Note: Descriptions are shown in the official language in which they were submitted.
~ ~V0 95/14381 ~ ~ ' ~ 2 ~ 7 6 7 1 5 PCT/US94/13428
A~P~IPT~TT TC DFRTVATTV~ OF GUA~INF,
CROSS-R~FFRF~C_ TO RF.T.ATF.l~ ,APPT.T~t.TlONS
S This application is a par~ of USSN 08/157,727, filed
November 24, 1993, which is a ~ ~^~ of USSN 07/991,935, filed
December 17, 1992, which disclosures are herein UIWl,~ ' ' by reference.
T~TRODUCTTO~
10 F1F~n OF TT~ INVE~r~
This invention relates to nitrogen containing l' ,' ' for use in the
of liposomes and other lipid~ontaining carriers of
D-ubstances, including nucleic acids used in gene therapy.
15 P.~ nDr~Tr~ OF TRP. INVE~1TION
Liposomes are one of a number of lipid-based materials used as biological
carriers and have been used effectivdy as carriers in a number of l
and other biological situations, I ' ',~, to introduce drugs, --r
agents, enz3rmes, viruses, i , ' factors and other cellular vectors into a
20 variety of cultured cell lines and anunals. Successful clinical trials have examined
_rf~.Wl~DD of ~i ' ' drug delivery for t^Drgeting
' drugs to specific tissues and specific cdl types. See, for
e,.: ~le, U.S. patent No. 5,264,618, which describes a number of techniques for
usirl~ lipid carriers, including the preparation of liposomes and L
, and the use of such: , in clinical situations. However,
'1 i t '~
WO 95/14381 -2 1 7 6 7 1 5 PCl[lUS94113428
while the basic ' ' 0~ for using 1- ' ' vectors is well
developed, ~... in the materials used in the methods, both in terms of
1ity and in terms of ~r~ of the carrier process, are still
desirable.
In particular, the expression of e~ogenous genes in humans and/or various
"~, important arlimals will ultimately permit the prevention and/or cure
of many important diseases and the ~ of animals with "y
important ~ Genes are high molecular weight, ~1~ molecules
for which carrier-mediated delivery usually is required for DNA ti ~ of
cells either in vitro or in viw. Therefore it is of interest to develop lipid
vectors which will enhance both the delivery and the ultimate
expression of the cloned gene in a tissue or cell of interest. Since in some
instances a treatment regimen will involve repeated - ' of a gene (or
other l ' ' product), it also is of interest that the lipid carricrs be
lS nontoxic to the host, even after repeated
RFT .r~.V~N r T .rr~R ~TITRI~.
Iiterature describing the use of liposomes as carricrs for DNA mclude the
following: tFriedmann (1989), supra; Brigham, et al., (1989) Am. J. Med. Sci.,
298:278-281; Nabel, et al. (l990) Science, 249:1285-1288; Ha~inski, et al. (l99l)
Am. J. Resp. Ccll Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc.
Natl. Acad. Sci. tUSA), 84:7851-7855); coupled to ~ p~;Plc, cation-based
trarlsport systems tWu and Wu (1988) J. Biol. Chem., 263:14621-14624) or the
use of naked DNA cxpression vcctors tNabel et al. tl990), supra; Wolff et al.
W095/14381 : 2 1 7 6 7 1 5 PCr/US94/13428
3
(1990) Science, 247:1465-1468). Direct injection of transgenic material into tissue
produced only localized expression (Rosenfeld (1992) supra); Rosenfeld et al.
(1991) supra). Brigham et al. (1989) supra; Nabel (1990) supra; and Ha~inski et
al. (1991) supra). The Brigham et al. group (Am. J. Med. Sci. (1989)
5 298:278-281 and Clinical Research (1991) 39 (abstract) have reported in YiYo
t. r '- restricted to lungs of mice following either ~ ui, or
- of a DNA liposome complex. &e also Stribling et al. Proc. Natl.
Acad. Sci. (USA) 89:11277-11281 (1992) which reports the use of liposomes as
carriers for aerosol delivery of transgenes to the lungs of mice and Yoshimura et
10 al. Nucleic Acids Research (1992) 20:3233-3240.
C.,~onic lipid carriers have been shown to mediate " ' delivery of
plasmid DNA (Felgner, et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416);
mRNA (Malone, et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081); and
purified i factors (Debs, et al., J. Biol. Chem. (1990)
15 265:10189-10192), in functional form.
SU~M~RY OF T~. TNVF~ ON
Non-toxic, novel,: , ' ' ' - derivatives of guanidine are provided as are
the methods of their use. The ,' ,' ' are capable of forming complexes
20 with nucleic acids, and other biological ~ and the nucleic acid
complexes are capable of ~ ~ ' cells. The , ' ' of the
invention are non-to~ic even when subjected to . ~ - enzymatic processes.
W095/14381 ` -` ` 2 1 76 7 1 5 PCT/US94/13428
DF~ RTIYrlON OF SPFCIFIC FMROD~MF~TS
r ~ b~ , ', ' " derivatives of guanidine are provided which are
useful as caniers for biologicaUy active molecules, such as antibiotics or nucleic
acids used in cell j r " processes. The use of the ' ' 1;,~ materials
5 as nucleic acid carriers is described in detail, since the ~l prepared
using the ' ' are ~ ul~ efficacious for this purpose. However, the
l' ,' ' are also useful in standard drug delivery regimens, such as for the
delivery of antibiotics to the lungs of a patient. In particular, complexes of the
, ' ' ' with DNA (for the j r '- of ceUs in " tissues) give
10 rise to reduced amounts of toxic cleavage products when subject to the metabolic
.l. c,~.l..;.... process
The invention in particular is directed to ' ,' ' derivatives of
guanidine which are nontoxic themselves and which yield by-products, such as
those produced by enz~nnatic cleavage, which a~e nontoxic to a host organism or
15 which are identical to substances ~ l- ,~,. -- ...~ to a host organism. These
,' ' ' thus offer the advantage that they can readily be used in humans,
since they can be used repeatedly without the ' of toxic by-products.
It wiU be apparent that the cations of the invention must be present in
association with one or more anions, e.g., hydroxide, chloride, or bromide ions or
20 more complex organic anions or bases. The particular anion associated with an
' ' ' cation is not critical to the formation or utility of the ' ' ' -
cation and may exchange (in whole or part) for other anions during use of the
/, the ' ' ' c ' of the invention are
described in this ~ ; - generally in terms of the cation without reference to
W095/14381 2 ~ 7 6 7 1 5 PCT/U594/13428
any particular anion. However, a number of specific examples are given, as well
as general guidance for selection of anions. For human ~ chloride is
the preferred anion; also acceptable are bromide or other ~ ;ologi~lly
acceptable anions including acetate, succinate and citrate. The cations are either
5 nontoxic themselves, and/or they yield by-products, for example, enzymatic
cleavage products, which are nontoxic to a host organism or which are
to a host organism. Generally, both the original lipids and their
products are nontoxic to a host organism.
The invention 1 'y relates to novel nitrogen-containing .l,l.;l,l.;l;
having the formula:
NH
~ ( 2)m ]n }2 NH2
wherein each R; ~ ly is a ! ' ',,- ' C~ , aliphatic l.~.L~l,.~l group of 5
15 to 29 carbon atoms inclusive, each X is -CH~- or -CO-, each m is an integer from
0 to 7 inclusive and each n is zero or 1, with the proviso that when n is 1, the
total number of carbon atoms in R and -(CH,)",- is at least 10, and when n is zero,
each R; i l~ l~ ly is a straight-chain, aliphatic l~ydlu~l)yl group of at least 11
carbon atoms inclusive. Preferred derivatives of the above formula I are those
20 wherein n is 1. Also preferred are those ~ r ~ of formula I wherein m is
from 1 to 5 inclusive, ~ uly 1. Also preferred are those derivatives wherein
each R , 'y has from 13 to 23 carbon atoms inclusive. The R groups
are saturated o} are I ' having one or more ethylenically I
linkages and are suitably the same or are different from each other. Also
wossil43sl ~ 1 7 6 7 1 5 PCT/US94/13428
preferred are those derivatives wherein X is -CO-, in which case illustrative R
groups together with the -CO- group to which it is attached (i.e., R-CO-) include
lauroyl, myristoyl, palmitoyl, stearoyl, linoleoyl, eicosanoyl, tricosanoyl and
- --Jyl (derived from the fatty acids of the . -., I L ~ , name: lauric,
5 myristic, etc.). Alternatively, X can be -CHr. When given system names for the
R groups alone, the c~ r names of the l~yd1u~b.~1 group derived from
lauric acid is undecyl; from myristic acid, tridecyl; from palmitic acid, pentadecyl;
from stearic acid, heptadecyl; from linoleic acid, cis,cis-~,ll h~ di~yl;
from eicosanoic acid, nonadecyl; from tricosanoic acid, dicosanyl; and from
10 ' yl, yl. This grouping of R groups is preferred when n is 1.
When n is 0, R is preferably the entire l1~d1u~b~1 portion of a fatty alcohol, such
as a lauryl, stearyl, or myristyl group.
In the -~ of the: . ' ' . ' of formula I wherein n is
zero, the compounds are of the formula
1~ (II)
NH
(R)2N - C - NH2
wherein R has the previously stated meaning. Illustrative of such compounds are
~0 N,N--I; :~'g ' : ' ~.ly named " ' ' .~' ',
N~N-dil~ ulidil1~ alt~ ti~.ly named: ' " ' yl~11il.~., and
N,N-di~y~ l,. ' . alt~ y named ~ d ~t~L~dd~l)amine. Other
illustrative compounds of the above formula II will be apparent from the formula
and the above meaning of R.
wo 95/14~81 ~ 2 ~ 7 6 7 1 5 PCTIUS94113428
In the ...~ i..,. of the ~ hirhii~ of formula I wherein n is 1, the
Wl_T_ are of the formula
(III)
NH
[R--X - O--CH2--(CH~ ]2N--C - N~2
wherein R, X and m have the previously stated meanings. Such . ' are
illustrated by N~ dit2-(~ fitu.~lu~.y)~lllyl]~ in~, ~It~..lld~ named
amidino-di[2-(1~ ....yluAy). ~l~l]amine, by N,N-di[2
(ol~vylu,.y)~..rllg ~~ ' alternative named
10 amidino[2-(9-u~ vylu~.y)~ .]amine, and by N,N--
di[~(i~t~u.~lu~y~ yl~g '~ ~ ly named
amidinodi[~ lu~.y`' yl]amme. Other illustrative compounds of
formula m will be apparent from the formula and the above meanings of R and m.
For cu~ n~e~ e, the ....~ . ' of the invention
by formula II can be viewed as N,N-di-R-guanidine derivatives amd the compounds
' by formula m can be visualized as N,N-di(R-carboxyaLIcyl`g '
derivatives, wherein R has the previously stated meaning, although as previously
stated the derivatives are not necessarily prepared from guanidine. In general, the
~nmro~ of formula m are prefelred over the: A ~ of formula ~.
There are a number of synthetic techniques im the art that have been
developed for the synthesis of ~ ' ; . '- A general synthesis that
can be used to produce . ' of the invention involves the conversion of a
.1;--1l ~ to a diacyl derivative (after protecting the amine), d~,.ut~Liù.~ of
the amine, and reaction of the resulting secondary amine with cyanamide in base
WO 95/14381 ` ' ` 2 1 7 6 7 1 5 P~ uS94/13428
to provide the desired proAuct. The initial i " ' - can be obtilineA
. ~Uy ( ' ' is readily available in quantity and is v~)
or csn be s~ ' by standard Ais1kyl ~ n reactions for the pro_uction of
æcondary amines from hydro~y-protected ~.~ h~ u~ " ," ' ' Omega-
S hy_roxyaUcyUlalida are themselYes available from the ~
1'-' ~dIUA~.W~ whieh can be readily prepared from ~' ' by ocidation
(e.g., with ozone) and ri duction. The acyl groups are available from the acid
halides (or anhydrides) of the ' ~ carbocylic acids, which, as previously
indicated, are preferably fatty acids and thus available ~ ".y.
On the other hand, æveral of the general methods are not suitable for
producing the: ' ' ' of the present invention. Fior example, the reaction of
secondary aliphatic amines with ' , ' wiU form the ~ ~ ' g
~ ' , which then has been repor~ to form a substituted guanidine on
reaction with ammonium chloride; however, the second step of this procedure did
15 not work for making the compounds described herein. It has also been reported
that aliphatic amines c,m also be reacted with cyanamide in either acetic acid or n-
but. nol to produce the r ~- guanidine derivative, although the reaction in
acetic acid did not work for making the compounds described herein. FinaUy,
re_ction of primary or secondary amines with 3,5: ;Ih.~ I
20 w~ nitrate in aqueous solution for several days has been shown to form
guanidines, although this method did not work for making the ~ 1, '
described herein. r~. either the method ou~ined in general above or
detiled examples provided in more detail in the Examples section below wiU be
sufficient to produce any of the ~ compounds witbin the scope of the
WO 95/14381 ` 2 t 7 6 7 1 5 PCr/US94/13428
~ .
invention.
The cationic lipids of the invention are typically used as carriers for various
biological molecules, such as antibiotics or nucleic acids. In particular, the
cationic lipids can be used alone or combined with other lipids im ' for
5 the I , of lipid vesicles or liposomes for use in - - delivery
systems. Uses ~ for the lipids of the invention include; r ~
procedures, r ~ to those presently known that use ~ ' lipids,
including those using ' cationic lipid I such as Lipofectin m~
and various other published techniques using Wll~ " ' cationic lipid technology
10 and methods. The cationic lipids of the invention can be used in 1'
to deliver therapeutic agents by various routes and to various sites in
an animal body to achieve a desired therapeutic effect.
Because such techniques are generally known im the ar~
; r ~ and basic techniques for the l~lC~/~UaliUI- of IJ~
15 ~ , containing lipids will not be repeated at this time. A reader
unfamiliarwith this I 'c~ ' ' is referred to theI ' unde}
the headimg Relevant Literature above and fur~her to U.S. Patent No. 5,264,618.
This last-cited patent describes a number of therapeutic ' ' and methods
in detail, including examples of the use of sp~cific cationic lipids (different from
20 those described here) that can be followed im detail by ~ the cationic
lipids of the present invention for those described in the patent. (' of
the present invention will minimally be useable in the manner described in the
patent, although operating parametcrs may need to be modified in order to achieYe
optimum results, using the specific ' provided for , ' of the
WO 95/14381 , 2 ~ 7 6 7 ~ 5 PCT/US94/13428 ~ -
invention in this ~ ... along with thc knowledge of a person skilled in the
arts of lipid prepaTation and use.
The lipids of the present invention are ~ , useful and ad~. ~
in the i ' of animal ceUs by genetic material. AdditionaUy, since these
A are non-toxic even when subjected to host enzymatic reactions, the
provide a number of advantages in the area of low toxicity when
compared to previously known cationic lipids. These and other advantages of the
invention are discussed in detail below. The remainder of this discussion is
directed principaUy to selection, L ' " , and use parameters for the cationic
lipids of the present invention that may not ~, be apparent to one of
ordinaty skiU in the art.
r ~, where it is desirable to target a lipid-DNA complex to a
particular ceU or tissue, a lipid mixture used as a carrier can be modified in avariety of ways. By a lipid mixture is intended a r ~ " prepared from the
cationic amphiphile of the invention, with or without additional agents such as
steroids, and includes liposomes, interleaved bilayers of lipid, and the lilce.
Steroids, e.g. cholesterol or ergosterol, can be used in ' with the
cationic ' ,' when used to prepare mixtures. In some; b~ " the
lipid mi cture wiU have from 0-67 mole percent steroid, preferably about 33 to 50
mole percent steroid. A lipid-DNA complex is the ~ obtained foUowing
' of DNA and a lipid mi cture. Non-lipid material (such as biological
molecules being delivered to an animal or plant ceU or ~ ~;r~ moieties)
can be conjugated through a lin~ing group to one or more h~.' l' ' groups,
e.g. using aUcyl chains containing from about 12 to 20 carbon atoms, either prior
~ W095/14381 2 1 7 6 7 1 5 PCT/US94113428
or subsequent to vesicle formation. Various linking groups can be used for join~ng
the lipid chains to the compound. r. of particular interest include
thioethers, disulfides" I ' : " ~ ethers, and the like, used
i~divid~Uy or in ~ ' The particular manner of linlcing the compound to
S a lipid group is not a critical part of this invention, as the literature provides a
great variety of such methods. Alternatively, some ' wiU have
h~ ' regions or domains, which wiU aUow for their association with the
lipid mi~;ture without covalent linking to one or more lipid groups.
For the most part, the active ~ ' to be bound to the lipid mixture
10 are ligands or receptors capable of binding to some biological molecule of interest
that is pres-~t in the target ceU. A ligand can be any compound of interest which
can s, ~ "~, bind to another compound, refer~ed to as a receptor, the ligand
and receptor forming a y pair. The active: ' bound to the
lipid mi~ ture can vary widely, from smaU haptens (molecular weights of about 125
15 to 2,000) to andgens which will generaUy have molecular weights of at least about
6,000 and generaUy less than about 1 million, more usuaUy less than about
300,000. Of pardcular interest are r ~ ' ligands and receptors that have
specific ~ binding partners on oeU surfaces. Illustrative active
' include chorionic ~ ' ' endorphin, luteinizing
20 hormone, morphine, , ,' interferon, ACTH, and ~l~;~dulh~ and
fragments of such , that retain the aoility to bind to the same ceU-
surface binding partners that bind the original (non-fragment) molecules.
The number of targeting molecules (either ligand or receptor) bound to a
lipid mixture wiU vary with the size of the liposome, the size of the molecule, the
WO 95/14381 ~ ; 2 1 7 6 7 1 5 PCT/US9411342~ ~
binding affinity of the molecule to the target cell receptor or ligand, and the like.
Usually, the bound active molecules will be present in the lipid mixture in from
about 0.05 to 2 mole percent, more usually from about 0.01 to I mole percent
based on the percent of bound molecules to the total number of molecules
5 available in the mixture for binding.
The surface membrane proteins which bind to specific effector molecules
(usually soluble molecules in the e~ternal ~ of the cell) are referred to
as receptors. In the present context, receptors include antibodies and
~ 0 ' - since these molecules are foumd on the surface of certain cells.
10 However, since antibodies are generally used to bind liposomes to receptor
molecules on target cells, the antibodies and ~ ' bound to a liposome
containing a cationic lipid of the invention can also be considered to be ligamds.
The l~, ' may be or polyclonal, preferably
Usually the ~- ' " wiU be IgG and IgM, although the other
~, ' ' may also find use, such as IgA, IgD, and IgE. The intact
~ O~ ' may be used or only fragments thereof, such as Fab, F(ab')2 Fd,
or F~, fragments as well as a complete light or heavy chain.
For antibodies used as cell-targeting ligands, antibodies of interest are those
that bind to surface membrane antigens such as those antigens comprising the
20 major l ' "~/ complex, ~culi~.uLL~ly the HLA-A, -B, -C amd -D. Other
surface amtigens include thy-l,leu-5, and la.
The cationic: , ' ' ' are ~ , useful as carriers for anionic
pol,~ ~ ~ such as nucleic acids.
Where the ~ are intended for use in vivo, ~ , in vivo in
~ WO 95/14381 ~ 2 ~ 7 6 7 1 5 PCTNS94/13428
humans, or where it is necessary to use the ' ' ' repeatedly, it is important
to screen the carriers for those which are ' ' ' to non-toxic by-products and
which themselves are not toxic or those which are eliminated from the body
without ~ ;..., The elimination of such cationic ~ ', ' from tissues
S can be d--- ' in animal; . An animal, such as a mouse, can be
' one or more doses of material containing between 0.5 and 10 pmole
of the lipid to be tested, complexed with an active component (such as DNA) if
desired. At various times after ' the animals are sacrificed, tissues
taken, total lipids extracted using an r~ solvent extraction system, and the
total lipid analyzed for the particular cationic lipid or its partial .1. c,~.l ~;.~
product using, for example, ~IPLC.
The cationic: , ' ' ' are positively charged, and a tight charge
complex can be formed between a cationic lipid carrier and a ~1~ nucleic
acid, resulting in a lipid ~1~ ~ ' acid complex which can be used directly
for systemic delivery to a mammal or ' cell. Where delivery is via
the charge complex will withstand both the forces of ~
and the o l~ within the lung air vays arld be capable of ~ , lung
cells after the ar~c~li7~ DNA:lipid carrier complex has been deposited in the
lung following imtranasal or intraoral delivery of the aerosolized complex.
To evaluate the efficacy of a particular cationic amphiphile for use a a
nucleic acid carrier in an ,.~ ;.... process, as well as to determine the
optimum of lipid carrier-nucleic wid complexes, involves a tvo-
step process. The first step is to identify lipid carriers and the . of
lipid carrier-nucleic acid complexes that do not aggregate when the
W09S/14381 ~ 2 ~ 767 1 5 PCT/US94/13428
are combined or during the significant agitation of the mixture that occurs during
the b~ step. The second step is to identify among those lipids that do not
aggregate those complexes that provide for a high level of i ' and
t~ ;' of a gene of interest in target oells in the lung. These techniques are
5 described in WO/US PCT/US92/11008 filed December 17, 1992, which disclosure
is hereby , ' by reference.
As an example, a reporter gene CAT (which encodes '
-' r ) can be inserted in an expression cassette and used to evaluate
each lipid carrier ~ of interest. The DNA:lipid carrier complexes are
10 mixed in solutions which do not themselves induce i6L.~ ;.... of the DNA:lipid
carrier complexes, such as sterile water. The expression cassette (DNA) is mixed
together with each of the lipid carriers to be tested in multiple different ratios,
ranging as an example from 4:1 to 1:10 ~ " of DNA to nanomoles of
cationic lipid or total ]ipid, if a lipid mixture is present). r of the
15 stability of the resulting mixtures provides - concerning which ratios
result in s~ 6~ ' of the DNA:lipid carrier complexes and are therefore not
useful for use in vivo, and which complexes remain in a form suitable for
" ,~ ;" The ratios which do not result in 5i~ 6,-~ are tested in animal
models to determine which of the DNA:lipid carrier ratios confer the highest level
20 of transgene e~pression in vivo. For example, for aerosol-based, ~ . the
optimal DNA:lipid carrier ratios for lipid mixtures such as N-[1-(2,3-dioleyloxy)-
propyl]-N,N,N-l~h,a..~' chloride(DOTMA);~Iiulw~
amine(DOPE) (the A ' of this mixture being present in a 1:1 weight ratio)
and dimethyl dioctadecyl ammonium bromide (DDAB):Cholesterol (1:1) are 1 to
~ wogs/14381 2 1 7 6 7 1 5 PCT/US94/13428
1. ~or O~thyl egg 1 ' ,' '~, ' ' (~EiPC) or especially O-ethyl
tU~1,' , ' '~; ' ' (E DMPC) in a 1:1 weight ratio with, ' ' 1,
the DNA:lipid carrier ratio is preferably in the range of from 1.5:1 to 2:1.
If the cationic amphiphile is used for injection, then it need be evaluated
5 only for whether it is effe~tive for i ~ of a target cell.
Particular cells can be targeted by the use of particular cationic lipids for
of the lipid-mixture carriers, for ex~unple, by the use of ~DMPC to
target lung cells r ' " Iy, or by modifying the ,', ' to direct them tû
particular types of cells using ! ' ; ~ molecules. Thus antibodies or ligands
lû for particular receptors may be employed, to target a cell associated with a
particular surface protein. A particular ligand or antibody can be conjugated to
the cationic amphiphile in accordance with co~ l techniques, either by
~ . the site-directing molecule to a lipid for; ~ into the lipid
bilayer or by providing a linking group on a lipid present in the bilayer for linking
15 to a ~ ' ~ of &e site-directing compound. Such techniques are well known
to those skilled in the art.
The various lipid carrier-nucleic acid complexes wherein the lipid carrier is
a liposome are prepared using methods well known in the art. Mi~ing conditions
can be optimized by visual of the resultant lipid-DNA mixture to
20 establish that no ~ occurs. To make the lipid-DNA complexes more
visible, the complexes can be stained with a dye which does not itself cause
'~.e" 6~1;"'', but which will stain either the DNA or the lipid. For example,
Sudan black (which stains lipid) can be used as an aid to examine the lipid-DNA
mixture to determine if ~ has occurred. Particle size also can be studied
WO 95/14381 , ; ' ` . ~ 1 7 6 7 1 5 PCT/U594/13428
16
with methods known in the art, including dectron , y, laser light
scattering, Coulter'Y ~ g, amd the like. Standard-size beads can be
included as markers for ~' _ the size of any liposomes or aggregates that
form. By "lipid ~l;~- ' - acid complex~ is meant a nucleic acid se~uence as
5 described above, generally bound to the surface of a lipid carrier l t~ as
discussed below. The lipid carrier preparation can also include other substances,
such as enzymes necessary for _ ~,; and translation or
cofactors. r, the lipid carrier-nucleic acid complex can include
targeting agents to deliver the complex to parlicular cell or tissue types.
lO GeneraUy, the nucleic acid material is added to a suspension of preformed
liposomes which may be " ' " vesicles (~Vs) or small ~ ' "
vesicles (SWs), usually SWs formed by sonication. The liposomes themselves
are prepared from a dried lipid film that is "- '`l 1 .) im an 3~r ~ ' mixing
solution such as sterile water or an isotonic buffer solution such as lOmM
l5 Tris/NaCl or 5 % dextrose in sterile water amd sonicated to form the liposomes.
Then the preformed lipid carriers are mLxed directly with the DNA.
Mixing and preparing of the lipid-DNA complex csm be critically affected
by the sequence in which the lipid and DNA are combined. Generally, it is
preferable (to minimize 'L,L'` L'";"") to add the lipid to the DNA at ratios of
20 DNA:lipid of up to 1:2 inclusive (~ DN~ ' cationic lipid).
Where the ratio of DNA:lipid is 1:4 or higher, better results are generaUy
obtained by adding the DNA to the lipid. In either case, mixing should be rapidly
achieved by shaking or vortexing for small volumes and by use of rapid mixing
systems for large volumes. The lipid carrier amd DNA form a very stable
Wo 95114381 ` ~ 2 1 7 ~ 7 1 5 PCT/Uss4/l342s
complex due to binding of the negatively charged DNA to the cationic lipid
carriers. SWs flnd use with small nucleic acid fragments ~s well as with large
regions of DNA (2250kb).
~ preparing the lipid carrier-nucleic acid complex for care
S should be taken to exclude any compounds from the mixing solution which
promote the formation of aggregates of the lipid carrier-nucleic acid complexes.
I~ge particles generally will not be aerosolized by the nebuliær, and even if
~ ,. .1; . .1 would be too large to penetrate beyond the large airways. A~r~g~inn
of the lipid carrier-nucleic acid complex is prevented by controlling the ratio of
10 DNA to lipid carrier, - - ~ the overall, of DNA:lipid carrier
comple~ in solution, usually less than S mg DNA/8 ml solution, and avoiding the
use of chelating agents such as EDTA andlor significant amounts of salt, either of
which tends to promote ~L~' ' L~ ;"" The preferred excipient is water,
d~llu~J~._ or another solution having low or æro ionic strength. Further, the
15 volume should be adjusted to the minimum necessary for deposition in the lungs of
the host mammal, while at the same time taking care not to make the solution too
' so that aggregates form. Increasing the volume of the solution is to
be avoided if possible due to the need to increase the inhalation time for the host
animal to ' the increased volume. In some cases, it may be preferable
20 to Iyophilize the lipid carrier-nucleic acid complexes for inhalation. Snch
materials are prepared as complexes as described above, except that a
~ly~l such as mannitol or trehalose is included in the buffer solution
which is used for ~ of the lipid carrier-DNA comple~es. Any glucose
generally included in such a buffer is preferably omitted. The lipid carrier
WO 95/14381 ~ ~ 7 ~ 7 ~ 5 PcTluS94/13428
complex is rapidly freeze dricd following mixing of the lipid and DNA. The
mi~ture can be lc ' with sterile water to yield a ~ which is
ready for to a host animal.
Where the - .i h;.~ form liposomes, the liposomes may be sized in
5 accordance with c~,...wlliul~l techniques, depending upon the desired size. In
some instances, a large ]iposome injected ir~to the ' ' ' of an animal has
. igher affmity for lung cells as compared to liver cells. Therefore, the particular
size range may be evaluated in accordance with any intended target tissue by
~ i acid complexes of varying particle sizes to a host
animal and ~ the size of particle which provides the desired results.
The cationic ,' ' ' comple~ed with nucleic acid of this invention can
be - ' ' in a variety of ways to a host, such as
y~ topically, ~ ".~.,
" by aerosol, following ' and th~e lil~e. Normally, the
15 ,' ' ' will be injectcd in solution where the . of compound
bound to or entrapped in the liposome will dictate the amount to be
This amount will vary with the ~rr~,i._.~ of the compound being ~ ' l,
the required - for the desired effc^ct, the number of: ' s,
and the li~e. In some inst~nces, ~ for aerosol ' the
20 lipid-DNA comple~es can be ' ' in the fonn of a Iyophilized powder.
Upon _ ' of the ~ , ' ' ' when a targeting moiety is used,
the ' ,~ l~c ~ F ,~ -t; 11y bind to a cell surface factor ' y to the
' bound to the liposome. If no targeting moiety is bolmd to the
liposome, then it binds to cell surface by lipophilic The liposomes
2 ~ 767 ~ 5
u~o 95/14381 ~ . PCT~S94113428
: :;
19
normally are transferred mto the cell by ClldC~
The cationic ,', ' IP~ find use for . ' ~ with nucleic acid or
protein for t~ , .. these ' ' in vivo. The nucleic acid can
include DNA, RNA, antisense RNA or other antisense molecules. Cationic
5 ,~ that form liposomes also find use in drug delivery, wherc the drug
can bc entrapped within the liposome or bound to the outside.
Il~e followin~ e~amples are offered by way of illustration and not by way
of limitation.
- . .J
wo 95/14381 2 1 7 o 7 1 5 PCT/US94/l3428 ~ .
~XAMPI
~xam~le 1
(a) Pr~ara~iQn of N-~mir~inn-O O-di~y~ ",~lA",;",~
OH OH
~ BOC2O ~ : ~ RCOCI/NEt
HN BOCN ~ 3
\~\ \
OH OH
OCOR OCOR
~/ HCI/Dy ~/ H N-CN
BOCN - ~ HCI HN 2
\~ \~
OCOR OCOR
3 4
OCOR
/
HCI H N-C-N
NH \~\ OCOR
Commercially available ll;r~ "r,l,"";"f was protected on nitrogen using
di-tert-butyl-l,JIu, ~IIJ~ r, acylated with appropriate acyl chloride, N-BOC
protection group was cleaved with 4M HCI in dioxane, r~sulting hydrochloride
salt was reacted with cyanamide in n-BuOX to afford the desired
20 N-amidino-O,O-diacyl,l;. I~lll~lrllll,~r
Example: Synthesis of N-amidino-O,O-dipal",ituylrl;rl:,~,lnl~minr~ 5.
N-BOC r~iP~h~nnl~minr~_
To a solution of 10 ml (0.1 mol) rlir~ih~nnl7minr~ in 1~0 ml of acetonitrile
were added 22.8 g (0.105 mol) of di-tert-butyl-~ uc~u"~ and mixture was
SUBSTITUTE SHEET (RULE Z6)
~ W095/14381 ~- 2176715 PCT,US94"3428
21
stirred at R.T. overnight. The resulting solution was e ~ the residue
dissolved in ethyl acetate/hexane (7/3) and passed through a plug of silica. After
of solvent to get 16.4 g (80%) of N-BOC ~' ' '
S N-BOC ester :~.
To a solution of 2.0 g (0.0097 mol) of_ in 100 ml of CH2CI2 at OC were
added 3.4 ml (0.024 mol) of ~ a.J' , then in 10 min with stirring were
added 6.2 ml (0.02 mol) of palmitoyl chloride. The mixture was stirred at OC
for 30 min, then at R.T. for 45 min. The resulting solution was washed with 10%
10 citric acid (50 mlx2), with 10% aqueous solution of sodium 1.;~ (50 mlx2),
dried over MgS0~, filtered, filtrate evaporated on rotavapor and the rest was
' . ~ , ' ' on silica gel using 0-15% EtOAc/Hexane to get 6.4 g (95%) of
N-BOC ester ~.
15 Amino ester 4.
To 3.5 g (0.086 mol) of N-BOC ester ;~ were added 20 ml of 4M solution
of HCI in dioxane and mixture was stirred at R.T. for 2 hrs. The resulting
suspension was evaporated on rotavapor, diluted with ether (50 ml), filtered,
washed with ether (25 mk2) and dried in vacuum to get 3.1 g (97%) of amino
20 ester .
Amidino ester 5.
To 0.22 g (0.00036 mol) of amino ester 4 was added 0.5 ml of n-BuOH
and 0.022 g (0.00054 mov of cyan unide. The mixture was stirred at 110C for I
- i~
WO 95/14381 ' ' ' 2 1 7 6 7 1 5 PCIIUS94/13428
22
hr, diluted with chloroform (lOml) and evaporated on rotavapor. White precipitate
formed was washed with ether (lOml x 2), then with water (lOml x 2) on filter
and dried in vacuum to get O.l90 g (79%) of amidino ester _.
5 (b) T r ~ r~ m~ N ' ~ 0.0-
npDE~. N . o.o-~ vT'i '1~
(AnODE~. ' rT~ Ans).
Liposomes cont~uning ADODE, ADPDE or ADS m a 1:1 molar ratio with
cholesterol were tested as DNA carriers for gene transfer and expression in mice.
10 The plasmid used was pZN51. The methods and plasmids used are described in
more detail in W093/24640. The liposomes were im a lOmM stocl~ in 5%
dextrose. The li~ , ' DNA ratios were screened for the presence of
'LL'`~;~I;'''' Ratios from 1:2 to 1:7 ~Lg plasmid DNA to nanomoles cationic
lipid) were screened. DNA:]iposome ratios that did not prcduce ~c~- a~ were
then tested in mice. 100 ~Lg of pZN51 was comple~ed to 500 nanomoles of
DDAF ' ' liposomes as a positive contrcl and an uninjected mouse served
as the negative control (N).
ICR female m. iCe (25 g) were used for the in YiYo studies. A dose of 100
~Lg plasmid DNA in 200 ILl 5% de~trose in water was injected by tail vein per
20 mouse.
The lung, heart, liver, kidney and spleen were removed after 24 hours.
Each organ was ~ ~ ' in 0.3 ml of 0.25 M Tris-HCl pH7.8, 5 mM
EDTA, amd the resulting extract was centrifuged and then subjected to 3 cycles of
~ :' and then treated to 65 C for 20 min. The prctein of
~ Wo 95/14381 2 ~ 7 6 7 1 5 PCTluS94/1342s
23
IUDg, heart, liver and kidney extracts were quantitated UsiDg a ~
protein assay (13io-Rad, Berkeley, CA), and salDe amount of total protein from
each tissue extract was added in the CAT assay, together with 10 fll of 20 mM
acetyl CoA+12 ~1 of l~c ~ ~ ' (25 ~Ci/ml, SS mCi/mmole,
5 Amersham)), at 37 C for 13 hrs.
ADODE:CHOL liposomes in a 1:6 ratio produced the highest levels of
CAT activity iD the lung, liver and heart. However, CAT activity was lower than
that produced by DDAB:CHOL in a 1:5 ratio. There was almost no CAT activity
iD the kidney and spleen in ADODE:CHOI,treated mice.
ADPDE:CHOL liposomes in a 1:6 ratio produced the highest levels of
CAT activity in the lung, heart, liver, kidney and spleeD. The level of CAT
activity was similar to DDAB:CHOL in a 1:5 ratio in these orgaDs.
ADS:CHOL liposomes in a 1:5 ratio produced the highest levels of CAT
activity iD the lung and liver. The CAT activity was lower thaD that produced by
15 DDAB:CHOL at the 1:5 ratio. ID heart, spleen and l~idney ADS:CHOL produces
lit~e to no CAT ac~ivity.
All ~ hl;. ~';.. ` and patent ~ mentioDed iD this ~ are
herein I ' by reference to the same exteDt as if each individual
0 publication or pateDt application was "~;r~lly and ~.1;~ , indicated to be
' by reference.
The invention now beiDg fully described, it will be apparent to one of
ordiDary sl~ll in the art that many changes aDd "~ - can be made thereto
without departing from the spirit or scope of the appended claims.
