Lipids and complexes for the delivery of biologically-active material to cells
11098006 · 2021-08-24
Assignee
Inventors
- Helen Claire Hailes (London, GB)
- Alethea Bernice Tabor (London, GB)
- Mohn Firouz Mohd Mustapa (London, GB)
- Stephen Lewis Hart (London, GB)
- Aristides Tagalakis (London, GB)
Cpc classification
A61K47/543
HUMAN NECESSITIES
C07C217/28
CHEMISTRY; METALLURGY
International classification
C07C217/28
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
Abstract
A lipid comprising a tri-chain cation having a cationic head group and three or more C.sub.7-24 hydrocarbyl groups for use in non-viral gene delivery systems, for example in the formation of lipopolyplex transfection vectors. Exceptionally good nucleic acid transfection is observed when nucleic acid and targeting peptides are formulated with the lipid of the invention (or lipid formulated with a co-lipid) into a LPD complex.
Claims
1. An ionic compound of formula (Ia): ##STR00016## wherein: each of X and Y are the same or different and is selected from —O—, and —O—C(O)— where the carbonyl carbon is bonded to group R.sup.1 or R.sup.2; R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.12-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from the group consisting of hydroxy, halogen and OR.sup.A, wherein R.sup.A is a C.sub.1-6 hydrocarbyl group; R.sup.3 and R.sup.4 are the same or different and are each independently a C.sub.1-10 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from the group consisting of hydroxy, halogen, —OR, —C(O)OH, —CN, —N(R′).sub.2, and —C(O)R, wherein each R is the same or different and is a C.sub.1-6 hydrocarbyl group; Sp and W are together a bond; or Sp is a C.sub.1-8 alkylene group which is unsubstituted or substituted by one or more substituents selected from the group consisting of hydroxy, halogen and OR, wherein R is a C.sub.1-6 hydrocarbyl group; and W is selected from the group consisting of a bond, —O—C(O)—, —C(O)—O— and —O—; each B is the same or different and is a C.sub.1-6 alkylene group which is unsubstituted or substituted by one or more substituents selected from the group consisting of hydroxy, halogen, —OR.sup.A, —NR.sup.AR.sup.A and —OC(O)R.sup.A; wherein each R.sup.A is independently selected from C.sub.1-4 hydrocarbyl; in is an integer from 1 to 8; and Q is —OR.sup.5 or —O—C(O)—R.sup.5, wherein R.sup.5 is selected from a C.sub.12-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR.sup.A, wherein R.sup.A is a C.sub.1-6 hydrocarbyl group.
2. The ionic compound of claim 1 wherein R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-22 hydrocarbyl group having one or more double bonds.
3. The ionic compound of claim 1 wherein either Sp and W are both bond, or Sp is an unsubstituted C.sub.1-8 alkylene group and W is bond, —C(O)—O— or —O—.
4. The ionic compound of claim 1 wherein R.sup.3 and R.sup.4 may be the same or different and each is a straight or branched, unsubstituted C.sub.1-4 alkyl group.
5. The ionic compound of claim 1 wherein each B is selected from an unsubstituted C.sub.1-3 alkylene group.
6. The ionic compound of claim 1 wherein m is 1, 2, 3, 4, 5 or 6.
7. The ionic compound of claim 1, wherein R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-22 hydrocarbyl group having one double bond; Sp and W are both bond; R.sup.3 and R.sup.4 are each a straight or branched, unsubstituted C.sub.1-4 alkyl group; each B is selected from an unsubstituted C.sub.1-3alkylene group; m is 2, 3, 4, 5 or 6; and Q is selected from —OR.sup.5 and —O—C(O)—R.sup.5 and R.sup.5 is a C.sub.14-22 hydrocarbyl group having one double bond.
8. The ionic compound of claim 1 comprising a cation of the formula (Id): ##STR00017## wherein each of X—R.sup.1 and Y—R.sup.2 and Q is —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 or —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3; and m is an integer from 2 to 5.
9. The ionic compound of claim 1, wherein each of X—R.sup.1 and Y—R.sup.2 are the same and are —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2)CH.sub.3 or —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3, Q is selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 or —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3, and m is 3 or 4.
10. A non-viral transfection complex that comprises (i) a ionic compound as defined in claim 1 and a nucleic acid.
11. A non-viral transfection complex of claim 10 that further comprises (iii) a polycationic nucleic acid-binding component and (iv) a cell surface receptor binding component.
12. A pharmaceutical composition which comprises a transfection complex of claim 10 in admixture or conjunction with a pharmaceutically suitable carrier.
13. A vaccine comprising the transfection complex of claim 10.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
DETAILED DESCRIPTION OF THE INVENTION
(8) Lipids
(9) In a first aspect, the present invention provides an ionic compound of formula (Ia):
(10) ##STR00005##
wherein: Each of X and Y are the same or different and is selected from —O—, and —O—C(O)— where the carbonyl carbon is bonded to group R.sup.1 or R.sup.2 R.sup.1 and R.sup.2 are the same or different and are each independently selected from a C.sub.12-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR.sup.A, wherein R.sup.A is a C.sub.1-6 hydrocarbyl group; R.sup.3 and R.sup.4 are the same or different and are each independently selected from a C.sub.1-10 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR′, —C(O)OH, —CN, —N(R′).sub.2, and —C(O)R′, wherein each R′ is the same or different and is a C.sub.1-6 hydrocarbyl group; Sp and W are together a bond; or Sp is a C.sub.1-8 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C.sub.1-6 hydrocarbyl group; and W is selected from bond, —O—C(O)—, —C(O)—O— and —O—; each B is the same or different and is a C.sub.1-6 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR.sup.A, —NR.sup.AR.sup.A and —OC(O)R.sup.A; wherein each R.sup.A is independently selected from C.sub.1-4 hydrocarbyl; m is an integer from 1 to 8; and Q is selected from —OR.sup.5 and —O—C(O)—R.sup.5, wherein R.sup.5 is selected from a C.sub.12-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR.sup.A, wherein R.sup.A is a C.sub.1-6 hydrocarbyl group.
(11) Optionally, the ionic compound of the first aspect of the invention is of the formula (Ia) wherein either Sp and W are both bond, or Sp is an unsubstituted C.sub.1-8 alkylene group and W is bond, —O—C(O)—, —C(O)—O— or —O—, especially bond, —C(O)—O— or —O—. Advantageously, Sp and W are both bond, or Sp is an unsubstituted C.sub.1-8 alkylene group and W is —C(O)—O—.
(12) The term “alkylene” as used herein refers to a divalent radical derived from removing two hydrogen atoms from an alkane, also known as an alkanediyl group, for example, —CH(CH.sub.3)CH.sub.2-(propane-1,2-diyl).
(13) The term “hydrocarbyl” as used herein refers to a univalent group formed by removing a hydrogen atom from a hydrocarbon, for example, ethyl or phenyl.
(14) The ionic compound of the first aspect of the invention is optionally of formula (b):
(15) ##STR00006##
wherein: Each of X and Y are the same or different and is selected from —O—, and —O—C(O)— where the carbonyl carbon is bonded to group R.sup.1 or R.sup.2 R.sup.1 and R.sup.2 are the same or different and are each independently selected from a C.sub.12-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR.sup.A, wherein R.sup.A is a C.sub.1-6 hydrocarbyl group; R.sup.3 and R.sup.4 are the same or different and are each independently selected from a C.sub.1-10 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR′, —C(O)OH, —CN, —N(R′).sub.2, and —C(O)R′, wherein each R′ is the same or different and is a C.sub.1-6 hydrocarbyl group; each B is the same or different and is a C.sub.1-6 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR.sup.A, —NR.sup.AR.sup.A and —OC(O)R.sup.A, and is preferably unsubstituted; wherein each R.sup.A is independently selected from C.sub.1-4 hydrocarbyl; m is an integer from 1 to 8; and Q is selected from —OR and —O—C(O)—R.sup.5, C.sub.12-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR.sup.A, wherein R.sup.A is a C.sub.1-6 hydrocarbyl group.
(16) Optionally, the ionic compound of the first aspect of the invention is of the formula (Ta) or (Ib), wherein each B is selected from an unsubstituted C.sub.1-3alkylene group, especially ethylene.
(17) The ionic compound of the first aspect of the invention is optionally of formula (Ic):
(18) ##STR00007##
wherein: Each of X and Y are the same or different and is selected from —O—, and —O—C(O)— where the carbonyl carbon is bonded to group R.sup.1 or R.sup.2 R.sup.1 and R.sup.2 are the same or different and are each independently selected from a C.sub.12-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR.sup.A, wherein R.sup.A is a C.sub.1-6 hydrocarbyl group; R.sup.3 and R.sup.4 are the same or different and are each independently selected from a C.sub.1-10 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR′, —C(O)OH, —CN, —N(R′).sub.2, and —C(O)R′, wherein each R′ is the same or different and is a C.sub.1-6 hydrocarbyl group; m is an integer from 1 to 8; and Q is selected from —OR and —O—C(O)—R.sup.5, wherein R.sup.5 is selected from a C.sub.12-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR.sup.A, wherein R.sup.A is a C.sub.1-6 hydrocarbyl group.
(19) The term “hydrocarbyl” as used herein refers to straight or branched, saturated or unsaturated groups unless otherwise specified.
(20) Optionally, the ionic compound of the first aspect of the invention is of the formula (Ia), (Ib) or (Ic), wherein R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-22 hydrocarbyl group, for example, an unsaturated C.sub.14-22 alkenyl group having one, two or three double bonds, especially on double bond. Advantageously, R.sup.1 and R.sup.2 are the same or different and are each independently a straight chain, unsaturated C.sub.16 or C.sub.18 alkenyl group having one double bond. Optionally R.sup.1 and R.sup.2 are selected from —(CH.sub.2).sub.5-10CH═CH(CH.sub.2).sub.5-9CH.sub.3, especially —(CH.sub.2).sub.6-9CH═CH(CH.sub.2).sub.6-8CH.sub.3, such as —(CH.sub.2).sub.7CH═CH(CH.sub.2).sub.7CH.sub.3 or —(CH.sub.2).sub.8CH═CH(CH.sub.2).sub.7CH.sub.3. Preferably, the double bond is cis and R.sup.1 and R.sup.2 are selected from —(CH.sub.2).sub.5-10CH[Z]═CH(CH.sub.2).sub.5-9CH.sub.3, especially —(CH.sub.2).sub.6-9CH[Z]═CH(CH.sub.2).sub.6-8—CH.sub.3, such as —(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 or —(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3. Preferably, X—R.sup.1 and Y—R.sup.2 are selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3. Preferably, each of X and Y are the same. Preferably, each of R.sup.1 and R.sup.2 are the same. Preferably, each of X—R.sup.1 and Y—R.sup.2 are the same. Preferably, each of X—R.sup.1 and Y—R.sup.2 are the same and are selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3.
(21) Optionally, the ionic compound of the first aspect of the invention is of the formula (Ia), (Ib) or (Ic), wherein R.sup.3 and R.sup.4 are the same or different and are each independently selected from a straight or branched, unsubstituted C.sub.1-10 alkyl group, for example, a straight or branched, unsubstituted C.sub.1-6 alkyl group, especially a straight or branched, unsubstituted C.sub.1-4 alkyl group, such as methyl or ethyl. Advantageously, R.sup.3 and R.sup.4 are both the same, for example, both methyl or both ethyl, especially both methyl.
(22) Optionally, the ionic compound of the first aspect of the invention is of the formula (Ia), (Ib) or (Ic), wherein m is selected from 1, 2, 3, 4, 5, 6 or 7, for example 2, 3, 4, 5 or 6, especially 2, 3, 4 or 5. In a further embodiment of the first aspect of the invention, m is 3 or 4.
(23) Optionally, the ionic compound of the first aspect of the invention is of the formula (Ia), (Ib) or (Ic), wherein Q is selected from —OR.sup.5 and —O—C(O)—R.sup.5 and R.sup.5 is selected from a C.sub.12-24 hydrocarbyl group, such as a C.sub.14-22 hydrocarbyl group, for example, an unsaturated C.sub.14-22 alkenyl group having one, two or three double bonds, especially a straight chain, unsaturated C.sub.16 or C.sub.18 alkenyl group having one double bond, especially a cis double bond. Optionally R.sup.5 is —(CH.sub.2).sub.5-10CH═CH(CH.sub.2).sub.5-9CH.sub.3, especially —(CH.sub.2).sub.6-9CH═CH(CH.sub.2).sub.6-8CH.sub.3, such as —(CH.sub.2).sub.7CH═CH(CH.sub.2).sub.7CH.sub.3 or —(CH.sub.2).sub.8CH═CH(CH.sub.2).sub.7CH.sub.3. Preferably the double bond is cis and R.sup.5 is —(CH.sub.2).sub.5-10CH[Z]═CH(CH.sub.2).sub.5-9CH.sub.3, especially —(CH.sub.2).sub.6-9CH[Z]═CH(CH.sub.2).sub.6-8CH.sub.3, such as —(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 or —(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3. Preferably, Q is selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3.
(24) For the avoidance of doubt any of the optional elements of the lipids of the first aspect of the invention may be combined in further embodiments of the first aspect of the invention. For example, in one embodiment of the first aspect of the invention, the ionic compound is of the formula (Ia) where R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-22 hydrocarbyl group; Sp and W are both bond; R.sup.3 and R.sup.4 each straight or branched, unsubstituted C.sub.1-6 alkyl group; each B is selected from an unsubstituted C.sub.1-3alkylene group; and m is 3, 4, 5, 6 or 7. Similarly, in one embodiment of the first aspect of the invention, the ionic compound is of the formula (Ib) where R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-22 hydrocarbyl group; R.sup.3 and R.sup.4 each straight or branched, unsubstituted C.sub.1-4alkyl group; each B is selected from an unsubstituted C.sub.1-3alkylene group; and m is 3, 4, 5, 6 or 7. Again similarly, in one embodiment of the first aspect of the invention, the ionic compound is of the formula (Ic) where R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-22 hydrocarbyl group; R.sup.3 and R.sup.4 each straight or branched, unsubstituted C.sub.1-4alkyl group; and m is 3, 4, 5, 6 or 7. In a further embodiment of the first aspect of the invention, the ionic compound is of the formula (Ia) where R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-20 hydrocarbyl group, for example, an unsaturated C.sub.14-20 alkenyl group, having one two or three double bonds; Sp and W are both bond; R.sup.3 and R.sup.4 each straight or branched, unsubstituted C.sub.1-4 alkyl group; each B is selected from an unsubstituted C.sub.1-3alkylene group; m is 3, 4, 5 or 6; and R.sup.5 is selected from a C.sub.14-20 hydrocarbyl group, for example, an unsaturated C.sub.14-20 alkenyl group, having one, two or three double bonds. Similarly, in a further embodiment of the first aspect of the invention, the ionic compound is of the formula (Ib) where R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-20 hydrocarbyl group, for example, an unsaturated C.sub.14-20 alkenyl group, having one two or three double bonds; R.sup.3 and R.sup.4 each straight or branched, unsubstituted C.sub.1-4 alkyl group; each B is selected from an unsubstituted C.sub.1-3alkylene group; m is 3, 4, 5 or 6; and R′ is selected from a C.sub.14-20 hydrocarbyl group, for example, an unsaturated C.sub.14-20 alkenyl group, having one, two or three double bonds. Again similarly, in a further embodiment of the first aspect of the invention, the ionic compound is of the formula (Ic) where R.sup.1 and R.sup.2 are the same or different and are each independently a C.sub.14-20 hydrocarbyl group, for example, an unsaturated C.sub.14-20 alkenyl group, having one two or three double bonds; R.sup.3 and R.sup.4 each straight or branched, unsubstituted C.sub.1-4 alkyl group; m is 3, 4, 5 or 6; and R.sup.5 is selected from a C.sub.14-20 hydrocarbyl group, for example, an unsaturated C.sub.14-20 alkenyl group, having one, two or three double bonds. In a yet further embodiment of the first aspect of the invention, the ionic compound is of the formula (Ia) where R.sup.1 and R.sup.2 are the same or different and are each independently a straight chain, unsaturated C.sub.16 or C.sub.18 alkenyl group having one double bond; Sp and W are both bond; R.sup.3 and R.sup.4 each methyl or ethyl; each B is an ethylene group; m is 3, 4 or 5; and R.sup.5 is a straight chain, unsaturated C.sub.16 or C.sub.18 alkenyl group having one double bond. Similarly, in a yet further embodiment of the first aspect of the invention, the ionic compound is of the formula (Ib) where R.sup.1 and R.sup.2 are the same or different and are each independently a straight chain, unsaturated C.sub.16 or C.sub.18 alkenyl group having one double bond; R.sup.3 and R.sup.4 each methyl or ethyl; each B is an ethylene group; m is 3, 4 or 5; and R.sup.5 is a straight chain, unsaturated C.sub.16 or C.sub.18 alkenyl group having one double bond. Again similarly, in a yet further embodiment of the first aspect of the invention, the ionic compound is of the formula (Ic) where R.sup.1 and R.sup.2 are the same or different and are each independently a straight chain, unsaturated C.sub.16 or C.sub.18 alkenyl group having one double bond; R.sup.3 and R.sup.4 each methyl or ethyl; m is 3, 4 or 5; and R.sup.5 is a straight chain, unsaturated C.sub.16 or C.sub.18 alkenyl group having one double bond.
(25) The ionic compound of the first aspect of the invention is optionally of formula (Id):
(26) ##STR00008##
wherein: each of X—R.sup.1 and Y—R.sup.2 and Q are selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3; and m is an integer from 2 to 5.
(27) Optionally, the ionic compound of the first aspect of the invention is of the formula (Id), wherein m is 3 or 4.
(28) Optionally, the ionic compound of the first aspect of the invention is of the formula (Id), wherein each of X—R.sup.1 and Y—R.sup.2 are the same and are selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3.
(29) Optionally, the ionic compound of the first aspect of the invention is of the formula (Id), wherein each of X—R.sup.1 and Y—R.sup.2 are the same and are selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3, Q is selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3, and m is 3 or 4.
(30) Optionally, the ionic compound of the first aspect of the invention is of the formula (Id), wherein each of X—R.sup.1 and Y—R.sup.2 are the same and are selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3, and Q is —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3. Optionally, the lipid of the first aspect of the invention comprises a cation of the formula (Id), wherein each of X—R.sup.1 and Y—R.sup.2 are the same and are selected from —O—(CH.sub.2).sub.8CH[Z]═CH(CH.sub.2).sub.7CH.sub.3 and —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3, Q is —O—C(O)—(CH.sub.2).sub.7CH[Z]═CH(CH.sub.2).sub.7CH.sub.3, and m is 3 or 4.
(31) The ionic compound of the first aspect of the invention typically includes an inorganic counter ion, for example, a pharmaceutically acceptable anion such as chloride or bromide.
(32) Transfection Complexes
(33) In a second aspect, the invention provides a transfection complex that comprises (i) a ionic compound of the first aspect of the invention. The transfection complex of the second aspect of the invention optionally further comprises a (ii) a polycationic nucleic acid-binding component and (iii) a cell surface receptor binding component. Typically, the polycationic nucleic acid-binding component (ii), and the cell surface receptor binding component (iii) together form a peptide derivative. Advantageously, the transfection complex of the second aspect of the invention further comprises (iv) a nucleic acid.
(34) The transfection complex of the second aspect of the invention is typically a non-viral transfection complex, for example, LPD (or LID) complex.
(35) In an eleventh aspect, the invention provides a kit that comprises: (i) a nucleic acid, (ii) an ionic compound of the invention as defined above, (iii) a polycationic nucleic acid-binding component, and (iv) a cell surface receptor binding component.
(36) For example, the invention provides a kit that comprises: (i) an ionic compound of the first aspect of the invention nucleic acid; a peptide comprising (ii) a polycationic nucleic acid-binding component and (iii) a cell surface receptor binding component; and optionally (iv) a nucleic acid.
(37) The kit of the eleventh aspect of the invention may, for example, be used to assemble a transfection complex of the second aspect of the invention.
(38) In a third aspect, the invention provides a pharmaceutical composition which comprises the ionic compound of the first aspect of the invention or the transfection complex of the second aspect of the invention in admixture or conjunction with a pharmaceutically suitable carrier.
(39) Peptides
(40) In one embodiment, the peptide derivative of the second aspect of the invention is of the formula A-B-C wherein: A is a polycationic nucleic acid-binding component, B is a spacer element, and C is a cell surface receptor binding component.
(41) It has been found that transfection complexes of the second aspect of the invention which comprise spacer element peptide that is susceptible to cleavage within a cell are more efficient in bringing about transfection of a target cell than prior art complexes including peptides without the spacer that is susceptible to cleavage within a cell.
(42) The polycationic nucleic acid-binding component A is any polycation that is capable of binding to DNA or RNA. A polycation may be polycationic itself or it may have any number of cationic monomers provided the ability to bind to DNA or RNA is retained. For example, from 3 to 100 cationic monomers may be present, for example, from 10 to 20, for example from 14 to 18, for example, about 16.
(43) The term “polycationic nucleic acid-binding component” is well known in the art and refers to polymers having at least 3 repeat cationic amino acid residues or other cationic unit bearing positively charged groups, such polymers being capable of complexion with a nucleic acid under physiological conditions. An example of a nucleic acid-binding polycationic molecule is an oligopeptide comprising one or more cationic amino acids. Such an oligopeptide may, for example, be an oligo-lysine molecule, an oligo-histidine molecule, an oligo-arginine molecule, an oligo-ornithine molecule, an oligo diaminopropionic acid molecule, or an oligo-diaminobutyric acid molecule, or a combined oligomer comprising any combination of histidine, arginine, lysine, ornithine diaminopropionic acid, and diaminobutyric acid residues. Any of the above oligopeptides may have, for example, a total of from 3 to 35, for example, from 5 to 25 residues, preferably from 10 to 20 residues, for example, from 14 to 18 residues, for example 16 residues.
(44) An oligolysine is particularly preferred, for example, having from 3 to 35, for example, from 2 to 25, for example, form 10 to 20 lysine residues, for example, from 13 to 19, for example, from 14 to 18, for example, from 15 to 17 residues, for example, 16 residues i.e. [K].sub.16, “K” denoting lysine.
(45) Further examples of polycationic components include dendrimers and polyethylenimine. Polyethylenimine (PEI) is a non-toxic, cross-linked cationic polymer with gene delivery potential (Proc. Natl. Acad. Sci., 1995, 92, 7297-7301). Polyethylenimine is obtainable from Fluka (800 kDa) or from Sigma (50 kDa) or alternatively pre-diluted for transfection purposes from PolyPlus-tranfection (Illkirch, France). Typically, PEI is most efficient when used in a 9 fold excess over DNA, the excess ratio being calculated as PEI nitrogen:DNA phosphate, and at pH 5 to 8. Such parameters may be optimised in a manner familiar to the person skilled in the art.
(46) The spacer element peptide B is advantageously susceptible to cleavage within a cell. A spacer element peptide B that is susceptible to cleavage within a cell may be susceptible to cleavage within the endosome, lysosome, and/or cytoplasm of a cell. Susceptible to cleavage is understood herein to mean that the element is susceptible to cleavage over a timescale during which the components A and C remain in tact. Element B is cleaved more rapidly than the cellular peptide-degradation pathways take effect.
(47) Preferably, the spacer element peptide is susceptible to enzymatic cleavage, reductive cleavage, or pH-dependent cleavage e.g. hydrolysis. In the case of enzymatic cleavage, in one aspect of the invention, preferred peptides are those which are susceptible to cleavage by an enzyme selected from NOX (NADH-oxidase), GILT (gamma-interferon-inducible lysosomal thiol reductase) and PDI (protein disulfide isomerase). In another aspect of the invention, preferred peptides are those which are susceptible to cleavage by an enzyme which is present in the endosome, for example an endosomal protease, such as furin or cathepsin.
(48) Preferably the spacer element peptide B comprises a group selected from: a) a peptide chain comprising a disulfide-linkage; b) a peptide chain comprising an ester-linkage; c) an amino acid sequence susceptible to cleavage by furin; and d) an amino acid sequence susceptible to cleavage by a cathepsin enzyme
(49) The disulfide linkage is preferably one that is stable under normal atmospheric and physiological conditions, but that may be reductively cleaved in an endosome. Similarly, an ester linkage in a peptide chain of the invention is preferably one that is stable at neutral pH, but is cleaved in the acidic environment of an endosome (for example at pH below 6.0, preferably at pH below 5.5, or at pH below 5.0).
(50) For example, amino acid sequences susceptible to cleavage by furin include sequences selected from i) RX.sup.1KR; and ii) RX.sup.2RR; in which X.sup.1 and X.sup.2, which may be the same or different, each represents any amino acid residue (Zimmer et al., J. Biol. Chem., 2001, 276, 31642-31650; Nakayama, Biochem. J., 1997, 327, 625-635)
(51) Preferred amino acid residues X.sup.1 include Lys (K) and Val (V), for example Lys (K). Preferred amino acid residues X.sup.2 include Lys (K) and Val (V), for example Val (V).
(52) For example, the cathepsin enzyme may be any suitable cathepsin enzyme (see Pillay et al., Biochem. J., 2002, 363, 417-429). For example, it may be cathepsin B. For example, amino acid sequences susceptible to cleavage by cathepsin B (see Pechar et al., Macromol. Chem. Phys., 1997, 198, 1009-1020) include sequences selected from: iii) X.sup.3X.sup.4 where X.sup.3 is selected from Tyrosine (Tyr, Y), Phenylalanine (Phe, F), Leucine (Leu, L), Valine (Val, V) and Isoleucine (Ile, I) and X.sup.4 is selected from Glycine (Gly, G), Alanine (Ala, A) and Glutamic acid (Glu, E).
(53) Preferably, X.sup.3 is selected from Tyrosine (Tyr, Y), Phenylalanine (Phe, F) and Leucine (Leu, L).
(54) For example, the sequence X.sup.3X.sup.4 may be present as GFX.sup.3X.sup.4, for example as GFLG (as used in Pechar et al., Bioconjugate Chem., 2000, 11, 131-139)
(55) The spacer element peptide B may additionally comprise a linker, which is preferably either a peptide, that is to say, it comprises amino acid residues, or a polyethyleneglycol group, or a mixture of the two. The amino acids may be naturally occurring or non-naturally occurring. They may have L- or D-configuration. The linker may have two or more amino acids. It may, for example, comprise three or more amino acids, for example, four or more, for example, five or more, for example, up to ten amino acids or more. The amino acids may be the same or different, but the use of multiple lysine residues (or other cationic amino acids suitable for use in the polycationic nucleic acid-binding component of a vector complex) should generally be avoided in the spacer as oligo-lysine sequences have activity as a polycationic nucleic acid-binding component.
(56) The linker may be, for example, the dipeptide glycine-glycine (GG) or glycine-alanine (GA).
(57) The linker may be, or may include a polyethyleneglycol moiety. The polyethyleneglycol moiety may comprise from 1 to 30 ethylene glycol units, preferably from 1 to 15 units, more preferably from 1 to 8 units, for example from 2 to 6 units, for example 4 units.
(58) Preferably, the linker is at the end of the spacer element peptide B which is bonded to the polycationic nucleic acid-binding component A.
(59) Preferably the cell surface receptor binding component C comprises a peptide. Where cell surface receptor binding component C comprises a peptide, the peptide may be up to 20 amino acids in length, or may be longer. The peptide generally has at least 5 amino acids but may have fewer. Generally, the peptide has any number of amino acids from 6 to 20 inclusive. Generally, it is preferred for the peptide to have 15 amino acids or fewer, more preferably 12 amino acids or fewer, most preferably 10 amino acids or fewer. Generally, it is preferred for the peptide to have 5 or more amino acids, for example, 6 or more amino acids. Most preferably, the peptide has 7 amino acids.
(60) Preferably the cell surface receptor binding component C comprises a peptide comprising a cyclic region. Cyclic peptides may be formed by the provision of at least two cysteine residues in the peptide, thus enabling the formation of a disulphide bond. Accordingly, preferred cell surface receptor binding components C consist of or comprise a peptide having two or more cysteine residues that are capable of forming one or more disulphide bond(s). Preferably the cysteine residues flank the primary receptor binding portion.
(61) In one embodiment of the invention, the cell surface receptor binding component C comprises an integrin-binding peptide. An integrin-binding peptide is any peptide that is capable of binding specifically to integrins found on the surface of cells. The integrin-binding peptide may be a naturally occurring integrin-binding ligand, for example, an extracellular matrix protein, a viral capsid protein, the bacterial protein invasin, a snake venom disintegrin protein, or an integrin-binding fragment of any such protein. Such integrin-binding proteins and fragments thereof may be obtained from natural sources or by recombinant techniques. It is preferable to use integrin-binding peptides, in particular because of their ease of synthesis, purification and storage, their potential for chemical modification, and their potentially low immunogenicity in vivo. Preferred integrin-binding peptides are those such as described in WO 96/15811, and especially WO 98/54347. For example, an integrin-binding peptide may be specific for α4β1 integrins.
(62) In this embodiment, the cell surface receptor binding component C preferably comprises a peptide selected from: a) RGD; b) RRETAWA; c) LDV
(63) In a further embodiment of the invention, the cell surface receptor binding component C comprises a peptide which is capable of binding to human airway epithelial (HAE) cells. Preferred HAE cell-binding peptides are those such as described in WO 02/072616. In this embodiment, the cell surface receptor binding component C preferably comprises a peptide selected from a) X.sup.5SM; b) LX.sup.6HK; c) PSGX.sup.7ARA; d) SX.sup.8RSMNF; and e) LX.sup.9HKSMP;
in which X.sup.5 is a basic amino acid residue, X.sup.6 is Q or P, X.sup.7 is A or T, X.sup.8 is an acidic amino acid residue and X.sup.9 is P or Q.
(64) Preferably, the cell surface receptor binding component C comprises a peptide selected from a) X.sup.5SM; b) LX.sup.6HK; and c) PSGAARA,
in which X.sup.5 is a basic amino acid residue and X.sup.6 is Q or P.
(65) Preferably X.sup.5 is K or R. Preferably X.sup.6 is P. Preferably X.sup.7 is A. Preferably X.sup.8 is E or Q. More preferably X.sup.8 is E. Preferably X.sup.9 is P. Accordingly, preferred peptides are those comprising a sequence selected from LQHKSMP, LPHKSMP, VKSMVTH, SERSMNF, VGLPHKF, YGLPHKF, PSGAARA, SQRSMNF and PSGTARA.
(66) In another embodiment of the invention, the cell surface receptor binding component C comprises a peptide which is capable of binding to human dendritic cells. Preferred human dendritic cell-binding peptides are those such as described in WO 2004/108938. For example, such a peptide may be selected from a peptide comprising an amino acid sequence selected from: a) PX.sup.10X.sup.11X.sup.12T; b) PSX.sup.13S; c) QX.sup.14X.sup.15X.sup.16Q; d) SX.sup.17S,
in which X.sup.10, X.sup.11 and X.sup.12, which may be the same or different, each represents an amino acid residue;
X.sup.13 represents an amino acid residue;
X.sup.14 and X.sup.16, which may be the same or different, each represents an amino acid residue,
and X.sup.15 represents an amino acid residue having an amide side chain, for example, N or Q.
X.sup.1 represents an amino acid residue having an aliphatic side chain, for example, L or I.
(67) In a preferred embodiment, the cell surface receptor binding component C comprises a peptide selected from:
(68) TABLE-US-00001 a) CRGDCLG; b) CRGDCLG; c) ACDCRGDCFCG; d) CRGDMFGCA; e) CRRETAWACG; f) CRGEMFGCA; g) CSERSMNFCG; h) CYGLPHKFCG; and i) CLPHKSMPCG.
(69) The invention provides the use of a transfection complex of the second aspect of the invention in the formation of a lipopolyplex (LPD) transfection vector. The transfection vector may be used to target an entity to a cell, the entity being a nucleic acid or another molecule, for example, a therapeutically or pharmaceutically active molecule, or a molecule comprising a detectable label.
(70) Medical Uses
(71) It has further been found that an ionic compound of the invention improves the targeting of a vector complex to tumour cells, particularly when used in conjunction with a cleavable peptide. The ionic compound of the first invention thus finds use in the treatment of cancer, therapeutic or prophylactic immunisation, or anti-sense or RNAi therapy. The invention thus provides a method of treating cancer, therapeutic or prophylactic immunisation, or anti-sense or RNAi therapy comprising administering an ionic compound of the first aspect of the invention in a suitable complex to the patient in an effective amount. The transfection complexes of the second aspect of the invention thus find use in the treatment of cancers, for therapeutic or prophylactic immunisation, or for anti-sense or RNAi therapy. The invention thus provides a method of treating cancer, therapeutic or prophylactic immunisation, or anti-sense or RNAi therapy comprising administering a transfection complex of the second aspect of the invention to the patient in an effective amount. The ionic compound of the first aspect of the invention or the transfection complex of the second aspect of the invention may be administered in a pharmaceutical composition of the third aspect of the invention, which comprises the ionic compound or transfection complex in admixture or conjunction with a pharmaceutically suitable carrier.
(72) In a fourth aspect, the invention provides an ionic compound of the first aspect of the invention or a transfection complex of the second aspect of the invention for use in therapy. The invention further provides an ionic compound of the first aspect of the invention or a transfection complex of the second aspect invention for use as a medicament or a vaccine. For example, the fourth aspect of the invention provides an ionic compound of the first aspect of the invention or a transfection complex of the second aspect of the invention for use in the treatment or prophylaxis of condition caused by a defect and/or a deficiency in a gene, for use in the treatment of cancer, for therapeutic or prophylactic immunisation, or for anti-sense or RNAi therapy.
(73) The nucleic acid component (D) may be any suitable nucleic acid. It may be DNA or RNA or a chemically modified nucleic acid mimetic, for example a PNA molecule. It may, for example, code for a protein that has a utility in the target cell. It may be an anti-sense nucleic acid or an RNAi nucleic acid. RNAi is achieved by exposing the cellular messenger RNA (mRNA) molecules produced by the target gene to double-stranded RNA (dsRNA) molecules that contain sequences complementary to a short portion of the mRNA molecule. Inside the cell, the double-stranded RNA molecules are cleaved to produce short (21-23 nucleotides long) single and double-stranded fragments which can bind to the target mRNA molecules. Such binding leads to the cleavage of the target mRNA by nucleases, thus resulting in a reduction in the expression levels of the target gene. Thus the nucleic acid component may itself be an RNAi molecule (an “siRNA”); alternatively, the nucleic acid administered may be a DNA molecule that comprises a sequence that, when transcribed, produces an RNAi molecule, i.e. an RNA that is capable of suppressing the expression of a target gene via RNA interference.
(74) The invention also provides processes for the production of a transfection complex of the invention.
(75) In a fifth aspect, the invention provides a method for the treatment or prophylaxis of a condition caused in a human or in a non-human animal by a defect and/or a deficiency in a gene which comprises administering the ionic compound of the first aspect of the invention or the transfection complex of the second aspect invention the human or to the non-human animal.
(76) The term “a defect and/or a deficiency in a gene” as used herein denotes not only a defect or deficiency in the coding region of a gene, but a defect or deficiency in a control element for the gene, for example, a control element in trans or in cis, or a defect or deficiency in any other element that is involved in the transcription or translation of the gene, whether directly or indirectly.
(77) In a sixth aspect, the invention provides a method for therapeutic or prophylactic immunisation of a human or of a non-human animal, which comprises administering the ionic compound of the first aspect of the invention together with, or the transfection complex of the second aspect of the invention comprising, an anti-sense nucleic acid (for example anti-sense RNA) or a nucleic acid suitable for RNAi therapy to the human or to the non-human animal. The invention also provides a method of anti-sense therapy, which comprises administering the ionic compound of the first aspect of the invention together with a nucleic acid, or a transfection complex of the second aspect invention comprising nucleic acid, to a human or to a non-human animal in which the nucleic acid is a nucleic acid (for example RNA) suitable for use in anti-sense therapy or a nucleic acid suitable for RNAi therapy.
EXAMPLES
(78) Unless otherwise noted, solvents and reagents for synthesis were reagent grade from commercial suppliers and used without further purification. Dry CH.sub.2Cl.sub.2 was obtained using anhydrous alumina columns using the procedure described in Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15, 1518-1520. All moisture-sensitive reactions were performed under a nitrogen or argon atmosphere using oven-dried glassware. Reactions were monitored by TLC on Kieselgel 60 F.sub.254 plates with detection by UV, potassium permanganate, and phosphomolybdic acid stains. Flash column chromatography was carried out using silica gel (particle size 40-63 μm). .sup.1H NMR and .sup.13C NMR spectra were recorded on a Bruker AMX300 MHz, Avance-500 MHz and Avance-600 MHz machines. Coupling constants are measure in Hertz (Hz) and unless otherwise specified, spectra were acquired at 298 K. Mass spectra were recorded on Thermo Finnegan MAT 900XP, Micromass Quattro LC electrospray and VG70-SE mass spectrometers. Infrared spectra were recorded on a Shimadzu FTIR-8700 spectrometer.
(79) DODEG4 (13)
(80) {2,3-di[(Z))-octadecy-9-enyloxy]-propyl}-N-{2-[2-(2-{2-hydroxy-ethoxy}-ethoxy)-ethoxy]-ethyl}-N,N-dimethylammonium chloride (DODEG4) (13) (CH300) was synthesized as previously reported by Dori, Y.; Bianco-Peled, H.; Satija, S.; Fields, G. B.; McCarthy, J. B.; Tirrell, M. J. Biomed. Mater. Res. 2000, 50, 75-81, from (2,3-bis-octadec-9-enyloxypropyl)-dimethylamine and 4-EG bromide (HO—(CH.sub.2CH.sub.2—O).sub.3—CH.sub.2CH.sub.2—Br).
(81) ##STR00009##
(82) DODEG4 (13) (CH300) can also be synthesis as described in U.S. Pat. No. 7,598,421, Example 4.
(83) Analogues to DODEG4
(84) Salts similar to DODEG4 (13) (CH300) with R.sup.1 and R.sup.2 hydrocarbyl groups of differing chain lengths and with different values for m, i.e. differing polyethylene glycol chain lengths, may be prepared as described in U.S. Pat. No. 7,598,421 (see Examples 3, 5, 6 and 7 in particular). For example, DODEG3 can be synthesized by the protocol set of Example 3 of U.S. Pat. No. 7,598,421.
(85) ##STR00010##
DOesDEG4 (15)
(86) The diester analogue DOesDEG4 (15) was prepared similarly to DODEG4 (13) via the quaternisation of (2,3-bis-heptadec-9-enylcarboxypropyl)-dimethylamine with 4-EG bromide.
(87) Tertiary Amine (16)
(88) 2,3-di-((9Z)-octadecenyloxy)propyl-N,N-dimethylamine (16) is prepared as described in Hurley, C. A.; Wong, J. B.; Hailes, H. C.; Tabor, A. B. J. Org. Chem. 2004, 69, 980-983 from octadec-9-enyl mesylate and 3-dimethylamino-1,2-propanediol.
(89) 4-EG Bromide (17)
(90) 11-Bromo-3,6,9-trioxoundecan-1-ol (17) was prepared as described in Hurley, C. A.; Wong, J. B.; Ho, J.; Writer, M.; Irvine, S. A.; Lawrence, M. J.; Hart, S. L.; Tabor, A. B.; Hailes, H. C. Org. Biomol. Chem. 2008, 6, 2554-2559.
(91) 4-EG Oleoyl Ester (20)
(92) 4-EG oleoyl ester (20) was prepared by the reaction of 4-EG bromide (17) with oleic acid, in the presence of DMAP and DIC in CH.sub.2Cl.sub.2.
(93) ##STR00011##
(94) A solution of oleic acid (2.00 mL, 6.30 mmol), the 4-EG bromide 17 (1.50 g, 5.83 mmol), and DMAP (70 mg, 0.58 mmol) in anhydrous dichloromethane (50 mL) were stirred for at rt for 5 min. After cooling to 0° C., N,N′-diisopropylcarbodiimide (DIC) (1.20 mL, 7.75 mmol) was added dropwise and the mixture stirred at rt for 18 h. The dichloromethane was removed in vacuo, ethyl acetate (60 mL) was added, and the mixture washed with sodium hydrogencarbonate (2×60 mL), brine (60 mL), dried (Na.sub.2SO.sub.4) and concentrated in vacuo. Purification by silica flash chromatography (EtOAc/hexane, 1:4) yielded 20 as a yellow oil (1.65 g, 54%). R.sub.F 0.19 (EtOAc/hexane, 1:4); v.sub.max(neat)/cm.sup.−1 2923, 1736, 1457; .sup.1H NMR (300 MHz; CDCl.sub.3) δ 0.84 (t, J=6.7 Hz, 3H), 1.18-1.30 (m, 20H), 1.58 (m, 2H), 1.93-2.00 (m, 4H), 2.29 (t, J=7.6 Hz, 2H), 3.43 (t, J=6.3 Hz, 2H), 3.59-3.67 (m, 10H), 3.77 (t, J=6.3 Hz, 2H), 4.19 (t, J=4.8 Hz, 2H), 5.27-5.36 (m, 2H); .sup.13C NMR (75 MHz; CDCl.sub.3) δ 14.1, 22.6, 24.9, 27.2, 29.1-29.8 (signals superimposed), 30.4, 31.9, 34.0, 63.3, 69.2, 70-4-70.6 (signals superimposed), 71.2, 129.7, 130.0, 173.6; m/z [HRMS ES+] found [MNa].sup.+ 543.2671. C.sub.26H.sub.49O.sub.5.sup.79BrNa requires 543.2661.
(95) TC-DODEG4 (14)
(96) TriChain ionic compound (9Z)—N-(2,3-bis((9Z)-octadecenyloxy)propyl)-N,N-dimethyl-13-oxo-3,6,9,12-tetraoxatriacont-21-en-1-aminium bromide (TC-DODEG4) (14) was synthesised by the quaternisation of (2,3-bis-octadec-9-enyloxypropyl)-dimethylamine (16) with the 4-EG oleoyl ester (20).
(97) ##STR00012##
(98) A solution of the amine (16) (0.260 g, 0.420 mmol) and the 4-EG oleoyl ester (20) (0.240 g, 0.460 mmol) in acetone (2 mL) were stirred in a sealed tube at 90° C. for 48 h. The acetone was removed in vacuo. Purification by flash silica chromatography (CH.sub.2Cl.sub.2/MeOH, 19:1) yielded TC-DODEG4 (14) as a pale yellow oil (171 mg, 36%). R.sub.F 0.40 (CH.sub.2Cl.sub.2/MeOH, 9:1); v.sub.max (neat)/cm.sup.−1 2950, 2859, 1740, 1464; H NMR (600 MHz; CDCl.sub.3) δ 0.83 (t, J=7.0 Hz, 9H), 1.13-1.22 (m, 64H), 1.51 (m, 4H), 1.57 (m, 2H), 1.92-1.97 (m, 12H), 2.29 (t, J=7.7 Hz, 2H), 3.38-3.51 (m, 14H), 3.51 (m, 6H), 3.58 (m, 4H), 3.92-4.05 (m, 4H), 4.08 (m, 1H), 4.18 (m, 2H), 5.27 (m, 6H); .sup.13C NMR (150 MHz; CDCl.sub.3) δ 14.2, 22.8, 25.0, 26.1, 26.3, 27.26, 27.30, 29.0, 29.1-29.9 (signals superimposed), 30.1, 32.1, 32.7, 34.2, 53.3, 53.4, 63.3, 65.0, 65.2, 66.8, 68.6, 69.29, 69.32, 70.3, 70.4, 70.5, 70.6, 72.1, 73.5, 127.9-130.5 (signals superimposed), 173.9; m/z [HRMS ES+] found [M-Br].sup.+ 1060.9904. C.sub.67H.sub.130NO.sub.7 requires 1060.9847; m/z (+ES) 1061 ([M-Br].sup.+, 100.sup.0/), 980 (60), 931 (70), 843 (65), 306 (63).
(99) Tertiary Amine (18)
(100) (9Z)-3-(dimethylamino)propane-1,2-diyl dioleate (18) was prepared as described in Narang, A. S.; Thoma, L.; Miller, D. D.; Mahato, R. I. Bioconjugate Chem. 2005, 16, 156-168.
(101) TC-DOesDEG4 (19)
(102) TriChain ionic compound (9Z)—N,N-dimethyl-N-(3-(oleoyloxy)-2-((9Z)-2-oxooctadecenyloxy) propyl)-13-oxo-3,6,9,12-tetraoxatriacont-21-en-1-aminium bromide (TC-DOesDEG4) (19) was synthesised by the quaternisation of (2,3-bis-heptadec-9-enylcarboxypropyl)-dimethylamine (18) with the 4-EG oleoyl ester (20).
(103) ##STR00013##
(104) A solution of the amine 18 (0.610 g, 0.941 mmol) and the 4-EG oleoyl ester 20 (0.640 g, 1.22 mmol) in acetone (2 mL) were stirred in a sealed tube at 80° C. for 48 h. The acetone was removed in vacuo. Purification by flash silica chromatography (CH.sub.2Cl.sub.2/MeOH, 19:1) yielded 19 as a pale yellow oil (118 mg, 11%). R.sub.F 0.24 (CH.sub.2Cl.sub.2/MeOH, 19:1); v.sub.max (neat)/cm.sup.−1 2924, 2854, 1740, 1465; .sup.1H NMR (500 MHz; CDCl.sub.3) δ 0.86 (t, J=6.8 Hz, 9H), 1.25-1.32 (m, 60H), 1.58 (m, 6H), 1.99 (m, 12H), 2.30 (t, J=7.5 Hz, 4H), 2.34 (m, 2H), 3.44 (s, 3H), 3.51 (s, 3H), 3.64-3.68 (m, 11H), 3.82 (dd, J=13.5 and 10.0 Hz, 1H), 3.94 (m, 2H), 4.05 (m, 2H), 4.13 (m, 1H), 4.20 (t, J=5.0 Hz, 2H), 4.35 (d, J=13.5 Hz, 1H), 4.48 (dd, 0.1=12.3 and 3.3 Hz, 1H), 5.28-5.35 (m, 6H), 5.64 (m, 1H); .sup.13C NMR (125 MHz; CDCl.sub.3) δ 14.2, 22.7, 24.7, 24.8, 25.0, 25.7, 27.2, 27.3, 29.1-29.8, 31.6, 32.0, 34.0, 34.3, 52.8, 52.9, 63.2, 63.3, 64.0, 64.9, 65.6, 65.8, 69.3, 70.1, 70.45, 70.48, 70.6, 129.7-130.1 (signals superimposed), 172.9, 173.2, 173.8; m/z [HRMS ES+] found [M-Br].sup.+ 1088.9382. C.sub.67H.sub.126NO.sub.9 requires 1088.9433; m/z (+ES) 1089 ([M-Br].sup.+, 100%).
(105) Analogues to TC-DOesDEG4
(106) Tri-chain lipids TC-DODEG3 and TC-DOesDEG3, in which m=2 may similarly be synthesised by the quaternisation of (2,3-bis-octadec-9-enyloxypropyl)-dimethylamine (16) and (2,3-bis-heptadec-9-enylcarboxypropyl)-dimethylamine (18) respectively with the 3-EG oleoyl ester.
(107) Lipid ME42 (Comparative)
(108) (2,3-Bis-octadec-9-enyloxypropyl)(8-hydroxy-3,6-dioxooctyloxycarbonylbutyl)-dimethylammonium bromide (ME42) was prepared as described in WO 2007/138324 (see pages 41 and 42).
(109) ##STR00014##
Lipid FMM32 (Comparative)
(110) ##STR00015##
Lipid DHDTMA (106a) (Comparative)
(111) DHDTMA is a cationic lipid based on a glycerol backbone with two unsaturated C16 alkyl chains linked by diether linkages described in Writer M, Hurley C A, Sarkar S, Copeman D M, Wong J B, et al. Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo. J Liposome Res. 2006; 16:373-389.
(112) Peptide Synthesis
(113) The peptides described (Table IA) were synthesized using standard instruments and techniques.
(114) TABLE-US-00002 TABLE IA Peptide sequence Peptide Sequence K16 KKKKKKKKKKKKKKKK ME27 (K).sub.16RVRRGACRGDCLG K16CY (K).sub.16RVRRGACYGLPHKFCG K16Y (K).sub.16GACYGLPHKFCG K16P (K).sub.16GACLPHKSMPCG
(115) TABLE-US-00003 TABLE IB Peptide mass Peptide Mass (g .Math. mol.sup.−1) K16 2068 ME27 3467.5 K16CY 3871 K16Y 3326.8 K16P 3148.1
(116) ME27 was synthesized on a SYRO automated peptide synthesizer.
(117) Linear peptide sequences: The peptide was synthesized on a 20 μmol scale using 2 ml syringes with Teflon frits and 500 μl coupling volume. Fmoc-Gly preloaded NovaSyn TGT resin or Fmoc-Gly-2-Cl-Trt-resin were used for these sequences. Fmoc-Peg4-COOH was synthesized following a procedure reported previously (see synthesis of Fmoc-Haa4-COOH at page 82 of WO 2005/117985—Fmoc-Haa4-COOH was the name given to Fmoc-Peg4-COOH in that specification). The TGT resin was initially swelled for 10 min, however the 2-Cl-Trt resin needed a prolonged initial swelling time (some hours) in DMF. Routine coupling was performed with HBTU (in DMF) and DIPEA (in NMP) using a fourfold excess of reagents. Fmoc was cleaved with a 40% solution of piperidine in DMF for 3 min and a 20% solution for 10 min. Synthesis cycles consisted of 40 min coupling time, 3 min for Fmoc deprotection with 40% piperidine, another 10 min for Fmoc deprotection with 20% piperidine and washing steps. After synthesis and the last wash cycle with DMF, peptides were washed with DCM, methanol and diethyl ether (3 times each) using the “manual”/“empty” function of the Syro. Suction was applied for some more time to help evaporate the ether.
(118) On-resin disulfide bond formation: To form disulphide bridges on resin, the resin was placed in a syringe with PE frit and swelled in DMF. After removal of excess DMF a freshly prepared solution of iodine in a minimum amount of DMF (e.g. 500 μl for a 2 ml syringe, 10 eq iodine to resin loading) was added and the syringe was vortexed during 4 h for 20 s every 4 min. The reagent solution was removed and the resin was washed 10 to 20 times with DMF, and 3 times each with DCM, methanol and ether.
(119) Cleavage and deprotection: The syringes were transferred to the fume hood for cleavage. Cleavage was performed with a cocktail of 95% TFA, 2.5% TIS and 2.5% H.sub.2O. A minimum amount of freshly prepared cocktail was added to cover the resin (e.g. <500 μl in the 2 ml syringes). After 4 h the cleavage solutions were passed into polypropylene (PP) tubes using a plunger and the resins were washed with another small amount of cleavage cocktail (e.g. 200 μl in the 2 ml syringes). Then the peptides were precipitated with ether (e.g. to the combined fractions of the 2 ml syringes some 4 ml of diethyl ether were added). The PP tubes were kept in the freezer for at least 15 min, then centrifuged at 3000 rpm for 3 min and solution was decanted from the peptide pellet. Centrifugation and decantation were repeated twice with about 2 ml of ether. Finally the peptides were dissolved in water or tBuOH/water (4:1) and freeze-dried. Some peptide sequences showed very poor solubility and sometimes several lyophilisation/dissolution processes with different solvent mixtures (water, tBuOH or acetonitrile) were necessary to obtain a fluffy peptide.
(120) The peptide was analyzed by reverse phase HPLC and purified by reverse phase HPLC to >90% pure. Mass spectra were recorded using the Micromass Quattro ES-MS (Software: Masslynx) and the masses are recorded in the table IB.
(121) K16CY, K16Y and K16P were purchased from AMS Bio Ltd., Birmingham, UK, and synthesised using semi-automated peptide synthesis chemistry. The peptide was analysed by reverse phase HPLC and purified where necessary by reverse phase HPLC to 85% pure. Relative molecular masses are given in the table IB.
(122) K16 was purchased as described previously (Hart et al., Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector. Hum Gene Ther., 1998, 9, 575-585). Relative molecular masses are given in the table IB.
(123) All these freeze-dried peptides were diluted at 10 mg/ml in water and stored at −20° C. during several months. Once thawing, aliquots of peptides are kept at 4° C. during several weeks.
(124) Plasmid DNA
(125) The plasmid pCI-Luc (5.7 kb) consists of pCI (Promega, Southampton, UK) containing the luciferase gene driven by the cytomegalovirus (CMV) immediate/early promoter-enhancer. The plasmids were grown in Escherichia coli DH5α and purified, after bacterial alkaline lysis, on resin columns (Qiagen Ltd., Crawley, UK). Isopropanol-precipitated DNA pellets were washed with 70% ethanol, then dissolved in water at 1 mg/ml.
(126) In Vitro Transfection
(127) Cells tested included 1HAEo-human airway epithelial cells, NIH3T3 murine fibroblasts, and Neuro2A murine neuroblastoma cells. Cells were seeded into 96-well plates at approximately 2×10.sup.4 cells per well, then incubated overnight at 37° C. in complete growth medium. The following day, lipopolyplex (LPD) formulations were prepared essentially as described previously (Hart et al., 1998), by mixing the components in the following order: 50 μl of lipid (L) at 80 μg/ml in OptiMEM, 70 μl of peptide (P) at 110 μg/ml in OptiMEM and 50 μl of plasmid pCI-Luc (D) at 40 μg/ml in OptiMEM corresponding to a weight ratio of 2:4:1, respectively. All the complexes were mixed by pipetting briefly, kept for 1 h at room temperature and then diluted in OptiMEM to a final volume of 1.57 ml. Two hundred microlitres of complexes corresponding at 0.25 g of plasmid DNA were added to each culture well after removal of the complete growth medium. All the transfections were carried out in 6 wells each. A centrifugation (1500 rpm, during 5 minutes) could be performed to promote the complex sedimentation and cell contact. The cells were incubated with the complexes for 4 h at 37° C. before replacing with fresh media for 24 h, after which reporter gene expression was analyzed by luciferase assay (Promega, Madison, Wis., USA).
(128) Unless otherwise specified, cationic liposomes comprising mixtures of two ionic compounds, e.g. FMM30 and DOPE, comprise the ionic compounds in a 1:1 ratio.
(129) NIH3T3 and 1HAEo-cells were transfected with LPD formulations containing peptide ME27 and plasmid pCI-Luc and one of the lipid formulations: CH300/DOPE in a molar ratio of 1:1, FMM30/DOPE in a molar ratio of 1:1, CH300/FMM30/DOPE in a molar ratio of 4:1:5, CH300/FMM30/DOPE in a molar ratio of 1:1:2, FMM32/DOPE in a molar ratio of 1:1.
(130) As a positive control cells were transfected with the commercial transfection reagent Lipofectamine 2000 (L2K purchased from Life Technologies Inc.). Total lipid:peptide:DNA ratios were 1:4:1, 2:4:1 or 4:4:1. LPD formulations were all prepared by mixing in the order L:P:D as described above. The results are displayed in
(131) LPD formulations containing different combinations of lipids were prepared with four different peptides, ME27, K16CY, K16Y and K16P, and used to transfect NIH3T3 cells with pCI-Luc as described above. The lipids used were: CH300/DOPE in a molar ratio of 1:1, FMM30/DOPE in a molar ratio of 1:1, CH300/FMM30/DOPE in a molar ratio of 4:1:5, CH300/FMM30/DOPE in a molar ratio of 1:1:2, FMM32/DOPE in a molar ratio of 1:1, 106a (DHDTMA)/DOPE in a molar ratio of 1:1, and DOTMA/DOPE in a molar ratio of 1:1.
(132) The commercial reagents DOTAP, Lipofectin and Lipofectamine 2000 (L2K) were also compared in LPD combination with peptides and DNA. All L:P:D weight ratios were 2:4:1 and wells each received 0.25 μg of pCI-Luc. The results are displayed in
(133) LPD formulations containing different lipids ME42/DOPE (ME42), CH300/DOPE(CH300) or FMM30/DOPE were prepared with either the targeting peptides ME27 or K16Y and used to transfect Neuro-2A murine neuroblastoma cells. The results are displayed in
(134) Luciferase and Protein Assays
(135) Cells were washed once with PBS before the addition of 20 μl of 1× Reporter Lysis Buffer (Promega, Madison, Wis., USA) to the cells for 20 min at 4° C. before freezing at −80° C. for at least 30 min, followed by thawing at room temperature. Then the luciferase activity was measured during 10 seconds using the Luciferase Assay System (Promega, Madison, Wis., USA) and an Optima Fluostar plate reader (BMG Labtech). The amount of protein present in each transfection lysate was determined with the Bio-Rad (Hercules, Calif., USA) protein assay reagent by the manufacturer's instructions, adding 20 μl from the luciferase test to 180 μl of the reagent diluted 1 in 5 and incubating at room temperature for 10 min before comparing the OD590 to a range of BSA standards. In vitro luciferase activity was expressed as Relative Light Units (RLU) per milligram of protein (RLU/mg).
(136) Particle Imaging
(137) Transmission Electon Microscope images of LPD particles comprising FMM30 with peptide ME27 at a weight ratio of 0.75:4:1 (L:P:D) is shown in
(138) The size of LPD nanoparticles containing peptides ME27 with lipids FMM30 or FMM30/DPE were determined at different weight ratios of liposome to DNA (w/w) by dynamic light scattering using a NanoZS Zetasizer (Malvern). The results are shown in
(139) Results and Discussion
(140) In Vitro Transfection Efficiency of Lipids in NIH3T3 and 1HAEo-Cells
(141)
(142)
(143) In Vitro Transfection Efficiency of Lipids in Neuro 2A Cell Lines
(144) Previous results had shown that ME27/ME42 was optimal for Neuro-2A murine neuroblastoma cells. The results presented in
(145) LPD Particle Imaging
(146) Both nanoparticles comprising FMM30 with peptide ME27 at a weight ratio of 0.75:4:1 (L:P:D) shown in
(147) As shown in