WO2011115862A1 - Endosomolytic poly(amidoamine) disulfide polymers for the delivery of oligonucleotides - Google Patents

Endosomolytic poly(amidoamine) disulfide polymers for the delivery of oligonucleotides Download PDF

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WO2011115862A1
WO2011115862A1 PCT/US2011/028260 US2011028260W WO2011115862A1 WO 2011115862 A1 WO2011115862 A1 WO 2011115862A1 US 2011028260 W US2011028260 W US 2011028260W WO 2011115862 A1 WO2011115862 A1 WO 2011115862A1
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polymer
formula
substituents
ratios
polymers
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PCT/US2011/028260
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French (fr)
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Marina Busuek
Steven L. Colletti
Rubina G. Parmar
Weimin Wang
J. Michael Williams
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Merck Sharp & Dohme Corp.
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Priority to EP11756781A priority Critical patent/EP2547345A1/en
Priority to US13/634,579 priority patent/US20130005797A1/en
Publication of WO2011115862A1 publication Critical patent/WO2011115862A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L79/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
    • C08L79/02—Polyamines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G75/00—Macromolecular compounds obtained by reactions forming a linkage containing sulfur with or without nitrogen, oxygen, or carbon in the main chain of the macromolecule
    • C08G75/14—Polysulfides

Definitions

  • Oligonucleotides conjugated to polymers are known. Further, the delivery of oligonucleotides conjugated to polymers (polyconjugates) for therapeutic purposes is also known. See WO2000/34343; WO2008/022309; and Rozema et al. PNAS (2008) 104, 32: 12982- 12987.
  • Poly(amidoamine) disulfide polymers are known. Ou et al. Bioconjugate Chem. (2008) 19: 626-633 and Lin et al. J. of Controlled Release (2008) 126: 166-174.
  • Polyconjugates have numerous toxicities associated with extensive circulation half-life. It is thus an object of the invention to provide polyconjugates that are biodegradable.
  • the poly(amidoamine) disulfide polymers of the instant invention are novel and contain both cationic and aliphatic functional moieties. These polymers are biodegradable and are therefore less toxic.
  • the present invention provides endosomolytic poly(amidoamine) disulfide polymers, polyconjugates, compositions and methods for the delivery of oligonucleotides for therapeutic purposes.
  • FIG. 1 RBC Hemolysis Data of Polymers at pH 5.5.
  • FIG. 2. RBC Hemolysis Data of Masked Polyconjugate.
  • FIG. 3. In-Vitro Data.
  • FIG. 4. In-Vivo Data.
  • n 2 to 250;
  • R is independently selected from A, B and C, wherein at least two of A, B and C are present in the polymer and the polymer must include at least one cationic component (A or B) and at least one aliphatic component (B or C);
  • A is a first cationic component
  • B is a second cationic component or a second aliphatic component
  • C is a first aliphatic component
  • n 2 to 250
  • R is independently selected from A, B and C, wherein at least two of A, B and C are present in the polymer and the polymer must include at least one cationic component (A or B) and at least one aliphatic component (B or C);
  • A is a first cationic component selected from an amine (primary, secondary, tertiary or quaternary), a nitrogen heterocycle, an aldimine, a hydrazide and a hydrazone;
  • B is a second cationic component selected from an amine (primary, secondary, tertiary or quaternary), a nitrogen heterocycle, an aldimine, a hydrazide and a hydrazone, or a second aliphatic component; and
  • C is a first aliphatic component
  • n 2 to 250
  • R is independently selected from A, B and C, wherein A, B and C are all present in the polymer;
  • A is 2-(2-aminoethoxy)ethyl
  • B is 2-(1H-imidazol-4»y!ethyl; and C is dodecyl;
  • R is independently selected from A, B and C, wherein A, B and C are all present in the polymer and wherein A, B and C are different from one another.
  • R is independently selected from A, B and C, wherein A, B and C are all present in the polymer and wherein A and B are different from one another.
  • R is independently selected from A, B and C, wherein A, B and C are all present in the polymer and wherein B and C are different from one another.
  • substituent A must comprise at least 50% of the polymer.
  • substituent B must comprise at least 50% of the polymer.
  • substituent C must comprise at least 50% of the polymer.
  • compositions comprising a polymer of Formula Z and an oligonucleotide.
  • composition comprising a polymer of
  • Formula Z a masking agent and an oligonucleotide.
  • compositions comprising a polymer of Formula Z, a masking agent, a targeting agent and an oligonucleotide.
  • Another aspect of the instant invention is a method of treating a disease in a patient by administering a composition of the instant invention.
  • “Cationic component” means a chemical moiety that carries a positive charge.
  • a cationic component includes amines, nitrogen heterocycles, aldimines, hydrazides and hydrazones.
  • Cationic component is further defined as a “first cationic component” and a “second cationic component”. This further delineation is meant to show that the first cationic component is different than the second cationic component.
  • Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group.
  • Neitrogen heterocycle means an organic compound containing at least one atom of carbon and at least one atom on nitrogen within a ring structure. These structures may comprise either simple aromatic rings or non-aromatic rings.
  • Aldimine means a class of organic compounds with the general formula R—
  • Hydrazide means a class of organic compounds sharing a common functional group characterized by a nitrogen to nitrogen covalent bond with 4 substituents with at least one of them being an acyl group.
  • “Hydrazone” means a class of organic compounds with the structure
  • R 1 R 2 C NNH 2 .
  • Aliphatic component means a compound composed of carbon and hydrogen.
  • Aliphatic compounds can be cyclic, like cyclohexane, or acyclic, like hexane.
  • Aliphatic compounds can be saturated, like hexane, or unsaturated, like hexene.
  • Aliphatic compounds can be straight chains, branched chains, or non-aromatic rings (in which case they are called alicyclic).
  • Aliphatic compounds can be joined by single bonds (alkalies), double bonds (alkenes), or triple bonds (alkynes).
  • An aliphatic component includes aromatic components and steriods.
  • Aliphatic component is further defined as a "first aliphatic component” and a "second aliphatic component”. This further delineation is meant to show that the first aliphatic component is different than the second aliphatic component.
  • Aromatic component means a compound composed of hydrocarbon with a conjugated cyclic molecular structure.
  • Steproid includes, for example, cholesterol.
  • “Masking agent” means a molecule which, when linked to a polymer, shields, inhibits or inactivates one or more properties (biophysical or biochemical characteristics) of the polymer. See WO2008/022309 for a more detailed description of masking agents.
  • Targeting agent means an agent that can deliver a polymer or polyconjugate to target cells or tissues, or specific cells types. Targeting agents enhance the association of molecules with a target cell. Thus, targeting agents can enhance the pharmacokinetic or biodistribution properties of a polyconjugate to which they are attached to improve cellular distribution and cellular uptake of the conjugate. See WO2008/022309 for a more detailed description of targeting agents.
  • Polymer means a molecule built up by repetitive bonding together of smaller units called monomers.
  • a polymer can be linear, branched network, star, comb, or ladder type.
  • a polymer can be a homopolymer in which a single monomer is used or a polymer can be copolymer in which two or more different monomers are used. Copolymers may by alternating, random (statistical), block and graft (comb). The monomers in random copolymers have no definite order or arrangement along any given chain.
  • the general compositions of such polymers are reflective of the ratio of input monomers. However, the exact ratio of one monomer to another may differ between chains. The distribution of monomers may also differ along the length of a single polymer.
  • the chemical properties of a monomer may affect its rate of incorporation into a random copolymer and its distribution within the polymer.
  • the ratio of monomers in a random polymer is dependent on the input ratio of monomer, the input ratio may not match exactly the ratio of incorporated monomers. See WO2008/022309 for a more detailed description of polymers.
  • Oligonucleotide means deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinations of DNA, RNA and other natural and synthetic nucleotides.
  • DNA maybe in form of cDNA, in vitro polymerized DNA, plasmid DNA, parts of a plasmid DNA, genetic material derived from a virus, linear DNA, vectors (PI, PAC, BAC, YAC, and artificial chromosomes), expression vectors, expression cassettes, chimeric sequences, recombinant DNA, chromosomal DNA, anti-sense DNA, or derivatives of these groups.
  • RNA may be in the form of messengerRNA (mRNA), in vitro polymerized RNA, recombinant RNA, transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), chimeric sequences, anti-sense RNA, interfering RNA, small interfering RNA (siRNA), microRNA (miRNA), ribozymes, external guide sequences, small non-messenger RNAs (snmRNA), untranslatedRNA (utRNA), snoRNAs (24-mers, modified snmRNA that act by an anti-sense mechanism), tiny non-coding RNAs (tncRNAs), small hairpin RNA (shRNA), or derivatives of these groups.
  • mRNA messengerRNA
  • tRNA transfer RNA
  • snRNA small nuclear RNA
  • rRNA ribosomal RNA
  • RNA anti-sense RNA
  • interfering RNA small interfering RNA
  • miRNA small interfer
  • DNA and RNA may be single, double, triple, or quadraple stranded.
  • Double, triple, and quadruple stranded polynucleotide may contain both RNA and DNA or other combinations of natural and/or synthetic nucleic acids.
  • Oligonucleotides can be chemically modified. The use of chemically modified oligonucleotides can improve various properties of the oligonucleotides including, but not limited to: resistance to nuclease degradation in vivo, cellular uptake, activity, and sequence- specific hybridization. Non-limiting examples of such chemical modifications include:
  • Patient means a mammal, typically a human, in need of treatment for a disease.
  • Disease means a disorder or incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors; illness; sickness; ailment.
  • a cationic component is selected from an amine (primary, secondary, tertiary or quaternary), a nitrogen heterocycle, an aldimine, a hydrazide and a hydrazone.
  • a cationic component is selected from, 2-(2- aminoethoxy)ethyl, 2-(1H-imidazol-4 ⁇ yl)ethyl, 2-[2-(2-aminoethoxy)ethoxy]ethyl, 3-amino-2- hydroxypropyl, 2-aminoethyl, 4-aminobutyl, 6-aminohexyl, 8-aminooctyl and 10-aminodecyl.
  • a cationic component is selected 2-(2-aminoethoxy)ethyl and 2-(1H- imidazol-4-yl)ethyl.
  • an aliphatic (hydrophobic) component is selected from steroids, an alkyl group, an alkenyl group and an alkynyl group, all of which may be branched or cyclic or acyclic or aromatic.
  • an aliphatic component is selected from methyl, ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecy], cholesterol, lipid chains and benzyl.
  • an aliphatic component is selected from dodecyl, octyl and octadecyl.
  • a masking agent is selected from a disubstituted maleic anhydride derivative
  • a targeting agent is selected from compounds with affinity to cell surface molecules, cell receptor ligands, and antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules.
  • a targeting agent is selected from carbohydrates, glycans, saccharides (including, but not limited to: galactose, galactose derivatives, mannose, and mannose derivatives), vitamins, folate, biotin, aptamers, and peptides (including, but not limited to: RGD-containing peptides, insulin, EGF, and transferrin).
  • a targeting agent is selected from N-acetylgalactosamine, mannose and glucose.
  • an oligonucleotide is selected from siRNA, miRNA and antisense. In another embodiment, an oligonucleotide is an siRNA.
  • n 2 to 250.
  • n is 2 to 225.
  • n 2 to 200.
  • n 2 to 175.
  • n 2 to 150.
  • n is 2 to 125.
  • n is 2 to 100.
  • n 2 to 75.
  • n 2 to 50.
  • n 2 to 25.
  • n is 5 to 250.
  • n is 5 to 225.
  • n is 5 to 200.
  • n is 5 to 175.
  • n is 5 to 150.
  • n is 5 to 125.
  • n is 5 to 100.
  • n is 5 to 75.
  • n is 5 to 50.
  • n is 5 to 25.
  • n is 10 to 60. In another embodiment of Formula Z, n is 15 to 55.
  • n 20 to 50.
  • n 25 to 45.
  • n is 30 to 40.
  • substituents A and B are selected from, 2-(2- aminoethoxy)ethyl, 2-(1H-imidazol-4-yl)ethyl, 2-[2-(2-aminoethoxy)ethoxy]ethyl, 3-amino-2- hydroxypropyl, 2-aminoethyl, 4-aminobutyl, 6-aminohexyl, 8-aminooctyl and 10-aminodecyl.
  • substituents B and C are selected from steroids, an alkyl group, an alkenyl group and an alkynyl group, all of which may be branched or cyclic or acyclic or aromatic .
  • substituents B and C are selected from methyl, ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, cholesterol, lipid chains and benzyl.
  • substituents B and C are selected from methyl, ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl and cholesterol.
  • substituents B and C are selected from dodecyl, octyl and octadecyl.
  • substituent A is 2-(2-aminoethoxy)ethyl.
  • substituent B is 2-(1H-imidazol-4-yl)etliyl.
  • substituent C is dodecyl
  • the polyconjugate (composition of the polymer of Formula Z and an oligonucleotide) is formed by covalently linking the oligonucleotide to the polymer. Conjugation of the oligonucleotide to the polymer can be performed in the presence of an excess of polymer. Because the oligonucleotide and the polymer may be of opposite charge during conjugation, the presence of excess polymer can reduce or eliminate aggregation of the polyconjugate. Excess polymer can be removed from the polyconjugate prior to administration of the polyconjugate to a patient. Alternatively, excess polymer can be co-administered with the polyconjugate to the patient.
  • the polymer can be conjugated to a masking agent in the presence of an excess of polymer or masking agent. Because the oligonucleotide and the polymer may be of opposite charge during conjugation, the presence of excess polymer can reduce or eliminate aggregation of the polyconjugate. Excess polymer can be removed from the polyconjugate prior to administration of the polyconjugate to a patient. Alternatively, excess polymer can be coadministered with the polyconjugate to the patient. The polymer can be modified prior to or subsequent to conjugation of the oligonucleotide to the polymer.
  • Parenteral routes of administration include intravascular (intravenous, interarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, intrathecal, subdural, epidural, and intralympliatic injections that use a syringe and a needle or catheter.
  • Intravascular herein means within a tubular structure called a vessel that is connected to a tissue or organ within the body.
  • a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile.
  • CSF cerebrospinal fluid
  • lymphatic fluid or bile.
  • vessels examples include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, bile ducts, and ducts of the salivary or other exocrine glands.
  • the intravascular route includes delivery through the blood vessels such as an artery or a vein.
  • the blood circulatory system provides systemic spread of the pharmaceutical.
  • An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes.
  • Intraparenchymal includes direct injection into a tissue such as liver, lung, heart, muscle (skeletal muscle or diaphragm), spleen, pancreas, brain (including intraventricular), spinal cord, ganglion, lymph nodes, adipose tissues, thyroid tissue, adrenal glands, kidneys, prostate, and tumors.
  • a tissue such as liver, lung, heart, muscle (skeletal muscle or diaphragm), spleen, pancreas, brain (including intraventricular), spinal cord, ganglion, lymph nodes, adipose tissues, thyroid tissue, adrenal glands, kidneys, prostate, and tumors.
  • epithelial routes include oral, nasal, respiratory, rectum, and vaginal routes of administration.
  • the polyconjugates can be injected in a pharmaceutically acceptable carrier solution.
  • Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view.
  • pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a patient.
  • the term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • polyconjugates compositions of a polymer of Formula Z and an oligonucleotide
  • compositions of a polymer of Formula Z and an oligonucleotide may be used for research purposes or to produce a change in a cell that can be therapeutic.
  • the use of polyconjugates for therapeutic purposes is known. See WO2000/34343; WO2008/022309; and Rozema et al. PNAS (2008) 104, 32: 12982- 12987.
  • the monomers were weighed and brought up in 30% ethanol solution in water.
  • reaction mixture was stirred at 55 °C in dark for 5 days under nitrogen atmosphere. After 5 days, polymerization was quenched by adding 10 mol% excess of amine to consume any unreacted acrylate. After that, polymer was precipitated with diethylether and dried. Polymers with Boc-protected oligoamines were deprotected by TF A. The crude polymer was precipitated again in diethylether. The polymer was further purified by dialysis.
  • cystaminebisacrylamide 125 mg, 0.48 mmol, 1 equiv
  • teft-butyl [2-(2-aminoethoxy)ethyl]caibamate 39 mg, 0.192 mmol, 0.4 equiv
  • histamine 16 mg, 0.144 mmol, 0.3 equiv
  • dodecylamine 27 mg, 0.144 mmol, 0.3 equiv
  • the reaction mixture was stirred at 55 °C for 5 days in dark under nitrogen atmosphere. After 5 days, polymerization was quenched by adding 10 mol% excess of dodecylamine to consume any unreacted bisacrylamide group. After that, polymer was precipitated with 100 ml diethylether and dried.
  • Boc-amine was carried out by dissolving protected polymer in 2 ml of TFA/TIS/H2O 95/2.5/2.5 solutions for 30 min at room temperature. The crude polymer was precipitated out again in 100 ml diethylether and dried. It was further purified by dialysis using 2k cut off membrane against Milli-Q water and then lyophilized.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 1 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 2 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 3 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 4 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 5 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 6 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 8 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 10 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 1 1 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 12 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 14 include substituents A, B and C in the identified % ratios.
  • Polymers of the instant invention include:
  • x, y and z are between 0 to 50 and individual polymers in Table 15 include substituents A, B and C in the identified % ratios.
  • polymers of Formula Z and the specific examples shown above were synthesized for use in the following conjugation steps to ultimately create the polyconjugates of the instant invention.
  • the polymers of Formula Z and the specific examples disclosed are useful in the preparation of polyconjugates which are, in turn, useful for the delivery of nucleic acids, specifically the delivery of siRNA.
  • Other methods for the synthesis of polyconjugates are described in WO2008/022309.
  • Step 1 Activation of polymer
  • Step 2 Activation of siRNA
  • siRNA (lg, 0.0714 mmol) is dissolved in 0.1M sodium bicarbonate buffer (20 ml, 50 mg/mL) in a vial with magnetic stir bar and cooled to 0-5 °C in an ice water bath.
  • N-Succinimidyl-S-acetylthioacetate (83 mg, 0.357 mmol, 5 equivalents) is dissolved in 0.78 ml DMSO. The SATA solution is added over lmin and the clear, colorless reaction mixture stirred at 0-5 °C for 2h. After 2h, the reaction mixture is sampled and analyzed by UPLC or HPLC for completion of the conjugation.
  • the activated polymer is diluted with additional 5mM TAPS 5% glucose buffer pH 9 resulting in a final polymer concentration of - 1.9 mg/mL.
  • About 0.66 mg of siRNA is added to the acvitated polymer solution and stirred at room temperature overnight and proceeded to final masking step.
  • CDM-PEG carboxydimethylmaleic anhydride-polyethyleneglycol
  • CDM-NAG carboxydimethylmaleic anhydride-N-acetylgalactosamine
  • Red blood cells were washed three times in either 150mM NaCl/20mM MES, pH 5.4, or 150mM NaC3/20mM HEPES, pH 7.5 by centrifuging at 1700 x g for 3 min and resuspending in the same buffer to yield the initial volume. RBCs were then diluted in appropriate pH buffer to yield 10 cells in suspension.
  • a lOx stock concentration of each test agent (Polymer 1, Polymer 2, Polymer 3 and Polymer 4) was prepared and a 10 point, 2-fold dilution was performed in appropriate pH buffers.
  • the diluted test agents were added to the RBCs in appropriate pH buffers in Costar 3368 flat-bottom 96 well plates. Solutions were mixed 6 to 8 times and the microliter plate was covered with a low evaporation lid and incubated in a 37°C warm room or incubator for 30 minutes to induce hemolysis. The plate was then centrifuged at 1700 x g for 5 min and 150 ⁇ supernatants were transferred to a Costar 3632 clear bottom 96 well plate. Hemoglobin absorbance was read at 541nM using a Tecan Safire plate reader and percent hemolysis was calculated assuming 100% lysis to be measured by the hemoglobin released by RBCs in 1% Triton X-100.
  • the data demonstrate that the polymers are lytic at endosomal pH 5.4.
  • the ratio of hydrophobic group (dodecyl) increases in the polymer from 20% to 50% the lytic activity increases.
  • Incorporation of histidine in the polymers further enhanced the lytic acitivity of polymer.
  • the data demonstrates that in extracellular environment at pH 7.5, the polymer is masked with CDM and do not have any lytic activity. At endosomal pH 5.4, after the demasking of CDM, the polymer retains its lytic properties.
  • HepG2 cells were plated in 96- well microtiter plates at 6000 cells/well and incubated overnight at 37 °C to allow cell adherence.
  • lOx stock of PCs (polyconjugates) were prepared in media and 20 ⁇ 1 lOx PC was added to 180 ⁇ media already in wells resulting in lx final treatment and a 300-0 nM 10-point half log titration, based on siRNA concentration.
  • Cells were incubated with PCs in 37 degrees C0 2 incubator for 24 -72h.
  • MTS Toxicity Assay was performed on 24h - 72h treated cells and cytotoxicity was assessed by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega #G3581 S Madison, WI).
  • ApoB mRNA knockdown was determined using Quantigene 1.0 bDNA Assay (Panomics # QG0002 Lot # 51 CW36, Fremont, CA), a kit designed to quantitate RNA using a set of target-specific oligonucleotide probes.
  • DLM diluted lysis mixture
  • PBS Nuclease Free water
  • Wash buffer add 3ml Component 1 and 5ml Component 2 to 1L distilled water. (Wash Buffer is stable at Room Temperature for up to 6 months)
  • CD1 mice were tail vein injected with the freshly prepared siRNA containing polymer conjugates at a dose of 3 mg/kg in a volume of 0.2 mL, lOOmM TRIS 5% glucose, pH9, vehicle. Forty-eight hours post dose, mice were sacrificed and liver tissue samples were immediately preserved in RNALater (Ambion). Preserved liver tissue was homogenized and total RNA isolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNA isolation kit following the manufacturer's instructions. Liver ApoB mRNA levels were determined by quantitative RT-PCR. Message was amplified from purified RNA utilizing primers against the mouse ApoB mRNA (Applied Biosystems Cat. No.
  • the PCR reaction was run on an ABI 7500 instrument with a 96- well Fast Block.
  • the ApoB mRNA level is normalized to the housekeeping PPIB mRNA and GAPDH.
  • PPIB and GAPDH mRNA levels were determined by RT-PCR using a commercial probe set (Applied Biosytems Cat. No.
  • Results are expressed as a ratio of ApoB mRNA/ PPIB / GAPDH mRNA. All mRNA data is expressed relative to the vehicle control.

Abstract

The present invention provides endosomolytic poly(amidoamine) disulfide polymers, polyconjugates, compositions and methods for the delivery of oligonucleotides for therapeutic purposes.

Description

TITLE OF THE INVENTION
ENDOSOMOLYTIC POLY(AMIDO AMINE) DISULFIDE POLYMERS FOR THE
DELIVERY OF OLIGONUCLEOTIDES
BACKGROUND OF THE INVENTION
Oligonucleotides conjugated to polymers are known. Further, the delivery of oligonucleotides conjugated to polymers (polyconjugates) for therapeutic purposes is also known. See WO2000/34343; WO2008/022309; and Rozema et al. PNAS (2008) 104, 32: 12982- 12987.
Poly(amidoamine) disulfide polymers are known. Ou et al. Bioconjugate Chem. (2008) 19: 626-633 and Lin et al. J. of Controlled Release (2008) 126: 166-174.
Polyconjugates have numerous toxicities associated with extensive circulation half-life. It is thus an object of the invention to provide polyconjugates that are biodegradable. Herein, we disclose and describe novel endosomolytic poly(amidoamine) disulfide polymers and polyconjugates useful for the delivery of oligonucleotides for therapeutic purposes. The poly(amidoamine) disulfide polymers of the instant invention are novel and contain both cationic and aliphatic functional moieties. These polymers are biodegradable and are therefore less toxic.
SUMMARY OF THE INVENTION
The present invention provides endosomolytic poly(amidoamine) disulfide polymers, polyconjugates, compositions and methods for the delivery of oligonucleotides for therapeutic purposes.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. RBC Hemolysis Data of Polymers at pH 5.5.
FIG. 2. RBC Hemolysis Data of Masked Polyconjugate.
FIG. 3. In-Vitro Data.
FIG. 4. In-Vivo Data.
DETAILED DESCRIPTION OF THE INVENTION
An embodiment of the instant invention is a polymer of Formula Z:
Figure imgf000003_0001
Z wherein:
n is 2 to 250; R is independently selected from A, B and C, wherein at least two of A, B and C are present in the polymer and the polymer must include at least one cationic component (A or B) and at least one aliphatic component (B or C);
A is a first cationic component;
B is a second cationic component or a second aliphatic component; and
C is a first aliphatic component;
or stereoisomer thereof.
In a second embodiment of the instant invention is a polymer of Formula Z:
Figure imgf000004_0001
wherein:
n is 2 to 250;
R is independently selected from A, B and C, wherein at least two of A, B and C are present in the polymer and the polymer must include at least one cationic component (A or B) and at least one aliphatic component (B or C);
A is a first cationic component selected from an amine (primary, secondary, tertiary or quaternary), a nitrogen heterocycle, an aldimine, a hydrazide and a hydrazone;
B is a second cationic component selected from an amine (primary, secondary, tertiary or quaternary), a nitrogen heterocycle, an aldimine, a hydrazide and a hydrazone, or a second aliphatic component; and
C is a first aliphatic component;
or stereoisomer thereof.
In a third embodiment of the instant invention is a polymer of Formula Z:
Figure imgf000004_0002
wherein:
n is 2 to 250;
R is independently selected from A, B and C, wherein A, B and C are all present in the polymer;
A is 2-(2-aminoethoxy)ethyl;
B is 2-(1H-imidazol-4»y!)ethyl; and C is dodecyl;
or a stereoisomer thereof.
In another embodiment of the polymer of Formula Z, R is independently selected from A, B and C, wherein A, B and C are all present in the polymer and wherein A, B and C are different from one another.
In another embodiment of the polymer of Formula Z, R is independently selected from A, B and C, wherein A, B and C are all present in the polymer and wherein A and B are different from one another.
In another embodiment of the polymer of Formula Z, R is independently selected from A, B and C, wherein A, B and C are all present in the polymer and wherein B and C are different from one another.
In another embodiment of the polymer of Formula Z, substituent A must comprise at least 50% of the polymer.
In another embodiment of the polymer of Formula Z, substituent B must comprise at least 50% of the polymer.
In another embodiment of the polymer of Formula Z, substituent C must comprise at least 50% of the polymer.
Another aspect of the instant invention is a composition comprising a polymer of Formula Z and an oligonucleotide.
Another aspect of the instant invention is a composition comprising a polymer of
Formula Z, a masking agent and an oligonucleotide.
Another aspect of the instant invention is a composition comprising a polymer of Formula Z, a masking agent, a targeting agent and an oligonucleotide.
Another aspect of the instant invention is a method of treating a disease in a patient by administering a composition of the instant invention.
DEFINITIONS
"Cationic component" means a chemical moiety that carries a positive charge. A cationic component includes amines, nitrogen heterocycles, aldimines, hydrazides and hydrazones. Cationic component is further defined as a "first cationic component" and a "second cationic component". This further delineation is meant to show that the first cationic component is different than the second cationic component.
"Amine (primary, secondary, tertiary or quaternary)" means organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group.
"Nitrogen heterocycle" means an organic compound containing at least one atom of carbon and at least one atom on nitrogen within a ring structure. These structures may comprise either simple aromatic rings or non-aromatic rings. "Aldimine" means a class of organic compounds with the general formula R—
CH=N-R'.
"Hydrazide" means a class of organic compounds sharing a common functional group characterized by a nitrogen to nitrogen covalent bond with 4 substituents with at least one of them being an acyl group. The general structure for a hydrazide is (Ri=0)R2-N-N~R3R4.
"Hydrazone" means a class of organic compounds with the structure
R1R2C=NNH2.
"Aliphatic component" means a compound composed of carbon and hydrogen. Aliphatic compounds can be cyclic, like cyclohexane, or acyclic, like hexane. Aliphatic compounds can be saturated, like hexane, or unsaturated, like hexene. Aliphatic compounds can be straight chains, branched chains, or non-aromatic rings (in which case they are called alicyclic). Aliphatic compounds can be joined by single bonds (alkalies), double bonds (alkenes), or triple bonds (alkynes). An aliphatic component includes aromatic components and steriods. Aliphatic component is further defined as a "first aliphatic component" and a "second aliphatic component". This further delineation is meant to show that the first aliphatic component is different than the second aliphatic component.
"Aromatic component" means a compound composed of hydrocarbon with a conjugated cyclic molecular structure.
"Steroid" includes, for example, cholesterol.
"Masking agent" means a molecule which, when linked to a polymer, shields, inhibits or inactivates one or more properties (biophysical or biochemical characteristics) of the polymer. See WO2008/022309 for a more detailed description of masking agents.
"Targeting agent" means an agent that can deliver a polymer or polyconjugate to target cells or tissues, or specific cells types. Targeting agents enhance the association of molecules with a target cell. Thus, targeting agents can enhance the pharmacokinetic or biodistribution properties of a polyconjugate to which they are attached to improve cellular distribution and cellular uptake of the conjugate. See WO2008/022309 for a more detailed description of targeting agents.
"Polymer" means a molecule built up by repetitive bonding together of smaller units called monomers. A polymer can be linear, branched network, star, comb, or ladder type. A polymer can be a homopolymer in which a single monomer is used or a polymer can be copolymer in which two or more different monomers are used. Copolymers may by alternating, random (statistical), block and graft (comb). The monomers in random copolymers have no definite order or arrangement along any given chain. The general compositions of such polymers are reflective of the ratio of input monomers. However, the exact ratio of one monomer to another may differ between chains. The distribution of monomers may also differ along the length of a single polymer. Also, the chemical properties of a monomer may affect its rate of incorporation into a random copolymer and its distribution within the polymer. Thus, while the ratio of monomers in a random polymer is dependent on the input ratio of monomer, the input ratio may not match exactly the ratio of incorporated monomers. See WO2008/022309 for a more detailed description of polymers.
"Oligonucleotide" means deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinations of DNA, RNA and other natural and synthetic nucleotides. DNA maybe in form of cDNA, in vitro polymerized DNA, plasmid DNA, parts of a plasmid DNA, genetic material derived from a virus, linear DNA, vectors (PI, PAC, BAC, YAC, and artificial chromosomes), expression vectors, expression cassettes, chimeric sequences, recombinant DNA, chromosomal DNA, anti-sense DNA, or derivatives of these groups. RNA may be in the form of messengerRNA (mRNA), in vitro polymerized RNA, recombinant RNA, transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), chimeric sequences, anti-sense RNA, interfering RNA, small interfering RNA (siRNA), microRNA (miRNA), ribozymes, external guide sequences, small non-messenger RNAs (snmRNA), untranslatedRNA (utRNA), snoRNAs (24-mers, modified snmRNA that act by an anti-sense mechanism), tiny non-coding RNAs (tncRNAs), small hairpin RNA (shRNA), or derivatives of these groups. In addition, DNA and RNA may be single, double, triple, or quadraple stranded. Double, triple, and quadruple stranded polynucleotide may contain both RNA and DNA or other combinations of natural and/or synthetic nucleic acids. Oligonucleotides can be chemically modified. The use of chemically modified oligonucleotides can improve various properties of the oligonucleotides including, but not limited to: resistance to nuclease degradation in vivo, cellular uptake, activity, and sequence- specific hybridization. Non-limiting examples of such chemical modifications include:
phosphorothioate internucleotide linkages, LNA, 2 -O-methyI ribonucleotides, 2'-deoxy-2'-fiuoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. These chemical modifications, when used in various oligonucleotide constructs, are shown to preserve oligonucleotide activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Chemically modified siRNA can also minimize the possibility of activating interferon activity in humans. See WO2008/022309 for a more detailed description of oligonucleotides.
"Patient" means a mammal, typically a human, in need of treatment for a disease. "Disease" means a disorder or incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors; illness; sickness; ailment.
In an embodiment, a cationic component is selected from an amine (primary, secondary, tertiary or quaternary), a nitrogen heterocycle, an aldimine, a hydrazide and a hydrazone. In another embodiment, a cationic component is selected from, 2-(2- aminoethoxy)ethyl, 2-(1H-imidazol-4~yl)ethyl, 2-[2-(2-aminoethoxy)ethoxy]ethyl, 3-amino-2- hydroxypropyl, 2-aminoethyl, 4-aminobutyl, 6-aminohexyl, 8-aminooctyl and 10-aminodecyl. In another embodiment, a cationic component is selected 2-(2-aminoethoxy)ethyl and 2-(1H- imidazol-4-yl)ethyl. In an embodiment, an aliphatic (hydrophobic) component is selected from steroids, an alkyl group, an alkenyl group and an alkynyl group, all of which may be branched or cyclic or acyclic or aromatic. In another embodiment, an aliphatic component is selected from methyl, ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecy], cholesterol, lipid chains and benzyl. In another embodiment, an aliphatic component is selected from dodecyl, octyl and octadecyl.
In an embodiment, a masking agent is selected from a disubstituted maleic anhydride derivative
In an embodiment, a targeting agent is selected from compounds with affinity to cell surface molecules, cell receptor ligands, and antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules. In another embodiment, a targeting agent is selected from carbohydrates, glycans, saccharides (including, but not limited to: galactose, galactose derivatives, mannose, and mannose derivatives), vitamins, folate, biotin, aptamers, and peptides (including, but not limited to: RGD-containing peptides, insulin, EGF, and transferrin). In another embodiment, a targeting agent is selected from N-acetylgalactosamine, mannose and glucose.
In an embodiment, an oligonucleotide is selected from siRNA, miRNA and antisense. In another embodiment, an oligonucleotide is an siRNA.'
In an embodiment of Formula Z, n is 2 to 250.
In another embodiment of Formula Z, n is 2 to 225.
In another embodiment of Formula Z, n is 2 to 200.
In another embodiment of Formula Z, n is 2 to 175.
In another embodiment of Formula Z, n is 2 to 150.
In another embodiment of Formula Z, n is 2 to 125.
In another embodiment of Formula Z, n is 2 to 100.
In another embodiment of Formula Z, n is 2 to 75.
In another embodiment of Formula Z, n is 2 to 50.
In another embodiment of Formula Z, n is 2 to 25.
In an embodiment of Formula Z, n is 5 to 250.
In another embodiment of Formula Z, n is 5 to 225.
In another embodiment of Formula Z, n is 5 to 200.
In another embodiment of Formula Z, n is 5 to 175.
In another embodiment of Formula Z, n is 5 to 150.
In another embodiment of Formula Z, n is 5 to 125.
In another embodiment of Formula Z, n is 5 to 100.
In another embodiment of Formula Z, n is 5 to 75.
In another embodiment of Formula Z, n is 5 to 50.
In another embodiment of Formula Z, n is 5 to 25.
In another embodiment of Formula Z, n is 10 to 60. In another embodiment of Formula Z, n is 15 to 55.
In another embodiment of Formula Z, n is 20 to 50.
In another embodiment of Formula Z, n is 25 to 45.
In another embodiment of Formula Z, n is 30 to 40.
In an embodiment of Formula Z, substituents A and B are selected from, 2-(2- aminoethoxy)ethyl, 2-(1H-imidazol-4-yl)ethyl, 2-[2-(2-aminoethoxy)ethoxy]ethyl, 3-amino-2- hydroxypropyl, 2-aminoethyl, 4-aminobutyl, 6-aminohexyl, 8-aminooctyl and 10-aminodecyl.
In an embodiment of Formula Z, substituents B and C are selected from steroids, an alkyl group, an alkenyl group and an alkynyl group, all of which may be branched or cyclic or acyclic or aromatic .
In another embodiment of Formula Z, substituents B and C are selected from methyl, ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, cholesterol, lipid chains and benzyl.
In another embodiment of Formula Z, substituents B and C are selected from methyl, ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl and cholesterol.
In another embodiment of Formula Z, substituents B and C are selected from dodecyl, octyl and octadecyl.
In an embodiment of Formula Z, substituent A is 2-(2-aminoethoxy)ethyl.
In an embodiment of Formula Z, substituent B is 2-(1H-imidazol-4-yl)etliyl.
In an embodiment of Formula Z, substituent C is dodecyl.
FORMULATION
The polyconjugate (composition of the polymer of Formula Z and an oligonucleotide) is formed by covalently linking the oligonucleotide to the polymer. Conjugation of the oligonucleotide to the polymer can be performed in the presence of an excess of polymer. Because the oligonucleotide and the polymer may be of opposite charge during conjugation, the presence of excess polymer can reduce or eliminate aggregation of the polyconjugate. Excess polymer can be removed from the polyconjugate prior to administration of the polyconjugate to a patient. Alternatively, excess polymer can be co-administered with the polyconjugate to the patient.
Similarly, the polymer can be conjugated to a masking agent in the presence of an excess of polymer or masking agent. Because the oligonucleotide and the polymer may be of opposite charge during conjugation, the presence of excess polymer can reduce or eliminate aggregation of the polyconjugate. Excess polymer can be removed from the polyconjugate prior to administration of the polyconjugate to a patient. Alternatively, excess polymer can be coadministered with the polyconjugate to the patient. The polymer can be modified prior to or subsequent to conjugation of the oligonucleotide to the polymer.
Parenteral routes of administration include intravascular (intravenous, interarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, intrathecal, subdural, epidural, and intralympliatic injections that use a syringe and a needle or catheter. Intravascular herein means within a tubular structure called a vessel that is connected to a tissue or organ within the body. Within the cavity of the tubular structure, a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, bile ducts, and ducts of the salivary or other exocrine glands. The intravascular route includes delivery through the blood vessels such as an artery or a vein. The blood circulatory system provides systemic spread of the pharmaceutical. An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes. Intraparenchymal includes direct injection into a tissue such as liver, lung, heart, muscle (skeletal muscle or diaphragm), spleen, pancreas, brain (including intraventricular), spinal cord, ganglion, lymph nodes, adipose tissues, thyroid tissue, adrenal glands, kidneys, prostate, and tumors. Transdermal routes of
administration have been affected by patches and iontophoresis. Other epithelial routes include oral, nasal, respiratory, rectum, and vaginal routes of administration.
The polyconjugates can be injected in a pharmaceutically acceptable carrier solution. Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a patient. Preferably, as used herein, the term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
UTILITY
The polyconjugates (compositions of a polymer of Formula Z and an oligonucleotide) of the instant invention may be used for research purposes or to produce a change in a cell that can be therapeutic. The use of polyconjugates for therapeutic purposes is known. See WO2000/34343; WO2008/022309; and Rozema et al. PNAS (2008) 104, 32: 12982- 12987.
EXAMPLES
Examples and schemes provided are intended to assist in a further understanding of the invention. Particular materials employed, species and conditions are intended to be further illustrative of the invention and not limitative of the reasonable scope thereof.
GENERAL POLYMER SYNTHESIS
The monomers were weighed and brought up in 30% ethanol solution in water.
The reaction mixture was stirred at 55 °C in dark for 5 days under nitrogen atmosphere. After 5 days, polymerization was quenched by adding 10 mol% excess of amine to consume any unreacted acrylate. After that, polymer was precipitated with diethylether and dried. Polymers with Boc-protected oligoamines were deprotected by TF A. The crude polymer was precipitated again in diethylether. The polymer was further purified by dialysis.
Figure imgf000011_0001
POLYMER SYNTHESIS (Scheme 1)
In a typical experiment, cystaminebisacrylamide (125 mg, 0.48 mmol, 1 equiv), teft-butyl [2-(2-aminoethoxy)ethyl]caibamate: 39 mg, 0.192 mmol, 0.4 equiv), histamine (16 mg, 0.144 mmol, 0.3 equiv) and dodecylamine ( 27 mg, 0.144 mmol, 0.3 equiv) were weighed in the reaction flask and 1 ml of 30% ethanol solution in water was added to it. The reaction mixture was stirred at 55 °C for 5 days in dark under nitrogen atmosphere. After 5 days, polymerization was quenched by adding 10 mol% excess of dodecylamine to consume any unreacted bisacrylamide group. After that, polymer was precipitated with 100 ml diethylether and dried.
The deprotection of Boc-amine was carried out by dissolving protected polymer in 2 ml of TFA/TIS/H2O 95/2.5/2.5 solutions for 30 min at room temperature. The crude polymer was precipitated out again in 100 ml diethylether and dried. It was further purified by dialysis using 2k cut off membrane against Milli-Q water and then lyophilized.
5 days
in H20,
Figure imgf000012_0001
1H and 13C NMR spectra were recorded on Varian spectrometer operating at 500 MHz. 1H NMR spectra were in rail accordance with the expected structures. No signals were present in the region between 5 and 7 ppm, corresponding to the acrylamide group, indicating that these polymers have amino-capped end groups. All NMR spectra were taken in deuterated methanol. The molecular weight and polydispersity (Mw/Mn) of the synthesized polymers were determined by GPC relative to polystyrene standards (Sigma- Aldrich) using a Waters 2695, Waters 2414 RI detector and TSK-GEL Alpha-2500 column. Total amine content and the incorporation ratios of monomers in the polymers were calculated by treating the polymers with excess of DTT and then analysed by LC-MS. POLYMER SYNTHESIS (Scheme 2)
Figure imgf000013_0001
The following polymers were prepared according to the General Reaction Scheme and Schemes above.
Polymer 1
Polymers of the instant invention include:
Figure imgf000014_0001
wherein x, y and z are between 0 to 50 and individual polymers in Table 1 include substituents A, B and C in the identified % ratios.
Figure imgf000014_0003
Polymer 2
Polymers of the instant invention include:
Figure imgf000014_0002
wherein x, y and z are between 0 to 50 and individual polymers in Table 2 include substituents A, B and C in the identified % ratios.
Figure imgf000015_0003
Polymer 3
Polymers of the instant invention include:
Figure imgf000015_0001
wherein x, y and z are between 0 to 50 and individual polymers in Table 3 include substituents A, B and C in the identified % ratios.
Figure imgf000015_0004
Polymer 4
Polymers of the instant invention include:
Figure imgf000015_0002
wherein x, y and z are between 0 to 50 and individual polymers in Table 4 include substituents A, B and C in the identified % ratios.
Figure imgf000015_0005
Figure imgf000016_0001
Polymer 5
Polymers of the instant invention include:
Figure imgf000016_0002
wherein x, y and z are between 0 to 50 and individual polymers in Table 5 include substituents A, B and C in the identified % ratios.
Table 5
Figure imgf000016_0004
Polymer 6
Polymers of the instant invention include:
Figure imgf000016_0003
wherein x, y and z are between 0 to 50 and individual polymers in Table 6 include substituents A, B and C in the identified % ratios.
Figure imgf000017_0003
Polymer 7
Polymers of the instant invention include:
Figure imgf000017_0001
wherein x, y and z are between 0 to 50 and individual polymers in Table 7 include substituents A. B and C in the identified % ratios.
Figure imgf000017_0004
Polymer 8
Polymers of the instant invention include:
Figure imgf000017_0002
wherein x, y and z are between 0 to 50 and individual polymers in Table 8 include substituents A, B and C in the identified % ratios.
Figure imgf000017_0005
Figure imgf000018_0005
Polymer 9
Polymers of the instant invention include:
Figure imgf000018_0001
wherein x, y and z are between 0 to 50 and individual polymers in Table 9 include substituents A, B and C in the identified % ratios.
Figure imgf000018_0003
Polymer 10
Polymers of the instant invention include:
Figure imgf000018_0002
wherein x, y and z are between 0 to 50 and individual polymers in Table 10 include substituents A, B and C in the identified % ratios.
Figure imgf000018_0004
Polymer 11
Polymers of the instant invention include:
Figure imgf000019_0002
wherein x, y and z are between 0 to 50 and individual polymers in Table 1 1 include substituents A, B and C in the identified % ratios.
Figure imgf000019_0003
Polymer 12
Polymers of the instant invention include:
Figure imgf000019_0001
wherein x, y and z are between 0 to 50 and individual polymers in Table 12 include substituents A, B and C in the identified % ratios.
Figure imgf000019_0004
Polymer 13
Polymers of the instant invention include:
Figure imgf000020_0001
wherein x, y and z are between 0 to 50 and individual polymers in Table 13 include substituents A, B and C in the identified % ratios. able 13
Figure imgf000020_0003
Polymer 14
Polymers of the instant invention include:
Figure imgf000020_0002
wherein x, y and z are between 0 to 50 and individual polymers in Table 14 include substituents A, B and C in the identified % ratios.
Figure imgf000020_0004
Polymer 15
Polymers of the instant invention include:
Figure imgf000021_0001
wherein x, y and z are between 0 to 50 and individual polymers in Table 15 include substituents A, B and C in the identified % ratios.
Figure imgf000021_0002
CONJUGATION (Scheme 3)
The polymers of Formula Z and the specific examples shown above were synthesized for use in the following conjugation steps to ultimately create the polyconjugates of the instant invention. The polymers of Formula Z and the specific examples disclosed are useful in the preparation of polyconjugates which are, in turn, useful for the delivery of nucleic acids, specifically the delivery of siRNA. Other methods for the synthesis of polyconjugates are described in WO2008/022309.
Step 1 : Activation of polymer
About 2.6 mg of polymer in a 4 mL vial is added with ~ 87 μΐ, of 5 mM TAPS, pH 9 buffer and stirred until the polymer is dissolved. To this solution was added 3.9 of (4- succinimidyloxycarbonyl-{-methyl-(-[2-pyridyldithio]toluene) solution (lmg/100 μΐ in DMSO) corresponding to 1.5 wt% with respect to the polymer weight.
Step 2: Activation of siRNA
siRNA (lg, 0.0714 mmol) is dissolved in 0.1M sodium bicarbonate buffer (20 ml, 50 mg/mL) in a vial with magnetic stir bar and cooled to 0-5 °C in an ice water bath. In a separate vial N-Succinimidyl-S-acetylthioacetate (SATA) (83 mg, 0.357 mmol, 5 equivalents) is dissolved in 0.78 ml DMSO. The SATA solution is added over lmin and the clear, colorless reaction mixture stirred at 0-5 °C for 2h. After 2h, the reaction mixture is sampled and analyzed by UPLC or HPLC for completion of the conjugation. If >5 % siRNA remains unreacted, another charge of SATA in DMSO (2.0 equivalents) is added and the reaction aged at 0-5 °C for completion of the SATA conjugation (confirmation by HPLC or UPLC). When there is < 5% unreacted siRNA remaining by UPLC or HPLC, the reaction mixture is purified by TFF dialysis using water (~ 2L) or PD10 column to remove any remaining SATA/succinimides. The recovered purified solution is lyophilized to a white fluffy solid. The recovery is typically around 95% and the purity is greater than 70% by UPLC.
Step 3: PoIvmer-siRNA conjugation
The activated polymer is diluted with additional 5mM TAPS 5% glucose buffer pH 9 resulting in a final polymer concentration of - 1.9 mg/mL. About 0.66 mg of siRNA is added to the acvitated polymer solution and stirred at room temperature overnight and proceeded to final masking step.
Step 4: Masking of polymer conjugate
To the polymer-siRNA conjugate add 1.15 mg of TAPS free base. In a separate vial, 0.4 mg of carboxydimethylmaleic anhydride-polyethyleneglycol (CDM-PEG) is weighed and 0.775 mg of carboxydimethylmaleic anhydride-N-acetylgalactosamine (CDM- NAG) is added to this. The siRNA-polymer conjugate solution is then transferred into this vial containing CDM-PEG and CDM-NAG and stirred for lhr at room temperature.
Figure imgf000023_0001
-21 - EXAMPLE 1
RBC Hemolysis Assay:
Human blood was collected in 10ml EDTA Vacutainer tubes. A small aliquot was assessed for evidence of hemolysis by centrifugation at 15000 RCF for 2 min and non- hemolyzed samples were carried forward into the assay. Red blood cells (RBCs) were washed three times in either 150mM NaCl/20mM MES, pH 5.4, or 150mM NaC3/20mM HEPES, pH 7.5 by centrifuging at 1700 x g for 3 min and resuspending in the same buffer to yield the initial volume. RBCs were then diluted in appropriate pH buffer to yield 10 cells in suspension. A lOx stock concentration of each test agent (Polymer 1, Polymer 2, Polymer 3 and Polymer 4) was prepared and a 10 point, 2-fold dilution was performed in appropriate pH buffers. The diluted test agents were added to the RBCs in appropriate pH buffers in Costar 3368 flat-bottom 96 well plates. Solutions were mixed 6 to 8 times and the microliter plate was covered with a low evaporation lid and incubated in a 37°C warm room or incubator for 30 minutes to induce hemolysis. The plate was then centrifuged at 1700 x g for 5 min and 150 μΐ supernatants were transferred to a Costar 3632 clear bottom 96 well plate. Hemoglobin absorbance was read at 541nM using a Tecan Safire plate reader and percent hemolysis was calculated assuming 100% lysis to be measured by the hemoglobin released by RBCs in 1% Triton X-100.
As shown in Figure 1, the data demonstrate that the polymers are lytic at endosomal pH 5.4. As the ratio of hydrophobic group (dodecyl) increases in the polymer from 20% to 50% the lytic activity increases. Incorporation of histidine in the polymers further enhanced the lytic acitivity of polymer.
As shown in Figure 2, the data demonstrates that in extracellular environment at pH 7.5, the polymer is masked with CDM and do not have any lytic activity. At endosomal pH 5.4, after the demasking of CDM, the polymer retains its lytic properties.
EXAMPLE 2
HepG2 gene silencing and toxicity data:
HepG2 cells were plated in 96- well microtiter plates at 6000 cells/well and incubated overnight at 37 °C to allow cell adherence. lOx stock of PCs (polyconjugates) were prepared in media and 20μ1 lOx PC was added to 180 μΐ media already in wells resulting in lx final treatment and a 300-0 nM 10-point half log titration, based on siRNA concentration. Cells were incubated with PCs in 37 degrees C02 incubator for 24 -72h. MTS Toxicity Assay was performed on 24h - 72h treated cells and cytotoxicity was assessed by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega #G3581S Madison, WI). 40μ1 MTS Solution was added, incubated in 37 degrees C02 incubator 1 hour, absorbance at 490nm was read on Tecan Safire. Cells were then washed 3x in PBS and 150μ1Λνβ11 bDNA DLM Lysis Buffer (Panomics "Quantigene" 1.0 bDNA kit #QG0002, Fremont, CA) was added. Plate was then incubated at 37 degrees in Warm Room 30 min. Lysates were removed and frozen at -70 degrees C overnight. The next day, all cell lysates were thawed at RT and 20μ1 of each lysate was removed and used for determination of total protein using Micro BCA Protein Assay kit (Pierce #23235, through Thermo Scientific, Rockford, IL). Absorbance was measured on Tecan Safire: Wavelength = 562nM, Plate = Costar96ft, Number of Reads = 100, Time between Reads = 5. 50μ1 each lysate was also used to determine mRNA expression levels in cells treated with SSB siRNA.
ApoB mRNA knockdown was determined using Quantigene 1.0 bDNA Assay (Panomics # QG0002 Lot # 51 CW36, Fremont, CA), a kit designed to quantitate RNA using a set of target-specific oligonucleotide probes.
Zimmerman Apo B siRNA was utilized in the experiments. Zimmermann et al., (2006) Nature, 441(7089): 111-114 or doi:10.1038/nature04688 (see supplementary information). Panomics Quantigene bDNA Kit # QG0002 - protocol for 96 well plate:
Day 1
Make diluted lysis mixture (DLM) by mixing 1 volume of lysis mixture with 2 volumes of Nuclease Free water (Ambion cat # AM9930). Aspirate (PBS) from plate. Add 150μ1 DLM to each well and mix. (Include Column 1 as Buffer Alone Background). Incubate at 37°C for 30 minutes. (After heating, Lysates can be placed in the -70°C freezer until analysis is performed. If lysates are frozen, thaw at Room Temperature and incubate at 37°C for 30 minutes and mix well before adding the samples to the capture plate.) Bring all reagents to Room Temperature before use, including the capture plates. Dilute CE, LE and BL probe set components: Ο.ΐ μΐ/well each into DLM. Add (100-X) μΐ diluted probe set/well. Add ( X ) μΐ cell lysate/well. Cover with foil plate sealer. Incubate at 53°C for 16-20 hrs. Note: If assay contains multiple plates, perform steps 7, 8, 9 on 2-3 plates at a time and place at 53°C before going on to next 2-3 plates.
Day 2
Bring Amplifier, Label Probe and Substrate to Room Temperature. Vortex and briefly centrifuge the tubes of Amplifier and Label Probe to bring the contents to the bottom of the tube. Prepare Wash buffer: add 3ml Component 1 and 5ml Component 2 to 1L distilled water. (Wash Buffer is stable at Room Temperature for up to 6 months)
Prepare as needed: Amplifier Working solution, Label Probe Working Solution, and Substrate Working Solution:
Amplifier Working Solution - 1 :1000 dilution into Amplifier/Label Probe diluent.
Label Probe Working solution - 1 :1000 dilution into Amplifier/Label Probe diluent.
Substrate Working Solution - 1:333 dilution of 10% Lithium Lauryl Sulfate Substrate into Substrate Solution (protect from light).
Add 200μ1 /well of wash buffer to overnight hybridization mixture. Repeat washes 3x with 300μ1 of Wash Buffer. *Do not let the capture plates stand dry for longer than 5 minutes. Add ΙΟΟμΙ/well of Amplifier Working Solution. Seal plate with clear seal and incubate at 53°C for 30 minutes. Wash plate 3x with 300μ1 of Wash Buffer. Add ΙΟΟμΙ/well of Label Probe Working Solution. Seal plate with clear seal and incubate at 53°C for 30 minutes. Wash plate 3x with 300μ1 of Wash Buffer. Add Ι ΟΟμΙ/well Substrate Working Solution. Seal plate with foil seal and incubate at 53 °C for 15 minutes. Let plate stand at Room Temperature for 10 minutes. Read in luminometer with integration time set to 0.2 seconds. bDNA data was normalized to protein and graphed using GraphPad Prism Program using non-linear regression curve fit analysis.
In Vivo Evaluation of Efficacy
CD1 mice were tail vein injected with the freshly prepared siRNA containing polymer conjugates at a dose of 3 mg/kg in a volume of 0.2 mL, lOOmM TRIS 5% glucose, pH9, vehicle. Forty-eight hours post dose, mice were sacrificed and liver tissue samples were immediately preserved in RNALater (Ambion). Preserved liver tissue was homogenized and total RNA isolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNA isolation kit following the manufacturer's instructions. Liver ApoB mRNA levels were determined by quantitative RT-PCR. Message was amplified from purified RNA utilizing primers against the mouse ApoB mRNA (Applied Biosystems Cat. No. Mm01545156_ml). The PCR reaction was run on an ABI 7500 instrument with a 96- well Fast Block. The ApoB mRNA level is normalized to the housekeeping PPIB mRNA and GAPDH. PPIB and GAPDH mRNA levels were determined by RT-PCR using a commercial probe set (Applied Biosytems Cat. No.
Mm00478295_ml and Mm4352339E_m1). Results are expressed as a ratio of ApoB mRNA/ PPIB / GAPDH mRNA. All mRNA data is expressed relative to the vehicle control.
As shown in Figures 3 and 4, the data demonstrate that the polyconjugates of the instant invention can deliver siRNA both in vitro and in vivo. In mice, 65-80% knockdown of ApoB was observed with 3 mg/kg dose.

Claims

WHAT IS CLAIMED IS:
1. A olymer according to the Formula Z:
Figure imgf000027_0001
Z wherein: n is 2 to 250;
R is independently selected from A, B and Cf wherein at least two of A, B and C are present in the polymer and the polymer must include at least one cationic component (A or B) and at least one aliphatic component (B or C);
A is a first cationic component;
B is a second cationic component or a second aliphatic component; and
C is a first aliphatic component; or stereoisomer thereof,
2, A polymer according to Claim 1 of the Formula Z:
Figure imgf000027_0002
z wherein: n is 2 to 250;
R is independently selected from A, B and C, wherein at least two of A, B and C are present in the polymer and the polymer must include at least one catiomc component (A or B) and at least one aliphatic component (B or C); A is a first cationic component selected from an amine (primary, secondary, tertiary or quaternary), a nitrogen heterocycle, an aldimine, a hydrazide and a hydrazone;
B is a second cationic component selected from an amine (primary, secondary, tertiary or quaternary), a nitrogen heterocycle, an aldimine, a hydrazide and a hydrazone, or a second aliphatic component; and
C is a first aliphatic component; or stereoisomer thereof.
3. A polymer according to Claim 2 of the Formula Z:
Figure imgf000028_0001
Z wherein: n is 2 to 250;
R is independently selected from A, B and C, wherein A, B and C are all present in the polymer;
A is 2-(2-aminoethoxy)ethyl; B is 2-(1H-imidazoI-4-yl)ethyI; and C is dodecyl; or a stereoisomer thereof.
4. A polymer according to Claim 1 of the Formula Z selected from
Figure imgf000029_0001
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 1 ;
Figure imgf000029_0002
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 2;
Figure imgf000029_0003
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 3;
Figure imgf000029_0004
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 4;
Figure imgf000030_0001
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 5;
Figure imgf000030_0002
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined Table 6;
Figure imgf000030_0003
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 7;
Figure imgf000031_0001
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 8;
Figure imgf000031_0002
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 9;
Figure imgf000031_0003
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 10;
Figure imgf000031_0004
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 11 ;
Figure imgf000032_0001
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 12;
Figure imgf000032_0002
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined Table 13;
Figure imgf000032_0003
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 14; and
Figure imgf000033_0001
wherein x, y and z are between 0 to 50 and the % ratios of substituents A, B and C are defined in Table 15; or stereoisomer thereof.
5. The polymer according to Claim 1 of the Formula Z wherein substituents A, B and C are all present in the polymer and wherein A, B and C are different from one another.
6. The polymer according to Claim 1 of the Formula Z wherein substituent A must comprise at least 50% of the polymer.
7. The polymer according to Claim 1 of the Formula Z wherein substituent B must comprise at least 50% of the polymer.
8. The polymer according to Claim 1 of the Formula Z wherein substituent C must comprise at least 50% of the polymer.
9. A composition comprising a polymer of Formula Z and an oligonucleotide.
10. A composition according to Claim 9 further comprising a masking agent.
11. A composition according to Claim 10 further comprising a targeting agent.
12. A method of treating a disease in a patient by administering a composition of Claim 9.
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