EP1499627A2 - Oligonucleotides chimeres resistants a la nuclease - Google Patents

Oligonucleotides chimeres resistants a la nuclease

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Publication number
EP1499627A2
EP1499627A2 EP02746811A EP02746811A EP1499627A2 EP 1499627 A2 EP1499627 A2 EP 1499627A2 EP 02746811 A EP02746811 A EP 02746811A EP 02746811 A EP02746811 A EP 02746811A EP 1499627 A2 EP1499627 A2 EP 1499627A2
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EP
European Patent Office
Prior art keywords
group
alkyl
independently
substituted
oligomeric compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02746811A
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German (de)
English (en)
Inventor
Muthiah Manoharan
Martin A. Maier
Thazha P. Prakash
Kallanthottathil Gopalan Rajeev
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Ionis Pharmaceuticals Inc
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Isis Pharmaceuticals Inc
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Priority claimed from US09/996,292 external-priority patent/US20030158403A1/en
Priority claimed from US10/013,295 external-priority patent/US20030175906A1/en
Application filed by Isis Pharmaceuticals Inc filed Critical Isis Pharmaceuticals Inc
Publication of EP1499627A2 publication Critical patent/EP1499627A2/fr
Withdrawn legal-status Critical Current

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    • 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

Definitions

  • the present invention relates to novel nuclease-resistant oligomeric compounds and to novel methods for increasing the nuclease resistance of 15 oligomeric compounds.
  • Efficacy and sequence specific behavior of antisense oligonucleotides (ONs) in biological systems depend upon their resistance to enzymatic
  • heterocyclic modifications have been shown to enhance the binding affinity of nucleic acids through increased hydrogen bonding and/or base stacking interactions.
  • heterocyclic modifications include 2,6- diaminopurine, which allows for a third hydrogen bond with thymidine and 5 replacement of the hydrogen atom at the C5 position of pyrimidine bases with a propynyl group, resulting in increased stacking interactions (Chollet, A.; Chollet- Damerius, A.; Kawashima, E. H. Chem. Scripta 1986, 26, 37-40; Wagner, R. W.; Matteucci, M. D.; Lewis, J. G.; Guttierrez, A. J.; Moulds, C; Froehler, B. C. Science 1993, 260, 1510-1513).
  • cytosine analogs such as phenoxazine, phenothiazine (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and tetrafluorophenoxazin (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388), have been developed and have been shown to hybridize to guanine and, in case of tetrafluorophenoxazin, also with
  • the tricyclic cytosine analogs have also been shown to enliance helical thermal stability by extended stacking interactions.
  • cytosine analogs are further improved with G-clamp, a cytosine analog with an aminoethoxy moiety attached to the rigid phenoxazine scaffold (Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc.
  • oligonucleotides as the T m data indicate an even greater discrimination between the perfectly matched and mismatched sequences as compared to dC5 me .
  • the tethered amino group may serve as an additional hydrogen bond donor that interacts with the Hoogsteen face, namely the O6, of a complementary guanine.
  • the increased affinity of G-clamp is thus most likely mediated by the combination
  • oligonucleotides in biological systems are dependent, in part, upon their nuclease stability. Resistance to the many nucleases present in biological systems is best achieved by modified oligonucleotides. It is therefore essential, when designing modified nucleotides, to evaluate and optimize their resistance to enzymatic degradation.
  • the present invention relates to novel nuclease-resistant oligomeric compounds and to novel methods for increasing the nuclease resistance of oligomeric compounds.
  • the compounds of the invention relate to oligomeric compounds of formula V:
  • n is from 3 to about 50; each Yi is, independently, an intemucleoside linking group; Y 2 is oxygen or an intemucleoside linking group; Y 3 is oxygen or an intemucleoside linking group; each Bx is an optionally protected heterocyclic base moiety; each Ai is, independently, hydrogen or a sugar substituent group; Wi is hydrogen, a hydroxyl protecting group or a modified nucleoside selected from the group consisting of
  • W is hydrogen, a hydroxyl protecting group or a modified nucleoside selected from the group consisting of
  • the intemucleoside linking groups of the compounds of formula V are phosphorus-containing intemucleoside linking groups.
  • at least one intemucleoside linking group of the compounds of formula V is other than phosphodiester, and more preferably, greater than 90% of the intemucleoside linking groups of the compounds of formula V are non-phosphorous containing intemucleoside linking groups.
  • greater than 90% of the intemucleoside linking group of the compounds of formula V are phosphorothioate linking groups.
  • the oligomeric compounds of formula V comprise gapmers, hemimers or inverted gapmers.
  • the oligomeric compounds of formula V comprise at least one 2'-O-CH 2 CH 2 -O-CH 3 sugar substituent group in at least one region of the gapmer, hemimer of inverted gapmer.
  • the oligomeric compounds of formula V comprise at least one nucleoside wherein Bx is a polycyclic heterocyclic base moiety.
  • the oligomeric compounds of formula V comprise at least one nucleoside wherein Bx is, independently, of the formula:
  • a 6 is O or S
  • a 7 is CH 2 , N-CH 3 , O or S; each Ag and A 9 is hydrogen or one of A 8 and A 9 is hydrogen and the other of Ag and A is selected from the group consisting of:
  • each Q 2 is, independently, H or Pg;
  • a 10 is H, Pg, substituted or unsubstituted C1-C10 alkyl, acetyl, benzyl, -(CH 2 ) p3 NH 2 , -(CH 2 ) p3 N(H)Pg, a D or L ⁇ -amino acid, or a peptide derived from D, L or racemic ⁇ -amino acids;
  • Pg is a nitrogen, oxygen or thiol protecting group; each pi is, independently, from 2 to about 6; p2 is from 1 to about 3; and p3 is from 1 to about 4.
  • Y 3 of formula V is an intemucleoside likning group and Wi of formula V is a modified nucleoside.
  • Y 2 of formula V is an intemucleoside linking group and W 2 of formula V is a modified nucleoside.
  • the invention relates to methods of enhancing the nuclease resistance of an oligomeric compound comprising providing at least one modified nucleoside at either the 3' or 5' terminus of the oligomeric compound to give a modified oligomeric compound of formula V, such that at least one of Wi and W 2 of formula V is not hydrogen or a hydorxyl protecting group.
  • Figure 1 A depicts the structure of the tricyclic cytosine analog G-clamp
  • Figure 1 B depicts guanidinyl G-clamp hybridized to complementary guanosine
  • Figure 1 C depicts a palindromic decamer duplex that was used for x-ray crystallography.
  • the five hydrogen bonds formed between C* and G are indicated by horizontal lines.
  • C* refers to guanidinyl G-clamp and T refers to 2'- O-MOE-T.
  • Figure 2 depicts a Fourier (2F 0 -F C ) sum electron density map (contoured at
  • Figure 3 depicts the base stacking that occurs between a guanidmyl G- clamp nucleobase analog and guanosine viewed approximately along the vertical to the phenoxazine rings.
  • Figure 4 depicts the degradation of oligonucleotides 157 (open triangles) and 158 (closed circles) with SVPD as a function of incubation time and compared to degradation of an unmodified control oligonucleotide 159 (closed diamonds) as determined by CGE analysis.
  • Figure 5 depicts the velocity of the hydrolysis of oligonucleotide 159 with
  • BIPD as a function of the concentration of co-incubated oligonucleotides 157 (open triangles) and 158 (closed circles).
  • Figure 6A depicts the relative units of a full-length L/D chimeric oligonucleotide before administration to BalbC mice
  • Figure 6 B depicts the relative units of a full-length L/D chimeric oligonucleotide that was present in the liver one hour after administration of a 25 mg/kg dose by IV bolus into BalbC mice
  • Figure 6 C depicts the relative units of a full-length L/D chimeric oligonucleotide that was present in the kidney one hour after administration of a 25 mg/kg dose by IV bolus into BalbC mice
  • Figure 6 D depicts the relative units of a full-length L/D chimeric oligonucleotide that was present in the spleen one hour after administration of a 25 mg/kg dose by IV bolus into BalbC mice
  • Figure 6 E depicts the relative units of a full-length L/D chimeric oligonucleotide that was present in the lung one hour after administration of
  • Figure 7A depicts the relative units of a full-length L/D chimeric oligonucleotide before administration to BalbC mice
  • Figure 7 B depicts the relative units of a full-length L/D chimeric oligonucleotide that was present in the liver 24 housr after administration of a 25 mg/kg dose by IV bolus into BalbC mice
  • Figure 7 C depicts the relative units of a full-length L/D chimeric oligonucleotide that was present in the kidney 24 hours after administration of a 25 mg/kg dose by IV bolus into BalbC mice
  • Figure 7 D depicts the relative units of a full-length L/D chimeric oligonucleotide that was present in the spleen 24 hours after administration of a 25 mg/kg dose by IV bolus into BalbC mice
  • Figure 7 E depicts the relative units of a full-length L/D chimeric oligonucleotide that was present in the lung 24 hours after
  • oligomer and “oligomeric compound” refer to a plurality of naturally-occurring or non-naturally-occurring nucleosides joined together in a specific sequence.
  • oligomer and oligomeric compound include oligonucleotides, oligonucleotide analogs, oligonucleosides and chimeric oligomeric compounds where there are more than one type of intemucleoside linkages dividing the oligomeric compound into regions.
  • Oligomeric compounds are typically structurally distinguishable from, yet functionally interchangeable with, naturally-occurring or synthetic wild-type oligonucleotides.
  • oligomeric compounds include all such structures that function effectively to mimic the stmcture and/or function of a desired RNA or DNA strand, for example, by hybridizing to a target.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides composed of naturally- occurring nucleobases, sugars and covalent intemucleoside (backbone) linkages as well as oligonucleotides having non-iiaturally-occurring portions that function similarly.
  • Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
  • nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • this linear polymeric stmcture can be further joined to form a circular stmcture.
  • open linear structures are generally preferred.
  • the phosphate groups are commonly referred to as forming the intemucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • modified backbones include those having a phosphoms atom in the backbone and those that do not have a phosphoms atom in the backbone.
  • modified oligonucleotides that do not have a phosphoms atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorofhioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a 3' to 3', 5' to 5 ' or 2' to 2' linkage.
  • Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most intemucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts, mixed salts and free acid forms are also included.
  • Preferred modified backbones that do not include a phosphoms atom therein are those that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • oligonucleotide mimetics both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • a peptide nucleic acid PNA
  • PNA compounds the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
  • oligonucleotides with phosphorothioate backbones and oligonucleotides with heteroatom backbones and in particular -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 - [known as a methylene (methylimino), MMI backbone or more generally as methyleneimino], -CH2-O-N(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -O- N(CH 3 )-CH 2 -CH 2 - of the above referenced U.S.
  • Bx is intended to indicate a heterocyclic base moiety.
  • Heterocyclic base moieties (often referred to in the art simply as a “bases” or a “nucleobases”) amenable to the present invention include naturally or non- naturally occurring nucleobases. One or more functionalities of the base can bear a protecting group.
  • “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-G ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and gu
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidme(lH-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted 5 phenoxazine cytidine (e.g.
  • nucleobases include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-
  • nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30,
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted 0 purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more 5 particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • oligomeric compounds are prepared having one or more heterocyclic base moieties comprising a polycyclic 5 heterocyclic base moiety.
  • polycyclic heterocyclic base moiety is intended to include compounds comprising at least 3 or more fused rings.
  • a number of tricyclic and some tetracyclic heterocyclic compounds have been prepared and substituted for naturally ocurring heterocyclic base moieties in oligomeric compounds.
  • the resulting oligomeric compounds have been used in 10 antisense applications to increase the binding properties of for example a modified strand to a target strand. The more studied modifications have been targeted to guanosines and are commonly referred to as cytidine analogs.
  • a polycyclic heterocyclic base moiety has the formula:
  • cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand or elsewhere in the same strand include 1,3-
  • the gain in helical stability does not compromise the specificity of the oligonucleotides.
  • the T m data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5 me .
  • the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the O6, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding.
  • polycyclic heterocyclic base moieties comprising tricyclic and tetracyclic heteroaryl compounds amenable to the present invention include those having the formulas:
  • R 14 is NO 2 or both R ⁇ 4 and R ⁇ 2 are independently -CH 3 .
  • the synthesis of these compounds is dicslosed in United States Patent Serial Number 5,434,257, which issued on July 18, 1995, United States Patent Serial Number 5,502,177, which issued on March 26, 1996, and United States Patent Serial Number 5,646, 269, which issued on July 8, 1997, the contents of which are commonly assigned with this application and are incorporated herein in their entirety.
  • a and b are independently 0 or 1 with the total of a and b being 0 or 1;
  • A is N, C or CH;
  • Z is taken together with A to form an aryl or heteroaryl ring stmcture comprising 5 or 6 ring atoms wherein the heteroaryl ring comprises a single O ring heteroatom, a single N ring heteroatom, a single S ring heteroatom, a single O and a single N ring heteroatom separated by a carbon atom, a single S and a single N ring heteroatom separated by a C atom, 2 N ring heteroatoms separated by a carbon atom, or 3 N ring heteroatoms at least 2 of which are separated by a carbon atom, and wherein the aryl or heteroaryl ring carbon atoms are unsubstituted with other than H or at least 1 nonbridging ring carbon atom is fubstituted with R 20 or
  • R 6 is independently H, C ]-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, NO 2 , N(R 3 ) 2 ,
  • R is , independently, H, C 1-6 alkyl, C 2-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 1 70 9ft
  • R 21 is, independently, H or a protecting group
  • R 3 is a protecting group or H; and tautomers, solvates and salts thereof.
  • each Rj 6 is, independently, selected from hydrogen and various substituent groups.
  • the present invention provides oligomeric compounds comprising a plurality of linked nucleosides wherein the preferred intemucleoside linkage is a 3 ',5 '-linkage.
  • the preferred intemucleoside linkage is a 3 ',5 '-linkage.
  • 2',5'-linkages can be used (as described in U.S.
  • a 2*,5'-linkage is one that covalently connects the 2'-position of the sugar portion of one nucleotide subunit with the 5'-position of the sugar portion of an adjacent nucleotide subunit.
  • the compounds described herein may have asymmetric centers. Unless otherwise indicated, all chiral, diastereomeric, and racemic forms are included in the present invention. Geometric isomers may also be present in the compounds described herein, and all such stable isomers are contemplated by the present invention. It will be appreciated that compounds in accordance with the present invention that contain asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms or by synthesis.
  • the present invention includes all isotopes of atoms occurring in the intermediates or final compounds.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium.
  • sucrose substituent group refers to optionally protected groups that are attached to selected sugar moieties at the 2', 3', or 5'- position. Sugar substituent groups have also been attached to heterocyclic base moieties for example by attachment at amino functionalities.
  • a representative list of sugar substituent groups amenable to the present invention include hydroxyl, C ⁇ -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, C 5 -C 20 aryl, O-alkyl, O-alkenyl, O-alkynyl, O-alkylamino, O-alkylalkoxy, O- alkylaminoalkyl, O-alkyl imidazole, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH- alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, halogen (particularly fluoro), amino, thiol, keto, carboxyl, nitro, nitroso, nitrile, triflu
  • polyethers linear and cyclic polyethylene glycols (PEGs), and (PEG)-containing groups, such as crown ethers and those which are disclosed by Ouchi et al. (Drug Design 5 and Discovery 1992, 9, 93), Ravasio et al (J. Org. Chem. 1991, 56, 4329) and Delgardo et. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249), each of which is herein incorporated by reference in its entirety. Further sugar modifications are disclosed in Cook, P.D., Anti-Cancer Drug Design, 1991, 6, 585-607. Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O-
  • each Ri is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl.
  • 2'-S-R 1 nucleosides are disclosed in United States Patent No. 5,670,633, issued September 23, 1997, hereby incorporated by reference in its entirety. The incorporation of 2'- S Ri monomer synthons are disclosed by Hamm et al, J. Org. Chem., 1997, 62,
  • sugar substituent groups can include groups having the structure of one of formula I or II:
  • Z 0 is O, S or NH
  • each R 8 , R 9 , Rn and R ⁇ 2 is, independently, hydrogen, C(O)R ⁇ 3 , substituted or unsubstituted Ci-Cio alkyl, substituted or unsubstituted C 2 -C ⁇ o alkenyl, substituted or unsubstituted C 2 -C ⁇ o alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; or optionally, R ⁇ and R ⁇ 2 , together form a phthalimido moiety with the nitrogen atom to which they are attached; each R ⁇ 3 is, independently, substituted or unsubstituted -Cio alkyl, tri
  • R 5 is hydrogen, a nitrogen protecting group or -T-L
  • R 5a is hydrogen, a nitrogen protecting group or -T-L
  • T is a bond or a linking moiety
  • L is a chemical functional group, a conjugate group or a solid support material; each R 6 and R 7 is, independently, H, a nitrogen protecting group, substituted or unsubstituted Ci-Cio alkyl, substituted or unsubstituted C 2 -C ⁇ 0 alkenyl, substituted or unsubstituted C 2 -C ⁇ 0 alkynyl, wherein said substitution is hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH 3 + , N(R ⁇ )(R 15 ), guanidino or acyl where said acyl is an acid amide or an ester; or R 6 and R 7 , together, are a nitrogen protecting group, are joined in a ring structure that optionally includes an additional heteroatom selected from N and O or are a chemical functional group;
  • R ⁇ 6 is H or C ⁇ -C 8 alkyl
  • Zi, Z 2 and Z 3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
  • Z 5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R 5 )(R 6 ) OR 5 , halo, SR 5 or CN; each qi is, independently, an integer from 1 to 10; each q 2 is, independently, 0 or 1; q 3 is 0 or an integer from 1 to 10; q 4 is an integer from 1 to 10; provided that when q 3 is 0, q 4 is greater than 1.
  • Representative sugar substituent groups of Formula I are disclosed in United States Patent Application Serial No. 09/130,973, filed August 7, 1998, entitled “Capped 2'-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety.
  • Representative cyclic sugar substituent groups of Formula II are disclosed in United States Patent Application Serial No. 09/123,108, filed July 27, 1998, entitled “RNA Targeted 2'-Modified Oligonucleotides that are Conformationally Preorganized,” hereby inco orated by reference in its entirety.
  • Particularly preferred sugar substituent groups include O[(CH 2 ) p ⁇ O] p2 CH 3 , O(CH 2 ) p ⁇ OCH 3 , O(CH 2 ) p iNH 2; O(CH 2 ) pl CH 3; O(CH 2 ) p ⁇ ONH 2> and O(CH 2 ) piON[(CH 2 ) p iCH )] 2; where pi and p2 are from 1 to about 10.
  • Some preferred oligomeric compounds of the invention contain at least one nucleoside having one of the following sugar substituent groups: d to do lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3> SOCH 3) SO 2 CH 3 , ONO 2)
  • a preferred modification includes 2'-methoxyethoxy [2'-O-CH 2 CH 2 OCH 3 , also known as 2'- O-(2-methoxyethyl) or 2'-MOE] (Martin et al, Helv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group.
  • a further preferred modification is 2'- dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'- DMAOE.
  • nucleosides and oligomers Similar modifications may also be made at other positions on nucleosides and oligomers, particularly the 3' position of the sugar on the 3' terminal nucleoside or at a 3' ⁇ position of a nucleoside that has a linkage from the 2'-position such as a 2'-5' linked oligomer and at the 5' position of a 5' terminal nucleoside. Oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S.
  • a further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
  • the linkage is preferably a methelyne (- CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in WO 98/39352 and WO
  • the present invention also includes oligomeric compounds that are chimeric compounds.
  • "Chimeric" oligomeric compounds or “chimeras,” in the context of this invention are oligomeric compounds, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • Chimeric oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid upon the oligonucleotide.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • Chimeric oligomeric compounds of the invention maybe formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
  • the oligomeric compounds of the invention can be chimeric oligonucleotides, including " gapmers,” “inverted gapmers,” or “hemimers.”
  • a single terminal (either 5' or 3') region of the oligonucleotide contains modified nucleosides.
  • the oligonucleotide is called a "gapmer” and the modified 5'- and 3'-terminal regions are referred to as "wings".
  • the 5' and 3' wings can contain nucleosides modified in the same or different manner.
  • an "inverted gapmer” a central region of the oligonucleotide contains modified nucleosides.
  • the present invention provides compounds and methods that are useful for enhancing the nuclease resistance of oligomeric compounds. More specifically, the present invention is directed to oligomeric compounds that exhibit enhanced nuclease resistance, and to methods for improving the nuclease stability of oligomeric compounds.
  • resistance to enzymatic degradation is an important feature of antisense oligonucleotide therapeutics, and the efficacy of antisense oligonucleotide drags has been hampered by the activity of nucleases present in biological systems.
  • certain modifications of oligomeric compounds enhance their nuclease stability. Novel methods for increasing the nuclease stability of oligomeric compounds involving the incorporation of modified nucleosides have also been discovered.
  • the present invention is directed to nuclease-resistant oligomeric compounds that may be useful as pharmaceuticals.
  • Antisense oligonucleotides can be designed to bind in predictable ways to certain nucleic acid target sequences, which can cause selective inhibition of the expression of genes whose products lead to disease.
  • Antisense oligonucleotides can bind to specific complementary regions on mRNA, thereby inhibiting protein biosynthesis through the disruption of processes such as splicing, polyadenylation, correct RNA folding, translocation and initiation of translation of mRNA, or ribosome movement along the mRNA.
  • the oligomeric compounds of the invention typically exhibit enhanced nuclease resistance and can be used as effective antisense oligonucleotides in therapeutic applications for the treatment of specific diseases.
  • the methods of the invention can also be used to increase the efficacy of antisense oligonucleotides as therapeutics through enhancement of the nuclease resistance of oligomeric compounds.
  • Preferred embodiments of the invention include nuclease resistant oligomeric compounds that comprise at least one modified 5' or 3' terminal nucleoside or nucleotide and at least one intemucleoside linking group other than phosphodiester, and optionally comprise modified 2' substituent groups in the gapmer, hemimer, and inverted gapmer configuration and one or more modified nucleobases.
  • the tricyclic cytosine analogs phenoxazine and 9- (aminoethoxy)phenoxazine (G-clamp) have been shown to significantly enhance the nuclease resistance of oligonucleotides.
  • Phenoxazine and G-clamp were incorporated into model oligomers with a natural phosphodiester backbone and enzymatic degradation was monitored after treatment with snake venom phosphodiesterase.
  • a single incorporation of either phenoxazine or G-clamp at the 3' terminus completely protected the oligonucleotides against 3' exonuclease attack.
  • nuclease resistance of oligonucleotides containing phenoxazine and G-clamp is not believed to be caused by low binding affinity for the enzyme's active site, as the modified oligonucleotides are capable of slowing down the degradation of a natural DNA fragment by bovine intestinal mucosal phosphodiesterase in a dose-dependent manner. No significant difference was observed between phenoxazine and G-clamp in terms of their effects on nuclease resistance and their capacity to inhibit nuclease activity.
  • a guanidinyl moiety can be added to an oligonucleotide by postsynthetic guanidinylation of a primary amino group tethered to either the 2 '-position or to the phenoxazine ring system of a tricyclic cytosine analog (G-clamp).
  • the former amino group can be selectively deprotected and guanidinylated on the solid support, while the aminoethoxy tether of G-clamp can be guanidinylated in aqueous solution after deprotection and cleavage of the oligonucleotide from the support. Both methods have been successfully used to synthesize and characterize various guanidinyl-modified oligonucleotides.
  • Support media is used to attach a first nucleoside or larger nucleosidic synthon which is then iteratively elongated to give a final oligomeric compound.
  • Support media can be selected to be insoluble or have variable solubility in different solvents to allow the growing oligomer to be kept out of or in solution as desired.
  • Traditional solid supports are insoluble and are routinely placed in a reaction vessel while reagents and solvents react and or wash the growing chain until cleavage frees the final oligomer.
  • soluble supports including soluble polymer supports to allow precipitating and dissolving the bound oligomer at desired points in the synthesis
  • Representative support media that are amenable to the methods of the present invention include without limitation: controlled pore glass (CPG); oxalyl- controlled pore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527); TENTAGEL Support, (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373); or POROS, a copolymer of polystyrene/divinylbenzene available from Perceptive Biosystems.
  • CPG controlled pore glass
  • oxalyl- controlled pore glass see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527
  • TENTAGEL Support see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 33
  • a soluble support media poly(ethylene glycol), with molecular weights between 5 and 20 kDa
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • Activated phosphorus compositions may be used in coupling reactions for the synthesis of oligomeric compounds.
  • the term "activated phosphoms composition” includes monomers and oligomers that have an activated phosphorus-containing substituent group that is reactive with a hydroxyl group of another monomeric or oligomeric compound to form a phosphorus- containing intemucleotide linkage.
  • Such activated phosphoms groups contain activated phosphorus atoms in P m valence state.
  • Such activated phosphorus atoms are known in the art and include, but are not limited to, phosphoramidite, H- phosphonate, phosphate triesters and chiral auxiliaries.
  • a preferred synthetic solid phase synthesis utilizes phosphoramidites as activated phosphates.
  • the phosphoramidites utilize P m chemistry.
  • the intemiediate phosphite compounds are subsequently oxidized to the P v state using known methods to yield, in a preferred embodiment, phosphodiester or phosphorothioate intemucleotide linkages. Additional activated phosphates and phosphites are disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).
  • a representative list of activated phosphoms containing monomers or oligomers include those having the formula:
  • each Bx is, independently, a heterocyclic base moiety or a blocked heterocyclic base moiety; and each R ⁇ 7 is, independently, H, a blocked hydroxyl group, a sugar substituent group, or a blocked substituent group;
  • W 3 is an hydroxyl protecting group, a nucleoside, a nucleotide, an oligonucleoside or an oligonucleotide;
  • R 18 is (L L 2; each L and L is, independently, C 1-6 alkyl; or Li and L 2 are joined together to form a 4- to 7-membered heterocyclic ring system including the nitrogen atom to which L ⁇ and L 2 are attached, wherein said ring system optionally includes at least one additional heteroatom selected from O, N and S; and R ⁇ 9 is X ⁇ ;
  • Xi is Pg-O-, Pg-S-, Ci-Cio straight or branched chain alkyl, CH 3 (CH 2 ) p5 - O- or R 20 R 2 ⁇ N-; p5 is from 0 to 10; Pg is a protecting group; each R 20 and R 2 ⁇ is, independently, hydrogen, Ci-Cio alkyl, cycloalkyl or aryl; or optionally, R 2 o and R 21 , together with the nitrogen atom to which they are attached form a cyclic moiety that may include an additional heteroatom selected from O, S and N; or
  • R 18 and R 19 together with the phosphoms atom to which R ⁇ 8 and R 19 are attached form a chiral auxiliary.
  • Groups that are attached to the phosphoms atom of intemucleotide linkages before and after oxidation can include nitrogen containing cyclic moieties such as morpholine.
  • Such oxidized intemucleoside linkages include a phosphoromorpholidothioate linkage (Wilk et al, Nucleosides and nucleotides, 1991, 10, 319-322).
  • cyclic moieties amenable to the present invention include mono-, bi- or tricyclic ring moieties which may be substituted with groups such as oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, sulfone, sulfonamide, thiol and thioalkoxy.
  • groups such as oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, hal
  • alkyl generally C1-C20
  • alkenyl generally C2-C20
  • alkynyl generally C2-C20
  • alkyl generally C1-C20
  • alkenyl generally C2-C20
  • alkynyl generally C2-C20
  • alkyl groups include but are not limited to substituted and unsubstituted straight chain, branch chain, and alicyclic hydrocarbons, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl and other higher carbon alkyl groups.
  • Further examples include 2-methylpropyl, 2-methyl-4-ethylbutyl, 2,4- diethylbutyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl, 6-propyl-6- butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl and other branched chain groups, allyl, crotyl, propargyl, 2-pentenyl and other unsaturated groups containing a pi bond, cyclohexane, cyclopentane, adamantane as well as other alicyclic groups, 3-penten-2-one, 3-methyl-2-butanol, 2-cyanooctyl, 3- methoxy-4-heptanal, 3-nitrobutyl, 4-isopropoxydodecyl, 4-azido-2-nitrodecyl, 5- mercaptononyl
  • a number of chemical functional groups can be introduced into compounds of the invention in a blocked form and subsequently deblocked to form a final, desired compound.
  • Protecting groups can be selected to block functional groups located in a growing oligomeric compound during iterative oligonucleotide synthesis while other positions can be selectively deblocked as needed.
  • a blocking group renders a chemical functionality of a larger molecule inert to specific reaction conditions and can later be removed from such functionality without substantially damaging the remainder of the molecule (Greene and Wuts, Protective Groups in Organic Synthesis, 3rd ed, John Wiley & Sons, New York, 1999).
  • the nitrogen atom of amino groups can be blocked as phthalimido groups, as 9- fluorenylmethoxycarbonyl (FMOC) groups, and with triphenylmethylsulfenyl, t- BOC or benzyl groups.
  • Carboxyl groups can be blocked as acetyl groups. Representative hydroxyl protecting groups are described by Beaucage et al., Tetrahedron 1992, 48, 2223.
  • Preferred hydroxyl protecting groups are acid-labile, such as the trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9- phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).
  • Chemical functional groups can also be "blocked” by including them in a precursor form.
  • an azido group can be used considered as a "blocked” form of an amine since the azido group is easily converted to the amine.
  • thiol (sulfur) protecting groups include, but are not limited to, benzyl, substituted benzyls, diphenylmethly, phenyl, t-butyl, methoxymethyl, thiazolidines, acetyl and benzoyl. Further thiol protecting groups are illustrated in Greene and Wuts, ibid.
  • Additional amino-protecting groups include but are not limited to, carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1- methyl-l-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyl- oxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2- nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl. Equivalents of these amino-protecting groups are also encompassed by the compounds and methods of the present invention.
  • Some prefened amino-protecting groups are stable to acid treatment and can be selectively removed with base treatment which make reactive amino groups selectively available for substitution.
  • Examples of such groups are the Fmoc (E. Atherton and R.C. Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p.l), and various substituted sulfonylethyl carbamates exemplified by the Nsc group (Samukov et al., Tetrahedron Lett, 1994, 35:7821; Verhart and Tesser, Rec. Trav. Chim. Pays- Bas, 1987, 107:621).
  • the nucleoside components of the oligomeric compounds are connected to each other by optionally protected phosphorothioate intemucleoside linkages.
  • Representative protecting groups for phosphoms containing intemucleoside linkages such as phosphite, phosphodiester and phosphorothioate linages include
  • Patents Nos. 4,725,677 and Re. 34,069 (/3-cyanoethyl); Beaucage, S.L. and Iyer, R.P., Tetrahedron, 49 No. 10, pp. 1925-1963 (1993); Beaucage, S.L. and Iyer, R.P., Tetrahedron, 49 No. 46, pp. 10441-10488 (1993); Beaucage, S.L. and Iyer, R.P., Tetrahedron, 48 No. 12, pp.
  • the present invention also includes pharmaceutical compositions and formulations that include the oligomeric compounds of the invention.
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • Preferred topical formulations include those in wliich the oligomeric compounds of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include neutral (e.g.
  • dioleoylphosphatidyl DOPE ethanolamine dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
  • Oligomeric compounds of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligomeric compounds may be complexed to lipids, in particular to cationic lipids.
  • Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a C 1 0 &yl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
  • arachidonic acid oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, lino
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Preferred oral formulations are those in which oligomeric compounds of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Prefenred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate.
  • Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium).
  • arachidonic acid arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, g
  • penetration enhancers for example, fatty acids/salts in combination with bile acids/salts.
  • a particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligomeric compounds of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.
  • Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino- methylethylene P(TDAE), polyaminostyrene (e.g.
  • PLGA poly(DL-lactic-co- glycolic acid
  • PEG polyethyleneglycol
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non- aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • the preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • the compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ m in diameter.
  • Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other, hi general, emulsions may be either water-in-oil (w/o) or of the oil-in- water (o/w) variety.
  • w/o water-in-oil
  • o/w oil-in- water
  • an oily phase when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion.
  • Emulsions may contain additional components in addition to the dispersed phases and the active drag which may be present as a solution in either the aqueous 0 phase, oily phase or itself as a separate phase.
  • Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed.
  • compositions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in- water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
  • Such complex 5 formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability.
  • the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation.
  • Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams.
  • Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.
  • Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
  • Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion.
  • HLB hydrophile/lipophile balance
  • surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
  • Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations.
  • polar inorganic solids such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions.
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
  • the compositions of oligomeric compounds and nucleic acids are formulated as microemulsions.
  • a microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
  • microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system.
  • microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drags: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).
  • Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the stmcture and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1985, p. 271).
  • microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants.
  • the cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1- butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.
  • the aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil. Microemulsions are particularly of interest from the standpoint of drag solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions have been proposed to enhance the oral bioavailability of drags, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205).
  • Microemulsions afford advantages of improved drug solubilization, protection of drag from enzymatic hydrolysis, possible enhancement of drag absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
  • microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides.
  • Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligomeric compounds and nucleic acids of the present invention.
  • Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories - surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
  • liposome means a vesicle composed of amphiphilic lipids ananged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered.
  • Cationic liposomes possess the advantage of being able to fuse to the cell wall.
  • Non-cationic liposomes although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo, hi order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
  • Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
  • Liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drag, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drags, both hydrophilic and hydrophobic, into the skin.
  • liposomes to deliver agents including high-molecular weight DNA into the skin.
  • Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are raptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985). Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs.
  • pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
  • liposomal composition includes phospho lipids other than naturally-derived phosphatidylcholine.
  • Neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non- ionic surfactant and cholesterol.
  • Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/ cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
  • Liposomes also include "sterically stabilized" liposomes, a tenn which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G MI , or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • Liposomes comprising (1) sphingomyelin and (2) the ganglioside G MI or a galactocerebroside sulfate ester.
  • U.S. Patent No. 5,543,152 discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn- dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.). Many liposomes comprising lipids derivatized with one or more 5 hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al.
  • Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Patent Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Patent No. 5,213,804 and European Patent No. EP 0 496 813 Bl).
  • Woodle et al. U.S. Patent Nos. 5,013,556 and 5,356,633
  • Martin et al. U.S. Patent No. 5,213,804 and European Patent No. EP 0 496 813 Bl
  • Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S.
  • Patent No. 5,225,212 both to Martin et al.
  • WO 94/20073 Zalipsky et al.
  • Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).
  • U.S. Patent Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
  • WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes.
  • U.S. Patent No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA.
  • U.S. Patent No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes.
  • WO 97/04787 to Love et al. discloses liposomes comprising 5 antisense oligonucleotides targeted to the raf gene.
  • Transfersomes are yet another type of liposomes, and are highly defonnable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller
  • Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self- loading.
  • surface edge-activators usually surfactants
  • hydrophile/lipophile balance HLB
  • hydrophile/lipophile balance The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.
  • Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic
  • surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • the most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • the surfactant molecule If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides. The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).
  • the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligomeric compounds, to the skin of animals.
  • nucleic acids particularly oligomeric compounds
  • Most dmgs are present in solution in both ionized and nonionized forms.
  • usually only lipid soluble or lipophilic drags readily cross cell membranes.
  • penetration enhancers also enhance the permeability of lipophilic drags.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
  • surfactants or "surface-active agents" are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that abso ⁇ tion of oligonucleotides through the mucosa is enhanced.
  • these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drag Carrier Systems, 1991, p.92); and perf iorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
  • fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac- glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, l-dodecylazacycloheptan-2-one, acylcamitines, acylcho lines, d-io alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee e.
  • bile salts include any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • the bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid 5 (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al, Critical Reviews in Therapeutic Drag Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences,
  • Chelating agents as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming
  • chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr.,
  • Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drag Carrier Systems,
  • non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of
  • This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1 -alkyl- and 1- alkenylazacyclo-alkanone derivatives (Lee et al, Critical Reviews in Therapeutic Drag Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
  • Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (Junichi et al, U.S. Patent No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • carrier compound or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • a nucleic acid and a carrier compound can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
  • the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
  • a "pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal.
  • the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxy
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • the compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipraritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • Representative United States patents that teach the preparation of such uptake, distribution and/or abso ⁇ tion assisting formulations include, but are not limited to, U.S.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291;
  • the oligomeric compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • prodrag indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drag) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published December 9, 1993 or in WO 94/26764 and U.S. 5,770,713 to Imbach et al.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • metals used as cations are sodium, potassium, magnesium, calcium, and the like.
  • suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. of Pharma Sci., 1977, 66, 1-19).
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for pu ⁇ oses of the present invention.
  • a "pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines.
  • Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.
  • Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic
  • Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation.
  • Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
  • prefened examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid,
  • the succinyl derivative (0.19 g, 0.25 mmol) was dried over P 2 O 5 in vacuo at 40° C overnight.
  • Anhydrous DMF (0.62 mL) was added followed by 2-(lH- benzotriazole)-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (0.081 g, 0.25 mmol) and N-methylmo ⁇ holine (55 ⁇ L, 0.5 mmol). Vortexed to give a clear solution.
  • anhydrous DMF (2.4 mL) and activated CPG (1.08 g, 115.2 mmol/g, particle size 120/200, mean pore diameter 520 A) were added.
  • N 4 -benzoyl adenine This is then coupled with N 4 -benzoyl adenine under Vorbruggen condition to give the N 4 -benzoyl-5',3'-tolyl-l-adenosine.
  • Deprotection of the tolyl group with methylamine gives L-adenosine.
  • TMSC1 benzoyl chloride
  • TMSC1 pyridine
  • aqueous ammonia 5'-Tritylation in presence of DMTC1, in pyridine and phosphitylation at the 3 '-position gives compound 5.
  • Compound 2 is converted into 5'-O-DMT-L-5-methylcytidine according to literature procedure [Divakar K. J. et. al. J. Chem. Soc. Perk. Trans. 1 1982, 1171- 5 1176]. It is then converted into iv -benzoyl derivative according to literature procedure [Bhat, V. et. al. Nucleosides Nucleotides 1989, 8, 179-183]. This is then phosphitylated at the 3'- position to give compound 6.
  • 5'-0-DMT-L-5-(l-propynyl)uridine (prepared following the procedure described for compound 7) is converted into 5'- ⁇ -DMT-L-5-(l-propynyl cytidine according to literature procedure [Divakar K. J. et. al. J. Chem. Soc. Perk. Trans. 1 1982, 1171-1176]. This is phosphitylated at the 3'-position to give compound 9.
  • L-5-Bromouridine is obtained from 5-bromo uridine and l-Chloro-5,3- bis(tolyl)-2-deoxy L-ribose under Vorbraggen conditions.
  • EXAMPLE 10 5'-0-DMT-L-iV 4 -benzoyl-2'-deoxyadenosine-3 , -0-succinyl CPG (11). S'-O-DMT-L- ⁇ -benzoyl ⁇ '-deoxyadenosine (prepared as described in the synthesis of compound 5) is converted into 3'-O-succinyl derivative in the presence of succinic anhydride and DMAP in dichloroethane at 60 °C.
  • the succinyl derivative is coupled to amino alkyl CPG in presence of 2-(lH- benzotriazole)-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate and N- methylmo ⁇ holine in DMF to give compound 11.
  • 5'-O-DMT-L-A ⁇ -benzoyl-5-methyl-2'-deoxycytidine (prepared as described in the synthesis of compound 6) is converted into 3'-0-succinyl derivative in the presence of succinic anhydride and DMAP in dichloroethane at 10 60 °C.
  • the succinyl derivative is coupled to amino alkyl CPG in presence of 2- (lH-benzotriazole)-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate and N- methylmo ⁇ holine in DMF to yield the compound 12.
  • EXAMPLE 12 15 5'-0-DMT-L-N sobutyryl-2'-deoxyguanosine-3'-0-succinyl CPG (13).
  • 5'-O-DMT-L-N 2 -isobutyryl-2'-deoxyguanosine (prepared as described in the synthesis of compound 7) is converted into 3'-O-succinyl derivative in the presence of succinic anhydride and DMAP in dichloroethane at 60 °C.
  • the succinyl derivative is coupled to amino alkyl CPG in presence of 2-(lH- 0 benzotriazole)-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate and N- methylmo ⁇ holine in DMF to yield the compound 13.
  • EXAMPLE 13 5'-0-DMT-L-5-(l-propynyl)uridine-3'-0-succinyl CPG (14).
  • 5 5'-O-DMT-L-5-(l-propynyl)uridine (prepared as described in the synthesis of compound 8) is converted into 3'-O-succinyl derivative in the presence of succinic anhydride and DMAP in dichloroethane at 60 °C.
  • the succinyl derivative is coupled to amino alkyl CPG in presence of 2-(lH-benzotriazole)-l- yl)-l,l,3,3-tetramethyluronium tetrafluoroborate and N-methylmo ⁇ holine in
  • 5'-O-DMT-L-5-(l-propynyl)cytidine (prepared as described in the synthesis of compound 8) is converted into 3'-0-succinyl derivative in the presence of succinic anhydride and DMAP in dichloroethane at 60 °C.
  • the succinyl derivative is coupled to amino alkyl CPG in the presence of 2-(lH- benzotriazole)-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate and N- methylmo ⁇ holine in DMF to yield the compound 15.
  • 5'-O-DMT-L-3(2-deoxy- ⁇ -D-erythro-pentofuranosyl)(9I)-lH- pyrimido[5,4-b]benzoxazin-2(3H)-one (prepared as described in the synthesis of compound 10) is converted into 3'-O-succinyl derivative in the presence of succinic anhydride and DMAP in dichloroethane at 60 °C.
  • the succinyl derivative is coupled to amino alkyl CPG in presence of 2-(lH-benzotriazole)-l- yl)-l,l,3,3-tetramethyluronium tetrafluoroborate and N-methylmo ⁇ holine in DMF to yield the compound 16.
  • the amidite 3 was dissolved in anhydrous acetonitrile to give a 0.1 M solution and loaded on to a Expedite Nucleic Acid Synthesis system (Millipore 8909) to synthesize the oligonucleotides.
  • the coupling efficiencies were more than 98%.
  • the CPG was suspended in aqueous ammonia (30 wt %) and at room temperature for 2 h to deprotect oligonucleotides form the CPG.
  • T* L-Thymidine
  • T* L-Thymidine
  • C* L-Cytidine
  • A* L-Adenosine
  • All P S
  • C° 2'-O-MOE 5 J M M e e / C ⁇ t
  • T° 2'-O-MOE 5Me U
  • G° 2'-O- MOE G.
  • B phenoxazine 16.
  • B phenoxazine
  • 2',3'-dideoxycytidine 26 [Prepared according to the literature procedure Horwitz, J. P. et. al. J. Org. Chem. 1967, 32, 817-818] is converted into 5'-O-silyl derivative in presence of TBDMSC1 and pyridine. This is then treated with 4- (hydroxymethyl)benzoylchloride in pyridine to give compound 27 (Scheme 3).
  • Compound 27 is treated with succinic anhydride and DMAP in 1,2-dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4-methylmo ⁇ holine in DMF to give 28.
  • Compound 28 is desilylated with triethylamine trihydrofluoride and triethylamine in THF. It is then tritylated with DMTCl in pyridine and DMAP to give compound 29.
  • 2',3'-Dideoxyadenosine 30 [Prepared according to the literature procedure Horwitz, J. P. et. al. J. Org. Chem. 1967, 32, 817-818] is converted into 5'-O-silyl derivative in presence of TBDMSC1 and pyridine. This is then treated with 4- (hydroxymethyl)benzoylchloride in pyridine to give compound 31 (Scheme 4).
  • Compound 31 is treated with succinic anhydride, DMAP in 1,2-dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4-methylmo ⁇ holine in DMF to give 32.
  • Compound 32 is desilylated with triethylamine trihydrofluoride and triethylamine in THF. It is then tritylated with DMT chloride in pyridine and DMAP to give compound 33.
  • Oligonucleotides 34 (SEQ ID NO: 16) and 35 (SEQ ID NO: 17) are prepared accoding to the procedure used for the synthesis of componds 17-25 (SEQ ID NOS:8-15) using solid support 29 and 33 respectively.
  • 2',3'-Dideox-2',3'-didehydroycytidine 36 [prepared according to the reported procedure, Chu, C. K. et. al. J. Org. Chem. 1989, 54, 217-225] is converted into 5'-O-silyl derivative in presence of TBDMSCl in pyridine. This is then treated with 4-(hydroxymethyl)benzoylchloride in pyridine to give compound 37 (Scheme 5).
  • Compound 37 is treated with succinic anhydride, DMAP in 1,2- dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4-methylmo ⁇ holine in DMF to give 38.
  • Compound 38 is desilylated with triethylamine trihydro fluoride and triethylamine in THF. It is then tritylated with DMT chloride in pyridine and DMAP to give compound 39.
  • 2',3'-Dideoxy-2'-3'-didehydroadenosine 40 [prepared according to the reported procedure, Chu, C. K. et. al. J. Org. Chem. 1989, 54, 217-225] is converted into 5'-O-silyl derivative in presence of TBDMSCl and pyridine. This is then treated with 4-(hydroxymethyl)benzoylchloride in pyridine to give compound 41 (Scheme 6).
  • Compound 41 is treated with succinic anhydride, DMAP in 1,2-dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in the presence of TBTU and 4- methylmo ⁇ holine in DMF to give 42.
  • Compound 42 is desilylated with triethylamine trihydrofluoride and triethylamine in THF. It is then tritylated with DMTCl in pyridine and DMAP to give compound 43.
  • 2',3'-Dideoxy-2'-fluro uridine 46 [prepared as reported, Martin J. A. et. al. J. Med. Chem. 1990, 33, 2137-2145] is converted into 2',3'-dideoxy-2'- flurocytidine 47 (Scheme 7) according to the reported procedure [Reference :- Divakar, K. J. et. al. J. Chem. Soc. Perk. Trans. 1 1982, 1171-1176].
  • Compound 47 is converted into 5'-O-silyl derivative in presence of TBDMSCl and pyridine. This is then treated with 4-(hydroxymethyl)benzoylchloride in pyridine to give compound 48.
  • Compound 48 is treated with succinic anhydride, DMAP in 1,2- dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4-methylmo ⁇ holine in DMF to give 49.
  • Compound 49 is desilylated with triethylamine trihydrofluoride and triethylamine in THF. It is then tritylated with DMTCl in pyridine and DMAP to give compound 50.
  • 2',3'-Dideoxy-3'-fluro uridine 52 [prepared according to thereported procedure Zaitseva, G. V. et. al. Bioorg. Khim. 1988, 14, 1275-1281] is converted into 2',3'-dideoxy-3'-fluorocytidine 53 (Scheme 8) according to the reported procedure [Reference:-. Divakar, K. J. et. al. J. Chem. Soc. Perk. Trans. 1 1982, 1171-1176].
  • Compound 53 is converted into 5'-O-silyl derivative in presence of TBDMSCl and pyridine. This is then treated with 4-(hydroxymethyl)benzoyl chloride in pyridine to give compound 54.
  • Compound 54 is treated with succinic anhydride, DMAP in 1,2-dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4- methylmo ⁇ holine in DMF to give 55.
  • Compound 55 is desilylated with triethylamine trihydrofluoride and triethylamine in THF. It is then tritylated with DMT chloride in pyridine and DMAP to give compound 56.
  • Compound 60 is treated with succinic anhydride, DMAP in 1,2- dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4-methylmorpholine in DMF to give 61.
  • Compound 61 is desilylated with triethylamine trihydro fluoride and triethylamine in THF. It is then tritylated with DMTCl in pyridine and DMAP to give compound 62.
  • Compound 64 is synthesized according to the literature procedure [Huwe, C. M. et. al. Synthesis, 1997, 1, 61-67]. It is then converted into trifluromethyl derivative 65 in presence of ethyl trifluroacetate in ethanol. Compound 65 is tritylated to give compound 66. Compound 66 is phosphitylated to give the compound 67.
  • EXAMPLE 35 3-0-(CPG-succinyl)-N-trifluoroacetyI-pyrrolidine-2-(DMT)methanoI (68).
  • Compound 66 is treated with succinic anhydride, DMAP in 1,2- dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4-methylmorpholine in DMF to give 68.
  • Compound 73 is silylated with 1,3-dichloro-l, 1,3,3- tetraisopropyldisiloxane in pyridine to give compound 77. This is then acetylated with acetyl chloride in pyridine to give compound 78.
  • Compound 78 is desilylated with TEA.3HF and TEA in THF. This is tritylated with DMTCl, DMAP and pyridine followed by phosphitylation give compound 79.
  • Compound 82 is synthesized according to literature procedure [Nake, T. et. al. J. Am. Chem. Soc. 2000, 122, 7233-7243]. This is converted into 83 by following a reported procedure for cleavage of vicinlal diols and subsequent reduction of aldehyde thus obtained [Bessodes, M. et. al. Tetrahedron Lett. 1985, 25(10), 1305-1306].
  • Compound 83 is silylated with 1,3-dichloro-l, 1,3,3- tetraisopropyldisiloxane in pyridine to give compound 84.
  • Compound 92 is prepared according to the procedure reported in the literature (Reference :-Krenitsky, T. A. et. al. J. Med. Chem. 1983, 26(6), 891- 895). This is then selectively tritylated with DMTCl and pyridine to give the 5'- O-DMT derivative which is acetylated to give acetylated product. Selective removal of the acetyl group at ⁇ -position with aqueous ammonia at room temperature gives compound 93. This is then treated with 4- (hydroxymethyl)benzoyl chloride in pyridine to give compound 94.
  • C* 2',3'-dideoxy-3'-(amino)cytidine
  • 2'-Deoxy-3'-S-phenyl-3'-thiouridine 97 [prepared as reported in Kawakami, H. et. al. Heterocycles, 1991, 32(12), 2451-2470] is converted into 2'- deoxy-3-S-phenyl-3-thiocytidine 98 (Scheme 7) according to the reported procedure [Divakar, K. J. et. al. J. Chem. Soc. Perk. Trans. 1 1982, 1171-1176].
  • Compound 98 is converted into 5'-O-silyl derivative in presence of TBDMSCl and pyridine. This is then treated with 4-(hydroxymethyl)benzoylchloride in pyridine to give compound 99.
  • Compound 99 is treated with succinic anhydride, DMAP in 1,2-dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4- methylmorpholine in DMF to give 100.
  • Compound 100 is desilylated with triethylamine trihydrofluoride and triethylamine in THF. It is then tritylated with DMTCl in pyridine and DMAP to give compound 101.
  • 3'-Deoxy-2'-S-phenyl-2'-thiouridine 103 [prepared as reported , Kawakami, H. et. al. Heterocycles, 1991, 32(12), 2451-2470] is converted into 2',3'-dideoxy-2'-fiurocytidine 104 (Scheme 17) according to the reported procedure [Divakar, K. J. et. al. J. Chem. Soc. Perk. Trans. 1 1982, 1171-1176].
  • Compound 104 is converted into 5'-O-silyl derivative in presence of TBDMSCl and pyridine. This is then treated with 4-(hydroxymethyl)benzoylchloride in pyridine to give compound 105.
  • Compound 105 is treated with succinic anhydride, DMAP in 1,2-dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4- methylmorpholine in DMF to give 106.
  • Compound 106 is desilylated with triethylamine trihydrofluoride and triethylamine in THF. It is then tritylated with DMTCl in pyridine and DMAP to give compound 107.
  • succinyl derivative 15 with succinic anhydride, DMAP in 1,2-dichloroethane to give the succinyl derivative.
  • the succinyl derivative is coupled with aminoalkyl CPG in presence of TBTU and 4-methylmorpholine in DMF to give 112.
  • Compound 112 is desilylated with triethylamine trihydrofluoride and triethylamine in THF. It is then tritylated with DMT chloride in pyridine and DMAP to give compound 113.
  • G-Clamp Nucleoside (G-Clamp), G-Clamp Succinate 154
  • Solid phase syntheses of oligonucleotides containing G-clamp and phenoxazine units were carried out using standard phosphoramidite chemistry and an Applied Biosystems (Perkin Elmer Corp.) DNA/RNA synthesizer 380B. Cleavage and deprotection of the oligonucleotides was performed using a solution of 40% aq. Me_NH 2 and 28-30% aq. NH 3 (1:1) at r.t. for 4 h.
  • the oligonucleotides were purified by reversed phase HPLC using a 306 Piston Pump System, a 81 IC Dynamic Mixer, a 170 Diode Array Detector and a 215 Liquid Handler together with the Unipoint Software from Gilson (Middleton, Wi).
  • the HPLC conditions were as follows: Column: Waters Deltapak C ⁇ 8 reversed phase (300x3.9 mm, 15 ⁇ , 300 A); Solvent A: 0.1 M NH 4 OAc in H 2 0; solvent B: 0.1 M NH 4 OAc in CH 3 CN/H 2 0 (80:20); Gradient: 0-40 min 0-50% B. After chromatographic purification the oligonucleotides were desalted by RP-HPLC, lyophilized, and stored at -20°C.
  • the support-bound oligonucleotides were washed with DCM, acetone, sodium N,N-diethyldithiocarbamate (ddtc Na + ), H 2 O, acetone, DCM, diethyl ether and dried in vacuo.
  • the resin Prior to guanidinylation, the resin was suspended in a solution of 10%> DIEA in DMF, shaken for 5 min, and washed with DMF followed by DCM. Subsequently, a 1.0 M solution of lH-pyrazole-1- carboxamidine hydrochloride and DIEA in DMF was added to the support-bound oligonucleotides and the suspension was shaken at r.t.
  • the resin was treated with cone, aqueous ammonia at 55°C for 1 h. After separation from the CPG support and evaporation of ammonia, the aqueous solution was filtered through a 0.45 ⁇ m Nylon-66 filter and stored frozen at -20°C for further analysis.
  • (A) Reaction conditions: (i) 1.0 mL of 10 mg Pd 2 [(Ph-CH CH) 2 CO] 3 , 26 mg P(Ph) 3 in 1.2 M nBuNH 2 /HCOOH in THF, 50°C, 1.5 h; (ii) washing with DCM, acetone, sodium N,N-diethyldithiocarbamate (ddtc Na + ), H 2 O, acetone, DCM, diethyl ether; (iii) 1.0 M of lH-pyrazole-1 -carboxamidine hydrochloride and DIEA in DMF, r.t., 5 h. (B) (i) 40% aq.
  • the oligonucleotides were deprotected and cleaved from the solid support prior to guanidinylation by using a 1:1 mixture of 40%> aqueous CH 3 -NH 2 and cone, aqueous ammonia (AMA), which prevents the formation of acyl- or acrylonitrile adducts with the highly nucleophilic primary amino group.
  • AMA aqueous ammonia
  • N-acetyl- instead of N-benzoyl-protected C was used for oligonucleotide synthesis.
  • the primary amino group of G-clamp was guanidinylated by treating the oligonucleotides with 1-2 ⁇ mol of 2 mmol (297 mg) of lH-pyrazole- 1 -carboxamidine hydrochloride in 2 mL of a 1.0 M aqueous Na 2 CO 3 solution at r.t. for 3 h.
  • oligonucleotides were purified by gel chromatography (Sephadex G25) followed by RP-HPLC and analyzed by capillary gel electrophoresis (CGE) and electrospray mass specfrometry (ES-MS).
  • CGE capillary gel electrophoresis
  • ES-MS electrospray mass specfrometry
  • the guanidinyl G-clamp modification was designed to allow for additional hydrogen bonds to the O6 and N7 Hoogsteen binding sites of guanosine (Figure IB). Binding studies of DNA oligomers containing a single unit to a RNA target revealed an increase in the melting temperature of 16°C relative to the wildtype DNA, slightly lower than the ⁇ T m observed for the original G-clamp modification.
  • the hydrogen bond lengths are 2.88 A and 2.86 A and the lengths of the corresponding hydrogen bonds in base pair C2*-G19 are 2.92 A and 2.87 A, respectively.
  • the quality of the electron density around individual atoms of the phenoxazine ring and tethered group demonstrate that this modification is well ordered and does not assume random conformations. There is some buckling of modified base pairs relative to the other base pairs in the duplex.
  • This out-of-plane distortion of the base pair between the G-clamp and G may be a consequence of the requirement to optimize the geometry of both the Watson-Crick and Hoogsteen-type hydrogen bonds within the geometric boundaries provided by a guanidinium-ethoxy moiety, hi addition, the observed arrangements help avoid a steric contact between O6 of G and the ethoxy-linker oxygen of the G-clamp ( Figures 1 and 2).
  • Presence of the G-clamp results in a considerable improvement of intra- strand stacking at the GpC* step compared with stacking between cytosine and the 5 '-adjacent base (Gl and Gl 1, respectively).
  • the overlap between Gl and C2* is depicted in Figure 3. While the "cytosine core" displays relatively little stacking to the guanosine base, the remainder of the phenoxazine ring system virtually covers the entire guanosine base. However, while stacking between G-clamp and the base to the 5 '-side is improved, stacking to the 3 '-adjacent base is not affected by incorporation of the modified base.
  • the present structure of the guanidyl G-clamp is similar to the bidentate hydrogen bonding of the arginine fork with the N7 and O6 positions of guanine in protein- nucleic acids interactions [Clarke, N. D.; Beamer, L. J.; Goldberg, H. R.; Berkower, C; Pabo, C. O. Science 1991, 254, 267-270; Rich, A. In The Chemical Bond: Structure and Dynamics; Zewail, A. Ed.; Academic Press, New York,
  • the in vivo stability of selected modified oligonucleotides synthesized is determined in B ALB/c mice. Following a single i.v. administration of 5 mg/kg of oligonucleotide, blood samples are drawn at various time intervals and analyzed by CGE.
  • mice For each oligonucleotide tested, 9 male BALB/c mice (Charles River, Wilmington, MA) weighing about 25 g are used. Following a one week acclimatization the mice received a single tail- vein injection of oligonucleotide (5 mg/kg) administered in phosphate buffered saline (PBS), pH 7.0. One retro- orbital bleed (either at 0.25, 0.5, 2 or 4 h post-dose) and a terminal bleed (either 1, 3, 8, or 24 h post-dose) are collected from each group. The terminal bleed (approximately 0.6-0.8 mL) is collected by cardiac puncture following ketamine/xylazine anasthesia.
  • PBS phosphate buffered saline
  • the blood is transferred to an EDTA-coated collection tube and centrifuged to obtain plasma.
  • the liver and kidneys are collected from each mouse.
  • Plasma and tissue homogenates are used for analysis to determine intact oligonucleotide content by CGE. All samples are immediately frozen on dry ice after collection and stored at -80°C until analysis.
  • oligonucleotides with longer durations of action can be designed by incorporating both the G-clamp modification and other analogous motifs into their structure.
  • a plot of the percentage of full length oligonucleotide remaining intact in plasma one hour following administration of an i.v. bolus of 5 mg/kg oligonucleotide is determined to evaluate the stability in plasma.
  • a plot of the percentage of full length oligonucleotide remaining intact in tissue 24 hours following administration of an i.v. bolus of 5 mg/kg oligonucleotide is determined.
  • CGE traces of test oligonucleotides and a standard phosphorothioate oligonucleotide in both mouse liver samples and mouse kidney samples after 24 hours are evaluated.
  • the maximum stability is seen when both 5' and 3' ends are capped with C*. - Ill -
  • an in vitro cell culture assay is used that measures the cellular levels of c-raf expression in bEND cells.
  • the bEnd.3 cell line a brain endothelioma, is obtained from Dr. Werner Risau (Max-Planck Institute).
  • Opti-MEM, trypsin-EDTA and DMEM with high glucose are purchased from Gibco-BRL (Grand Island, NY).
  • Dulbecco s PBS is purchased from Irvine Scientific (Irvine, CA). Sterile, 12 well tissue culture plates and Facsflow solution are purchased from Becton Dickinson (Mansfield, MA). Ultrapure formaldehyde is purchased from Polysciences (Wamngton, PA). NAP-5 columns are purchased from Pharmacia (Uppsala, Sweden).
  • Oligonucleotide Treatment Cells are grown to approximately 75 % confluency in 12 well plates with DMEM containing 4.5g/L glucose and 10 %> FBS. Cells are washed 3 times with Opti-MEM pre-warmed to 37°C. Oligonucleotide is premixed with a cationic lipid (Lipofectin reagent, (GIBCO/BRL) and, serially diluted to desired concentrations and transferred on to washed cells for a 4 hour incubation at 37°C. Media is then removed and replaced with normal growth media for 24 hours for northern blot analysis of mRNA.
  • a cationic lipid Lipofectin reagent, (GIBCO/BRL)
  • RNA is prepared from cells by the guanidinium isothiocyanate procedure [Monia et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 15481-15484] 24 h after initiation of oligonucleotide treatment. Total RNA is isolated by centrifugation of the cell lysates over a CsCl cushion. Northern blot analysis, RNA quantitation and normalization to G3PDH mRNA levels are done according to the reported procedure [Dean and McKay, Proc. Natl. Acad. Sci. USA, 1994, 91, 11762- 11766].
  • the G-clamp oligonucleotides showed reduction of c- r ⁇ /message activity as a function of concentration.
  • the fact that these modified oligonucleotides retained activity promises reduced frequency of dosing with these oligonucleotides which also show increased in vivo nuclease resistance.
  • All G-clamp modified oligonucleotides retained the activity of the parent 11061 oligonucleotide (SEQ ID NO:42) and improved the activity even further.
  • Example 2 for the synthesis of compound 3 yields compound 206a.
  • Compound 207 is obtained from compound 250, 1,1'- thiocarbonyldiimidazole and methylamine under similar reaction conditions as described for the synthesis of compound 206 in Example 68.
  • Phenoxazine nucleoside 252 with desired tether X is synthesized in five steps from 5-bromo-3'-O-TBDMS-5'-O-DMT-dU (251) according to the literature procedure by [Lin and Matteucci J. Am. Chem. Soc, 1998, 120, 8531- 8532].
  • Compound 216a is synthesized from compound 255, l,l'-thiocarbonyl- diimidazole and methylamine as described in Example 86 for the synthesis of compound 215a.
  • Compound 256 is prepared from compound 255 (1 mmol) and N-benzyl- oxycarbonyl-2-aminoethanol-O-methane sulfonate (1 mmol) as described in Example 72.
  • Compound 217a is prepared from compound 257 as described in Example 89 for the preparation of compound 211 a.
  • the phosphoramidite 218a is synthesized from compound 258 under identical conditions described in Examples 81 and 83 for the preparation of compound 210a from compound 253.
  • Tether of choice is attached to the hydroxyl function of compound 263 in presence of Ph P and DEAD (Mitsunobu alkylation) to obtain compound 264.
  • Compound 267 is synthesized from compound 266 and compound 265 according to reported procedures [Lin et. al, J. Am. Chem. Soc, 1995, 117, 3873- 3874].
  • Tricyclic nucleoside 268 is prepared from compound 267 according to the reported procedure [Lin and Matteucci, J Am. Chem. Soc, 1998, 120, 8531 - 8532].
  • R' H, Me, Et or any R or s - either from R or s or
  • the desired compound 242 is obtained by reacting the free amino group formed with CDl and methylamine as described in Examples 68 and 69.
  • Phenoxazine 151 and G-clamp 152 nucleosides were prepared by modifying previously published procedures [Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874; Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532].
  • the succinates 153 and 154 and the corresponding substituted solid supports 155 and 156 were prepared as outlined in Scheme 19.
  • Solid phase oligonucleotide syntheses was carried out using standard phosphoramidite chemistry.
  • G-clamp containing oligonucleotide 158 was performed with a 1:1 solution of MeNH 2 (40%, aq.) and NH 3 (28-30%, aq.) at r.t. for 4 h.
  • the oligonucleotides were purified and desalted by reversed phase HPLC.
  • SVPD snake venom phosphodiesterase
  • BIPD bovine intestinal mucosal phosphodiesterase
  • CGE capillary gel electrophoresis
  • oligonucleotide samples were incubated with BIPD (0.55 units/ ⁇ mol substrate) in 50 mM Tris-HCl, 8 mM MgCl 2 , pH 7.5 at 37°C. At certain time points aliquots of 10 ⁇ l were withdrawn and diluted directly into 200 ⁇ L dH2O before CGE analysis. The influence of the modified oligonucleotides on the nucleolytic activity was determined by looking at the overall velocity of the enzymatic reaction.
  • Phenoxazine 151 and G-clamp 152 nucleosides were prepared by modifying previously published procedures [Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874; Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532].
  • the succinates 153 and 154 and the corresponding substituted solid supports 155 and 156 were prepared as outlined in Scheme 19.
  • Solid phase oligonucleotide syntheses was carried out using standard phosphoramidite chemistry. Deprotection of G-clamp containing oligonucleotide 158 was performed with a 1:1 solution of MeNH 2 (40%, aq.) and NH 3 (28-30%, aq.) at r.t. for 4 h.
  • oligonucleotides were purified and desalted by reversed phase HPLC.
  • Snake venom phosphodiesterase (SVPD) and bovine intestinal mucosal phosphodiesterase (BIPD) were utilized as the hydrolytic enzymes for in vitro nuclease resistance studies. Both enzymes predominantly exhibit 3' exonuclease activity.
  • An unmodied 19mer oligothymidylate (oligonucleotide 159) SEQ ID NO:64 was used as a control.
  • Oligonucleotide samples were incubated with SVPD (2.5 units/ ⁇ mol substrate) or BIPD (0.55 units/ ⁇ mol substrate) in 50 mM Tris-HCl, 8 mM MgCl 2 buffer, pH 7.5 at 37°C. At certain time points aliquots of 10 ⁇ l were removed and heated in boiling water for 2 min to inactivate the enzyme. Subsequently, the samples were desalted by membrane dialysis against Nanopure deionized water using Millipore 0.025 ⁇ m VS membranes and stored frozen until they were analysed. The progress of enzymatic degradation was monitored by capillary gel electrophoresis (CGE).
  • CGE capillary gel electrophoresis
  • the amino tether of a G-clamp residue protrudes into the major groove, while the 2' modification points into the shallow groove of a duplex. Whether or not the positive charge of the latter can interfere with the metal binding of an exonuclease remains to be investigated.
  • Oligonucleotides at a final concentration of 2 ⁇ M, were incubated with snake venom phosphodiesterase (.005 U/ml) in 50 mM Tris-HCl, pH 7.5, 8 mM MgCl 2 at 37°C. The total reaction volume was 100 ⁇ L. At each time point 10 ⁇ L aliquots of each reaction mixture were placed in a 500 ⁇ L microfuge tube and put in a boiling water bath for two minutes. The sample was then cooled on ice, quick spun to bring the entire volume to the bottom of the tube, and desalted on a Millipore .025 micron filter disk (Bedford, MA) that was floating in water in a 60 mm petrie dish.
  • snake venom phosphodiesterase .005 U/ml
  • oligonucleotide and metabolites were separated and analyzed using the Beckman P/ACE MDQ capillary electrophoresis instrument using a 100 ⁇ m ID 30 cm coated capillary (Beckman No. 477477) with eCAP ssDNA 100-R gel (Beckman No. 477621) and Tris-Borate Urea buffer (Beckman No. 338481).
  • the samples were injected electrokinetically using a field strength of between 5-10 kV for a duration of between 5 and 10 seconds. Separation wash achieved at 40°C with an applied voltage of 15kV.
  • the percentage of full length oligonucleotide was calculated by integration using Caesar v. 6 software (Senetec Software, New Jersey) followed by correction for differences in extinction coefficient for oligonucleotides of different length.

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Abstract

L'invention concerne de nouveaux composés oligomères résistants à la nucléase, et de nouveaux procédés permettant d'accroître la résistance des composés oligomères. Dans les modes de réalisation préférés, ces composés oligomères comprennent au moins un nucléoside modifié contenant une fraction sucre modifiée au niveau de l'extrémité 3' ou 5' du composé oligomère, ainsi qu'au moins un groupe de liaison internucléosides autre que le phosphodiester. D'autres formes de réalisation préférées comprennent des procédés permettant d'augmenter la résistance à la nucléase des composés oligomères, consistant à incorporer au moins un nucléoside modifié contenant une fraction sucre modifié dans l'extrémité 3' ou 5' d'un composé oligomère.
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US10/013,295 US20030175906A1 (en) 2001-07-03 2001-12-10 Nuclease resistant chimeric oligonucleotides
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