 1-20 mole percent of PE derivatized with PEG); Martin et al, International application publication No WO 91/05545 and U.S. patent No. 5,225,212 and in Zalipsky et al.
  • Liposomes that include nucleic acids have been described, for example, in Thierry et al., International application publication No WO 96/40062 (methods for encapsulating high molecular weight nucleic acids in liposomes); Tagawa et al, U.S. patent No 5,264,221 (protein-bonded liposomes containing RNA); Rahman et al, U.S. patent No 5,665,710 (methods of encapsulating oligodeoxynucleotides in liposomes); Love et al. , International application publication No WO 97/04787 (liposomes that include antisense oligonucleotides).
  • Transfersomes Another type of liposome, transfersomes, is highly deformable lipid aggregates which are attractive for drug delivery vehicles.
  • Transfersomes may be described as lipid droplets which are so highly deformable that they can penetrate through pores which are smaller than the droplet.
  • Transfersomes are adaptable to the environment in which they are used, for example, they are shape adaptive, self-repairing, frequently reach their targets without fragmenting, and often self-loading. Transfersomes can be made, for example, by adding surface edge- activators, usually surfactants, to a standard liposomal composition.
  • surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes.
  • HLB hydrophile/lipophile balance
  • the nature of the hydrophilic group also known as the "head" provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
  • the surfactant molecule is not ionized, it is classified as a nonionic surfactant.
  • Nonionic surfactants are widely used in pharmaceutical and cosmetic products and are usable over a wide range of pH values, and with typical HLB values from 2 to about 18 depending on structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters; and nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most commonly used members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isothionates, acyl laurates and sulfosuccinates, and phosphates.
  • the alkyl sulfates and soaps are the most commonly used anionic surfactants.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines, with the quaternary ammonium salts used most often.
  • Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • penetration enhancers are used in or with a composition to increase the delivery of nucleic acids, particularly ONs across membranes of animals.
  • Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating nonsurfactants (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of these classes of penetration enhancers is described below in greater detail.
  • surfactants are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of ONs through the mucosa is enhanced.
  • penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al. (J. Pharm. Pharmacol., 1988, 40, 252), each of which is incorporated herein by reference in its entirety.
  • Fatty acids Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1 -monocaprate, 1 -dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, Ci-io alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and diglycerides thereof (L e.
  • oleic acid lauric acid
  • capric acid n-decanoic acid
  • myristic acid palmitic acid
  • bile salts in the present invention includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • the bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25- dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18
  • chelating agents can be regarded as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of ONs through the mucosa is enhanced.
  • chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Cliromatogr., 1993, 618, 315-339).
  • chelating agents include disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N- amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al, J. Control ReI., 1990, 14,
  • non-chelating non- surfactant penetration enhancing compounds are compounds that do not demonstrate significant chelating agent or surfactant activity, but still enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).
  • penetration enhancers examples include unsaturated cyclic ureas, 1 -alkyl- and 1- alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et ah, J. Pharm. Pharmacol., 1987, 39, 621-626).
  • nucleic acids include glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • glycols such as ethylene glycol and propylene glycol
  • pyrrols such as 2-pyrrol
  • azones such as 2-pyrrol
  • terpenes such as limonene and menthone.
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • carrier compound or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • carrier compound can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • the co administration of a nucleic acid and a carrier compound often with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney
  • the recovery of a partially phosphorothioated ON in hepatic tissue can be reduced when it is co administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido- 4'isothiocyano-stilbene-2,2-disulfonic acid (Miyao e? ⁇ /.,AntisenseRes. Dev., 1995, 5, 115-121; Takakura et al, Antisense & NucL Acid Drug Dev., 1996, 6, 177-183), each of which is incorporated herein by reference in its entirety.
  • a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal, and is typically liquid or solid.
  • a pharmaceutical carrier is generally selected to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition, in view of the intended administration mode.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxyprop
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • compositions may additionally contain other components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran, and/or stabilizers.
  • a pharmaceutical composition containing (a) one or more anti amyloid-related disease ONs and (b) one or more other chemotherapeutic agents used which function by similar or different mechanisms.
  • chemotherapeutic agents include but are not limited to maltose tetrapalmitate, maltose tripalmitate, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethytmetamine
  • chemotherapeutic agents When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and an ON), sequentially (e.g., 5-FU and an ON for a period of time followed by MTX and ON), or in combination with one or more other such chemotherapeutic agents (e.g., 5-EU, MTX and an ON, or 5-FU, radiotherapy and an ON).
  • Chemotherapeutic agents can be anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids. Other specific agents used to treat amyloidogenic disorders include donepezil, rivastigmine, and galantamine, the cholinesterase inhibitor tacrine vitamin E, selegiline and estrogen in postmenopausal women.
  • the pharmaceutical composition or ON formulation of the invention may further contain other drugs for the treatment of amyloid-related diseases.
  • additional factors and/or agents may be included in the pharmaceutical composition, for example, to produce a synergistic effect with the ONs of the invention.
  • anti amyloid-related disease ONs demonstrating low, preferably the lowest possible, homology with the human (or other subject organism's) genome is designed.
  • One goal is to obtain an ON that will show the lowest toxicity due to interactions with human or animal genome sequence(s) and/or mRNAs.
  • the first step is to produce the desired length sequence of the ON, e.g., by aligning nucleotides A, C, G, T/U in a random fashion, manually or, more commonly, using a computer program.
  • the second step is to compare the ON sequence with a library of human sequences such as GenBank and/or the Ensemble Human Genome Database.
  • sequence generation and comparison can be performed repetitively, if desired, to identify a sequence or sequences having a desired low homology level with the subject genome. It is desirable for the ON sequence to have the lowest homology possible with the entire genome, while also minimizing self interaction.
  • the last step is to test the ON in an assay to measure anti amyloid-related disease activity.
  • sequence independent ON sequence portion(s) is/are coupled with antisense sequence portion(s) to increase the activity of the final ON.
  • the non-specific portion of the ON is described in the present invention.
  • the antisense portion can be complementary to a amyloid pathogeneses related gene mRNA or to other genes important for the progression of amyloid-related diseases.
  • sequence independent sequence portion(s) is/are coupled with a G-rich motif ON portion(s) to improve the activity of the final ON.
  • the non-specific portion of the ON is described in the present invention.
  • the G-rich motif portion can, as non-limiting examples, include, CpG, Gquartet, and/or CG that are described in the literature as stimulators of the immune system.
  • Another approach is to use an ON composed of one or more types of non- Watson-Crick nucleotides/nucleosides.
  • Such ONs can mimic PS-ONs and other modifications with some of the following characteristics similar to PS-ONs: a) the total charge; b) the space between the units; c) the length of the chain; d) a net dipole with accumulation of negative charge on one side; e) the ability to bind to proteins f) the ability to be used with delivery systems, h) an acceptable therapeutic index, i) an anti amyloid-related disease activity.
  • the ON can have a phosphorothioate backbone but is not limited to it.
  • Non- Watson-Crick nucleotides/nucleosides are described in Kool, 2002, Ace. Chem. Res. 35:936-943; and Takeshita et al. , (1987) J Biol. Chem. 262:10171-10179 where ONs containing synthetic abasic sites are described.
  • Another embodiment of the present invention is to use a polymer mimicking the activity of ONs described in the present invention to obtain inhibition of amyloid-related disease activity. As described in the literature, several anionic polymers were shown to bind to proteins.
  • polysaccharides include sulfate esters of polysaccharides (dextrin and dextran sulfates; cellulose sulfate); (2) polymers containing sulfonated benzene or naphthalene rings and naphthalene sulfonate polymers; (3) polycarboxylates (acrylic acid polymers); and acetyl phthaloyl cellulose (Neurath et al. (2002) BMC Infect Dis 2:27); and (4) abasic ONs (Takeshita et al. , 1987, J. Biol. Chem. 262:10171-10179).
  • Other examples of non-nucleotide protein binding polymers are described in the literature.
  • the polymers described herein can mimic ONs described in this invention and may have some or all of the following characteristics similar to ONs: a) the length of the chain; b) a net dipole with accumulation of negative charge on one side; c) the ability to bind to proteins; d) the ability to be encapsulated by a delivery system, e) an acceptable therapeutic index, f) an anti amyloid-related disease activity.
  • the anti amyloid-related disease polymer may preferably be a polyanion displaying similar space between its units as compared to a PS-ON.
  • the anti amyloid-related disease polymer may display a similar hydrophobicity than PS-ON.
  • the binding of PS-ONs to amyloid beta (1-42) is dependent on size and sulfur modification.
  • FL refer to a fluorescent linker (FITC label) attached to the ONs.
  • PS-ON randomers have potent, sequence-independent size dependent, anti-prion activity
  • PS-ON randomers were diluted into PBS prior to being introduced to the cell medium. 5 ⁇ L of solutions were added to the cell medium. After PS-ON randomers were added, the cells were incubated for 5 days at 37° C in a CO 2 incubator before being lysed.
  • lysis buffer was composed of 0.5% (w/v) Triton X-100TM, 0.5% (w/v) sodium deoxycholate, 5 mM tris-HCl, pH 7.4 at 4 0 C, 5 mM EDTA, and 150 mM NaCl.
  • 25 ⁇ L of 0.1 mg/mL PK (Calbiochem) in TBS was added to each well and incubated at 37° C for 50 minutes.
  • 225 ⁇ L of 1 mM PefablocTM (Boehringer Mannheim) was then added to each well to inhibit PK activity. 250 ⁇ L of 1 mM Pefabloc was added to samples that were not PK-treated.
  • the GdnSCN was removed by 5 PBS rinses and the membrane blocked in 5% (w/v) milk, 0.05% (v/v) Tween 20TM (Sigma) in TBS (TBST-milk) for 30 minutes.
  • Example 4 The anti scrapie activity of ONs is dependent on sulfur modification
  • PS-ONs are effective prophylactic agents against scrapie in vivo and in vivo activity is dependent on sulfur modification.
  • Tg7 mice were first dosed with 10mg/kg GMP-REP 2006 (SEQ ID NO: 6) in 5% dextrose subcutaneously (sc) or intraperitoneally (ip) daily for 3 days. Also on the third day immediately after the third dose of cGMP-Randomer 1, animals were inoculated ip with 50 ⁇ L of 1% 263K hamster scrapie-infected brain homogenate.
  • mice were dosed Monday, Wednesday, and Friday for either the next 4 or 10 weeks with the above mentioned amount of cGMP-REP2006 (SEQ ID NO: 6).
  • Groups of Tg7 mice were also inoculated ip with 50 ⁇ L of 1% 263K hamster scrapie-infected brain homogenate and dosed with 5% dextrose either sc or ip as a control.
  • REP 2006 (SEQ ID NO: 6) as a scrapie prophylactic in Tg7 mice inoculated ip with 1%
  • Untreated 10% (wt/vol) homogenates of 263K hamster scrapie-infected brains were diluted with PBS to 1, 0.1, 0.01, or 0.001% (wt/vol) and incubated at 37° C for one hour. 50 ⁇ L of each of these diluted homogenates were then injected intracerebrally into Tg7 mice. Separate 10% 263K-infected brain homogenate solutions were diluted to 1% with PBS and ImM randomer solution to the desired final concentrations. These mixtures of brain homogenate and randomer were also incubated at 37° C for one hour. As with control homogenates, 50 ⁇ L of each of these were injected intracerebrally (ic) into Tg7 mice. Table 7
  • Anti amyloid-related disease ONs with increased pH resistance, lower serum protein binding and superior nuclease resistance.
  • a phosphorothioate randomer labeled at the 3' end with FITC (the bait) is diluted to 2nM in assay buffer (1OmM Tris, pH7.2, 8OmM NaCl, 1OmM EDTA, 10OmM b-mercaptoethanol and 1% tween 20). This oligo is then mixed with the appropriate amount of non heat-inactivated FBS. Following randomer-FBS interaction, the complexes are challenged with various unlabelled randomers to assess their ability to displace the bait from its complex. Displaced bait is measured by fluorescence polarization. The displacement curve was used to determine Kd.
  • pH resistance was determined by incubation of randomers in PBS adjusted to the appropriate pH with HCl. 24 hours after incubation, samples were neutralized with IM TRIS, pH 7.4 and run on denaturing acryalmide gels and visualized following EtBr staining.
  • N unmodified deoxyribonucleotide
  • unmodified linkage N unmodified deoxyribonucleotide
  • phosphorothiate linkage N 2'-0 methyl modified ribose
  • unmodified linkage N 2'-0 methyl modified ribose + phosphorothioate linkage
  • REP 2107 has a lower affinity to serum proteins than REP 2006 (SEQ ID NO: 6) or REP 2024 (SEQ ID NO: 24) (see Table 9 in this example) and that there was no interaction detected between REP 2086 (SEQ ID NO: 83) and serum proteins.
  • REP 2107 SEQ ID NO: 103 was less effective at displacing bound bait than REP 2006 (SEQ ID NO: 6) or REP 2024 (SEQ ID NO: 24) (see Table 10 in this example).
  • REP2107 (SEQ ID NO: 103) R R R R
  • REP2086 (SEQ ID NO: 83) R R S R
  • Anti amyloid-related disease phosphorothioated polypyrimidine ONs exhibit acid and nuclease resistance.
  • the phosphorothioated 40mer ONs containing only the pyrimidine nucleotides cytosine (polyC, REP 2031; SEQ ID NO: 31) or thymidine (polyT, REP 2030; SEQ ID NO: 30) or the polyTC heteropolymer (REP 2056; SEQ ID NO: 52) had equivalent acid resistance compared to the fully 2'-O-methylated randomers whether phosphorothioated (REP 2107; SEQ ID NO: 103) or not (REP 2086; SEQ ID NO: 83).
  • phosphorothioated oligonucleotides containing only the purine nucleotide adenosine (polyA, REP 2029; SEQ ID NO: 29) or any adenosine or guanosine nucleotides (REP 2033, SEQ ID NO: 33; REP 2055, SEQ ID NO: 51; REP 2057, SEQ ID NO: 53) showed no greater acid resistance compared to unmodified DNA.
  • PII phosphodiesterase 11
  • Sl Sl nuclease
  • Exol Exonuclease 1
  • PS all linkages phosphorothioated
  • various 40 base ONs having different nucleotide compositions and modifications were incubated in the presence of various endo and exonucleases for 4 hours at 37 deg C. The degradation of these ONs was assessed by urea-polyacryamide gel electrophoresis.
  • PII phosphodiesterase 11
  • Sl Sl nuclease
  • Exol Exonuclease 1
  • PS all linkages phosphorothioated
  • 2'OMe all riboses are 2'0 methylated.
  • - complete degredation
  • ++++ no degredation
  • PS phosphorothioate
  • 2'0Me 2'-O-methyl modification of the ribose.
  • the pyrimidine content of such an oligonucleotide is more than 50%, more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100%.
  • these results show the potential of a method of treatment using oral administration of a therapeutically effective amount of at least one pharmacologically acceptable ON composed of pyrimidine nucleotides.
  • These results also show the potential of ONs containing high levels of pyrimidine nucleotides as a component of an ON formulation.
  • TEST #1 Effect of partial degeneracy of a candidate on its anti amyloid-related disease efficacy
  • L the number of bases in the candidate ON.
  • X the number of bases on each end of the oligo to be made degenerate (but having the same chemistry as the candidate ON).
  • X must be equal to or greater than 4.
  • the anti-amyloidogenic activity of the candidate and partially degenerate ON shall be determined by the cell free binding activity to amyloid related peptides or in vitro anti-scrapie-activity using the assays described herein.
  • IC 50 values for binding (Kd) or activity
  • IC 50 values shall be generated using a minimum of seven concentrations of compound, with three or more points in the linear range of the dose response curve. Using these tests, the IC50 of the candidate ON shall be compared to its degenerate counterpart. If the IC50 of the partially degenerate ON is less than 5-fold greater than the original candidate ON (based on minimum triplicate measurements, standard deviation not to exceed 15% of mean) then the ON shall be deemed to have sequence independent activity.
  • This test serves to compare the anti amyloid-related disease efficacy of a candidate ON with the anti amyloid-related disease efficacy of a randomer ON of equivalent size and chemistry.
  • the anti-amyloidogenic activity of the candidate and partially degenerate ON shall be determined by the cell free binding activity to amyloid related peptides or in vitro anti-scrapie-activity using the assays described herein.
  • IC 50 values binding (Kd) or activity
  • IC 50 values binding (Kd) or activity
  • the IC50 of the candidate ON shall be compared to an ON randomer of equivalent size and chemistry. If the IC50 of the ON randomer is less than 5- fold greater than the candidate ON (based on minimum triplicate measurements, standard deviation not to exceed 15% of mean) then the candidate ON shall be deemed to have sequence-independent activity.
  • the thresholds described in tests 1 and 2 above are the default thresholds. If specifically indicated, other thresholds can be used in the comparison tests. Thus for example, if specifically indicated, the threshold for determining whether an ON is acting with sequence-independent activity can be any of 10-fold, 8-fold, 6-fold, 5 -fold, 4-fold, 3-fold, 2-fold, 1.5-fold, or equal..
  • Sulfur modified nucleic acids have a therapeutic activity in slowing the progression of plaque formation in a mouse model of Alzheimer's disease.
  • FL refer to a fluorescent linker (FITC label) attached to the ONs
  • FL refer to a fluorescent linker (FITC label) attached to the ONs
  • any nucleotide sequence could be used to generate an oligonucleotide which could interact with amyloid beta and thus may prevent amyloid fibril (plaque formation) as long it is of sufficient length (greater than 20 bases in length) and consists of at least one sulfur containing modification including but not restricted to phosphorothioation, diphosphorothioation or a sulfur modification to the base moiety at any suitable position.

Abstract

Formulation d'oligonucléotides comprenant des oligonucléotides agissant contre une maladie liée à l'amyloïde qui agissent au moyen d'un mécanisme indépendant de la séquence. L'activité contre la maladie liée à l'amyloïde est plus grande pour les grands oligonucléotides et pour les oligonucléotides présentant une modification soufrée. L'invention concerne aussi les oligonucléotides et leur utilisation comme agents thérapeutiques, et plus particulièrement leur utilisation et leurs formulations pour le traitement de maladies liées à l'amyloïde. Les oligonucléotides modifiés sont compris dans la présente invention, par exemple, ceux contenant des squelettes modifiés.
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US9381208B2 (en) 2006-08-08 2016-07-05 Rheinische Friedrich-Wilhelms-Universität Structure and use of 5′ phosphate oligonucleotides
US9399658B2 (en) 2011-03-28 2016-07-26 Rheinische Friedrich-Wilhelms-Universität Bonn Purification of triphosphorylated oligonucleotides using capture tags
WO2016187217A2 (fr) 2015-05-18 2016-11-24 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions permettant de traiter des troubles associés au vieillissement
US9738680B2 (en) 2008-05-21 2017-08-22 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US10059943B2 (en) 2012-09-27 2018-08-28 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
US11166976B2 (en) 2018-11-08 2021-11-09 Aligos Therapeutics, Inc. S-antigen transport inhibiting oligonucleotide polymers and methods

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US9381208B2 (en) 2006-08-08 2016-07-05 Rheinische Friedrich-Wilhelms-Universität Structure and use of 5′ phosphate oligonucleotides
US10238682B2 (en) 2006-08-08 2019-03-26 Rheinische Friedrich-Wilhelms-Universität Bonn Structure and use of 5′ phosphate oligonucleotides
US10036021B2 (en) 2008-05-21 2018-07-31 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US10196638B2 (en) 2008-05-21 2019-02-05 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
US9738680B2 (en) 2008-05-21 2017-08-22 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
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US9896689B2 (en) 2011-03-28 2018-02-20 Rheinische Friedrich-Wilhelms-Universität Bonn Purification of triphosphorylated oligonucleotides using capture tags
US9399658B2 (en) 2011-03-28 2016-07-26 Rheinische Friedrich-Wilhelms-Universität Bonn Purification of triphosphorylated oligonucleotides using capture tags
US10059943B2 (en) 2012-09-27 2018-08-28 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
US10072262B2 (en) 2012-09-27 2018-09-11 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
US11142763B2 (en) 2012-09-27 2021-10-12 Rheinische Friedrich-Wilhelms-Universität Bonn RIG-I ligands and methods for producing them
WO2016187217A2 (fr) 2015-05-18 2016-11-24 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions permettant de traiter des troubles associés au vieillissement
EP3892315A1 (fr) 2015-05-18 2021-10-13 The Board of Trustees of the Leland Stanford Junior University Méthodes et compositions permettant de traiter des troubles associés au vieillissement
US11166976B2 (en) 2018-11-08 2021-11-09 Aligos Therapeutics, Inc. S-antigen transport inhibiting oligonucleotide polymers and methods

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