Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
  • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
  • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
  • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D207/36—Oxygen or sulfur atoms
  • C07D207/40—2,5-Pyrrolidine-diones
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Bifunctional molecules commonly referred to as crosslinkers. are used to connect two molecules together Bifunctional molecules can contain homo or heterobifunctionalitv
• the disulfide linkage (RSSR') may be used within bifunctional molecules
• the reversibility of disulfide bond formation makes them useful tools for the transient attachment of two molecules Disulfides have been used to attach a bioactive compound and another compound (Thorpe, P E J Natl Cancer Inst 1987. 79, 1101)
• the disulfide bond is reduced thereby releasing the bioacftve compound Disulfide bonds may also be used in the formation of polymers (Kishore. K ⁇ , Ganesh. K. in Advances in Polvmer Science, Vol 21, Saegusa, T Ed , 1993)
• cystine is reduced 3-15 times faster than oxidized glutathione
• Another example of a relatively acidic thiol is 5- th ⁇ o-2-n ⁇ trobenzo ⁇ c acid. pKa 5 Its acidity is due to resonance stabilization and inductive effects Its disulfide is rapidly reduced by all standard alkyl thiols and its colored thiolate makes it a convenient assay for thiol concentration
• Descnbed in a preferred embodiment is a process for the delivery of a compound to a cell, compnsing associating a compound, containing a disulfide bond that can be cleaved under physiological conditions, with a polymer, then delivering the polymer to the cell
• the polymer may compnse a first polvmer and a second polymer
• the first polymer and the second polymer mav compnse nucleic acids, proteins, genes, antisense polymers. DNNRNA hyb ⁇ ds. or synthetic polymers
• a biologically active compound is associated with a disulfide-containing compound, compnsing the disulfide-containing compound having a labile disulfide bond that is selected from the group consisting of (a) a disulfide bond that is cleaved more rapidly than oxidized glutathione and (b) a disulfide bond constructed from thiols m which one of the constituent thiols has a lower pKa than glutathione and (c) a disulfide bond that is activated by intramolecular attack from a free thiol
• a compound for inserting into an organism, compnsing the compound having a disulfide bond that is labile under physiologic conditions selected from the group consisting of (a) a disulfide bond that is cleaved more rapidly than oxidized glutathione and (b) a disulfide bond constructed from thiols in which one of the constituent thiols has a lower pKa than glutathione and (c) a disulfide bond that is activated by intramolecular attack from a free thiol
• a process for forming a compound having a labile disulfide bond for use with an organism, comprising forming the compound having a disulfide bond selected from the group consisting of (I) a disulfide bond that is cleaved more rapidly than oxidized glutathione, and (n) a disulfide bond constmcted from thiols in which one of the constituent thiols has a lower pKa than glutathione. and (in) a disulfide bond that is activated by intramolecular attack from a free thiol. inserting the compound into the organism
• a process is desc ⁇ bed for compacting a nucleic acid for delivery to a cell, compnsing associating a polymer containing a disulfide bond with a nucleic acid and delivenng the nucleic acid to the cell
• a process is descnbed for compacting a nucleic acid for delivery to a cell compnsing associating a polymer with the nucleic acid, then associating a compound containing a disulfide bond that can be cleaved under physiological conditions with the nucleic acid polymer complex, then delivenng the complex to a cell
• a process is desc ⁇ bed for compacting a nucleic acid for delivery to a cell, compnsing associating a polymer containing a disulfide bond with a nucleic acid, then associating another polymer with the disulfide containing polymer - nucleic acid complex, then delivenng the complex to the cell
• a process is descnbed for compacting a nucleic acid for delivery to a cell compnsing associating a polymer with the nucleic acid, then associating a compound containing a disulfide bond that can be cleaved under physiological conditions with the nucleic acid polymer complex, then associating another polymer with the complex, then delivenng the complex to a cell
• a compound is desc ⁇ bed which contains a disulfide bond that can be cleaved under physiological conditions and possesses heterobifunctional or homobifunctional groups
• a compound can be descnbed as a disulfide containing bifunctional molecule
• a compound that contains an aliphatic disulfide bond with one or more electronegative (electron withdrawing groups) substituted alpha or beta to one or both of the sulfur atoms serve to lower the pK a of the constituent thiols
• the spacer may be charge positive, charge negative, charge neutral, or zwitte ⁇ omc Ai and A are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as m a heterobifunctional bifunctional molecule
• the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions
• Such reactive groups include (but not limited to) isothiocyanate, isocyanate.
• acyl azide acid halide, O-acyl urea, N-hydroxysuccimmide esters, succinimide esters, amide, urea, sulfonyl chlonde, aldehyde, ketone.
• ether epoxide, carbonate, alkyl halide, lmidoester, carboxylate. alkylphosphate. arylhalides (e g difluoro-dinitrobenzene) or anhydndes
• A1,A2 can react with (but not restncted to) an activated carboxyhc acid, isothiocyanate, isocyanate.
• amide, carboxylate, or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or anhydndes In other terms when function A1,A2 is an amine, then an acylating or alkylating agent can react with the amine
• A1.A2 can react with (but not restncted to) a haloacetyl denvative. activated carboxvhc acid, maleimide, azindine denvative, acryloyl denvative. fluorobenzene de ⁇ vatives. or disulfide denvative (such as a py ⁇ dyl disulfide or 5-thio-2-nitrobenzoic acidjTNB ⁇ de ⁇ vatives)
• A1,A2 can react with (but not restncted to) a diazoacetate, alcohol, thiol or an amine once the acid has been activated
• A1.A2 can react with (but not restncted to) an activated carboxyhc acid, epoxide. oxirane. or an amine in which carbonvldiimidazole is used If functional group A1.A2 is an aldehyde or ketone then A1.A2 can react with (but not restncted to) an hydrazine, hydrazide denvative. amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal
• A1,A2 can react with (but not restncted to) an amine. a hvdroxyl, hydrazine. hydrazide, or sulfhydryl group
• A1,A2 an activated carboxyhc acid, haloacetyl denvative, maleimide, azindine denvative. acryloyl denvative. fluorobenzene denvatives, or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-mtrobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ denvatives) then A1,A2 can react with (but not restncted to) a sulfhydryl
• A1.A2 is an aldehyde, ketone, epoxide, oxirane, or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then A1.A2 can react with (but not restncted to) a hvdroxyl
• A1.A2 is a hvdrazine, hydrazide denvative. or amine (p ⁇ marv or secondary) then A1.A2 can react with (but not restncted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3)
• a compound which contains an aromatic disulfide bond in which the sulfur atom is bonded directlv to the aromatic nng
• the ring may contain 5 or more atoms
• the substitution pattern on the nng may be vaned to alter the reduction potential of the disulfide bond
• the substiuents may be selected from the group that includes but is not limited to OH. OR (an ether), NH 2 ,(also secondary, tertiary, and quaternary amines), S0 3 " , COOH. COOR (an ester), CONH 2 , CONR 2 (substituted amide), a halogen (F. Cl.
• L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups L may or may not be present and may be chosen from a group that includes alkanes, alkenes. esters, ethers, glycerol, amide, saccha ⁇ des, polysaccha ⁇ des, heteroatoms such as oxygen, sulfur, or nitrogen.
• the spacer may be charge positive, charge negative, charge neutral, or zwitte ⁇ onic R5, Rio - are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as in a heterobifunctional bifunctional molecule
• the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions
• Such reactive groups include lsothiocynanate, lsocynanate, acyl azide. N-hydroxysuccinimide esters, succinimide esters, sulfonyl chlonde. aldehyde, epoxide. carbonate, imidoester, carboxylate. alkylphosphate. arylhalides (e g difluoro-dinitrobenzene) or succinic anhyd ⁇ de
• RIO is an amine
• R5. can react with (but not restricted to) an activated carboxyhc acid, isothiocyanate. isocyanate, acyl azide, alkyl halide. acid halide. N-hydroxysuccinimide ester, sulfonyl chlonde, aldehyde, ketone. epoxide. carbonate, imidoester. amide, carboxylate. or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or anhydndes
• an acvlating or alkvlating agent can react with the amine
• RIO is a sulfhydryl then R5, RIO can react with (but not restncted to) a haloacetvl denvative. activated carboxyhc acid, maleimide. azindine denvative, acryloyl denvative. fluorobenzene denvatives. or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-mtrobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ de ⁇ vatives)
• RIO is carboxylate then R5. RIO can react with (but not restncted to) a diazoacetate, alcohol, thiol or an amine once the acid has been activated
• RIO is an hvdroxyl then R5, RIO can react with (but not restncted to) an activated carboxyhc acid, epoxide. oxirane, or an amine m which carbonyldiimidazole is used
• RIO is an aldehyde or ketone then R5, RIO can react with (but not restncted to) an hydrazine. hydrazide denvative. amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal
• RIO is activated carboxyhc acid, isothiocyanate. isocyanate, acyl azide, N-hydroxysuccinimide ester, sulfonyl chlonde, aldehyde, ketone, epoxide, carbonate, imidoester, alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhyd ⁇ de, alkyl halide.
• R5 can react with (but not restncted to) an amine.
• RIO an activated carboxyhc acid, haloacetvl denvative. maleimide, azindine denvative. acryloyl denvative. fluorobenzene denvatives, or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ denvatives) then R5. RIO can react with (but not restncted to) a sulfhydryl
• R10 is an aldehvde. ketone, epoxide, oxirane. or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then R5. R10 can react with (but not restncted to) a hvdroxy 1 If functional group R5. RIO is a hydrazine. hydrazide denvative, or amine (p ⁇ mary or secondary) then R5. RIO can react with (but not restncted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3)
• the heterocychc nng may be aromatic or aliphatic
• the heterocychc nng may contain 5 or more atoms of which 1 or more is a heteroatom (O, N, S, P), and the rest being carbon atoms
• H is a heteroatom selected from the group including sulfur, oxygen, nitrogen, or phosphorus
• R ⁇ -R , R5-R7 are substiuents that may be selected from the group that includes but is not limited to OH, OR (an ether), NH 2 ,(also secondary, tertiary, and quaternary amines), S0 3 " , COOH, COOR (an ester), CONH 2 , CONR 2 (substituted amide), a halogen (F, CI, Br, I), N0 2 , CH 3 (or longer branched or straight chain, saturated, or unsaturated aliphatic group)
• the substitution pattern on the aromatic nng may be va ⁇ ed to alter the reduction potential of the disulfide bond
• L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups L may or may not be present and may be chosen from a group that includes alkanes.
• the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions
• reactive groups include isothiocynanate. lsocynanate. acyl azide, N-hydroxysuccimmide esters, succmimide esters, sulfonyl chlonde. aldehyde, epoxide. carbonate, imidoester, carboxylate. alkylphosphate. arylhalides (e g difluoro-dinitrobenzene) or succinic anhyd ⁇ de
• R8 is an amine then R4. R8 can react with (but not restocted to) an activated carboxyhc acid, isothiocyanate. isocyanate, acyl azide, alkyl halide. acid halide, N-hydroxysuccinimide ester, sulfonyl chlonde. aldehyde, ketone, epoxide. carbonate, imidoester. amide, carboxylate, or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or anhydndes
• an acylating or alkylating agent can react with the amine
• R8 is a sulfhydryl then R4. R8 can react with (but not restncted to) a haloacetvl denvative, activated carboxyhc acid, maleimide, azindine denvative, acryloyl denvative. fluorobenzene de ⁇ vatives, or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ denvatives)
• R8 is carboxylate then R4, R8 can react with (but not restncted to) a diazoacetate. alcohol, thiol or an amine once the acid has been activated
• R8 is an hvdroxyl then R4, R8 can react with (but not restncted to) an activated carboxyhc acid, epoxide, oxirane. or an amine m which carbonyldiimidazole is used
• R8 is an aldehyde or ketone then R4. R8 can react with (but not restocted to) an hvdrazine. hydrazide denvative, amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal
• R8 is activated carboxyhc acid, isothiocyanate, isocyanate, acyl azide, N-hydroxysuccimmide ester, sulfonyl chlonde. aldehyde, ketone. epoxide. carbonate, imidoester. alkylphosphate. arylhalides (difluoro-dinitrobenzene), anhyd ⁇ de. alkyl halide. or acid halide, p-mtrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyndinium.
• R8 can react with (but not restocted to) an amine. a hydroxvl. hydrazine. hydrazide, or sulfhydryl group
• R8 an activated carboxyhc acid, haloacetyl denvative, maleimide. azindine denvative. acryloyl denvative. fluorobenzene de ⁇ vatives, or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ denvatives) then R4. R8 can react with (but not restncted to) a sulfhydryl
• R8 is an aldehyde, ketone, epoxide, oxirane, or an amine in which carbonyldiimidazole or N, N'-disuccinimidyl carbonate is used, then R4, R8 can react with (but not restocted to) a hvdroxyl
• R8 is a hydrazine. hydrazide denvative. or amine (p ⁇ mary or secondary) then R4. R8 can react with (but not restocted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3)
• the cyclic nng may contain 5 or more atoms
• R1-R4 are substiuents selected from the group that includes but is not limited to H, OH. OR (an ether). NH 2 (also secondary, tertiary, and quaternary amines), S0 3 " ,
• COOR an ester
• a halogen F, Cl. Br, I
• N0 2 , CH 3 or longer branched or straight chain, saturated, or unsaturated aliphatic group
• L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups L may or may not be present and may be chosen from a group that includes alkanes. alkenes, esters, ethers, glycerol, amide, saccha ⁇ des, polysacchandes.
• the spacer may be charge positive, charge negative, charge neutral, or zwitte ⁇ omc R5, and Rio are reactive groups that may be identical as in a homobifunctional bifunctional molecule, or different as m a heterobifunctional bifunctional molecule
• the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions
• Such reactive groups include lsothiocynanate. lsocynanate, acyl azide. N-hydroxysuccmimide esters, succinimide esters, sulfonyl chlonde. aldehyde, epoxide, carbonate, imidoester. carboxylate, alkylphosphate, arylhalides (e g difluoro-dinitrobenzene) or succinic anhyd ⁇ de
• RIO is an amine then R5.
• RIO can react with (but not restocted to) an activated carboxyhc acid, isothiocyanate. isocyanate.
• acyl aade alkyl halide. acid halide. N-hydroxysuccinimide ester, sulfonyl chlonde, aldehyde, ketone, epoxide. carbonate, imidoester. amide, carboxylate. or alkylphosphate. arylhalides (difluoro-dinitrobenzene) or anhydndes
• RIO is an amine. then an acylating or alkylating agent can react with the amine
• RIO is a sulfhydryl then R5. RIO can react with (but not restocted to) a haloacetyl denvative. activated carboxyhc acid, maleimide. azindine denvative. acryloyl denvative. fluorobenzene de ⁇ vatives. or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ de ⁇ vatives)
• RIO is carboxvlate then R5. RIO can react with (but not restocted to) a diazoacetate. alcohol, thiol or an amine once the acid has been activated If functional group R5. RIO is an hvdroxyl then R5. RIO can react with (but not restocted to) an activated carboxyhc acid, epoxide. oxirane. or an amine in which carbonyldiimidazole is used
• RIO is an aldehyde or ketone then R5. RIO can react with (but not restocted to) an hydrazine. hydrazide denvative, amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal
• RIO is activated carboxyhc acid, isothiocyanate, isocyanate.
• acyl azide N-hydroxysuccmimide ester, sulfonyl chlonde, aldehyde, ketone, epoxide, carbonate, imidoester. alkylphosphate. arylhalides (difluoro-dinitrobenzene), anhyd ⁇ de. alkyl halide. or acid halide.
• p-mtrophenyl ester o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole.
• R5 can react with (but not restncted to) an amine.
• RIO an activated carboxyhc acid, haloacetyl denvative, maleimide, azindine denvative. acryloyl denvative, fluorobenzene de ⁇ vatives, or disulfide denvative (such as a pyndyl disulfide or 5-thio-2-mtrobenzoic ac ⁇ d ⁇ TNB ⁇ de ⁇ vatives) then R5. RIO can react with (but not restocted to) a sulfhydryl
• RIO is an aldehvde. ketone. epoxide. oxirane. or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then R5. RIO can react with (but not restncted to) a hvdroxyl
• RIO is a hydrazine. hydrazide denvative, or amine (pnmary or secondary) then R5. RIO can react with (but not restncted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3)
• the heterocychc nng may contain 5 or more atoms of which 1 or more is a heteroatom (0. N. S. P) or combinations of heteroatoms. and the rest bemg carbon atoms
• H is a heteroatom selected from the group including sulfur, oxygen, nitrogen, or phosphorus
• N0 2 L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups.
• Ai and R9 L may or may not be present and may be chosen from a group that includes alkanes, alkenes, alkynes. esters, ethers, glycerol. amide, urea, saccha ⁇ des, polysaccha ⁇ des, heteroatoms such as oxygen, sulfur, or nitrogen
• the spacer may be charge positive.
• charge negative, charge neutral, or zwitte ⁇ onic Ai and R9 are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as in a heterobifunctional bifunctional molecule
• the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions
• Such reactive groups include (but not limited to) lsothiocynanate. lsocynanate, acyl azide. acid halide.
• A1.R9 can react with (but not restncted to) an activated carboxyhc acid, isothiocyanate. lsocvanate. acyl azide, alkyl halide. acid halide. N-hydroxysuccinimide ester, sulfonyl chlonde. aldehyde, ketone, epoxide. carbonate, imidoester, amide, carboxvlate. or alkylphosphate. arylhalides (difluoro-dimtrobenzene) or anhydndes In other terms when function A1.R9 is an amine. then an acylating or alkvlating agent can react with the amine
• A1.R9 is a sulfhydryl then A1.R9 can react with (but not restncted to) a haloacetyl denvative. activated carboxyhc acid, maleimide, azindine denvative, acryloyl denvative. fluorobenzene denvatives. or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ denvatives)
• A1.R9 can react with (but not restocted to) a diazoacetate. alcohol, thiol or an amine once the acid has been activated
• A1,R9 is an hvdroxyl then A1,R9 can react with (but not restncted to) an activated carboxyhc acid, epoxide. oxirane. or an amine in which carbonvldiimidazole is used
• A1,R9 can react with (but not restocted to) an hydrazine, hydrazide denvative. amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal
• A1,R9 can react with (but not restocted to) an amine. a hvdroxyl. hydrazine. hydrazide. or sulfhydryl group
• A1.R9 an activated carboxyhc acid, haloacetyl denvative, maleimide. azindine denvative. acryloyl denvative. fluorobenzene de ⁇ vatives, or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ de ⁇ vatives) then A1.R9 can react with (but not restncted to) a sulfhydryl If functional group A1.R9 is an aldehyde, ketone. epoxide. oxirane, or an amine in which carbonyldiimidazole or N. N'-disuccinimidyl carbonate is used, then A1.R9 can react with (but not restncted to) a hvdroxyl
• A1.R9 is a hydrazine. hydrazide denvative. or amine (p ⁇ mary or secondary) then A1.R9 can react with (but not restncted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3)
• a compound which contains a disulfide bond that is connected directly to a heterocychc nng system (aromatic or non-aromatic) through one of the sulfur atoms and to an aromatic nng system through the other sulfur atom
• the heterocychc nng may contain 5 or more atoms of which 1 or more is a heteroatom (O, N. S, P) or combinations of heteroatoms. and the rest being carbon atoms
• H is a heteroatom selected from the group including sulfur, oxygen, nitrogen, or phosphorus
• Ri, R 2 , R 3 , R », R5, Re, R 7 , Re , Rio, R11, R ⁇ , R13- at least one of which is an electronegative atom or functionality such as OH.
• OR an ether
• S0 3 " .
• COOH. COOR an ester
• N0 2 L is defined as a linker or spacer group that provides a connection between the disulfide and the reactive heterobifunctional or homobifunctional groups.
• R9 and R ⁇ 4 L may or may not be present and may be chosen from a group that includes alkanes, alkenes, alkynes. esters, ethers, glycerol, amide, urea, sacchandes, polysaccha ⁇ des, heteroatoms such as oxygen, sulfur, or nitrogen
• the spacer may be charge positive, charge negative, charge neutral, or zwitte ⁇ onic
• R 9 and R ⁇ are reactive groups they may be identical as in a homobifunctional bifunctional molecule, or different as in a heterobifunctional bifunctional molecule
• the disulfide compounds contain reactive groups that can undergo acylation or alkylation reactions Such reactive groups include (but not limited to) lsothiocynanate, lsocynanate, acyl azide.
• O-acyl urea N-hydroxysuccinimide esters, succinimide esters, amide, urea, sulfonyl chlonde, aldehyde, ketone.
• ether epoxide, carbonate, alkyl halide. imidoester. carboxylate, alkylphosphate, arylhalides (e g difluoro- dimtrobenzene) or anhydndes
• R9.R14 can react with (but not restncted to) an activated carboxyhc acid, isothiocyanate. isocyanate. acyl azide, alkyl halide, acid halide. N-hydroxysuccinimide ester, sulfonyl chlonde, aldehyde, ketone, epoxide. carbonate, imidoester, amide, carboxylate, or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or anhydndes In other terms when function R9,R14 is an amine. then an acylating or alkylatmg agent can react with the amine
• R9,R14 can react with (but not restocted to) a haloacetyl denvative, activated carboxyhc acid, maleimide, azindine denvative. acryloyl denvative. fluorobenzene denvatives, or disulfide denvative (such as a pyndyl disulfide or 5-thio-2-nitrobenzoic ac ⁇ d ⁇ TNB ⁇ de ⁇ vatives)
• R9,R14 can react with (but not restocted to) a diazoacetate. alcohol, thiol or an amme once the acid has been activated
• R9.R14 can react with (but not restocted to) an act ⁇ ated carboxyhc acid, epoxide. oxirane. or an amine in which carbonyldiimidazole is used
• R9.R14 is an aldehyde or ketone then R9.R14 can react with (but not restocted to) an hydrazine, hydrazide denvative, amine (to form a Schiff Base that may or may not be subsequently reduced by reducing agents such as NaCNBH3), or a diol to form an acetal or ketal
• R9.R14 is activated carboxyhc acid, isothiocyanate. isocyanate. acvl azide. N-hydroxvsuccinimide ester, sulfonyl chlonde, aldehyde, ketone. epoxide. carbonate, imidoester. alkylphosphate. arylhalides (difluoro-dinitrobenzene), anhydnde. alkyl halide. or acid halide.
• R9,R14 can react with (but not restocted to) an amine. a hydroxvl. hydrazine. hydrazide. or sulfhydryl group
• R9.R14 an activated carboxyhc acid, haloacetyl denvative, maleimide. azindine denvative, acryloyl denvative, fluorobenzene de ⁇ vatives, or disulfide denvative (such as a pyndyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ denvatives) then R9.R14 can react with (but not restocted to) a sulfhydryl
• R9,R14 can react with (but not restocted to) a hydroxyl
• R9.R14 is a hydrazine, hydrazide denvative, or amine (p ⁇ mary or secondary) then R9.R14 can react with (but not restocted to) an aldehyde or ketone (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3)
• the object of the current invention is to synthesize labile disulfide molecules
• disulfides are pnmanly reduced by the cysteine-based thiol glutathione ( ⁇ -glutamylcystylglycine). which is present in millimolar concentrations in the cell
• cysteine-based thiol glutathione ⁇ -glutamylcystylglycine
• Bifunctional molecules possessing either homo or heterobifunctionality (commonly refe ⁇ ed to as crosshnkers). are used to connect two molecules together
• the disulfide linkage (RSSR') may be used within bifunctional molecules
• the reversibility of disulfide bond formation makes them useful tools for the transient attachment of two molecules Physiologically, disulfides are reduced by glutathione
• a disulfide bond that is labile under physiological conditions means the disulfide bond is cleaved more rapidly than oxidized glutathione or any disulfide constmcted from thiols in which one of the constituent thiols is more acidic, lower pKa, than glutathione or is activated by intramolecular attack by a free thiol
• Constituent in this case means the thiols that are bonded together in the disulfide bond
• Cleavable means that a chemical bond between atoms is broken
• the present invention descnbes physiologically labile disulfide bond containing bifunctional molecules
• the present invention is also meant to include constructs prepared from the bifunctional molecules, including polymers, peptides, proteins, nucleic acids, polymer nucleic acid complexes
• Construct means any compound resulting from the chemical reaction of at least one of the reactive centers of the bifunctional molecule resulting in new chemical bond other that that resulting from hydrolysis of both reactive centers of the bifunctional molecule Further chemical modification may occur after the formation of the construct
• Cross nkmg refers to the chemical attachment of two or more molecules with a bifunctional reagent
• a bifunctional reagent is a molecule with two reactive ends The reactive ends can be identical as in a homobifunctional molecule, or different as in a heterobifunctional molecule
• Polymers A polymer is a molecule built up b ⁇ repetitive bonding together of smaller units called monomers
• the term polvmer includes both ohgomers which have two to about 80 monomers and poh mers having more than 80 monomers
• the polymer can be linear, branched network, star. comb, or ladder types of polymer
• the polymer can be a homopolymer in which a single monomer is used or can be copolvmer in which two or more monomers are used
• Types of copolymers include alternating, random, block and graft
• Step Polymenzation In step polymenzation. the polymenzation occurs in a stepwise fashion Polvmer growth occurs by reaction between monomers, o gomers and polymers No initiator is needed since there is the same reaction throughout and there is no termination step so that the end groups are still reactive The polymenzation rate decreases as the functional groups are consumed
• step polyme ⁇ zation is done either of two different ways
• the monomer has both reactive functional groups (A and B) in the same molecule so that A-B yields -[A-B]-Or the other approach is to have two bifunctional monomers A-A + B-B yields -[A-A-B-B]-Generally.
• these reactions can involve acylation or alkylation
• Acylation is defined as the mtroduction of an acyl group (-COR) onto a molecule
• Alkylation is defined as the introduction of an alkyl group onto a molecule If functional group A is an amine then B can be (but not restocted to) an isothiocyanate. isocyanate. acyl azide.
• N-hydroxysuccimmide N-hydroxysuccimmide. sulfonyl chlonde, aldehyde (including formaldehyde and glutaraldehyde). ketone, epoxide. carbonate, lmidoester. carboxylate activated with a carbodnmide. alkylphosphate. arylhalides (difluoro-dinitrobenzene). anhvdnde. or acid halide. p-nitrophenyl ester, o- nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole. carbonyl pyndimum.
• function B can be acylating or alkylating agent or amination agent If functional group A is a sulfhydryl then function B can be (but not restncted to) an lodoacetvl denvative. maleimide. azindine denvative. acrylovl denvative. fluorobenzene denvatives.
• function B can be (but not restocted to) a diazoacetate or an amine in which a carbodiimide is used
• Other additives may be utilized such as carbonyldiimidazole. dimethylamino py ⁇ dine (DMAP), N- hydroxysuccinimide or alcohol using carbodiimide and DMAP
• DMAP dimethylamino py ⁇ dine
• oxirane oxirane
• function B can be (but not restocted to) an hydrazine.
• hydrazide denvative, amine to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3 ) or hydroxyl compound to form a ketal or acetal
• function A is a sulfhydryl group then it can be converted to disulfide bonds by oxidizing agents such as iodine (12 ) or NaI04 (sodium penodate), or oxygen (02 )
• Function A can also be an amine that is converted to a sulfhydryl group by reaction with 2-Im ⁇ noth ⁇ olate (Traut's reagent) which then undergoes oxidation and disulfide formation
• Disulfide denvatives such as a pyndyl disulfide or 5-thio-2-nitiObenzoic ac ⁇ d ⁇ TNB ⁇ de ⁇ vatives
• Functional group A or B in any of the above examples could also be a photoreactive group such as aryl azide (including halogenated aryl azide), diazo , benzophenone. alkyne or diazinne de ⁇ v ative
• Monomers containing (but not limited to) vinyl, acrylate. methacrylate, acrylamide, methacrylamide groups can undergo chain reaction which can be radical, anionic , or catiomc Cham polyme ⁇ zation can also be accomplished by cycle or nng opening polyme ⁇ zation
• chain reaction can be radical, anionic , or catiomc Cham polyme ⁇ zation can also be accomplished by cycle or nng opening polyme ⁇ zation
• free radical initiators include peroxides, hydroxy peroxides, and azo compounds such as 2,2'-Azob ⁇ s(- amidinopropane) dihydrochlo ⁇ de (AAP)
• a wide vanety of monomers can be used in the polymenzation processes These mclude positive charged organic monomers such as amme salts, lmidine, guanidine, imine. hydroxylamine. hydrozyine. heterocycle (salts) like imidazole, py ⁇ dine. morphohne. py ⁇ midine.
• the amines could be pH-sensitive in that the pKa of the amine is within the physiologic range of 4 to 8 Specific amines include spermine, spermidine, N.N'-b ⁇ s(2-am ⁇ noethyl)-l,3-propaned ⁇ am ⁇ ne (AEPD), and 3.3'- Diamino-N.N-dimethyldipropylammonium bromide
• Monomers can also be hydrophobic.
• hydrophi c or amphipathic Amphipathic compounds have both hydrophihc (water-soluble) and hydrophobic (water-insoluble) parts
• Hydrophihc groups indicate in qualitative terms that the chemical moiety is water-prefernng Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water Examples of hydrophihc groups include compounds with the following chemical moieties, carbohydrates, polyoxyethylene. peptides. oligonucleotides and groups containing amines, amides, alkoxy amides, carboxyhc acids, sulfurs. or hydroxyls
• Hydrophobic groups indicate m qualitative terms that the chemical moiety is water-avoiding Typically, such chemical groups are not water soluble, and tend not to hvdrogen bonds Hydrocarbons are hydrophobic groups
• Monomers can also be intercalating agents such as acndine.
• thiazole organge, or ethidium bromide Monomers can also contain chemical moieties that can be modified before or after the polyme ⁇ zation including (but not limited to) amines (p ⁇ mary. secondary, and tertiary), amides, carboxv c acid, ester, hydroxyl, hydrazine, alkyl halide. aldehyde . and ketone
• the polymers have other groups that increase their utility These groups can be incorporated into monomers pnor to polymer formation or attached to the polymer after its formation These groups include targeting groups, signals, reporter or marker molecules, spacers, ste ⁇ c stabilizers, chelators. polycations, polyanions, and polymers
• Targeting groups are used for targeting the polymer-nucleic acid complexes to specific cells or tissues
• targeting agents include agents that target to the asialoglycoprotein receptor bv using asiologlycoproteins or galactose residues Proteins such as insulin. EGF.
• Protein refers to a molecule made up of 2 or more amino acid residues connected one to another by peptide bonds between the alpha-ammo group and carboxyl group of contiguous ammo acid residues as in a polypeptide
• the amino acids may be naturally occur ⁇ ng or synthetic Peptides that include the RGD sequence can be used to target many cells
• Peptide refers to a linear senes of amino acid residues connected to one another by peptide bonds between the alpha-amino group and carboxyl group of contiguous ammo acid residues
• Polypeptide includes proteins and peptides. modified proteins and peptides. and non-natural proteins and peptides
• Chemical groups that react w ith sulfhydryl or disulfide groups on cells can also be used to target manv types of cells Folate and other vitamins can also be used for targeting Other targeting groups include molecules that interact with membranes such as fattv acids, cholesterol, dansvl compounds, and amphote ⁇ cin denvatives
• targeting groups can be used to increase the delivery of the drug or nucleic acid to certain parts of the cell
• agents can be used to disrupt endosomes and a nuclear localizing signal (NLS) can be used to target the nucleus
• NLS nuclear localizing signal
• the ligand mav seek a target within the cell membrane, on the cell membrane or near a cell Binding of gands to receptors typically initiates endocytosis
• Ligands could also be used for DNA delivery that bind to receptors that are not endocytosed
• peptides containing RGD peptide sequence that bmd integnn receptor could be used
• viral proteins could be used to bind the complex to cells
• Lipids and steroids could be used to directly insert a complex mto cellular membranes
• the polymers can also contain cleavable groups within themselves When attached to the targeting group, cleavage leads to reduce interaction between the complex and the receptor for the targeting group C
• a chemical reaction can be used to attach a signal to a nucleic acid complex
• the signal is defined in this specification as a molecule that modifies the nucleic acid complex and can direct it to a cell location (such as tissue cells) or location in a cell (such as the nucleus) either in culture or in a whole organism
• a cell location such as tissue cells
• a cell such as the nucleus
• the signal can be a protein, peptide. lipid. steroid, sugar, carbohydrate, nucleic acid or synthetic compound
• the signals enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other mtracellular vesicles
• Nuclear localizing signals enhance the targeting of the gene mto proximity of the nucleus and/or its entrv into the nucleus
• Such nuclear transport signals can be a protein or a peptide such as the SV40 large T ag NLS or the nucleoplasmin NLS
• These nuclear localizing signals interact with a vanetv of nuclear transport factors such as the NLS receptor (karyophenn alpha) which then interacts with karyophenn beta.
• the nuclear transport proteins themselves could also function as NLS's since they are targeted to the nuclear pore and nucleus
• Signals that enhance release from mtracellular compartments can cause DNA release from mtracellular compartments such as endosomes (early and late), lysosomes. phagosomes. vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network (TGN).
• sarcoplasmic reticulum Release includes movement out of an mtracellular compartment into cytoplasm or into an organelle such as the nucleus Releasing signals include chemicals such as chloroquine, bafilomycin or Brefeldin Al and the ER-retaining signal (KDEL sequence), viral components such as influenza virus hemagglutimn subumt HA-2 peptides and other types of amphipathic peptides
• Cellular receptor signals are any signal that enhances the association of the gene or particle with a cell This can be accomplished by either increasing the binding of the gene to the cell surface and/or its association with an lntracellular compartment, for example hgands that enhance endocytosis by enhancing binding the cell surface This includes agents that target to the asialoglycoprotein receptor by using asiologlycoproteins or galactose residues Other proteins such as insulin, EGF, or transfernn can be used for targeting Peptides that include the RGD sequence can be used to target many cells Chemical groups that react with sulfhydr
• Reporter or marker molecules are compounds that can be easily detected Typically they are fluorescent compounds such as fluorescein, rhodamme, Texas red, cy 5, cy 3 or dansyl compounds They can be molecules that can be detected by UV or visible spectroscopy or by antibody interactions or by electron spin resonance Biotin is another reporter molecule that can be detected by labeled avidin Biotin could also be used to attach targeting groups
• a spacer is any linker known to those skilled in the art to enable one to join one moiety to another moiety
• the moieties can be hydrophihc or hydrophobic
• Prefened spacer groups include, but are not limited to C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl. C6-C18 aralkyl. C6-C18 aralkenyl. C6-C18 aralkynyl. ester, ether, ketone. alcohol, polyol. amide, amine. polyglycol. polvamine. thiol. thio ether, thioester. phosphorous containing, and heterocvchc
• a Ste ⁇ c stabilizer is a long chain hvdrophi c group that prevents aggregation of final polymer by stencallv hinde ⁇ ng particle to particle electrostatic interactions
• Examples include alkyl groups. PEG chains, polysacchandes, hydrogen molecules, alkyl amines Electrostatic interactions are the non-covalent association of two or more substances due to attractive forces between positive and negative charges
• a polvcation is a polymer containing a net positive charge, for example poly-L-lysme hydrobromide
• the polvcation can contain monomer units that are charge positive, charge neutral, or charge negative, however, the net charge of the polymer must be positive
• a polvcation also can mean a non-polyme ⁇ c molecule that contains two or more positive charges
• a polyamon is a polymer containing a net negative charge, for example polyglutamic acid
• the polyamon can contain monomer units that are charge negative, charge neutral, or charge positive, however, the net charge on the polymer must be negative
• a polyamon can also mean a non-polyme ⁇ c molecule that contains two or more negative charges
• the term polyion includes polvcation, polyamon. zwittenonic polymers, and neutral polymers
• the term zwittenonic refers to the product (salt) of the reaction between an acidic group and a basic group that are part of the same
• Salts are ionic compounds that dissociate into cations and anions when dissolved in solution Salts increase the lomc strength of a solution, and consequently decrease interactions between nucleic acids with other cations
• a chelator is a poly dentate ligand. a molecule that can occupy more than one site in the coordination sphere of an ion. particularly a metal ion, p ⁇ mary amine, or single proton
• examples of chelators include crown ethers, cryptates. and non-cyclic polydentate molecules
• the X and CR1-2 moieties can be substituted, or at a different oxidation states
• X can be oxygen, nitrogen, or sulfur, carbon, phosphorous or any combination thereof
• R can be H, C, O, S, N.
• the beginning X atom of the strand is an X atom in the (-X-(CRl-2)n)m unit, and the terminal CH2 of the new strand is bonded to a second X atom in the (-X- (CRl-2)n)m umt
• the X and CR1-2 moieties can be substituted, or at a different oxidation states X can be oxygen, nitrogen, or sulfur, carbon, phosphorous or am combination thereof
• a polychelator is a polymer associated with a plurality of chelators by an ionic or co ⁇ alent bond and can include a spacer The polymer can be catiomc.
• anionic. zwittenonic. neutral or contain any combination of catiomc, anionic. zwittenonic. or neutral groups with a net charge bemg catiomc, anionic or neutral, and may contain ste ⁇ c stabilizers, peptides, proteins, signals, or amphipathic compound for the formation of micellar. reverse rmcellar, or umlamellar structures
• the amphipathic compound can ha e a hydrophihc segment that is catiomc. anionic. or zwittenonic. and can contain polyme ⁇ zable groups, and a hydrophobic segment that can contain a polvmenzable group
• the present invention provides for the transfer of polynucleotides, and biologically active compounds into parenchymal cells within tissues in situ and in vivo, utilizing disulfide bonds that can be cleaved under physialogicval condidtions, and delivered lntravasculary (U S patent application senal number 08/571,536), lntrarte ⁇ ally, intravenous, oralh intraduodenaly. v la the jejunum (or lleum or colon), rectally, transdermall . subcutaneously, lntramuscularlv .
• lntrapentoneally lntraparenterally, via direct injections into tissues such as the liver, lung, heart, muscle, spleen, pancreas, brain (including lntraventncular), spinal cord, ganglion, lymph nodes, lymphatic system, adipose tissues, thrvoid tissue, adrenal glands, kidneys, prostate, blood cells, bone marrow cells, cancer cells, tumors, eye retina, via the bile duct, or via mucosal membranes such as in the mouth, nose, throat, vagina or rectum or into ducts of the sali arv or other exoc ⁇ ne glands
• “Delivered” means that the polynucleotide becomes associated with the cell
• the polynucleotide can be on the membrane of the cell or inside the cytoplasm, nucleus. or other organelle of the cell
• the process of deliv enng a polynucleotide to a cell has been commonly termed “transfection” or the process of "transfecting” and also it has been termed “transformation”
• the poh nucleotide could be used to produce a change m a cell that can be therapeutic
• the deliv en of polynucleotides or genetic mate ⁇ al for therapeutic and research purposes is commonh called "gene therapy”
• the polynucleotides or genetic matenal being deliv ered are generally mixed with transfection reagents prior to deliverv
• a biologically activ e compound is a compound having the potential to react with biological components More particularh biologically active compounds utilized in this specification are designed to change the natural processes associated with a living cell
• a cellular natural process is a process that is associated with a cell before delivery of a biologically active compound
• the cellular production of. or inhibition of a mate ⁇ al. such as a protein, caused bv a human assisting a molecule to an in vivo cell is an example of a delivered biologically active compound
• Pharmaceuticals, proteins, peptides. polypeptides. hormones, cytokines. antigens, viruses, oligonucleotides, and nucleic acids are examples of biologicallv active compounds
• Bioactive compounds may be used interchangeably with biologically active compound for purposes of this application
• nucleic acid is a term of art that refers to a polymer containing at least two nucleotides "Nucleotides” contain a sugar deoxv ⁇ bose (DNA) or ⁇ bose (RNA), a base, and a phosphate group Nucleotides are linked together through the phosphate groups "Bases” include pu ⁇ nes and pynmidines. which further mclude natural compounds ademne. thvmine. guanine. cytosine.
• Nucleotides are the monome ⁇ c units of nucleic acid polymers
• a "polynucleotide” is distinguished here from an "oligonucleotide” bv containing more than 80 monome ⁇ c umts, oligonucleotides contain from 2 to 80 nucleotides
• nuclei acid includes deoxynbonucleic acid (DNA) and nbonucleic acid (RNA) DNA may be in the form of anti-sense, plasmid DNA. parts of a plasmid DNA. vectors (PI. PAC, BAC, YAC.
• R ⁇ A mav be in the form of oligonucleotide R ⁇ N tR ⁇ A (transfer R ⁇ A).
• snR ⁇ A small nuclear R ⁇ A
• rR ⁇ A nbosomal R ⁇ A
• rnR ⁇ A messenger R ⁇ A
• anti-sense R ⁇ A ⁇ bozvmes.
• Anti-sense is a polv nucleotide that interferes with the function of D ⁇ A and/or R ⁇ A This may result in suppression of expression
• Natural nucleic acids have a phosphate backbone
• artificial nucleic acids may contain other types of backbones and bases These include PNAs (peptide nucleic acids), phosphothionates. and other v ariants of the phosphate backbone of native nucleic acids In addition.
• DNA and RNA may be single double, t ⁇ ple, or quadruple stranded
• “Expression cassette” refers to a natural or recombinantly produced polynucleotide molecule which is capable of expressing protem(s)
• a DNA expression cassette tv picallv includes a promoter (allowing transcnption initiation), and a sequence encoding one or more proteins
• the expression cassette may include trancnptional enhancers, non-coding sequences, splicing signals, transcnption termination signals, and polyadenylation signals
• An RNA expression cassette typically includes a translation imtiation codon (allowing translation imtiation), and a sequence encoding one or more proteins
• the expression cassette may include translation termination signals, a polyadenosine sequence, internal nbosome entrv sites (IRES), and non-coding sequences
• naked polynucleotides indicates that the polynucleotides are not associated with a transfection reagent or other delivery vehicle that is required for the polynucleotide to be deliv ered to the cardiac muscle cell
• a "transfection reagent” or “delivery vehicle” is a compound or compounds used in the pnor art that b ⁇ nd(s) to or complex(es) with oligonucleotides or polynucleotides. and mediates their entry into cells The transfection reagent also mediates the binding and lntema zation of polynucleotides into cells Examples of transfection reagents include catiomc liposomes and lipids. polyamines.
• the transfection reagent has a net positive charge that binds to the polynucleotide's negative charge
• the transfection reagent mediates binding of polynucleotides to cell via its positive charge (that binds to the cell membrane's negative charge) or via hgands that bind to receptors in the cell
• catiomc liposomes or polylysine complexes have net positive charges that enable them to bind to DNA or RNA
• Other deliverv vehicles are also used, in the pnor art, to transfer genes into cells These include complexmg the polynucleotides on particles that are then accelerated into the cell This is termed "bio stic" or "gun” techniques
• Ionic (electrostatic) interactions are the non-covalent association of two or more substances due to attractiv e forces between positive and negative charges, or partial positive and partial negativ e charges
• Condensed Nucleic Acids A method of condensing a polymer is defined as decreasing its linear length, also called compacting Condensing a polymer also means decreasing the v olume that the polymer occupies
• An example of condensing nucleic acid is the condensation of DNA that occurs in cells
• the DNA from a human cell is approximately one meter in length but is condensed to fit in a cell nucleus that has a diameter of approximately 10 microns
• the cells condense (or compacts) DNA by a senes of packaging mechanisms involving the histones and other chromosomal proteins to form nucleosomes and chromatin
• the DNA within these structures is rendered partially resistant to nuclease DNase) action
• the process of condensing polymers can be used for delivenng them into cells of an organism
• a delivered polymer can stay within the cytoplasm or nucleus apart from the endogenous genetic matenal Alternatively, the polymer could recombine (become a
• Intravascular An mtrav ascular route of administration enables a polymer or polynucleotide to be delivered to cells more evenly dist ⁇ ubbed and more efficiently expressed than direct injections
• 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
• bodily fluid include blood, lymphatic fluid, or bile
• vessels include artenes, arte ⁇ oles. capilla ⁇ es, venules, sinusoids, veins, lymphatics, and bile ducts
• the mtrav ascular route includes delivery through the blood vessels such as an artery
• An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes
• Buffers are made from a weak acid or weak base and their salts Buffer solutions resist changes in pH hen additional acid or base is added to the solution
• Biomolecule refers to peptides. polypeptides. proteins, enzymes, polynucleotides. oligonucleotides. viruses, antigens, carbohydrates (and conjugates), lipids.
• Enzvmes are proteins ev ol ed by the cells of living organisms for the specific function of catalyzing chemical reactions
• a chemical reaction is defined as the formation or cleavage of covalent or ionic bonds
• a polymer is defined as a compound containing more than two monomers
• a monomer is a compound that can be attached to itself or another monomer and thus a form a polymer
• Transdermal refers to application to mammal skin in which drug delivery occurs by crossing the dermal layer
• Alkyl means containing sp 3 hyb ⁇ dized carbon atoms, alkenyl means containing two or more sp 2 hybridized carbon atoms, aklkynyl means containing two or more sp hyb ⁇ dized carbon atoms, aralkyl means containing one or more aromatic ⁇ ng(s) in addition containing sp 3 hybndized carbon atoms, aralkenyl means containing one or more aromatic ⁇ ng(s) in addition to containing two or more sp 2 hyb ⁇ dized carbon atoms, aralkvnyl means containing one or more aromatic ⁇ ng(s) in addition to containing two or more sp hybndized carbon atoms, steroid includes natural and unnatural steroids and steroid de ⁇ vatives
• a steroid denvative means a sterol. a sterol in which the hydroxyl moity has been modified (for example, acylated) or a steroid hormone, or an analog thereof
• Carbohydrates include natural and unnatural sugars (for example glucose), and sugar denvatives (a sugar denvative means a system in which one or more of the hydroxyl groups on the sugar moiety has been modified (for example acylated), or a system in which one or more of the hv drow 1 groups is not present)
• sugar denvatives a sugar denvative means a system in which one or more of the hydroxyl groups on the sugar moiety has been modified (for example acylated), or a system in which one or more of the hv drow 1 groups is not present
• R is meant to be anv compatible group, for example hydrogen, alkyl, alkenyl, alkynyl. aralkyl. aral env 1. or aralkvnvl. and can include heteroatoms (N. O. S), and carbon l groups
• a compound is a material made up of t o or more elements
• Electron withdrawing group is anv chemical group or atom composed of electronegative atom(s), that is atoms that tend to attract electrons
• Resonance stabilization is the ability to distnbute charge on multiple atoms through pi bonds
• the inductive effective, in a molecule, is a shift of electron density due to the polanzation of a bond by a nearbv electronegative or electropositive atom
• Stenc hindrance, or stencs. is the prevention or retardation of a chemical reaction because of neighbonng groups on the same molecule
• An activated carboxylate is a carboxyhc acid denvative that reacts with nucleophiles to form a new covalent bond
• Nucleophiles include nitrogen, oxygen and sulfur- containing compounds to produce ureas, amides, carbonates, esters, and thioesters
• the carboxvhc acid may be activated bv va ⁇ ous agents including carbodiimides, carbonates, phosphoniums. uroniums to produce activated carboxylates acyl ureas, acylphosphonates.
• carboxyhc acid may be used in conjunction with hydroxy and amine-containing compounds to produce activated carboxylates N-hydroxysuccinimide esters, hydroxybenzotnazole esters, N-hydroxy- 5-norbornene-endo-2.3-dicarboximide esters, p-nitrophenyl esters, pentafluorophenyl esters. 4-d ⁇ methvlam ⁇ nopynd ⁇ n ⁇ um amides, and acyl lmidazoles
• a nucleophile is a species possessing one or more electron-nch sites, such as an unshared pair of electrons, the negative end of a polar bond, or pi electrons
• Template polvmenzation was earned out in 25 mM HEPES buffer, pH 8 0 N-(2-Am ⁇ noethv l)-1.3-propaned ⁇ am ⁇ ne (48 ⁇ g, 0 3 mM. Aldnch Chemical Company) was added to a 0 5 mL solution of pCIluc DNA (25 mg. 075 mM in phosphate. 2 6 ug/uL pCIluc. prepared according to Danko. I . Williams. P . Herweijer, H et al Hum Mol Genetics (1997) in press) Dimethyl 5 5"-d ⁇ th ⁇ ob ⁇ s(2- n ⁇ trobenzoate)prop ⁇ on ⁇ m ⁇ date -2 HCl (500 ⁇ g.
• pDNA/ Poly-L-lysine hydrobromide complexes were prepared by combining plasmid DNA (25 ⁇ g) with Poly-L-lysine hydrobromide (95 ⁇ g, MW 35 kDa, Aldrich
• the example indicates that dimethy 1 5.5 ' -d ⁇ th ⁇ obis(2-n ⁇ trobenzoate)propionimidate-2 HCl caged DNA
• the particles formed are stable in physiological salt, and are under lOO nm in size.
• pDNA (pCI Luc)/poly ethyleneimine (25 kDa. Aldrich Chemical Company)/ dimethyl 3.3 '-dithiobispropionimi date and pDNA/polyethyleneimine/dimethyl 5,5'-dithiobis(2- nitrobenzoate)prop ⁇ on ⁇ m ⁇ date-2 HCl complexes were prepared in 25 mM HEPES buffer pH 8 0 All complexes were prepared at pDNA/polyethyleneimine ratios of 1/3.
• the complexes were diluted in Opti-MEM and added by drops. 5 0 ⁇ g DNA/well. to the cells. After a 4 hour incubation period at 37 °C. the media containing the complexes was aspirated from the cells, and replaced ith complete growth media. DMEM with 10% fetal bovine serum (Sigma) After an additional incubation of 42 hours, the cells were harvested and the lysate was assayed for luciferase expression (Wolff, J.A.. Malone, R.W.. Williams. P . Chong. W . Acsadi. G . Jani. A and Feigner. P.L. Direct gene transfer into mouse muscle in ivo Science. 1465-1468. 1990.). A Lumat LB 9507 (EG&G Berthold. Bad-Wildbad. Germany) luminometer was used.
• physiologically labile disulfide bonds present in the dimethyl 5,5'-d ⁇ th ⁇ ob ⁇ s(2- mtrobenzoate)prop ⁇ on ⁇ m ⁇ date-2 HCl complexes can be reduced by cultured cells, while the disulfide bonds present in the dimethyl 3.3'-d ⁇ th ⁇ ob ⁇ sprop ⁇ omm ⁇ date complexes cannot be reduced.
• N N'-B ⁇ s(t-BOC)-L-cv stine 85 mg. 0 15 mmol
• ethyl acetate 20 mL
• N N'-dicy clohexylcarbodiimide 108 mg. 0 5 mmol
• N- hydroxv succinimide 60 mg.
• reaction w as allowed to stir at room temperature for 16 h and then the aqueous solution was dialyzed in a 1 .000 MW cutoff tubing against water (2 X 2 1) for 24 h.
• the solution w as then removed from dialysis tubing, filtered through 5 ⁇ M nylon syringe filter and then dned by lyophihzation to yield 5 mg of polymer.
• Example 13 The particle size of pD A- L-cystine - 1.4-bis(3- am ⁇ nopropyl)p ⁇ peraz ⁇ ne copolymer and DNA-guan ⁇ dino-L-cyst ⁇ nel.4-bis(3- aminopropyl)p ⁇ peraz ⁇ ne copolymer complexes
• Example 14 Condensation of DNA ith L-cystine - 1.4-bis(3- aminopropy piperazine copolymer and decondensation of DNA upon addition of glutathione
• Fluorescein labeled DNA w as used for the determination of DNA condensation in complexes with L-cystine - 1.4-b ⁇ s( 3 -aminopropyOpiperazine copolymer.
• pDNA was modified to a level of 1 fluorescein per 100 bases using Minis' LabelltTM Fluorescein kit.
• the fluorescence was determined using a fluorescence spectrophotometer (Shimadzu RF-1501 spectrofluorometer) at an excitation wavelength of 495 nm and an emission wavelength of 530 nm (Trubetskoy. V S.. Slattum. P. M., Hagstrom, J. E., Wolff. J. A . Budker. V G.. "Quantitative Assessment of DNA Condensation," Anal. Biochem (1999) incorporated by reference).
• the intensity of the fluorescence of the fluorescein-labeled DNA ( 10 ⁇ g/mL) in 0.5 mL of 25 mM HEPES buffer pH 7 5 w as 300 units.
• 10 ⁇ g/mL of L- cystine - 1.4-b ⁇ s(3-am ⁇ nopropyl)p ⁇ peraz ⁇ ne copolymer Upon addition of 10 ⁇ g/mL of L- cystine - 1.4-b ⁇ s(3-am ⁇ nopropyl)p ⁇ peraz ⁇ ne copolymer. the intensity decreased to 100 units.
• To this DNA-polycation sample was added 1 mM glutathione and the intensity of the fluorescence was measured An increase in intensity was measured to the level observed for the DNA sample alone The half life of this increase in fluorescence was 8 minutes
• the expenment indicates that DNA complexes with physiologically -labile disulfide- containing polymers are cleavable in trie presence of the biological reductant glutathione.
• the expe ⁇ ment indicates that DNA complexes with the physiologically-labile disulfide-containing poly mers are capable of being broken, thereby allowing the luciferase gene to be expressed
• Plasmid delivery mtro rat leg was performed as descnbed (Wolff, J.A., Malone. R.W .
• the expenment indicates that DNA complexes with the physiologically-labile disulfide-comaining poly mers are capable of being broken, thereby allowing the luciferase gene to be expressed
• Example 17 Injection of DNA-L-cy stine - 1.4-b ⁇ s(3-am ⁇ nopropyl)p ⁇ peraz ⁇ ne copolymer complex and pDNA (pCI Luc)/5.5'-D ⁇ th ⁇ ob ⁇ s(2-mtrobenzo ⁇ c acid) - 1,4- B ⁇ s(3-am ⁇ nopropy Opiperazine Copolymer Complex and pDNA (pCI Luc)/5,5'- d ⁇ th ⁇ ob ⁇ s(2-mtrobenzo ⁇ c acid) - L4-bis(3-aminopropyl)piperazine -Folate Copolymer Complexes into the intestinal lumen of mice
• Intestinal cells were transfected by injecting pDNA solutions into the mesentenc vasculature A 3-cm section of the small intestines was clamped, blocking both vascular inflow and outflow A volume of 250 ⁇ l containing 50 ⁇ g pCILuc and 50 ⁇ g poly(ethylen ⁇ m ⁇ ne) (Aldnch Chemical Co MW 25.000 MW), L-cystine - l,4-b ⁇ s(3- aminopropy Opiperazine copolymer.
• the expenment indicates that DNA complexes with labile disulfide-contaimng polymers are capable of being broken, thereby allowing the luciferase gene to be expressed
• the resulting solution was heated to 80 TJ and diisopropylethylamine (35 ⁇ L, 0.20 mmol. Aldnch Chemical Company) was added by drops After 16 hr, the solution was cooled, diluted with 3 mL H 2 0. and dialyzed in 12.000 - 14,000 MW cutoff tubing against water (2 X 2 L) for 24 h. The solution was then removed from dialysis tubing and dned by ly ophili/.ation to yield 23 mg (82%) of 5.5'-dithiobis(2- mtrobenzoic acid) - 1.4-b ⁇ s(3-am ⁇ nopropy Opiperazine copolymer.
• Results indicate an increased level of luciferase expression in pDNA / 5.5'- Dithiob ⁇ s(2-n ⁇ trobenzo ⁇ c acid) - 1.4-B ⁇ s(3 -amino propy Opiperazine Copolymer complexes over pCI Luc DNA itself .
• pCI Luc DNA/poly-L-lysine complexes and pCI Luc DNA/ Histone HI complexes
• results also indicate that the pDNA is being released from the pDNA / 5.5 " -Dith ⁇ obis(2-mtrobenzoic acid) - 1,4-Bis(3- aminopropyOpiperazine Copolymer complexes, and is accessible for transcription.
• Tetraethv lenepentamine ( 3 2 ⁇ L. 0 017 mmol. Aldnch Chemical Company) was taken up in 1 0 mL dichloromethane and HCl (1 mL. 1 M in Et 2 0, Aldnch Chemical Companv ) was added Et 2 0 as added and the resulting HCl salt was collected by filtration The salt was taken up in 1 mL DMF and 5.5'-d ⁇ th ⁇ ob ⁇ s[succ ⁇ n ⁇ m ⁇ dyl (2- nitrobenzoate) I ( 1 mg 0 017 mmol) w as added The resulting solution was heated to 80°C and dnsopropv lethv lamine (15 ⁇ L 0 085 mmol.
• Tetraethylenepentamine ( 2 3 ⁇ L. 0 012 mmol. Aldnch Chemical Company) and 1 ⁇ L. 0 0034 mmol. Aldrich Chemical Company) were taken up in 0 5 mL methanol and HCl ( 1 mL. 1 M in Et 2 0, Aldrich Chemical Company) was added EbO w as added and the resulting HCl salt was collected by filtration The salt as taken up in 1 mL DMF and 5.5 " -dithiobis[succinimidyl (2- nitrobenzoate)] ( 10 mg. 0 017 mmol) w as added The resulting solution was heated to 80°C and diisopropylethy lamine (15 ⁇ L.
• Example 28 Synthes ⁇ s of 5. ' -D ⁇ th ⁇ ob ⁇ s(2-n ⁇ trobenzoic acid) - N,N'-Bis(2- aminoethy l)-1.3-propaned ⁇ am ⁇ ne - T ⁇ s(2-am ⁇ noethyl)amine Copolymer
• N,N'-Bis(2-am ⁇ noethyD-1.3-propaned ⁇ am ⁇ ne ( 2.0 ⁇ L, 0.012 mmol, Aldrich Chemical Company ) and tris(2-aminoeihyl)amine (0 51 ⁇ L. 0.0034 mmol. Aldrich Chemical Company ) were taken up in 0 5 mL methanol and HCl (1 mL. 1 M in Et 2 0, Aldrich Chemical Company ) w as added Et 2 0 w as added and the resulting HCl salt was collected by filtration The salt was taken up in 1 mL DMF and 5,5'- dithiobis[succ ⁇ n ⁇ m ⁇ dv l(2-n ⁇ robenzoate)] (10 mg.
• Example 30 Intramuscular Injections of Complexes from pDNA (pCI Luc)/Phys ⁇ olog ⁇ cally Labile Disulfide Bond Containing Polymers on Mouse.
• the complexes prepai ed 10 pC I Luc D ⁇ .V Physiologically Labile Disulfide Bond Containing Poly mers ore ei i ctn e in direct muscle injections.
• the luciferase expression indicates that the pDNA is being released from the complex and is accessible for transcnption
• Complexes prepared with 5.5'-Dithiobis(2-nitrobenzoic acid) - 1.4-B ⁇ s(3-am ⁇ nopropyl)p ⁇ peraz ⁇ ne Copolymer were the most effective, giving luciferase expression levels 2 to 10 times as high as pDNA
• Pentaethylenehexamine ( 4 2 ⁇ L. 0 017 mmol. Aldnch Chemical Company) was taken up in 1 0 mL dichloromethane and HCl (1 mL, 1 M in Et 0. Aldnch Chemical Company) was added Et 2 0 was added and the resulting HCl salt was collected by filtration The salt was taken up in 1 mL DMF and 5.5'-d ⁇ th ⁇ ob ⁇ s[succ ⁇ mm ⁇ dyl(2- mtrobenzoate)] (10 mg, 0 017 mmol) was added The resulting solution was heated to 80°C and dnsopropylethylamine (12 ⁇ L.
• Pentaethylenehexamine 2 9 ⁇ L. 0 012 mmol. Aldnch Chemical Company
• tns(2-am ⁇ noethyl)am ⁇ ne (0 51 ⁇ L, 0 0034 mmol. Aldnch Chemical Company) were taken up in 0 5 mL methanol and HCl (1 mL. 1 M in Et 2 0.
• Folate-PEG(3400 MW)-NH2 was prepared according to the known procedure (Lee, R J . Low, P S Biochimica et Biophysica Acta 1233, 1995. 134-144) Folate-PEG- NH2 was acylated with succinylated N-(3-(BOC)aminopropyl)-1.3- propaneam ⁇ ne(BOC)am ⁇ ne Removal of the BOC protecting groups afforded the Folate monomer
• H 2 N-EEEEEEEE-NHCH 2 CH 2 NH 2 (5 0 mg, 0 0052 mmol, Genosis) was taken up m 0 1 mL HEPES (250 mM, pH 7 5) 5.5 ' -d ⁇ th ⁇ ob ⁇ s[succ ⁇ mm ⁇ dyl(2-n ⁇ trobenzoate)] (3 1 mg, 0 0052) was added with 0 2 mL DMSO and the mixture was stined ovemight at room temperature After 16 hr the solution was heated to 70°C for 10 mm. cooled to room temperature and diluted to 1 10 mL with DMSO
• Fluorescein labeled DNA was used for the determination of DNA condensation m complexes with 5,5'-D ⁇ th ⁇ ob ⁇ s(2-mtrobenzo ⁇ c acid) - Poly-Glutamicacid (8mer) Copolymer pDNA was modified to a level of 1 fluorescein per 20 bases using Minis' LabellTTM Fluorescein kit The fluorescence was determined using a fluorescence spectrophotometer (Shimadzo RF-1501 Fluorescence Spectrophotometer), at an excitation wavelength of 497 nm. and an emission wavelength of 520 nm
• LT-1TM (12 ⁇ g. Minis Corporation) followed by pDNA (pCI Luc. 4 ⁇ g), and Poly-Glutamicacid (4 ⁇ g, MW 49000, Sigma
• the expe ⁇ ment demonstrates rapid cleavage of the disulfide bond of 5.5 ' -d ⁇ th ⁇ ob ⁇ s(2- mtrobenzoic acid) - contaimng copolymers with the physiological reducing agent glutathione

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