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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
  • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
• • 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
• • 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
  • A61K47/60—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 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

• the invention provides ester-based biodegradable linkers for the delivery of oligonucleotides in vivo.
• oligonucleotides especially oligonucleotides that are complementary to a specific target messenger RNA (mRNA) sequence.
• mRNA target messenger RNA
• antisense nucleic acid sequences complementary to the products of gene transcription ⁇ e.g., mRNA
• sense nucleic acid sequences having the same sequence as the transcript or being produced as the transcript.
• An antisense oligonucleotide can be selected to hybridize to all or part of a gene, in such a way as to modulate expression of the gene.
• oligonucleotide compounds Molecular strategies are being developed to down-regulate unwanted gene expression. Recently, the use of modified oligonucleotide compounds has developed into a promising method of treatment against such diseases as viral infections, inflammatory and genetic disorder and significantly, cancer.
• Antisense DNAs were first conceived as alkylating complementary oligodeoxynucleotides directed against naturally occurring nucleic acids (Belikova, etal., Tetrahedron Lett. 37:3557-3562, 1967). Zamecnik and Stephenson were the first to propose the use of synthetic antisense oligonucleotides for therapeutic purposes. (Zamecnik & Stephenson, 1978, Proc. Natl. Acad. ScI U.S.A., 75:285-289; Zamecnik &
• Oligonucleotides have also found use in among others, diagnostic tests, research reagents e.g. primers in PCR technology and other laboratory procedures. Oligonucleotides can be custom synthesized to contain properties that are tailored to fit a desired use. Thus numerous chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities.
• oligonucleotides especially antisense oligonucleotides show promise as therapeutic agents, they are very susceptible to nucleases and can be rapidly degraded before and after they enter the target cells making unmodified antisense oligonucleotides unsuitable for use in in vivo systems. Because the enzymes responsible for the degradation are found in most tissues, modifications to the oligonucleotides have been made in an attempt to stabilize the compounds and remedy this problem. The most widely tested modifications have been made to the back-bone portion of the oligonucleotide compounds. See generally Uhlmann and Peymann, 1990, Chemical Reviews 90, at pages 545-561 and references cited therein.
• oligonucleotides tend to form secondary and high-order solution structures. Once these structures are formed, they become targets of various enzymes, proteins, RNA, and DNA for binding. This results in nonspecific side effects and reduced amounts of active compound binding to mRNA.
• Other attempts to improve oligonucleotide therapy have included adding a linking moiety and polyethylene glycol. See for example, Kawaguchi, etal., Stability, Specific Binding Activity, and Plasma Concentration in Mice of an Oligodeoxynucleotide
• the present invention provides compounds for the in vivo delivery of polynucleotides, such as oligonucleotides, that include a structure according to Formula (I)
• A is a capping group
• R 1 is a substantially non-antigenic water-soluble polymer
• L 2 and L' 2 are independently selected bif ⁇ nctional linkers; Yi and Y ⁇ are independently O, S, OrNR 5 ;
• X and X' are independently O or S;
• R 2 , R' 2 , R 3 , R' 3 and R 5 are independently selected from among hydrogen, C 1-6 alkyl, C 2 _6 alkenyl, C 2-6 alkynyl, C 3-19 branched alkyl, C 3-8 cycloalkyl, Ci -6 substituted alkyl, C 2-6 substituted alkenyl, C 2-6 substituted alkynyl, C 3 - 8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, Ci -6 heteroalkyl, substituted Q-sheteroalkyl, C 1-6 alkoxy, aryloxy, C 1-6 heteroalkoxy, heteroaryloxy, C 2-6 alkanoyl, arylcarbonyl, C 2-6 alkoxycarbonyl, aryloxycarbonyl, C 2-6 alkanoyloxy, arylcarbonyloxy, C 2-6 substituted alkanoyl, substituted arylcarbonyl, C
• R 4 and R' 4 are independently selected polynucleotides and derivatives thereof; (p) and (p') are independently zero or a positive integer, preferably zero or an integer from about 1 to about 3, more preferably zero or 1 ; and (q) and (q') are independently zero or 1, provided that R 3 is a substituted or unsubstituted hydrocarbon having at least three carbons when R 2 is H, and further provided that L 1 is not the same as C(R 2 )(R 3 ).
• the substantially non- antigenic polymer is a polyalkylene oxide and is more preferably polyethylene glycol (hereinafter PEG), hi other aspects, the PEG is either capped on one terminal with a CH 3 group, i.e. mPEG, while in other embodiments, bis-activated PEGs are provided such as those corresponding to the formula:
• Further aspects of the invention include methods of methods of making conjugates containing the hindered ester as well as methods of treatment based on administering effective amounts of conjugates containing a biologically active moiety to a patient (mammal) in need thereof. Methods of delivering the conjugate to cells requiring such treatment are also included.
• the polymeric delivery systems described herein include novel linkers which can form a releasable bond such as an ester bond between the polymer and biologically active moiety such as oligonucleotides. While the hindered ester of oligonucleotides is stable during the storage, it can release the native oligonucleotides without any tails by hydrolyzing the phosphodiester or phosphothioester bonds. In addition, the polymeric compound of the invention can facilitate hydrolysis of the stable hindered ester bond by anchimeric assistance from the linkers.
• the polymeric delivery systems have improved stability.
• the ester bond in a sterically hindered environment between the polymer and a moiety such as an oligonucleotide can inhibit the ester linkage from being exposed to basic aqueous medium or enzymes, and thereby stabilizes the covalent linkage.
• the stability of the polymeric systems allows longer shelf life for the polymeric conjugate. The improved stability increases cost efficiency.
• the polymeric delivery systems described herein are especially well suited for use with oligonucleotides and related antisense, short-interfering RNA (siRNA), or locked nucleic acid (LNA) compounds.
• siRNA short-interfering RNA
• LNA locked nucleic acid
• the presence of the hindered ester group in proximity to the oligonucleotide attached thereto provides improved stability and resistance to nuclease degradation. It also helps decrease toxicity and increase binding affinity to mRNA of oligonucleotide compounds.
• Conjugates made in accordance with the invention provide a means for protecting antisense oligonucleotide compounds against degradation, preventing the formation of high-order structures.
• the polymer conjugates allow the artisan to deliver sufficient amounts of active antisense oligonucleotide compounds to the target.
• the inventive linker is stable under all the buffer conditions suitable for animal or human intravenous administration in aqueous form.
• the inventive linker will hydrolyze to release the intact oligonucleotide in plasma in the presence of plasma enzymes. Variation of the steric hinderance on the linker will modify the rate of hydrolysis, as required for particular delivery systems.
• Another advantage of the activated polymers containing the' hindered esters is that it allows the artisan to more easily conjugate oligonucleotides of choice. There is no need to modify the oligonucleotide or target moiety with the hindered ester before PEGylation.
• the oligonucleotides is taken as is and PEGylated with the activated PEG linker which contains the desired hindered ester protective group thereon.
• inventive linker can be conjugated with any of the nucleotides (A, G, C, T, U etc) and then converted to its phosphoamidite, for example.
• the phosphoamidite can then be employed under normal solid phase oligonucleotide synthesis conditions to make oligonucleotide molecules.
• the linkage between the linker and the oligonucleotide is stable under the conditions needed for synthesis and purification.
• the term "residue” shall be understood to mean that portion of a biologically active compound, such as an oligonucleotide, which remains after it has undergone a reaction in which the prodrug carrier portion has been attached by modification of e.g., an available hydroxyl or amino group, to form, for example, an ester or amide group, respectively.
• the residue of a substantially non-antigenic polymer e.g., a polyalkylene oxide polymer, is that portion of the polymer that remains after it has undergone a reaction in which the polymer has been attached to a linker, spacer and/or biologically active compound or residues thereof.
• alkyl shall be understood to include straight, branched, substituted, e.g.
• substituted shall be understood to include adding or replacing one or more atoms contained within a functional group or compound with one or more different atoms;
• substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls and mercaptoalkyls;
• substituted cycloalkyls include moieties such as 4-chlorocyclohexyl; aryls include moieties such as napthyl; substituted aryls include moieties such as 3-bromophenyl; aralkyls include moieties such as toluyl; heteroalkyls include moieties such as ethylthiophene; the term "substituted heteroalkyl
• FIG. 1 schematically illustrates methods of synthesis described in Example 1-9.
• FIG. 2 schematically illustrates methods of synthesis described in Example 10.
• FIG. 3 schematically illustrates methods of synthesis described in Examples 11-13.
• FIG. 4 schematically illustrates methods of synthesis described in Example 14.
• the invention provides hindered ester-based biodegradable linkers for oligonucleotide delivery in vivo.
• the present invention provides for polymer-linked oligonucleotide prodrugs useful having many practical uses, including uses as diagnostic and analytic reagents, as research and investigational tools, both in vitro and in vivo, and as therapeutic agents.
• A is a capping group
• R 1 is a substantially non-antigenic water-soluble polymer
• L 2 and L' 2 are independently selected bifunctional linkers
• Y 1 and Y'I are independently O, S, or NR 5 ;
• X and X' are independently O or S;
• R 2 , R' 2 , R 3 , R' 3 and R 5 are independently selected from among hydrogen, Ci -6 alkyl, C 2 - 6 alkenyl, C 2-6 alkynyl, C 3 . 49 branched alkyl, C 3 -s cycloalkyl, C 1-6 substituted alkyl, C 2 _ 6 substituted alkenyl, C 2 - 6 substituted alkynyl, C 3 _g substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C f-6 hetero alkyl, substituted C 1-6 hetero alkyl, C 1-6 alkoxy, aryloxy, C 1-6 heteroalkoxy, hetero aryloxy, C 2-6 alkanoyl, arylcarbonyl, C 2-6 alkoxycarbonyl, aryloxycarbonyl, C 2 _ 6 alkanoyloxy, arylcarbonyloxy, C 2-6 substituted alkanoyl,
• R 4 and R' 4 are independently selected polynucleotides and derivatives thereof; (p) and (p') are independently zero or a positive integer, preferably zero or an integer from about 1 to about 3, more preferably zero or 1 ; and
• (q) and (q') are independently zero or 1, provided that R 3 is a substituted or unsubstituted hydrocarbon having at least three carbons when R 2 is H, and further provided that L 1 is not the same as C(R 2 )(R 3 ).
• the compounds described herein contain polymers according to Formula (Ia):
• the substantially non- antigenic polymer is a polyalkylene oxide and is more preferably polyethylene glycol (hereinafter PEG).
• PEG polyethylene glycol
• the PEG is either capped on one terminal with a CH 3 group, i.e. mPEG.
• bis-activated PEGs are provided such as those corresponding to
• the substituents contemplated for substitution can include, for example, acyl, amino, amido, amidine, ara-alkyl, aryl, azido, alkylmercapto, arylmercapto, carbonyl, carboxylate, cyano, ester, etiier, formyl, halogen, heteroaryl, heterocycloalkyl, hydroxy, imino, nitro, thiocarbonyl, thioester, thioacetate, thioformate, alkoxy, phosphoryl, phosphonate, phosphinate, silyl, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamide, and sulfonyl.
• the polynucleotides include oligonucleotides, preferably from about 2 to about 100 oligomers, more preferably from about 3 to about 50 oligomers, most preferably from about 5 to about 30 oligomers.
• A can be selected from among H, NH 2 , OH, CO 2 H, C 1-6 alkoxy, and Ci -6 alkyls.
• A can be methyl, ethyl, methoxy, ethoxy, H, and OH.
• A is more preferably methyl or methoxy.
• the present invention provides intermediates to extend the polynucleotide.
• the compounds of Formula (I) further include N 5 N- tetraisopropyl-cyanoethyl phosphoramidite and form compounds of formula (Ib):
• Polymers employed in the polymeric delivery systems described herein are preferably water soluble polymers and substantially non-antigenic such as polyalkylene oxides (PAO 's).
• the compounds described herein include a linear, terminally branched or multi-armed polyalkylene oxide.
• the polyalkylene oxide includes polyethylene glycol and polypropylene glycol.
• the polyalkylene oxide has an average molecular weight from about 2,000 to about 100,000 daltons, preferably from about 5,000 to about 60,000 daltons.
• the polyalkylene oxide can be from about 5,000 to about 25,000, and preferably from about 12,000 to about 20,000 daltons when proteins or oligonucleotides are attached or alternatively from about 20,000 to about 45,000 daltons, and preferably from about 30,000 to about 40,000 daltons when pharmaceutically active compounds (small molecules) are employed in the compounds described herein.
• the polyalkylene oxide includes polyethylene glycols and polypropylene glycols. More preferably, the polyalkylene oxide includes polyethylene glycol (PEG).
• PEG is generally represented by the structure:
• the polyethylene glycol (PEG) residue portion of the invention can be selected from among: -Y 71 -(CH 2 CH 2 O) n -CH 2 CH 2 Y 71 - ,
• Y 71 and Y 73 are independently O, S, SO 5 SO 2 , NR 73 or a bond;
• Y 72 is O, S, or NR 74 ;
• R 71-74 are independently selected from among hydrogen, Ci -6 alkyl, C 2-6 alkenyl, C 2 - 6 alkynyl, C 3-19 branched alkyl, C 3-8 cycloalkyl, C 1-6 substituted alkyl, C 2 - ⁇ substituted alkenyl, C 2-6 substituted alkynyl, C 3-8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C 1 6 heteroalkyl, substituted C 1-6 heteroalkyl, Ci -6 alkoxy, aryloxy, C ⁇ heteroalkoxy, hetero aryloxy, C 2-6 alkanoyl, arylcarbonyl, C 2-6 alkoxycarbonyl, aryloxycarbonyl, C 2-6 alkanoyloxy, arylcarbonyloxy, C 2-6 substituted alkanoyl, substituted arylcarbonyl, C 2-6 substituted alkanoyloxy, substituted
• Y 6 i -62 are independently O, S OrNR 61 ; Y 63 is O 5 NR 62 , S 5 SO or SO 2
• the polymers include multi-arm PEG-OH or "star-PEG" products such as those described in NOF Corp. Drug Delivery System catalog, Ver. 8, April 2006, the disclosure of which is incorporated herein by reference.
• the multi-arm polymer conjugates contain four or more polymer arms and preferably four or eight polymer arms.
• the multi-arm polyethylene glycol (PEG) residue can be H 2 C 0-(CH 2 CH 2 O) n H
• the multi-arm PEG has the structure:
• the polymers have a total molecular weight of from about 5,000 Da to about 60,000 Da, and preferably from 12,000 Da to 40,000 Da.
• the multi-arm PEG has the structure: wherein n is a positive integer.
• the degree of polymerization for the multi-arm polymer (n) is from about 28 to about 350 to provide polymers having a total molecular weight of from about 5,000 Da to about 60,000 Da, and preferably from about 65 to about 270 to provide polymers having a total molecular weight of from 12,000 Da to 45,000 Da. This represents the number of repeating units in the polymer chain and is dependent on the molecular weight of the polymer.
• the polymers can be converted into a suitably activated polymer, using tihe activation techniques described in U.S. Patent Nos. 5,122,614 or 5,808,096 patents.
• PEG can be of the formula:
• (u') is an integer from about 4 to about 455; and up to 3 terminal portions of the residue is/are capped with a methyl or other lower alkyl.
• all four of the PEG arms can be converted to suitable activating groups, for facilitating attachment to aromatic groups.
• Such compounds prior to conversion include:
• the polymeric substances included herein are preferably water-soluble at room temperature.
• a n ⁇ n-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
• PEG polyethylene glycol
• PAO-based polymers one or more effectively non-antigenic materials such as dextran, polyvinyl alcohols, carbohydrate-based polymers, hydroxypropylmethacrylamide (HPMA), polyalkylene oxides, and/or copolymers thereof can be used. See also commonly assigned U.S. Patent No.
• polymers having terminal amine groups can be employed to make the compounds described herein.
• the methods of preparing polymers containing terminal amines in high purity are described in U.S. Patent Application Nos. 11/508,507 and 11/537,172, the contents of each of which are incorporated by reference.
• polymers having azides react with phosphine-based reducing agent such as triphenylphosphine or an alkali metal borohydride reducing agent such as NaBH 4 .
• polymers including leaving groups react with protected amine salts such as potassium salt of methyl-tert-butyl imidodicarbonate (KNMeBoc) or the potassium salt of di-tert-butyl imidodicarbonate (KNB0C2) followed by deprotecting the protected amine group.
• protected amine salts such as potassium salt of methyl-tert-butyl imidodicarbonate (KNMeBoc) or the potassium salt of di-tert-butyl imidodicarbonate (KNB0C2) followed by deprotecting the protected amine group.
• KNMeBoc methyl-tert-butyl imidodicarbonate
• KNB0C2 di-tert-butyl imidodicarbonate
• polymers having terminal carboxylic acid groups can be employed in the polymeric delivery systems described herein. Methods of preparing polymers having terminal carboxylic acids in high purity are described in U.S. Patent
• the methods include first preparing a tertiary alkyl ester of a polyalkylene oxide followed by conversion to the carboxylic acid derivative thereof.
• the first step of the preparation of the PAO carboxylic acids of the process includes forming an intermediate such as f-butyl ester of polyalkylene oxide carboxylic acid. This intermediate is formed by reacting a PAO with a t- butyl haloacetate in the presence of a base such as potassium ⁇ -butoxide.
• the R 2 , R' 2 , R 3 , R' 3 and R 5 can be selected from among hydrogen, C 1-6 alkyl, C2-6 alkenyl, C 2-6 alkynyl, C 3-19 branched alkyl, C 3 _g cycloalkyl, C 1-6 substituted alkyl, C 2-6 substituted alkenyl, C 2 - 6 substituted alkynyl, C 3-8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C 1-6 heteroalkyl, substituted C 1-6 heteroalkyl, d-ealkoxy, aryloxy, Q-fjheteroalkoxy, heteroaryloxy, C 2-6 alkanoyl, arylcarbonyl, C 2-6 alkoxycarbonyl, aryloxycarbonyl, C 2-6 alkanoyloxy, arylcarbonyloxy, C 2-6 substituted alkanoyl, substituted arylcarbony
• R 2 and R 3 can be used so long as both R 2 and R 3 (R' 2 and R' 3 ) are not simultaneously H.
• R 2 and R 3 R' 2 and R' 3
• the other contains at least three hydrocarbons.
• R 2 , R' 2 , R 3 and R' 3 include methyl, ethyl and isopropyl.
• R 2 together with R 3 and R' 2 together with R' 3 can form a substituted or unsubsituted non-aromatic cyclohydrocarbon containing at least three carbons.
• free electron pairs of the L 1 and L' t spacers linked to the CR 2 R 3 and CR' 2 R' 3 moieties provide enchimeric effects.
• the L 1 and L' ⁇ spacers can be selected from among: wherein:
• R 11 -R 16 are independently selected from among hydrogen, amino, substituted amino, azido, carboxy, cyano, halo, hydroxyl, nitro, silyl ether, sulfonyl, mercapto, C 1-6 alkylmercapto, aryhnercapto, substituted arylmercapto, substituted C 1-6 alkylthio, C 1-6 alkyls, C 2-6 alkenyl, C 2-6 alkynyl, C 3-19 branched alkyl, C 3-8 cycloalkyl, C 1-6 substituted alkyl, C 2-6 substituted alkenyl, C 2-6 substituted alkynyl, C 3-8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroary ⁇ , C 1-6 heteroalkyl, substituted C 1-6 heteroalkyl, C 1-6 alkoxy, aryloxy, C 1-6 heteroalkoxy, heteroaryloxy, C 2-6 alkanoy
• (s) and (s') are independently zero or a positive integer, preferably from about 1 to about 4;
• (r) is O or l.
• Li and L'i rou s can be selected from among:
• (p) is an integer from about 1 to about 12, preferably from about 1 to about 8, more preferably from about2 to about 5;
• (q) are independently a positive integer, preferably from about 1 to about 8, and more preferably from about 1 to about 4.
• L 1 and L'j preferably include -(CH 2 ) ⁇ 2 i- Or-(CH 2 )X 21 -W-(CH 2 )X 22 -, wherein (x21) and (x22) are integers ranging in value from 1 to 7, and W is O or NH.
• the polymeric delivery systems described herein include that R 3 is a substituted or unsubstituted hydrocarbon having at least three carbons when R 2 is H, and L 1 is not the same as C(R 2 )(R 3 ).
• the compounds described herein can include bifunctional linkers.
• the bifunctional linkers include amino acids or amino acid derivatives.
• the amino acids can be among naturally occurring and non-naturally occurring amino acids.
• Derivatives and analogs of the naturally occurring amino acids, as well as various art-known non-naturally occurring amino acids (D or L), hydrophobic or non-hydrophobic, are also contemplated to be within the scope of the invention.
• a suitable non-limiting list of the non-naturally occurring amino acids includes 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-aminobutyric acid, desmosine, 2,2-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, 3-hydroxyprolme, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, sarcosine, N-methyl-isoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, and
• R 21 - 29 are independently selected from the group consisting of hydrogen, C 1-6 alkyls, C 3-12 branched alkyls, C 3-8 cycloalkyls, C 1-6 substituted alkyls, C 3-8 substituted cyloalkyls, aryls, substituted aryls, aralkyls, C 1-6 heteroalkyls, substituted C 1-6 heteroalkyls, C 1-6 alkoxy, phenoxy and C 1-6 heteroalkoxy; (t) and (t') are independently zero or a positive integer, preferably zero or an integer from about 1 to about 12, more preferably an integer from about 1 to about 8, and most preferably 1 or 2; and
• L 2 and L' 2 can be selected from among:
• Y ⁇ -i 9 are independently O, S or NR 4S ;
• R- 3M8 , R 50 -51 and A 51 are independently selected from the group consisting of hydrogen,
• C 1-6 alkyls C 3-I2 branched alkyls, C 3 - S cycloalkyls, C 1-6 substituted alkyls, C 3-8 substituted cyloalkyls, aryls, substituted aryls, aralkyls, C 1-6 heteroalkyls, substituted Ci-sheteroalkyls, phenoxy and C 1-6 heteroalkoxy;
• Ar is an aryl or heteroaryl moiety
• L 1 us are independently selected bifunctional spacers
• J 3 and J' 3 are independently selected from selected from among moieties actively transported into a target cell, hydrophobic moieties, bifunctional linking moieties and combinations thereof;
• leaving groups are to be understood as those groups which are capable of reacting with a nucleophile found on the desired target, i.e. an oligonucleotide, a bifunctional spacer, intermediate, etc.
• the targets thus contain a group for displacement, such as OH or SH groups found on oligonucleotides.
• Leaving groups attached to the hindered ester allows covalent reaction to the biologically active moiety of choice, i.e. pharmaceutically active compounds (small molecular weight compounds), oligonucleotides, etc.
• Suitable leaving groups include, without limitations, halogen (Br, Cl), activated esters, cyclic imide thione, N-hydroxysuccinimidyl, N-hydroxyphtalimidyl, N-hydroxybenzotriazolyl, imidazole, tosylate, mesylate, tresylate, nosylate, C]-Ce alkyloxy, Ci-C 6 alkanoyloxy, arylcarbonyloxy, ortho-nifrophenoxy, para-nitrophenoxy, pentafluorophenoxy, 1,3,5-trichlorophenoxy, and 1,3,5-trifluorophenoxy or other suitable leaving groups as will be apparent to those of ordinary skill.
• the leaving groups can be selected from among OH, methoxy, tert-butoxy, para-nitrophenoxy and N- hydroxysuccinimidyL
• nucleic acid or “nucleotide” apply to deoxyribonucleic acid ("DNA”), ribonucleic acid, ("RNA) whether single-stranded or double-stranded, unless otherwise specified, and any chemical modifications thereof-
• oligonucleotide is generally a relatively short polynucleotide, e.g., ranging in size from about 2 to about 200 nucleotides, or more preferably from about 10 to about 30 nucleotides in length.
• the oligonucleotides according to the invention are generally synthetic nucleic acids, and are single stranded, unless otherwise specified.
• polynucleotide and “polynucleic acid” may also be used synonymously herein.
• antisense refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence that encodes a gene product or that encodes a control sequence.
• antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense" strand.
• the sense strand of a DNA molecule is the strand that encodes polypeptides and/or other gene products.
• the sense strand serves as a template for synthesis of a messenger RNA (“mRNA”) transcript (an antisense strand) which, in turn, directs synthesis of any encoded gene product.
• mRNA messenger RNA
• Antisense nucleic acid molecules may be produced by any art- known methods, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant pheno types may be generated.
• the antisense oligonucleotide is introduced into a cell. Once introduced into a cell, the antisense oligonucleotide hybridizes to the corresponding mRNA sequence through Watson-Crick binding, forming a heteroduplex. Once the duplex is formed, translation of the protein coded by the sequence of bound mRNA is inhibited.
• antisense oligonucleotides are also employed in the art as probes, e.g., hybridization probes, generally linked to a tag or label, as well as being used to provide precise downregulation of the expression of specific cellular products or genetic regulatory elements for both investigational and therapeutic purposes.
• probes e.g., hybridization probes
• polynucleotide moieties can be attached to the activated polymers described herein.
• the polynucleotides are suitable for medicinal or diagnostic use in the treatment of animals, e.g., mammals, including humans, for conditions for which such treatment is desired.
• hydroxyl- or thiol-containing polynucleotides are within the scope of the present invention.
• the only limitations on the types of the biologically active moieties suitable for inclusion herein is that there is available at least one hydroxyl- or thiol- group which can react and link with a carrier portion and that there is not substantial loss of bioactivity in the form of conjugated to the polymeric delivery systems described herein.
• parent compounds suitable for incorporation into the polymeric transport conjugate compounds of the invention maybe active after hydrolytic release from the linked compound, or not active after hydrolytic release but which will become active after undergoing a further chemical process/reaction.
• an anticancer drug that is delivered to the bloodstream by the polymeric transport system may remain inactive until entering a cancer or tumor cell, whereupon it is activated by the cancer or tumor cell chemistry, e.g., by an enzymatic reaction unique to that cell.
• the choice for conjugation is an oligonucleotide and after conjugation, the target is referred to as a residue of an oligonucleotide.
• the oligonucleotides can be selected from among any of the known oligonucleotides and oligodeoxynucleotides with phosphorodiester backbones or phosphorothioate backbones, locked nucleic acid(LNA), nucleic acid with peptide backbone(PNA), tricyclo-DNA, double stranded oligonucleotide (decoy ODN) 5 catalytic RNA sequence (RNAi), ribozymes, aptmers, and CpG oligomers.
• LNA locked nucleic acid
• PNA nucleic acid with peptide backbone
• tricyclo-DNA tricyclo-DNA
• double stranded oligonucleotide decoy ODN
• RNAi catalytic RNA sequence
• RNAi rib
• the polynucleotides include 2 to 100 oligomer oligonucleotides, more preferably 3 to 50 oligomers and most preferably 10 to 30 oligomers. All other suitable size of the oligonucleotides is also contemplated.
• polynucleotides of the compounds described herein can be single stranded or double stranded including phosphorodiester backbone or phosphorothioate backbone.
• the "polynucleotide” includes oligonucleotides and oligodeoxynucleotides, including, for example, an oligonucleotide that has the same or substantially similar nucleotide sequence as does Genasense (a/k/a oblimersen sodium, produced by Genta Inc., Berkeley Heights, NJ).
• Genasense is an 18-mer phosphorothioate antisense oligonucleotide, TCTCCCAGCGTGCGCCAT (SEQ ID NO: 4), that is complementary to the first six codons of the initiating sequence of the human bcl-2 mRNA (human bcl-2 mRNA is art-known, and is described, e.g., as SEQ ID NO: 19 in U.S. Patent No. 6,414,134, incorporated by reference herein).
• the U.S. Food and Drug Administration (FDA) gave Genasense Orphan Drug status in August 2000.
• oligonucleotides and oligodeoxynucleotides useful according to the invention include, but are not limited to, the following: Oligonucleotides and oligodeoxynucleotides with natural phosphorodiester backbone or phosphorothioate backbone or any other modified backbone analogues;
• PNA nucleic acid with peptide backbone
• tricyclo-DNA decoy ODN (double stranded oligonucleotide); catalytic RNA sequence; ribozymes; spiegelmers (L-conformational oligonucleotides);
• CpG oligomers and the like, such as those disclosed at: Tides 2002, Oligonucleotide and Peptide Technology Conferences, May 6-8, 2002,
• Oligonucleotides according to the invention can also optionally include any suitable art-known nucleotide analogs and derivatives, including those listed by Table 1, below:
• Modifications to the oligonucleotides contemplated in the invention include, for example, the addition to or substitution of selected nucleotides with functional groups or moieties that permit covalent linkage of an oligonucleotide to a desirable polymer, and/or the addition or substitution of functional moieties that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to an oligonucleotide.
• Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5- iodouracil, backbone modifications, methylations, base-pairing combinations such as the isobases isocytidine and isoguanidine, and analogous combinations.
• Oligonucleotide modifications can also include 3' and 5' modifications such as capping. Structures of illustrative nucleoside analogs are provided below.
• R 4 or R' 4 include all suitable polynucleotides known to benefit from PEG or polymer attachment.
• the oligonucleotide is involved in targeted tumor cells or downregulating a protein implicated in the resistance of tumor cells to anticancer therapeutics.
• a protein implicated in the resistance of tumor cells to anticancer therapeutics for example, any art-known cellular proteins such as BCL-2 for downregulation by antisense oligonucleotides, for cancer therapy, can be used for the present invention. See U.S. Patent Application No. 10/822,205 filed April 9, 2004, the contents of which are incorporated by reference herein.
• a non-limiting list of preferred therapeutic oligonucleotides includes antisense HIF-Ia oligonucleotides and antisense Survivin oligonucleotides. Preferred embodiments include:
• antisense Survivin LNA (SEQ ID NO: 1) mC 3 -T s - m C 3 -A s .a s -t s -c s -c s -a s -t s -g s -g s - m C 3 -A ⁇ -G s -c ; where the upper case letter represents LNA, the "s" represents a phosphorothioate backbone; (iii) antisense Bcl2 siRNA:
• Genasense (phosphorothioate antisense oligonucleotide): (SEQ ID NO: 4) t s -c s -t s -c s- c s -c s -a s -g s -c s -g s -t s -g s -c s -g s -c s -c s -c s -a s -t where the lower case letter represents DNA and and "s" represents phosphorothioate backbone; (iv) antisense HIF 1 ⁇ LNA
• LNA includes 2'-O, 4'-C methylene bicyclomicleotide as shown below:
• the compounds described herein can include oligonucleotides modified with hindered ester-containing (CH 2 ) W amino linkers at 5' or 3' end of the oligonucleotides, where w in this aspect is a positive integer of preferably from about 1 to about 10, preferably about 6.
• the polymeric compounds can release the oligonucleotides without amino tail.
• the oligonucleotides can have the structure:
• w is a positive integer from about 1 to about 10, preferably about 6.
• oligonucleotides can include (CH 2 ) W sulfliydryl linkers (thio oligonucleotides).
• the thio oligonucletides can be used for conjugating directly to cysteine of the positively charge peptide or via maleimidyl group.
• the thio oligonucleotides can have the structure:
• a further aspect of the invention provides the conjugate compounds optionally prepared with a diagnostic tag linked to the polymeric delivery system described herein, wherein the tag is selected for diagnostic or imaging purposes.
• a suitable tag is prepared by linking any suitable moiety, e.g., an amino acid residue, to any art-standard emitting isotope, radio-opaque label, magnetic resonance label, or other non-radioactive isotopic labels suitable for magnetic resonance imaging, fluorescence-type labels, labels exhibiting visible colors and/or capable of fluorescing under ultraviolet, infrared or electrochemical stimulation, to allow for imaging tumor tissue during surgical procedures, and so forth.
• the diagnostic tag is incorporated into and/or linked to a conjugated therapeutic moiety, allowing for monitoring of the distribution of a therapeutic biologically active material within an animal or human patient.
• the inventive tagged conjugates are readily prepared, by art-known methods, with any suitable label, including, e.g., radioisotope labels.
• radioisotope labels include 13I Iodine, 125 Iodine, 99m Technetium and/or 1 'indium to produce radioimmunoscintigraphic agents for selective uptake into tumor cells, in vivo.
• there are a number of art-known methods of linking peptide to Tc-99m including, simply by way of example, those shown by U.S. Patent Nos. 5,328,679; 5,888,474; 5,997,844; and 5,997,845, incorporated by reference herein.
• the compound according to Formula (I) is covalently conjugated to a substantially nonantigenic polymer, e.g., a polyalkylene oxide.
• a substantially nonantigenic polymer e.g., a polyalkylene oxide.
• the compound according to Formula (I) includes the following:
• R 4 is selected from among sense oligonucleotides, antisense oligonucleotides, locked nucleic acids (LNA), short interfering RNA (siRNA), microRNA (miRNA), aptamers, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotides (PMO), tricyclo-DNA, double stranded oligonucleotide (decoy ODN), catalytic RNA (RNAi), aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers, aptamers,
• (z) is a positive integer from about 1 to about 10;
• (z') is zero or a positive integer from about 1 to about 4;
• mPEG has the formula: CH 3 -O(CH 2 CH 2 O) n -;
• PEG has the formula -0(CH 2 CH 2 O) n -;
• (n) is a positive integer from about 10 to about 2,300.
• Preferred polymeric compounds according to the present invention include:
• R 4 One preferred embodiment for R 4 includes:
• antisense Survivin LNA SEQ E) NO: 1 ) mC 3 -T s - m C 3 -A s- a s -t s -c s -c s -a s -t s -g s -g s - m C 3 -A s -G s -C; where the upper case letter represents LNA, the "s" represents a phosphorothioate backbone; (ii) antisense Bcl2 siRNA:
• Genasense (phosphorothioate antisense oligonucleotide): (SEQ ID NO: 4) t s -c s -t s ⁇ c s .c s -c s -a s -g s -c s -g s -t s -g s -c s -g s -c s -c s -c s -a s -t where the lower case letter represents DNA and and "s" represents phosphorothioate backbone; (iv) antisense HIF 1 ⁇ LNA (SEQ ID NO: 5) where the upper case letter represents LNA and the "s" represents phosphorothioate backbone.
• Genasense (SEQ ID NO: 4) is described as TCTCCCAGCGTGCGCCAT or 5 -t s c s t s c s g s c s g s c s c s c s a s t -3'.
• the polymeric compound having hindered ester can be prepared by conjugating a polymeric compound having an OH or a leaving group at the terminal end with a nucleophile having a protected hindered ester or a hindered acid at the distal end. Further deprotecting and activating the resulting polymeric compound will provide the compound of the current invention.
• the terminal group of the current invention can be either carboxylic acid form ready to be coupled with OH or SH containing moiety or an activated form which can be replaced upon conjugating with OH or SH containing moiety.
• OH or SH containing compound can be conjugated to form a hindered ester intermediate, which in turn reacted with an activated polymer for the polymeric conjugate having a hindered ester with a biologically active moiety.
• the methods of preparing a hindered acyl or ester moiety- containing polymeric conjugate include: reacting a compound of Formula (III):
• Ai is a capping group or Mi
• a 2 is a capping group
• M 1 is a leaving group such as halogens, activated carbonates, isocyanate, N- hydroxysuccinimidyl, tosylate, mesylate, tresylate, nosylate, ortho-nitrophenoxy, imidazole and other leaving groups known by those of ordinary skill in the art;
• M 2 is -OH, -SH, or -NHRi 01 ;
• Rioo is OH or OR 10 1; wherein, Rioi is selected from among hydrogen, Q -6 alkyl, C2-6 alkenyl, C2- 6 alkynyl, C 3-19 branched alkyl, C 3-8 cycloalkyl, C 1-6 substituted alkyl, C2-6 substituted alkenyl, C 2-6 substituted alkynyl, C 3- 8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted hetero aryl, C 1 ⁇ heteroalkyl, substituted C ⁇ heteroalkyl, C ⁇ ealkoxy, aryloxy, C 1-6 heteroalkoxy, heteroaryloxy, C2-6 alkanoyl, arylcarbonyl,
• hindered ester moiety according to Formula (IV) to the PEG or other polymer can be done using standard chemical synthetic techniques well known to those of ordinary skill.
• the activated polymer portion such as SC-PEG, PEG-amine, PEG acids, etc. can be obtained from either commercial sources or synthesized by the artisan without undue experimentation.
• a non-limiting list of such hindered ester moiety includes:
• the compounds of Formula (V) can further react with a -OH or -SH containing moiety in the presence of base and a coupling agent under conditions sufficient to form a compound of Formula (Ia):
• a 3 is a capping group
• R 1O3 is selected from among targeting agents, diagnostic agents and biologically active moieties; and all other variables are previously defined.
• the R 1O3 shall be understood as the portion of the OH or SH containing moiety which remains after it has undergone a reaction with the compound of Formula (V).
• the compounds described herein can be prepared by methods including: reacting a compound of Formula (VI):
• a 4 is a capping group or M 4 ;
• M 3 is -OH, SH, or -NHRi 05 ;
• M 4 is a leaving group such as halogens, activated carbonates, isocyanate, N-hydroxysuccinimidyl, tosylate, mesylate, tresylate, nosylate, ortho-nitrophenoxy, imidazole and other leaving groups known by those of ordinary skill in the art;
• R 1O4 iselected from biologically active moieties, targeting groups and diagnostic agents
• R 105 is selected from among hydrogen, C h alky! C 2-6 alkenyl, C 2 - 6 alkynyl, C 3 ⁇ 9 branched alkyl, C 3-8 cycloalkyl, C 1-6 substituted alkyl, C2-6 substituted alkenyl, C 2-6 substituted alkynyl, C 3-8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C 1-6 heteroalkyl, substituted Q-sheteroalkyl, C ⁇ e alkoxy, aryloxy, C ⁇ heteroalkoxy, heteroaryloxy, C 2 _ 6 alkanoyl, arylcarbonyl, C 2-6 alkoxycarbonyl, aryloxycarbonyl, C 2 ⁇ 6 alkanoyloxy, arylcarbonyloxy, C 2-6 substituted alkanoyl, substituted arylcarbonyl, C 2 - 6 substituted alkanoyloxy,
• Attachment of the hindered ester containing group to the polymer portion is preferably carried out in the presence of a coupling agent.
• suitable coupling agents include 1,3-diisopropylcarbodiimide (DIPC), any suitable dialkyl carbodiir ⁇ ides, 2-halo-l- alkyl-pyridinium halides, (Mukaiyama reagents), 1 -(3 -dimethyl aminopropyl)-3 -ethyl carbodiimide (EDC), propane phosphonic acid cyclic anhydride (PPACA) and phenyl dichlorophosphates, etc. which are available, for example from commercial sources such as Sigma- Aldrich Chemical, or synthesized using known techniques.
• DIPC 1,3-diisopropylcarbodiimide
• EDC 1 -(3 -dimethyl aminopropyl)-3 -ethyl carbodiimide
• PPACA propane phosphonic acid cyclic anhydride
• the reactions are carried out in an inert solvent such as methylene chloride, chloroform, DMF or mixtures thereof.
• the reactions can be preferably conducted in the presence of abase, such as dimethylaminopyridine (DMAP), diisopropylethylamine, pyridine, triethylamine, etc. to neutralize any acids generated.
• abase such as dimethylaminopyridine (DMAP), diisopropylethylamine, pyridine, triethylamine, etc. to neutralize any acids generated.
• DMAP dimethylaminopyridine
• the reactions can be carried out at a temperature from about O 0 C up to about 22 0 C (room temperature).
• Another aspect of the present invention provides methods of treatment for various medical conditions in mammals.
• the methods include administering, to the mammal in need of such treatment, an effective amount of a compound described herein.
• the polymeric conjugate compounds are useful for, among other things, treating diseases which are similar to those which are treated with the parent compound, e.g. enzyme replacement therapy, neoplastic disease, reducing tumor burden, preventing metastasis of neoplasms and preventing recurrences of tumor/neoplastic growths in mammals.
• the amount of the polymeric conjugate that is administered will depend upon the amount of the parent molecule included therein. Generally, the amount of polymeric conjugate used in the treatment methods is that amount which effectively achieves the desired therapeutic result in mammals. Naturally, the dosages of the various polymeric conjugate compounds will vary somewhat depending upon the parent compound, molecular weight of the polymer, rate of in vivo hydrolysis, etc. Those skilled in the art will determine the optimal dosing of the polymeric transport conjugates selected based on clinical experience and the treatment indication. Actual dosages will be apparent to the artisan without undue experimentation.
• the compounds of the present invention can be included in one or more suitable pharmaceutical compositions for administration to mammals.
• the pharmaceutical compositions may be in the form of a solution, suspension, tablet, capsule or the like, prepared according to methods well known in the art. It is also contemplated that administration of such compositions may be by the oral and/or parenteral routes depending upon the needs of the artisan.
• a solution and/or suspension of the composition may be utilized, for example, as a carrier vehicle for injection or infiltration of the composition by any art known methods, e.g., by intravenous, intramuscular, intraperitoneal, subcutaneous injection and the like.
• Such administration may also be by infusion into a body space or cavity, as well as by inhalation and/or intranasal routes, hi preferred aspects of the invention, however, the polymeric conjugates are parenterally administered to mammals in need thereof.
• oligonucleotides preferably antisense oligonucleotides to mammalian cells.
• the methods include delivering an effective amount of a conjugate prepared as described herein to the condition being treated will depend upon the polynucleotides efficacy for such conditions.
• the method would include delivering a polymer conjugate containing the oligonucleotides to the cells having susceptibility to the native oligonucleotides.
• the delivery can be made in vivo as part of a suitable pharmaceutical composition or directly to the cells in an ex vivo environment.
• the polymeric conjugates including oligonucleotides can be used.
• Ethyl 7-bromo ⁇ 2,2-dimethylheptanoate (compound 3, 26.5 g) was heated with sodium azide (13 g) in DMF (500 mL) at 100 °C for 2 hours. The mixture was concentrated and the residue was purified by a silica gel column, eluted with 10% ethyl acetate in hexane to give the desired product as a liquid (20.5 g, yield 90.3%).
• Ethyl 7-azido-2,2-drmethylheptanoate (compound 4, 20.5 g) was heated with sodium hydroxide (10 g, 85%) in ethanol (500 mL) under reflux for 2 hours. The mixture was concentrated and water (400 mL) was added. The mixture was acidified with concentrated hydrochloric acid to pH 2 and extracted with ethyl acetate (500 mL). The organic layer was concentrated and the residue was purified by a silica gel column, eluted with 50% ethyl acetate in hexane to give the desired product as a liquid (17.1 g, yield 95%).
• Compound 10 was transferred to Trilink Biotechnologies, CA to use as the last monomer in the oligo synthesis.
• the Mmt group was deprotected after the synthesis and the oligo was purified by RP-HPLC and compound 11 as the free amine was obtained for PEG conjugation.
• the sequence of oligonucleotide was TCTCCCAGCGTGCGCCAT (SEQ ID NO. 4).
• Example 9 Preparation of PEG-HE-Oligo, Compounds (13) To a solution of compound 11 (10 mg, 1.7 ⁇ mol) in PBS buffer (5 mL, pH 7.8) was added SC-PEG (compound 12, Mw 30 kDa, 520 mg, 17 ⁇ mol) and stirred at room temperature for 5 hrs. The reaction mixture was diluted to 50 mL with water and loaded on a Poros HQ, strong anion exchange column (10 mm x 1.5 mm, bed volume - 16 mL) which was pre-equilibrated with 20 mM Tris-HCl buffer, pH 7.4 (buffer A). The column was washed with 3-4 column volumes of buffer A to remove the excess PEG linker.
• the product was eluted with a gradient of 0 tol 00 % 1 M NaCl in 20 mM Tris-HCl buffer, pH 7.4, buffer B in 10 min, followed by 100 % buffer B for 10 min at a flow rate of 10 mL/min.
• the eluted product was desalted using HiPrep desalting column (50 mL) and lyophilized to give 6 mg of the product.
• the equivalent of oligonucleotide in the conjugate measured by UV was 60%, wt/wt.
• PEG-Linker-NHS compound 14, Mw 30 kDa, 520 mg, 17 ⁇ mol
• the reaction mixture was diluted to 50 mL with water and loaded on a Poros HQ, strong anion exchange column (10 mm x 1.5 mm, bed volume - 16 mL) which was pre-equilibrated with 20 mM Tris-HCl buffer, pH 7.4 (buffer A). The column was washed with 3-4 column volumes of buffer A to remove the excess PEG linker.
• the product was eluted with a gradient of 0 to 100 % 1 M NaCl in 20 mM Tris-HCl buffer, pH 7.4, buffer B in 10 min, followed by 100 % buffer B for 10 min at a flow rate of 10 mL/min.
• the eluted product was desalted using HiPrep desalting column (50 mL) and lyophilized to solid to give 5 mg of the desired product.
• the equivalent of oligonucleotide in the conjugate measured by UV was 50%, wt/wt.
• Example 13 Preparation of PEG-HE-T, Compounds (21) mPEG-Linker-NHS (compound 20, Mw. 20k, 0.50g, 0.0246 mmol) and compound 19 (26mg, 0.0738 mmol) were dissolved in a mixture of DCM (5 mL) and DMF (1 mL), and DMAP (15mg, 0.0123 mmol) was added to the solution. Reaction mixture was stirred at room temperature for 2.5 hours. Solvent was removed in vacuo, and the crude product was precipitated by the addition of ethyl ether.
• Example 14 Preparation of PEG-HE-T, Compounds (23) mPEG-NHS (compound 22, Mw. 20k, Ig, 0.0492 mmol) and compound 19 (26mg, 0.1476 mmol) were dissolved in a mixture of DCM (10 mL) and DMF (2 mL), and DMAP (30mg, 0.246 mmol) was added to the solution. Reaction mixture was stirred at room temperature for 2.5 hours. Solvent was removed in vacuo, and the crude product was precipitated by the addition of ethyl ether.
• the rates of hydrolysis were obtained by employing a C 8 reversed phase column (Zorbax ® SB-C8) using a gradient mobile phase consisting of (a) 0.1 M triethylammonium acetate buffer and (b) acetonitrile. A flow rate of 1 mL/min was used, and chromatograms were monitored using a UV detector at 227 nm for paclitaxel and 260 nm for oligonucleotides.
• PEG derivatives were dissolved in 0.1 M pH 7.4 PBS or water at a concentration of 5 mg/mL, while for hydrolysis in plasma, the derivatives were dissolved in distilled water at a concentration of 20 mg / 100 ⁇ L and 900 ⁇ L of rat plasma was added to this solution.
• the mixture was vortexed for 2 min and divided into 2 mL glass vials with 100 ⁇ L of the aliquot per each vial.
• the solutions were incubated at 37 0 C for various periods of time.

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