Polyconjugates for delivery of RNAi triggers to tumor cells in vivo
View Patent ↗The present invention is directed compositions for delivery of RNA interference (RNAi) triggers to integrin positive tumor cells in vivo. The compositions comprise RGD ligand-targeted amphipathic membrane active polyamines reversibly modified with enzyme cleavable dipeptide-amidobenzyl-carbonate masking agents. Modification masks membrane activity of the polymer while reversibility provides physiological responsiveness. The reversibly modified polyamines (dynamic polyconjugate or conjugate) are further covalently linked to an RNAi trigger.
1. A compound for delivering an RNAi trigger to an integrin positive tumor cell in vivo comprising the structure represented by:
R-A 1 A 2 -amidobenzyl-carbamate-P-RNAi trigger
wherein, R comprises an RGD mimic, A 1 is a hydrophobic amino acid, A 2 is a hydrophilic uncharged amino acid linked to A 1 via an amide bond, wherein said hydrophilic uncharged amino acid is uncharged at neutral pH, and P comprises a membrane active polyamine.
2. The compound of claim 1 wherein A 1 is selected from the group consisting of: alanine, phenylalanine, valine, leucine, isoleucine, and tryptophan.
3. The compound of claim 1 wherein A 2 is selected from the group consisting of: citrulline, threonine, asparagine, and glutamine.
4. The compound of claim 1 wherein the integrin is an α v β 3 integrin.
5. The compound of claim 1 wherein the R-A 1 A 2 -amidobenzyl-carbamate-P has the structure represented by:
wherein R 4 comprises an RGD mimic, R 1 is the side group of a hydrophobic amino acid, R 2 is the side group of a hydrophilic uncharged amino acid and polyamine is the membrane active polyamine P.
6. The compound of claim 1 wherein the RGD mimic comprises a guanidinium group linked to a glycine-aspartate dipeptide via an amide bond.
7. The compound of claim 6 wherein the RGD mimic comprises the structure represented by:
8. The compound of claim 7 wherein the guanidinium is selected from
and its resonance structures, or
and its resonance structures.
9. The compound of claim 1 wherein R comprises:
RGD mimic-PEG1-diaryl hydrazone-PEG2
wherein:
PEG1 comprises (CH 2 —CH 2 —O) n ,
PEG2 comprises (CH 2 —CH 2 —O) m ,
n and m are independently integers greater than or equal to 4,
the sum of n+m is 12-48, and
RGD mimic consists of the structure represented by
wherein R14 is selected from the group consisting of:
and its resonance structures, and
and its resonance structures.
10. The compound of claim 1 further comprising a polyethylene glycol linked to the membrane active polyamine via a reversible physiologically labile covalent linkage.
11. The compound of claim 10 wherein an RNAi trigger is covalently linked to the membrane active polyamine.
12. The compound of claim 11 wherein the compound has the structure represented by:
wherein N is the RNAi trigger, L 1 is a physiologically labile linkage, PEG comprises polyethylene glycol, L 2 is A 1 A 2 -amidobenzyl-carbamate, y is an integer greater than zero, z is an integer greater than zero, and the value of the sum of y and z is greater than 50% of the number of amines present on membrane active polyamine P as determine by the number of amines on the unmodified membrane active polyamine.
13. The compound of claim 12 wherein P-L 2 -R comprises:
R-PEG′-diaryl hydrazone-PEG″-AA-amidobenzyl carbamate-polyamine
wherein:
R has the structure represented by:
PEG′ has the structure represented by:
wherein n=4-44,
diaryl hydrazone has the structure represented by:
PEG″ has the structure represented by:
wherein m is 4-44, and
AA-amidobenzyl carbamate-polyamine has the structure represented by:
wherein polyamine is the membrane active polyamine P.
14. A method for reversibly modifying a membrane active polyamine comprising:
a) reversibly modifying one or more amines on the polyamine by reacting the one or more amines with one or more first compounds each having the structure represented by:
wherein R 3 comprises an amine reactive carbonate moiety capable of reacting with the amine to form a carbamate, R 7 comprises a first reactive group less amine reactive than the amine reactive carbonate, R 1 is a side group of a hydrophobic amino acid, R 2 is a side chain of a hydrophilic uncharged amino acid, and
b) reacting R 7 with a second compound having the structure represented by:
wherein R 14 is selected from the group consisting of:
and A comprises a polyethylene glycol having 4-44 ethoxy units linked to a second reactive group capable of reacting with the first reactive group of R7 to form a covalent linkage.
15. The process of claim 14 wherein R 7 further comprises a polyethylene glycol group having 4 to 44 ethoxy units.
16. The process of claim 14 comprising further reversibly modifying one or more amines on the polyamine by reacting the one or more amines with a one or more compounds each having the structure represented by:
PEG-A 1 A 2 -amidobenzyl-carbonate
PEG comprises a polyethylene glycol, A 1 is a hydrophobic amino acid, A 2 is a hydrophilic uncharged amino acid linked to A 1 via an amide bond, wherein said hydrophilic uncharged amino acid is uncharged at neutral pH.
17. The process of claim 16 wherein the 80% or more of the number of amines on the polyamine are reversibly modified and the modified polyamine is not membrane active.
18. The process of claim 17 wherein an RNAi trigger molecule is covalently linked to the polyamine via a physiologically labile bond.