Lipids

Details Lipids Lipids

2000-11-20

2003-06-24

Carr, Deborah D. (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Fatty compounds having an acid moiety which contains the...

C554S068000, C514S613000

Reexamination Certificate

active

06583301

ABSTRACT:

This invention relates to a series of bipolar lipids and to their use to deliver bioactive substances to cells.
To be effective, many pharmaceutical agents need to be efficiently delivered to the cytoplasm of a eucaryotic cell. For many low molecular weight compounds of low to moderate polarity this is not a problem since such molecules can pass directly through the plasma membrane of the cell and into the cytoplasm. Direct passage is not available to other compounds of greater polarity or high molecular weight and these generally enter the cell by receptor mediated endocytosis or phagocytosis. These mechanisms are not efficient however with all sizes and types of molecule. In particular, large, polyanionic compounds are not readily taken up by cells when delivered to them in aqueous solution.
One general solution to this problem is to couple any poorly transported pharmaceutical agent to a carrier which itself is readily taken up into the cytoplasm of a cell. This is not always satisfactory however, since coupling to the carrier may have an undesirable effect on the metabolism and/or antigenicity of the pharmaceutical agent and/or it may be difficult to recover the desired biological activity from the resulting conjugate once inside the cell.
An alternative solution is to formulate the pharmaceutical agent with a delivery vehicle which is soluble in aqueous solutions but which can also mimic naturally occurring cell membrane constituents. This encourages fusion of the vehicle with a cell membrane and subsequent delivery of any associated pharmaceutical agent to the cytoplasm.
Amphiphilic lipids have frequently been used for this purpose. These typically have a hydrophobic backbone composed of one or more hydrocarbons and a hydrophilic polar head group containing one or more ionisable groups, to facilitate the transport of macromolecules to and across the plasma membrane of cells and into the cytoplasm. The polarity of the head group may be controlled by the selection of the number and/or type of ionisable groups to achieve a range of negatively charged (anionic), neutral or positively charged (cationic) lipids.
For the delivery of polyanions it is generally advantageous to use cationic lipids. The advent of gene therapy and the need to deliver anionic molecules such as nucleic acids to mammalian cells has provided much impetus to the development of this class of lipids. First generation compounds include those with a monocation head group such as N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride [DOTMA; Felgner, P L and Ringold, G M, Nature, 337 387-388 (1989)], 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide [DMRIE; Zabner, J et al J. Biol. Chem, 270, 18997-19007 (1995)] and 3&bgr;[N-(N
1
,N
1
-dimethylaminoethane)carbamoyl]cholesterol [DC-Chol; Farhood, H et al, Biochim. Biophys. Acta. 1111, 239-246 (1992)] and those with a polycation head group such as dioctadecylamidoglycylspermine [DOGS; Behr, J-P, et al, Proc. Natl. Acad. Sci. 86, 6982-6986 (1989)].
In an effort to improve the properties of these early compounds for in vivo delivery of polyanions many more cationic lipids have been developed in which the nature and size of the hydrophobic backbone and/or the cationic head group have been varied (see for example International Patent Specifications Nos. WO95/21931, WO96/10038, WO96/17823, WO96/18273, WO96/25508, WO96/26179, WO96/41606, WO97/18185, WO97/25339, WO97/3010 and WO97/31934).
The goal in the development of cationic lipids for in vivo use is to provide a molecule which is simple to use in a clinical setting; which is robust; which forms small stable complexes over wide pH and ionic strength ranges; which is non-toxic; and which is capable of delivering a high concentration of polyanion to a cell.
We have now developed a class of lipid which meets these requirements. Importantly, our lipids are capable of self-assembly and will form stable complexes in aqueous solutions. The lipids are able to efficiently compact polyanions to give defined particle sizes of less than 500 nm. The lipid-polyanion complex remains associated over wide pH and ionic strength ranges and is able to efficiently deliver high concentrations of polyanions to cells.
Thus according to one aspect of the invention we provide a bipolar lipid comprising a cationic head (1) a hydrophobic backbone (2) and a hydrophilic tail (3) in which:
(A) the cationic head comprises two or more cationic centres, each centre being covalently linked to one or more others by one or more carbon containing spacer groups;
(B) the hydrophobic backbone comprises one or more hydrocarbon chains; and
(C) the hydrophilic tail comprises one or more hydrophilic hydrocarbons each containing two or more atoms or groups capable of being solvated by water;
each of said components (1) to (3) being covalently linked head (1) to backbone (2) to tail (3) and arranged such that at least one hydrocarbon chain in the hydrophobic backbone (2) is covalently linked to a carbon atom of a spacer group in the cationic head (1) and each hydrophilic hydrocarbon in the hydrophilic tail (3) is covalently linked to a chain in the backbone (2) to achieve at least a ten atom spacing along the chain between the tail (3) and the head (1).
In the lipids according to the invention, each cationic centre in the cationic head (1) may be provided by one or more heteroatoms capable of retaining a positive charge at a pH in the range from around pH 2.0 to around pH 10.0. In practice, whether a heteroatom will retain a positive charge in this pH range will depend on the nature and number of any other atoms or groups attached to it. Thus particular examples of suitable cationic centres include primary, secondary, tertiary and quaternary amino groups, sulphonium and phosphonium groups.
The number of cationic centres may be varied as desired depending on the intended use of the lipid of the invention. At least two centres will be present, but three, four, five, six, seven, eight or more may be optionally incorporated. More than one type of centre may be present, for example mixtures of amino groups may be accommodated, and/or sulphonium and/or phosphonium groups.
In one general preference each cationic centre is an amino group. Particularly useful amino groups include primary and secondary amino groups. The number of cationic centres in the cationic head (1) will preferably be from three to six.
Each cationic centre will in general be separated from any other centre by spacer groups arranged to link the centres in a linear (straight and/or branched) or cyclic fashion. The overall effect may be a cationic head (1) which has a straight and/or branched linear structure, a cyclic structure, or a mixture of straight and/or branched linear and cyclic structures. More than one type of spacer group may be present in a cationic head (1). Where desired a spacer group may form a terminal group on the cationic head (1), acting as a substituent on a cationic centre rather than a group connecting centres together.
Each spacer group will in general be non-ionic and contain at least one carbon atom. Suitable groups include optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic or heteroaromatic groups.
Particular examples of optionally substituted aliphatic spacer groups include optionally substituted C
1-10
aliphatic chains such as optionally substituted straight or branched C
1-6
alkylene, C
2-6
alkenylene or C
2-6
alkynylene chains.
Heteroaliphatic spacer groups include the aliphatic chains just described but with each chain additionally containing one, two, three or four heteroatoms or heteroatom-containing groups. Particular heteroatoms or groups include atoms or groups L
2
where L
2
is as defined below for L
1
when L
1
is a linker atom or group. Each L
2
atom or group may interrupt the aliphatic chain, or may be positioned at its terminal carbon atom to connect the chain to the atom or group R
1
.
Particular examples of aliphatic spacer group

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