Granted Patent B2

US 8,193,246 · App. 12/004,154 · Granted Jun 5, 2012

Abstract

Claims (42)

1. A formulation comprising a composition of at least one lipid moiety covalently linked to one or more transfection enhancer elements (TEE's), wherein the at least one lipid moiety comprises a sterol and wherein the one or more TEE's are defined according to the formula:

hydrophobic moiety-pH sensitive hydrophilic moiety  (I)

wherein the pH sensitive hydrophilic moiety of each of the one or more TEE's is a weak acid having a pKa of between 2 and 6 and is located distally from the link between the lipid moiety and the TEE, and wherein the hydrophobic moiety of each of the one or more TEE's is independently an alkyl carbon chain comprising 12 to 20 carbon atoms;

wherein the composition comprises the general formula (XIV)

wherein Xa is bound at the 3′ position of the sterol and is —OPO 3 —; —OPO 2 —CH 2 —; -pyrophosphate-; —OSO 3 —; —OSO 2 —; —O—; —NH—; —S—; —N(H)C(O)—; —C(O)—, —C(O)N(H)—; —OC(O)N(H)—; —N(H)C(O)O—; —OC(O)—; —C(O)O—; —N═CH—; —CH═N—; —CH 2 —; —S—S—; —N(R 13 )(R 14 )—;

wherein R 13 and R 14 are independently H or lower alkyl and wherein one of R 13 or R 14 may be absent;

Ya is absent or a non-branched, branched or cyclic substituted or unsubstituted alkyl, alkenyl, alkylene, alkynyl, or an aryl group with 1 to 8 carbon atoms, wherein Ya is substituted with one or more groups are selected from —OH; —NH 2 ; —N(R 13 )(R 14 ); —COOH; a sugar; an amino acid; a di- or tripeptide; an α-hydroxyacid, a β-hydroxyacid or a dihydroxyacid, wherein R 13 and R 14 are defined above;

Za is absent or selected from —O—; —S—; —N(H)C(O)—; —C(O)N(H)—; —C(O)O—; —OC(O)N(H)—; —C(O)—; —C(O)N(H)—; —N(H)C(O)O—; —NH—; —N(R 13 )(R 14 )—; —CH═N—; —OC(O)—; —N═CH—; —S—S—; —NH 2 ; —(NH 3 )—; —(N(R 13 )(R 14 )(R 15 ))—; —C(O)OH; —OH; —C(O)H; —C(O)OR 16 ; —C(O)NH 2 ; wherein R 15 is defined as R 13 and R 14 above and wherein R 16 is lower alkyl;

wherein the sterol is selected from cholesterol, sitosterol, campesterol, desmosterol, fucosterol, 22-ketosterol, 20-hydroxysterol, stigmasterol, 22-hydroxycholesterol, 25-hydroxycholesterol, lanosterol, 7-dehydrocholesterol, dihydrocholesterol, 19-hydroxycholesterol, 5α-cholest-7-en3β-ol, 7-hydroxycholesterol, epicholesterol, ergosterol, and/or dehydroergosterol; and

wherein the dotted lines represent possible graft positions of the one or more TEE's.

2. The formulation as claimed in claim 1 , wherein the composition forms a lipid assembly selected from liposomes, micelles, inverted micelles, cubic or hexagonal lipid phases, cochleates, emulsions, double emulsions and other multimeric assemblies that are constituted substantially of lipids, oils and/or amphiphiles.

3. The formulation as claimed in claim 1 , further comprising neutral and/or cationic and/or anionic lipids.

4. The formulation as claimed in claim 3 , wherein the neutral lipids are selected from phosphatidylcholines; phosphatidylethanolamines; sphingolipids; ceramides; cerebrosides; sterol-based lipids; and/or derivatives thereof.

5. The formulation as claimed in claim 4 , wherein the neutral lipids are selected from DMPC, DPPC, DSPC, POPC, DOPC, DMPE, DPPE, DSPE, POPE, DOPE, Diphythanoyl-PE, sphingomyelin, ceramide and/or cholesterol.

6. The formulation as claimed in claim 3 , wherein the cationic lipids are selected from DOTAP, DMTAP, DPTAP, DC-Chol, DAC-Chol, DODAP, DOEPC, TC-Chol, DOTMA, DORIE, DDAP, CTAB, CPyC, DPIM, CHIM, MoChol, HisChol, BGSC, BGTC, DOSPER, DOSC, DOGSDO, DmC4Mo2, DmC3Mo2, C3Mo2, C3Mo3, C4Mo4, C5Mo2, C6Mo2, C8Mo2, PipC2Chol, MoC2Chol, MoC3Chol, N-methyl-PipChol, PyrroC2Chol, PyC2Chol, ImC3Chol, PipeC2Chol and derivatives thereof.

7. The formulation as claimed in claim 3 , wherein the anionic lipids are selected from phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, CHEMS, anionic sterol-derivatives, cetylphosphate, diacylglycerol hemisuccinates, cardiolipins and/or derivatives thereof.

8. The formulation as claimed in claim 1 , wherein the amount of the composition is between 0.1 mol % and 90 mol %.

9. The formulation as claimed in claim 2 , wherein the lipid assembly is an amphoteric liposome.

10. The formulation as claimed in claim 9 , further comprising one or more amphoteric lipids.

11. The formulation as claimed in claim 10 , wherein the amphoteric lipids are selected from HistChol, HistDG, isoHistSuccDG, Acylcarnosin, HCChol, Hist-PS and EDTA-Chol.

12. The formulation as claimed in claim 9 , further comprising an amphoteric lipid mixture selected from (i) a stable cationic lipid and a chargeable anionic lipid, (ii) a chargeable cationic lipid and chargeable anionic lipid or (iii) a stable anionic lipid and a chargeable cationic lipid, and optionally comprising one or more neutral or zwitterionic lipids.

13. The formulation as claimed in claim 12 , comprising between 20 and 90 mol % of the amphoteric lipid mixture and between 2 and 20 mol % of the composition.

14. The formulation as claimed in claim 12 , wherein the molar ratio of the stable cationic lipid to the chargeable anionic lipid (C/A ratio) is between 0.3 and 0.7.

15. The formulation as claimed in claim 12 , wherein the molar ratio of the chargeable cationic lipid to the chargeable anionic lipid (C/A ratio) is between 0.3 and 3.

16. The formulation as claimed in claim 12 , wherein the amphoteric lipid mixture is selected from DOTAP/Chems, DOTAP/DMG-Succ, DOTAP/DOG-Succ, DODAP/Chems, DODAP/DMG-Succ, DODAP/DOG-Succ, DDAB/Chems, DDAB/DMG-Succ, DDAB/DOG-Succ, DC-Chol/Chems, DC-Chol/DMG-Succ, DC-Chol/DOG-Succ, DAC-Chol/Chems, DAC-Chol/DMG-Succ, DAC-Chol/DOG-Succ, TC-Chol/Chems, TC-Chol/DMG-Succ, TC-Chol/DOG-Succ, MoChol/Chems, MoChol/DMG-Succ, HisChol/DOG-Succ, HisChol/Chems, HisChol/DMG-Succ, HisChol/DOG-Succ, Chim/Chems, Chim/DMG-Succ, Chim/DOG-Succ, DmC4Mo2/Chems, DmC4Mo2/DMG-Succ and DmC4Mo2/DOG-Succ.

17. The formulation as claimed in claim 9 , wherein the amphoteric liposome is unilamellar, oligolamellar or multilamellar and the size of the amphoteric liposome varies between 50 and 1000 nm.

18. The formulation as claimed in claim 1 , further comprising at least one active pharmaceutical ingredient.

19. The formulation as claimed in claim 18 , wherein the active pharmaceutical ingredient is a nucleic acid-based drug, selected from DNA plasmids, polynucleotides and oligonucleotides.

20. The formulation as claimed in claim 19 , wherein the nucleic acid-based drug is capable of being transcribed in a vertebrate cell into one or more RNAs, wherein the RNAs are mRNAs, shRNAs, miRNAs or ribozymes, and wherein the mRNAs code for one or more proteins or polypeptides.

21. The formulation as claimed in claim 19 , wherein the nucleic acid-based drug is a circular DNA plasmid, a linear DNA construct or an mRNA.

22. The formulation as claimed in claim 19 , wherein the nucleic acid-based drug is an oligonucleotide.

23. The formulation as claimed in claim 22 , wherein the oligonucleotide is a decoy oligonucleotide, an antisense oligonucleotide, an siRNA, an agent influencing transcription, an agent influencing splicing, Ribozymes, DNAzymes or Aptamers.

24. The formulation as claimed in claim 22 , wherein the oligonucleotide is directed against tumor targets.

25. The formulation as claimed in claim 24 , wherein the tumor targets are selected from CEACAM6, BCL-2, EPHA2, CTNNB1, RhoA, PLK1, XIAP, Telomerase, Cyclin D1, K-Ras, EG5, Ki67, androgen receptor, FoxM1, Akt1, VEGF, KSB and CDC25B.

26. A pharmaceutical composition comprising the formulation as claimed in claim 18 and a pharmaceutically acceptable vehicle therefor.

27. The formulation as claimed in claim 1 , wherein the pH sensitive hydrophilic moiety of the one or more TEE's is a carboxylic acid or comprises the following structure:

wherein the dotted line represents an optional double bond;

R 1 , R 2 , and R 3 are independently the hydrophobic moiety of the one or more TEE's, a hydrogen, a linear, branched or cyclic, unsubstituted or substituted C 1 -C 10 alkyl, alkylene, alkoxy-, alkoxyalkyl-, alkylthio-, alkylthioalkyl-, hydroxy-, mercapto-, oxo-, formyl-, nitro-, cyano-, halo-, trihalomethyl group or a heteroalkyl group having 0-5 sites of unsaturation, or an aryl group, wherein any of R 1 , R 2 , or R 3 optionally comprise 0-5 heteroatoms selected from —O— or —S—;

D is a carbon atom or an unsubstituted or substituted cyclic alkyl or aryl group having 0-3 sites of unsaturation and comprising 0-5 heteroatoms selected from —O—, —S—, and the substituents of D are selected from alkyl-, alkylene-, alkenyl-, alkynyl-, alkoxy-, alkoxyalkyl-, alkylthio-, alkylthioalkyl-, hydroxy-, mercapto-, oxo-, formyl-, cyano-, halo- or a trihalomethyl group.

28. The formulation as claimed in claim 1 , wherein the composition comprises the following structure:

wherein m=12-40.

29. The formulation as claimed in claim 1 , wherein the composition comprises the following structure:

Assignments (6)